Note: Descriptions are shown in the official language in which they were submitted.
~ 93/23743 ` 2 1 3 5 8 8 2 Pcr/us93/o3g86
Me~hod of ~re~arinq a diaqnostic aaent fQr detectinq inflamrn~tions
The invention relates to a method of preparing a dia~nostic agent for detecting
and locating inflammations in a warm-blooded living bein~ by attachin~ a detsctabls
label to an immunospecific protein or proteinaceous substance. Th~ invention furthcr
relates to a diagnostic agent obtained by using said method, to a pharmac~uticalcomposition comprising said diagnostic aQent, to the usc of said composition and to
a kit for preparing said pharmaceutic~l composition.
Inflammations in ths body of a warm-blooded living being cause many diseases
and disorders, and may even turn out to be iife-threatening. In order eo b~ abl0 to
achieve a specific therapy, the detection and location o~ the inflamrnation site~s) in
an earty stage is o~ the utmost importance. A good dia~nostic a~ent is also
indispensable for supporting the therapy used. Various requir~rnents havo ~o be
imposed on such a diagnostic agent, for example, non-toxic, no adverse influenceon the host resistance andlor therapeutic treatment, well d~tec~abla and selective.
The required high selectivity means that the dia~nostic agent, aftet having beenintroduced into the body, must accumulate selectively at the site of the inflammaticn
to be detected. In order to be detectable from outside the body, the diagnos~ic agent
2û should be labelled, preOerably with a radionuclide or wi~h a paramagnetic metal
;~ ~ isotope. In the former case, the radioactive radiation can be detected by usin~ a
suitable detector (scanning~. Modern techniques in this field vse emission
tomo~raphy; when gamma radiating isotopes are used, the so-called single photon
emission computerized tomography ~SPECTI may be applied. The use of
paramagnetic dia~nostic a~ents enables a detection by means of imaging by
magnetic resonance (magnetic resonance imagin~
Europearl Patent Application 241106 describes the us~ of labelled
immunoglobulins or fragments thereof for dia9nosing i~flammations. As is stated in
said paten~ specification, irnmunoglobulins tend to accumulate more strongly in the
inflamed sites than in the non-inflamed sites. As a res~lt of this seleetivity, i.e. a
comparatively stronger concentration at the sit~ of the infiammation, labelled
irnmunoglobulins may be used for diagnostic imaging. From the examples of said
patent specification a notable preference of radioactive-labelled immunog~obulins
indeed appears ~or accumul~tion in inflamed sites over uptake in ~-on-inflamed sites
of the body.
However, the selectivity, i.c. the specific accumulation of the abov~-described
labelled immunoglobulins in the inflamed sites, is still unsatisfactory in practice.
SUBSTi~ 3-E SH~E t
W0 93/237~3 2~3S S ~ Z PCI/US93/03586
Selectivity is to bo und~rstood to mean the target-to-non- taraet ratio. This means
that a comparativel~l lar~e amount of radioactivity is found in tha non-tar~ot tissues,
-8 in ths ~lood, as a result of which the image of the inflammation to bc examined
is obscured. Moreover, when a considsrable quantity of radioactivity remains
circulating, tha locating of inflammations is dis~urbed in cas0 these are present in
certain sites of the body. As a result of this a spod imaging, in particular in an early
stage of the inflammationl is disturbed so that a correct diagnosis is impeded
Therefore there exists a need for a~ents for dia~nosing inflammations with a better
selectivity than the above-described labelled immunoglobulins known for this
1 0 purpose.
It has now been found surprisingly that said need can be met by using a protein
or proteinaceous substance, which prior to tho labelling procedwe has been pvrified
by using a s~fstem compr;sing an anion exchange ssparatin~ substance
Conseq~lently the present invention relates to a method of preparing a diagnostic
agent as defined in the opening paragraph, and is characterized in that prior to the
labell;ng procedure the protein or proteinaceous~ substance is purified by using a
system comprising an anion exchange separatir~g substance.
Suitable anion exchange separating substances which can be used for the
method of the invention are, in particular, 3rion exchangs resins. The use of anion
20 ~ exchange resins instead of bacterial proteins such as protein-A is advantageous in
that no detrimental bacterial endotoxins or othet pyro~ens are applied by ;ntravenous
administration in a human being.
An example of a suitable system comprising an anion exchange resin is Mono-
: ~a,
According to a preferred procedure, the purification is carried out by subjectin~
a solution of the protein or ~proteinaceous substance to be purified, preferably ;n a
suitsble aqueous buffer solu:tion, to anion exchang~ chromatography. In the
chromatographic process said system, comprising an anion exchange resin, is usedas the im~nobile phase and the solution ot the protein or proteinaceous substance as
the mobile phase. By eluting the irnmobile phase with a suitable eluent under
appropria~e conditions the purified protein or proteinaceous substance can be
isolated and then be labelled at eaGh desired instant.
After purification as~described above, the protein or proteinaeeous substance
should be labelled by attaching a detectable label thereto. The detectable label may
be a radionuclide. prefer~biy selected from the ~roup consistin~ of 1-123,1-131, Br-
75, Br-76, Tc-~9m, Pb-~t)3, Ga-67, Ga-68, As-72, In-l 11, In-l 1 3m, Ru-97, Cu-62,
Cu-64, Cu-67, Fe-52, Mn-52m and Cr-51, or a paramagnetic label, preferably
.
~l~BST~T~T F S~E~T
~ 2i~882
J 93/23743
PCI /US93/~3586
selected from the group consisting of F-19, D, Na-23, P-31, Gd-157, Mn-55, Dy-
162, Cr-52 and Fe-56. The label may be attached to th~ purified protein or
pro~einaceous substance directly, if desired after a raductivo treatment ot saidprotein or proteinaceous substance, or through a suitable linker.
Direct labelling ot th~ protein with radioactiv~ halogen can be carried out
by reacting the protein with a suitable compound of the radionuclide in a preferably
aqueous solution, if desired in the presenca of an oxidant. Suitable radioactivehalogen cornpounds are radioactive alkali halogenides or radioactive halo-substituted
aromates, such as tytosin~.
The direct labell;n~ of tha prot~in with a metal radionuclide has two
disadvantages, via (i\ that the biologically active site of the protein needed for a good
selectivity can easily be blocked by said r~action so that the normal behaviour of the
biologic macromolecule is disturbed, and (ii) that the affinity between metal isotope
and macromolecul~ is often insufficiEnt as a result of which the formed bond is
insu~ficiently stable to remain intact under physiological conditions. Therefore in that
case a reductive treatment of the protein, as sug~ested eØ in European patent
applications 237150 and 271806, is pre~erred. In such a pretreatment the disulphide
bonds in lhe - protein are reduced with a suitable agent, e.g. dithiothreitol,
dithioetythritol, 2-mercaptoethanol or 2-mercaptoethylamine, after which the
reducod protein, now comprising free rtlercapto groups, may be reacted with a salt
ot chelate of the desired metal isotope. Stabiliz~tion of the reduced protein or the
final complex may be desired. It is generally recognized, however, that the reductive
pretreatnnent of the protein may exert an unfavourable influence on the biological
behaviour of the protein.
To avoid this disadvantage, it is proposed in th~ Netherlands patent
application 9001t37 in the namB of Academisch Ziekenhuis Leiden to use an
effective amount of a borohydride in the labelling with technetium-99rn, in order to
obtain a Tc-99m labeiled~protein or proteinaceovs substance which is excellentlysuitabls for the purpose in~view. Equally suitable is the use of a suitable linker or
coupling agent for preparing~a metal-rad;onuclide-labelled protein or proteinaceous
substanc~, e~g. as described in the Intetn~ paterlt application ~PCT) W0 89tO7456
and the European patent application 178125, both in the name of Maliinckrodt Inc.
The protein or proteinacsous substanee to be labelled is then first modified by `'~
tre~ting with a couplin~ a9ent, after which the resulting protein conjugate is btought
into a complexing reaction with a salt w chelate of the desired metal isotopes. In
literaturc various different coupling agents are desc-ibed, via eompounds which after
coupling with the protein can complex the metal isotope by an N2Sa-, N3S- or N"-
~UB~IT~ITE SH~ET
W093t23743 7 13S882
~CI/US93/03586
tetradentat~ ring structure, N-containing di- or polyacatic acids or their derivativ~s
such as EDl A, DTPA, NTA, etc., amino-containin~ compounds such as the
maleimide d~rivatives disclosed in tha above EP 1~8125, peptide-derivatiws, and
compounds comprising chelating ~roups such as isocyanat~, formyl, diazonium,
isothiocyanate, alkoxycarbimidoyl groups and the lik~. For the modification of the
pro~ein or proteinaceous substanc~, purified according to the invention, however, a
coupling agent is preferred whish is described in the above W0 8~/07456 and which
may g~nerally be represented by the formula
, - R--~
`- S--Y--~
wherein
is a branched or non-branched, optionally subst;tuted hydrocarbyl radical, whiohmay be interrupted by on~ or more hetero-atoms selected from N, 0 and S andlor
by one or more NH groups, and
Y i$ a group which is capable of reacting with a functional group of the protein and
which is preferably selected frorn the group consisting of carbonyl, carbimidoyl,
N-~C~-Co) alkylcarbimidoyf, N-hydroxycarbimidoyl and
N-(C,-C~,) alkoxycarbimidoyl, or a water-soluble salt of this ring cornpound. Examples
of suitable coupling agents described in said Intern. patent application are substituted
20 ~ or unsubstituted 2-iminothiolanes and 2-iminothiacyclohexanes.
The modification of the protein or proteinaceous substance, i.e. the reaction
with the coup!ing agent resulting in the protein~conju8ate~ can generally be carried
out in a simple manner. In the subsequent complex ~orming reaction, the metal
isotope is presented to the protein conjugate in the form of a salt or chelate. In the
latter oaso relatively weak chelators are used, e.g. a phosphonate or
polyphosphonate, an oxinate, a carboxylate, a hydroxycar~oxylate, an
aminocarboxylate or an enolat~. Then the~ desired complex is formed by ligand
exchange. The complex forming reactions can ~enerally be carried out in a simplernanner and under condition which spare the protein or proteinaceous substance.
The aboYe-mentioned labelling with To-9gm in the presence of a
borohydride can equally be carrieJ out in a simple manner and under very moderate
condi~ions, e.g. at room temperature.
The method of the invention relates more in particu1ar to the preparation of a
dia~nostic agent for detecting and locatin~ inflammations, by labelling human
immunoglobulin, a subclass of human immuno~lobulin or a suitable ~ragment of
human immunoglobulin, said immuno~lobulin Ifragment~ being purified by using a
system comprising an anion exchange separating substance.
SUBS~ITIJTE ~HEFT
93/23743 2 1 3 S ~ 8 2 PCI/US93/03586
The invention further r~la~s to a diagnostic a~ent obtain~d by usin~ the m~thod
as defined above and to a pharrnaceutical composition which comprises, in addition
to a pharmaceutically acceptable carrier, a diagnostic agent obtained as defined
above. Such a pharmaceutical composition is int~nded for diagnostic application. If
desired th~ composition so obtained can be brought into a form mor~ suitable forintravenous or subcutaneous application, e.g. by addin~ a pharmaceutically
acceptable liquid cartier material. For intravenous or subcutan~ous application tha
solution should of course be in a sterilc condition.
For performing a diagnostic examination the composition, as doscribed above,
if desired aftef dilution with a pharmacsutically acceptable liquid, pteferabl~,r a
physiological saline solution, can be administered to a warm-blooded living being in
a quantity suff;cient for externally imaging said baing to detect and locate an
inflammation in said being. In case a radioactive labelled protein or ptoteinaceous
substance is used as a diagnostic agent, the radioactive material is generally
administered to the living bein~ in a quantity from 1 to 2000 MBq, preferably ~rom
100 to 1200 Mbq, per 70 kg of body weight. Thereupon thc b0ing is subjected to
external imagin~ to detect ~ccurnulated radioactiv;ty and thus to d~terminc the
location thereof in the body of the being. Both sterile and as~erile inflamrnations can
be~detected by ~sin~ the above compositions. An example of the former is
rheumascinti~raphy: bacterial inflarnmations and abscesses are examples of asterile
inflammations.
In case a radioactive labelled protein or proteinaceous substance is used as a
diagnostic agent, it is frequently impossible to put the ready-for-use composition at
the disposal of the user, in connection with thc often poor shelf life of the
2 5 radiolabelled oompound andlor the short half-lifc of tha radionuclid~ used. In such
cases the us~r will carry out the labelling reaction with the radionuclide in the clinical
hospital or laboratory. ~For this purpose the various reaction ingrsdients are then
offered to the user in ~the iorm of a so-called "kit". It will be obvious that the
manipulations necessary to perform the desired reaction should bs as simple as
possible to enable th3 user to prepara from the kit tha radioactiv~ labelled
composition by using the facilities that are at his disposal. Therefor~ the invention
~Iso relates to a kit for preparing a radiopharmaceutical cornposition,
Such a kit according to th~ present invention may comprise (i) an
immunospecific protein or proteinaceous substance purified as described above, to
3~ which substanc~, if desired, an inert pharmaceutically acceptabie carrier andlor
~ormulating agents and/or auxiliaty substances islar~ added, ~ii) a solution of a
compound of a radionvclide, and ~iii) instructions for use with a prescription for
SUBSTITUTE S~ T
WO93/23743 ~ 13~8~2 PCI/US93/~)35~6
reacting th~ ingredients present in the kit. If in such a kit th~ radionuclide is
radioactiv~ halogen, preferably a halogen compound as defin~d hereinbefore is used
as an ingredient of th~ kit.
Alternatively, such a kit accordin~ to ~he pr~sent invention may comprise (i) a
substance obtained by ~reating a human irnmuno-sp~cific protein or prot0inaceoussubstance, putified as described above, w~h a suitable disulphid~-reducing aQen~ as
defined hereinbefore, to which substance, if desired, an inert pharmaceuticaliy
acceptable carrier and/or formulation agents andlor auxiliary substances is/are added,
(ii) a solution of a salt or chelate of a metal-radionuclide, and ~ instruction for us~
with a prescription for reacting the ingredients pres0nt in th~ kit. A suitable carrier
is ~ physiological saline solution. Examplss of auxiliary sub~tances arc s~abilizers,
antioxidants and filling agents. Suitable metal-radionuclides have been mentioned
. hereinbefore. As state~ abov~, the desired radionuclidc for this complex formin~
reaction prescribed may be presented to the reduced protein in the form of a chelate,
~ bound to a cornparatively weak cheiator, in which the reaction may take place in a
neutral environment, e.g. a buffered substantially aqueous solution. The kit to be
supplied to th~ user may also comprise th~ in~redient(s) defined sub fi~ above,
toge~h~r with instructions for use, whereas the solution of the salt or chelate of th~
radionuclide, defined sub tii) above, which solution has ~ limited shelf life, may be
put to the disposal of the user separa~ely.
In case the kit serves to prepare a radiopharmaceutical com~osition labeiled with
technetium-99m, such a kit according to the preserlt invention may comprise, in
addition to the ingredient~s~ defined sub (i) above, lii) a pertechnetate-reducing a~ent
and, if desired, a chelator, and ~ii;) instructions for use with a prescription for
reacting the in~redients of th2 kit with technetium-99rn in the ~orm of
pertechnetat~ sotution. If desired, ths ingrsdients of tha kit rnay be combined,provided they are compatible. The kit shoutd compriss a reducin~ a~ent to reducethe pertechnetate, for example, a dithionite, a metallic reducing a~ent or a complex-
stabilizing reducing agent, e.~. Sn~il3-tartrat~, Sn(lll-phosphQnats or -pyrophosphats,
or Sn(ll)-~lucoheptonate. The pertechnetate can ~imply be obtained by the ~ser from
a suitable generator. Examples of suitabla chelators haw been described
hereinbefore.
In a preferred embodiment the kit according to the prssen~ invention and
intended for the preparation of a Tc-~9m labelled composition comprises in addition
a borohydride in a quantity effective ~or the labeiling procedure.
In an eq-cally preferred embodiment the kit acoording tv the present invention
comprises, instead of a Feduced protein or proteinaceous substance, a protein
SUBST~TUT~ SH~:ET
....... ... . ...... .. ..... . .. .. .... ..
~ 2i35882
93/23743 PCr/US93/03~i~6
conjugatt~, obtained by modifyin~ an immunospscific prot~in or proteinaceous
substancc, purified as described hereinbefore, by 3 traatrnent with a couplin~ a~ent.
Suitable coupling a00nts have been described hereinbefor~. The use of a compoundof the gen~ral formula
, --R----~
~ y J
wher~in the symbols have thc meanings ~iven hereinbeforc; as a coupling agent isto be preferred.
When the radionuclido is present in the kit itself, tht~ complex formirlg reaction
with the protein ~onju~at~ can simply be produced by combining the components
in a neutral medium and causin~ them to react. For that purpose the radionuclide is
prefetably presented to the protein conjugate in tht~ form of a chelats bonded to a
. comparatively weak chelator. Examplss of suitable chelators for tht~ radionuclid~ ara
8-hydroxquinolin~ or derivatives ht~resf; dicarboxylic acids, polyc~rboxylic acids or
hydroxycarboxylic acids, for ~xampl~, oxalic acid, malonic acid, succinic aoid, maleic
acid, orthophtalic acid, malic acid, lactic acid, tartaric acid, CitfiC acid, ~corbic acid,
salicylic acid or d~rivatives of thess acids; pyrophosphat~s; phosphona~es or
polyphosphonates, for example, methylene diphosphonate, hydroxyethylena
disphosphonate or hydroxyrnsthylene diphosphonate; or enolat~, f¢r exampls, wi~h~: 20 a ~-diketone, for e%ample, acetyl acetone, furoyl ac0tone, ~henoyl aceton~, benzoyl
acetone, dibenzoyl methane, tropolone or derivatives of these diketones. For this
purpose are to be considsred in particular 8-hydroxyquinoline, citric acid, tartaric
acid, ascorbic acid, glucoheptonic acid or a derivative thereof, or acetyl acetone as
ohelators because it h~s been found that a chelate of a radionuclide, for example,
indium-111 or lead-203, with one of thesc chelators in a suitabls medium, preferably
a buffered aqueous solution, easily reacts at a physiological pH with a protein
conjugate as defined hereinbe~or~, the desired radionuclid~ compl0x bein~ formsdin a high yield and purity by ligan~ exchange. A buffer~d aqueou~ indium-1111-
tropolonate solvtion which may be used ~or the desired complex formation and îs
suitable fw this purpose is described in European patent applicaeion no.
131327. The supplied kit may also consist of the constituent ~efined sub ~l) with
instructions ~or use, while the sclution mentioned sub ~2), which is bound to anexpiration date, can be placed at the user's disposal separately.
When ~he kit in addition comprises a borohydride, said borohydride is
preferably NaBH,, or NaBH3CN. A quantity of O-Q1-t ug of borohydride is generally
suffieient for labelling ().1 mg o~ the protein or prote;naeeou8 svbstance. Pre~erably
also a pertechnetats-reducing agent is present in such a kit, e.g. Sn(l!).
SUB~T~TllTF SH~ET
WO 93/23743 '~ 1 3 S 8 ~ 2 PCI /US93~035B6
Wh~n th~ kit comprises a protsin conju~at~ as d~fin~ hereinbefora and is
intended for th~ preparation of a radiopharrnaceutical composition, labell~d with
technetium 99m, the radionuclide will pref~rably be~added saparately in the form of
a pertechnetate solution. In that cas~ the kit will comprise a pertechnetate-rsducin~
agent and, if desired, a chelator, the former to reduca tha pertechnetata. As ~
pertechnetate-reducing agent may b0 ~Issd, for exampte, a dithionite or a metallic
reducing agenti The in~redients may optionally be combined, provided th~y ar~
compatible. Such a rnonocomponent kit, in which the combined in~redients are
preferably freeze-dried, is exc~llently suitable for bein~ r~acted, by the user, with the
t 0 radionuclide solution. As a r~ducin~ agent for th~ above-mentionad kits is pre~erably
used a metallic reducing a~ent, fot example, Sn~ll), Fe(ll~, Cu~l), Ti~ or Sb~lll); Snlll)
is excellently suitabl~ The protein constituent of the above-mentioned kits, iØ
preferably the protein coniu3ate, rnay be supplied as a solution, for example, in the
form of a physiolo~ical saline solution, or in some buffer ~olution, but is preferably
present in a dry state, for example, in th~ freeze-dried state. When used as a
component for an injection liquid it should be sterile, in which, when th~ constituent
is in ~h~ dry state, the user should preferably use a sterile physiolo9ical saline
solution as a solv0nt. If d~sired, th~ above-men~ioned constitu~nt may b~ stabilizad
in the conventional n anner with suitablc stabilizers, for exampla, ascorbic acid,
ZO gentisic acid or salts of these acids, or it may comprise other auxiliary a~ents, for
example, fillers, svch as glucosc, lactosc, mannitol, an the like.
The invention will~ now be described in ~reater detail with reference to the
ensuing specific example.
EXAMPLE
To show the results vf purification of human immunoglobuiin, by using Mono~
as the system comprising an anion exchange resin, the following experiments are
carried out.
To be able to determina the results of the purification proc~dure, a protein i~
used, iabelled with Tc-99m as described in the Intern. patent application W0
89iO7456, mentioned hereinbefore. This product is prepared from a Iyophilized kit
containing 1 mg 2-iminothiolane-modified polyc~onal human immunoglobulin and
stannous tartratc, marketed by Mallinckrodt Medical B-V- under the ~egist2red :rade
name Technescan HIG, and technetium-99m in the form of a pertechnetate solution,obtained from ~ molybdenum-technetium generator.,
The above labelled human immunoglobulin ~13~ is purified by anion exchangs
chromato~raphy by using a Mono d~)cotumn. This column i5 regenerated with 100
S~BST~,~U I E ~HEET
.
~ 21~88~
~3/23743 PCl`/US93/0358~
ml 2M NaOH solution and then flushed with an elution buffer, viz. TRIS 20 mM.
1 mg of human ;mmuno~lobulin (T~chnescan 1 IIG~ is labelled with 100 MBq of
99mTc-pertechnetate in 1 ml, at room temperature accordin~ to the product insert.
The labelled protein is brought on the regenerated Mono Q~Dcolumn and the
protein is fixed ~n the column with a 20 mM TRIS solution. The protein fractions are
eluted from ~h~ column using mixtures of 25/75%, 50/50% and 0/100% of 20 mM
TRiS and 1 M NaCI, resp.
Th0 fractions obtained ar~ ;ncubated with 3.8x107 CFU of Staphylococcus
Aureus iSt. Aureus) ATCC 25923 in 2 ml growth medium for 1.2 and 5 hou-s. The
- 1û labellin~ of the bact~rial pellet is measured in a wsll-typ~ scintillation counter and
reported as percentages of total activity bound. The followin~ results are obtain~d:
Table A. -~
Table A
t -- 60' 120' 300'
control 12.5 2156
25f7~~ 22 2744
50/50% 34 ~g54
OJ100% 29 6873
The con rol is non-purified labelled HIG.
Separately, the fractions obtained are injected in mice, infected with 2x10' CFUof St. Aursus 25923 in on~ thigh 18 hours before. Aft0r 15 minutes, 1 hour, 4
hours and 24 hours, via 3 region of interest (ROI) method, the ratios of inJected-to-
norrnat thi~h are determined.
As a control, non-purified l~belled HIG is used. The results are recorded ir) Table B
below.
Tab!e B
t = 15 min. 1 hour 4 hours 24 hours
_ .
contfo! 2.6 3.3 3.8 3.5
2517556 3.4 4.~ 5.5 5.3
~iO~50% 3.6. 4.9 5.6 2.3
0/1 00% 2.5 3.7 4.7 5.0
SUBST~TUTE Sh'EET
