WO2012175702A1

WO2012175702A1 - Rimfampicin analogue useful for infection imaging

Info

Publication number: WO2012175702A1
Application number: PCT/EP2012/062130
Authority: WO
Inventors: Vijaya Raj Kuniyil Kulangara; Ravi Hegde; Peter Brian Iveson
Original assignee: Ge Healthcare Limited
Priority date: 2011-06-24
Filing date: 2012-06-22
Publication date: 2012-12-27
Also published as: GB201110767D0

Abstract

The present invention provides radiolabelled RIF derivatives that are suitable for in vivo imaging of bacterial infection. The in vivo imaging agent of the invention is preferably provided as a radiopharmaceutical composition, which forms a further aspect of the invention. Also provided by the present invention is a precursor compound useful in the preparation of the in vivo imaging agent of the invention, a method for the preparation of the in vivo imaging agent of the invention comprising reaction of the precursor compound with a suitable source of the desired radiolabel, and methods for the use of the in vivo imaging compound.

Description

RIMFAMPICIN ANALOGUE USEFUL FOR INFECTION IMAGING

Technical Field of the Invention

The present invention relates to in vivo imaging and m particular to in vivo imaging of infection. The invention provides radiolabeled compounds that are useful for in vivo imaging of infection, along with methods and intermediates useful for the preparation of these compounds. The invention also provides methods for using the compounds of the invention.

Description of Related Art

Pulmonary tuberculosis (TB) is an airborne infection caused by Mycobacterium tuberculosis (MTB) that causes high mortality and morbidity, particularly in developing countries (Dye et al JAMA 1999; 282(7): 677-686). A recent factsheet produced by the World Health Organisation reported that the number of new cases of TB continues to increase each year in South-East Asia, the Eastern Mediterranean and Africa

(http ://www. who . int/mediacentre/factsheets/fs 104/en/ rint.html) . Accurate and prompt diagnosis is crucial in the control of TB and also in ensuring the most appropriate therapy for infected patients. Definitive diagnosis of TB requires culture of MTB from a sample taken from a patient. Patients with clear signs and symptoms of pulmonary disease with a sputum smear-positive result present no problems to diagnose. However, there can be difficulty culturing the slow-growing MTB organism in the laboratory. Furthermore the emergence of HIV has resulted in a decreased likelihood of sputum smear positivity and an increase in non-respiratory disease, such that ease of diagnosis is more difficult in these cases (see reviews by Jeong & Lee Am J Roent 2008; 191: 834-844; Davies & Pai Int J Tuberc Lung Dis 2008; 12(11): 1226-1234; and, Lange & Mori Respirology 2010; 15: 220-240). Rifampicin (RTF; also known as rifampin) is a bactericidal antibiotic drug of the rifamycin group and is the most popular broad- spectrum antibacterial agent in this class. Various alterations of the core structure have been reviewed (De Souza et al 2006 Anti- Infective Drug Discov; 1 : 33-44) but the RIF form, with a 4-methyl-l-piperazinaminyl group, is by far the most clinically effective. RTF is used, typically in combination with other antibiotics, in the treatment of a variety of infections, including: tuberculosis, leprosy, infection caused by methicillin-resistant Staphylococcus aureus (MRSA), Listeria, Neisseria gonorrhoeae, Haemophilus influenzae and Legionella pneumophila (Drapeau et al 2010 Int J Antimicrob Agents; 35 : 39-44). RIF inhibits bacterial ribonucleic acid (RNA) synthesis by binding to the β-subunit of the deoxyribonucleic acid-dependent (DNA-dependent) RNA polymerase. The RIF-RNA polymerase complex is extremely stable and as such an in vivo imaging based on RIF and retaining this property would have value in the diagnosis of a range of infections.

Liu et al (2010 J Med Chem; 53: 2882-2891) report labelling of RIF with "C:

[¹¹C]RIF

This "C-labelled RIF was administered intravenously to healthy baboons to investigate the biodistribution of RTF The ¹ 'C-labelled RIF had moderate distribution in the heart, lung, and kidneys and concentrated in the liver and gallbladder. In most organs, the concentration of injected RTF exceeded that in plasma over the study period except for the cortex of the kidney at 60 min. However, the short half-life of "C and high plasma protein binding are known drawbacks associated with ⁿC-labelled RTF.

R F directly labelled with ^99mTc was described by Shah et al (2010 Appl Radiat Isotop; 68: 2255) and reported to be useful as a radiotracer for MSRA infection imaging. A disadvantage of direct labelling is that ^99mTc can bind to donor groups in rifampcin which are necessary for the pharmacological activity. Moreover, Shah et al have studied this tracer only for MSRA infection imaging but do not report whether it is useful for imaging tuberculosis. There is therefore a need for improved tools and strategies for imaging infection.

Summary of the Invention

The present invention provides radio labelled RTF derivatives that are suitable for in vivo imaging of bacterial infection. The in vivo imaging agents of the present invention are indirectly labelled rather than directly labelled such that the radiolabel is less likely to bind to donor atoms in RIF, and potentially to the active site of RTF. Furthermore, data obtained by the present inventors demonstrates that the pharmacological activity of RTF is maintained even when a relatively bulky metal chelate complex comprising a radiometal suitable for in vivo imaging is attached to RIF. The in vivo imaging agent of the invention is preferably provided as a radiopharmaceutical composition, which forms a further aspect of the invention. Also provided by the present invention is a precursor compound useful in the preparation of the in vivo imaging agent of the invention, a method for the preparation of the in vivo imaging agent of the invention comprising reaction of the precursor compound with a suitable source of the desired radiolabel, and methods for the use of the in vivo imaging compound.

Detailed Description of the Invention

In Vivo Imagine Agent

In one aspect, the present invention provides an in vivo imaging agent of Formula I:

wherein: L¹ is a bivalent linker moiety comprising between 1 -15 linker groups wherein each linker group is independently selected from -C(=0)-, -CRV, -CR -CR'-, -C≡C-, - CRSCO2-, -CO2CR2-, -NR'-, -NR'CO-, -CO R'-, -NR^* (C=0)NR'-, -NR^*(C=S)NR'- , -SO2NR'-, -NR'S0₂-, -CR'aOCRV, -CR^SCR , -CRWCRV, a C₄.₈ cycloalkylene group, a C_4-g cycloheteroalkylene group, a C_5-i₂ arylene group, a C₃,_!2 heteroarylene group, or an amino acid; and, wherein each R' group is independently H, Ci-ioalkyl, C5-12 aryl, C_6-i₂ alkylaryl, C_2-|₀ alkoxyalkyl, Ci-iohydroxyalkyl, C_2-i₀ carboxyalkyl, C).j₀ aminoalkyl or Ci-iohaloalkyl;

Ch¹ is a chelator capable of complexing a radioactive metal ion to form a radioactive metal complex; and,

M comprises a radioactive metal ion complexed by Ch¹

An "in vivo imaging agent" in the context of the present invention is a radiolabeled compound suitable for in vivo imaging. An in vivo imaging agent can be detected external to the human body following administration to a subject. The term "in vivo imaging" as used herein refers to those techniques that noninvasively produce images of all or part of the internal aspect of a subject.

A "bivalent linker moiety" is a bivalent chain of linker groups that links RTF to the radioactive metal complex -Ch^!-M. Specifically excluded is wherein 2 or more carbonyl groups are linked together, or wherein 2 or more heteroatoms are linked together. The skilled person would understand that these are either not chemically feasible, or are too reactive or unstable to be suitable for use in the present invention. It is envisaged that one role of the bivalent linker moiety is to distance the relatively bulky radioactive metal complex from the active site of RTF so that binding to the target bacteria is not impaired. This can be achieved by a combination of flexibility (e.g.

simple alkyl chains), so that the bulky group has the freedom to position itself away from the active site and/or rigidity such as a cycloalkyl or aryl spacer which orientates the metal complex away from the active site. The nature of the bivalent linker moiety can also be used to modify the biodistribution of the resulting radiometal complex of the conjugate. Thus, e.g. the introduction of ether groups in the linker will help to minimise plasma protein binding. A minimum linker group backbone chain of 2 atoms confers the advantage that the chelator is well-separated from the biological targeting moiety so that any interaction is minimised. Preferably, said bivalent linker moiety comprises a backbone chain of between 2 and 50 atoms in length, preferably between 2 and 10 atoms. Preferred linker groups are -C(=0 , -C¾-, -NH-, -NHC(=0)-, -C(=0)NH-, - CH2-O-CH2-, and amino acids. Most preferred linker groups are -C(=0)-, -CH?-, and - NH-.

The term "cycloalkylene" refers to the corresponding bivalent radical of a cycloalkyl. The term "cycloalkyl" refers to a non-aromatic, saturated or partially unsaturated, hydrocarbon ring, non- limiting examples of which are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups.

The term "cycloheteroalkylene" refers to a cycloalkylene as defined above wherein one or more of the carbon atoms of the ring are replaced with a heteroatom selected from N, S or O.

The term "alkyl", alone or in combination, means a straight-chain or branched-chain alkyl radical having the general formula C_nH2_n+i wherein n is suitably an integer of between 1 and 10, preferably between 1 and 6. Examples of such radicals include methyl, ethyl, and isopropyl.

Unless otherwise specified, the term "alkoxyalkyl" means an alkyl radical as defined above comprising an ether linkage, and the term "ether linkage" refers to the group -C- 0-C-. Examples of suitable alkyl ether radicals include methoxy, ethoxy, and propoxy.

The term "hydroxyalkyl" refers to an alkyl radical as defined above substituted with a hydroxyl group. The term "hydroxyl" refers to the group -OH.

The term "carboxyalkyl" refers to an alkyl radical as defined above substituted with a carboxy group. The term "carboxy" refers to the group -COOH.

The term "aminoalkyl" refers to an alkyl radical as defined above substituted with an amino group. The term "amino" refers to the group -NH₂.

The term "haloalkyl" refers to an alkyl radical as defined above substituted with a halo group. The term "halo" or "halogen" means a substituent selected from fluorine, chlorine, bromine or iodine. A "fluoroalkyl" is therefore an alkyl radical substituted with a fluorine.

The term "aryl" is refers to aromatic rings or ring systems having 5 to 12 carbon atoms, preferably 5 to 6 carbon atoms, in the ring system, e.g. phenyl or naphthyl. The term "arylene" refers to the corresponding bivalent radical.

The term "heteroarylene" refers to an arylene as defined above wherein one or more of the carbon atoms of the ring are replaced with a heteroatom selected from N, S or O.

The term "alkylaryl" refers to alkyl-substituted aryls and aryl-substituted alkyls, wherem aryl and alkyl are as defined above.

By the term "amino acid" is meant an L- or D-amino acid, ammo acid analogue or amino acid mimetic which may be optically pure, i.e. a single enantiomer and hence chiral, or a mixture of enantiomers.

A suitable "radioactive metal ion" can be either a positron emitter such as ⁶⁴Cu, ⁴⁸V, ⁵²Fe, ⁵⁵Co, ^94mTc or ⁶⁸Ga; or a γ-emitter such as ^{9 m}Tc, ^{1 1} 'in, ^113mIn, or ⁶⁷Ga. Preferred radiometals are ^99mTc, ⁶⁴Cu, ⁶⁸Ga and ^{1 1} 'in. Most preferred radiometals are ⁶⁸Ga and ^99mTc, and especially ^99mTc. The term "comprises a radioactive metal ion" is intended to encompass the situation where M is a radioactive metal ion coordinated to Ch¹, as well as the situation wherein M is a radioactive metal ion coordinated in part to Ch¹ and in part to another entity, e.g. particularly envisaged in this latter case is wherein the radioactive metal ion is ^mTc coordinated by means of ^99mTc(CO)₃ radiochemistry.

By the term "radioactive metal complex" is meant a coordination complex of the radioactive metal ion M with the chelator Ch' . It is strongly preferred that the radioactive metal complex is "resistant to transchelation", i.e. does not readily undergo ligand exchange with other potentially competing ligands for the metal coordination sites. Potentially competing ligands include excipients in any associated

radiopharmaceutical composition (e.g. radioprotectants or antimicrobial preservatives used in the preparation), or endogenous compounds in vivo (e.g. glutathione, transferrin or plasma proteins). Suitable chelators for use in the present invention which form radioactive metal complexes resistant to transchelation include chelators where 2-6, preferably 2-4, metal donor atoms are arranged such that 5- or 6-membered chelate rings result (by having a non-coordinating backbone of either carbon atoms or non- coordinating heteroatoms linking the metal donor atoms). Examples of donor atom types which bind well to radioactive metal ions as part of a chelator include: amines, thiols, amides, oximes and phosphines. Phosphines form such strong radioactive metal complexes. Examples of suitable phosphines include tetrofosmin, and monodentate phosphines such as im(3-methoxypropyl)phosphine. Examples of suitable diazenides include the HYNIC series of ligands i.e. hydrazine-substituted pyridines or

nicotinamides. the radioactive metal complex may also be formed by means of M(CO)3 radiochemistry (wherein M is the radioactive metal of Formula I), as described more fully by Schibli in Chapter 2.2 of "Technetium-99m Pharmaceuticals: Preparation and Quality Control in Nuclear Medicine" (2007 Springer; Zolle, Ed.). A benefit of this chemistry is that it can further reduce the likelihood of non-specific binding of the radioactive metal M to RTF. The radioactive metal complex using this chemistry would therefore take the form of Ch¹ being a tridentate chelate linked to M(CO)₃ (hence the designation that M

"comprises" the radioactive metal ion M).

Other examples of preferred chelators for use in the present invention are:

(i) diaminedioximes;

(ii) N₃S ligands having a thioltriamide donor set such as MAG₃

(mercaptoacetyltriglycine) and related ligands; or having a diamidepyridinethiol donor set such as Pica;

(iii) N₂S₂ ligands having a diaminedithiol donor set such as BAT or ECD (i.e.

ethylcysteinate dimer), or an amideaminedithiol donor set such as MAMA;

(iv) N₄ ligands which are open chain or macrocyclic ligands having a tetramine, amidetriamine or diamidediamine donor set, such as cyclam, monoxocyclam or dioxocyclam; and,

(v) N2O2 ligands having a diaminediphenol donor set. The above described chelators are described more fully by Junsson et al (Chem Rev 1999: 99: 2205-2218). 2S2 dithiosemicarbazone chelators are described by Arano et al (1991 Chem Pharm Bull; 39: 104-107). N₂S? phenylenediaminethioetherthiol chelators are described by McBride et al (1993 J Med.Chem; 36: 81-6). Diaminedioximes are described by Nanjappan et al (1994 Tetrahedron; 50: 8617-8632). N₃S chelators having a diamidepyridinethiol donor set such as Pica are described by Bryson et al (1990 Inorg Chem; 29: 2948-2951). N₂0₂ ligands having a diaminediphenol donor set are described by Pillai et al (1990 Appl Rad Isot; 41 : 557-561).

Preferred diaminedioximes are of Formula III:

wherein:

E' -E⁶ are each independently an R* group wherein each R* group is H or Cj.io alkyl, C . 10 alkylaryl, C2-10 alkoxyalkyl, Cuo hydroxyalkyl, CH O haloalkyl, Q.io carboxyalkyl or Ci-io aminoalkyl, or two or more R* groups together with the atoms to which they are attached form a carbocyclic, heterocyclic, saturated or unsaturated ring; and Q' is a bridging group of formula -(J - wherein e is 3, 4 or 5 and each J' is independently -0-, -NR*- or -C(R*)₂- provided that -(J')_e-contains a maximum of one J' group which is -O- or -NR*-.

For Formula ΓΠ, each of the terms alkyl, alkylaryl, alkoxyalkyl, hydroxyalkyl, haloalkyl,

1

Preferred Q' groups are selected from -(CH₂)(CHR*)(CH₂)~ (i.e. propyleneamine oxime or PnAO derivatives), -(CH₂)2(CITR*)(CH2)2- (i.e. pentyleneamine oxime or PentAO derivatives), and -(CH₂)₂NR*(CH₂)2-. E¹ to E⁶ are each preferably a Cu alkyl, and most preferably CH3.

The diaminedioxime is preferably conjugated to RIF via the bivalent linker group at one of E¹ to E⁶, or an R* group of the Q' moiety. Most preferably, RIF is conjugated via the bivalent linker group at an R* group of the Q' moiety. When conjugated at an R* group of the Q¹ moiety, the R* group is preferably at the bridgehead position. In that case, Q' is preferably -(CH₂)(CHR*)(CH₂)- , -(CH₂)₂(CHR*)(CH₂)₂- or -(CH₂)₂NR*(CH₂)₂-, most preferably -(CH₂)₂(CHR*)(CH₂)₂-. An especially preferred diaminedioxime has the Formula Ilia:

wherein:

E⁷-E²⁰ are each independently an R* group as defined above; G is N or CR*; and

Y' is the point of attachment to the bivalent linker moiety. A preferred chelator of Formula nia is of Formula Illb:

where G is as defined above, and is preferably CH. Said bivalent linker moiety is conjugated via said free N¾ group. This chelator is also referred to herein as cPn216, and a method for its preparation is disclosed in WO 03/006070.

Preferred tetraamine chelators are of Formula IV:

wherein:

Y" is the point of attachment to RIF via said bivalent linker moiety; and,

E²¹ to E²⁶ are as suitably and preferably defined above for the E groups of Formula III.

Λ most preferred tetraamine chelate is of Formula IVa:

wherein Y" is as defined for Formula IV. A method for the synthesis of a chelator of Formula IVa is disclosed in WO 06/008496.

A preferred tetradentate chelator having an N₂S₂ diaminedithiol or amideaminedithiol donor set is of Formula V:

wherein: each of E³¹-E³⁵ is as suitably and preferably defined above for the E groups of Formula ΙΠ; and Q" is a bridging group of formula -J"(CR*₂)r where f is 1 or 2, and J" is -CR*₂- or 0=0 and wherein R* is as suitably and preferably defined above in relation to Formula ΙΠ; and,

P¹ and P² are independently H or a thiol protecting group.

By the term "protecting group" is meant a group which inhibits or suppresses undesirable chemical reactions (e.g. oxidation of the free thiol to the corresponding disulphide), but which is designed to be sufficiently reactive that it may be cleaved from the thiol under mild enough conditions that do not modify the rest of the molecule during radiolabelling of the conjugate. Thiol protecting groups are well known to those skilled in the art and include but are not limited to: trityl, 4-methoxybenzyl, benzyl, tetrahydropyranyl, methyltetrahydrofuranyl (MTHF), acetamidomethyl and ethoxyethyl. The use of further thiol protecting groups is described in 'Protective Groups in Organic Synthesis', Theorodora W. Greene and Peter G. M. Wuts, (John Wiley & Sons, 1991). In Formula V, P¹ and P² are preferably both H.

Preferred Q^" groups are as follows: -CH₂CH₂- , -CH₂C¾CH₂- or -(C=0)CH₂-, most preferably N₂S₂ diaminedithiol chelators where Q" is -CH₂CH₂- or -CH₂CH₂CH₂-, with -CH₂CH₂- (i.e. BAT type chelators) being especially preferred.

E³¹ to E³⁵ are preferably chosen from: H, Ci^ alkyl, Ci_-3 alkoxyalkyl, C|„₃ hydroxyalkyl or C 1 -3 fluoroalkyl, wherein each of the terms alkyl, alkoxyalkyl, hydroxyalkyl or fluoroalkyl are as previously defined. Most preferably, each E^jl to E³⁴ group is H, and E ^S is C, -3 alkyl.

A most particularly preferred chelator of Formula V is the N2S2 diaminedithiol chelator of Formula V where: Q" is -CH₂CH₂- , E³¹ to E³⁵ are all H and P¹ = P² = H. The tetradentate chelating agents of Formula V are preferably attached to RIF via the bivalent linker moiety either at the bridging group Q" or the E⁵ group, most preferably the E⁵ group.

A preferred in vivo imaging agent of the present invention is of Formula la.

wherein:

L^la is a bivalent linker group as defined herein wherein said linker groups are selected

Ch^!a is a chelator of any one of Formulae ΓΠ-V as suitably and preferably defined above; and, M^a is a gamma-emitting isotope of technetium, preferably ^99mTc.

Non-limiting examples of preferred in vivo imaging agents of the present invention are:

Examples 1-3 describe the synthesis and testing of in vivo imaging agent 1 above, also referred to herein as ^99mTc-labelled Rifampicm-cPn216. hi the Examples it is shown that two active peaks are present in the formulated ^99mTc-labelled Rifampicin-cPn216. Given that the rifampicin analogue contains donor groups which could coordinate to the ^99mTc in addition and in preference to the cPn216 chelate it may be that this active peak represents directly-labelled RIF, such as disclosed by Shah et al (2010 Appl Radiat Isotop; 68: 2255). Given that this directly-labelled RTF has good properties for imaging, its presence in the formulation is not believed to be problematic. In Example 2 it was found that the MIC value for ^99mTc-labelled Rifampicin-cPn216 was increased against ATCC 29213 Staphylococcus aureus strain as compared wit RTF. However, from the perspective of in vivo imaging ^99mTc-labelled Rifampicm- cPn216 is still interesting since the inhibition is very good, as indicated by a MIC value less than 1.0. Precursor Compound

In another aspect, the present invention provides a precursor compound of Formula II

wherein:

L² is as defined above for L¹ of Formula I; and,

Ch² is as defined above for Ch¹ of Formula I. The embodiments set out above as preferred in connection with L¹ and Ch¹ apply equally to L² and Ch², respectively. Preferred precursors of the invention have the structures presented above for the preferred in vivo imaging agents of the invention, except that the radioactive metal ion is not complexed to the chelator.

Method to Prepare the In Vivo Imaging Agent In a further aspect, the present invention provides a method for the preparation of the in vivo imaging agent of the invention comprising:

(i) providing a precursor compound of the invention;

(ii) reacting said precursor compound with a suitable source of the radioactive metal ion as suitably and preferably defined above in connection with the in vivo imaging agent of the invention.

Depending on the nature of the radioactive metal ion for use in the method of the invention, it is obtained either using an appropriate cyclotron or generator. A general presentation about how a number of radioactive isotopes can be obtained can be found in Chapters 1 and 2 of "Handbook of Radiopharmaceuticals: Radiochemistry and Applications" (2003 Wiley, Welch and Redvaniey, Eds.). The precursor compound of the invention is preferably reacted with the suitable source of radioactive metal ion at the appropriate pH in the presence of the radioprotectant, in solution in a suitable solvent. The suitable source of radioactive metal ion is the radioactive metal ion in the appropriate oxidation state to permit complexation with the chelator. The radioprotectant may be supplied either together with the precursor compound or with the solution of the radioactive metal ion. Preferably, the radioprotectant is pre- mixed with the precursor compound, and this composition is subsequently reacted with the radioactive metal ion. The precursor compound solution may preferably contain a hgand which complexes weakly but rapidly to the radioactive metal ion, such as gluconate or citrate i.e. the radioactive metal complex is prepared by hgand exchange or transchelation. Such conditions are useful to suppress undesirable side reactions such as hydrolysis of the radioactive metal ion. General methods for the preparation of in vivo imaging agents comprising radioactive metal complexes of technetium, gallium, indium and copper are presented in Chapters 10-12 of "Handbook of Radiopharmaceuticals: Radiochemistry and Applications" (2003 Wiley, Welch and Redvanley, Eds.).

In a preferred embodiment, the reacting step of the method of the invention comprises reaction of said precursor compound with a source of technetium. When the radioactive metal ion is technetium, the usual source of technetium for radiolabelling is pertechnetate, i.e. Tc0₄ , which is technetium in the Tc(VII) oxidation state, and which is obtained from a "Mo generator. Pertechnetate itself does not readily form complexes, hence the preparation of technetium complexes usually requires the addition of a suitable reducing agent such as stannous ion to facilitate complexation by reducing the oxidation state of the technetium to the lower oxidation states, usually Tc(I) to Tc(V). The solvent may be organic or aqueous, or mixtures thereof. When the solvent comprises an organic solvent, the organic solvent is preferably a biocompatible solvent, such as ethanol or dimethylsulfoxide (DMSO). Preferably the solvent is aqueous, and is most preferably isotonic saline.

M

In a yet further aspect, the present invention provides a kit to facilitate the preparation of an in vivo imaging agent of the invention, said kit comprising a precursor compound of the invention, so that reaction with a sterile source of the radioactive metal ion gives the desired in vivo imaging agent with the minimum number of manipulations. Such considerations are particularly important where the radioactive metal ion has a relatively short half-life, and for ease of handling and hence reduced radiation dose for the radiopharmacist. The precursor compound is preferably present in the kit in lyophilized form, and the reaction medium for reconstitution of such kits is preferably a biocompatible carrier. Suitable and preferred embodiments of the precursor compound for the kit of the invention are as provided above for the precursor compound of the invention. A "biocompatible carrier" is a fluid, especially a liquid, in which the resultant in vivo imaging agent of the invention is suspended or dissolved, such that the composition is physiologically tolerable, i.e. can be administered to the mammalian body without toxicity or undue discomfort. The biocompatible carrier is suitably an injectable carrier liquid such as sterile, pyrogen- free water for injection; an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is either isotonic or not hypotonic); an aqueous solution of one or more tonicity-adjusting substances (e.g. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol), or other non-ionic polyol materials (e.g. polyethyleneglycols, propylene glycols and the like). The biocompatible carrier may also comprise biocompatible organic solvents such as ethanol. Such organic solvents are useful to solubilise more lipophilic compounds or formulations. Preferably the biocompatible carrier is pyrogen-free water for injection, isotonic saline or an aqueous ethanoi solution. The pH of the

biocompatible carrier for intravenous injection is suitably m the range 4.0 to 10.5. In the kit of the invention, the precursor compound is preferably presented in a sealed container which permits maintenance of sterile integrity and/or radioactive safety, plus optionally an inert headspace gas (e.g. nitrogen or argon), whilst pennitting addition and withdrawal of solutions by syringe. A preferred sealed container is a septum-sealed vial, wherein the gas-tight closure is crimped on with an overseal (typically of aluminium). Such sealed containers have the additional advantage that the closure can withstand vacuum if desired e.g. to change the headspace gas or degas solutions.

The precursor compound for use in the kit may be employed under aseptic manufacture conditions to give the desired sterile, non-pyrogenic material. The precursor compound may alternatively be employed under non-sterile conditions, followed by terminal sterilisation using e.g. gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide). Preferably, the precursor compound is provided in sterile, non-pyrogenic form. Most preferably the sterile, non-pyrogenic precursor compound is provided in the sealed container as described above.

Preferably, all components of the kit are disposable to minimise the possibilities of contamination between runs and to ensure sterility and quality assurance.

The preferred embodiments indicated for the precursor compound above apply equally to the kit of the invention.

Radiopharmaceutical Composition hi another aspect, the present invention provides a radiopharmaceutical composition compnsmg the in vivo imaging agent as suitably and preferably defined herein together with a biocompatible carrier in a form suitable for mammalian administration. The definitions and embodiments provided for the biocompatible carrier in relation to the kit of the invention apply equally to the radiopharmaceutical composition of the invention.

The radiopharmaceutical composition may be administered parenterally, i.e. by injection, and is most preferably an aqueous solution. Such a composition may optionally contain further ingredients such as buffers; pharmaceutically acceptable solubilisers (e.g. cyclodextrins or surfactants such as Pluronic, Tween or

phospholipids); pharmaceutically acceptable stabilisers or antioxidants (such as ascorbic acid, gentisic acid or />ara-aminobenzoic acid). Where the in vivo imaging agent of the invention is provided as a radiopharmaceutical composition, the method for preparation of said in vivo imaging agent may further comprise the steps required to obtain a radiopharmaceutical composition, e.g. removal of organic solvent, addition of a biocompatible buffer and any optional further ingredients. For parenteral administration, steps to ensure that the radiopharmaceutical composition is sterile and apyrogenic also need to be taken.

In Vivo Imaging and Diagnosis

In a further aspect, the present invention provides an in vivo imaging method comprising: (a) administration to a subject of the in vivo imaging agent as suitably and preferably defined herein;

(b) allowing said in vivo imaging agent to bind to infectious gram positive and gram negative bacteria present in said subject;

(c) detecting by an in vivo imaging procedure signals emitted by the radioisotope comprised in said in vivo imaging agent;

(d) generating an image representative of the location and/or amount of said signals; and,

(e) determining the distribution of mycobacteria in said subject wherein said distribution is directly correlated with said signals emitted by said in vivo imaging agent.

The step of "administering" the in vivo imaging agent is preferably carried out parenterally, and most preferably intravenously. The intravenous route represents the most efficient way to deliver the in vivo imaging agent throughout the body of the subject, and also does not represent a substantial physical intervention on the body of the subject. By the term "substantial" is meant an intervention which requires professional medical expertise to be carried out, or which entails a substantial health risk even when carried out with the required professional care and expertise. The in vivo imaging agent of the invention is preferably administered as the pharmaceutical composition of the invention, as defined herein. The in vivo imaging method of the invention can also be understood as comprising the above-defined steps (b)-(e) carried out on a subject to whom the in vivo imaging agent of the invention has been pre-administered. The in vivo imaging agent is preferably administered as the radiopharmaceutical composition of the invention. For this aspect of the invention it may equally be understood that first step (a) is the alternative step of providing said subject wherein said in vivo imaging agent has been previously administered to said subject, and then carrying out steps (b)-(e).

Following the administering step and preceding the detecting step, the in vivo imaging agent is allowed to bind to infectious gram positive or infectious gram negative bacteria within said subject. For example, when the subject is an intact mammal, the in vivo imaging agent will dynamically move through the mammal's body, coming into contact with various tissues therein. Once the in vivo imaging agent comes into contact with the target, the two entities bind such that clearance of the in vivo imaging agent from tissue in which the infectious bacteria are present takes longer than from tissue without any mycobacteria present. A certain point in time will be reached when detection of in vivo imaging agent specifically bound to infectious bacteria is enabled as a result of the ratio between in vivo imaging agent bound to tissue with the infectious bacteria versus that bound in tissue without any infectious bacteria. This is the optimal time for the detecting step to be carried out. Preferably, the infectious bacteria are mycobacteria, most preferably Mycobacterium tuberculosis.

The "detecting" step of the method of the invention involves detection of signals emitted by the radioisotope by means of a detector sensitive to said signals. This detection step can also be understood as the acquisition of signal data. Single-photon emission tomography (SPECT) and positron-emission tomography (PET) are preferred in vivo imaging procedures for use in the method of the invention, with SPECT being most preferred.

The "generating" step of the method of the invention is carried out by a computer which applies a reconstruction algorithm to the acquired signal data to yield a dataset. This dataset is then manipulated to generate images showing the location and/or amount of signals emitted by the radioisotope which is comprised in the in vivo imaging agent. The signals emitted directly correlate with the presence of the infectious bacteria such that the "determining" step can be made by evaluating the generated image.

The "subject" of the invention can be any human or animal subject. Preferably the subject of the invention is a mammal. Most preferably, said subject is an intact mammalian body in vivo. In an especially preferred embodiment, the subject of the invention is a human. The in vivo imaging method is preferably used in subjects known or suspected to have a pathological condition associated with a mycobacterial infection. Preferably, said method relates to the in vivo imaging of a subject known or suspected to have tuberculosis caused by Mycobacrerinm tuberculosis, and therefore has utility in a method for the diagnosis of said condition. Where a subject is known to have tuberculosis caused by Mycobacterium tuberculosis, the in vivo imaging method of the invention may be carried out repeatedly during the course of a treatment regimen for said subject, said regimen comprising administration of a drug to combat tuberculosis caused by Mycobacterium tuberculosis. The present invention additionally provides a method for diagnosis of abacterial infection in a subject wherein said method comprises the in vivo imaging method as defined herein, together with a further step (vi) of attributing the distribution of bacteria to a bacterial infection. The method of diagnosis is preferably used to diagnose a mycobacterial infection, and most preferably to diagnose tuberculosis caused by Mycobacterium tuberculosis.

Γη a yet further aspect, the present invention provides the radiopharmaceutical composition as suitably and preferably defined herein for use in a method of in vivo imaging wherein said method of in vivo imaging is as suitably and preferably defined herein.

The present invention also provides the radiopharmaceutical composition as suitably and preferably defined herein for use in a method of diagnosis wherein said method of diagnosis is as suitably and preferably defined herein.

Alternatively the present invention provides for use of the in vivo imaging agent of the invention in the manufacture of the radiopharmaceutical composition of the invention for use in either the method of in vivo imaging of the invention or the method of diagnosis of the invention.

The embodiments for the in vivo imaging agent of the invention and for the

radiopharmaceutical composition of the invention apply equally to the in vivo imaging and diagnostic methods of the invention. Brief Description of the Examples

Example 1 described the synthesis of Rifampicin-glu-cPn2I 6.

Example 2 describes the in vitro MIC test used to evaluate compounds of the invention. Example 3 descnbes radiolabeling of Rifampicin-cPn216. List of Abbreviations used in the Examples eq. equivalent(s)

DIPEA N,N-diisopropylethylamine

ESI electrospray ionization

EtOH ethanol HPLC high-performance liquid chromatography

LCMS liquid chromatography mass spectrometry

MIC minimum inhibitory concentration

NMR nuclear magnetic resonance

RCP radiochemical purity TFA trifluoroacetic acid

THF tetrahydrofura

UV ultraviolet

Examples

3-formy] rifampicin was obtained from Sigma Aldrich, and N-aminopiperazine was obtained from Apollo Scientific. All other reagents and solvents were obtained from Sigma Aldrich. Example 1: Synthesis of Rifampicin-glu-cPn216

1.1 Synthesis of Schiff's base of Rifampicin aldehyde with N- Amino piper azine

Rifampicin aldehyde

3-formyl rifampicin (50 mg, 0.072 mmol, 1 eq.), p-toluenesulfonic acid (1.8 mg, 9.5 μΐΏθΙ, 0.13 eq.), and 6 mL of dry THF were added with molecular sieves to N- aminopiperazine (29 mg, 0.288 mmol, 4 eq.). The reaction mixture was stirred under nitrogen and at room temperature overnight. The mixture was filtered through celite and the filtrate concentrated in vacuo. The final product after prep purification and freeze drying was a red solid with >97 % purity by HPLC. The product yield was 26 mg (47%).

LCMS (Μ+Η)⁺· m/z calcd, 809.9; found, 809.6.

Ή-NMR (500 MHz, CDCI₃) δ_Η: 13.15 (s, IH, NH (1)), 12.18 (s, IH, CH (29)), 8.44 (s, IH, CH_(4)), 6.58 (dd, J = 15.6, 1 1.4 Hz, IH, CH (16)), 6.39 (d, J = 1 1.4 Hz, IH, CH (5)), 6.18 (d, J = 12.6 Hz, IH, CH (6)), 5.94 (dd, 3=15.6 Hz, IH, CH (15)), 5.10 (dd, J=12.6, 6.6 Hz, IH, CH (14)), 4.93 (d, J=10.8 Hz, IH, CH (12)), 3.75 (d, J=9.0 Hz, IH, CH (10)), 3.49 (d, J=6.6 Hz, IH, CH (8)), 3.2-3.46 (m, 8H, CH₂ (30, 31, 33, 34)), 3.04 (s, 3H, CH₃ (40)), 2.97-3.06 (m, IH, CH (11)), 2.36-2.41 (m, IH, NH (32)), 2.24 (s, 3H, CH₃ (39)), 2.08 (s, 3H, CH₃ (43)), 2.07 (s, 3H, CH₃ (35)), 1.81 (s, 3H, CH₃ (41)), 1.66- 1.74 (m, 1 H, CH (13)), 1.47-1.58 (m, IH, CH (7)), 1.30-1.40 (m, IH, CH (9)), 1.01 (d, J=7.2Hz, 3H, CH₃, (36)), 0.88 (d, J=7.2Hz, 3H, CH₃ (44)), 0.60 (d, J=6.6 Hz, 3H, CH3 (42)), -0.30 (d, J = 6.6 Hz, 3H, CH₃ (37)). ^{l 3}C~NMR (500 MHz, CDC1₃) 5_C: 196.10 (18), 174.78 (38), 172.38 (2), 169.79 (20), 169.15 (22), 162.7 (27), 149.06 (29), 143.15 (16), 142.55 (4), 138.74 (3), 138.59 (24), 135.08 (5), 129.91 (6), 123.41 (25), 120.42 (15), 1 18.57 (28), 1 18.25 (23), 1 13.79 (19), 109.67 (17), 108.91 (21 ), 107.08 (14), 104.75 (26), 77.41 , 77.17 (CDC1₃), 76.91 (10), 74.41 (12), 70.91 (8), 57.32 (40), 47.79 (30, 34), 42.55 (31, 33), 39.48 (7), 38.52 (13), 37.55 (9), 33.45 (11), 21.44 (35), 20.90 (41), 20.83 (39), 17.61 (36), 1 1.01 (42), 9.07 (43), 8.62 (44), 7.74 (37).

The compound was analyzed by LCMS for the purity and mass identification. The chromatographic conditions was using Zorbax Eclipse CI 8, 4.6 x 50 mm, 1.8 μ column, Acetonitrile (B) and 0.01% TFA in water (A) as mobile phase, gradient 5% B for 3 min, 5-90% by 15 min with flow of 0.8mL/min. Rt~12.9 min. HPLC purity was >97 % and found (M+H)⁺, 809.6, calculated 809.9

The crude product was purified by prep HPLC with a reverse-phase Phenomenex Luna C 18 (2), 21.1 -x250 mm, 10 μ column and a gradient of acetonitrile (mobile phase B) and 0.05% TF A/water mixture (mobile phase A). Starting conditions of 20% B were maintained for the first 3 minutes and then increased to the percentage of B from 20% to 100% over the next 25 min (flow rate =12 iL/min). Maintained 100% B for the next 7 mm before bringing the percentage of B back to 20% in 1 minute and conditioning with 20% B for the next 4 minutes (flow rate = 16 mL/min). The product was observed as a broad peak at 18 min.

1.2 Synthesis of Rifampicin-glut-cPn216

A solution of the Schiff s base of Rifampicin aldehyde with N-Amino piperazine ( 10 mg, 0.12 mmol), DIPEA (5pL, 0.25 mmol) and cPn216-Glu-OTfp (11 mg, 0.018 mmol) in DMF (2 mL) was stirred at ambient temperature for 23 hours. As unreacted starting material was observed, an additional portion of the Otfp ester (11 mg, 0.018 mmol) was added and 3 hours after that LC/MS showed complete consumption of the starting material. The mixture was evaporated to dryness and the residue purified by preparative HPLC.

Preparative HPLC was carried out using Shimadzu, Phenomenex Luna CI 8 5u (2) 250 x 21.20 mm) 5 micron, gradient 30-95% B over 60 min, IR: 30 mm.

After freeze-drying, a red compound was obtained. Yield: 14 mg (93%).

LCMS was carried out using a Thermo Finnegan Surveyor MSQ plus instrument with ESI, positive mode, flow lmL/min, gradient: 30-95%B over 5 min, tR: 3.44 min, found (M+H)⁺: 1248.5, expected: 1248.7

Example 2: In Vitro MIC Test

The MIC testing was performed from 128.0 - 0.00^g/ml and 64.0 - 0.0005 pg/ml of the test compound and standard antimicrobial agent against ATCC 29213

Staphylococcus aureus using standard microbroth MIC testing method described in "Methods for Dilution Antimicrobial Susceptibility Test for Bacteria That Grow

Aerobically. 6th Edition. M07-A6. Vol.23, No.2" The MIC value of Rifampicin-cPn216 for ATCC 29213 S. aureus was observed as 0.062 μg/ml. Table 1 shows the MIC value of Rifarnpicin standard and Rifampicin-cPn216 conjugate.

Example 3: Radiolabeling of Rifampicin-cPn 216 mTc labelling of Rifampicin-cPn216 was carried out at room temperature using 100μg (80 nanomoles) to give a final concentration of 72μΜ for the precursor.

HPLC analysis after 20 minutes reaction time revealed one major active peak together with a much smaller active following peak (see Figure 1 : solid trace represents activity and dotted trace UV). RCP values for the unpurified ^{9 m}Tc-labelled Rifampicin-cPn216 preparations were 88-94%.

The major active ^99mTc-labelled Rifampicin-cPn216 peak was purified by HPLC from other active peaks including the small following peak but not from Rifampicm-cPn216. 9^9mTc-labelled Rifampicin-cPn216 was then formulated in 10% EtOH / 25mM phosphate buffer (pH 7).

HPLC analysis of the purified, formulated ^99mTc-labelled Rifampicin-cPn216 showed it to be stable 163 minutes post formulation (see Figure 2: solid trace represents activity and dotted trace UV).

Claims

An in vivo imaging agent of Formula 1:

wherein:

L¹ is a bivalent linker moiety comprising between 1-15 linker groups wherein each linker group is independently selected from -C(=0 , -CRV, -CR -CR'-, -C≡C-, - CR'₂C02-, -CO2CR2-, -NR-, -NR'CO-, -CONR'-, -NR'(C=0)NR'-, -NR'(C=S)NR\ -S0₂NR'-, -NR'S0₂-, -CR'₂OCR'₂-, -CR'₂SCR'₂-, -CR'₂NR'CR'₂-, a C₄.₈ cycloalkylene group, a C₄_8 cycloheteroalkylene group, a C5_]₂ arylene group, a C3-12 heteroarylene group, or an amino acid; and, wherein eachR' group is independently H, Ci-ioalkyl, C_5-i₂ aryl, C_6-i2 alkylaryl, C_2-]0 alkoxyalkyl, C_M0 hydroxyalkyl, C_2-io carboxyalkyl, Cj.io aminoalkyl, or CMO haloalkyl;

M comprises a radioactive metal ion complexed by Ch¹.

(2) The in vivo imaging agent as defined in Claim 1 wherein each linker group of L¹ is independently selected from -C(=0)-, -CH₂~, -NH-, -NHC(=OK -C(=0)NH-, -CH₂- O-CH2-, and an amino acid.

(3) The in vivo imaging agent as defined in either Claim 1 or Claim 2 wherein Ch¹ is selected from: a) diaminedioximes; b) N₃S ligands having a thioltnamide or a diamidepyridinethiol donor set; c) N₂S₂ ligands having a diaminedithiol or an amideaminedithiol donor set; d) N₄ ligands which are open chain or macrocyclic ligands having a tetramrae, amidetriamine or diamidediamine donor set; e) N₂O₂ ligands having a diaminediphenol donor set; or, f) is a tridentate chelate linked to M wherein M is M(CO)₃.

(4) The in vivo imagmg agent as defined in any one of Claims 1 -3 wherein said radioactive metal ion is selected from ⁶⁴Cu, ^4SV, ⁵²Fe, ⁵⁵Co, ^94mTc, ⁶⁸Ga, ^99mTc, iⁿIn, ^{n 3m}rn, and ⁶⁷Ga.

(5) The in vivo imaging agent as defined in Claim 4 wherein said radioactive metal ion

wherein:

L² is as defined in either Claim 1 or Claim 2; and,

Ch² is a chelator as defined in either Claim 1 or Claim 3. (7) A method for the preparation of the in vivo imaging agent as defined in any one of Claims 1 -5 wherein said method comprises:

(i) providing a precursor compound as defined in Claim 6;

(ii) reacting said precursor compound with a suitable source of the radioactive metal ion as defined in any one of Claims 1 , 4 and 5

(8) The method as defined in Claim 7 wherein said suitable source of radioactive metal ion is a source of technetium.

(9) A kit for carrying out the method as defined in either Claim 7 or Claim 8 wherein said kit comprises a vial containing the precursor compound as defined in Claim 6.

(10) A radiopharmaceutical composition comprising the in vivo imaging agent as defined in any one of Claims 1-5 together with a biocompatible carrier in a form suitable for mammalian administration.

(1 1) An in vivo imaging method comprising:

(a) administration to a subject of the in vivo imaging agent as defined in any one of

Claims 1-5;

(c) detecting by an in vivo imaging procedure signals emitted by said radioisotope suitable for in vivo imaging comprised in said in vivo imaging agent;

(e) determining the distribution of bacteria in said subject wherein said distribution is directly correlated with said signals.

(12) The in vivo imaging method as defined in Claim 11 wherein said administration step is carried out by intravenous injection. (13) The in vivo imaging method as defined in either Claim 1 1 or Claim 12 wherein said in vivo imaging agent is administered as the radiopharmaceutical composition as defined in Claim 10.

(1 ) The in vivo imaging method as defined in any one of Claims 1 1-13 wherein said bacteria are Mycobacterium tuberculosis.

( 15) The in vivo imaging method as defined in Claim 14 which is carried out repeatedly during the course of a treatment regimen for said subject, said regimen comprising administration of a drug to combat tuberculosis caused by Mycobacterium tuberculosis. (16) A method for diagnosis of a bacterial infection in a subject wherein said method comprises the in vivo imaging method as defined in any one of Claims 11-15, together with a further step (vi) of attributing the distribution of bacterium to a bacterial infection.

(17) The method of diagnosis as defined in Claim 16 wherein said bacterial infection is tuberculosis caused by Mycobacterium tuberculosis.

(18) The radiopharmaceutical composition as defined in Claim 10 for use in a method of in vivo imaging wherein said method of in vivo imaging is as defined in any one of Claims 11-15

(19) The radiopharmaceutical composition as defined in Claim 10 for use in a method of diagnosis wherein said method of diagnosis is as defined in either Claim 16 or Claim 17.