AU743255B2 - Biotin compounds for targeting tumors and sites of infection - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): David R ELMALEH Invention Title: BIOTIN COMPOUNDS FOR TARGETING TUMORS AND SITES OF INFECTION S. The following statement is a full description of this invention, including the best method of performing it known to me/us: S.

C

S

12/11 2001 15:17 FAX 61 3 92438333 GRIFFITH HACK 1o007 -la Biotin Compounds for Targeting Tumors and Sites of Infection Background of the Invention All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents i: 15 forms part of the common general knowledge in the art, in Australia or in any other country.

SHigh coptrast imaging of tumors can be achieved by allowing a nonradiolabelled antibody to localise and S• clear from the circulation prior to administration of a 20 low molecular weight, radiolabelled moiety with high affinity for the pretargeted antibody (Paganelli, G. et al., J. Nucl. Med. Comm. 12:211-234 (1991); Green, NM Biochem. J. 89:585-91 (1963); Hnatowich DJ et al., J.

Nucl. Med. 28:1294-1302 (1987)). One such method utilises the high affinity of avidin, a cationic glycoprotein found in egg whites, for biotin, a naturally occurring vitamin.

Avidin is capable of binding four biotin molecules and forming an avidin-biotin complex with a very high affinity. (Kd=10- 1

M)

Two basic approaches for targeting tumors with avidin-biotin systems have been used in patients and animals. In the first method, avidin (or streptavidin)conjugated antibodies are injected and days later, when antibody-tumor binding is maximised, a radioactive biotin derivative is injected to localise the tumor.

Unfortunately, incomplete clearance of unbound antibody from the blood circulation can obscure visualisation of mel b l l *sl\ 4\cS~ incr \Keep\*paci\2782.99.flag 12/11/01 12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11 2001 15:17 FAX 61 3 92438333 GRIFFITH HACK oo008 the target site. In the second method, blood background is reduced by injecting biotinylated antibodies followed three days later by cold avidin. The resultant circulating biotinylated antibody-avidin complexes are sequestered from the blood by the liver. Radioactive biotin is then injected and binds to the antibody-biotinavidin complexes already localised in the tumor. However, by employing "pretargeting" steps, both approaches for targeting tumors require that a subject be available to undergo multiple procedures over the course of a few days.

A study by Morrel et al., reported uptake of In- 111 labelled IgG and human serum albumin (HSA) in an E.

coli infected rat model. The accumulation of both labelled proteins was found to be sufficient to produce clear images of the invention site (Morrel, EM et al., J.

Nucl. Med. 30:1538-1545 (1989)).

Simple, rapid methods for more specifically targeting therapeutic or imaging agents to tumors and sites of infection in vivo are needed.

\\ma Ei I \hon$\cinrac\Kaep\apic\23 7..99.ao c 12/11/01 12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11 2,001 15:17 FAX 61 3 92438333 GRIFFITH RACK Q009 -2- Summary of the Invention In one aspect, the present invention features biotin compounds that have a high specificity for target sites when administered to a subject in vivo. Preferred biotin compounds are biotin amide analogs and preferred target sites include tumors and sites of bacterial, viral or fungal infection. Particularly preferred biotin compounds show a target to non-target ratio of at least 5:1, are stable in vivo and substantially localise to target within 1 hour after administration- An especially preferred biotin amide analog is [3aS-(3aa, 4p, 6aa)]hexahydro-2-oxo-lH-thieno[3,4d]imidazole-4-(N-3- (if luoropropyl) pentanamide.

In another aspect, the invention features 15 pharmaceutical compositions comprised of a biotin compound. Preferred pharmaceutical compositions are biotin amide analogs having a therapeutic agent in the R6 position. In a further aspect, the invention features new uses for the biotin compounds in treating or preventing the establishment or growth of tumors or sites of infection by pathogenic bacteria, viruses or fungi)., In a further aspect, the present invention features biotin compounds further comprisingT an imaging agent and uses for the compounds in detecting and/or monitoring tumors or sites of infection in a subject. In one embodiment, the biotin compound imaging agent is administered in vivo and monitored using a means appropriate for the label. Preferred methods for detecting and/or monitoring a biotin compound imaging agent in vivo include Positron Emission Tomography (PET), and Single Photon Emission Computer Tomography (SPECT) and Magnetic Resonance Imaging (MRI)- The instant biotin compounds and in vivo methods for targeting therapeutic or imaging agents to a tumor or site of infection do not require an initial pretargeting step. In addition, localisation times are generally under 1 hour. Therefore, therapy or imaging can be accomplished k\'im-bfi1e,\hom cjncLmte \XeeD.aci\2$'742 .99.doc 12t11101.

12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11 2001 15:17 FAX 61 3 92438333 GRIFFITH RACK 9OlO 2a in one short procedure. In addition, biotin compounds, which are natural small molecules that are rapidly cleared in vivo, are unlikely to have toxic side effects at the levels required for therapy or imaging.

Additional features and advantages of the invention will become more apparent from the following detailed description and claims.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

Detailed Description of the invention As used herein, the following terms and phrases 15 shall have the meanings set forth below.

"Biotin compound" shall mean "biotin" (hexahydro- 2 -oxo-IH-thieno[3.4-dj-imidazoline-4-valeric acid); a 244 dalton vitamin, conjugated to an imaging agent or therapeutic agent; or a "biotin anide analog", which is a compound having either of the following two structural formulas: e

S

S

S

S

S. *5

*S

S

12/11 '01 MON 15:14 [TX/RX NO 6292] -3wherein,

R

1

-R

5 2-10; H, acyl, alkyl, alkylene, alkenylene, alkynylene,alkenyl or.

alkynyl groups; H, acyl, alkyl, alkylene, alkenylene, alkynylene,alkenyl or alkynyl groups; or a conjugated therapeutic agent or imaging agent; and C=O, S=0 or wherein, n= 2-10; H, acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl and alkynvi groups; H, acyl, alkyl, alkyvlene, alkenyvlene, alkynylene, alkenyl and alkynyl groups; or a conjugated therapeutic agrent or imaging agent C=O, S=0 or C=NH; and 0, S. or N positions 2, 3 or 4= -4- The biotin amide analogs include racemic compounds and all possible enantiomers. The alkylene, alkenylene, alkynylene, alkenyl and alkynyl groups (hereinafter hydrocarbon groups) comprising the R groups can be either straight or branched chains, saturated or unsaturated. Unsaturated groups may have a single site of unsaturation or a plurality of unsaturated sites. The hydrocarbon groups preferably have up to about ten carbons, more preferably up to about six carbons, and most preferably up to about three carbons. A hydrocarbon group having three carbon atoms or less is considered to be a lower hydrocarbon group. For example, an alkyl group having three carbon atoms or less is a lower alkyl. Examples of lower hydrocarbon groups which may be used in the present invention include methyl, methylene, ethyl, ethylene, ethenyl, ethenylene, ethynl, ethynylene, propyl, propylene, propenyl, propenylene, propynyl, and propynylene. Examples of higher hydrocarbon groups (from four to about ten carbons) include butyl, t-butyl, butenyl, butenylene, and butynyl, butynylene, nonyl, nonylene, nonenyl, nonenylene, nonynyl. and nonynylene.

The alkyl or alkylene groups may be substituted with one or more oxygen or halogen atom to form alkoxy, haloalkyl, alkoxyene, and haloalkylene groups. The alkoxy and haloalkyl groups also may be straight or branched chain and preferably are made up of up to about ten atoms (including carbon, oxygen or halogen), preferably up to about six atoms, and most preferably up to about three atoms. The term halogen is art-recognized and includes chlorine, fluorine, bromine, astatine and iodine. Examples of substituted hydrocarbon groups which are useful within this invention are similar to hydrocarbon groups set forth above except for the incorporation of oxygen(s) or halogen(s) into the groups.

Substitutions, which result in more lipophilic biotin compounds are preferred for targetting of tumors and sites of infection in the brain and spinal cord.

25 Especially preferred biotin compounds exhibit a high target to non-target ratio when administered in vivo. Preferably the ratio is> 5:1. A biotin compound can be glycosylated with a sugar moiety glucose, fucose, galactose, mannose) that is recognized by a target specific receptor to further increase the target specificity of a compound. For example, biotin compounds can be glycosylated with mannose residues attached as Cglycosides to a free nitrogen) to yield biotin compounds having higher affinity binding to tumors expressing mannose receptors glioblastomas and gangliocytomas); and bacteria, which are also known to express mannose receptors (Bertozzi, CR and MD Bednarski Carbohydrate Research 223:243 (1992); J. Am. Chem. Soc. 114:2242,5543 (1992)) as well as potentially other infectious agents.

"conjugated" shall mean ionically or covalently attached (e.g.via a crosslinking agent).

An "imaging agent" shall mean a composition capable of generating a detectable image upon binding with a target and shall include radionuclides (e.g.In-111, Tc- 99m, 1-123, 1-125 F-18, Ga-67, Ga-68, and for Positron Emission Tomography (PET) and Single Photon Emission Tomography (SPECT), unpair spin atoms and free radicals Fe.

lanthides and Gd) and contrast agents chelated (DTPA) manganese) for Magnetic Resonance Imaging (MRI).

A "therapeutic agent" shall mean an agent capable of having a biological effect on a host. Preferred therapeutic agents are capable of preventing the establishment or growth (systemic or local) of a tumor or infection. Examples include drugs antibiotics, anti-virals, antifungals), toxins ricin) radionuclides 1-131, Re-186, Re-188, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-67 and Cu-64), hormone antagonists (e.g.

tamoxifen), heavy metal complexes cisplatin), oligonucleotides antisense oligonucleotides that bind to a target nucleic acid sequence mRNA sequence)), chemotherapeutic nucleotides, peptides, non-specific (non-antibody) proteins sugar oligomers), boron containing compound carborane), photodynamic agents (e.g.

rhodamine 123), enediynes calicheamicins, esperamicins, dynemicin, neocarzinostatin chromophore, and kedarcidin chromophore) and transcription based pharmaceuticals. In a preferred embodiment for treating or preventing the establishment or growth of a tumor, the therapeutic agent is a radionuclide, toxin, hormone antagonist, heavy metal complex, oligonucleotide, chemotherapeutic nucleotide, peptide, non-specific (non-antibody) protein,a boron compound or an enediyne. In a preferred embodiment for treating or preventing the establishment or growth of a bacterial infection, the therapeutic agent is an antibiotic, radionuclide or oligonucleotide. In a preferred embodiment for treating or preventing the establishment or growth of a viral infection, the therapeutic agent is an antiviral compound, radionuclide or oligonucleotide. In a preferred embodiment for treating or preventing the establishment or growth of a fungal infection, the therapeutic agent is an antifungal compound radionuclide or oligonucleotide.

25 A "target" shall mean an in vivo site to which biotin compounds bind. A preferred target is a tumor tumors of the brain, lung (small cell and non-small cell), ovary, prostate, breast and colon as well as other carcinomas and sarcomas). Another preferred target is a site of infection by bacteria, viruses HIV, herpes, hepatitis) and pathogenic fungi (Candida Particularly preferred target infectious organisms are those S* 30 that are drug resistant Enterobacteriaceae, Enterococcus, Haemophilus influenza, SMycobacterium tuberculosis, Neisseria, gonorrhoeae. Plasmodiumfalciparum, Pseudomonas ~aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Streptococcus pneumoniae). The localization of biotin at a site of infection, may be related to that fact that it.is an essential nutrient of many bacteria, viruses and fungi.

"Subject" shall mean a human or animal rat, mouse, cow, pig, horse.

sheep, monkey, cat, dog, goat etc) *e eee e* Method for Making Biotin Compounds A. variety of coupling or crosslinking agents such as protein A, carboiimide, dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), N-succinimidyl-S-acetyl-thioacetate (SATA), and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), 6 -hydrazinonicotimide (HYNIC), N 3 S and N 2

S

2 can be used in well-known procedures to synthesize biotin amide analogs or biotin compounds. For example, biotin can be conjugated via DTPA using the bicyclic anhydride method of Hnatowich et al Int. J. Appl. Radiat. Isotop. 33: 327 (1982).

In addition sulfosuccinimidyl 6-(biotinamido) hexanoate (NHS-LC-biotin (which can be purchased from Pierce Chemical Co. Rockford, IL), "biocytin", a lysine conjugate of biotin, can be useful for making biotin compounds due to the availability of a primary amine. In addition, corresponding biotin acid chloride or acid precursors can be coupled with an aminoderivative of the therapeutic agent by known methods (See e.g.

methods described in Examples 1 and 2).

Synthesized biotin compounds and biotin amide analogs can be characterized using standard methods of high field NMR spectra as well as IR, MS and optical rotation.

Elemental analysis, TLC and/or HPLC can be used as a measure of purity. A purity of>98% is preferred. TLC and/or HPLC can also be used to characterize more lipophilic compounds.

Once prepared, candidate biotin derivatives can be screened for ability to bind avidin e.g. as described in the attached Example for in vivo binding to sites of infection as described in Example 4) or in vitro or in vivo binding to tumors. In addition, stability can be tested by administering the compound to a subject, obtaining blood samples at various time periods 30 min, 1 hour, 24 hours) and analyzing the blood samples for the biotin compound and/or metabolites.

25 Two 18 F biotin derivatives: an amide containing derivative (biotin 1) and an alkyl containing derivative (biotin 2) were prepared as described in Examples 1 and 2. Biotin was easily synthesized from the sulfonate ester in good yield with high specific activity.

Biotin 1 retains the basic structure of biotin. Biotin 2 was prepared to elucidate the role of the side chain on biotin binding to avidin and infection sites.

The two biotin derivatives were evaluated for binding to avidin in the presence of varying concentrations of unlabeled d-biotin as described in Example 3. Biotin 1 was found to bind to avidin and could be displaced by cold d-biotin. A scatchard transformation displacement curve yielded a Kd of 3.12 x 10-14 M with a Bmax of 5.57 x 10 -16 M. In contrast, compound 2 showed no specific binding to avidin.

35 Both compounds were then evaluated for localization at sites of infection in E.coli infected rats as described in Example 4. Half of the infected rats were treated with avidin 24 hours prior to i.v. injection of the 18 F-labeled biotin analogs. Biotin 1, without avidin pretreatment, showed a high selectivity (6.08 1.12) for infected compared to normal -7muscle at 60 minutes post injection. This ratio increased only slightly by administration of avidin 24 hr prior to injection of 1 8 F-labeled biotin 1 (6.39± 0.96).

In contrast, the biodistribution of biotin 2 indicated more binding to normal muscle with an infection/normal muscle ratio of 0.58 0.07. This lack of selectivity illustrates the importance of the side-chain amide group in infection localization. Some defluorination of both biotin 1 and biotin 2 occured in vivo as evidenced by increased 1 8

F

bone uptake after 60 min.: 2.94 0.37 and 1.17 0.21 respectively.

Use of Biotin Compounds as Therapeutics For use in therapy, an effective amount of an appropriate biotin compound can be administered to a subject by any mode, which allows the compound to be taken up by the appropriate target. Preferred routes of administration include oral and transdermal via a patch). Examples of other routes of administration include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.). The injection can be in a bolus or a continuous infusion. Depending on the route of administration, the biotin compound,therapeutic agent can be coated with or disposed in a selected material (e.g.

positively or negatively charged liposomes), to protect it from natural conditions which may detrimentally effect its ability to perform its intended function, increase its in vivo availability or increase its uptake by a specific organ.

A biotin compound therapeutic agent can be administered alone, or in conjunction with a pharmaceutically acceptable carrier. As used herein, the phrase "pharmaceutically acceptable carrier" is intended to include substances that can be coadministered with a biotin compound therapeutic agent and allows the compound to 25 perform its intended function Examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of such media for pharmaceutically active substances are well known in the art. Any other conventional carrier suitable for use with the biotin compounds also falls within the scope of the present ;invention.

The language "effective amount" of a biotin compound therapeutic agent refers to that amount necessary or sufficient to eliminate, reduce or maintain (e.g prevent the spread of) an infection, tumor or other target. The effective amount can vary depending on such factors as the disease or condition being treated, the particular biotin compound being administered, the size of the subject, or the severity of the disease or condition. One of 35 ordinary skill in the art can empirically determine the effective amount of a particular compound without necessitating undue experimentation.

A preferred dose for treating or preventing a tumor or site of infection is in the range of 5 jg- 100mg.. However, the exact dose depends to a great extent on the toxicity of •the therapeutic agent being administered. For example, a subject can not withstand more than -8a milligram dose of bleomycin. In addition, certain chemotherapeutic peptides cause hemophilia and other blood disorders when given to a subject in microgram amounts.

However, the selective targeting of a therapeutic agent by the instant biotin compounds decreases their otherwise toxic effects on normal body cells.

Use of Biotin Compounds as Imaging Agents Biotin compounds can be labeled with a variety of imaging agents, which are known in the art and which will depend to some extent on the means used to detect or monitor the compound in vivo or in vitro Preferred imaging agents for performing positron emission tomography (PET), and single photon emission computer tomography

(SPECT)

include F-18, Tc-99m, and 1-123. Preferred imaging agents for magnetic resonance imaging (MRI) include an appropriate atom with unpaired spin electrons or a free radical. An imaging agent can be complexed with a biotin compound by a variety of techniques that are wellknown in the art. In a preferred embodiment, the imaging agent is attached via the amine to a HYNIC, DTPA or other chelating agent.

Biotin compounds that have been labeled with an appropriate imaging agent can be added to a particular tumor cell line or bacterial, viral or fungal infected tissue culture to test the binding affinity of a particular candidate biotin compound therapeutic.

Labeled biotin compounds can also be injected into an appropriate subject monkey, dog, pig, cow) and its binding with tumors or sites of infection in vivo as described in the following Example 4).

The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references 25 (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

.e 'Example 1: Synthesis of Two F 18 -Labeled Biotin Analogs Analysis for carbon and hydrogen was performed by Galbraith Laboratories (Knoxville, TN). Melting points were measured with a Fisher-Johns apparatus and are uncorrected. Proton NMR spectra (d 6 -DMSO) were acquired with a Bruker AM 500 instrument; the chemical shifts are reported in parts per million (delta) down field from 35 tetramethylsilane. All reagents were purchased from Aldrich Chemical Co., (Milwaukee, WI) and were used without further purification. Acetonitrile ws purchased from Pierce (Rockford, IL) and used as shipped.

S

Thin layer chromatography was performed on silica gel Gf 250 plates (Analtech, Inc., Newark, DE). Chromatograms of the radiolabeled compounds were counted -9with a Bioscan System 200 (Washington, High pressure liquid chromatography was performed on a Resolvex C18 column (4.6 mm x 25 cm, Fisher Scientific Co.) at a flow rate of 1 ml/min using water/acetonitrile (60:40) solvent system. Separation was monitored with a U.V. detector (X=185 nm) and a Nal (TI) radioactivity detector.

Synthesis of 3 aS-(3ac, 4p, 6 aa)]-hexahydro-2-oxo-lH-thieno [3, 4d]imidazole- 4-(N-3-(1-fluoropropyl))pentanamide (Biotin 1) (+)Biotin (Ig, 4.09 mmol) was added to 20 ml of a stirred 50/50 mixture of thionyl chloride and benzene and the mixture was refluxed under N2 for 2 h. Excess thionyl chloride was removed by distillation and the residue was cooled to room temperature under

N

2 dissolved in dry THF (10 ml) and added to a stirred solution of 3 -amino--propanol (I g) in THF (20 ml) at 25 0 C. The solvent was removed by roto-evaporation, and excess amino propanol was removed at 120 0 C under vacuum (1 torr). The crude material was purified by chromatography on a silica gel column (30 g) eluted with CH2CI2/MeOH/HCO 2

H

(20:80:0.5). Crystallization from MeOH gave 0.80g of product. This product showed a single spot (Rf=0.45) on TLC with CH2CI 2 /MeOH/HCO 2 H (20:80:0.5); mp=130-132 0

C.

1 H NMR (d 6 -DMSO) d2.05 (t,J=6.2 Hz, 2H, CH 2 3.2-3.5 6H, N-CH 2 6.35 1H, O=CNH-ring), 6.40 1H, O=CNH-ring), 7.75 J=5.6 Hz, 1H, O=CNH). Anal. Calcd. for

C

13

H

2 3

N

3 0 3 S: C, 51.80, H, 7.70. Found: C, 51.69; H, 7.51.

The product (100 mg, 0.33 mmol) was dissolved in 3 ml of dry pyridine by heating and stirring under N 2 The solution was cooled to room temperature, and 0.7 mL of a 0.87 M solution of methansulfonyl chloride in CH 2 C1 2 was added. After 30 min., the mixture was chromatographed on silica gel using acetone/acetonitrile (90:10). The product :25 (86 mg, 69%) showed a single spot on TLC (Rf=0.49) with MeOH/CH2Cl2/HCO 2

H

(10:90:0.5) that was visulaized with MoO 3

*H

3 P0 4 A 10-ml reactivial containing KF (48 mg), Kryptofix (300 mg), biotin mesylate (200 mg), and 7 ml of acetonitrile was heated at 110 0 C for 30 min. The solvent was roto-evaporated and the residue was chromatographed on silica gel (25 g) using CH2C1 2 /MeOH/HCO 2 H (85:15:0.5). This product, [3aS-(3aa., 4p, 6aa)]-hexahydro-2-oxo- H-thieno [3,4d]imidazole- 4 3 -(l-fluoropropyl))pentanamide (Biotin showed a single spot (Rf=0.38) by TLC with the same solvent. 1 H NMR (d 6 -DMSO) d3.2-3.5 6H.

N-CH

2 4.4 (dt, J=52, 6.5 Hz, 2H, CH 2 6.35 1H, O=CNH-ring), 6.40 1H, O=CNH-ring), 7.75 J=5.6 Hz, 1H, O=CNH). Anal. Calcd. for C 13

H

2 2N 3 02SF: C, 54.90, H, 7.80. Found: C, 52.19; H, 8.13.

Synthesis of [3aS-(3aa, 4p, 6aa)]-tetrahydro-4-(5-(1-[ 18 F]fluoro-pentyl)- 1H-thieno[3,4-dJimidazol-2(3H)-one (Biotin 2).

To a stirred suspension of lithium aluminum hydride (LAH) (1.0g, 25.8 mmol) in anhydrous ether (125 ml), a hot pyridine (25 ml) solution of(+)biotin (l.Og, 4.01 mmol) was added dropwise. After addition, the reaction mixture was stirred at room temperature for min. and then heated to reflux for an additional 30 min. The excess LAH was then destroyed by careful dropwise addition of water. Water and organic solvents were removed by roto-evaporation. The residue was acidified (pH 2) with 6 N HCI and the water was removed by roto-evaporation. The residue was extracted with chloroform (5 x 20 mL) and the choroform layer evaporated in vacuo to give a white solid. Recrystallization from methanol gave 0.44 g of the reduced product; mp 164-167 0 C (reference 11, mp 164- 169 0 1 H NMR (d 6 -DMSO)d 4.35 J=6 Hz, 2H, O-CH 2 6.35 1H, O=CNH-ring), 6.40 1H, O=CNH-ring).

To a solution of(+)Biotinol (100 mg, 0.43 mmol) in pyridine (3 mi) was added 0.6 mL of a 0.87 M solution of thionyl chloride in CH 2 CI2. The mixture was stirred for 30 min. followed by chromatography on silica gel using acetone/acetonitrile 90:10. The product (96 mg, 75%) showed a single spot on TLC (Rf=0.42) with EtOAc/CH-C 2 /acetone/HCO 2 H (10:80:10:0.5) that was visualized with MoO 3

*H

3

PO

4 Biotin 2 was prepared from biotinol mesylate in the same manner as described for [3aS-(3aa, 4p, 6ac)]-hexahydro-2-oxo-lH-thieno [3,4d]imidazole fluoropropyl))pentanamide above. [3aS-(3aa, 4p, 6aa)]-tetrahydro-4-(5-(1-[1 8 F]fluoropentyl)- I H-thieno[3,4-d]imidazol-2(3H)-one was purified on silica gel using EtOAc/CH 2 Cl 2 /acetone/HCO 2 H (10:80:10:0.5) (Rf=0.48); mp 110-112 0 C. I H NMR (d 6 DMSO) d 4.6 (dt, J=55, 6.5 Hz, F-CH 2 6.35 1H, O=CNH-ring), 6.40 IH, O=CNHring). Anal. Calcd. for C 10

H

16

N

2 0SF: C, 56.31, H, 7.57. Found: C, 56.59; H, 7.79.

Preparation of 18 F-Labeled Biotin 1 and Biotin 2 18 F-Labeled Biotin 1. [1 8 F] Fluoride was produced with a cyclotron by the S.

I

18 0(p,n) 18 F nuclear reaction on 18 O-enriched water in a silver plated target at 17 MeV, (Scanditronix, Sweden, MC17F) (Kilbourn MR, Jerabek PA, Welch MJ. An improved [1 8 0]water target for [1 8 F]fluoride production. Int JAppl Radiar Isor 36:327-328 35 (1985).

A 5-ml reactivial containing 18 F in H 2 1 80 (50 mCi, 1 mL), Kryptofix (3 mg), and K 2 C0 3 (1 mg) ws evaporated to dryness at 100 0 C under N 2 The contents were dried by addition of acetonitrile (4 x 2 mL) with heating at I 10 OC under a stream of Biotin mesylate (2 mg) in acetonitrile (1 mL) was added and the vial was sealed and heated at I 10 0

C

-1 for 10 min. Solvent was removed and the labelled product purified by chromatography on a short column of silica gel (10 g) using MeOH/CH 2 C12/HCO 2 H (20:80:0.5). The time required for synthesis and purification was 90 min. (from the end of bombardrfent and the radiochemical yield was 3.4 to 5.9 mCi (12-21%, EOS). HPLC analysis of the final product showed a single radioactive peak with a retention time (Rt) 2.8 min. corresponding to that of the 19 F]fluoro-biotin The chemical purity of [3aS-(3aa, 4p, 6aa)]-hexahydro-2-oxo-

H-

thieno 4-d]imidazole-4-(nnN-3-(1l-[l 8 F fluoropropyl))pentanamide was 18 F-Labeled Biotin 2. 1 8 F-labeled Biotin 2 was prepared in the same manner as 18 F-labeled Biotin 1. 18F-labeled Biotin 2 was purified on silica gel (10 g) using EtOAc/CH2Cl 2 /acetone/HCO 2 H (10:80:10:0.5). The radiochemical yield was 5.6 mCi EOS) and radiochemical purity was determined to be greater than 98%. The chemical purity of 3aS-(3ac, 43, 6aa)]-tetrahydro-4-(5-(1-[1 8 F] fluoropentyl)-IH-thieno-[3, 4d]imidazol-2(3H)-one was >98% by HPLC.

Example 2: Competitive Binding ofF-18 Labeled Biotin 1 and Biotin 2 to Avidin vs. d-Biotin A preliminary measure of the binding characteristics of the fluoro analogs of biotin were performed. The binding of these novel F-18 labeled biotin analogs to avidin was evaluated by measuring the binding of F-18 labeled biotin analogs to avidin in the presence of varying concentrations of unlabeled d-biotin. Serial dilutions of d-biotin were prepared in phopshate buffered saline pH 7.4 (PBS). A fixed quantity of the no-carrier added F-18 labeled biotin 1 or 2 (10-100pCi) were mixed with each d-biotin dilution so that the range of concentrations of cold biotin were 3 orders of magnitude greater and less than the 25 concentration of avidin (10- 16 A 200 pl aliquot of the F-18 biotin/cold biotin solution was added to 200 pl of avidin in PBS. The solution was vortexed and allowed to incubate for 1 hour after which time a 5 pl aliquot of the solution was analysed by instant thin layer chromatography (Gelman ILTC-SG) using 0.1 M acetate buffer pH 6.0. In this chromatographic system the F-18 biotin analogs bound to avidin origin while the unbound F- 18-biotin analogs move to the solvent front. Percent binding of F-18 biotin analog was calculated as [(counts at origin/total counts)* 100]. For each F-18-biotin analog potential i. mpurities remaining at the origin were determined and the results of binding corrected accordingly.

e, 12- Example 3: Biodistribution Studies in Rats A clinical isolate of E. Coli was stored at -70 0 C in a freezing media containing glycerol and 80% dextrose phosphate until use. Aliquots of bacteria were defrosted and colony counts performed on serial dilutions grown overnight on BBL Brucella agar plates.

Based on the colony count, freshly thawed bacteria suspensions were washed and diluted with saline to a final concentration of 8 x 1010 organisms/ml. Aliquots (0.1 ml) of bacterial suspensions (8 x 109 organisms/ml) were injected into the right thigh muscle of 48 male Sprague-Dawley rats (125-150 g, Charles River Breeding Laboratories, Burlington, MD).

Twenty-four rats were used for each labeled biotin compound. Twenty-four hours after injection, swelling in the right thigh readily appeared, and 24 rats each received 1.67 mg of avidin in saline via the tail vein Forty-eight hours after bacterial inoculations, 80 to 150 mCi of 18 F-biotin 1 or 2 was administered. Rats were sacrificed at 5 and 60 min. postinjection, six rats per time point. Blood samples were obtained by cardiac puncture.

Syringes were weighed before and after injection to determine the volume delivered. The activity per unit volume was obtained from standards. A total of eleven different tissues were excised, weighed, and counted. These tissues included blood; bone; lung; liver; adrenal; spleen; kidneys; heart; muscle (thigh); teste; and infected muscle (thigh). The excised tissues were blotted, weighed, and counted in a scintillation well counter. The raw counts were decay corrected. The results were expressed as percent injected dose per gram, and infectionto-normal muscle ratios (mean The biodistribution results were evaluated by analysis of variance (one-way ANOVA). The effect of avidin pretretment on biodistribution was evaluated by student t-test.

Table 1 shows the biodistribution of 18 F-labeled biotin 1 at 5 and 60 min.

25 after injection in rats with and without avidin pretreatment (24 hr). Analysis of variance demonstrated a significant main effects of organs (P<0.001) at 5 and 60 min. on biotin I accumulation. At 5 min. accumulation of 1 in kidney was greater than in all other organs (4.5-fold higher than blood) (P<0.001). Accumulation of 1 in liver and spleen were greater than normal muscle Sufficient clearance of radioactivity had occurred within min. in all other organs relative to infection except for bone (P<0.001). At 60 min., the selectivity for infected compared to normal muscle was 6.08± 1.12 (Table accumulation in infected muscle was greater than kidney blood spleen liver (P<0.001). and normal muscle (P<0.001). The distribution of biotin 1 in all organs did not change significantly with avidin-pretreatment at 5 min. At 60 min. with pretreatment, 35 accumulation in bone (P<0.01) was less than without avidin-pretreatment. Compared to untreated rats, accumulation of 1 in avidin-treated rats was greater in kidney blood and normal muscle (P<0.002).

i' Table 1. Tissue distribution of 1 8 F-Iabeled biotin I in E. co/i infected rats Without Avidin 5 Mi. 60 Min 5 Mi.

Tissue With Avidin 60 Min.

Blood Heart Lung Liver Spleen Kidney Adrenal Teste Norr mus lnf mus Bone 1.07±0.31 1.-00-±0.3 4 1.2 1±0.13' 1.39±0.16 1.40±-0.30 4.78±0.80 1.00±0.20 0.57±0.02 0.83±0.09 1.22--0.36 1.10±0.16 0.14±0.02 0.14±0.02 0-11±0.02 0.13±0.03 0.14±0.03 0.23±0.05 0.09±0.02 0.16±0.03 0.78±0.02 0.43±0.10 2.94±0.37 1.13±0.11 0.99±0.12 1 .07±0.21 1.29--0.17 1.52±0.32 4.0 1±0.77 0.85±0.13 0.50±0.11 0.82±0. 12 0.99±0.11 1.19±0.23 0.19±0.04 0.09±0.01 0. 13±0.02 0. 16±0.02 0. 19±0.0 0.32±0.04 0.11±0.02 0.19±0.04 0. 10±0.02 0.65±0.14 2.29±0.14 Table 1. Tissue distribution of 1 8 F-labeled biotin 1 in E. coli infected rats Without Avidin 5 Min. 60 Min. 5 Min.

With Avidin 60 Min.

Tissue Blood Heart Lung Liver Spleen Kidney Adrenal Teste Norm mus Inf mus, .Bone 1.07±0.31 1.00±0.34 1.21±0.13 1.39±0. 16 1.40±0.30 4.78±0.80 1.00±0.20 0.5 7±0. 02 0.83±0.09 1.22±0.3)6 1. 10±0. 16 0-14±0.02 0.14±0.02 0.11-±0.02 0.13±0.03 0. 14±0.03 0.23±0.05 0.09±0.02 0.16±0.03' 0.078±.02 0.43 10 2.94±0.3 7 1. 13±0. 11 0-99±0. 12 1.07±0.21 1 .29±0.17 1.52±0.3)2 4.0 1±0.77 0.85±0.13) 0.50±0.11 0.82±0. 12 0.99±0.11 1.19±0.23 0. 19±0.04 0.09±0.01 0. 13±0.02 0. 16±0.02 0. 19±0.0 0.32±0.04 0.11±0.02 0. 19±0.04 0. 10±0.02 0.65±0. 14 2.29±0. 14 (P<0.00 kidney (P<0.0 and normal muscle (P<0.05) relative to untreated rats. Avidin pretreatment did not change the infected/normal tissue ratio.

Table 3. Tissue distribution of 18 F-labeled biotin 2 in E. coli infected rats.

Without Avidin With Avidin Tissue 5 Min. 60 Min. 5 Min. 60 Min.

Blood Heart Lung Liver Spleen Kidney Adrenal ***.Teste Norm mus Inf mus bone 0.88±0.07 0.80±0.06 1.38±0.21 1.63±0.27 1.70±0.68 1.62±0.18 1.56±0.14 0.59±0.08 0.62±0.09 0.49±0.05 0. 73 ±0.09 0.30±0.02 0.25±0.03 0.58±0.10 0.42±0.03 0.26±0.01 0.57±0.04 0.92±0.21 0.20±0.03 0.3 5±0.09 0.20±0.02 1.17±0.21 0.85±0.04 0.96±0.10 1.5 9±0.16 1.91±0.22 0.99±0.10 2.70-±0.48 2.00±0.24 0.74±0.11 0.69±0.10 0.44±0.05 0.86±0.12 0.25±0.03 0.20±0.03 0.55±0.12 0.55±0.13 0.51±0.09 1.39±-0.21 0.72±0.12 0.15±0.02 0.28±0.04 0.18±0.02 1. 10±-0.20 -16- Table 4. Infected to normal tissue ratios of 1 8 F-labeled biotin 2 in E. coli infected rats.

Without Avidin With Avidin Tissue 5 Min. 60 Min. 5 Min. 60 Min.

Blood 0.56±0.04 0.68±0.06 0.51±0.07 0.71±0.05 Heart 0.61±0.03 0.81-±0.05 0.46±0.03 0.91±0.05 Lung 0-36±0.05 0.43±0.20 0.28±0.02 0.'33±0.06 Liver 0.31±0.04 0.48±0.33 0.23±0.02 0.33'±0.05 Spleen 0.70±0.04 0.78±0.08 0.44±0.05 0.35±0.04 Kidney 0.30±0.02 0.35±0.03 0.16±0.02 0.13±0.02 Adrenal 0.31±0.02 0.23±0.05 0.22±0.03 0.214±0.03 :Teste 0.84±0.10 1.04±0.11 0.59±0.03 1-15±0.06 V..Normn mus 0.80±0.12 0.58±0.07 0.65-±0.10 0.64±0.07 *bone 0.67±0.05 0.17±0.03 0.51±0.03 0. 17±0.03 17- Table 5. Comnparison of Infected thigh to normal tissue ratios.

Tissue lllln-IgG* 1111n-Streptavidin* Streptavidin/ I1I1 In-Biotin"' 18F-biotinl normal muscle 4.5 4.4 13.0 6.1 Blood 0.3 0.8 6.2 3.1 Liver 1.1 1.2 9.3 3. Kidney 0.8 0.3 0.8 *As reported in Rusckowski M, Fritz B, Hnatowich DJ. Localization of infection using streptavidin and biotin: an alternative to nonspecific polyclonal immunoglobulin. J. Nuci.

Med 33: 1810-1815 (1992).

18- Table 6. Comparison of E. coli infected thigh to normal thigh ratios for various radiopharinaceuticals.

Infected/normal Agents muscle ratio Time Reference 67 Ga-citrate 2.6±0.6 4 99 MTc-HSA 4.1±0.6 4 *1 12 5 1MTNT-l 4 99 MTc-TNT-~ 1 3.4±0.08 4 *1 9 9 MTc..JgG 3.0±1.1 4 *1 I I I1n-IgG 4.5 6 *2 1 11 In-Streptavidin 4.4 6 *2 Streptavidin 1 1 1 n-Biotin 13 3 *2 In-Biotin minimal a*2 1 8 F-Biotin 1 6.1±1.1 All values are reported for E coli infection in right thigh. TNT =antinucleus antibody I Hnatowich DJ et al., (1992) J1 Nuci. A'ed 33: 934- 935; *2 Rusckowski, M et al., (1992)].

Nuci. Med 33:1810-1815.

19- Example 4: Biotin Compounds for Treating Malaria and Tuberculosis Antisense oligonucleotides to genes involved in the growth aspartate semialdehyde dehydrogenase (asu) or aspartakinase (ask) or cell wall or mvcolic acid synthesis of Mycobacterium tuberculosis and Plasmodiumfalciparum can be conjugated to a biotin compound as described above and administered to a subject to prevent or treat tuberculosis or malaria.

Euivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The entire disclosure in the complete specification of our Australian Patent Application No.26991/95 (700864) is by this cross-reference incorporated into the present specification.

Claims (30)

1. A method for directly targeting a therapeutic or imaging agent to a site of infection comprising the steps of: a) conjugating the agent, that is not an antibody, to biotin via a non-alkyl linkage, thereby forming a biotin compound that has a high specificity for the site of infection; and b) without pre- or post- administering avidin or streptavidin, administering an effective amount of the biotin compound to the subject, so that the biotin compound becomes localised at the site of infection.

2. A method according to claim 1, wherein step b), the biotin compound becomes localised at the site of infection within 1 hour after being administered.

3. A method according to claim 1 or claim 2, wherein step the biotin compound becomes localised at the site of infection at a target to non-target ratio of at least 5:1.

4. A method according to any one of claims 1-3, wherein the agent is a therapeutic agent A method according to claim 4, wherein the therapeutic agent is selected from the group consisting of a drug, toxin, radionuclide, hormone antagonist, heavy metal complex, oligonucleotide, chemotherapeutic nucleotide, peptide, non-specific (non-antibody) protein, boron containing compound and an enediyne-

6. A method according to claim 5, wherein the radionuclide is selected from the group consisting of: I- 131, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-67 and Cu-64. 12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11 2001 15:18 FAX 61 3 92438333 GRIFFITH RACK L9j012 -21

7. A method according to claims 1 to 6, wherein the site of infection is selected from the group consisting of: a bacterial, viral, fungal. or protozoal infection.

8. A method according to any one of claims 1 to 3, wherein the agent is an imaging agent.

9. A method according to claim 8, wherein the imaging agent is selected from the group of consisting of: In-ill, Tc-99m, 1-123, 1-125, F-18, Ga-67, Ga-68, a paramagnetic atom and a contrast agent. A method for directly targeting a therapeutic or imaging agent to a tumor comprising the steps of: conjugating the agent, that is not an antibody, to biotin via a non-alkyl linkage, thereby forming a biotin compound that has a high specificity for the tumor; and b) without pre- or post administering avidin or streptavidin, administering an effective amount of the biotin compound to the subject, so that the biotin 4 compound becomes localised at the tumor.

11. A method according to claim 10, wherein step b), the biotin compound becomes localised at the site of infection within 1 hour after being administered-

12. A method according to claim 10 or 11, wherein step the biotin compound becomes localised at the site of infection at a target to non-target ratio of at least 5:1.

13. A method according to any one of claims 10 to 12, wherein the agent is a therapeutic agent.

14. A method according to claim 13, wherein the RA hm$NiMCKQA~~\34.9.dc1/1D 12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11, 2001 15:18 FAX 61 3 92438333 GRIFFITH HACK ~013 22 therapeutic agent is selected from the group consisting of a drug, toxin, radionuclide, hormone antagonist, heavy metal complex, oligonucleotide, chemotherapeutic nucleotide, peptide, non-specific (non-antibody) protein, boron containing compound and an enediyne. A method according to claim 14, wherein the radionuclide is selected from the group consisting of: I- 131, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89, Ilo-166, Sm-153, Cu-67 and Cu-64.

16. A method according to any one of claims 10 to wherein the tumor is selected from the group consisting of a minor of the brain, lung, ovary, prostate, breast and colon.

17. A method according to any one of claims 10 to 12, wherein the agent is an imaging agent. 20 18. A method according to claim 17, wherein the imaging agent is selected from the group consisting of: In-ill, Tc-99m, 1-123, 1-125, F-18, Ga-67, Ga-68, a paramagnetic atom and a contrast agent.

19. A method according to any one of claims 1 to 18, wherein the biotin compound is a biotin amide analog. A method according to any one of claims 1 to 9, wherein step the agent is conjugated to biotin using a crosslinking agent selected from the group consisting of lysine or 6-hydrazinonicotimide (HYNIC).

21. A method according to any one of claims 10 to 19, wherein step the agent is conjugated to biotin using a crosslinking agent selected from the group consisting of lysine or 6-hydrazinonicotimide (HYNIC). 12/\\elb_fileB\hceS\1intae\le/p\c i\2372 ?.da 1/11/01 12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11,2001 15:18 FAX 61 3 92438333 GRIFFITH HACK Z014 23

22. A method according to any one of claims 1 to 21, wherein step the biotin compound is administered orally or transdermally.

23. A biotin amide analog compound having the structural formula: R 4 R, N NR S wherein, n =5-10; R 1 -R 5 H, acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkynyl groups; Rs H, acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkynyl groups; or a conjugated therapeutic agent or a diagnostic agent; and X C=O, S=O or C=NH. analog compound having the

24. A biotin amide structural formula: X 'N FP \\m*~bfiles\hom6\eincncr\K**p\cpeci\?97 1P/11101 12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11 2001 15:19 FAX 61 3 92438333 GRIFFITH HACK U01o5 24 wherein, ii 2-10; R 1 -R.S H, acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkynyl groups; R6=H, acyl, alkyl, alkylene, alkenylene, alkynylene, alkeny. or alkynyl groups; or a conjugated therapeutic agent or a diagnostic agent; and X S=0 or C=NH, analog compound having the S *5 S S *5 S S S A biotin amide structural formula: wherein, n= 2-10; R 1 acyl, alkyl, alkylene, alkenylene, alkynylene, alkeriyl or alkyny. groups; R-S= H, acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or aikynyl groups; R 6 H, acyl, alkyl., alkylene, alkenylene, alky-nylene, alkenyl or alkynyl groups; or a conjugated therapeutic agent or a diagnostic agent; and X S=0 or C=NA. -o 12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11 2001 15:19 FAX 61 3 92438333GRFIHAC j01 GRIFFITH RACK IM 016 25

26. A biotin amide analog compound having the structural formula; 5 p wherein, N H 1 R 0 n =2-10; R 1 R 3 -R 5 H, aCYl, alkyl, alkylene, alkenyleie, alky-nylene, alkenyl or alkynyl groups; R= acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkynyl groups; R= H, acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkynyl groups; or a conjugated therapeutic agent or a diagnostic agent; and X C=0, S=O or C=NH. analog compound having the

27. A biotin amide structural formula: 12/11/01 12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11,2001 15:19 FAX 61 3 92438333GRFIH AC GRIFFITH HACK [aj 017 26 wherein, n 2-10;- R 1 R 2 ,R 4 Rs H, acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkynyl groups; R 3 acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkyriyl groups; R 6 acyl, alkyl, alkylenie, alkenylele. alkynylene, alkenyl or alkyriyl groups; or a conjugated therapeutic agent or a diagnostic agent; and X C=O, S0-- or C=NH.- analog compound having the 9 *9 .*9 S 9*9.S9 S .9.9 9 S *9 .9

28. A biotin amnide structural formula: 20 S (CH 2 wherein, n 2-10; Ri.-R 3 R 5 H, acyl, a2lkyl, alkylente, alkenylene, alkynylene, alkenyl or a.lkynyl groups; R= acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkyny1. groups; R 6 acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkynyl. groups; or a conjugated therapeutic agent or K 4' 12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11 2001 15:19 FAX 61 3 92438333 GRIFFITH HACK o 018 27 a diagnostic agent; and X C=O, S=0 or C=NH.

29. A biotin amide analog compound having the structural formula: 0e 0** *0 0 0 0 0 0* 4 *0 N H R wherein, 20

30. A biotin amide structural formula: n 2-10; R1-R4 H, acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkynyl groups; R 5 acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkynyl groups; R6 H, acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkynyl groups; or a conjugated therapeutic agent or a diagnostic agent; and X C=O, S=O or C=NH. analog compound having the \\mel~filc~ho~r~cinae\RC2/fl/O1~l 12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11 2001 15:19 FAX 61 3 92438333 GRIFFITH RACK [aj 019 28 .00. *.0 00 a. wherein, ni 2-10; R 1 -RS= H, acyl, alkyl, alkylene, alkenylene, alkynylene, alkenyl or alkynyl groups; R= H, acyl, alky2lene, alkenylene, alkynylene, alkenyl or alky-nyl groups; or a conjugated therapeutic agent or a diagnostic agent; and X C=O, S=O or C=NW.

31. A compound according to any one of claims 23 to wherein R 6 is a therapeutic agent and a conjugating moiety.

32. A compound according to any one of claims 23 to wherein R 6 is a diagnostic agent and a conjugating moiety.

33. The compound according to claim 31 or claim 32, wherein the conjugating moiety is selected from the group consisting of protein A, carboiimide, dirnaleimide, dithio- bis-nitrobenzoic acid (DTNB), N-succinimdiyl-S-acetyl- thioacetate (SATA), and N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), 6-hydrazinonicotimide (HYNIC), N3S and N2S2. \.10lb-V I log \Zkonw-s C 1-2- \X.=\aVmvi\23742,99-11-0 12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11 2001 15:20 FAX 61 3 92438333GIFIH AC GRIFFITH HACK IZj020 29

34- A composition comprising a compound of any one of claims 23 to 33, and a pharmaceutically acceptable carrier. A composition according to claim 34, wherein the therapeutic agent is selected from the groujp consisting of drugs, toxins, radionuclides, hormone agonsits, heavy metal complexes, oligonucleotides, chemotherapeutic nucleotides, peptides, non-specific (non-antibody) proteins, boron containing compounds and enediynes. *636. A method for treating or preventing the establishment or growth of a tumor or site of infection in *15 a subject, comprising administering to the subject an effective amount of a composition of claim 34 or claim

37. A method according to claim 36, wherein the composition is administered intraperitoneally, *ego 20 intravenously, orally or transderznally. 6 It 06

638. A method according to claim 36 or 37, wherein the 6@infection is a act.erial infection. 39. A method according to claim 38, wherein the bacterial infection is an E. coli infection- A composition according to any one of claims 34 and 37 to 39, wherein the agent is selected from the group consisting of Tn-ill, Tc-99m, 1-123, 1-125, F-18, Ga-67, Ga-68, paramagnetic atoms and contrast agents. 41. A method foT imaging a tumor or site of infection in a subject comprising administering to the subject a composition according to claim 40 and detecting or monitoring the composition using an appropriate means. 12/11 '01 MON 15:14 [TX/RX NO 6292] 12/11 2001 15:20 FAX 61 3 92438333 GRIFFITH HACK I021 30 42. A method according to claim 1, 10 or 36, substantially a~s herein described with reference to the Examples. 43. A compound according to any one of claims 23 to substantially as herein described with reference to the Examples.- 44. A composition according to claim 34, substantially as herein described with reference to the Examples. Dated this 12th dlay of November 2001 DAVID R ELMALEH By their Patent Attorneys GRIFFITH RACK Fellows Institute of Patent and Trade Mark Attorneys of Australia RAS *446 121 '0 O351 T/XN 22