Abstract:
This invention relates to radiodiagnostic agents and reagents for preparing such agents, and also methods for producing radiolabeled radiodiagnostic agents. Specifically, the invention relates to technetium-99m ( 99m  Tc) labeled agents, methods and kits for making the agents, and methods for using the agents to image pathological sites, including sites of infection, inflammation, cancer and atherosclerosis in a mammalian body. Specifically the agents and reagents are derivatives of oligosaccharides, more specifically β-glucans.

Description:
This application is a 371 of PCT/US 94/08501 filed Jul. 28, 1994 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to radiodiagnostic agents and reagents for preparing such agents, and also methods for producing radiolabeled radiodiagnostic agents. Specifically, the invention relates to technetium-99m ( 99m  Tc) labeled agents, methods and kits for making the agents, and methods for using the agents to image pathological sites, including sites of infection, inflammation, cancer and atherosclerosis in a mammalian body. Specifically the agents and reagents are derivatives of oligosaccharides, more specifically β-glucans. 
     2. Description of the Prior Art 
     In the field of nuclear medicine, certain pathological conditions can be localized or the extent of such conditions determined by imaging the internal distribution of administered radioactively-labeled tracer compounds (i.e. radiotracers or radiopharmaceuticals) that accumulate specifically at the pathological site. This type of procedure is commonly known as radioimaging or scintigraphic imaging. Radioimaging has particular advantages over other methods of diagnosis in that it is essentially non-invasive, highly sensitive, highly specific, can be used to scan the entire body and is relatively cost-effective. A variety of radionuclides are known to be useful for radioimaging, including  67  Ga,  68  Ga,  99m  Tc,  111  In,  123  I,  125  I or  201  Tl. 
     There is a clinical need to be able to determine the location and/or extent of sites of focal or localized infection. In a substantial number of cases conventional methods of diagnosis (such as physical examination, x-ray, CT and ultrasonography) fail to identify such sites (e.g., an abscess). In some cases, biopsy may be resorted to, but is preferably avoided at least until it is necessary in order to identify the pathogen responsible for an abscess at a known location. Identifying the site of such &#34;occult&#34; infection is important because rapid localization of the problem is critical to effective therapeutic intervention. 
     An abscess may be caused by any one of many possible pathogens, so that a radiotracer specific for a particular pathogen would have limited scope. On the other hand, infection is almost invariably accompanied by inflammation, which is a general response of the body to tissue injury. Therefore, a radiotracer specific for sites of inflammation would be expected to be useful in localizing sites of infection caused by any pathogen. 
     One of the main phenomena associated with inflammation is the localization of leukocytes (white blood cells), including macrophages, monocytes and neutrophils, at the site of inflammation. A radiotracer specific for leukocytes would be useful in detecting leukocytes at the site of a localized infection. 
     Currently approved nuclear medicine procedures for imaging sites of infection use either indium-111 labeled leukocytes ( 111  In-WBC) (see, e.g. Peters, 1992, J. Nucl. Med. 33: 65-67) or gallium-67 ( 67  Ga) citrate (see, e.g. Ebright et al., 1982, Arch. Int. Med. 142: 246-254). 
     A major disadvantage of using  111  In-labeled WBCs is that the preparation of the radiotracer requires sterile removal of autologous blood, sterile isolation of the leukocytes from the blood, sterile labeling of the leukocytes using conditions that do not damage the cells (since damaged WBC are taken up by the reticuloendothelial system when re-injected) and return (re-injection) of the (now labeled) leukocytes to the patient. Furthermore, a delay of 12 to 48 hours between injection and imaging may be required for optimal images. While  99m  Tc labeled leukocytes have been used to shorten this delay period (see, e.g. Vorne et al., 1989, J. Nucl. Med. 30: 1332-1336), ex-corporeal labeling is still required. A preferred radiotracer would be one that does not require removal and manipulation of autologous blood components. 
       67  Ga- citrate can be administered by intravenous injection. However, this compound is not specific for sites of infection or inflammation. Moreover, a delay of up to 72 hours is often required between injection of the radiotracer and imaging. In addition, the γ- (gamma) emission energies of  67  Ga are not well suited to conventional gamma cameras. 
     Radiolabeled monoclonal and polyclonal antibodies raised against human leukocytes (including monocytes, neutrophils, granulocytes and others) have been developed.  99m  Tc labeled antigranulocyte monoclonal antibodies (see, e.g. Lind et al., 1990, J. Nucl. Med. 31: 417-473) and  111  In-labeled non-specific human immunoglobulin (see, e.g. LaMuraglia et al., 1989, J. Vasc. Surg. 10: 20-28) have been tested for the detection of inflammation secondary. to infection.  111  In-labeled IgG shares the disadvantages of  111  In-labeled WBC, in that 24-48 hours are required between injection and optimal imaging. In addition, antibodies are difficult to produce and are associated with safety concerns regarding potential contamination with biological pathogens (e.g. retroviruses). 
     In addition, the effective treatment of cancer by surgery or radiation therapy requires knowledge of the localization and extent of the disease. A means of monitoring the progression/regression of tumors following or during any form of therapy is also highly desirable. 
     Advances in high-resolution imaging modalities such as CT and MRI allow the detection of many neoplasms. However certain tumors and their metastases are small and difficult to localize by these methods. Nuclear medicine offers a potentially more sensitive alternative. 
     A radiotracer that selectively binds to or localizes to any and all cancerous tissue, sufficiently to allow easy external detection, might be considered to be the ultimate goal of radiodiagnostic oncology. 
     Also, despite remarkable advances in cardiology, coronary artery disease remains the leading cause of death in the U.S. The final event in this disease is usually fatal myocardial infarction caused by occlusive thrombosis of one or more coronary arteries usually at the site of a complicated atherosclerotic plaque. Therefore a means, preferably non-invasive, of determining the localization and/or extent of atherosclerotic plaque is highly desirable as an aid to selecting appropriate patient management. One of the most notable characteristics of atherosclerotic plaque is the accumulation of foam cells which are lipid-laden macrophages. 
     β-Glucans are oligoglucosides, which comprise 1,3 and 1,6 linked β-D-glucose residues, originally discovered as components of yeast and fungal cell walls (Bartnicki-Garcia in Ann Rev Microbiol. 1968, 22, 87). Originally obtained in an insoluble form, β-glucans have since been obtained as soluble, low molecular weight oligomers (Janusz et al., 1989, J. Immunol. 142: 959-965). They have been shown to be active in enhancing the host defense mechanisms of mammals by activating the alternative complement pathway through their specific binding to receptors (called β-glucan receptors) found on the cell-surfaces of monocytes, macrophages and neutrophils (Czop and Kay, 1991, J. Exp. Med. 173: 1511-1520; Czop et al., 1989, Biochemistry of the Acute Allergic Reactions: Fifth International Symposium, pp. 287-296; Czop, 1986, Pathol. Immunopathol. Res 5: 286-296; Czop and Austen, 1985, J. Immunol. 134: 2588-2593). The in vivo administration of particulate β-glucans has been shown to provide protection from many pathogens including bacteria, viruses and fungi as well as reducing tumor growth (Czop et al., 1989, Biochemistry of the Acute Allergic Reactions: Fifth International Symposium, pp. 287-296). The smallest active β-glucan reported so far is a heptaglucoside (Janusz et al., 1989, J. Immunol. 142: 959). Onderdonk and co-workers (1992, Infection &amp; Immunity, 60: 1642-1647) describe the anti-infective properties of this small β-glucan. The β-glucans have also been shown to exhibit an anti-tumor growth effect, believed to occur by increasing the number of macrophages localizing to tumors (Di Luzio, in Pathophysiology of the Reticuloendothelial System, (Altruo and Saba, eds.), Raven Press, New York, pp. 209-224). 
     Czop and Janusz, U.S. Pat. No. 5,057,503 (1991), claim a heptaglucoside capable of reacting with β-glucan receptors, their isolation and their therapeutic use. 
     Jamas et al., PCT/US90/03440 claim β-glucans as drug delivery vehicles and as adjuvants. 
     Jamas et al., PCT/US90/05022 claim a method of activating the immune system by administering β-glucans. 
     Jamas et al., PCT/US90/05041 claim a method of producing a soluble β-glucan. 
     Methods for preparing radiolabel-binding moieties and of labeling them with  99m  Tc are disclosed in U.S. Pat. applications Ser. Nos. 07/653,012, now abandoned, which issued as U.S. Pat. No. 5,654,272; 07/757,470, now U.S. Pat. No. 5,225,180; 07/807,062, now U.S. Pat. No. 5,443,815; 07/851,074, now abandoned, which issued as U.S. Pat. No. 5,711,931; 07/871,282, a divisional of which issued as U.S. Pat. No. 5,720,934; 07/886,752, now abandoned, a continuation of which has been allowed as U.S. Ser. No. 08/273,274; 07/893,981, now U.S. Pat. No. 5,508,020; 07/955,466; 07/977,628, now U.S. Pat. No. 5,405,597; 08/019,525, now U.S. Pat. No. 5,552,525; 08/044,825, now abandoned, which issued as U.S. Pat. No. 5,645,815; and 08/073,577, now U.S. Pat. No. 5,561,220; 08/092,355; 08/095,760, now U.S. Pat. No. 5,620,675; 08/098,206, now abandoned, a divisional of which has been allowed as U.S. Ser. Nos. 08/476,687; 08/210,822, now abandoned; 08/236,402; 08/241,625, now allowed; 08/244,336; 08/253,973; 08/253,317, now allowed; and 08/253,678 and PCT International Applications PCT/US92/00757, PCT/US92/10716, PCT/US93/02320, PCT/US93/03687, PCT/US93/04794, PCT/US93/06029, PCT/US93/09387, PCT/US94/01894, PCT/US94/05895, and PCT/US94/06274, which are hereby incorporated by reference. 
     SUMMARY OF THE INVENTION 
     The present invention provides scintigraphic imaging agents that are β-glucans which are radiolabeled with a radioisotope or are β-glucan-derived reagents radioactively-labeled with a radioisotope. The β-glucan-derived reagents of the invention are comprised of a β-glucan covalently linked to a radiolabel binding moiety. The scintigraphic imaging agents of this invention are useful for imaging pathological sites within a mammalian body including sites of infection, inflammation, cancer and atherosclerosis. 
     A first aspect of the invention comprises reagents for preparing scintigraphic imaging agents for imaging sites within a mammalian body, said reagents comprising a β-glucan having a 1,3- and 1,6-linked D-glucoside sequence, of molecular weight of up to about 2,000 kDa and a radiolabel-binding moiety. 
     In a second aspect, the scintigraphic imaging agent of the invention comprises a soluble β-glucan. 
     In a third aspect, the scintigraphic imaging agent of the invention comprises the radioisotope  99m  Tc. 
     In another aspect of the invention the radiolabel-binding moiety is linked to the β-glucan via a 1-amino, 1-hydrazino, or 1-thio substituent. 
     In yet another aspect, the reagents of the invention comprise a β-glucan and a radiolabel-binding moiety of formula 
     
         Cp(aa)Cp                                                   I 
    
     wherein Cp is a protected or unprotected ysteine residue and (aa) stands for any α- or β-amino acid, and wherein the radiolabel-binding moiety is covalently linked to the β-glucan. In a preferred embodiment, the amino acid is glycine. In another preferred embodiment, the radiolabel-binding moiety is linked to the β-glucan via a linker which forms either an ether, thioether or amine bond to the β-glucan. 
     In another aspect, the invention provides reagents comprising a a single thiol-containing radiolabel-binding moiety having the following structure: 
     
         A--CZ(B)--(C(R.sup.1 R.sup.2)).sub.n --X                   II 
    
     wherein A is H, HOOC, H 2  NOC, (amino acid or peptide)--NHOC, (β-glucan) -(linker)-(peptide)--NHOC, (amino acid or peptide)--OOC, (β-glucan)-(linker) -(peptide)--OOC or R 4  ; Z is H or R 4  ; B is H, SH or --NHR 3 , --N(R 3 )-(peptide) -(linker)-(β-glucan), --N(R 3 )-(amino acid or peptide) or R 4  ; X is SH or --NHR 3 , --N(R 3 )-(peptide)-(linker)-(β-glucan), --N(R 3 )-(amino acid or peptide) or R 4  ; R 1 , R 2 , R 3  and R 4  are independently H or straight or branched chain or cyclic lower alkyl; n is 0, 1 or 2; and: (1) where B is --NHR 3  or --N(R 3 )-(peptide)-(linker)-(β-glucan), X is SH and n is 1 or 2; (2) where X is --NHR 3  or --N(R 3 )-(peptide)-(linker)-(β-glucan), B is SH and n is 1 or 2; (3) where B is H or R 4 , A is HOOC, H 2  NOC, (β-glucan)-(linker)-(peptide)--NHOC or (β-glucan)-(linker)-(peptide)--OOC, X is SH and n is 0 or 1; (4) where A is H or R 4 , then where B is SH, X is --NHR 3  or --N(R 3 )-(peptide)-(linker)-(β-glucan) and where X is SH, B is --NHR 3  or --N(R 3 ) -(peptide)-(linker)-(β-glucan); (5) where X is H or R 4 , A is HOOC, H 2  NOC, (β-glucan) -(linker)-(peptide)--NHOC or (β-glucan)-(linker)-(peptide)--OOC and B is SH; (6) where Z is methyl, X is methyl, A is HOOC, H 2  NOC, (β-glucan) -(linker)-(peptide)--NHOC or (β-glucan)-(linker)-(peptide)--OOC and B is SH and n is 0; and wherein the thiol moiety is in the reduced form and wherein (amino acid) is any primary α- or β-amino acid not containing a thiol group. 
     In particular embodiments of this aspect of the invention, the radiolabel-complexing moiety has a formula that is: 
     
         (amino acid).sup.1 -(amino acid).sup.2 -{A--CZ(B)--(C(R.sup.1 R.sup.2)).sub.n,--X},                                     IIa. 
    
     
         {A--CZ(B)--(C(R.sup.1 R.sup.2)).sub.n,--X}-(amino acid).sup.1 -(amino acid).sup.2,                                              IIb. 
    
     
         (a primary α,ω-or β,ω-diamino acid)-(amino acid).sup.1 - {A--CZ(B)--(C(R.sup.1 R.sup.2)).sub.n --X}, orIIc. 
    
     
         {A--CZ(B)--(C(R.sup.1 R.sup.2)).sub.n --X}-(amino acid).sup.1 -(a primary α,ω- or β,ω-diamino acid),         IId. 
    
     wherein (amino acid) 1  and (amino acid) 2  are each independently any naturally-ocurring, modified, substituted or altered α- or β-amino acid not containing a thiol group: A is H, HOOC, H 2  NOC, (β-glucan)-(linker)--NHOC, (β-glucan)-(linker)--OOC or R 4  ; Z is H or R 4  ; B is H, SH or --NHR 3 , --N(R 3 )-(linker)-(β-glucan) or R 4  ; X is SH or --NHR 3 , --N(R 3 )-(linker)-(β-glucan) or R 4  ; R 1 , R 2 , R 3  and R 4  are independently H or straight or branched chain or cyclic lower alkyl; n is an integer that is either 0, 1 or 2; and: (1) where B is --NHR 3  or --N(R 3 )-(linker) -(β-glucan), X is SH and n is 1 or 2; (2) where X is --NHR 3  or --N(R 3 )-(linker)-(β-glucan), B is SH and n is 1 or 2; (3) where B is H or R 4 , A is HOOC, H 2  NOC, (β-glucan)-(linker)--NHOC, (β-glucan)-(linker)--OOC, X is SH and n is 0 or 1; (4) where A is H or R 4 , then where B is SH, X is --NHR 3  or --N(R 3 )-(linker) -(β-glucan) and where X is SH, B is --NHR 3  or --N(R 3 )--(linker)-(β-glucan); (5) where X is H or R 4 , A is HOOC, H 2  NOC, (β-glucan)-(linker)--NHOC, (β-glucan) -(linker)--OOC and B is SH; (6) where Z is methyl, X is methyl, A is HOOC, H 2  NOC, (β-glucan)-(linker)&#39;NHOC, (β-glucan)-(linker)--OOC and B is SH and n is 0; and (7) where Z is SH and X is SH, n is not 0; and wherein the thiol group is in the reduced form. 
     In yet another aspect, the present invention provides reagents comprising β-glucans covalently linked to a radiolabel-binding moiety having the following structure: ##STR1## For purposes of this invention, radiolabel-binding moieties having structure III will be referred to as picolinic acid (Pic)-based moieties; or ##STR2## For purposes of this invention, radiolabel-binding moieties having structure IV will be referred to as picolylamine (Pica)-based moieties; wherein X is H or a protecting group; (amino acid) is any amino acid. In a preferred embodiment, the amino acid is glycine and X is an acetamidomethyl protecting group. 
     In yet another embodiment of the invention, reagents are provided for preparing scintigraphic imaging agents for imaging sites within a mammalian body, comprising a β-glucan and a bisamino bisthiol radiolabel-binding moiety covalently linked to the β-glucan. The bisamino bisthiol radiolabel-binding moiety in this embodiment of the invention has a formula selected from the group consisting of: ##STR3## wherein each R 5  can be independently H, CH 3  or C 2  H 5  ; each (pgp) S  can be independently a thiol protecting group or H; m, n and p are independently 2 or 3; A is linear or cyclic lower alkyl, aryl, heterocyclyl, combinations or substituted derivatives thereof; and X is (linker)-β-glucan; ##STR4## wherein each R 5  is independently H, lower alkyl having 1 to 6 carbon atoms, phenyl, or phenyl substituted with lower alkyl or lower alkoxy; m, n and p are independently 1 or 2; A is linear or cyclic lower alkyl, aryl, heterocyclyl, combinations or substituted derivatives thereof; V is H or -CO-(linker)-β-glucan; R 6  is H or a (linker)-β-glucan; provided that when V is H, R 6  is a (linker)-β-glucan and when R 6  is H, V is a -CO-(linker)-β-glucan. For purposes of this invention, radiolabel-binding moieties having these structures will be referred to as &#34;BAT&#34; moieties. 
     The invention comprises scintigraphic imaging agents that are complexes between β-glucans or the reagents of the invention and  99m  Tc, and methods for radiolabeling the (β-glucans and reagents of the invention with  99m  Tc. Radiolabeled complexes provided by the invention may be formed by reacting β-glucans or the reagents of the invention with  99m  Tc in the presence of a reducing agent. Preferred reducing agents include but are not limited to dithionite ion, stannous ion and ferrous ion. Complexes of the invention are also formed by labeling β-glucans or the reagents of the invention with  99m  Tc by ligand exchange of a prereduced  99m  Tc complex as provided herein. 
     The invention also provides kits for preparing scintigraphic imaging agents that are β-glucans or the reagents of the invention radiolabeled with  99m  Tc. Kits for labeling the β-glucans or the reagents provided by the invention with  99m  Tc are comprised of a sealed vial containing a predetermined quantity of a β-glucan or a reagent of the invention and a sufficient amount of reducing agent to label the β-glucan or reagent with  99m  Tc. 
     This invention provides methods for using scintigraphic imaging agents that are radiolabeled β-glucans and reagents for imaging pathological sites, including infection, inflammation, cancer and atherosclerosis within a mammalian body by obtaining in vivo gamma scintigraphic images. These methods comprise administering an effective diagnostic amount of radiolabeled β-glucan or reagent of the invention and detecting the gamma radiation emitted by the radiolabel localized at the pathological site within the mammalian body. 
     Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The β-glucans of this invention have linear or branched 1,3 and 1,6 linked D-glucoside sequences. They comprise both insoluble and soluble molecular entities having molecular weights of up to about 2,000 kDa. In a preferred embodiment, the β-glucan is soluble. Most preferably the soluble β-glucan is a poly-β1-6-glucotriosyl-β1-3-glucopyranose. 
     In Cp(aa)Cp-containing β-glucan reagents, the Cp is a protected cysteine where the S-protecting groups are the same or different and may be but are not limited to: 
     Ch 2  --aryl (aryl is phenyl or alkyl or alkyloxy substituted phenyl); 
     CH--(aryl) 2 , (aryl is phenyl or alkyl or alkyloxy substituted phenyl); 
     C--(aryl) 3 , (aryl is phenyl or alkyl or alkyloxy substituted phenyl); 
     CH 2  --(4-methoxyphenyl); 
     CH--(4-pyridyl)(phenyl) 2  ; 
     C(CH 3 ) 3   
     9-phenylfluorenyl; 
     CH 2  NHCOR (R is unsubstituted or substituted alkyl or aryl); 
     CH 2  --NHCOOR (R is unsubstituted or substituted alkyl or aryl); 
     CONHR (R is unsubstituted or substituted alkyl or aryl); 
     CH 2  --S--CH 2  -phenyl 
     The preferred protecting group has the formula --CH 2  --NHCOR wherein R is a lower alkyl having 1 and 8 carbon atoms, phenyl or phenyl-substituted with lower alkyl, hydroxyl, lower alkoxy, carboxy, or lower alkoxycarbonyl. 
     β-Glucans of the present invention can be obtained from natural sources, such as yeast, by methods well known in the art (e.g., see Manners et al., 1974, J. Gen. Microbiol. 80: 411-417). Small soluble β-glucans can be obtained from larger β-glucans by methods known in the art (e.g. see Janusz et al., 1989, J. Immunol. 142: 959-965 and Jamas et al., PCT/US90/05041) or can be obtained by chemical synthesis. Preferred soluble β-glucans are poly-β1-6-glucotriosyl-β1-3-glucopyranoses including those that are heptaglucosides. 
     The term soluble β-glucan is used herein to mean soluble in a physiologically compatible solution to about 10 mg/mL. 
     The reagents of this invention comprise a β-glucan covalently attached to a radiolabel-binding moiety. The radiolabel binding moiety can be attached directly to the β-glucan or it can be attached via a linker. The direct attachment of the radiolabel-binding moiety may be advantageously made by a 1-thioether, 1-hydrazino, or 1-amino group, or via an ester or ether bond to any hydroxyl group of the β-glucan (see, for example, Her et al., 1987, J. Carbohydrate Chem. 6: 129-139; Bogwald et al., 1986, Carbohydrate Res. 148: 101-107). The linker is normally a small entity, of less than about 500 Da formula weight and may advantageously be a small (up to about 10 carbon atoms) linear or branched chain divalent alkyl, alkaryl or aryl group, optionally comprising a multiplicity of hetero atoms, preferably oxygens, and optionally substituted, preferably with hydrophilic moieties. The radiolabeling moiety may also be attached to the β-glucan following partial oxidation of the β-glucan. Partial oxidation of the β-glucan exposes additional aldehyde groups on the β-glucan, thereby allowing conjugation of a greater amount of radiolabel-binding moiety per β-glucan molecule, without substantially decreasing the receptor-binding affinity of the β-glucan. 
     In forming a complex of radioactive technetium with the β-glucans and the reagents of this invention, the technetium complex, preferably a salt of  99m  Tc pertechnetate, is reacted with the β-glucan or reagent in the presence of a reducing agent. Preferred reducing agents are dithionite, stannous and ferrous ions; the most preferred reducing agent is stannous chloride. Means for preparing such complexes are conveniently provided in a kit form comprising a sealed vial containing a predetermined quantity of a β-glucan or reagent of the invention to be labeled and a sufficient amount of reducing agent to label the reagent with  99m  Tc. Alternatively, the complex may be formed by reacting a β-glucan or reagent of this invention with a pre-formed labile complex of technetium and another compound known as a transfer ligand. This process is known as ligand exchange and is well known to those skilled in the art. The labile complex may be formed using such transfer ligands as tartrate, citrate, gluconate or mannitol, for example. Among the  99m  Tc pertechnetate salts useful with the present invention are included the alkali metal salts such as the sodium salt, or ammonium salts or lower alkyl ammonium salts. 
     The reaction of β-glucans and reagents of this invention with Tc-pertechnetate or preformed  99m  Tc labile complex can be carried out in an aqueous medium at room temperature or with heating for a short period (from 5 to about 60 minutes). When an anionic complex having a charge of  -1! is formed in the aqueous medium in the form of a salt with a suitable cation such as sodium cation, ammonium cation, mono, di- or tri-lower alkyl amine cation, etc. Any conventional salt of the anionic complex with a pharmaceutically acceptable cation can be used in accordance with this invention. 
     In a preferred embodiment of the invention, a kit for preparing  99m  Tc-labeled β-glucans and β-glucan reagents is provided. An appropriate amount of the β-glucan or reagent is introduced into a vial containing a reducing agent, such as stannous chloride, in an amount sufficient to label the β-glucan or reagent with  99m  Tc. An appropriate amount of a transfer ligand as described (such as tartrate, citrate, gluconate or mannitol, for example) can also be included. In forming the  99m  Tc complexes, it is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 millicurie (mCi) to 100 mCi per ml. 
     Scintigraphic imaging agents of this invention can also be prepared by incubating radiolabeled β-glucans or radiolabeled β-glucan reagents with leukocytes, wherein the leukocytes take up the radiolabeled species and can then be administered as radiolabeled leukocytes. 
     The radiolabeled scintigraphic imaging agents provided by the present invention can be used for visualizing pathological sites including sites of inflammation and infection, including abscesses and sites of &#34;occult&#34; infection and inflammatory bowel disease. The imaging agents provided can also be used to image sites of atherosclerotic plaque and also tumors. In accordance with this invention, the scintigraphic imaging agents are administered in a single unit injectable dose. Any of the common carriers known to those with skill in the art, such as sterile saline solution or plasma, can be utilized after radiolabeling for preparing the injectable solution to diagnostically image various organs, tumors and the like in accordance with this invention. Generally, the unit dose to be administered has a radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi. The solution to be injected at unit dosage is from about 0.01 ml to about 10 ml. After intravenous administration, imaging of the organ or tumor in vivo can take place in a matter of a few minutes. However, imaging can take place, if desired, in hours or even longer, after injecting into patients. In most instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour to permit the taking of scintiphotos. Any conventional method of scintigraphic imaging for diagnostic purposes can be utilized in accordance with this invention. 
     The scintigraphic imaging agents provided by the invention may be administered intravenously in any conventional medium for intravenous injection such as an aqueous saline medium, or in blood plasma medium. Such medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. Among the preferred media are normal saline and plasma. 
    
    
     The methods for making and labeling these compounds are more fully illustrated in the following Examples. These Examples illustrate certain aspects of the above-described method and advantageous results. These Examples are shown by way of illustration and not by way of limitation. 
     EXAMPLE 1 
     Reagent Synthesis 
     It will be understood in the synthetic schema described in this example that the abbreviation DMSO stands for dimethyl sulfoxide, DMF stands for N,N-dimethylformamide and DIEA stands for N,N-diisopropylethylamine. 
     Poly-β1-6-glucotriosyl-β1-3-glucopyranose (PGG) is obtained using the procedures described by Jamas et al. (International Patent Application No. PCT/US90/05041). 
     Selective oxidation of β-glucans is accomplished using the procedures described by Hay et al., (1965, Meth. Carbohydrate Chem. 5: 357-361). 
     N-α-Boc-lysyl-glycyl-(S-trityl)cysteine amide, glycyl-glycyl-(S-trityl) cysteine amnide and chloroacetyl-(S,S&#39;-bis-acetamidomethyl)cysteinyl-glycyl-cysteine amide are prepared by solid phase or solution phase peptide synthesis and are purified by reverse phase HPLC or diafiltration. 
     A conjugate with N 1 ,N 4  -bis(2-mercapto-2-methylpropyl)-1,4, 10-triazadecane is obtained by reacting a β-glucan (e.g., PGG) at from about 1 to 100 mg/mL with about 1.5 mmol N 1  -(t-butoxycarbonyl)-N 1 ,N 4  -bis (2-methyl-2-triphenylmethylthiopropyl)-1,4, 10-triazadecane in water, Cellosolve or mixtures thereof at approximately pH 7 at about 65° C. for from 1 to about 10 hours, followed by reduction with NaBH 3  CN followed by deprotection with trifluoroacetic acid. The product is purified by preparative HPLC or diafiltration. 
     Similarly conjugates of ε-(lysyl-glycyl-cysteine amide) and glycyl-glycyl-cysteine amide are prepared from N-α-Boc-lysyl-glycyl-(S-trityl)cysteine amide and glycyl-glycyl-(S-trityl)cysteine amide respectively. 
     A conjugate of N 6 ,N 9  -bis(2-mercapto-2-methylpropyl)-6,9-diazanonanoic acid is prepared by reacting β-glucan (e.g. PGG) at from about 1 to 100 mg/mL in water, DMSO or DMF containing about 1.5 mmol DIEA and optionally containing about 0.15 mmol 4-dimethylaminopyridine, with about 1.5 mmol of the N-hydroxysuccinimide ester of N 9  -(t-butoxycarbonyl)-N 6 , N 9  -bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanoic acid, at room temperature, followed by deprotection with TFA and purification by HPLC or diafiltration. 
     A conjugate of (S,S&#39;-bis-acetamidomethyl)cysteinyl-glycyl-cysteine amide is prepared by reacting β-glucan (e.g. PGG) at from about 1 to 100mg/mL in DMSO, with sodium methylsulfinylmethanide, or another suitable base, (approx. 1.6 mmol base/100 mg β-glucan) for from 1 to about 24 hours and reacting the resultant mixture with approx. 1.6 mmol chloroacetyl-(S,S&#39;-bis-acetamidomethyl) cysteinyl-glycyl-cysteine amide for about 1 to 5 hours at between 20° and 50° C., followed by purification by HPLC or diafiltration. 
     Potency of β-glucan-radiolabel binding moiety conjugates is determined using the methods disclosed by Janusz et al. (1989, J. Immunol. 142: 959-965). 
     EXAMPLE 2 
     A General Method for Radiolabeling with Tc-99m 
     1. About 0.1 mg of a β-glucan or a reagent prepared as in Example 1 is dissolved in 0.1 mL of water or 50/50 ethanol/water. Approximately 100 μg stannous salt as stannous chloride pre-dissolved in methanol, or stannous tartrate pre-dissolved in water is added followed by 1-10 mCi 99m  Tc pertechnetate in approximately 0.1 mL. The mixture is allowed to stand for 15-30 minutes at room temperature or at 100° C. For soluble β-glucans the preparation is then filtered through a 0.2 μm filter and the Tc-99m labeled product purity is determined by HPLC. The purity of insoluble β-glucan products is assessed by ITLC developed in saline. 
     2. About 0.1 mg of β-glucan or reagent prepared as described in Example 1 is dissolved in 0.1 mL of water or 50/50 ethanol/water or phosphate-buffered saline or 50 mM potassium phosphate buffer (pH=5, 6 or 7.4). Tc-99m gluceptate was prepared by reconstituting a Glucoscan vial (E. I. DuPont de Nemours, Inc.) with 1.0 mL of Tc-99m sodium pertechnetate containing up to 200 mCi and allowed to stand for 15 minutes at room temperature. 25 μl of Tc-99m gluceptate was then added to the peptide and the reaction allowed to proceed at room temperature or at 100° C. for 15-30 min. For soluble β-glucans the preparation is then filtered through a 0.2 μm filter and the Tc-99m labeled product purity is determined by HPLC. The purity of insoluble β-glucan products is assessed by ITLC developed in saline. 
     EXAMPLE 3 
     Preparation and Radiolabeling of a β-Glucan -(Lys(CO(CH 2 ) 3  CONHNH 2 )-Glv-Cvs-Phe.amide) Adduct 
     To a solution of β-glucan (having a nominal molecuar weight of 100,000 daltons; 0.05 μmol in 0.208 mL of an aqueous NaCl solution) was added the peptide Lys(CO(CH 2 ) 3  CONHNH 2 )-Gly-Cys-Phe.amide (29 mg, 50 μmol), prepared by solid phase peptide synthesis using Fmoc-Phe, Fmoc-Cys(Trt), Fmoc-Gly and Fmoc-Lys(CO(CH 2 ) 3  CONHNHBoc) amino acid precursors. This mixture was heated at 65° C. for about 36 hours. Dithiothreitol (70 mg, 450 μmol) dissolved in 0.5 mL phosphate buffer containing 0.5 mM EDTA was added to the mixture, which was then allowed to stand at room temperature for about 18 hours. The resulting mixture was filtered using a 10,000 nominal molecular weight limit Microcon 10 unit (Amicon, Beverly, Mass.) and the residue was washed three times with 0.15 M NaCl. Gel-filtration HPLC analysis (using TSK-Gel® GMPW XL , 0/79×30 cm column with a PW XL , 0.6×4 cm guard column, equipped with in-line refractive index and UV 214  detectors, eluted at 10 mL/min with 0.15 M NaCl) showed a single peak detected by both the refractive index monitor and UV spectroscopy and having the same retention time (about 9.4 mm) through the column as the starting β-glucan material. 
     The βglucan adduct thus prepared was radiolabeled with Tc-99m as follows. To a 0.1 mL solution of a β-glucan-(Lys(CO(CH 2 ) 3  CONHNH 2 )-Gly-Cys-Phe.amide adduct (approximately 1 mg in 0.15 M NaCl) was added 50 μL of a solution of Tc-99m gluceptate, prepared by reconstituting a Glucoscan® kit with 1.0 mL Tc-99m generastor eluate. This solution was incubated at room temperature for 15 min. Gel-filtration HPLC, as described above, showed a single radioactive peak at 9.9 min, approximately the position of the unlabeled starting material, indicating essentially complete labeling of the conjugate. 
     EXAMPLE 4 
     Scintigraphic Imaging and Biodistribution of Tc-99m Labeled Peptides 
     In order to demonstrate the effectiveness of Tc-99m labeled β-glucan reagents as provided above, New Zealand white rabbits are innoculated intramuscularly in the left calf with a potent stain of E. coli. After 24 h, the animals are sedated by i.m. injection of ketamine and xylazine, and then injected i.v. with Tc-99m labeled β-glucan (≦150 μg, 2-10 mCi). The animals are positioned supine in the field of view of a gamma camera (LEAP collimator/photopeaked for Tc-99m) and imaged over the first hour post-injection, and then at approximately 1 h intervals over the next three hours post injection. Animals are allowed to recover between image acquisitions and re-anesthetized as needed. 
     Upon completion of the final imaging, each animal is sacrificed by overdose of phenobarbital i.v. and dissected to obtain samples of blood and of infected and control muscle tissue. The tissue samples are weighed, and along with a standard amount of the injected dose, are counted using a gamma counter, and the percent injected dose (per gram of tissue) remaining in the tissues is determined. Ratios of percent of injected dose per gram of infected versus non-infected muscle tissue, and of infected muscle tissue versus blood, are calculated for each peptide. Scintiphotos of whole body and leg images of a rabbit injected with a Tc-99m labeled reagent of the invention are thereby obtained. 
     It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.