Note: Descriptions are shown in the official language in which they were submitted.
PATENT DOCKET 536
1 335725
METHOD OF DIAGNOSING BLOOD CLOTS
USING FIBRIN-BINDING PROTEINS
~çhnil~l Field
The present invention relates to mPth~l~ for det~ting fibrin
depositc within the body. Fibrin-binding proteins having det~' le
subs~n~s, e.g. radiolluçlides, ~tt -hed thereto are pravided for diagnosing
fibrin deposit~, such as blood clots, and for monitoring the di~sol~tion thereofduring therapy.
Bac~und Ar
Fibrin is an insoluble protein which is produced at the site of
a wound through a chain reaction involving formation and activation of certain
v?~Cul~r proteins. A fibrous netw~ of fibrin forms at the wound site and
co,nbines with blood pl~tf~.let~:, thus producin~ a fibrin-platelet clot which stops
the flow of blood from the wound. Fibrin-platelet clot formation is thus
e~n~i~l for the survival of hum~n~ and other ~nim~ls However, fibrin-
platelet clot formation elsewhere in the body (i.e., at locations other than
wound sites) causes a dangerous, potentially life-
1 335725 ``
threatening restriction of blood flow. Blood clots, also known as
thrombi, may remain at the original point of formation or may
dislodge and travel through the bloodstream to a new oite where the
clot causes a sudden blocking of blood flow. Examples of the
medical problems caused by abnormal fibrin-platelet clots include
venous and arterial thromboses, heart attacks caused by thrombi in
heart vessels, as well as pulmonary and cerebral thromboembolism.
In addition, fibrin-platelet clots have been reported to occur at
sites of infarcts and tumors, wherein fibrin may surround the
damaged tissue or tumor, thus further aggravating the patient's
condition. (See U.S. Patent No. 4,418,052.) In view of the high
incidence of medical problems associated with abnormal fibrin-
platelet clots, much effort has been directed to development of
techniques for diagnosing such conditions. Unfortunately, many of
these techniques 6uffer from lack of 6pecificity or reliability,
while others require unacceptable lengths of time to complete the
~ testing. Still other methods are designed to detect thrombi in the
process of forming, but not preexisting clots.
Among the diagnostic methods which have been attempted is
the use of radiolabeled proteins, ~uch as e.~ -s, which either
bind to or become incorporated within a clot, 60 that the clot can
be imaged using techniques for detection of the radioisotope within
the body. Such proteins include streptokinase, urokinase, tissue
pl~ ~n~gen activator, fibrokinase, streptokinase-activated human
plasmin, fibrin and certain fragments thereof. (See, for example,
U.S. Patents Nos. 4,427,646; 4,416,865; 4,418,052; and 4,663,146.)
However, such problems as low 6pecificity or affinity of the
radiolabeled protein for a clot, denaturation of the protein during
the radiolabeling procedures, and unstable attPc~ -~t of the
radioisotope to the protein have been associated with certain of
these proposed diagnostic techniques. Thus, a need ~ ~Inc for an
accurate, convenient method for early detection of abnormal fibrin-
platelet clots within the body.
LC8x165g.mhg
1 335725
Summary of the Invention
The present invention provides a method for detecting a
fibrin deposit iB vivo, comprising the steps of:
(a) administering to a patient suspected of having a
fibrin-platelet clot a labeled thrombolytic protein, wherein the
thrombolytic protein's clot-dissolving activity is reduced or
eliminated and the label is selectively sttached to a portion of
the thrombolytic protein other than the fibrin-binding domain; and
(b) detecting the pattern of biodistribution of the labeled
thrombolytic protein in the patient.
Thrombolytic proteins include such proteins as plasmin and
pl A~ inogen activators such as streptokinase, streptodornase and
urokinase. Preferably, the Al' ini5tered thrombolytic protein is
tissue-type plasminogen activator (t-PA) having reduced
_ pl~A~ insgen-activating activity, wherein the detectable substance
attached thereto is a radioisotope such as 99mTc in the form of a
chelate.
In one embodiment of the invention, the detectable
substance is attached to the thrombolytic protein through a linker
which may bind to a portion of the thrombolytic protein responsible
for the clot-dissolving activity or at such other portion of the
thrombolytic protein to reduce or eliminate the thrombolytic
activity. Reducing the activity prolongs localization of the
protein at the fibrin deposit in I vo and helps ini i 7e side
effects associated with Al' inistration of protein having clot-
dissolving activity.
The present invention also provides a method for making a
labeled thrombolytic protein, comprising attPehing a detectable
substance to the thrombolytic protein through a linker wherein the
attachment of the linker is to a portion of the thrombolytic
protein other than the fibrin-binding domain.
LC8x1659.mhg
1 335725
Also provided by the present invention are fibrin-binding
proteins having detectable substances attached thereto, wherein the
detectable substance is specifically attached to a portion of the
protein other thsn the fibrin-binding domain. Specifically, t-PA
having a rsdioisotope attached thereto through a linker which binds
specifically to the portion of the t-PA protein responsible for
plA! ~nogen activation is provided. This t-PA protein of the
invention has reduced or substantially eliminated plA! ~sgen-
activating activity (depending on how much of the linker is bound
thereto), and an intact fibrin-binding ~- ~in.
The proteins of the present invention are A~ ~n~ stered for
in vivo diagnosis of fibrin deposits such as blood clots and for
monitoring the dissolution thereof during therapy. ~its useful for
preparation of radiolabeled, fibrin-binding proteins of the
~ invention also are disclosed herein.
-
Brief Description of the Drawings
Figure 1 shows gamma camera images of rabbits taken at the
time points indicated, following in~ection of radiolabeled t-PA to
image blood clots in the An~ ~ls.
Figure 2 depicts a scheme for synthesizing a D-
PhenylAlAn~n~-Proline-Arginine-CH2Cl linker.
Figure 3 depicts the reaction of a chelating compound with
25 a D-Phenylalanine-Proline-Arginine-CH2Cl linker.
Figure 4 depicts the synthesis of two different tripeptide-
chloromethyl ketone derivatives which are useful as linkers.
Figure 5 shows two radioiodinated linkers that may be bound
to t-PA.
Figure 6 shows the image of a ~ugular vein fibrin-platelet
clot using a labeled thrombolytic protein.
Detailed Description of the Invention
The present invention provides a method for diagnosing
fibrin-platelet clots within the body and for monitoring the
LC8x1659.mhg
1 335725
dissolution of such clots durlng therapeutic treatnent. The method
comprises administration of a labeled thrombolytic protein, wherein
any clot-dissolving activity of the thrombolytic proteln has been
reduced or substantially eliminated prior to ~dministration. The
fibrin b~n~ing property of the protein i6 preserved by selectively
attAch~nE the detectable substance to a portion of the protein
- other than the fibrin-binding ~ n, Procedures for detection of
the distribution of the detectable substance within the body are
employed to locate any fibrin-platelet clots, such as fibrin-
platelet clots associated with myocardial infarcts or tumors.
Reduction or elimination of the clot-dissolving activity serves to
allow completlon of the detection step before the bound protein is
released from the fibrin-platelet clot through the process of clot
dissolution. In addition, use of a thrombolytic protein having
reduced or el~m~na.ed clot-dissolving activity aç a diagnostic
- agent allows a physician to avoid tlnnPcessArily inducing side
effects associated with A~minictration of clot-dissolving e, y -s
in a pAt~ ~t who may not be afflicted with fibrin-platelet clots.
~ollowing diagnosis of a clot, periodic ~mlni ~tration of the
labeled thrombolytic protein of the present invention during
therapy may be used to monitor varlous therapeutic trestments ln
order to ensure complete dissolution of the clot. ~hile the widest
use of this invention is in human medical therapy, the invention is
equally applicable in a veterinary setting.
25
The invention also provides a method for attachlnE a
detectable substance specifically to a non-fibrin-b~n~ing portion
of the thrombolytic protein, such that the fibrin-bln~n~ property
of the thrombolytic protein is not ~i inished by labeling with the
detectable substance. Reduction of the fibrin-bln~lng property of
a labeled thrombolytic protein may decrease its effectiveness as a
clot-~ ~EInE agent, since the clot residence time would be
shortened. In addition, higher "background on images might result
from the increased portion of the ~m inistered labeled protein
which would not be localized at the clot site. A detectable
. ,~0 ., ~ ~0 _~
1 335725
substance may be selectively attached outside the fibrin-binding
domain of a protein using linkers that are described below.
The thrombolytic protein which i8 ~ ~n~ ~tered in
accordance with the present invention is any thrombolytic protein
which binds to fibrin in vivo. The thrombolytic protein may be
naturally occurring or may have been e~g~nPered to contain a
fibrin-binding domain, e.g., through genetic engineering
techniques. Advantageously, the thrombolytic protein is naturally
occurring in the patient to which it i6 to be ~ ~ni6tered~ to
reduce the chances of an adverse immunological response. Known
naturally-occurring, fibrin-binding proteins include, but are not
limited to, the plP~ ~ogen activators, such a8 urokinase (UKS) and
ti6sue-type pla ~n~gen activator (t-PA). P1P~ ~n~gen activators
lS are enzymes which catalyze the conversion of the inactive precursor
plP ~nogen to plasmin, which also binds fibrin and has fibrin-
dissolving activity. Ti6sue pl~l ~nogen activator i6 a 6erine
protease recognized as having a much higher affinity for fibrin
than does urokinase, wherein the enzymatic activity of t-PA is
localized at fibrin depo6it ~ites, ~o t-PA is generally preferred
for use in the present invention. Methods of isolating t-PA from
certain cell lines (e.g., Bowes melanoma cells) have been
described, and methods for producing t-PA through recombinant DNA
technology also are known (see British patent application GB
2,119,804).
The protein is treated to reduce the clot-dissolving
activity thereof while maint~n~ng its fibrin-binding property.
The term "clot-dissolving activity, n as used herein, refers to any
biological activity of the protein which causes, directly or
indirectly, breakdown of fibrin and, therefore, dissolution of a
fibrin-platelet clot. The t-PA enzyme does not directly dissolve
clots, but catalyzes the formation of the clot-dissolving protein
plasmin from pl~r in~gen. Therefore, for t-PA, the plasminogen-
LC8x1659.mhg
~7~ 1 3 3 5 7 2 5
activating biological activity is considered to be the "clot-
dissolving activity," as used herein.
The clot-dissolving (i.e., fibrin-degrading) activity of
the protein is reduced to a degree sufficient to allow completion
of the detection step before the thrombolytic protein is released
from the clot site. In addition to prolonging localization of the
thrombolytic protein at the fibrin deposit site, reduction of the
clot-dissolving activity also serves to m~n~ ~7e side effects
associated with administration of proteins having such activity.
It has been discovered that reduction of the clot-dissolving
activity is advantageous in order to detect accurately the location
of a fibrin-platelet clot within the body. As described in more
tetail below, when a clot-dissolving protein (having its native,
lln~ ~n~ shPd clot-dissolving activity) binds to a clot in vivo,
~ localized degradation of the fibrin begins, which results in
release of the thrombolytic protein from the original fibrin-
platelet clot site, especially when the protein is bound primarily
to the outer surface of the clot. Therefore, the biodistribution
of the protein, and the detectable ~ub6tance attached thereto, will
become progressively more diffuse. There will only be a short
period of time in which a 6ufficient amount of the detectable
substance has accumulated at a fibrin-platelet clot to give a
strong localized signal or image before dispersion begins.
25 Therefore, the physician may conclude that there never was a
fibrin-platelet clot, and may sub;ect the patient unnecessarily to
further testing. Reduction or elimination of the enzymatic
activity of t-PA, when used in the method of the present invention,
is especially important, because the activity of t-PA is known to
increase dramatically in the presence of fibrin.
The clot-dissolving activity of the thrombolytic protein
may be reduced by any suitable means as long as the fibrin-binding
property is maintained. In the case of production through
recombinant DNA technology, the cloned gene which encodes the
LS8x1659.mhg
1 335725
protein may be sltered to d~ ~n~ch the clot-dissolving activity,
e.g., through known methods of creating insertions, deletions and
other mutations in the gene. For example, the isolated gene may be
subjected to restriction enzyme digestion, alone or in combination
with other enzymatic treatments, such as digestion with certain
nucleases, to excise a portion or all of the gene segment which
encodes the portion of the protein responsible for the clot-
dissolving activity. Other known procedures, such as site-directed
mutagenesis, may be used to inactivate the clot-dissolving
activity. (See Old and Primrose, Principles of Gene Manipulation,
2nd Ed., University of California Press, Los Angeles, page 164.)
Alternatively, the protein itself may be fragmented and the fibrin-
binding portion thereof purified by known techniques, ~uch as
affinity chromatography. Chemical treatment of the protein to
decrease the clot-dissolving activity thereof, while main~n~ng
- the fibrin-binding property, is yet another option. One embodiment
of the present invention, described in more detail below, involves
reacting the protein with a chemical compound which selectively
binds to the portion of the protein responsible for the fibrin-
platelet clot-dissolving activity, wherein binding of the chemical
compound to this portion of the protein reduces said activity. The
chemical compound may bind to the protein reversibly or
irreversibly, or may be a suicide inhibitor. When the protein is
to be administered in YiYQ. an irreversible inhibitor (that is
25 covalently bound to the protein) preferably is used.
The detectable substance attached to the thrombolytic
protein may be any substance which may be stably sttached to the
protein without significantly reducing the fibrin-binding property
thereof, safely administered to a patient, and detected by a
suitable known technique. The detectable substance may be attached
to the protein directly or through various linker or adaptor
molecules, including certain affinity lig~n~s, as di6cussed below.
Among the suitable detectable substances are nuclear magnetic
resonance contrast agents, X-ray contrast agents, and
LC8x1659.mhg
-9- 1 33~72~
radioisotopes, including, but not limited to, radioisotopes of
iodine (e.g., 131I or 123I), indium (e g lllIn) bromine (e
75Br or 76Br), or fluorine (e.g., 13F). These diagnostic agents
are detectable by external (non-invasive) means. A preferred
radioisotope for use in the present invention is the radionuclide
99mtechnetium (99mTc). The gix-hour half-life of 99mTc, as well as
its compatibility with gamma camera gcAnn~ne devices and its
availability in most hospitals and clinics, makes it a favored
radionuclide for use in diagnostic procedures.
Methods for radiolabeling proteins with various
radioisotopes are well known. These procedures include attAcl -nt
of the radioisotope directly to the protein, or attachment through
various chelators and other link~ne compounds which react with
various functional groups on the protein to bind the radioisotope
thereto. See, for example, U.S. Patents Nos. 4,652,440; 4,659,839;
and 4,472,509; and British patent application GB 2,109,407.
However, non-specific attachment of radiolabeled compounds to
fibrin-binding proteins may result in a decrease in the ability of
the protein to bind to fibrin, 6ince a portion of the radioisotopes
will be attached to the fibrin-binding domain. In accordance with
the present invention, the detectable substance (e.g. radioisotope)
preferably is selectively attached outside of the fibrin-binding
domain of the protein.
The labeled thrombolytic proteins of the present invention
are useful as diagnostic agents and for monitoring dissolution of a
fibrin-platelet clot during therapy. As would be known to the
ordinarily gkilled artisan, the amount in~ected into a particular
patient will depend on 6uch factors as the affinity of the
particular protein for fibrin, the nature of the detectable
substance attached thereto, and, when the ubgtance is a
radioisotope, the specific activity of the preparation. The amount
in~ected is sufficient for detection of the pattern of
biodistribution of the substance in vivo by appropriate detection
LC8x1659.mhg
-lo- 1 3 3 5 7 2 5
devices after administration to the patient. The labeled
thrombolytic protein may be in~ected in any 6uitable
physiologically acceptable carrier. Suitable carriers will not
denature-or otherwise alter the protein, or cau~e the protein to
precipitate from solution, and are nontoxic in the patient.
Suitable carriers include, but are not limited to, aqueous
solutions, preferably isotonic, comprising sodium chloride or other
salts, glucose, dextrose, or water for in~ections.
After in~ection of a labeled thrombolytic protein of the
invention into the patient, the detection procedure is delayed for
a sufficient length of time to a allow binding of the labeled
thrombolytic protein at the 6ite of any fibrin-platelet clots which
may be present. The appropriate length of time will depend on such
factors as the degree of specificity or affinity of the
thrombolytic protein for fibrin, the nature of the detectable
- substance (e.g., the half-life of a particular radio-isotope), the
efficiency with which in~ected labeled thrombolytic protein which
does not become bound to a fibrin-platelet clot is cleared from the
body, the site of in~ection and the resulting route the protein
must travel to the clot site, etc. In general, sufficient time is
allowed to pass to allow substantial clearance of the non-bound
portion of the protein from the bloodstream. Certain of the
thrombolytic proteins of the invention will be cleared from the
patient through a particular organ (e.g., the liver), and the route
of clearance from the body may vary according to the nature of the
thrombolytic protein. Such organs will not be interpreted as
fibrin deposit sites when the pattern of biodistribution is
detected.
Once the presence of a fibrin-platelet clot has been
diagnosed, therapy with any suitable agents, or mixtures thereof,
is begun. Known therapeutic reagents include the enzymes
streptokinase, urokinase and t-PA, and anticoagulants, such as
heparin. These agents are ~l' in~stered in accordance with
LC8x1659.mhg
1 335725
conventional procedures, in non-labeled form. ~sny of the current
methods for monitoring dissolution of blood clot8 during treatment
suffer from a lack of ability to distinguish bet~een part~al and
complete restoration of blood flow. Ihe proteins of the present
invention can be A~minictered in con~unction with administration of
therapeutic sgents to determine when the clot has been effectively
dissolved (i .e., when fibrin deposits are no longer detected in
accordance with the method of the invention) and treatment then can
be ended. Monitoring of treatment procedures in this msnner
reduces the incidence of premature tel inAtion of treatment, which
has been a problem in the past.
The present invention also provides a method for labeling a
thrombolytic protein while preserving the activity of the fibrin
binding of the thrombolytic protein, comprising attAching a
detectable substance to the thrombolytic protein through a linker,
~ wherein the attAr' ~.t of the linker is to a portion of the
thrombolytic protein other than the fibrin binding ~: D; n This
method is especially advantageous for throm~olytic proteins
conr~ning a clot dissolving ~omDin that i6 inactivated (or
susceptible to a reduction in the biological activity thereof) by
att~c t of detectable substances to that clot dissolving d: Din
Included in the invention are thrombolytic proteins having
an attached detectable substance, wherein the substance is attached
to the clot dissolving domain which reduces or eliminates
the clot dissolving activity while not affecting another activity,
e.g. fibrin bin~ing~ The protein may be a thrombolytic enzyme that
comprises both an enzymatic activity and a fibrin bin~;ng domain,
wherein preservation of the biological activity of the fibrin
b~n~jng do~D-jn is desired. A linker that binds ~pecifically to the
portion of the enzyme that is responsible for e~ tic activity is
used. The activity of other functional dom~nc (e.g., a substrate-
binding domDjn) thus is preserved after attachment of the
LC8x16S9. mhg
-12- 1 3 3 ~ 7 2 5
detectable substance to the thrombolytic protein through the
linker.
A number of compounds that bind to the portion of an enzyme
that confers the enzymatic activity are known, and may be used, or
modified for use, as linkers in accordance with the present
invention. Such compounds include but are not limited to, affinity
labeling reagents. These reagents are used for such purposes as
identification and characterization of enzymes, as well as the
inactivation of certain enzymes in in vitro assays. One group of
affinity labeling reagents includes oligopeptide chloromethyl
ketone compounds, which generally comprise from two to about four
amino acid residues and often are derived from a particular
enzyme's substrate. These compounds bind covalently (irrever6ibly)
to an enzyme's active site, thereby inactivating the enzyme.
Oligopeptide chloromethyl ketone compounds that inactivate certain
~ enzymes (e.g. serine proteases, especially trypsin-like serine
proteases) are known. Oligopeptide chloromethyl ketone
inactivators of kallikreins, plasmin, thrombin, urokinase, and
other proteases are described by Kettner and Shaw in Methods in
Fnzymolor~ Vol. 80, pp 826-842 (1981) and Biochemistry, Vol. 17,
pp. 4778-4784 (1978). These inhibitors, and the use thereof as
linkers, are further described below.
In one embodiment of the invention, a tetectable substance
is attached specifically to a thrombolytic protein having a fibrin-
binding domain, without ~ ~ni ~h~np. the fibrin-binding property of
the protein, by attflchlne the detectable 6ubstance to the protein
through a linker which binds specifically to a portion of the
thrombolytic protein other than the fibrin-binding domain. The
fibrin-binding domain is the portion of the protein which imparts
to the protein the ability to bind to fibrin. The linker may be
any suitable compound which binds the detectable ~ubstance, on the
one hand, and attaches to the protein at a site distant from the
fibrin-binding domain. Suitable linkers include, but are not
LC8x1659.mhg
1 335725
limited to, various affinity ligands which bind specifically with
portions of the protein other than the fibrin-bindin~ portion. For
example, the detectable substance may be attached to the portion of
the protein responsible for clot-dissolving activity, wherein this
att~rl -~.t causes a reduction in said activity, while the portion
of the protein responsible for fibrin binding remains unaffected.
One method of accomplishing this specific attachment
involves binding the detectable substance to the protein through
oli~opeptide derivative linker molecules, wherein the linkers
attach specifically to the portion of the protein responsible for
clot-dissolving activity, thereby d~ni shing said activity.
Likewise, the reduction in fibrin b~n~n~, which may result from
nonspecific attachment of a detectable substance to all portions of
a protein (including the portion responsible for fibrin binding),
is ~nim~ed by this spproach. Examples of such oligopeptide
- derivative linkers are those believed to inactivate a particular
enzyme by mimicking the portion of the particular polypeptide
substrate with which the enzyme interacts nsturally. Examples of
such linkers are ~chloromethyl ketoner tripeptide suicide enzyme
inhibitors. The chloromethyl ketone moiety of the inhibitor
molecule inactivates the enzyme by alkylating the histidine residue
within the enzyme's active site. One of several such inhibitors is
the tripeptide derivative glutamic acid-glycine-arginine-
chloromethyl ketone, which is commercially available from
Calbiochem Biochemicals, San Diego, as a urokinase inhibitor.
Another is D-phenylalanine-L-proline-L-arginine-chloromethyl
ketone, which is abbreviated as ~D-Phe-Pro-Arg-CH2Cl" hereinafter
and is sold as ~PPACK ~by Calbiochem as a thrombin inhibitor. This
thrombin inhibitor was described by Kettner and Sha~ (T~rombosis
~esearch 14: 969-973). It has been found that D-Phe-Pro-Arg-C~2Cl
also inhibits t-PA. (Mohler, M. et ~1., Thromb. and Haem.
52(2):160-164 [1986].) Another trlpeptide derivative that binds to
the portion of t-PA responsible for enzymatic activity is Tyr-Pro-
Arg-CH2-Cl.
'lQr' ~'
LC8x1659.mhg
- 14 - l 3 3 5 7 2 5
In accordance with one embodiment of the present invention,
the compound D-Phe-Pro-Arg-CH2Cl or Tyr-ProArg-CH2Cl is used as a
S linker for specific binding of a r~liol~beled mnlecule (e.g., a chelate
comprising a r~dionuçli~e metal) to the portion of the t-PA protein respon~ihle
for catalyzing the conversion of pl~minogen to p!~cmin. It has been found
that ~tt~hment of a ~iomlcli~le chelate to t-PA through this tripeptide linker
results in both stable co~alent ~tt~ hment of the radionuçlide to the protein and
10 rcducti~n of the pl~mino~on-activating activity of the enzyme, while the
fibrin-binding plo~,ly is ret~ined. Thus, one set of ch~omic~ tion~
accomplishes two goals, namely, specific radiolabeling of the protein and
~imlllt~n~usly reduçing the enzymatic activity.
The present invention provides co---powlds of the following
formula:
~
O ~ NHJ~HNH2
o a
wherein m is 0 or 1 and Q l~l~se~ a radiolabeled molecule. When m is 0,
the tripeptide chloromethyl ketone linker is D-Phe-L-Pro L-Arg-CH2Cl.
When m is 1, the linker is Tyr-L-Pro-L-Arg-CH2Cl. Among the many
radiolabeled molecules that these compounds may comprise are the
30 radionuclide metal c~el~tes and radiohalog~ated molecules described below.
Also pravided by the present invention is the protein t-PA having a
radiolabeled molecule ~tt~hed thereto through one of the above-described
tripeptide-CH2Cl linkers that binds to t-PA.
, t
- lS- 1 335725
Many çhel~tine compounds of various structure are known.
The c~ hle compound which is ~tt~hPd to the PPACK or Tyr-Pro-Arg-
CH2Cl linker may be any colllpound capable of reacting with the amino
5 t~ US of the linker to a forrn a bond thereto and which compri~Ps donor
atoms capable of rO. ..~h~ bonds with a ~dic-nucli~e to form a stable chelate
of the ra-lionucli~le~ The che~in~ colll~ nd may be bonded to the tripeptide
linker through a bifunctiQn~l adaptor mol~ule compri~in~ one functi-)n~l
group reactive with the free amino group on the phenyl~l~nine or tyrosine
10 residue of the linker molccllle and a second function~l group reactive with agroup of the çh.ol~tine colllpound. Many such adaptors are known, with th
noc~ity for an adaptor and the choice thereof being dep~nd~nt on the
chPmir~l structure of the ch~l~tine col--~und.
One of the many ch~1~tine colllpol~nds which may be bound to
15 the D-Phe-P~Arg-Ch2Cl or other tripeptide linker is a chPl~tine compound
having the following formula:
COOE
~ NH
~( ,)~
S S
T T
compri~in~ sulfurprotecting
group (T)
wherein "E" l~lesents an active ester group. This "N2S2" ch~l~ting
c~.llpound, which has been described in European patent application
publication no. 188,256, comprises an active ester group which will react with
30 the free amine of the tripeptide linker to form an amide bond. The tripeptidelinker may be synthesi7~d and the N2S2 ch~l~tine compound ~tt~h~ to the D-
Phe-Pro dipeptide before the Arg-CH2Cl portion of the linker is ~ hed, as
described in E~s rle 2 below. It has been found that when the chPl~ting
compound is reacted with
1 33572~
the intact tripeptide, a certain percentage of the chelating
compound reacts with a free amino group on the arginine residue
(which interferes with interaction of the r-~ulting tripeptide
derivative with t-PA) rather than reacting with the terminal NH2
group on the phenylPlAn~ residue. However, the pH of the
reaction mixture may be ad~usted (e.g., to about 5 to 7) to promote
selective reaction of the ester on the chelating compound with the
amine on the phenylalanine (rather than the arginine) residue. The
chelating compound thus may be reacted with the intact tripeptide.
The chelating compound may be reacted with a metal
radionuclide, such 99mTc, as described in the European application
no. 188,256 and in the examples below, to form the corresponding
chelate in which the radionuclide metal is held by four separate
covalent bonds to the two nitrogen and two sulfur donor atoms. The
_ tripeptide linker ha~ing the chelate attached thereto is then
~ reacted with t-PA, wherein the linker becomes attached to the
portion of the t-PA enzyme responsible for activation of
plP! inogen, as described above. The resulting radiolabeled t-PA
protein is Pl' ini ctered to diagnose fibrin deposits or to monitor
the progress of a therapeutic treatment.
A number of other radiolabeled molecules may be attached to
a fibrin-binding protein through a linker that binds outside the
fibrin-binding domain. Chelating compounds comprising various
combinations of sulfur, nitrogen, oxygen, and phosphorous donor
atoms may be used. Many such chelating compounds, as well as
methods for the synthesis and radiolabeling thereof to produce
metal radionuclide chelates, are known. In one embodiment of the
invention, the chelating compound comprises a total of four donor
atoms selected from nitrogen and sulfur atoms. During the
radiolabeling procedure, bonds form between the donor atoms and the
radionuclide metal. In addition to the N2S2 chelating compound
described above, compounds comprising three nitrogen and one sulfur
LC8x1659.mhg
- 17 - l 3 3 5 7 2 5
donor atoms may be used. FY~mp~es of such "N3S" co---pounds include those
of the following forrnula~
S
~ C~
~Q'
R~(~
where: T is sulfur protecting group, such as group that,
t~gethPr with a sulfur donor atom to which it is attached, defines a thi~r~p1
or hemithi~uP~l group;
each R indep~t~d~ntly lepr~sen~ H2 or - 0;
each R' independently ,~;~nt~ a substituent s~l~ted from the
15 group con~isting of hydrogen, a non-alkyl side chain of an amino acid other
than cysteine, alkyl, gemin~l dialkyl, and- (CH2)D-Z;
Z r~leser.ts -COOH or a functional group that will react with
a linker to join the ch~l~ting compound to the linker;
m ~c~l~nt~ O or 1, with the proviso that at most one m
,c;~
n is an integer of from 1 to about 4; and
R" is hydrogen; -(CH2)n-Z; or an alkyl group having one or
more polar groups s~Jbs~ ~ thereon;
wherein the co,..pound comprises at least one -(CH2)n-
substit~ent
Radiolabeling of this N3S chpl~ting co---pou- d in accor~ce
with the invention produces a radionuclide metal chelate of the following
formula:
5~ / ~1'~
; ~ R ~ (~
~.
- 18- l 335725
wherein M l~-esents a radionuclide metal or oxide thereof and
the other symbols are as described above.
s
l~etho~c for synthesi~ing various N3S chPl~tir~ compounds are
knawn. See, for ~mple, Eurvpeall patent appli~tion publication number
173,424.
Other chPl~tinp co.. ~ul-~s may have difrc~nl col-lbinations of
donor atoms. Such cGIllpounds include N2S4, N2S3, and N3S3 chPl~ting
colll~unds, among others. In addition, the N2S2 and N3S compoullds
p~senled above may comprise varying numbers of substituPntc such as
carooxylic acid groups and from 0 to 3 oxygen atoms (- 0) ~tt~ch~Pd to carbon
15 atoms of the chelate core.
Other eY~mples of radiolabeled molecules that may be ~tt~ch~Pd
to fibrin binding proteins in accol~ ce with the present invention include
radioh~loEe-u~ mol^cu1es
Radiohalogens useful for ~i~nostic im~ing include, but are not
limited to, l23I for i...~ing by s~nning the patient with a gamma camera, and
l8F, 75Br, or 76Br for posiLI~n tomo~phic im~ing
Examples of molecules that bind r~ioh~logPnc at the meta or
~ position on a phenyl ring are described in Europea~ patent application
publication number 203,764. These co.,.pounds may be lepresented by the
following formula:
*X - Ar - R
wherein
- -19 -
1 335725
*X is a radioisotope of iodine, bromine, fluorine, or
astatine;
Ar is an aromatic or heteroaromatic ring;
R is a chemical bond or a substituent cont~in~n~ 1 to 12
straight-chain carbon atoms that does not activate Ar toward
electrophilic substitution on the order produced by hydroxy or
amino substitution of the ring. The bond or substituent has
attached thereto a con~ugation group, which is a functional group
suitable for reaction with a linker to bind the radiohalogenated
molecule thereto. *I-para-iodophenyl compounds (in which *I
represents a radioisotope of iodine) may be prepared using the
procedures described in EP 203,764, which generally involve
substituting the organometallic group Sn(n-Bu)3 or SnMe3 on a
haloaromatic compound. A radioisotope of a halogen then is
6ubstituted for the organometallic group by halodemetalization.
Examples of radiohalogenated molecules that may be prepared using
such a procedure are represented by the following formulas:
*X ~ (CH2)n-z
O
*X ~ CNH (CH2)n ~Z
wherein n represents an integer from 0 to 3, Z represents a
conjugation group, and *X represents a radioisotope of a halogen.
In one embodiment of the invention, the conjugation group
is a group that will react with a linker that binds outside the
fibrin binding domain of a fibrin binding protein, e.g., a
tripeptide-chloromethyl ketone linker that binds to (and inhibits)
t-PA. The conjugation group may be an active ester that reacts
with a primary amine on the linker to form an smide bond. Among
the many suitable esters are 2,3,5,6-tetrafluorophenyl ester,
thiophenyl ester, and N-hydroxysuccinimidyl ester. The above-
described radiohalogenated molecules thus may be attached to t-PA
LC8x1659.mhg
- 20 - 1 335 725
outside the fibrin-binding domain through the aba~e-described tripeptide
derivative linkers.
~lt~ tively, the fibrin-binding protein may be radioio lin~t~
using a Bolton-Hunter reagent, i.e., N-succinimidyl-3-(4-
hydroxyphenyl)propionate or water-soluble derivatives thereof. Methods for
radioiodin~ g these reagents (~h~in the radioisotope is substituted ortho to
the hydroxyl on the aromatic ring) are known. See, for eY~mpl, Bolton and
Hunter (Biochem. J. 133, 529-539 [1973]) as well as page 295 of the Pierce
Chemic~l Company 1988 Handbook and General C~hlog- The res--lting
radioiodinated molecules are ~pl~ senled by the following formulas:
o
(~ o~
N-Succinimidyl-3(4-hydroxyphenyl)propionate
O ~ ~
~<~ I o r~3
(~ S~ O~
Suttnsuc~-inimi-lyl-3-(4-hy Il~Ay~he~lyl)propionate (water soluble)
wherein *I l~?~scn~ a radioisotope of iodine and m is 0 or 1 (with at least
one m being 1). Additional methylene groups may be inserted between the
aromatic ring and the ester group.
In accoldance with one embodiment of the present invention,
the succinimidyl ester group of the radioicYlin~d Bolton-Hunter reagent is
reacted with a free amine group on a linker that binds outside the fibrin-
binding domain of a fibrin-binding protein. The radioio~in~t~ reagent may
be joined to t-PA through one of the above-described tripeptide chloromethyl
ketone linkers, for example.
1 33572~
In another embodiment of the present invention, a
radiohalogen may be attached directly to a tripeptide linker. The
radiohalogen msy be substituted onto the aromatic ring of a
pheny~ nine or tyrosine residue in a tripeptide linker.
Procedures for producing such radiohalogenated linkers include
those presented in Examples 4 and 5 below.
The degree to which the clot-dissolving activity of a
particular protein is reduced is related to the amount of specific
linker compound (e.g., tripeptide linker) attached thereto. If
further reduction of enzymatic activity is desired, additional
labeled (e.g., radioisotope-labeled) or unlabeled linker compound
may be reacted with the protein.
In some cases, it may be desirable to avoid completely
~ destroying all plA! ~nngen-activating activity of the t-PA protein.
A low level of residual enzymatic activity may serve to "open up" a
clot sufficiently to allow binding of the radiolabeled t-PA within
the clot, as opposed to only the outer surface of the clot.
Improved images may result.
In one embodiment of the invention, a kit is provided for
use in preparing the radiolabeled, fibrin-binding protein of the
invention. An example of such a kit is one comprising a first vial
cont~in~ng t-PA. A second vial contains a lyophilized preparation
comprising three reagents:
(a) N2S2-D-Phe-Pro-Arg-CH2Cl (a molecule comprising an N2S2
chelating compound attached to the previously described D-Phe-Pro-
Arg-CH2Cl linker, which is synthesized as described in Example 2
below).
(b) A reducing agent effective in reducing pertechnetate
(99mTco4- which is in the +7 oxidation level) to a lower oxidation
state at a neutral to acidic pH so that a technetium exchange
complex can be formed. Many suitable reducing agents are known,
LC8x1659.mhg
-22- 1 ~ 3 5 ~ ~
including, but not limited to, stannous ion (e.g., in the form of
stannous salts, such as stsnnous chloride or ~t nnous fluoride),
metallic tin, formamidine sulfinic acid, ferrous chloride, ferrous
sulfate,- ferrous ascorbate, and alkali 6alts of borohydride.
Preferred reducing agents are stannous salts.
(c) An eYchAnee agent with which the reduced 99mTc will
form an eYchAn~e complex, thus protecting the 99mTc from
hydrolysis. In order to achieve efficient transfer or exchange of
the 99mTc from this complex to the chelating compound, the e~ch~nee
agent advantageously binds the radionuclide more weakly than the
chelating agent will. FYchAn~e agents which may be used include,
but are not limited to, gluconic acid, glucoheptonic acid,
methylene diphosphonate, glyceric acid, glycolic acid, mannitol,
oxalic acid, malonic acid, succinic acid, bicine, N,N'-bis(2-
hydroxyethyl) ethylene ~r ~n~, citric acid, ascorbic acid, and
gentisic acid. Good results are obtained using gluconic acid or
glucoheptonic acid as the exchange sgent.
Pertechnetate is combined, in aqueous solution, with the
contents of the second vial. The pertechnetate is reduced and
bound by the eYrhAnee agent, then transferred to the N2S2 chelating
compound to form a stable chelate. The resulting 99mTcN2S2-D-Phe-
Pro-Arg-CH2Cl is reacted with the t-PA under physiologically
acceptable conditions (i.e., reaction conditions which will not
25 denature the t-PA) to form the radiolabeled t-PA of the present
invention.
A stannous chloride reducing agent may be combined with a
gluconic acid eY-chAnee agent to form a stannous gluconate complex,
which therefore functions as ingredients (b) and (c). 99mTc-
radiolabeled t-PA is prepared, using such a kit, generally as
described in Example 2 below.
The kit optionally may comprise additional vials containing
various buffers, additional reagents used during the radiolabeling
LC8x1659.mhg
- 23 - I 3 3 5 7 2 5
procedures, stabiliærs, or other such co"~po~lnds. The procedures for
p~p~alion of a radiolabeled protein using the kits are col-ducb~ under sterile
cQn~ition~
S The following examples are pravided to illl-st~t~ certain
embo~;...ent~ of the present invention and are not int.~.n~ed to limit the scope of the claims which follow.
EXAMPLE I
Preparation of 99~c N2S2 chelate-t-PA conjugates,
with and without a D-Phe-Pro-Ar~-CH~C1 linker
A vial of freeæ-dried t-PA was recon~titut~d with sterile water
to abut S mg/ml. The buffer was eycll~nged by gel filt~tion into 0.25 M
NaPi, 0.3 M gu~ni~ine, pH 7.5. Gl~ni(line was added to keep the t-PA in
solution. Labeling was done with a p~ro.."ed N2S2 chelate comprising a
2,3,5,~tetrafluorophenyl active ester having the following formula:
~`~ ~ F F
The reaction I~ Ul~ was co~stitlltPd by:
1) drying the Tc-99m chelate into a vial
2) adding 1.5 ml t-PA (3 mg)
3) adding 0.45 ml lM gu~ni~line pH 7
4) adding 1.0 ml 0.5 NaPi pH 10
After 30 minutes at 37C, the reaction was applied to a
Sephadex* G-25 column equilibrated in an applupliate buffer. The fractions
(fiaw-through) cont~ining the purified, labelled t-PA were collected and
char~ri7~ by TLC.
* trade-mark
-24-
1 335725
Certain vsriations in this reaction mixture, a8 well as the
preparation of controls and the results achieved, were as follows:
Prep #l
3 mg t-PA
26.7 mCi chelate
pR 10, 37C, 30 min
reaction TLC 54.5%
Product: 10.7 mCi, 2.67 mCl/mg, TLC - 99.4%
Yield: radiochemical (uncorr) 40%, protein 100%
Fibrin binding: 37.9% at 1 mg/ml fibrinogen
Prep #2
1.5 mg Fab fragment of an antibody
4.22 mCi chelate
_ pH 10, 37C, 10 min
- reaction TLC 53.4%
Product: 2.59 mCi, 1.57 mCi/mg, TLC - 99.4%
Yield: radiochemical (uncorr) 59.7%, protein 93%
Prep #3
0.9 mg PPACK bound to t-PA
11.8 mCi chelate
pH 10, 37C, 15 min
reaction TLC not done
Product: 3.25 mCi, 2.81 mCi/mg, TLC - 99.2%
Yield: radiochemical (uncorr) 27.5%, protein 66.5%
Fibrin binding: 56.2% at 1.0 mg/ml fibrinogen
Prep #4
4.0 mg t-PA
40 mCi chelate
pH 10, 37C, 30 min
reaction TLC not done
Product: 14.63 mCi, 3.31 mCi/mg, TLC - 99.1%
LC8x1659.mhg
-25- 1 3 ~ 5 7 2 5
Yield: radiochemical (uncorr) 36.5%, protein 9896
Fibrin binding: 45.0% at l.0 mg/ml fibrinogen
Prep #5
1.2 mg t-PA
17.8 mCi chelate
pH 9, room temperature, 15 min, treat with lysine
reaction TLC 50.5%
Product: 3.5 mCi, 2.22 mCi/mg, TLC - 98.4%
Yield: radiochemical (uncorr) 19. 7%, protein 100%
Fibrin binding: 66.8% at l.0 mg/ml fibrinogen
Prep #6
0.2 mg PPACK bound to t-PA
21.8 mCi chelate
pH 9, room temperature, 25 min, treat with lysine
reaction TLC 47%
Product: l. 34 mCi, 6.28 mCi/mg, TLC -- 98.0%
Yield: radiochemical (uncorr) 6%, protein 93%
Fibrin binding: 65.5% at 1.0 mg/ml fibrinogen
Prep #7
1.0 mg PPACK bound to t-PA
41 mCi chelate
pH 9, room temperature, 30 min, trest with lysine
reaction TLC not done
Product: 9.96 mCi, 8.15 mCi/mg, TLC - 98.7%
Yield: radiochemical (uncorr) 24%, protein 4996
Fibrin binding: 65.2% at 1.0 mg/ml fibrinogen
The resulting preparations were P~ ini stered to rabbits
having artificially induced blood clots in the ~ugular vein. The
preparations were in~ected into the ear vein proxi~al the clot.
Unless noted otherwise, all preparations were diluted in~0 a
LC8x1659. ~hg
~ 335725
physiologically acceptable solution to a total volume of 8 ml and
infused into the rabbit over 10 min.
Rabbit B, Injection 1
Prep #3, 2.7 mCi, 0.97 mg PPACK t-PA
Rabbit B, Injection 2
Prep #1, 3.72 mCi, 1.8 mg t-PA
Rabbit C, Injection 1
Prep #4, 4.3 mCi, 1.3 mg t-PA
Rabbit C, Injection 2
Prep #4, 4.0 mCi, 1.3 mg t-PA
Rabbit D, Injection 1
Prep #5, 2.12 mCi, 0.9 mg t-PA
Rabbit D, Injection 2
Prep #6, 0.95 mCi, 0.15 mg PPACK t-PA
Rabbit D, Injection 3
Prep #5, 0.82 mCi, 0.45 mg t-PA,
- route is opposite ear
Rabbit F, Injection 1
Cold t-PA 1 mg
Rabbit F, Injection 2
Prep #7, 1.61 mCi, 0.26 mg PPACK t-PA
The rabbits were scPnn~d with a gamma camera at various
time points after injection to image the blood clots. Figure 1
represents four of the resulting scans, taken of rabbit C at the
indicated time points after injection #1: immediately after
injection and at 5, 10 and 15 minutes after injection. The site of
injection is indicated by the thin arrows, and the clot sites are
indicated by the heavier arrows. As can be seen, the clot image is
darkest at the 5- and 10-minute time points, and had become much
fainter only 15 minutes after in;ection. This animal was injected
with preparation #4, in which the N2S2 chelate was bound directly
to the t-PA protein through reaction of the ester group on the
chelate with free amine groups on the lysine residues of the
protein (i.e., without PPACK linkers). It is believed that the
image became faint so quickly due to release of the radiolabeled t-
LC8x1659.mhg
i
1 33572~
PA from the clot surface during clot dissolution, ~ince the t-PA
retains enzymatic activity.
In an effort to prolong the length of time during which
~ ~ging can take place, the linker D-Phe-Pro-Arg-CH2Cl (PPACK) was
bound to t-PA to inactivate the enzymatic activity thereof. The
resulting PPACK-t-PA was reacted with a 99mTcN2S2 chelate to form a
radiolabeled protein conjugate comprising a PPACK linker. This
conjugate was injected into rabbits B (injection #1), D (injection
#2), and F (injection #2), as indicated above. The scans for D and
F showed localized images of the clot for prolonged periods of time
(measured in hours rather than minutes), whereas scans for B did
not give very good images. While not wishing to be bound by
theory, it is believed that P~ ~n~tration of active t-PA as
injection #1 (unlabeled t-PA for F and 99mTcN2S2-labeled t-PA for
opened up" the clot slightly to provide additional binding
6ites for the 99mTcN2S2-PPACK-t-PA diagnostic agent, rather than
limiting binding of the diagnostic agent to sites only on the outer
surface of the clot.
Although prolonged time ~pans for ~ qgin~ were achieved by
binding PPACK to t-PA to inhibit the enzymatic activity thereof,
decreased fibrin binding remained a problem in all preparations.
This is believed to be attributable to covalent binding of the N2S2
chelate to free amine groups in each portion of the protein,
including the fibrin-binding portion. Thus, even though PPACK was
present on the t-PA in some preparations, binding of the chelate to
t-PA was nonspecific, i.e., was not limited to binding through the
PPACK linker.
In an effort to achieve specific binding of the
radionuclide to the non-fibrin-binding portions of t-PA, while
reducing the enzymatic activity thereof, procedure6 for ~oining the
N2S2 chelate to the PPACK linker prior to att~ nt to t-PA were
developed. These procedures are described in Example 2.
LC8x16 59 . mhg
-28- t 3357~5
EXAMPLE 2
~ltPrn~tive Method for P~ Lion of
599mTcN2S2-D-Phe-Pro-Arg-CH2Cl-t-PA Conjugates
An N2S2-D-Phe-Pro Arg-CH2Cl mo]~clllP is chemic~lly
synth~P~i7Pd and radiol~ l el~ with 99~Tc, then conjugated to t-PA, as follows:
Preparation of
t-butoxycarbonyl-D-phenyl~l~nine-L-proline-methyl ester (3)
e--o ~ ~,
~ ~ 2
~
One gm (6 mmole) L-proline-methyl ester HCl (Aldrich) was
added to 15 ml CH2Cl2. Then 866 1 (1 equiv.) triethylamine (TEA) was
25 added, followed by 1.6 gm (1 equiv.) of t-Boc-D-phenyl~l~nine (R~çhPm) and
1.6 gm (1.3 equiv.) dicyclohexylcarbodiimide (DCC, Aldrich). The reaction
was stirred at room ~",pe.dL~Ire for 3.5 hrs. 1 I,C (CH3CN:H20:AcOH,
94:5:1, ninhydrin stain) in~ te~ minor amounts of starting m~t~ri~l~ and a
major new product at an Rf of 0.95.
Dicyclohexylurea (DCU) was remaved by filtration and washed
with CH2Cl2. The organic filtrate was washed with 0.1 N HCl, 5%
-29- l 33~725
NaHCO3, and H2O, respectively. The CH2Cl2 layer was dried over MgSO4,
filtered, and evaporated under reduced pl~,S~ C, to yield an oil. This oil was
applied to a silica gel column (35 cm x 2 cm) and eluted with EtOAc:Hex
5 (4:6). Fractions were monilo~ed by TLC using the same solvent and a
KMnO4 stain. Initial fr~ctionc (Rf 0.9) contained a nonpolar i.,.~u.il~.
Following fr~rtionc conhined the desired co---pound (Ff 0.4) and were pooled
and e~dpoldlcd to yield 1.54 gm (70%) of a clear, sticky oil.
lH NMR (DCC13) 7.2 (s, SH, C6H5), 3.7 (s, 3H, OCH3), 1.9
(m, 4H, (C~2)2), 1.4 (s, 9H, C(C_3)3).
Plep~dtion of D-phenyl~l~nine-L-proline-methyl ester (4)
~
~o
To 1.54 gm (4 mmole) of t-Boc-D-Phe-L-P~Me (3) 35 ml of
trifluoracetic acid was added. This solution was stirred at 0C (H2O/ice) for
one hr. as the bath gr~dll~lly rose to room le---~ldlulc. TLC (EtOAc:Hex,
4:6) using the KMnO4 stain showed complete disdppe~dnce of starting
m~ttori~l. TLC (CH3CN:H20:AcOH, 94:5:1, ninhydrin stain) intlic~t~d one
product (Rf 0.7).
TFA was rema~ed using reduced plCSSu~c. The residue was
d with diethyl ether and filtered to yield 1.3 gm of a white crystalline
solid (819~ yield).
lH NMR (DCC13) 8.2 (br, lH, NH2), 7.2 (s, SH, C6H5), 3.5
(s, 3H, OC_3) 1.7 (m, 4H, (CH2)2.
1 335725
- 30 -
Preparation of succinimidyl-4,5-bis-(S-(l-etha~y)ethyl
mercapto)~cet~mido pentanoate (5)
~o ~
~-o~
~o o~
<
-
Compound S is an N2S2 ch~l~ting co.,.pound comprising (1-
ethoxy)ethyl sulfur-protecting groups and a succinimidyl ester group. The
synthesis of such chelating co---poullds, and the r~ r.l~eling thereof with
99~c to form the CO~l~ sponding chelate, is described in published European
patent application no. 188,256.
One gram (2.36 mmole) of 4,5-bis(s-(S-l-etha~cy) ethyl
nle.~;-dplû)ac~ .,ide pentanoic acid [bis-EOE carboxylic acid] was dissolved
in 10.0 ml anhydrous THF. 0.298 (2.59 mmole) of N-h~d~u~y-succinimide
was added, follawed by 0.584 9 (2.83 mmole) of dicyclohexylcarb~liimide.
The reaction was stirred at room ~ Jre a~ernight
TLC (96:4 EtOAc:HOAc p-~ni~ldehyde stain) analysis
indic~ted absence of bis-EOE-carboxylic acid (Rf - 0.5) and a new product (Rf
- 0.65).
Dicyclohexylurea (DCU) was removed by filtration and washed
with methylene chloride. The solvents were removed from the filtrate in
vacuo to leave an oil. The crude product was purified via flash
chru---d~og,dphy (SiO2, 2 cm x 45 cm) in 96:4 EtOAc:HOAc. Fractions
conl~inin~ product with an Rf of 0.65 were combined and
- 31 - l 3 3 ~ 7 2 ~5
e~rdl)oldled. Diethylether tntllrAtion and filtr~tion yielded a hy~lusclopic
white solid (0.97 9) in 79% yield.
IH NMR (DCC13): ~ 7.25 (m, 2H, NH x 2), 4.75 (g, 2H, SCH
x 2) 3.3 (S, 4H, SCH2 x 2), 2.85 (S, 4H, NHS (CH2)2), 1.55 (d, 6H, C_3CH
x 2), 1.2 (t, 6H, C_3CH2 x 2).
~dlion of
4,5-bis-(S-l-ethuxy)ethylmc~plo)~^P~mi~i~
pentanoyl-D-phenylalaninyl-L-prolyl methyl ester (6)
~_~p
~5 ~
~ o~
< >
To 849 mg (2.18 mmole) of D-He-Prt}Me(O, S ml of
anhydrous DMF was added. 314 1 triethylamine (1 equiv.) was added,
followed by 1.13 (1 equiv.) of bis-ethoxy-carboxylic acid NHS ester (~). The
reaction was stirred overnight
TLC (EtOAc:AcOH, 96:4 p-~ni~ldehyde) in-lic~t~d the
disappe~ce of the NHS ester (Rf 0.9) and the appealdnce of a new product
(Rf 0.8). DMF was removed under reduced p~S;~Ule and the residue taken up
in ELOAc. The EtOAc was washed with 0.1 N HCl 5% NaHCO3, and twice
with H20. The organic layer WdS aired over MgSO4, filtered, and evaporated
to yield 1.1 gm (63%) of an oil.
1 33572~
This oil was purified by flash chro~ Q~ rhy using a silica gel
column (45 cm x 2 cm). The desired co""~und was eluted using
S EtQ~c:AcOH (96:4, p-qniQqld~ohyde stain). Fr~q~cti~ ns cQntqining product were
combined to yield 600 mg (35%) of a clear oil.
IH NMR (DCCl3) 300 MH2 ~ 7.15 (m, SH, C~5) (s, 3H,
OC_3) 1.5 (m, 6H, C_3CH x 2) 1.18 (m, 6H, C~CH2).
~ ~K,dlion of 4,5-bis-(S-(l-etha~y)ethyl",e~d~
arRt-qmidopel,tdnoyl-D-phenylalaninyl-L-proline (71
_~)
~
6 ~ ~s sJ
~ ~ 0>_
> 7
To 600 mg (0.75 mmole) of bis-ethoxy-Phe-Pro Me (~), S ml
25 MeOH was added. .75 ml (1 equiv.) of 1 N NaOH was added and the
solution became cloudy. After 1 hr, a new product appeafed at an Rf of 0.2
by TLC (CH3CN:H20: AcOH, 94:5:1) with a large arnount of starting
mq~riql The reaction was run a~en-ight TLC in~ qt~ same starting
mq.~Priql still present. 350 1 (.5 equiv.) additional 1 N NaOH was added.
30 After 2 hrs, the new product was considered the major spot by TLC.
Solvents were removed to leave a white residue. This residue
was taken up in EtOAc and washed twice with 1 M AcOH and
33 l 3 3 5 7 2 5
twice with H2O. The organic layer was then dAed over MgSO4, filtered, and
(82%) evaporated to leave 484 mg of a sticky white solid.
The col~lpouild was purified by flash chro...~ rh~ using
silica gel (25 cm x 1.5 cm) and CH3CN:H2O:HOAc as an eluting solvent.
Once the desired co-lll)ound began coming off the column, the solvent ratio
was c~ eed to 92:6:2 and elution was continued until no more colllpuund wa
10 evident in the eluent by TLC. All solvents were removed and the final
product dried under high vacuum. Yleld: 460 mg (78%).
IH NMR (DCC13) 60 MHz ~ 7.2 (s, SH, C6~5), 3.3 (s, 4H,
SC_2 X 2), 1.6 (d, 6H, C~CH), 1.2 (t, 6HG, C_3CH2).
Preparation of 4,5-bis-(S-l-etha1~y)ethylmercapto)
~e~midopentanoyl-D-phenylalaninyl-prolyl-D-Y-N-nitro
ar~inine chloromethyl ketone (9)
n~'~~
e~ c~c*,e=e "~
< >
8 9
Bis-ethoxy-Phe-Pro (~) (0.1 g, 0.26 mmole) is reacted with N-
methylmorpholine (0.028 ml, 0.26 mmole) in 1.2 ml of THF for 10 min at -
20C. Cold THF (5 ml) containing isobutylchlon~fo"~late (0.035 ml, 0.26
mmole) is added to the mixed anhydride p-t;~a,dlion,
34 1 335725
and the mixture is imm~i~tPly added to H-Arg(NO2)CH2Cl HCl (~) (0.073
g, 0.26 mmole) dissolved in 1.2 ml of cold DMF (Kettner and Shaw,
Biochem. 17(22): 4780 (1978). The reaction is stirred for 1 hr at -20C and
2 hrs at room ~",~ldture, then filtered. The filtrate is evdpoldled to dryness,
and the residue is dissolved in 1 ml of mPth~nol. The solution is diluted to
24 ml with ethylacetate and then washed with 0.1 NHCl, 5% NaHCO3, and
c~J~led aqueous NaCl. The organic phase is dried over anhydrous Na2SO4
and concenllal~d _ vacuo to yield (~).
Plep~dlion of 4,5-bis-(S-(l-etha~y)ethylmercapto)
acPPmido pentanoyl-D-phenylalaninyl-L-Prolyl-D-~r~inine
chloromethyl ketone (10)
~ .~
~cs,
N2 ;~ )6D H ~1~
S S C 1 C)~ H
--<o o~ CDO-
>
The nitro-arginine derivative (9) is talcen up in MeOH and
treated under N2 with a freshly pl~a~d buffered solution of TiC13 made from
20% aqueous TiCl and 4 M aqueous ammonium acetate, dS described in
Frei~inEer et al., J. Or~. Chem. 43: 4800 (1978). Due to the propensity for
30 chloromethyl ketone groups to reduce in the presence of TiCl3, as described
by Clerici et al., Tet. Lett. 28: 1547 (1987), the pH of the ammonium acetate
may be lowered, using AcOH, to m~imi7~ reduction of the nitro group and
minimi7P chloromethyl ketone reduction.
,. , . ~
l 3 3 5 7 2 5
The resulting N2S2-D-Phe-Pr~-Arg(CH2Cl) molecule (i.e.,
compound ~) is radiolabeled with 99mTc as follow, to form a co-.lpound of the
following formula, comprising the colle~nding 99~TcN2S2 chPlqtç:
s
;O
`~`'~
One ml of sterile water for injection is added to a sterile vial
con~ining a stannous gluconate complex (50 mg sodium gluconate and 1.2 mg
stannous chloride dihydste, available from Merck Frosst, C-q-nq~1q in dry
solid form), and the vial is gently ~it-q-ted until the cont~ are dissolved. A
sterile insulin syringe is used to inject 0.1 ml of the reslllting stannous
gluc~nqtP solution into an empty sterile vial. So~ium pe~t~hn~ (0.75 ml,
75-100 mCi, eluted from a 99Mo/99Tc genestor pu~hased from DuPont,
lUedirh~,ics, M~qllincl~rodt, or E.R. Squibb) is added, and the vial is ~it-qb~
gently to mix the con~enl~, then incubqt~P~ at room k~l.pc~ l.G for 10 min to
form a 99~Tc-gluconate complex. This complex is an intermPAi~q~ or
~eYch-qrlge complex~ in which the 99~Tc radionuclide is bound tGIllpo.cuily
until it is eYch~nged into the N2S2 chPlqting compound.
0.87 ml of 100% isoplo~l alcohol is added to a vial contqining
co---pound 10, p~pa~Gd above, in dry solid form. The vial is shaken gently
to dissolve the compound. Next, 0.58 ml of this solution is transferred to a
vial con~;~ining 0.16 ml of glacial acetic acid/0.2 N HCl (2:14), and the vial
is gently a~ihtP~d. Of this acidified solution, 0.5 ml is transferred to the vial
conli~ining the 99mTc-gluconate complex, pl~a~Gd above. After gentle
agitation to mix, the vial is incubq-tPd in a 75Ci2C water
-36- 1 3 3 5 7 2 5
bath for 15 min, then ~diAtely transferred to a O-C ice bath for
2 min to stop the reaction, thereby forming (11)-
The 99mTcN2S2-D-Phe-Pro-Arg-CH2Cl compound (11) was
combined with t-PA in a buffered ~olution to produce the
radiolabeled t-PA complex as follows:
5 ml H20 was added to a 50 ng vial of Activase and allowed
to stand at room temperature for 20 min. The protein solution was
then eY~hAneed into a buffer cont~n~ng 0.2 M arginine, .01 M Na
Phosphate pH 7.2 using a gel filtration column. To the 99Tc-N2S2-
labeling mixture was added 1.0 M tris base to bring the pH of the
mixture to 7.7. The labeling mixture and the t-PA solution were
then combined in 1:1 molar ratio, and incubated at 37C for 10
minutes. (The pH of this final mixture should be no lower than
7.2, and no higher than 8.2.) Residual t-PA activity was
eliminated by adding a several-fold molar excess of PPACK. The
protein was then desalted on a gel filtration column which also
removes unincorporated 99Tc, PPACK, and 99Tc-N2S2 PPACK. This
column was equilibrated with 0.2 M Arginine, 10 mM Na Phosphate pH
7.2. The protein contA1n~n~ fraction was then used for i qglng
studies.
When compound 11 was Al' ~n~ stered over a 20 minute infusion
through the marginal ear vein of a rabbit in which a preformed
thrombus resided in the ~ugular vein, the thrombus was imaged
during the infusion. The image was still apparent 60 minutes after
the end of the infusion (figure 6).
F~MPLE 3
Synthesis of a D-Phe-Pro-Arg-CH2Cl Linker and
Attachment Thereof to an N2S2 Chelatin~ Com~ound
This synthetic scheme is depicted generally in Figure 2.
The linker is synthesized using a variation of the procedures
LC8x1659.mhg
I
1 33572~
described by Kettner and Shaw (Biochem$stry, 1~ [1978] p. 4780) for
the synthesis of tripeptide derivatives.
Synthesis of Com~ound 3
Cbz-D-Phe and Pro-Me are added to CH2C12. 1 equiv. of
dicyclohexyl carbodiimide (DCC) and triethyl amine (TEA) are added
and the reaction is stirred overnight. TLC in EtOAc: Hexane (1:1),
visualized by KMnO4 shows product at an Rf of 0.6.
Dicyclohexylurea (DCU) is filtered off and the organic layer is
washed with O.lN HCl, 54 NaHC03, and brine, respectively. The
CH2C12 is dried over MgS04, filtered, and evaporated to yield
Compound 3. Excess DCU can be removed by filtration from cold
CH3CN.
Synthesis of Compound 4
Compound 3 is 6tirred in methanol cont~in~ng 1.4 equiv. of
- lN NaOH overnight. The product can be seen on TLC (EtOAc: AcOH
96:4) at an Rf of 0.5 by visualization with PAA. After the
reaction is complete solvents are removed and the residue is taken
up in EtOAc. The solution is washed with O.lN HCl and H20. The
organic layer is dried over MgS04, filtered, and evaporated. The
product was purified on a silica gel column using 100% CH3 CN.
Synthesis of Compounds 5. 6. and 7.
Compounds 5, 6, and 7 were synthesized as described in the
publication by Kettner and Shaw supra (which is hereby incorporated
by reference) and as shown in Figure 2. The mixed an~d ide
product (6) was purified on a silica gel column using 100~ EtOAc.
Compound 7, which is D-Phe-Pro-Arg-CH2Cl (i.e., PPACK) is then
attached to a chelating compound.
Synthesis of Compound 8
As depicted in Figure 3, the PPACK linker is attached to an
"N2S2" chelating compound comprising ethoxyethyl (EOE) sulfur
protecting groups and an N-hydroxy succinimidyl ester. The ester
LC8x1659.mhg
-38-
I 335725
reacts with 8 primary amine group on the linker to ~oin the
chelating compound thereto. The reaction is as followg:
PPACK is taken up in H20 and 1 equiv. of NaHC03. The pH is
4 prior to the addition of NaHC03 and 6 after the addition. This
pH is gpecific for reaction at the Phe amino group over the
guanidium group of arginine. One equivalent of the chelating
compound i8 taken up in dimethoxyethane (DME) and added to the
aqueous solution over a period of 30 minutes. TLC in nBuOH: AcOH:
H20 (4:1:1) indicates a new 6pot at an Rf of 0.5, between the two
starting materials, that stains in both para-anisaldehyde and
ninhydrin. After 2 hours the golvents are removed. The compound 8
is purified directly on a silica gel column using deactivated
silica gel and eluted with CH2C12: MeOH: AcOH (85:13:2).
Radiolabelin~ and Bindin~ to t-PA
- Compound 8 is radiolabeled to produce a 99mTc-N2S2
radionuclide metal chelate ~oined to the PPACK linker. The
radiolabeling procedure is as described in Example 2 above. The
resulting 99mTc-N2S2-D-Phe-Pro-Arg-CH2Cl compound i8 combined with
t-PA in a buffered ~olution, whereupon the radionuclide metal
chelate is attached to t-PA through the linker.
Alternative T.inkPr
The amino acid tyrosine may be gubstituted for
phenylAlAninP in the above procedure to produce the linker Tyr-Pro-
Arg-CH2Cl. A radionuclide metal chelate may be attached to t-PA
through this linker as described above for the N2S2 chelate and the
D-Phe-Pro-Arg-CH2Cl linker.
LC8x1659.~hg
-39-
~ 335725
FXAMPLE 4
Production of Radioiodinated D-Phe-Pro-Arg-CH2Cl Linker
snd Bintin~ Thereof to t-PA
The synthesis procedure is generslly depicted in Figures 4
and 5.
Synthesis of
N-CBZ D - tri-n-butylstsnnyl phenylslanine (2)
To a solution of N-CBZ p-chloro phenyl Al ~n~ne 1 (one
equivalent) in anhydrous THF at 100C is added n-butyllithium (3.3
equiv.). After one hour, a solution of Bu3SnCl (excess) is added.
Other tri-alkyl-Sn compounds may be used in place of Bu3SnCl. The
solution is ~ -~ to 0C and quenched by the sddition of ssturated
NH4Cl. Extractive work-up into diethyl ether affords the desired
product.
-
Synthesis of
N-CBZ p-tri-n-butylstannyl Phe-Pro-~ethylester (3)
To a solution of N-CBZ p-tri-n-butylstannyl Phe (2) (1
equiv.) in ~ d~OUS THF at O-C is dded dicyclohexyl carbodiimide
(1.2 equiv.) followed by N-hydroxysuccini-mide (1.2 equiv.). The
resulting solution is stirred overnight. The mixture is filtered,
the filtrate concentrated, and the crude residue is
chromatographed. To a solution of the purified NHS ester in THF is
added a THF solution of proline methyl ester. The resulting
solution is stirred overnight. The mixture is filtered, the
filtrste concentrated and the crude residue is chromatographed to
afford N-CBZ p-tri-~-butylstannyl Phe-Pro-methylester. (3)
Synthesis of
N-CBZ p-tri-n-butylstannyl Phe-Pro-N02 Are (CMK) (4)
To a solution of N-CBZ p-tri-n-butylstannyl Phe-Pro-
methylester (3) in 95% ethanol is added KOH (5-10 equiv.). The
resulting solution is warmed slightly for several hours. The
LC8x1659.mhg
~40- 1 3 3 5 7 2 ~
solution is cooled to 0C and acidified with cold aqueous HCl.
Extractive work-up affords the desired carboxyllc acld. To a
solution of the carboxylic acld in THF i8 added isobutyl
chloroformate (1 equiv.) ln the presence of N-methylmorpholine (1
equiv.) and the mixture is reacted for an hour at -20C. Cold
triethylamine (1 equiv.) ls added and the resulting mixture is
i ?~i Ately added to a solution of nitroarginine chloromethyl-
ketone-HCl (1 equiv.) ln cold DMF. After stlrring for 1 hour at-
20C and 2 hours at room temperature, the reaction mixture is
filtered and concentrated to dryness. Extractive work-up into
ethyl acetate affords the desired product. (4)
Synthesis of
p-tri-n-butylstannyl Phe-Pro-Ar~ (CMK) (5)
To a solution of N-CBZ-p-tri-n-butylstannyl Phe-Pro-N02 Arg
(CMK) (4) in acetic acld/ethanol solutlon was added palladium on
~ actlvated charcoal. The resulting mixture was hydrogenated for
several days at 30 psi. The catalyst is removed by filtration
through Celite. The filtrate is diluted wlth water and washed with
ether. The aqueous phase is then lyophilized to afford the product
(5).
Radioiodination of
p-tri-n-butylstannyl Phe-Pro-Are (CMK) (5)
To a vial cont~in~ne Nal31I solution in 0.1 N NaOH (up to
10 mCi) ls added p-tri-n-butylstannyl Phe-Pro-Arg (CMK) (5) (50 g,
7.1 x 10-2 ~mol) ln PBS (phosphate buffered saline). To this
solution is added a solution of chloramine-T in water (160 ~g, 0.71
~mol in 160 ~1 water). After 3-5 minutes, Na2S20s is added (70 ~1
of a 1.0 mg/ml solution of Na2S20s in water). The resulting
radioiodinated PPACK linker is depicted in Figure 5. The 131I
radionuclide is substituted directly onto the aromatlc ring of the
phenylalanine residue of the linker.
LC8x1659.mhg
1 335725
The radioiodinated compound is combined with t-PA in a
buffered solution, whereupon it binds to the t-PA.
pT.F. 5
Production of Radioiodinated Tyr-Pro-Arg-CH2Cl
T.~n~r and Bindin~ Thereof to t-~A
The synthesis procedure is generally depicted in Figures 4
and 5.
Synthesis of Tyr-Pro-Ar~ (CMK) (9)
The synthesis of Tyr-Pro-Arg (CMK) (9) is accomplished as
described for p-tri-n-butylstannyl Phe-Pro-Arg (CMK) (5) by
replacement of N-CBZ-p-tri-n-butylstannyl Phe (2) with 0-benzyl-N-
CBZ tyrosine (6). This 0-benzyl-N-CBZ tyrosine (6) i8 coupled to
proline methyl ester, the resulting dipeptide is hydrolyzed to the
- acid and then coupled to N02 arginine CMK. Hydrogenation removes
the N02, benzyl, and CBZ protecting groups to afford the desired
compound (9).
Radioiodination of Tyr-Pro-Ar~ (CMK) (9)
Radioiodination of Tyr-Pro-Arg (CMK) (9) is accomplished as
described for p-tri-~-butylstannyl Phe-Pro-Arg (CMK) (5). The
resulting radioiodinated compound (shown in Figure 5) is reacted
with t-PA in a buffered solution, whereupon the compound binds to
t-PA.
LC8x1659.mhg
