Note: Descriptions are shown in the official language in which they were submitted.
This invention relates to the stabilization of radiolabeled
compounds, such as amino acids and nucleosides, and more par~icularly,
to stabilization of such compounds by the addition of soluble and
insoluble compounds to solutions of radiolabeled compounds.
This application is a division of Canadian Patent Application
Serial No. 356,336, filed December 8, 1930.
An increasing number of radiolabeled compounds are being
used in research, for medical diagnosis, and various other areas.
However, the radiolytic decomposition of such compounds has been a con-
stant problem. ~ithout the addition of some type of stabilizer7 a
solution of such a compound may become unusable due to decomposition
within a matter of weeks or less. This radiolytic clecomposition of
such compounds has been studied extensively. For example, the
radiation chemistry of amino acids is reviewed in an article by J.
Liebster and J. Kopeldova, Radiation Biol., 1, 157 (1~64) and the
self-decomposition of radioactively labeled compounds is discussed in
Atomic Energy Review, 10, 3-66 ~1972). While certain specific compounds
have been suggested for stabilization, problems still exist. The latter
article reviews the underlying causes and mechanisms of self-decompo-
sition, "which are very complex and in some cases not well understood."
(At p. 3). After discussing the principal mechanisms by which decom-
position occurs, the article notes generally at page 36 that buffers
such as ammonium bicarbonate help to stabilize radiolabeled compounds,
but care must be taken to insure that the buffer chosen does not
interfere with the later use of the labeled compound. For example,
phosphate buffers would interfere with phosphorylation reactions.
Other compounds which have been suggested at various times are listed
at page 35 and include benzyl alcohol
glycerol, cysteamine and sodium formaten However, each of
these are said to suffer due to their difficulty of removalO
Another compound mentioned is ethanol which is said to work
with many compounds. However, ethanol sometimes actually
sensitizes certain nucleosides to radiation decomposition
and thus it has been found not to be a universal panacea~
Furthermore, if it will interfere with the reaction in which
the radiolabeled compound lS to be u.sed, the ethanol must
be removed by evaporation which may also contribute to
decomposition.
Various compounds are sugges~ed in Atomic Enerqy
Review, above, for stabiliæation of radiolabeled compounds
prone to oxidation including antioxidants such as butylated~
hydroxytoluene, butylated-hydroxyanisole and mercaptoethanol.
While not mentioned for use with radiolabeled compounds, the
inhibition of autoxidation generally by certain amines has
also been described in the prior art. Recent reviews on the
inhibition of autoxidation are "Autoxidation" by R. Stroh,
p~ 1049 in Methoden der Or~nischen Chemie (Houben-Weyl),
ed. E. Muller and O. Bayer Vol. IV/Ib Oxidation Il~,
Georgthieme Verlag, 1975, and Encyclopedia o-f Chemical
Technolo~y, Kirk Othmer, Interscience Publishers, New York.
The prior art teaches the utility of secondary dialkyl
amines bearing full alpha-substitution (i.e~, containing
no hydrogens on the carbon atoms adjacent to the nitrogen)
and secondary diarylamines (also without alpha-hydrogens) as
antioxidants. However, the prior art does not teach the use
of primary, secondary and tertiary amines, those containing
alpha-hydrcgens, in this regard and in fact suggests that
they are not effective for this purpose~ However, such anti~
oxidants have many of the same problems as other of the
5 ~
~ompounds discussed above, including in addition generally
being insoluble in the solvents used to di~solve and store
radiolabeled compounds for use in biolo~ical studies
Accordingly, th~ra has been a continuing need for
alternati~es to the stabilizers known in the prior art
The synthesis and use of polystyr~ne supported
reayents for solid phase peptide preparation i~ k~own in the
t prior art. R. B. Merrifield, J. Am, Chem. Soc., 85, 2149
s (1963~; Crowley et al, Ac~t~. Ch,_m. Res., 9, 135 (1976),
G. Ro Stark (1971), Biochemical Aspects of Reactions on
501id Supports, Academic Press, ~.Y., U.S. Patents ~,9~8,293
and 4,029,706 describe thio~ydrocarbon polymers and their use
a~ borane chelators, However, neither the synthesis nor use
of the insoluble compounds of the present invention ~re
described in anly o thP above.
In accordance with the invention there is proYided a
~; '' .
compound for stabilizing a solution of a radiolabeled organic
compound that is subject to radiolytic degradation against
said degradation comprising a substantially insoluble resinous
substrate having pendant quaternary ammonium and sulfide groups.
In another aspect of the invention there is provided
a method of making the compounds of the invention which com-
prises reacting an alkyl thiol and a trialkylamine with a sub-
stantially insoluble resinous substrate having a reactive site.
In still another aspect of the invention there is
provided a composition comprising a solution of a radiolabeled
organic compound that is subject to radlolytic degradation,
said solution being maintained in contact with a compound hav-
ing a quaternary ammonium group bound to a substantially in-
soluble substrate, thereby stabilizing said radiolabeled
organic compound against said degradation.
- 3 -
35~
There is disclosed herein a meth~d of stabilizing a
~olution of a radiolabeled compound comprising adding to such
solution a compound having a substantially insoluble backbone,
preferably a resin, such asan ion exchange resin9 to which
has been bound a quaternary ammonium group; or a water soluble
primary, secondary or tertiary aliphatic amine which does not
interfere with ~he use contemplated for the particular radio-
labeled compound so stabilized. There is also disclosed a
solution of a radiolabeled compound maintained in contact
with such compound7 and as an artîcle of manufacture, a
sealed vial. containing such a solution~
It has been found that various amines and
quaternary ammonium compounds stabilize solutions of
radiolabeled compounds. A pri.or application, describes
the stabilization of radiolabeled
i~,7
~ 3 ~1 --
s~
compounds by maintaining a solution of such a labeled com-
pound in contact with a compound having a quaternary a~nonium
group bound to a substantially insoluble backbone Even
before the earlier application was filed the present inventors
had discovered that certain soluble amines would act as
stabilizers of radiolabeled compound~. That earlier
application describes one specie.
While originally believed to not only stabilize
solutions of radiolabeled compounds, but to do so without
adulterating such solutions, further studies using both
soluble am1nes and insoluble quaternary ammonium containing
compounds have shown that such quaternary ammonium containing
compounds stabilize such solutions at least in part by the
release of soluble amines. For example, the polystyrene-
divinyl benzene copolymer substituted by triethyl ammonium
as described in Example VII below provides stabilization, at
least in part, by the release of soluble triethyl amine, a
tertiary aliphatic amine. At the same time, it has also been
found that depending on how the radiolabeled compound is to be
used, the presence of such soluble amines is compatible with
the ultimate use for th~ labeled compound. While care must
be exercised to insure that the particular stabilizer does
not interfere with the ultimate use for the radiolabeled
compound, it has been found that soluble amines are generally
useful for stabilizing 301utions of such radiolabeled com-
pounds in the 9ame manner as amines which are released into
~olution from insoluble sub~trates to which they have pre-
viou~ly been bound. For the 3ake of convenlence, the species
~ill be described separately for the most part, although it
should be recognized that all the species stabilize, at least
in part, by virtue of their presence in solution.
r~
~ he substantially insoluble quaternary ammonium
containing compounds disclosed herei~ can be any o~
those well known in the prior art. A preferred class of
such compounds is that prepared by attaching quaternary
ammonium groups to an insoluble polystyrene-divinylbenzene
copolymer backbonel e.g., a copolymer formed by copolymerizing
about 1% to about l~/o by weight of divinylbenzene with
styreneO The preparation of such copolymers i5 well known in
the prior art and they are sold commercially for use as anion
exchange resins. Such commercially available copolymers are
! preferably treated by washing with ethanol and then methylene
chloride, followed by drying before being employed to
stabilize radiolabeled compounds~
The nitrogen of the quaternary ammonium group is
preferably substituted by hydroxyalkyl or alkyl chains,
preferably lower alkyl, and more preferably of from 1 to
5 carbon atoms.
The counterion (anion) of both the soluble and in-
soluble quaternary compounds of the present invention can be
any of those known in the prior art which do not significantly
detract from the stabilization provided by the quaternary
~mmon;um group, Preferred counterions are halides and
alkyl carboxylate ions of one to five carbon atoms, such as
formate and acetate. A particularly preferred counterion is
chloride.
Particularly preerred for use in the present
invention æ e novel compounds prepared by attaching a sulfide
or thiol group to the substantially insoluble stabilizing
compounds described above. Preferred sulfide groups are those
substituted by an alkyl group of one to five carbon atoms.
Insoluble ammonium sulfide compounds have been found to be
particularly effective in stabilizing solutions of radio-
labeled compounds.
s(~
With respect to amines and quaternary ammoniumcompounds soluble in the aqueous solutions usecl to store
radiolabeled compounds for biological use, it has been found
-that such compounds may be ~elected fxom the group consisting
of.
a) RNH2'
b) RR NH,
c) RRlR ~,
d) R3R N-R-~R3R4, and
e) R3~4N - I Rl-NR R
I 1 3R4
NR R n
and quaternized salts thereof, wherein R, Rl and
R2 are the same or different and are alkyl, cycloalkyl,
alkenyl, hydroxyalkyl, aminoalkyl, thioalkyl, carboxyalkyl,
or keto alkyl of one to eight carbon atoms, or R and
of RRl~H are combined to form a chain ~f foux to 5iX
carbon atoms at ached at each end to N, or one of the
carbon atoms of the chain may be replaced by 0 or N,
R3 and R are the same or different and are hydrogen
or R, R or R ; and
n is an integer between 0 and 1000.
Preferred soluble amines include triethylamine, tris
(hydroxymethyl) methyl amine, hydroxyethylpiperazine ethyl
sulfate, morpholine ethyl sulfate, and quaternary salts
derived therefrom, p~rticularly preferred such salts being
triethylamine hydrochloride and tris (hydroxymethyl) methyl
ammonium hydrochloride.
While any amount of the st~bilizer compounds
described above is beneficial in preventing the decomposition
of radiolabeled compounds, in the case of the quaternary
~ 6 --
s'~
ammonium subst~ntially insoluble stabilizers it is preferred
that the nitrogen be in excess equivalents of between about
and about 10 , more pre~erably between about 10 and 10,
and most preferably about 10 . When a sulfide or thiol
group is attached to the backbone, it is preferred that the
sulfur be present in equivalent excesses between about 10
and 105, preferably between about 103 and about 104, and
most pre-ferably about 104. By equivalent excess is meant,
an excess of the respective atom, e.g., nitrogen, in the
stabilizing compound over the equivalents of the radiolabeled
compound. Similarly in the case of soluble compounds, it is
preferred that the stabilizing compound be present at con-
centrations between about 1 millimolar through about one
molar depending on the specific activity of the radio-
labeled compound, the concentration of khe radiolabeled
compound in the solution, and the particular radioisotope
being employed as the label. In general, it is preferred
that the concentration of stabilizing agent be 103 to 10
times the concentration of labeled compound. For example,
a tritiated compound with a specific activity of 100 Ci/rnMole
would preferably contain between about 10 and about 20 mM
concentration of amine; a 10 excess. Sirnilarly, if the
label u~ed is phosphorus-32 which might produce a specific
activity of 1000 Ci/r~Mole, a one molar ~oncentration of
ar~ne would be preferred, a 105 excess.
In addition to the use of any of the above described
amines alone, in some instances, it is preferred to use the
soluble amines in combination. Pre~erred cornbinations are
tris ~hydroxymethyl) methyl arnmonium hydrochloride and either
ethylenediamine-tetraacetic acid (EDTA~ or diethylenetriamine-
pentaacetic acid (~ETAPAC). Such combinations are parti-
7 --
35(~93
cularly preferred for stabilizing the storage of nucleotides
and preferably employed in a ratio of about 10:1 of the
former to the latter with the overall ratio of labeled com~
pound to stabilized compound being as above. EDTA and
DETAPAC are also known to function as chelators of metal
ions and thus cannot he used when the stabilized cornpound
is to later be used in a reaction where chelation of metal
ions must be avoided.
The method disclosed herein can be used
with any o~ the solvents typically used to store radio-
labeled compounds such as water, ethanol, mixtures of water
and ethanol in any ratio, benzene, hexane, dilute mineral
and organic acids, and other such solvents employed in the
prior art~
The present invention can be used to prevent the
decomposition of radiolabeled compounds which have been
labeled with any of the radionuclides used for such purposes,
including tritium, carbon-14, phosphorus-32, phosphorus-33,
sulfur-35 and the various radioisotopes of iodine, including
iodine-125 and iodine-131.
The radiolabeled compound may be any of those
subject to radiolytic decomposition, such as radiolabeled
organic compoundq. Examples o-~ such organic cornpounds
include amino acids such as lycine, tyrosine, phenylalanine
and tryptophan.
; rThe stabilizing compounds are particularly effective
with methionine and leucine. Other swch organic compounds
include peptides; nucleoside.s, such as thymidine and uridine,
nucleotides, polynucleotides, lipidsl steroids and
catecholamines.
~;
~ 8 ~
Radiolabeled compound~ are typically commercially
distributed in closed vials containing a solution of the
particular radiolabeled compound. Stabilizing compounds
with an insoluble backbone can take various forms as long
as they are maintained in contact with the solution of
radiolabeled compound. An example of a possible form is
solid beads which are added to a solution of the radio-
labeled compound, in which case the solution would be
removed from the stabilizing compound by decanting sr
withdrawing by means of a syringe, Another potential means
of storage would be to pro~ide the stabilizing compound in
a form whereby the solution of the radiolabeled compound
would be absorbed by the stabiliæing compound.- In such a
case, the solution of radiolabeled compound would be eluted
from the mass of stabilzing compound by use of a suitable
solvent. Once separated, the solution of radiolabeled
compound is used in the same manner as unstabilized
solutions thereof. In the case of soluble amine~, the
sta~ilizing com~ound is simply added to a solution of the
radiolabeled compound which is typically shipped in a
sealed vial from which the stabilized compound is removed by
withdrawing by means of a syringe.
The invention will be further clarified by a con-
sideration of the following examples, which are intended to
be purely exempl~ry of the use of the invention.
EXAMPLE I
Preparation of Polystyrene-divinylbenzene
bound ammonium ~ulfide, chloride form
To a suspen~ion oE chloromethylated polystyrene
copolymerized with ]% divinylbenzene (10 g, 42 5 milli-
equivalents chloride by analysis) in methylene chloride
(200 ml) was added ethanethîol (15~7 ml, 213 mMol) and
triethylamine (29.6 ml, 213 mMol). A slight yellow color
began to develop after approximately 10 minutes and the
mixture was allowed to stir -for 60 hrs. under a nitrogen
atmosphere. The resultant suspension was filtered through
paper and continuously extracted with chloroform for 24 hrs.,
ethanol 3 hrs~, then washed with chloroform (5 x 40 ml),
ethanol (5 x 40 ml) and the white resin that remained
was dried in a vacuum oven at 60C/20 mm to leave 10 g of
the ammonium sulfide polymer. The analysis of C, 71.18,
H, 8.84; N, 215, S, 4.90, Cl, 5.26 indicates 1.53 mequivalents
- Sulfur/g, 1.53 mequivalents ~itrogen/g, and 1.48 mequivalents
Chloride/g of polymer resin.
EXAMPLE II
Preparation of the Ammonium Sulfide Polymer
in its acetate form
The ammonium chloride of Example I (3g) was
stirred with 1~ NaOH (100 ml) for 3 hours, filtered and
washed three times successively with methylene chloride
(50 ml) then ethanol (50 ml). The resultant resin was dried
ovexnight at 23C/20 mM to afford 2~7 g of a yellow product
which analysed for C, 73.39; H, 8~83; N, 1.90, S, 4.78
Cl, 3008. This analysis indicates 1~36 mequivalents
j Nitrogen/g, 1.49 mequivalents Sulfur/g, and 0.88 mequivalents
Chlcride/g of polymer resin~
The yellow product above was washed with l~/o
aqueous acetic acid (100 ml) then three times successively
, with methylene chloride (40 ml) followed by ethanol (40 ml).
The resultant white product was dried at 23/20 mM to
afford Example II as the acetate 2.7 g,
The following Examples demonstrate the use of various
insoluble quaternaxy ammonium containing compounds and soluble
amines to stabilize various radiolabeled compounds~ The
-- 10 -.
analytical method employs liquid ch.romatography for separation
followed by post column radioactivity quantitization. The
values given are an average of three separate determinations
from triplicate packagings.
EXAMPLES III - V
The following examples detail the storage of
tritium-labeled methionine in aqueous ethanol solution with
an initial radiochemical purity of 9~/O with the stabilizers
of E~Yamples I and II. The average change in purity is
based upon three ind1vidual determinations~
Average
Number ofChange in Molar
' Ex~ Stabilizer Days StoredPurity (%) Excess
;
None 47 6
62 11
87 13
: 118 19
III Compound 47 1 3
of Ex. I 62 3 1.2 x 10
87 4 of Sulfur
` 118 11 and Nitrogen
j . IV Compound 48 O 8.7 x 10
of Ex, I 62 O of Sulfur
87 O and Nitrogen
119 3
V Compound 47 2 1.2 x 10
of Ex. II 62 4 of Sulfur
87 5 and Nitroger
118 12
30 EXAMPLE VI
An aqueous solution of methionine having an initial
purity of 93% was divided into equal parts, One part was
stored without any sta~ilizer, while the other part was stored
over 4~5 x 10 molar excess of the compound of Example I.
Ater 36 days the change i.n radiochemical purity was 75% and
12% respectively~ After 66 days the change in radiochemical
purity was 85% and 19% respectively, and the biological
activity of the two solutions was tested by attempting to use
the stored solutions for protein translation~ The solution
of methionine stored without any stabilizer failed to
translate effectively, whereas the solution stored over
stabilizer underwent efficient protein translation.
Average
Number of Change in Molar
E Stabilizer Days Stored Purity (%) Excess
None 36 75
66 85
VI Compound 36 12 4.5 x 10
of Ex. 166 19 of Sulfur
and
Nitrogen
EXAMPLES VII and VIII
Examples VII and VIII illustrate the use o-f the
present invention with the stabilizer of Example I and poly-
styrenedivinyl benzene copolymer substituted hy triethyl
a-mmonium wi~h chloride as the counterion (TEAC) to stabilize
a solution of tritium labeled lysine with a starting purity
20 of g8.5%.
Average
Nur~er of Change in Molar
Ex~ Stabilizer Days Stored Purity (%) Exce~s
_
None 34 300
6.0
VII TEAC 34 0.5 105 x 10
1.5 of Sulfur
and
Nitrogen
VIII Compound 34 0~5 3
of Ex. 1 70 1.5 1.5 ~ 10
o-f Sulfur
and
Nitrogen
EXAMPLES IX-XII
Example~ IX - XII illustrate the use of the present
invention to stabilize a radiolabeled nucleoside, tritium-
labeled thymidine. Example~ IX and X employ th~ stabilizer
of Example I, whereas Examples XI and XII employ a commercially
- 12 -
available polystyrenedivinyl benzene copolymer anion e~change
r~sin to which has been bound a trimethyl ammonium group
with chloride as the counterion (TMAC) in accordance with
the present invention.
Average
Number of Change in Molar
Ex, Stabilizer Days Stored Purity (%) Excess
None 14 3
22 7
29 7
IX Compound14 0 10 Nitrogen
of Ex. 1 22 5 and Sulfur
29 3
X Compound 14 0 2 x 104
of Ex. 1 22 2 Nitrogen
29 2 and Sulfur
XI TMAC 14 0 10 Nitrogen
22
29 2
XII TMAC 14 0 2 x 10
22 3 Nitrogen
2g
EXAMPLES XIII - XV
The following examples detai.l the storage of
tritium labeled methionine (80 Ci/m~ol) in aqueous 7~/O
ethanol solution with an initial radiochemical purity of
98% to give 1 mCi/ml at -20C with amines as indicated~
EXAMPLE XIII
. Average
Number of Change in
StabilizerDays Stored Purity ~%) Molarity
~one 79 12
Cyclohexyl
isopropyl
amine 79 5 20 mM
- 13 -
EXAMPLE XIV
Average
Nur~er of Change in
5tabilizer Days Stored Purity (%) Molarity
None 45 6
114 12
1,5-Diaminopentane 50 1 20 mMolar
84
114 2
1,6-Diaminohexane51 1 20 mMolar
84 2
. 114
EXAMPLE XV
Average
Number of Change in
Stabilizer Days StoredPurity (%) Molarity
~one 45 6
114 12
triethyl amine 49 1 0.7 mM
83 4
114 5
EXAMPLE XVI
The following example details the storage of
tritium labeled methionine (80 Ci/mMol) in water with an
initial radiochemi.cal purity of 94% to give a concentration
of 1 mCi/ml at 4C with the amine as indicated~
14 -
s':~
EXAMPLE XVI
Average
Number of Change in
StabilizerDays Stored Purity (%) Molarity
None 6 21
16 35
22 ~
57
51 72
Triethylamine 6 3 lOmMolar
16 6
22 7
~3
51 11
EXAMPLES XVII - XVIII
The following examples detail the storage of
sulfur-35 labeled methionine (1000 Ci/mMol) in water with an
initial radiochemical purity of 95% to give a concentration
of 10 mCi/ml with the amines as indicated. Examples XVII and
XVIII were stored at 4C while Example XIX was stored at
-20C. Tr.is refers to tris (hydroxymethyl) methyl amine.
EXAMPLE XVII
Average
Number of Change in
StabilizerDays Stored Purity ~%) Molarity
None 1 17
4 37
8 57
triethyl amine 2 14 1 Molar
~ 1
8 16
19
triethyl amine 2 15 lOOmMolar
4 16
8 20
33
- 15 -
EXAMPLE XVIII
Average
Number of Change in
Stabilizer Days Stored Purity (%)Molarity
None 1 8
2 15
33
18 ~36
Tris*(as free ~ase) 1 4 1 Molar
2 6
12
~ 18 35
EXAMPLE XIX
Average
~umber of Change in
Stabilizer Days Stored Purity (%)Molarity
None 3 6
9 11
Tris.HCl 3 0 lOmMolar
9 4
EXAMPLE XX
Example XX details the storage of tritium labeled
Enk~phalin ~5-L-methionine) (30 Ci/mMol) in an aqueous
solution containing 70/O ethanol with an initial radio-
chemical purity of 98% to give a concentration of 1 mCi./ml
at -20 with various amines as indicated.
Average
Nu~ber of Change in
Stabilizer Days Stored Purity (%)Molarity
None 26 13
Triethylamine 26 0 33 mMolar
Hepes 26 4 3 mMolar
(hydroxyethyl
piperazine
ethyl
sulfate)
* Tris(hydroxymethyl)methyl amine
EXAMPLE XXI
Example XXI details the storage of tritium labeled
chemotactic peptide (57 Ci/mMol) in an aqueous solution con-
taining 50/O ethanol with an initial radiochemical purity of
99/O to ~ive a contentration of 1 mCi/ml at -20C with
polylysine.hydrobromide (av. M.W~ 20,000).
Average
Number of Change in
StabilizerDays Stored Purity (%) Molarity
None 54 4
216 13
Polylysine.HBr54 1 1 mMolar
216 5
EXAMPLES XXII - XXIII
Examples XXII and XXIII detail the storage of
deoxyguanosine-5'~triphosphate labeled with phosphate-32
(660 Ci/mMol) in water with an initial radiochemical purity
of 95% to give a concentration of 10 mCi/ml at -30C with
the amines indicated. EDTA is an abbreviation for ethylene
diamine tetracetic acid.
EXAMPLE XXII
Average
Number of Change in
StabilizerDays Stored Purity (%) Molarity
None 9 9
Tris (free base) 9 5 800 mMolar
Tris.HCl 9 5 800 mMolar
Triethylamine 2 3 800 mMolar
17 -
EXAMPLE XXIII
Average
Number of Change in
5tabilizer Days Stored Purity (%)Molarity
None 10 14
Tris.HC1 10 750 mMolar
Tris.HCl 10 810 mMolar
EDTA 10 71 mMolar
Tris.~Cl/EDTA 10 310mM/lmM
10EXAMPLE XXIV
Example XXIV details the storage of labeled
Adenosine-5~-triphosphate labeled with phosphorus-32 (8100
Ci/m~lol) in water at an initial radiochemical purity of 97%
to give a concentration of 3 mCi/rnl at -30C with triethyl-
amine,
Average
Number of Change in
Stabilizer Days Stored Purity (%) Molarity
None 13 7
Triethylamine 17 6 4 mMolar
Triethylamine 17 3 40 mMolar
Triethylamine 13 0 400 mMolar
Other embodiments of the invention will be apparentto those skilled in the art from a consideration of this
specification or practice of the invention disclosed herein.
It is intended that the specification and e~amples be con-
sidered as exemplary only, with the true scope and spirit
of the invention being indicated by the -followlng claims.
- 18
