CA1335725C - Method of diagnosing blood clots using fibrin-binding proteins - Google Patents

Abstract

A fibrin-binding protein such as t-PA is labeled with a detectable substance, such as a radionuclide, and administered to a patient for diagnosis of blood clots and for monitoring the dissolution thereof during therapy. The detectable substance preferably is attached to t-PA through linkers which specifically bind to the portion of the t-PA protein responsible for enzymatic activity, thereby diminishing this activity while leaving the fibrin-binding property of the protein intact.

Description

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 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 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 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.

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 _~

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 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 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 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 *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 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 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 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 ~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.

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 "~
< >

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, 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 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 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 ~ 335725 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 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

Claims (36)

WE CLAIM:

1. A method for detecting a fibrin-platelet clot in vivo, comprising the steps of:
(a) administering to a patient suspected of having a fibrin-platelet clot a thrombolytic protein labelled with a detectable substance, wherein the thrombolytic protein's clot-dissolving activity is reduced or eliminated and the label is selectively attached to a portion of the thrombolytic protein other than the fibrin binding domain; and (b) detecting the pattern of biodistribution of the labelled thrombolytic protein in the patient.

2. The method of claim 1 wherein said labeled thrombolytic protein is administered by injection into a patient's bloodstream.

3. The method of claim 1 wherein said labeled thrombolytic protein is t-PA.

4. The method of claim 1 or 3 wherein said detectable substance is a radioisotope.

5. The method of claim 4 wherein said radioisotope is a radionuclide metal in the form of a chelate.

6. The method of claim 5 wherein said rationuclite metal is 99mTc and detecting the biodistribution of the 99mTc is by scanning the patient with a gamma camera.

7. The method of claim 1 wherein said detectable substance is attached to the thrombolytic protein through a linker which specifically binds to the clot-dissolving portion of the thrombolytic protein.

8. The method of claim 7 wherein said thrombolytic protein is a fibrin-binding serine protease and the linker is an oligopeptide chloromethyl ketone that binds to the clot-dissolving portion of the serine protease.

9. The method of claim 8 wherein said thrombolytic protein is t-PA and said linker is selected from D-Phe-L-Pro-L-Arg-CH2Cl or Tyr-L-Pro-L-Arg-CH2Cl.

10. The method of claim 7, 8 or 9 wherein said detectable substance is a radioisotope.

11. The method of claim 10 wherein said radioisotope is a radionuclide metal in the form of a chelate.

12. The method of claim 1 wherein said thrombolytic activity is essentially eliminated.

13. A method for labeling a thrombolytic protein, comprising attaching 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.

14. The method of claim 13 wherein the linker is an oligopeptide chloromethyl ketone.

15. The method of claim 13 wherein said thrombolytic enzyme is t-PA and said linker binds specifically to the protease portion of t-PA.

16. The method of claim 15 wherein said linker is selected from D-Phe-L-Pro-L-Arg-CH2Cl and Tyr-L-Pro-L-Arg-CH2Cl.

17. The method of claim 16 wherein said detectable substance is a radioisotope.

18. The method of claim 17 wherein said ratioisotope is a radionuclide metal in the form of a chelate.

19. A kit for imaging a fibrin-platelet clot in vivo, comprising a thrombolytic protein, a linker and a detectable substance wherein the detectable substance is attached to the thrombolytic protein by the linker wherein the attachment is to a portion of the thrombolytic protein other than the fibrin-binding domain thereof.

20. The kit of claim 19 comprising a first vial wherein the thrombolytic protein is t-PA and a second vial containing a lyophilized preparation comprising:
(a) N2S2-D-Phe-L-Pro-L-Arg-CH2Cl or N2S2-Tyr-L-Pro-L-Arg-CH2Cl, (b) a reducing agent effective in reducing pertechnetate to an oxidation state at which an exchange complex will be formed, and (c) an exchange agent with which the reduced pertechnetate will form said exchange complex.

21. The kit of claim 20 wherein said reducing agent is stannous ion and said exchange agent is gluconic acid.

22. A compound of the formula:

wherein m is 0 or 1 and Q represents a radiolabeled molecule.

23. The compound of claim 22 wherein Q comprises a radionuclide metal chelate.

24. The compound of claim 23 wherein the radionuclide metal chelate comprises a total of four donor atoms selected from nitrogen and sulfur atoms.

25. The compound of claim 24 wherein the radionuclide metal chelate comprises one sulfur and three nitrogen donor atoms.

26. The compound of claim 24 wherein the radionuclide metal chelate comprises two sulfur and two nitrogen donor atoms and said compound is of the following formula:

wherein m is 0 or 1 and M represents a radionuclide or an oxide thereof.

27. The compound of claim 22 wherein said compound is of one of the following formulas:

wherein n is 0 to 3 and *X represents a radioisotope of a halogen.

28. The compound of claim 22 wherein said compound is of the formula:

wherein *I represents a radioisotope of iodine and m is 0 or 1, with at least one m being 1.

29. A compound of the formula:

wherein *X represents a radiohalogen.

30. A compound of the formula:

wherein *X represents a radiohalogen.

31. A radiolabeled t-PA protein comprising the compound of claim 22, 25, 26, 27, 28, 29, or 30 bound to t-PA.

32. A thrombolytic enzyme having a detectable substance attached thereto through a linker derived from an oligopeptide chloromethyl ketone inhibitor of said thrombolytic enzyme.

33. The enzyme of claim 32 wherein said enzyme is selected from the group consisting of kallikreins, plasmin, thrombin, urokinase, and t-PA.

34. The enzyme of claim 33 wherein said enzyme is t-PA and said linker binds specifically to the portion of the t-PA responsible for plasminogen activation.

35. The enzyme of claim 34 wherein the linker is selected from D-Phe-L-Pro-L-Arg-CH2C1 and Tyr-L-Pro-L-Arg-CH2C1.

36. The enzyme of claim 35 wherein said detectable substance is a radioisotope.