Note: Descriptions are shown in the official language in which they were submitted.
W O 91/l4664 20S82~ PC~r/GB9l/0044l
METAL COMPLEXES
This invention relates to novel radioactive metal complexes and
to their use in therapy and particularly diagnosis, especially in
the context of cell labelling.
Radioactive indium and gallium when labelled to blood cells
05 have been extensively used in diagnostic methods in the clinical
setting. The labelling of red cells has been used as a method of
splenic imaging or determination of red cell mass, platelet
labelling has been used for studies on platelet survival and
platelet kinetics, while white cell labelling has been used to
localise inflammatory foci in a variety of clinical conditions.
In a more general sense the efficient labelling of blood cells
has wide implications in the diagnosis of any disorder where
haemagglutination may occur, e.g. atherosclerosis, thrombocytopenia,
focal sepsis and in the location of tumour and metastatic tissues.
Radioactive indium and gallium are generally used in the form
of metal complexes. However, many of the agents currently used to
chelate these metals are toxic to the cells, e,g. oxine
(8-hydroxyquinoline) and tropolone which are two of the agents
which are widely used. We have now found that cell labelling with
radioactive indium and gallium may be carried out more effectively
using an alternative group of chelators.
Accordingly the present invention comprises a neutral 3:1
ligand:metal(III) complex, in which the trivalent metal cation is a
radioactive isotope of indium or gallium and each ligand is
separately provided by a compound being:
(1) a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms
attached to ring carbon atoms are replaced by an aliphatic
hydrocarbon group of one to six carbon atoms or such a group
substituted by one or more groups selected from fluoro,
hydroxy and aliphatiz hydrocarbyloxy groups but excluding
3-hydroxy-2-methyl-4-pyrone; or
:....... . : . . ... ... : - . . -
. . .
WO 91/14664 `~ J'~ PCr/GB91/0044~
ZO~ '`Y`''` ~
-- 2 --
(2) 3-hydroxypyridin-2-one or,a 3-hydroxypyridin-2-one in which the
hydrogen atom attached to the nitrogen atom is replaced by an
aliphatic acyl group, by an aliphatic hydrocarbon group of 1 to 6
carbon atoms, or by an aliphatic hydrocarbon group substituted by
OS one or more substituents selected from aliphatic acyl, alkoxy,
cycloalkoxy, aliphatic amide, aliphatic ester, halogen and hydroxy
groups and, optionally, in which one or more of the hydrogen atoms
attached to ring carbon atoms are replaced by one of said
substituents, by an aliphatic hydrocarbon group of 1 to 6 carbon
atoms, or by an aliphatic hydrocarbon group substituted by an
alkoxy, cycloalkoxy, aliphatic ester or hydroxy group or by one or
more halogen groups.
The use in the treatment of iron deficiency anaemia of iron
complexes containing certain of the ligands present in the metal
complexes of the present invention has been described in UK Patents
Nos. 2,117,766, 2,128,998 and 2,136,806. In 2,117,766 and
2,136,806 an alternative mode of nomenclature is used in which the
3-hydroxypyridin-2-ones are referred to as 3-hydroxypyridin-2-ones.
However, both designations have the same meaning indicating a ring
system as shown hereinafter in formula (V).
However, the suitabi1ity of a ligand for complexing with one
metal to provide a complex for use in one therapeutic context is no
indication of its value for complexing with a different metal to
provide a complex for use in a different therapeutic or diagnostic
context. In particular, as regards the primary, d;agnostic use of
the complexes of the present invention, it will be appreciated that
the problem to be solved in the present application is entirely
different from that with the iron complexes. With the iron
complexes the ligands are required to release iron which then
reassociates with apotransferrin. With the indium and gallium
complexes the complexes are required to bind to the cell and emit a
signal.
,,
~ ~ .
- ~
. ~, .
W O 91/14664 2~S~ P ~ /GB91/00441
-- 3 --
:, .~,;
The different requirements of the twb uses of the ligands are
illustrated by recent papers relating to certain selected
3-hydroxy-4-pyrones and 3-hydroxypyridin-4-ones but not to
3-hydroxypyridin-2-ones. Thus, Finnegan et al, Inorg. Chem., 1987,
05 26, 2171-2176 and Matsguke et al, Inorg. Chem., 1988, 27, 3935-3939
characterise various indium and gallium complexes. These studies
suggest that 3-hydroxy-4-pyrone and 3-hydroxy-2-methyl-4-pyrone, as
well as certain 3-hydroxypyridin-4-ones may be of use in chelating
these metals. However, although 3-hydroxy-2-methyl-4-pyrone
(maltol) is one of the preferred ligands for use in the iron
complexes of U.K. Patent No. 2,128,998 it is inferior to other
substituted 3-hydroxy-4-pyrones for use in indium and gallium
complexes as will be illustrated hereinafter.
Preferred radioactive isotopes are the isotopes which emit
X-rays or positrons. Specific isotopes which are preferred, are
for example the indium radioactive isotopes 111 and 113,
particularly 111, and the gallium isotopes 66, 67, 68 and 72,
particularly ~7 and 68.
Indium 111 is particularly suitable for use in the present
invention as it decays by emitting ~ radiation of 171 and 245 keV
and has a half life of 67.2 hours. Similarly Ga 67 is particularly
suitable as it decays by emitting y radiation of 185 and 300 keV
and has a half life of 78.26 hours. Gallium 66 and 68 are suitable
positron emitters.
The metal(III) complexes of use in the present invention
contain indium and gallium in the trivalent form. The metal(III)
complexes are neutral, i.e. there being an internal balance of
charges between the metal(III) cation and the ligand(s) bound
covalently thereto without the necessity for the presence of a
non-covalently bound ion or ions to achieve such balance. Thus,
these ligands are monobasic and bidentate, being formed through the
loss of a proton from the hydroxy group of the ligand forming
campound 50H --~ 0~). Metal(III) complexes containing a 3:1
proportion of monobasic, bidentate ligand:metal(III) are therefore
neutral.
: :. .,
,; :
- . ~: : : ,
,: , . .: , ~ , :
.:
: . . : .
.. . .
W O 91/14664 Z0S8~ P~/GB91/0044~
-- 4,;.,j~ `f . -
The complexes according to the present invention may containvarious different combinations of three ligands of the types (1)
and (2). Thus all three ligands within the complex may be chosen
from type (1), or all three from type (2), although optionally
05 differing from within these types. Alternatively the three ligands
may be chosen from both types in a 2:1 ratio, i.e. either two
ligands derived from the same or two different 3-hydroxy-4-pyrones
with one 3-hydroxypyridin-2-one ligand or two ligands derived from
the same or two different 3-hydroxypyridin-2-ones with one
3-hydroxy-4-pyrone ligand. However, complexes of particular
interest are those containing three ligands of type (2) or
particularly of type (1), these ligands conveniently being
identical.
As regards the 3-hydroxy-4-pyrone ligands of type ~1), these
3-hydroxy-4-pyrones may carry more than one type of substituent
group but substitution by one rather than two or three groups is
preferred except that substitution by two groups is of interest
when at least one of them is an aliphatic hydrocarbon group. The
term aliphatic hydrocarbon group is used herein to include both
acyclic and cyclic groups which may be unsaturated or saturated,
the acycl;c groups having a branched chain or especially a straight
chain. Groups of from 2 to 6 carbon atoms and particularly 3 to S
carbon atoms are of most interest except that methyl groups can be
of interest when an additional substituent is also present.
Saturated aliphatic hydrocarbon groups are preferred, these being
either cyclic groups such as the cycloalkyl groups cyclopropyl and
cyclohexyl or, more particularly, acyclic groups such as the alkyl
groups methyl and ethyl and especially propyl, butyl and pentyl
(and their branched chain analogues). Similar observations and
preferences apply to the aliphatic hydrocarbyl groups present in
substituents which are aliphatic hydrocarbon groups substituted by
aliphatic hydrocarbyloxy groups with the exception that the latter
may sometimes be a methoxy group. Moreover, the aliphatic
hydrocarbon group which is substituted by an aliphatic
hydrocarbyloxy group may often be a methyl group.
.,.~,........................... . . . .
'
WO 91/14664 -; ., i,.`'.~ .~ ? PC~r/GB91/00441
In this specification the term alkyl includes both straight and
branched chain groups but references to individual alkyl groups
such as "propyl" are specific for the straight chain group only.
An analogous convention applies to other generic terms.
05 When the 3-hydroxy-4-pyrone contains a substituted aliphatic
hydrocarbon group that group preferably conta;ns only one type of
substituent among fluoro, hydroxy and aliphatic hydrocarbyloxy
groups.
As an alternative, or in addition to, the use of aliphatic
lO hydrocarbon groups as substituents in the 3-hydroxy-4-pyrones,
fluoro substituted aliphatic hydrocarbon groups may be used, for
example any of the general types of or specific aliphatic
hydrocarbon groups as described hereinbefore but substituted by one
or more fluoro groups, for example up to five, nine or ten of such
l5 groups Fluoro substituted aliphatic hydrocarbon groups preferably
contain one, three or five fluoro atoms, specific examples of such
groups be;ng a l,l,2,2,2-pentafluoroethyl and 3,3,3-trifluoropropyl
group.
As a further alternative, or in addition to, the use of
20 aliphatic hydrocarbon groups as substituents in the 3-hydroxy-4-
pyrones hydrocarbyloxy substituted aliphatic hydrocarbon groups may
be used, or to a lesser extent hydroxy substituted aliphatic
hydrocarbon groups, particularly a substituted methyl group or
another aliphatic hydrocarbon group substituted on the carbon atom
25 of the aliphatic group which is attached to the pyrone ring. When
the substituted aliphatic hydrocarbon groups contain substituents
other than fluoro there is usually only one aliphatic
hydrocarbyloxy or hydroxy group present.
Substitutions at the 6 or especially the 2 position, and
30 sometimes at both, are of most interest although, when the ring is
substituted by the larger substituent groups, there may be an
advantage in avoiding substitution on a carbon atom alpha to
the - C - C ~ system. This system is involved in the complexing
O OH
with indium or gallium and the close proximity of one of the larger
,, i - .. . . . . .
:. :: -: .
: ' ,. ' - ,
: ,, ,
.
:
. ~ .: ::
. r,
WO 91/14664 ZOS8;;~ K~/GBgl/0044
-- 6 --
aliphatic hydrocarbon groups may lead to steric effects which
inhibit complex formation.
Examples of hydroxypyrones providing ligands which may be used
~In complexes according to the present invention have the
05 formula (I), specific hydroxypyrones of interest hav;ng the
formulae (II), (III) and (IV):
5 ~ 3~ 0H s ~ OH s ~ OH 5 ~ 0H
2 6 ~ 0 ~ R R ~ O ~ R 0 R
(I) (Il) (111) (IV)
in which each R separately (subject to the exclusion of the
compound 3-hydroxy-2-methyl-4-pyrone,) is an alkyl or cycloalkyl
group, or such a group substituted by three or five fluoro groups,
by an alkoxy group or by a hydroxy group, for example ethyl,
propyl, isopropyl, butyl or pentyl, or a fluoro substituted
derivative thereof containing three or five fluorine atoms,
particularly 1,1,2,2,2-pentafluoroethyl or 3,3,3-trifluoropropyl,
or a Cl_4 alkoxy substituted derivative thereof, particularly
methoxymethyl, ethoxymethyl, propoxymethyl or butoxymethyl, and n
is 1, 2 or 3. Preferred compounds are those which are 2-alkyl,
2,6-dialkyl, 2-fluoroalkyl, 2-alkoxyalkyl, 2-hydroxyalkyl or
6-alkyl-2-hydroxy- alkyl substituted.
Among these compounds 2-ethyl-3-hydroxy-4- pyrone and its
2-(1'-methylethyl), 2-propyl, 2-butyl and 2-pentyl analogues
[ethyl-maltol, isopropyl-maltol, propyl-maltol, butyl-maltol and
pentyl-maltol; II~ R = C2Hs, CH(CH3)2~ (CH2)2CH3~ (CH2)3CH3 or
(CH2)3CH3] are of particular interest as are its
1',1',2',2',2'-pentafluoroethyl and 3',3',3'-trifluoropropyl
analogues [II, R = CF2CF3 and R = CH2CH2CF3].
Also of particular interest are the compounds of formula (III)
in which R is an alkoxymethyl group and the compounds of
formula (IV) in which the group R at the 6-position is an alkyl
group and that at the 2-position is an alkyl or l~hydroxyalkyl
group, for example the compounds 3-hydroxy-6-methoxymethyl-4-pyrone,
..
. . .
: . .,........... . , ~
.~W O 91/14664 _ 7 _ , P ~ /GB91/00441
6-ethoxymethyl-3-hydroxy-4-~y;ron;e" i-hydroxy-6-propoxymethyl-4-
pyrone, 6-butoxymethyl-3-hydroxy-4-pyrone, 2~ethyl-3-hydroxy-
6-methyl-4-pyrone, 3-hydroxy-6-methyl-2-propyl-4-pyrone,
2-butyl-3-hydroxy-6-methyl-4-pyrone, 3-hydroxy-2-hydroxymethyl-6-
05 methyl-4-pyrone, 3-hydroxy-2-(1-hydroxyethyl)-6-methyl-4-pyrone,
3-hydroxy-2-(1-hydroxypropyl)-6-methyl-4-pyrone and
3-hydroxy-2-(1-hydroxybutyl)-6-methyl-4-pyrone.
As regards the 3-hydroxypyridin-2-one ligands of type (2),
these may be derived from hydroxypyridinones of the type described
in UK Patent No. 2,117,766 (corresponding to US Patent No. 4,550,101
and 3apanese Patent Application No. 83/049677) or of the type
described in UK Patent No. 2,136,806 (corresponding to US Patent
No. 4,810,491 and Japanese Patent Application No. 84/057186). ~he
former consist of a 3-hydroxypyridin-2-one in which the hydrogen
atom attached to the nitrogen atom is replaced by an aliphatic
hydrocarbon group of 1 to 6 carbon atoms and, optionally, in which
one or more of the hydrogen atoms attached to ring carbon atoms are
also replaced by the same or a different aliphatic hydrocarbon
group of 1 to 6 carbon atoms, whilst the latter include
3-hydroxypyridin-2-ones substituted as defined under (2)
hereinbefore but exclud;ng those compounds in which the replacement
of hydrogen atoms is effected only by aliphatic hydrocarbon groups,
these compounds being the substituted hydroxypyridinones of the
former patent It will be noted that the 3-hydroxypyridin-2-ones
may contain alkoxy and cycloalkoxy groups and that, as compared
with the compounds of U.K, Patent No. 2,136,806, an a1iphatic
hydrocarbon group on a carbon atom of the ring, as well as one on
the nitrogen atom of the ring, can be substituted by one or more
halogen groups. Hydroxypyridinones providing ligands which may be
used in complexes according to the present invention have the
formula (V)
~ 5 ~ OH
X
... . . . .
,
.
~, -. - ~, .- ~.
W O 91/14664 ~ ~ ~, ~ PC~r/GB~I/
in which X and Y are substituents as defined hereinbefore as
substituents on the nitrogen and carbon atoms of a
3-hydroxypyridin-2-one and n is 0, 1, 2 or 3.
Preferences as to the nature and position of the substituent
05 groups present in the hydroxypyridinones are broadly as expressed
in the aforementioned patents. Thus, substituted aliphatic
hydrocarbon groups present in the hydroxypyridinones may
conveniently contain 1 to 8 and particularly 3 to 6 carbon atoms
and, as indicated, may carry more than one substituent group,
although it is preferred that only one substituent group is
present. However, the simpler hydroxypyridinones of UK Patent
GB 2,118,176 containing only unsubstituted aliphatic hydrocarbon
groups are of the greatest interest so that a preferred type of
compound is a 3-hydroxypyridin-2-one in which the hydrogen atom
attached to the nitrogen atom is replaced by an aliphatic
hydrocarbon group of 1 to 6 carbon atoms and, optionally, in which
one or more of the hydrogen atoms attached to ring carbon atoms are
replaced by the same or a d;fferent aliphatic hydrocarbon group of
1 to 6 carbon atoms. The preferences among the aliphatic
hydrocarbon groups present in these hydroxypyridinones correspond
largely to those expressed hereinbefore in relation to the
hydroxypyrones, with methyl groups conveniently being used for
substitution on ring carbon atoms but larger alkyl and cycloalkyl
groups, particularly as described for the hydroxypyrones, being of
particular interest for substitution on the ring nitrogen atoms.
Substitution of the ring carbon atoms of the hydroxypyridinones
is again preferably by one rather than two or three groups, for
example aliphatic hydrocarbon groups, but 3-hydroxypyridin-2-ones
also of particular interest are those N-substituted without any
additional substituent on the ring carbon atoms.
Among the 3-hydroxypyridin-2-ones containing substituted
aliphatic hydrocarbon groups those in which the aliphatic
hydrocarbon group is substituted by one or more halogen groups,
particularly fluoro groups, are of especial interest. The
preferences for such halogen substituted aliphatic hydrocarbon
.. -
.:
.
W 0 9~/l4664 _ 9 _ PC~r~G~9l/Ul44
groups are largely as expressed hereinbefore in relation to the
fluoro substituted aliphatic hydrocarbon grcups of the
hydroxypyrones. In this case however, there is a greater
possibility of both an aliphatic hydrocarbon group and a halogen
05 substituted hydrocarbon group being present in the compound, with
one, for example the aliphatic hydrocarbon group, being on a carbon
atom and the other, for example the halogen substituted hydrocarbon
group, on the nitrogen atom. Specific hydroxypyridinones of
particular interest have formula (VI),
5 4 OH
6 ¢~ ( )
10 in which R is an alkyl or cycloalkyl group, or such a group
substituted by one, three or five fluoro groups, for example ethyl,
n-propyl, isopropyl, butyl, pentyl or hexyl, or a fluoro substituted
derivative thereof containing three or five fluorine atoms,
particularly 1,1,2,2,2-pentafluoroethyl or 3,3,3-trifluoropropyl.
15 Among such compounds l-ethyl-3-hydroxpyridin-2-one, 3-hydroxy-1-
propylpyridin-2-one, 3-hydroxy-1(1'-methylethyl)-pyridin-2-one,
l-butyl-3-hydroxpyridin-2-one and 3-hydroxy-1-pentylpyridin-2-one
are of particular interest.
Among the quite wide range of ligands described above certain
20 ligands or combinations of ligands will be of particular value and
some indication of these has already been given. One measure of
the value of the preferred complexes is provided by the value of
their partition coefficient (Kpart) between n-octanol and Tris
hydrochloride (20 mM, pH 7.4, Tris representing 2-amino-2-
25 hydroxymethylpropane 1,3-diol) at 200C, this being expressed as the
ratio (concentration in organic phase)/(concentration in aqueous
phase). Preferred complexes show a value of Kpart for each ligand
providing compound from 2 to 50, especially from 8 to 40, together
with a value of Kpart for the 3 1 indium(III) complex from 5 to 100,
.... ... . .
..
.. . . .
W 0 91/14664 ` ~ ~5~8~ P ~ /GB91/0044 ~
-- 10 --
especially from 30 to 70. The corresponding ranges for the 3:1
gallium(III) complexes are from S to 100 and 20 to 50, respectively.
Whilst these ranges of Kpart are given as a guide to preferred
complexes, ligands and/or complexes that have a Kpart value outside
05 these ranges can still be suitable for use.
These preferences for Kpart are reflected in the preferences
for particular ligands expressed hereinbefore. Examples of
specific complexes according to the present invention which are of
particular interest are the indium 111 and gallium 67 complexes of
hydroxypyrones and hydroxypyridinones indicated as being of
especial interest, for example
(butyl-maltol)3 In 111 and 6a 67
(isopropyl-maltol)3 In 111 and Ga 67
(3-hydroxy-6-propoxymethyl-4-pyrone)3 In 111 and Ga 67
(6-butoxymethyl-3-hydroxy-4-pyrone)3 In 111 and Ga 67
( ~ ) In 111 where R1 = butyl, pentyl or heryl
R1
The radioactive metal(III) complexes are conveniently prepared
by the reaction of the hydroxypyrone and/or hydroxypyr;dinone
ligand-providing compounds and radioactive metal ions, the latter
conveniently being derived from a metal(III) salt, particularly the
chloride salt. The reaction is conveniently effected in a suitable
mutual solvent, usually an aqueous medium. If desired, however, an
aqueous/solvent mixture may be used or an organic solvent, for
example ethanol, methanol or chloroform and mixtures of these
solvents together and/or with water where appropriate. In
particular, methanol or especially ethanol may be used where it is
desired to effect the separation of at least one major part of a
by-product such as sodium chloride by precipitation whilst the
indium or gallium complex is retained in solution.
.; . .
-
,W091/14664 '~8'a, PCI'/GB91/00441
Reaction is generally rapid and will usually have proceeded
substantially to completion at 200C in a few minutes. The
complexes of the present invention are often used in solution but,
where desired, may be prepared in solid form by evaporation of the
05 reaction mixture and freeze drying, followed by crystallization
from a suitable solvent ;f ~urther purification is re~uired.
It will be appreciated that the nature of the metal complex
obtained by the reaction of the ligand-providing compound(s) and
metal ions will depend both on the proportion of these reactants
and upon the pH of the reaction mixture. Thus for the production
of the 3:1 ligand:metal(llI) complex the hydroxypyrone and/or
hydroxypyridinone ligand-providing compound(s) and the metal salt
are conveniently mixed in solution in at least a 3:1 molar
proportion and the pH adjusted to a value in the range 6-9, for
example 7 or 8. If a similar excess of hydroxypyrone and/or
hydroxypyridinone:metal(III) is employed but no adjustment is made
of the acidic pH which results in the admixture of the
hydroxypyrone or hydroxypyridinone and a metal salt such as a
metal(III) chloride, then a mixture of the 2:1 and 1:1
ligand:metal(III) complexes will instead be obtained. To ensure
that all of the radioactive metal cations are combined with ligand
in complex form the ligand-providing compound:metal(III) ratio may
conveniently be greater than 3:1, for example being 100:1 or up
to 1000:1. However, providing the ratio is 3:1 or more, and the
previous requirements on reaction conditions are observed, the
product will consist essentially of the 3:1 ligand:metal(III)
complex. In some instances it will be preferable to incorporate
some "cold" non-radioactive metal with the radioactive metal when
forming the ligand:metal complex, the amount of ligand-providing
compound(s) being adjusted accordingly. This will result in some
non-radioactive complex being administered in combination with
radioactive complex.
'.
, ,. ~ : ~
.. . . .
.
:
wo 91/14664 zosazf3~ P~r/~B91/00441
'Y ~ 'J~
- 12 -
It will be apprecia~d~th~when the complex contains different
ligands the ligand-provid1ng compounds will conveniently be used in
a 2:1 or 1:1 ratio, as desired, although with complexes in which
the ligands are heterogeneous a mixture of different 3:1 complexes
05 will always result even when a 2:1 ratio is employed. For this
reason complexes in which the ligands are homogeneous are
preferred.
The radioactive indium or gallium cation is preferably obtained
from its chloride salt. The amount of radioactivity re~uired in
the complex is often within the range 10 to 60 mCi/~g, but will
ultimately depend on the proposed use of the complex as described
hereinafter.
The present invention thus further includes a process for the
preparation of a neutral 3:1 ligand:metal(III) complex of a
substituted 3~hydroxy-4-pyrone or 3-hydroxypyridin-2-one as defined
hereinbefore which comprises reacting the ligand providing compound
or compounds with the trivalent cation of the indium or gallium
radioactive isotope.
Certain substituted 3-hydroxy-4-pyrones, including
2-trifluoromethyl-3-hydroxy-4-pyrone, are commercially available.
For the preparation of substituted hydroxypyrones one convenient
starting material consists of 3-hydroxy-4-pyrone which is readily
obtainable by decarboxylation of 2,6-dicarboxy-3-hydroxy-4-pyrone
(meconic acid). Thus, for example, 3-hydroxy-4-pyrone may be
reacted with an aldehyde to insert a l-hydroxyalkyl group at the
2-position, which group may then be reduced to produce a
2-alkyl-3-hydroxy-4-pyrone. The preparation of 2-ethyl-3-
hydroxy-4-pyrone, etc., by this route is described in the published
US Application Serial No. 310,141 (series of 1960) referred to in
U.S. Patent No. 3,376,317.
The preparation of other substituted 3-hydroxy-4-pyrones may be
exemplified as follows. The hydrocarbyloxyalkyl substituted
3-hydroxy-4-pyrones may be prepared from 3-hydroxy-6-hydroxymethyl-
4-pyrqne (kojic acid) by protecting the 3-hydroxy group, for
example as a benzyloxy group, and reacting the protected compound
, ~ :
; .
. ,
.
W O 91/14664 ~ Z Q S 8 ? ~ PC~r/GB91/00441
- 13 -
with the corresponding alkyl halide, for example in
dimethylformamide using sodium hydride. Deprotection is then
effected, for example a benzyloxy group being converted to hydroxy
using concentrated hydrochloric acid, and the end product
05 ;solated. Using this method the novel compounds 3-hydroxy-6-
methoxymethyl-4-pyrone, 6-ethoxymethyl-3-hydroxy-4-pyrone,
3-hydroxy-6-propoxymethyl-4-pyrone and 6-butoxymethyl-3-hydroxy-4-
pyrone have been prepared. Additionally 3-hydroxy-4-pyrones
wherein two of the hydrogen atoms attached to the ring carbon atoms
have been substituted may also be prepared from kojic acid. For
example, kojic acid is converted to the chloro analogue using
thionyl chloride. This is then reacted with zinc dust and
hydrochloric acid to form 3-hydroxy-6-methyl-4-pyrone. This in
turn is reacted with the appropriate aldehyde to produce the
corresponding 2-hydroxyalkyl-3-hydroxy-6-methyl-4-pyrone. Further
reaction with zinc/hydrochlor;c acid yields the corresponding
2-alkyl-3-hydroxy-6-methyl-4-pyrone. Using this method the novel
compounds 2-ethyl-3-hydroxy-6-methyl-4-pyrone, 3-hydroxy-6-methyl-
2-propyl-4-pyrone, 2-butyl-3-hydroxy-6-methy1-4-pyrone,
2-hydroxymethyl~6-methyl-4-pyrone, 3-hydroxy-2-hydroxyethyl-6-
methyl-4-pyrone, 3-hydroxy-2-hydroxypropyl-6-methyl-4-pyrone and
3-hydroxy-2-hydroxybutyl-6-methyl-4-pyrone have been prepared.
The present invention thus includes a process for the
preparation of a 3-hydroxy-4-pyrone in which one or more of the
hydrogen atoms attached to the ring carbon atoms is replaced by an
aliphatic hydrocarbon group of one to six carbon atoms substituted
by an aliphatic hydrocarbyloxy group of one to six carbon atoms,
and one or more other hydrogen atoms are optionally replaced by an
aliphatic hydrocarbon group of one to six carbon atoms, which
comprises reacting an intermediate, in which the 3-hydroxy group is
protected and the or each aliphatic hydrocarbon group substituted
by an aliphatic hydrocarbyloxy group is replaced by the
corresponding hydroxy substituted aliphatic hydrocarbon group,
with an aliphatic hydrocarbyl halide containing the corresponding
aliphatic hydrocarbyl group and then deprotecting the 3-hydroxy
group.
W O 91/l4664 . ` ~ Z~828~ P ~ /GB9l/00441
.. . k~,,,
- 14 -
The present invention further includes a process for the
preparation of a 3-hydroxy-6-methyl-4-pyrone substituted at the
2-position by a Cl-6 aliphatic hydrocarbon group or such a group
substituted by a hydroxy group but excluding 3-hydroxy-2,6-
05 dimethyl-4~pyrone which comprises (a) reacting 3-hydroxy-6-methyl-
4-pyrone with the corresponding formyl substituted aliphatic
hydrocarbon to provide a 3-hydroxy-6-methyl-4-pyrone substituted at
the 2-position by a ~1-6 aliphatic hydrocarbon group itself
substituted at the l-position by a hydroxy group or (b~ effecting
step (a) and then reducing said hydroxy substituted Cl-6 aliphatic
hydrocarbon group to provide a 3-hydroxy-6-methyl-4-pyrone
substituted at the 2-position by a Cl-6 aliphatic hydrocarbon group.
Certain of the substituted 3-hydroxy-4-pyrone intermediates are
novel compounds and fall within the scope of the present invention.
The 3-hydroxypyridin-2-ones may be prepared by procedures
described in U.K. Patents Nos. 2,117,766 and 2,136,806, and by
variants thereon.
It will be appreciated that these are not the only routes
available to these compounds and their ind;um and gallium(III)
complexes and that various alternatives may be used as will be
apparent to those skilled in the art.
The radiolabelled metal(III) complexes of the invention are of
particular value in diagnosis when used as labels for blood cells.
This re~uires in vitro labelling of the desired blood cells which
are then injected back into the patient. The labelled cells will
circulate and their location can conveniently be monitored by use
of a gamma scintillation camera or PET scan. Alternatively, if
cell survival studies are being employed, a further blood sample
can be taken from the patient at a fixed time after the
radiolabelled cells have been injected and radioactive counts
measured to provide an index of cell survival.
As mentioned hereinbefore the labelling of different spec;es of
blood cell provides- a different diagnostic use. Accordingly
radiolabelled metal(III) complexes of the present invention can
with advantage be used to label granulocytes, platelets, red cells
.. .
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and lymphocytes. Radiolabelled leucocytes can be used clinically
to detect sites of abscess and inflammation. Radiolabelled
platelets may be used diagnostically to measure platelet survival
and distribution. Furthermore they may be used to visualize venous
05 and arterial accumulation of platelets in atherosclerotic lesions9
venous rhombi and thrombophlebitis. An additional use of
radiolabelled platelets is to provide an early indication of renal
transplant rejection and to assess the thrombogenicity of arterial
grafts. The circulation and distribution of radiolabelled
autologous lymphocytes can be visualized and analysed using the
complexes of the present invention. Labelling of red blood cells
is of diagnostic benefit in the following instances: cardiovascular
imaging, gated wall motion studies, measurement of ejection
function of the left ventricle, measurement of blood volume,
gastro-intestinal bleeding studies and for the labelling of heat
denatured red blood cells for splenic imaging.
In addition to the use of the complexes in diagnosis,
lymphocytes labelled with radioactive ind;um complexes, for example
of In 114, and potentially also gallium complexes, may be used in
therapy, principally in the treatment of lymphomas. The complexes
of the present invent;on are therefore also of interest in a
therapeutic context.
The dosage of radiolabelled metal(III) complex labelled blood
cell administered to the patient is dependent upon the radioactive
isotope, the species of blood cell, the diagnostic purpose it is
being employed for and the method of detection to be used. The
following information may, however, be provided by way of guidance.
The normal adult dose of indium 111 labelled leucocytes for
location of sites of infection is 500-1000 ~Ci (18.5 -~ 37 MBq).
For indium(III) complexes of the present invention dosages within
the range 300 to 1000 ~Ci, particularly 600 ~Ci, should generally
be suitable.
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For gallium(III) complexes of the present invention dosages
within the range 300 to 1000, 2000 or 2500 ~Ci, particularly 500,
1000 or ~500 yCi should generally be suitable. Thus the normal
adult dosage of radioactive gallium 67 is 1000-2000 ~Ci (37-74 MBq)
05 for detecting tumours in the lymphatic system. For tumour
v;sualization by scanning techniques a dosage of 1500-2500 ~Ci
(55.5-92.5 MBq) is recommended.
The dosages used for indium 111 and gallium 67 complexes in
other labelling contexts will be similar to those given above
subject to any variations generally recognised in the literature as
being suitable in that context. Moreover dosages for other
radioactive isotopes will be similar in terms of ~Ci although not
necessarily in terms of the amount of the complex.
Where the indium(III) or gallium(III) complexes are to be used
in vivo either diagnostically or therapeutically, then these are
best formulated into pharmaceutical compositions with a suitable
diluent or carrier as described in UK Patents Nos. 2,117,766,
2,128,998 or 2,136,806.
The labelling of the desired blood cells with the complexes of
the present invent;on can be conveniently achieved by methods
described in the literature for other complexes which are
illustrated in Examples 3 and 4.
The invention is illustrated by the following Examples,
Examples 1 to 3 of which relate to intermediates for the preparation
of the complexes. Certain of these intermediates are novel oer se
and are included within the scope of the present invention. The
nmr signals have been attributed to the protons to which they are
believed to relate.
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EXAMPLES
ExamPle 1 : Preparation of 3-hYd~xv'6-methoxvmethY1-4-pvrone
(A)
t1) 3-BenzYl-6-methoxYmethvl-4-Pvrone
05 3-Benzyloxy-6-hydroxymethyl-4-pyrone was prepared by heating
at 600C a mixture of 3-hydroxy-6-hydroxymethyl-4-pyrone and
a l.S molar excess of benzyl chloride with a 1.5 molar excess of
potassium carbonate in dimethylformamide for 24 hours. To the
resulting solution of 3-benzyloxy-6-hydroxymethyl-4-pyrone
(10 g, 0.023 mol) in dimethyl formamide was added iodomethane
(14 ml, 0.215 mol) and the solution stirred for half an hour.
Sodium hydride (2.6 9, 0.086 mol) was added and the mixture stirred
for 24 hours under nitrogen gas. The dimethylformamide was then
evaporated and the residue dissolved in water. The pH was then
adjusted to pH 12 using lOM NaOH before extraction into
dichloromethane (3 x lSO ml). The dichloromethane extracts were
combined, dried over anhydrous sodium sulphate and evaporated to
dryness. This yielded a yellow solid which was recrystallized from
hot ethanol to provide the title compound (5.8 9, 55X).
(2~ 3-HYdroxy-6-methoxvmethvl-4-pvrone
3-Benzyl-6-methoxymethyl-4-pyrone ~8 g, 0.032 mol) was refluxed
in 100 ml hydrochloric acid for 2 hours, After cooling, the pH of
the mixture was adjusted to pH 11, followed by extraction into
dichloromethane (2 x 50 ml). The aqueous layer was treated with
concentrated hydrochloric acid to pH 2 and further extracted into
dichloromethane (3 x 100 ml). The extracts were combined, dried
over anhydrous sodium sulphate and evaporated to dryness. This
yielded 3.5 g (70X) of a white solid that was recrystallized from
toluene to provide the title compound; melting point 79-800C;
~max (nujol) 1650, 1630, 1595, 1260, 1220, 1150, 9SO cm~l;
~H (d6-DMSO) 9.5 (lH, sbr, OH), 8.1 (lH, s, 2-H), 6.4 (lH, s, 5-H),
4.3 (2-H, s, CH2), ~.3 (3H, s, CH3).
(B) Other 3-hydroxy-6-alkoxymethyl-4-pyrones were prepared by the
same method as described above using a five molar excess of the
appropriate alkyl halide in step (1).
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6-Ethoxvmethvl-3-hydroxY-4-DYron~
Yield 8.2 9 (73.3X) in step (1) and 4.2 g (80%) in step (2);
Melting point 73-740C; VmaX (nujol) 1650, 1610, 1265, 1220, 1100,
900 tm~l;
05 ~H (d6-DMSO), 9.15 (lH, sbr, OH), 8.1 (lH, s, 2-H),
6.4 (lH, s, S-H), 4.3 (2H, s, CH2), 3.4 (2H, q, CH2CH3),
1.1 (3H, t, CH3).
3-Hydroxv-6-propoxvmethYl-4-pyrone
Yield 8.0 9 (68X) in step (1) and 4.6 9 (68.5%) in step (2);
Melting point 63-640C;
~max (nujol) 1650, 1620, 1580, 1265, 1210, 1150, 950 cm~l;
~H (d6-DMSO), 9.1 (lH, b, OH), 8.1 (lH, s, 2-H), 6.4 (lH, s, 5-H),
4.3 (2H, s, CH2), 3.4 (2H, b, CH2CH2CH3), 1.5 (2H, m, CH2CH2CH3),
0.9 (3H, t, CH3).
6-Butoxvmethvl-3-hvdroxv-4-PYrone
Yield 8.3 9 (83%) in step (1) and 5.4 9 (78.6%) in step (2);
Melting point 34-350C;
vmax (nujol~ 1650, 1630, 1300, 1210, 1100, 920 cm~l;
~H (d6-OMSO), 9.1 (lH, s, OH), 8,1 (lH, s, 2-H), 6.4 (lH, s, 5-H),
4.3 (2H, s, CH2), 3.4 (2H, t, CH2CH2CH2CH3)~
1.4 (4H, m, CH2CH2CH2CH3), 0.9 (3H, t, CH3).
ExamPle 2 : PreParation of 3-hvdroxv-2-hvdroxvmethvl-6-methvl-4-
Pvrone and other 3-hvdroxv-2-(1-hvdroxvalkvl)-6-methvl-4-pvrones
(A)
(1) 3-Hvdroxv-6-chloromethvl-4-Dvrone
3-Hydroxy-6-hydroxymethyl-4-pyrone (50 9, 0.35 mol) was
dissolved in 500 ml redistilled thionyl chloride and left to stand
for 2 hours. The title compound was precipitated from the
solution, filtered and washed in petroleum ether.
Recrystallisation from H20 gave a yield of 41.6 9 (74%);
~H (d6-DMSO), 9.3 (lH, s, OH), 8.1 (lH, s, 3H), 6.6 (lH, s, 5-H),
4.7 (2H, s, CH2).
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(2) 3-Hvdroxy-6-methvl-4-Pvrone
3-Hydroxy-6-chloromethyl-4-pyrone (20 9, 0.125 mol~ was added
to lSO ml H20 and heated to 400C. Zinc dust (16.29 9, 0.25 mol~
was added and the mixture stirred vigorously at 600C. Concentrated
05 hydrochloric acid (37 ml, 3 molar equivalent) was added dropwise
over a period of one hour. The slurry was left stirring for
2-3 hours at 600C. The excess zinc was removed by hot filtration
and the pH of the filtrate adjusted to 1 and extracted into
dichloromethane (3 x 100 ml). The combined extracts were dried
under anhydrous sodium sulphate and evaporated to dryness to give
the title compound (12.9 9, 86%). Recrystallisation was carried
out from hot propanol, melting point 152-1530C;
vmaX (nujol) 1650, 1610, 1575, 1220, 1150, 920 cm~l;
oH (d6-DMSO), 8.9 (lH, s, OH), 8.0 (lH, s, 2-H), 6.3 (lH, s, 5-H),
2.2 (3H, s, CH3).
(3) 3-Hvdroxv-2-hYdroxvmethvl-6-methyl-4-pvrone
3-Hydroxy-6-methyl-4-pyrone (9.4 g, 0.074 mol) was added
to 100 ml HzO. Forma1dehyde (5.5 ml, 0,074 mol) was added and the
final pH of the mixture adjusted to 10.5. The solution was stirred
at 25C for 24 hours. The pH ws then adjusted to 1 and the title
compound precipitated (10 9, 86.2%);
~H (d6-DMSO), 8.8 (lH, sbr, OH), 6.2 (lH, s, 5-H),
5.7 (lH, sbr, CH20H), 4.4 (2H, d, CH20H), 2.3 (3H, s, CH3).
(B) Using the appropriate aldehyde in a 1:1 molar ratio to the
3-hydroxy-6-methyl-4-pyrone in step (3) above the following
2-hydroxyalkyl compounds were prepared.
3-Hvdroxy-2-(l-hvdroxvethvl)-6-methYl-4-Dvrone
~H (d6-DMSO), 8.7 (lH, sbr, OH), 6.2 (lH, s, 5-H),
5.4 (lH, sbr, CHOH), 5.0 (lH, dbr, CH), 2.3 (3H, s, CH3),
1.4 (3H, d, CH3CH).
3-Hvdroxv-2-(1-hvdroxvProPvl)-6-methvl-4-Pvrone
- ~H (d6-DMSO), 8.7 (lH, sbr, OH), 6.2 (lH, s, 5-H),
5.3 (lH, d, CH-OH), 4.7 (lH, m, CH-OH), 2.3 (3H, s, CH3),
1.5 (2H, m, CHz), 0-9 (3H, t. CH2CH3)
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Example 3 : PreParation of 2.6-dimethYl-3-hydroxv-4-pYrone and
other 2-alkvl-6-methvl-4-Pvrones
LA) 3-Hydroxy-2-hydroxymethyl-6-methyl-4-pyrone (prepared as
described in Example 2) (2 9, 0.013 mol) was dissolved
05 in 100 ml H20. Zinc dust (1.7 g, 0.026 mol) was added and the
mixture was stirred vigorously at 600C. Concentrated HCl
(13 ml, 10 molar equivalent) was added dropwise over a period of
about an hour. The slurry was left stirring at 600C for 12 hours.
The excess zinc was then removed by hot filtration, the pH was
adjusted to 1 and the mixture was extracted into dichloromethane
(3 x 50 ml). The extracts were combined, dried over anhydrous
sodium sulphate and evaporated to dryness to yield the title
compound (0.8 g, 47X). This was recrystallized from toluene9
melting point 163-1640C;
VmaX (nuiol) 1660, 1630, 1580, 1220, 1080, 970, 930 cm~l;
~H (d6-DMSO), 8.5 (lH, sbr, OH), 6.2 (lH, s, 5-H),
2.2 (6H, s, CH3CH3).
~ Other 2-alkyl-6-methyl-4-pyrones may be prepared in a similar
manner from the corresponding 3-hydroxy-2-(1-hydroxyalkyl-6-
methyl-4-pyrones.
ExamPle 4 : PreParation of 3:1 liqand:indium(III) and qallium(III)
complexes
(A) PreParation of (ethvl-maltol)~-qallium 67 comPlex
Ethyl maltol is prepared as described in Example 1 of U.S.
Patent Application 310,141 referred to in U.S. Patent No. 3,376,317.
The ethyl maltol is made up in stock solutions at a concentration
of O.OlM in 20 mM HEPES/0.8.X NaCl, pH 7.4-7.5. The more
concentrated stock solution of ethyl-maltol is added dropwise to an
acidic solution of 67Ga-(GaC13) in 0.04 N HCl. The resulting
solution is neutralized to pH 7Ø
Complexes of other substituted 3-hydroxy-4-pyrones are prepared
in a similar manner.
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(B) Preparation of (l-ethvl-3-hydroxvPYridin-2-one)3-indium 111
complex
The synthesis of l-ethyl-3-hydroxypyridin-2-one is described in
Example 1 of European Patent No. 094149.
05 The l-ethyl-3-hydroxypyridin-2-one is made ùp into stock
solutions in 20 mM HEPES/0.8 NaCl pH 7.4-7.5 at a final
concentration of O.OlM. The 1-ethyl-3-hydroxypyridin-2-one
solution is added dropwise to an acidic solution of lllIn-(InC13)
in 0.04 N HCl. The resulting solution is neutralized to pH 7Ø
Complexes of other 3-hydroxypyridin-2-ones are prepared in a
similar manner.
In both the case of the hydroxypyrones and hydroxypyridinones
the ligands are preferably added in large excess as compared with
the metal salt, for example a 100:1 or 1000:1 rather than a 3:1
molar proportion of ligand providing compound:salt, in order to
ensure that all the radioactive metal cations combine with the
ligand providing compound in the form of the 3:1 complex.
ExamPle 5 : Partition coefficients
Partition coefficients of complexes were determined by addition
of 1 ml of a solution of the 67Ga-(III) and 1llIn-(III) 3:1 complex
prepared as described in Example 1 using a 3:1 molar proportion
to 1 ml of water-saturated octan-l-ol in a glass test tube.
The system was allowed to equilibrate at ambient temperature
for 15 minutes on a gently rotating mixer. The tubes were spun
at 3000 rpm (1500 9) for 10 minutes after which time 200 microliter
samples of each phase were taken, weighed using an analytical
balance and counted for radioactivity. Partition coefficients were
calculated using the following equation:
p c = counts per minute/qram octanol
counts per minute/gram HEPES/saline
The partition coefficients obtained for various 3-hydroxy-4-pyrone
(HP) complexes are shown in Table 1.
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TABLE 1
_
COMPOUNO PARTITION COEFFICIENT (Kpart)
67Ga lllIn
maltol 0.171.33 ~+0.04)
ethyl-maltol 4.2218.19 (+1.17)
isopropyl-maltol 20.9
butyl-maltol 72.5
The partition coefficients of various ligand-providing
compounds described in Examples 1 to 3 were also determined.
Solutions of the ligands were produced by dissolving the ligand
in aqueous tris hydrochloride of pH 7.4. Acid washed glassware was
05 used throughout and, following mixing of 5 ml of the 10-4M aqueous
solution with 5 ml of n-octanol for 1 minute, the aqueous n-octanol
m;xture was centrifuged at 1,000 9 for 30seconds. The two
resulting phases were separated for a concentration determination
by spectrophotometry on each. Values typical of those obtained are
shown in Table 2.
TABLE 2
COMPOUND PARTITION COEFFICIENT
( Kpart)
3-hydroxy-6-methyl-4-pyrone 0.261 + 0.013
3-hydroxy-2,6-dimethyl-4-pyrone 2.766 + 0.034
2-(trifluoromethyl)-3-hydroxy-4-pyrone 0.639 + 0.136
3-hydroxy-2-methoxymethyl-4-pyrone 0.238 + 0.047
2-ethoxymethyl-3-hydroxy-4-pyrone 0.417 + 0.062
3-hydroxy-2-propoxymethyl-4-pyrone 2.004 + 0.182
2-butoxymethyl-3-hydroxy-4-pyrone j 5.992 + 0.460
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Example 6: Red cell labellinq with r67Ga(III)-(3-hYdraxv-4-
pyrone)31 complexes
Red cell labelling was performed using whole blood taken from
normal adult human volunteers. The blood was initially spun
05 at 1500 rpm (400 91 for 15 minutes and the plasma removed. The
packed cells were washed with an equal volume of heparinised
saline, centrifuged to remove the saline and the wash step
repeated. The washed packed cells were pooled and briefly rotated
on a mixer-rotator before use. For cell labelling experiments 1 ml
of washed cells was added to a heparinised glass blood collection
tube. To this 1 ml of cells was added 0.5 ml of a solution of
the 67Ga(III) 3:1 3-hydroxy-4-pyrone complex prepared as described
in Example 1 using a 100:1 to 1000:1 molar proportion. The tubes
were capped and placed on a rotating mixer for the duration of the
study, except for brief sampling when required. One hundred
microliter samples of the cell suspensions were taken at various
time intervals and added to clean glass test tubes containing 2 ml
of saline. The sample suspension was mixed well and centrifuged
at 3000 rpm (1500 9) for 5 minutes. The supernatant was removed
and the cells washed for a second time with saline. After removal
of the second saline wash the cells were lysed using deionised
water and the final volume made up to match that of the previous
saline washes. Both cells and supernatant were counted using an
auto gamma counter set for 67Ga photon energies (95-300 keY).
The results are illustrated in the accompanying Figures 1 to ~.
Each figure shows the percentage of 67Ga-complex incorporated into
the red blood cells as a function of time,
Figure 1 represents the data for 67Ga(III)-(maltol)3 for
comparative purposes,
Figure 2 represents the data for 67Ga(III)-(ethyl-maltol)3,
Figure 3 for 67Ga(III)-(isopropyl-maltol)3, and
Figure 4 for 67Ga(III)-(butyl-maltol)3.
As can be seen from these figures the complexes conta;ning the
larger alkyl groups allow for more rapid uptake of 67Ga into the
red cells and achieve a higher absolute incorporation.
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Figure 5 shows the effect of ligand concentration on uptake
of 67Ga into eryth~ocy~es at 15 minutes for various complexes. The
graphs labelled i to 4 represent, respectively, the results for the
maltol, ethyl-maltol, isopropyl-maltol and butyl-maltol 67Ga
05 complexes (the first for comparative purposes). Again this
indicates that the complexes containing the larger alkyl groups
give increased incorporation of 67~a into red cells.
ExamPle 7 : Leucocvte labellin~ with [lllIn(III)-
( 3-hvdroxY-4-pyrones ) ~1
The effect of ligand concentration on white cell labelling
with lllIn(III) 3:1 3-hydroxy-4-pyrone complexes prepared as
described in Example 1 using a 100:1 to 1000:1 molar proportion was
performed using mixed whited cell populations suspended in buffer.
The white cells were harvested from 60 ml of whole blood obtained
from normal human volunteers. The blood was drawn into a 60 ml
syringe, anticoagulated using heparin (100 Units/10 ml whole blood)
and the red cells allowed to sediment by standing the syringe
upright using a ring-stand for 1 hour. At the end of 1 hour the
majority of the white cells were found in the supernatant plasma.
The supernatant was expressed into a sterile plastic tube using a
19 g bufferfly set. The cells were washed twice using 20 mM
HEPES-saline (0.8%) and resuspended in 5 ml buffer. A cell count
and differential was performed using a coulter-counter device and
the cell suspension concentration was adjusted to 9 x 106 white
cells/0.9 ml. Labelling was performed by adding 0.1 ml of a
solution of the appropriate lllIn(III) 3:1 complex and incubating
for 15 minutes. After 15 minutes incubation the cells were washed
twice with Z ml of buffer. The cells and separate washes were
counted using an automatic gamma counter with energy windows set
for lllIn.
Figure 6 shows the effect of ligand concentration on complex
incorporation into leucocytes, the labelling of the graphs being as
indicated above for Figure 5. These results are similar to those
observed for the gallium complexes in Example 3 in that increased
incorporation is obtained with the complexes containing the larger
alkyl groups.
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_xample 8 : The effect of cell concentration on radiolabelled
complex incorPoratiOn
The effect of white cell concentration was studied using
radiolabelled lllln-(111)-(2-butyl-3-hydroxypyridin-2-one)3
05 complex. Cells were labelled, separated and counted using the
procedures of Example 4. The effect of red cell volume was
investigated using the butyl-maltol 3:l Ga(IlI) complex.
0.5 ml of the complex prepared as in Example l using a lOO:l
to lOOO:l molar proportion was added to heparinised glass blood
collection tubes containing l, 2, 3, 4 or 5 ml of washed, packed
red cells obtained as in Example 3. lO0 ~l aliquots of cell
suspension were removed at time 5, lS, 30 and 45 minutes, added to
tubes containing 2 ml of O.9X saline and washed, separated and
counted using the procedures of Example 3.
lS Figure 7 shows the effect of leucocyte concentration on indium
labelled complex incorporation at O.OOOl M concentration of the
butyl-maltol 3:l ll In(III) complex, incubation being for
lS minutes. Cell concentration had no significant effect on
percentage uptake of the radiolabelled complex. These results were
paralleled in the red cell volume stud;es for gall;um labelled
complex incorporation of the butyl-maltol 3:l 67Ga(I11) complex
(results not shown).
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