WO2008003954A1

WO2008003954A1 - Dye imaging agents

Info

Publication number: WO2008003954A1
Application number: PCT/GB2007/002484
Authority: WO
Inventors: Sally-Ann Ricketts; Alexander Jackson; Bente Arbo; Michelle Avory
Original assignee: Ge Healthcare Limited
Priority date: 2006-07-05
Filing date: 2007-07-04
Publication date: 2008-01-10
Also published as: GB0613287D0

Abstract

The present invention relates to diagnostic imaging agents for in vivo imaging. The imaging agents are radiopharmaceuticals and comprise a class of xanthene dye labelled with a radiohalogen isotope or with a non-metallic positron-emitting radioisotope suitable for diagnostic imaging in vivo.

Description

Dye Imaging Agents.

Field of the Invention. The present invention relates to diagnostic imaging agents for in vivo imaging. The imaging agents are radiopharmaceuticals and comprise a class of xanthene dye labelled with radiohalogens or with non-metallic positron-emitting radioisotopes suitable for diagnostic imaging in vivo.

Background to the Invention.

Programmed cell death by apoptosis is a complex process, involving a large number of cellular processes with numerous levels of control. It is initiated by one of two pathways. The first is through an extrinsic pathway initiated via cell surface death receptors and the second is through intrinsic initiators, such as DNA damage by UV radiation. Both of these pathways culminate in the co-ordinated death of cells which requires energy and, unlike cell death by necrosis, does not involve an inflammatory response. Cells committed to apoptosis present 'eat me' signals on their cell surface, which invite other cells to consume them by phagocytosis.

Apoptosis is a critical event in numerous processes within the body. For example, embryonic development is totally reliant on apoptosis, and tissues that turnover rapidly require tight regulation to avoid serious pathological consequences. Failure to regulate apoptosis can give rise to cancers (insufficient cell death) and neuropathologies such as Alzheimer's disease (too much cell death). Furthermore, apoptosis can also be indicative of damaged tissues such as areas within the heart following ischaemia/reperfusion insults.

Annexin-5 is an endogenous human protein (RMM 36 kDa) which binds to the phosphatidylserine (PS) on the outer membrane of apoptotic cells with an affinity of around 10^~9 M. ^99mTc-labelled Annexin-5 has been used to image apoptosis in vivo [Blankenberg et al, J.Nucl.Med., 40, 184-191 (1999)]. There are, however, several problems with this approach. First, Annexin-5 can also enter necrotic cells to bind PS exposed on the inner leaflet of the cell membrane, which could lead to false-positive results. Second is the high blood pool activity, which is maintained for at least two hours after injection of labelled Annexin-5. This means that the optimal timing of imaging is between 10 and 15 h after injection [Reutelingsperger et al, J.Immunol.Meth., 265 (1-2), 123-32 (2002)], making it unsuitable for clinical decision making in patients with acute coronary syndromes. Furthermore, the clearance of Annexin-5 occurs via the kidney and the liver, with a very strong background signal in the abdominal regions. This makes imaging of abdominal cell death (eg. in kidney transplants and tumour monitoring) impossible.

Radiopharmaceuticals for apoptosis imaging have been reviewed by Lahorte et al [Eur.J.Nucl.Med., 31, 887-919 (2004)].

Rose Bengal (RB) has been radioiodinated with ¹²³I or ¹³¹I and used as a radiopharmaceutical for the in vivo imaging of hepatic function [Serafini et al, J.Nucl.Med., 16(7) 629-632 (1975) and references therein]:

Rose Bengal

US 4,298,591 discloses a method and kit for the room temperature radioiodination of Rose Bengal.

WO 2006/001925 discloses halogenated xanthenes labelled with a positron emitting radioisotope for imaging human and animal tissue. WO 2006/001925 teaches that the halogenated xanthene is radiolabeled with "...a positron emitting isotope of carbon, oxygen, fluorine, chlorine, bromine, or iodine, including ¹⁰C, ¹¹C, ¹³0, ¹⁴0, ¹⁵0, ¹⁷F,

18τ F₇, 3 "2,Cl, 7 "5τB>_r, 7 '₆ ⁰ _rB_>_r, 7 "7_DB_r, 7 '8⁵ _DB_r, I 'l"7_τ 8_τ I, 1 ^l2^z0^υI_T, 1 "21¹ _TI, I ¹22_τ

I, I "1⁶I, H "9^y _T ^ZZI, 1¹2^Z4⁴ _TI, I ¹2^/6⁰ _τI, and ¹²⁸I". Labelling with radioisotopes of bromine or iodine is said to be achievable using standard methods, such as that given by Serafini (above). For the remaining isotopes, radiolabeled starting materials (eg. radiolabeled resorcinol) are said to be used. The halogenated xanthenes of WO 2006/001925 are shown in Table 1 (below):

Table 1 : halogenated xanthenes of WO 2006/001925

WO 2006/001925 states that the unlabelled halogenated xanthene dyes exhibit selective retention in certain types of tissue, especially cancerous or pre-cancerous conditions (ie. neoplasia, dysplasia and hyperplasia). No evidence is provided that any positron-labelled halogenated xanthene dyes have been prepared, or that they have useful diagnostic imaging properties. It is also known to radioiodinate fluorescein with ¹³¹I to give di(iodo-¹³¹I)fluorescein:

131 J 131 J

[JP 38014143 B4; Kaltygina et al Metody Poluch.Izm.Radioakt, p. 188-191 (1960); Chiota et al, Revist.Chim., 14(3), 173 (1963)].

Fluorescein has also been radiobrominated at multiple positions with either ⁷⁶Br or ⁷⁷Br [US 4256727 and Gobuty et al, J.Lab.Comp.Radiophar., JJ(I), P- 73-80 (1980)].

There is therefore still a need for an apoptosis imaging agent which permits rapid imaging (eg. within one hour of injection), and with good clearance from blood and background organs.

The Present Invention.

The present invention provides radiopharmaceutical imaging agents which comprise halogenated xanthene dyes labelled at a single, defined position with radioisotope imaging moieties suitable for in vivo imaging. The imaging agents are particularly useful for the diagnostic imaging of apoptosis. Also disclosed are non-radioactive precursors useful for the preparation of the imaging agents, plus viable methods of producing said imaging agents from the precursors. The present invention also provides pharmaceutical compositions containing the imaging agents, together with kits for the preparation of the compositions. Also described are methods of imaging apoptosis in vivo using the imaging agents or pharmaceutical compositions of the invention.

The Serafini route for the radioiodination of Rose Bengal uses the so-called "halogen exchange", or more specifically "isotopic exchange" route in which ¹²³I- or ¹³¹I- labelled iodide ion is exchanged with the non-radioactive iodine (¹²⁷I) substituents of the parent molecule. A problem with this route is that it typically provides low specific activity product, wherein the ¹²⁷I-labeled (non-radioactive) material is always present and can compete for sites in vivo with the ¹²³I- or ¹³¹I-labelled imaging agent. A problem with this, and prior art radiohalogenated fluorescein derivatives, is the lack of control over both where the radiohalogen atom is located in the labelled product as well as the number of radiohalogen atoms incorporated. The present invention provides precursors which enable both high specific activity preparations and site- specific radiolabelling, together with improved radiochemical purity (RCP). Other advantages include the facility to label with different radionuclides and to tune the chemistry of the radiosynthesis to minimise the number of radioactive processing steps.

The only synthesis disclosed by WO 2006/001925 for halogenated xanthene dyes labelled with positron-emitting radioisotopes other than radiobromine or radioiodine involves incorporation of the radiolabel from the outset, ie. using a multi-step preparation. The halogen exchange route is not viable for radiolabelling with radiofluorine, and ¹⁸F is the main isotope used for clinical PET imaging. In addition, for short-lived PET radioisotopes having half-lives of the order of minutes, a multi- step preparation is not viable since the time elapsed during the multi-step synthesis and purification would mean that most of the product would be lost due to radioactive decay. For PET synthesis, the ideal is to incorporate the radioisotope at the last step with the minimum of purification involved. This minimises the time during which radioactive decay occurs, and thus maximises the radioactive yield.

Detailed Description of the Invention.

In a first aspect, the present invention provides an imaging agent which comprises a xanthene dye of Formula (Ia) or (Ib) labelled with an imaging moiety:

(Ia) (Ib) where:

X¹ to X⁴ are X groups, where each X is independently chosen from Br, I or H, such that at least two of X¹ to X⁴ are either Br or I;

Y¹ to Y⁴ are Y groups, where each Y is independently chosen from H or Hal;

E¹ to E³ are E groups, where each E is independently chosen from H or M; Z¹ is -OH, -OM, -OR^a or -NR^a ₂;

Z^a is -O- or -NR^as

M is a biocompatible cation;

R^a is independently chosen from H, Ci_-1O alkyl, C_3-I0 cycloalkyl, CMO fluoroalkyl, an Ar¹ group or -(Ci_-3 alkyl) Ar¹ where Ar¹ is a C_3-I2 aryl or heteroaryl ring;

Im^G is an imaging moiety which is attached at a single position only of

Formula Ia or Ib, where said position is chosen from X¹ to X⁴, Y¹ to Y⁴, E¹ to

E³ or Z^!, and Im^G comprises a gamma-emitting radioactive halogen or a positron-emitting radioactive non-metal, wherein following administration of said labelled xanthene dye to the mammalian body in vivo, the imaging moiety can be detected externally in a non-invasive manner; with the proviso that when Y¹ to Y⁴ are all Cl, X¹ to X⁴ are all I or Im^G, E is H or M and Z¹ is -OH or -OM, then Im^G is not the gamma-emitting radioactive halogens ¹²³I Or ¹³¹I. The term "xanthene dye" has its conventional meaning in the art, although the compounds of the present invention are limited to the bromine and/or iodine- substituted dyes defined by X and Y in Formulae Ia and Ib. The proviso excludes ¹²³I and ¹³¹I-labelled Rose Bengal, which are prior art compounds.

Preferred imaging agents do not undergo facile metabolism in vivo, and hence most preferably exhibit a half- life in vivo of 60 to 240 mins in humans. The imaging agent is preferably excreted via the kidney (ie. exhibits urinary excretion).

The molecular weight of the imaging agent is suitably up to 5000 Daltons. Preferably, the molecular weight is in the range 150 to 3000 Daltons, most preferably 1000 to 2000.

Preferably, at least two of the X groups (X¹ to X⁴) are chosen to be iodine atoms (J). When two such iodine atoms are present, they are preferably located at the X¹ and X⁴ positions. Most preferably, all four of the X groups are Br or I. When all four of the X groups are Br or I, preferably X¹ to X⁴ are the same. When four such iodine atoms are present, one may optionally be the imaging moiety (Im^G), by being a radioactive isotope of iodine. Direct radioiodination of the ring is least favoured at the X¹ or X⁴ positions due to steric constraints, and is thus more facile at the X² and X³ positions.

Preferably at least two of the Y groups (Y¹ to Y⁴) are chosen to be H or Cl. Most preferably, all four of the Y groups are H or Cl.

When Z¹ is -OR^a or -NR^a ₂ and R^a is not H, the xanthene dye is effectively fixed in the open chain form (Formula Ia). When Z¹ is -OH or -OM, Formulae Ia and Ib represent equilibrium forms of the same molecule in aqueous solution, with the predominant form dependent on the conditions of pH, temperature etc. Acidic pH favours the cyclic form (Ib, lactone or lactam) and pH 7.4 or higher favours the ring opened form (Ia), as observed from the dye colour change. Z¹ is preferably -OH or -OM. Z^a is preferably -O-. The "biocompatible cation" (M) is a positively charged counterion which forms a salt with an ionised, negatively charged group, where said positively charged counterion is also non-toxic and hence suitable for administration to the mammalian body, especially the human body. Examples of suitable biocompatible cations include: the alkali metals sodium or potassium; the alkaline earth metals calcium and magnesium; and the ammonium ion. Preferred biocompatible cations (M) are sodium and potassium, most preferably sodium.

R^a is preferably fluoroalkyl or alkyl-(iodoaryl).

The term "labelled with" means that either a functional group of the xanthene dye comprises the imaging moiety (Im^G), or the imaging moiety is attached as part of an additional species or substituent. When a functional group comprises the imaging moiety, this means that Im^G forms an intrinsic part of the chemical structure, and is a radioactive isotope present at a level significantly above the natural abundance level of said isotope. Such elevated or enriched levels of isotope are suitably at least 5 times, preferably at least 10 times, most preferably at least 20 times; and ideally either at least 50 times the natural abundance level of the isotope in question, or present at a level where the level of enrichment of the isotope in question is 90 to 100%. Examples of such functional groups include CH₃ groups with elevated levels of the positron-emitting radioisotope ¹¹C, or an aryl iodide where the iodine atom is radioactive (eg. ¹²³I), such that the imaging moiety is the isotopically labelled atom within an existing chemical structure.

When the imaging moiety (Im^G) forms part of a substituent, this means that the basic chemical structure of the xanthene dye is modified to accommodate the Im^G in a suitably chemically stable environment. When Im^G is attached in this way, it replaces the X¹ to X⁴, Y¹ to Y⁴ or E¹ to E³ substituent of the xanthene dye completely. Examples of this are fluoroalkyl groups with elevated levels of ¹⁸F in place of a methyl group; or use of an aryl iodide or vinyl iodide substituent to introduce a radioiodine Im^G. Further examples of suitable substituents are discussed together with methods of preparation of the imaging agents of the invention. When the imaging moiety (Im c ) is a gamm ⁹a-emitting radioactive halogen, the radiohalogen is suitably chosen from ¹²³I, ¹³¹I or ⁷⁷Br. A preferred gamma-emitting radioactive halogen is ¹²³I. When the imaging moiety is a positron-emitting radioactive non-metal, the imaging agent would be suitable for Positron Emission Tomography (PET). Suitable such positron emitters include: ¹¹C, ¹³N, ¹⁷F, ¹⁸F, ⁷⁵Br, ⁷⁶Br or ¹²⁴I. Preferred positron-emitting radioactive non-metals are ^{1 1}C, ¹³N, ¹²⁴I and ¹⁸F, especially ¹¹C and ¹⁸F, most especially ¹⁸F.

Preferred imaging moieties (Im^G) of the invention are ¹²³I and ¹⁸F.

The imaging moiety is preferably a positron-emitting radioactive non-metal. The use of a PET imaging moiety has certain technical advantages, including:

(i) the development of PET/CT cameras allowing easy co-registration of functional (PET) and anatomical (CT) images for improved diagnostic information;

(ii) the facility to quantify PET images to allow accurate assessment for staging and therapy monitoring; (iii) increased sensitivity and resolution to allow visualisation of smaller target tissues.

The imaging agents of the present invention thus have a single imaging moiety which is attached at a defined position of the xanthene dye of Formulae Ia or Ib.

When Im^G is attached at X¹ to X⁴, Im^G is preferably a gamma-emitting radiohalogen chosen from ⁷⁷Br or ¹²³I, most preferably ¹²³I. When Im^G is attached at E¹ to E³, Im^G is preferably -^{1 1}CH₃, -CH₂ ¹⁸F, -CH₂CH₂ ¹⁸F or -CH₂CH₂CH₂ ¹⁸F.

The imaging moiety (Im^G) is preferably attached at the Z¹, Y¹ to Y⁴ or X positions, most preferably at the Z'or Y¹ to Y⁴ positions, with Z¹ being especially preferred. The imaging agent of the present invention preferably further comprises a linker group and is of Formula II: [ {xanthene HA)_n]-Im^G

(H) where: Im^G is as defined above;

{xanthene} is the xanthene dye of Formulae (Ia or Ib), wherein (A)_n is attached in place of one of the substituents at the X¹ to X⁴, Y¹ to Y⁴, E¹ to E³ or Z¹ positions;

-(A)_n- is a linker group wherein each A is independently -CR₂- , -CR=CR- , -C≡C- , -CR₂CO₂- , -CO₂CR₂- , -NRCO- , -CONR- , -NR(C=O)NR-,

-NR(C=S)NR-, -SO₂NR- , -NRSO₂- , -CR₂OCR₂- , -CR₂SCR₂- , -CR₂NRCR₂- , a C_4-8 cycloheteroalkylene group, a C_4-8 cycloalkylene group, a C_5-I2 arylene group, or a C_3-12 heteroarylene group, an amino acid, a sugar or a monodisperse polyethyleneglycol (PEG) building block; each R is independently chosen from H, Ci_-4 alkyl, C_2-4 alkenyl, C₂.₄ alkynyl,

Ci_-4 alkoxyalkyl or C_1-4 hydroxyalkyl; n is an integer of value 1 to 10.

By the term "amino acid" is meant an L- or D-amino acid, amino acid analogue (eg. napthylalanine) or amino acid mimetic which may be naturally occurring or of purely synthetic origin, and may be optically pure, i.e. a single enantiomer and hence chiral, or a mixture of enantiomers. Conventional 3 -letter or single letter abbreviations for amino acids are used herein. Preferably the amino acids of the present invention are optically pure.

The linker group is attached at a single defined position of the xanthene dye of

Formula Ia or Ib. This has the advantage that the resulting imaging agent product has the imaging moiety (Im^G) attached at a defined location.

It is envisaged that one of the roles of the linker group -(A)_n- of Formula II is to distance Im^G from the site of interaction of the xanthene dye. This may be important when the imaging moiety is relatively bulky (eg. a radioiodine atom). This can be achieved by a combination of flexibility (eg. simple alkyl chains), so that the bulky group has the freedom to position itself away from the interaction site and/or rigidity such as a cycloalkyl or aryl spacer which orientate the Im^G away from the active site. More importantly, the nature of the linker group can also be used to modify the biodistribution and clearance of the imaging agent. Thus, eg. the introduction of ether groups in the linker will help to minimise plasma protein binding. When -(A)_n- comprises a polyethyleneglycol (PEG) building block or a peptide chain of 1 to 10 amino acid residues, the linker group may function to modify the pharmacokinetics and blood clearance rates of the imaging agent in vivo. Such "biomodifier" linker groups may accelerate the clearance of the imaging agent from background tissue, such as muscle or liver, and/or from the blood, thus giving a better diagnostic image due to less background interference. A biomodifier linker group may also be used to favour a particular route of excretion, eg. via the kidneys as opposed to via the liver.

By the term "sugar" is meant a mono-, di- or tri- saccharide. Suitable sugars include: glucose, galactose, maltose, mannose, and lactose. Optionally, the sugar may be functionalised to permit facile coupling to amino acids. Thus, eg. a glucosamine derivative of an amino acid can be conjugated to other amino acids via peptide bonds. The glucosamine derivative of asparagine (commercially available from Novabiochem) is one example of this:

When -(A)_n- comprises a peptide chain of 1 to 10 amino acid residues, the amino acid residues are preferably chosen from glycine, lysine, arginine or serine. When -(A)_n- comprises a PEG moiety, it preferably comprises units derived from oligomerisation of the monodisperse PEG-like structures of Formulae A or B:

17-amino-5-oxo-6-aza-3, 9, 12, 15-tetraoxaheptadecanoic acid of Formula A

(A)

wherein p is an integer from 1 to 10 and where the C-terminal unit (*) is connected to the imaging moiety. Alternatively, a PEG-like structure based on a propionic acid derivative of Formula B can be used:

(B) where p is as defined for Formula ILA and q is an integer from 3 to 15. In Formula B, p is preferably 1 or 2, and q is preferably 3 to 12, most preferably 4 to 6.

When the linker group does not comprise PEG or a peptide chain, preferred -(A)_n- groups have a backbone chain of linked atoms which make up the -(A)_n- moiety of 2 to 10 atoms, most preferably 2 to 5 atoms. A minimum linker group backbone chain of 2 atoms confers the advantage that the imaging moiety is well-separated so that any undesirable interaction is minimised.

Non-peptide linker groups such as alkylene groups or arylene groups have the advantage that there are no significant hydrogen bonding interactions with the conjugated xanthene dye, so that the linker does not wrap round onto the dye. Preferred alkylene spacer groups are -(CH?^- where d is 2 to 5. Preferred arylene spacers are of formula:

where: a and b are independently 0, 1 or 2.

Linker groups of the present invention preferably have n = 1 to 6, most preferably n = 1 to 4. The linker group -(A)_n- preferably comprises a polyethyleneglycol based unit, a PEG-like unit of Formula A or B or a poly(amino acid). Most preferred linker groups comprise a peptide chain of 1 to 10 amino acid residues, where the amino acid residues are preferably chosen from glycine, lysine, arginine or serine. Preferred such amino acids are glycine, lysine and arginine.

Various xanthene dyes are commercially available: Rose Bengal methyl ester and 2,4,5,7-tetraiodo-6-hydroxy-3-fluorone from Spectra (USA); Rose Bengal G from CHEMSRV-AS USA); Rose Bengal lactone and acid, Erythrosin B, Eosin Y, Eosin ethyl ester and 4',5'-Diiodofluorescein from Aldrich; 2',4',5'-Triiodofluorescein from ICN.

Other xanthene dyes can be synthesised by the methods of Martinez-Utrilla et al

[Dyes Pigments, K), 57-61 (1988) and U, 249-272 (1990)], Yang et al [J.Org.Chem., 70, 6907-6912 (2005)]; Graebe [Ann. der Chemie, 238, 333 (1887)]; Cihelnik et al [Collect. Czech Chem. Commun., Vol. 67, 1779-1789 (2002)] and Orndorff et al [J. Am. Chem. Soc. 680-725 (1914)].

The imaging agents of the present invention are suitably prepared by reaction of the appropriate source of the radioisotope of the first embodiment with a precursor, as described in the second and third embodiments below.

In a second aspect, the present invention provides a method of preparation of the imaging agent of the first embodiment, which comprises reaction of:

(i) a non-radioactive precursor which comprises the xanthene dye of

Formula Ia or Ib of the first embodiment having attached thereto in place of one of the substituents at the X¹ to X⁴, Y¹ to Y⁴, E¹ to E³ or Z¹ positions a non-radioactive group (Q^a) which comprises a substituent capable of reaction with a source of the positron- emitting radioactive non-metal or gamma-emitting radioactive halogen of the first embodiment to give said imaging agent; with (ii) a source of the positron-emitting radioactive non-metal or gamma- emitting radioactive halogen of the first embodiment.

Preferred embodiments of the xanthene dye of the precursor are as described for the first aspect above. The "precursor" suitably comprises a non-radioactive derivative of the xanthene dye, having a "substituent capable of reaction" as part of the Q^a group. The reactive substituent is designed so that chemical reaction with a convenient chemical form of the desired radioisotope can be conducted in the minimum number of steps (ideally a single step), and without the need for significant purification (ideally no further purification) to give the desired radioactive product. Such precursors are synthetic and can conveniently be obtained in good chemical purity. The "precursor" may optionally comprise one or more protecting groups (P^GP) for certain functional groups of the xanthene dye. Suitable precursors are described by Bolton, J.Lab.Comp.Radiopharm., 45, 485-528 (2002).

By the term "protecting group" (P^GP) is meant a group which inhibits or suppresses undesirable chemical reactions, but which is designed to be sufficiently reactive that it may be cleaved from the functional group in question under mild enough conditions that do not modify the rest of the molecule. After deprotection the desired product is obtained. Protecting groups are well known to those skilled in the art and are suitably chosen from, for amine groups: Boc (where Boc is terϊ-butyloxycarbonyl), Fmoc (where Fmoc is fiuorenylmethoxycarbonyl), trifluoroacetyl, allyloxycarbonyl, Dde [i.e. l-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl] or Npys (i.e. 3-nitro-2-pyridine sulfenyl); and for carboxyl groups: methyl ester, tert-butyl ester or benzyl ester. For hydroxyl groups, suitable protecting groups are: methyl, ethyl or tert-buty\; alkoxymethyl or alkoxyethyl; benzyl; acetyl; benzoyl; trityl (Trt) or trialkylsilyl such as terf-butyldimethylsilyl. For thiol groups, suitable protecting groups are: trityl and 4-methoxybenzyl. The use of further protecting groups are described in 'Protective Groups in Organic Synthesis', Theorodora W. Greene and Peter G. M. VVuts, (Third Edition, John Wiley & Sons, 1999). The source of the positron-emitting radioactive non-metal or gamma-emitting radioactive halogen is preferably that which is most readily available and is preferably chosen from:

(i) halide ion;

(ii) F or I⁺;

(iii) an alkylating agent chosen from an alkyl or fluoroalkyl halide, tosylate, triflate or mesylate.

The precursor of the method is preferably of Formula Ilia or HIb:

X⁶ X⁷ X⁶ X⁷

where:

X to X are independently chosen from Br, I, H or Q^a, such that at least two of the X groups are either Br or I;

Y⁵ to Y⁸ are independently chosen from H, Hal or Q^a;

E¹ to E³ are independently chosen from H, M, P^GP or Q^a;

P^GP is a protecting group;

Z² is -OH, -OM, -OR^a, -NR^a ₂, P^GP or Q^a;

M, R^a and Z^a are as defined for the first embodiment;

Q^a is a non-radioactive group which comprises a substituent capable of reaction with a source of the positron-emitting radioactive non-metal or gamma-emitting radioactive halogen of the imaging moiety (Im^G) to give the labelled xanthene dye product; with the proviso that said precursor contains one Q^a group. Preferred precursors are those wherein me reactive substituent of Q^a comprises a derivative which either undergoes direct electrophilic or nucleophilic halogenation; undergoes facile alkylation with a labelled alkylating agent chosen from an alkyl or fluoroalkyl halide, tosylate, triflate (ie. trifiuoromethanesulphonate), mesylate, maleimide or a labelled N-haloacetyl moiety; alkylates thiol moieties to form thioether linkages; or undergoes condensation with a labelled active ester, aldehyde or ketone. Examples of the first category are:

(a) organometallic derivatives such as a trialkylstannane (eg. trimethylstannyl or tributylstannyl), or a trialkylsilane (eg. trimethylsilyl); (b) a non-radioactive alkyl iodide or alkyl bromide for halogen exchange and alkyl tosylate, mesylate or triflate for nucleophilic halogenation; (c) aromatic rings activated towards electrophilic halogenation (eg. phenols) and aromatic rings activated towards nucleophilic halogenation (eg. aryl iodonium, aryl diazonium, aryl trialkylammonium salts or nitroaryl derivatives).

Preferred derivatives which undergo facile alkylation are alcohols, phenols, amine or thiol groups, especially thiols and sterically-unhindered primary or secondary amines. Preferred derivatives which alkylate thiol-containing radioisotope reactants are maleimide derivatives or N-haloacetyl groups. Preferred examples of the latter are N- chloroacetyl and N-bromoacetyl derivatives.

Preferred derivatives which undergo condensation with a labelled active ester moiety are amines, especially sterically-unhindered primary or secondary amines.

Preferred derivatives which undergo condensation with a labelled aldehyde or ketone are aminoxy and hydrazide groups, especially aminoxy derivatives.

For F, the xanthene dye can be radiofluorinated by direct displacement of one of the Y⁵-Y⁸ groups of Formulae Ilia or HIb when Y⁵- Y⁸ is Hal, especially when Y⁵ = Y⁶ = Y⁷ = Y⁸ = Cl. When Y⁵-Y⁸ are all Cl, Y⁶ is expected to be preferentially displaced, since it will be activated by the presence of the para acid group and further by the presence of the other chlorine substituents. It is expected that the hydroxy groups on the xanthene moiety will need to be protected (ie. E⁴ = E³ = E⁶ = P^GP). For the specific case of ^{l 8}F-labelled Rose Bengal (RB), suitable reaction schemes and precursors are shown in Scheme 1 : Deprotect

[F-18]-6-fluoro-RB

Deprotect

[F-18]-6-fluoro-RB

Scheme 1 - Synthesis of [F-18]-6-fluoro-RB (lactone form top and acid form bottom)

It is preferred to introduce the fluoride into the acid form of RB (Formula Ilia) as opposed to the lactone form (Formula HIb). In Scheme 1, the radio fluorination reaction conditions would be ¹⁸F-fluoride at 210 ⁰C in the presence of tetraphenylphosphonium bromide (TPPB) and 18-crown-6 (each 0.1 equivalent) without solvent. Model systems for this approach are described by Yoshida et al [J.Fluor.Chem., 53(2), 301-305 (1991)], and suggest that a protecting group for the carboxylic acid (Z² = P^GP) is important in obtaining good radiochemical yields. It was shown that the presence of a bulky alkyl group ester e.g. neopentyl had the effect of suppressing the side reaction of attack of the fluoride on the carboxyl group which leads to undesired side products. Neopentyl esters were particularly effective. It was also shown that more heavily chlorinated starting materials are better substrates for fluoridation.

Nitro-substituted precursors, wherein one of Y¹ to Y³ preferably Y⁶ is NO₂ are preferred for the ¹⁸F labelling of RB, and can be prepared by the method of Yang et al [J.Org.Chem., 70, 6907-6912 (2005)]. FFoorr tthhee pprreeppaarraattiioonn ooff iimmaaggiinngg agents of Formula II of the first embodiment, preferred precursors are of Formula IV:

[{Compound III}-(A)_n]-Q^a

(IV) where {Compound III} is the compound of Formulae Ilia or HIb; Q^a is as defined for Formulae Ilia or HIb, and A and n are as defined for Formula II above.

The "precursor" may optionally be supplied covalently attached to a solid support matrix. In that way, the desired imaging agent product forms in solution, whereas starting materials and impurities remain bound to the solid phase. Precursors for solid phase electrophilic fluorination with ¹⁸F-fluoride are described in WO 03/002489. Precursors for solid phase nucleophilic fluorination with ¹⁸F-fluoride are described in WO 03/002157. The solid support-bound precursor may therefore be provided as a kit cartridge which can be plugged into a suitably adapted automated synthesizer. The cartridge may contain, apart from the solid support- bound precursor, a column to remove unwanted fluoride ion, and an appropriate vessel connected so as to allow the reaction mixture to be evaporated and allow the product to be formulated as required. The reagents and solvents and other consumables required for the synthesis may also be included together with a compact disc carrying the software which allows the synthesiser to be operated in a way so as to meet the customer requirements for radioactive concentration, volumes, time of delivery etc. Conveniently, all components of the kit are disposable to minimise the possibility of contamination between runs and will be sterile and quality assured.

When the imaging moiety comprises a radioactive halogen, such as iodine, Q^a suitably comprises the following reactive groups: a non-radioactive precursor halogen atom such as an aryl iodide or bromide (to permit radioiodine exchange); an activated precursor aryl ring (e.g. phenol or aniline groups); an imidazole ring; an indole ring; an organometallic precursor compound (eg. trialkyltin or trialkylsilyl); or an organic precursor such as triazenes or a good leaving group for nucleophilic substitution such as an iodonium salt. Methods of introducing radioactive halogens (including ¹²³I and ¹⁸F) are described by Bolton [J.Lab.Comp.Radiopharm., 45, 485-528 (2002)]. E Exxaammpplleess ooff ssuuiittaabbllee pprreeccuurrssoorr aarryyll ggrrooiups to which radioactive halogens, especially iodine can be attached are given below:

Both contain substituents which permit facile radioiodine substitution onto the aromatic ring. Alternative substituents containing radioactive iodine can be synthesised by direct iodination via radiohalogen exchange, e.g. -

When the imaging moiety comprises a radioactive isotope of iodine the radioiodine atom is preferably attached via a direct covalent bond to an aromatic ring such as a benzene ring, or a vinyl group since it is known that iodine atoms bound to saturated aliphatic systems are prone to in vivo metabolism and hence loss of the radioiodine. An iodine atom bound to an activated aryl ring like phenol has also, under certain circumstances, been observed to have limited in vivo stability.

When the imaging moiety comprises a radioactive halogen, such as ¹²³I and ¹⁸F, Q^a preferably comprises a functional group that will react selectively with a radiolabeled synthon and thus upon conjugation gives the imaging agent of Formula (Ia or Ib). By the term "radiolabelled synthon" is meant a small, synthetic organic molecule which is:

(i) already radiolabelled such that the radiolabel is bound to the synthon in a stable manner; (ii) comprises a functional group designed to react selectively and specifically with a corresponding functional group which is part of the desired compound to be radiolabelled. This approach gives better opportunities to generate imaging agents with improved in vivo stability of the radiolabel relative to direct radiolabelling approaches.

A synthon approach also allows greater flexibility in the conditions used for the introduction of the imaging moiety.

Examples of precursors suitable for the generation of imaging agents of the present invention are those where the reactive group of Q^a comprises an aminoxy group, a thiol group, an amine group, a maleimide group or an N-haloacetyl group. A preferred method for selective labelling is to employ aminoxy derivatives as precursors, as taught by Poethko et al [J.Nuc.Med., 45, 892-902 (2004)]. Such precursors are then condensed with a radiohalogenated-benzaldehyde synthon under acidic conditions (eg. pH 2 to 4), to give the desired radiohalogenated imaging agent via a stable oxime ether linkage. Q^a therefore preferably comprises an aminoxy group of formula -NH(C=O)CH₂-O-NH₂. Another preferred method of labelling is when Q^a comprises a thiol group which is alkylated with radiohalogenated maleimide- containing synthon under neutral conditions (pH 6.5-7.5) eg. as taught by Toyokuni et al [Bioconj. Chem. 14, 1253-1259 (2003)] to label thiols.

An additional preferred method of labelling is when Q^a comprises an amine group which is condensed with the synthon iV-succinimidyl 4-[¹²³I]iodobenzoate at pH 7.5- 8.5 to give amide bond linked products. The use of N-hydroxysuccinimide ester to label peptides is taught by Vaidyanathan et al [Nucl.Med.Biol, 19(3), 275-281 (1992)] and Johnstrom et al [Clin.Sci., iO3_(Suppl. 48), 45-85 (2002)].

When the imaging moiety comprises a radioactive isotope of fluorine the radiofiuorine atom may form part of a fluoroalkyl or fluoroalkoxy group, since alkyl fluorides are resistant to in vivo metabolism. Radiofluorination may be carried out via direct labelling using the reaction of ¹⁸F- fluoride with a suitable precursor having a good leaving group, such as an alkyl bromide, alkyl mesylate or alkyl tosylate.

Alternatively, the radiofluorine atom may be attached via a direct covalent bond to an aromatic ring such as a benzene ring. For such aryl systems, the precursor suitably comprises an activated nitroaryl ring, an aryl diazonium salt, or an aryl trialkylammonium salt. The direct radiofluorination of biomolecules is, however, often detrimental to sensitive functional groups since these nucleophilic reactions are carried out with anhydrous [¹⁸F] fluoride ion in polar aprotic solvents under strong basic conditions. Some precursors of Formula (Ilia or IHb) may exhibit instability under basic conditions. Therefore direct radiofluorination of precursors of the imaging agent of the present invention is not a preferred labelling method. Examples of preferred methods for radiofluorination involve the use of radiolabeled synthons that are conjugated selectively to precursors of the imaging agent of Formula (Ilia or HIb), as discussed above for the labelling of radiohalogens in general.

¹⁸F can also be introduced by N-alkylation of amine precursors with alkylating agents such as ¹⁸F(CH₂)₃OMs (where Ms is mesylate) to give N-(CH₂)₃ ¹⁸F, O-alkylation of hydroxyl groups with ¹⁸F(CH₂)₃OMs, ¹⁸F(CH₂)₃OTs or ¹⁸F(CH₂)₃Br or S-alkylation of thiol groups with ¹⁸F(CH₂)₃OMs or ¹⁸F(CH₂)₃Br. ¹⁸F can also be introduced by alkylation of N-haloacetyl groups with a F(CH₂)₃OH reactant, to give -NH(CO)CH₂O(CH₂)₃ ¹⁸F derivatives or with a ¹⁸F(CH₂)₃SH reactant, to give -NH(CO)CH₂S(CH₂);!¹⁸F derivatives. ¹⁸F can also be introduced by reaction of maleimide-containing precursors with F(CH₂)₃SH. For aryl systems, F-fluoride nucleophilic displacement from an aryl diazonium salt, an aryl nitro compound or an aryl quaternary ammonium salt are suitable routes to aryl-¹⁸F labelled synthons useful for conjugation to precursors of the imaging agent.

Precursors of Formula (Ilia or HIb) wherein Q^a comprises a primary amine group can also be labelled with ¹⁸F by reductive amination using ¹⁸F-C₆H₄-CHO as taught by Kahn et al [J.Lab.Comp.Radiopharm. 45, 1045-1053 (2002)] and Borch et al [J. Am. Chem. Soc. 93, 2897 (1971)]. This approach can also usefully be applied to aryl primary amines, such as compounds comprising phenyl-NH₂ or phenyl-CH₂NH₂ groups.

An especially preferred method for F-labelling of precursors of Formula (Ilia or HIb) is when Q^a comprises an aminoxy group of formula -NH(C=O)CH₂-O-NH₂ which is condensed with ¹⁸F-C₆H₄-CHO under acidic conditions (eg. pH 2 to 4). This method is particularly useful for precursors which are base-sensitive. Further details of synthetic routes to 18 F-la ²b²elled derivatives are described by Bolton, J.Lab.Comp.Radiopharm., 45, 485-528 (2002). Examples of specific precursors and the associated products are given below.

Benzyl-, acetyl- and methyl-protected Rose Bengal derivatives are commercially available from Spectra Group Limited, Aldrich and Spectra Group Limited respectively. Amine-functionalised RB precursors can be prepared as shown in an analogous manner to Carreon et al [Org. Lett., 7(1), 99-102 (2004)] Scheme 2:

Scheme 2 - Synthesis of RB conjugates via an ester linkage

where: amine = H₂N(CH₂)₂NH₂

An alternative amide-linked precursor is as follows:

The primary amine-functionalised precursor product can then be used to prepare imaging agents via coupling with active esters or converted to aminoxy derivatives -NH(C=O)CH₂-O-NH₂ for conjugation with a suitable radioactive aldehyde as described above. Amide-derivatised xanthene dyes are described by Adamczyk et al [Tet. Lett., 41, 807-809 (2000)]. These amide derivatised dyes are most likely to exist in the spirolactam form. The spirolactam formation can be prevented when Z¹ is -NR^a ₂ and neither R^a group is H. Examples of such useful secondary amines are cyclic amines, proline or N-methyl amino acids as described by Nguyen et al [Org.Lett., 5, 3245-3248 (2003)].

A precursor incorporating a linker group can be used for either iodination via conjugation with a suitable active ester, or converted into an aminooxy synthon for ¹⁸F-fluorobenzaldehyde conjugation:

Suitable precursors can also be prepared by reaction of commercially available functionalised fluorescein derivatives, such as fluorescein-6 N-hydroxysuccinimide ester (Fluka, Sigma) with an appropriately functionalised amine (eg. diamines as described above), to give an amide-linked derivative. Subsequent halogenation with bromine or iodine under standard conditions then gives the xanthene dye precursors of the present invention. Similar logic can be applied to commercially available fluorescein-5-isothiocyanate (Aldrich, Fluka).

In a third aspect, the present invention provides a non-radioactive precursor for the preparation of the imaging agent of the first embodiment, which comprises the xanthene dye of Formula Ia or Ib having attached thereto at one of the positions X¹ to X⁴, Y¹ to Y⁴ or E¹ to E³ a non-radioactive group (Q^a) which comprises a substituent capable of reaction with a source of the positron-emitting radioactive non-metal or gamma-emitting radioactive halogen of the imaging moiety (Im ) to give the imaging agent of the first embodiment; with the proviso that:

(i) when Q^a is attached to X¹ to X⁴ or Y¹ to Y⁴, Q^a is not Hal. Preferred precursors are as defined in the method of preparation of the second embodiment. An especially preferred precursor is of Formula IV above, but subject to the above proviso (i).

In a fourth aspect, the present invention provides a pharmaceutical composition which comprises the imaging agent of the first embodiment, together with a biocompatible carrier, in a form suitable for mammalian administration. The "biocompatible carrier" is a fluid, especially a liquid, in which the imaging agent can be suspended or dissolved, such that the composition is physiologically tolerable, ie. 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 isotonic); an aqueous solution of one or more tonicity- adjusting substances (eg. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (eg. sorbitol or mannitol), glycols (eg. glycerol), or other non-ionic polyol materials (eg. polyethyleneglycols, propylene glycols and the like).

Preferably the biocompatible carrier is pyrogen- free water for injection or isotonic saline.

Such radiopharmaceutical compositions are suitably supplied in either a container which is provided with a seal which is suitable for single or multiple puncturing with a hypodermic needle (e.g. a crimped-on septum seal closure) whilst maintaining sterile integrity. Such containers may contain single or multiple patient doses. Preferred multiple dose containers comprise a single bulk vial (e.g. of 10 to 30 cm³ volume) which contains multiple patient doses, whereby single patient doses can thus be withdrawn into clinical grade syringes at various time intervals during the viable lifetime of the preparation to suit the clinical situation. Pre-filled syringes are designed to contain a single human dose, or "unit dose" and are therefore preferably a disposable or other syringe suitable for clinical use. The pre-filled syringe may optionally be provided with a syringe shield to protect the operator from radioactive dose. Suitable such radiopharmaceutical syringe shields are known in the art and preferably comprise either lead or tungsten. The pharmaceutical compositions of the present invention preferably have a radioactive dose suitable for a single patient and are provided in a suitable syringe or container, as described above.

The pharmaceutical compositions of the present invention may be prepared from kits, as is described in the fifth embodiment below. Alternatively, the pharmaceutical compositions may be prepared under aseptic manufacture conditions to give the desired sterile product. The radiopharmaceuticals may also be prepared 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 pharmaceutical compositions of the present invention are prepared from kits.

In a fifth aspect, the present invention provides a kit for the preparation of the pharmaceutical composition of the fourth embodiment. Such kits comprise the "precursor" of the method of the second embodiment, so that reaction with a sterile source of the radioisotope gives the desired radiopharmaceutical with the minimum number of manipulations.

The precursor of the kit is preferably the precursor of the third embodiment. The precursor of the kit is preferably in sterile, non-pyrogenic form. When the kit precursor is in sterile form it is most preferably in lyophilised form and designed to dissolve readily in the solvent for reconstitution.

Such considerations are particularly important for radiopharmaceuticals where the radioisotope has a relatively short half- life, and for ease of handling and hence reduced radiation dose for the radiopharmacist. Hence, the reaction medium for reconstitution of such kits is preferably a "biocompatible carrier" as defined above, and is most preferably aqueous. The chemical form of the positron-emitting radioactive no-metal or gamma-emitting radioactive halogen is chosen to be that which is most readily available, preferably chosen from: (i) halide ion;

(ii) F⁺ or I⁺;

Suitable kit containers comprise a sealed container which permits maintenance of sterile integrity and/or radioactive safety, plus optionally an inert headspace gas (eg. nitrogen or argon), whilst permitting addition and withdrawal of solutions by syringe. A preferred such container is a septum-sealed vial, wherein the gas-tight closure is crimped on with an overseal (typically of aluminium). Such containers have the additional advantage that the closure can withstand vacuum if desired eg. to change the headspace gas or degas solutions.

The non-radioactive kits may optionally further comprise additional components such as a radioprotectant, antimicrobial preservative, pH-adjusting agent or filler.

By the term "radioprotectant" is meant a compound which inhibits degradation reactions, such as redox processes, by trapping highly-reactive free radicals, such as oxygen-containing free radicals arising from the radiolysis of water. The radioprotectants of the present invention are suitably chosen from: ascorbic acid, /><2rø-aminobenzoic acid (ie. 4-aminobenzoic acid), gentisic acid (ie. 2,5- dihydroxybenzoic acid) and salts thereof with a biocompatible cation. The term "biocompatible cation" and preferred embodiments thereof are as defined in the first embodiment for M.

By the term "antimicrobial preservative" is meant an agent which inhibits the growth of potentially harmful micro-organisms such as bacteria, yeasts or moulds. The antimicrobial preservative may also exhibit some bactericidal properties, depending on the dose. The main role of the antimicrobial preservative(s) of the present invention is to inhibit the growth of any such micro-organism in the radiopharmaceutical composition post-reconstitution, ie. in the radioactive diagnostic product itself. The antimicrobial preservative may, however, also optionally be used to inhibit the growth of potentially harmful micro-organisms in one or more components of the non-radioactive kit of the present invention prior to reconstitution. Suitable antimicrobial preservative(s) include: the parabens, ie. methyl, ethyl, propyl or butyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal. Preferred antimicrobial preservative(s) are the parabens.

The term "pH-adjusting agent" means a compound or mixture of compounds useful to ensure that the pH of the reconstituted kit is within acceptable limits (approximately pH 4.0 to 10.5) for human or mammalian administration. Suitable such pH-adjusting agents include pharmaceutically acceptable buffers, such as tricine, phosphate or TRIS [ie. fra(hydroxymethyl)aminomethane], and pharmaceutically acceptable bases such as sodium carbonate, sodium bicarbonate or mixtures thereof. When the conjugate is employed in acid salt form, the pH adjusting agent may optionally be provided in a separate vial or container, so that the user of the kit can adjust the pH as part of a multi-step procedure.

By the term "filler" is meant a pharmaceutically acceptable bulking agent which may facilitate material handling during production and lyophilisation. Suitable fillers include inorganic salts such as sodium chloride, and water soluble sugars or sugar alcohols such as sucrose, maltose, mannitol or trehalose.

Preferred aspects of the "precursor" when employed in the kit are as described for the second and third embodiments above. The precursors for use in the kit may be employed under aseptic manufacture conditions to give the desired sterile, non- pyrogenic material. The kit precursors may also 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 precursors are employed in sterile, non-pyrogenic form. Most preferably the sterile, non-pyrogenic precursors are employed in the sealed container as described above. The "precursor" of the kit is preferably supplied covalently attached to a solid support matrix as described for the third embodiment.

In a sixth aspect, the present invention provides the use of the imaging agent of the first embodiment, the pharmaceutical composition of the fourth embodiment or the kit of the fifth embodiment in the diagnostic imaging of apoptosis in vivo. Said diagnostic imaging refers to non-invasive, diagnostic imaging of the mammalian, preferably human, body.

The imaging agent preferably exhibits a signal-to-background ratio at apoptotic foci of at least 1.5, most preferably at least 5, with at least 10 being especially preferred. Clearance of one half of the peak level of imaging agent which is either non- specifically bound or free in vivo, preferably occurs over a time period less than or equal to the radioactive decay half-life of the radioisotope of the imaging moiety.

The use in diagnostic imaging is suitably carried out such that the mammal had been previously administered with either the imaging agent or the radiopharmaceutical composition. By "previously administered" is meant that the step involving the clinician, wherein the imaging agent is administered to the patient eg. intravenous injection, has already been carried out. This embodiment includes the use of the imaging agent of the first embodiment for the manufacture of diagnostic agents for the diagnostic imaging in vivo of disease states of the mammalian body where apoptosis is implicated.

Such non-invasive imaging would relate to abnormal apoptosis, and is believed valuable in pathologies where cell proliferation and apoptosis are high, eg. myocardial infarction, aggressive tumours and transplant rejection. Such imaging would also be of value in the monitoring of chemotherapeutic drug therapy for these conditions. In other diseases where apoptosis is thought to be important, but the number of apoptotic events is relatively rare such as in Alzheimer's disease, the available cell pool would be small and hence much more difficult to visualise. It is therefore believed likely that the apoptosis imaging agents of the present invention are best applied to pathologies where apoptosis is relatively acute, such as that seen in myocardial infarctions, aggressive tumours and transplant rejection. For those diseases in which apoptosis is more chronic, such as neuropathologies and less aggressive tumours, there may be insufficient apoptotic cells to register above background.

Essentially all treatments for cancer, including radiotherapy, chemotherapy or immunotherapy, are intended to induce apoptosis in their tumour cell targets. The imaging of apoptosis may have the capability for providing rapid, direct assessment or monitoring of the effectiveness of tumour treatment which may fundamentally alter the way cancer patients are managed. It is anticipated that patients whose tumours are responding to therapy will show significantly increased uptake of the imaging agent due to the elevated apoptotic response in the tumour. Patients whose tumours will not respond to further treatment may be identified by the failure of their tumours to increase uptake of the imaging agent post-treatment.

Excessive apoptosis is associated with a wide range of human diseases, and the importance of caspases in the progression of many of these disorders has been demonstrated. Hence, the imaging agents of the present invention are useful for the in vivo diagnostic imaging and or therapy monitoring in a range of disease states, which include:

(a) acute disorders, such as response to cardiac and cerebral ischaemia/reperfusion injury (eg. myocardial infarction or stroke respectively), spinal cord injury, traumatic brain injury, organ rejection during transplantation, liver degeneration (eg. hepatitis), sepsis and bacterial meningitis;

(b) chronic disorders such as neurodegenerative diseases (eg. Alzheimer's disease, Huntington's Disease, Down's Syndrome, spinal muscular atrophy, multiple sclerosis, Parkinson's disease), immunodeficiency diseases (eg. HIV), arthritis, atherosclerosis and diabetes;

The monitoring of efficacy for agents used to induce apoptosis in cancers such as: bladder, breast, colon, endometrial, head and neck, leukaemia, lung, melanoma, non- Hodgkins lymphoma, ovarian, prostate and rectal.

The evaluation of therapeutic intervention in cancer patients with measurable disease has several applications:

• the evaluation of the anti-neoplastic activity of new anti-cancer drugs; • to determine efficacious therapeutic regimens;

• the identification of the optimal dose and dosing schedules for new anticancer drugs; • the identification of optimal dose and dosing schedules for existing anticancer drugs and drug combinations;

• the more efficient stratification of cancer patients in clinical trials into responders and non-responders of therapeutic regimens; • the efficient and timely evaluation of response of individual patients to established therapeutic anti-cancer regimens. The invention is illustrated by the non-limiting Examples detailed below.

Example 1 provides a general procedure for the synthesis of xanthene dyes of the present invention incorporating amine-functionalised linker groups. Such precursors are suitable for radiolabelling with eg. ¹⁸F or ¹²³I via conjugation reactions, ie. the synthon route described. The synthesis of a radiolabelling precursor incorporating a small PEG linker is provided in Example 2. Examples 3 describes the conjugation of the corresponding non-radioactive ¹²⁷I-iodo-labelled analogues of imaging agents of the present invention to the amine precursor of Example 2. Examples 4 and 5 provide a conjugation reaction for an ¹⁹F derivative. Example 6 provides the synthesis of partially iodinated tetrachlorofluorescein derivatives. Example 7 shows the selectivity of the xanthene dyes of the present invention for apoptopic cells, and demonstrates that the X¹ to X⁴ groups can be Br or I and still retain selectivity.

The following abbreviations are used:

Boc = tert-butyloxycarbonyl.

DCM = dichloromethane.

DIEA = Diisopropylethylamine. DMF = N.N'-dimethylformamide.

NMM = 4-methylmorpholine.

PBS = phosphate-buffered saline.

PEG = polyethylene glycol.

PyAOP = 7-Azabenzotriazol-l-yloxytris (pyrrolidino)-phosphonium- hexafluorophosphate.

RCP = radiochemical purity.

TES = N-Ms(hydroxyτnethyl)methyl-2-aminoethane sulfonic acid.

TFA = trifluoroacetic acid.

TIS = triisopropylsilane. Example 1 : General procedure for the synthesis of amine-functionalised precursors for radiolabelling.

A xanthene dye is activated at its carboxylic acid group and conjugated to a diamine linker attached to a trityl resin in DMF-NMM mixture. The amine-functionalized xanthene dye is cleaved from the solid support using 5% TFA in DCM and isolated by evaporation of the solvents. A specific example is provided in Example 2.

Example 2: Synthesis of Rose BengaHfasfaminoethvDethylene glycol).

Rose Bengal Na⁺ salt (3 eq.) was dissolved in DMF (2ml) and NMM (12 eq.) was added followed by PyAOP (3 eq.). After activation for 5 min the mixture was added to O-Bis-(aminoethyl)ethylene glycol trityl resin (1 eq.; NovaBiochem) pre-swollen in DMF. The coupling reaction wrapped in foil was allowed to proceed overnight on a nitrogen bubbler apparatus. The resulting red-burgundy coloured resin was filtered and washed well with DMF. The resin was treated with 20% piperidine in DMF to hydrolyze any unwanted acylation on the phenolic hydroxyl groups. The resin was washed well with DMF, DCM and dried. The product was liberated from the resin by treatment with TFA-5% TIS for 20 min. The resin was washed well with DMF and DCM until the resin appeared colourless. The strongly red-coloured filtrate was concentrated down as much as possible and then analyzed by RP-HPLC using a Cl 8 column (Luna (Phenomenex), 4.6 x 250 mm, 5μ). The column was eluted with a gradient of 30-70% acetonitrile-water containing 0.1% TFA, over 20 min at 1 ml/min: Rt 17.4 min. The product was further analyzed by LC-MS using a Cl 8 column Luna (Phenomenex) 2 x 20 mm, 3μ. The column was eluted with a gradient of 30-70% acetonitrile-water containing 0.1% TFA, over 5 min at 0.6 ml/min: Rt 2.96 min, MH+ expected 1102.6 m/z, found 1102.7 m/z. Example 3: Synthesis of Rose Bengal-PEG_rN-(3-iodo(127)-benzoyl).

3-iodo-benzoic acid (3 eq., Aldrich) was activated with 3eq. PyAOP and 6 eq. NMM in DMF (1 ml) and added to the Rose Bengal-PEG₂- Amine solution (from Example 2, leq, 2ml). Additional NMM was added to bring the pH up to about 8. The reaction was run overnight wrapped in foil. The reddish product was isolated by addition of water and collecting the red precipitate by centrifugation. The product was analyzed RP-HPLC using a C18 column (Luna (Phenomenex), 4.6 x 250 mm, 5μ). Eluted with a gradient of 40-95% acetonitrile-water containing 0.1% TFA, over 20 min at 1 ml/min: Rt 18.1 min. The product was further analyzed by LC-MS using a Cl 8 column Luna (Phenomenex) 2 x 20 mm, 3μ. The column was eluted with a gradient of 30-80% acetonitrile-water containing 0.1% TFA, over 5 min at 0.6 ml/min: Rt 5.1 min, MNa+ expected 1354.5 m/z, found 1354.5m/z. The product was purified by preparative RP- HPLC. The column (218TP 1022 Vydac 22 x 250 mm, lOμ.) was eluted at 10 mL/min using a gradient over 60 min of 40-99%.acetonitrile- water containing 0.1% TFA. The desired peak fractions were pooled and lyophilised affording a light pink almost colourless product. The purified product was characterized by LC-MS using a Cl 8 column Luna (Phenomenex) 2 x 20 mm, 3μ. The column was eluted with a gradient of 40-95 % acetonitrile-water containing 0.1% TFA, over 5 min at 0.6 ml/min: Rt 3.63 min, MH+ expected 1332.6 m/z, found 1332.4 m/z. NMR-analysis of the final purified product confirmed the spirolactam form.

Example 4: Synthesis of Rose Bengal-(PϋG;>-N-(Boc-aniinooxyacetyl) spirolactam.

Rose Bengal-(bis(aminoethyl)ethylene glycol) (from Example 2; 2.3 mg) was dissolved in DMF and added to pre-activated Boc-Aminoxyacetic acid (PyAOP, NMM, 5 eq). The reaction was allowed to proceed overnight to yield the desired product. LC-MS analysis: C18 column Luna (Phenomenex) 2 x 20 mm, 3μ. eluted with a gradient of 40- 90% acetonitrile-water containing 0.1% TFA, over 5 min at 0.6 ml/min: Rt 3.1 min, MH+ expected 1275.7 m/z, found 1275.3 m/z.

Example 5. Synthesis of Rose Bengal-(PEG2-N-(4-fluorobenzylidene) aminooxyacetyl) spirolactam.

Rose Bengal-(PEG₂-N-(Boc-aminooxyacetyl)spirolactam (Example 4) was Boc- deprotected (TFA) and conjugated to ¹⁹F-Benzaldehyde in DMF in an overnight reaction to yield the desired product. LC-MS analysis: C18 column Luna (Phenomenex) 2 x 20 mm, 3μ. eluted with a gradient of 40-95% acetonitrile-water containing 0.1% TFA, over 5 min at 0.6 ml/min: Rt 3.1 min, MH+ expected 1281.7 m/z, found 1281.4 m/z

Example 6: Synthesis of partially iodinated 4,5,6,7-tetrachlorofluorescein derivatives.

4,5,6,7-Tetrachlorofluorescein (100 mg, 0.21 mmol) was suspended in water (5 mL) and sodium hydroxide solution (2M) was added dropwise until the solids had fully dissolved. A solution of iodine (108 mg, 0.43 mmol) in methanol (0.5 mL) was added and the solution was made acidic by dropwise addition of glacial acetic acid. The mixture was then heated at reflux for 18 hours. The mixture was concentrated to dryness, water added (2 mL) followed by triethylamine (10 eq. to 4,5,6,7- tetrachlorofluorescein).The resulting solution mixture was purified by preparative HPLC (column was Phenomenex Luna C18(2) 21.2x150 mm eluting at 15 mL/min with solvent A = 1% triethylamine in water, B = 1% triethylamine in acetonitrile using a gradient). The isolated fractions were analysed by LCMS and freeze dried to give 4'-iodo-4,5,6,7-tetrachloro fluorescein, 4',5'-diiodo-4,5,6,7-tetrachloro fluorescein and 2',4',5'-triiodo-4,5,6,7-tetrachlorofluorescein respectively.

Example 7: Apoptopic to Normal Cell Selectivity In Vitro.

Jurkat cells [Hirt UA, Leist M. Cell Death Differ., JJ)(IO): 1156-64 (2003)] were washed and cultivated 24 hours prior to use. Cells were washed in phosphate- buffered saline (PBS) and diluted to give a concentration of 5 x 10 ⁶IwX. An aliquot of the cell suspension (300 μL) was added per well onto a 24 well plate. The cells were then incubated with a solution of Staurosporine (4 μg/ml) for 3.5 hours at 37⁰C with shaking (rpm=100) to induce apoptosis.

The compound to be tested was dissolved in DMSO and 3μl of each concentration was added to wells containing either normal or apoptotic cells (previously incubated with Staurosporine) to give final concentration ranging from 100 to 1 μg/ml and further incubated for 30 minutes at 37⁰C with shaking (rpm=100). The cells were washed with PBS (X3) at 4⁰C, and then flow cytometry carried out using fluorescence detection. The fluorescence detection is based on an excitation at 488nm and emission at 575nm for all the compounds with exception of 3,3,5,5- tetraiodophenolsulfone-phthalein witch have an excitation at 580nm and emission at 640nm.

Uptake into apoptotic compared to normal cells was expressed as a ratio between the two populations, where ~1 means that no selective apoptopic cell binding was observed.

The compounds tested and results are given in Tables 2 and 3 (-1 means that no selective apoptopic cell binding was observed): Table 2: Compound Structures

Where:

Compound 1 is Rose Bengal lactone;

Compound 2 is Rose Bengal acid;

Compound 3 is Erythrosm B;

Compound 4 is Eosin Y;

Compound 5 is 2',4',5'-Tπ-iodo-fluorescem.

Table 3: Apoptopic to Normal Cell Selectivity

Note: the chemical structures of the xanthene dyes are given in Table 1.

Claims

CLAIMS.

1. An imaging agent which comprises a xanthene dye of Formula (Ia) or (Ib) labelled with an imaging moiety (Im ):

where:

Y¹ to Y⁴ are Y groups, where each Y is independently chosen from H or Hal; E¹ to E³ are E groups, where each E is independently chosen from H or M; Z¹ is -OH, -OM, -OR^a or -NR^a ₂; Z^a is -O- or -NR^a-; M is a biocompatible cation;

R^a is independently chosen from H, C_M o alkyl, C_3-I0 cycloalkyl, C _MO fluoroalkyl, an Ar¹ group or -(Ci_-3 alkyl)Ar' where Ar¹ is a C_3-I2 aryl or heteroaryl ring;

Im^G is an imaging moiety which is attached at a single position only of Formula Ia or Ib, where said position is chosen from X¹ to X⁴, Y¹ to Y⁴, E¹ to E³ or Z¹, and Im comprises a gamma- emitting radioactive halogen or a positron-emitting radioactive non-metal, wherein following administration of said labelled xanthene dye to the mammalian body in vivo, the imaging moiety can be detected externally in a non-invasive manner; with the proviso that when Y¹ to Y⁴ are all Cl, X¹ to X⁴ are all I or Im^G, E is H or M and Z is -OH or -OM, then Im ,G _; is not the gamma-emitting radioactive halogens 123τ I

Or ^{1 31} I.

2. The imaging agent of Claim 1, where at least 2 of the X groups are I.

3. The imaging agent of Claim 2, where all 4 of the X groups are Br or I.

4. The imaging agent of any one of Claims 1 to 3, where at least 2 of the Y groups are H or Cl.

5. The imaging agent of Claim 4, where all 4 of the Y groups are chosen from H or Cl.

6. The imaging agent of any one of Claims 1 to 5, where Im^G is attached at the Z¹ , Y¹ to Y⁴ or E positions.

7. The imaging agent of any one of Claims 1 to 6, where the positron-emitting radioactive non-metal is chosen from ¹⁸F, ¹¹C, ¹³N or ¹²⁴I.

8. The imaging agent of any one of Claims 1 to 6, where the gamma-emitting radioactive halogen imaging moiety is I.

9. The imaging agent of any one of Claims 1 to 8, which further comprises a linker group and is of Formula II:

[ {xanthene} -(A)_nJ-Im⁰ (H) where:

{xanthene} is the xanthene dye of Formula (Ia or Ib) of Claim 1, wherein (A)_n is attached in place of one of the substituents at the X¹ to X⁴, Y¹ to Y⁴, E¹ to E³ or Z¹ positions; -(A)_n- is a linker group wherein each A is independently -CR₂- , -CR=CR- , -C≡C- , -CR₂CO₂- , -CO₂CR₂- , -NRCO- , -CONR- , -NR(C=O)NR-,

-NR(C=S)NR-, -SO₂NR- , -NRSO₂- , -CR₂OCR₂- , -CR₂SCR₂- , -CR₂NRCR₂- . a C4-₈ cycloheteroalkylene group, a C4-S cycloalkylene group, a C_{5-I 2} arylene group, or a C_3-12 heteroarylene group, an amino acid, a sugar or a monodisperse polyethyleneglycol (PEG) building block; each R is independently chosen from H, C_{1 -4} alkyl, C_2-4 alkenyl, C_2-4 alkynyl,

C ₁-₄ alkoxyalkyl or Ci_-4 hydroxyalkyl; n is an integer of value 1 to 10.

10. The imaging agent of Claim 9, where n is 1 to 6.

11. A method of preparation of the imaging agent of Claims 1 to 10, which comprises reaction of:

(i) a non-radioactive precursor which comprises the xanthene dye of Formula Ia or Ib of Claims 1 to 10 having attached thereto in place of one of the substituents at the X¹ to X⁴, Y¹ to Y⁴, E¹ to E³ or Z¹ positions a nonradioactive group (Q^a) which comprises a substituent capable of reaction with a source of the positron-emitting radioactive non-metal or gamma- emitting radioactive halogen of Claims 1, 7 or 8 to give the imaging agent of Claims 1 to 10; with

(ii) a source of the positron-emitting radioactive non-metal or gamma- emitting radioactive halogen.

12. The method of Claim 11, where the precursor is of Formula Ilia or HIb:

Ilia HIb

where: X³ to X^s are independently chosen from Br, I, H or Q^a, such that at least two of the X groups are either Br or I;

Y³ to Y⁸ are independently chosen from H, Hal or Q^a; E¹ to E³ are independently chosen from H, M, P^GP or Q^a; P^GP is a protecting group;

Z² is -OH, -OM, -OR^a, -NR^a ₂, P^GP or Q^a; M, R^a and Z^a are as defined in Claim 1 ;

Q^a is a non-radioactive group which comprises a substituent capable of reaction with a source of the positron-emitting radioactive non-metal or gamma-emitting radioactive halogen of the imaging moiety (Im^G) to give the labelled xanthene dye of Claims 1 to 10; with the proviso that said precursor contains one Q^a group.

13. The method of Claims 11 or 12, wherein the reactive substituent of the Q^a group is chosen from:

(i) an organometallic derivative such as a trialkylstannane or a trialkylsilane; (ii) a derivative containing an alkyl halide, alkyl tosylate or alkyl mesylate for nucleophilic substitution; (iii) a derivative containing an aromatic ring activated towards nucleophilic or electrophilic substitution; (iv) a derivative containing a functional group which undergoes facile alkylation;

(v) a derivative which alkylates thiol-containing compounds to give a thioether-containing product;

(vi) a derivative which undergoes condensation with an aldehyde or ketone; (vii) a derivative which is acylated by an active ester group.

14. The method of any one of Claims 11 to 13, where the precursor is in sterile, apyrogenic form.

15. The method of any one of Claims 1 1 to 14, where the precursor is bound to a solid phase.

16. The method of any one of Claims 1 1 to 15, where the source of the positron- emitting radioactive non-metal or gamma-emitting radioactive halogen is chosen from:

(i) halide ion; (ii) F⁺ or I⁺;

17. The method of any one of Claims 11 to 16, wherein the imaging agent is of Formula II of Claim 9, and the precursor further comprises a linker group as defined in Formula IV:

[ {Compound III} -(A)_n]-Q^a

(IV) where {Compound III} is the compound of Formula Ilia or HIb of Claim 12, and A and n are as defined for Formula II in Claim 9.

18. A non-radioactive precursor as defined in Claims 11 to 17, with the proviso that:

(i) when Q^a is attached to X¹ to X⁴ or Y¹ to Y⁴, Q^a is not Hal.

19. A pharmaceutical composition which comprises the imaging agent of Claims 1 to 10 together with a biocompatible carrier, in a form suitable for mammalian administration.

20. The pharmaceutical composition of Claim 19, which has a radioactive dose suitable for a single patient and is provided in a suitable syringe or container.

21. A kit for the preparation of the pharmaceutical composition of Claims 19 or 20, which comprises the precursor as defined in Claims 11 to 18.

22. The kit of Claim 21, where the precursor is in sterile, apyrogenic form.

23. Use of the imaging agent of Claims 1 to 10, the pharmaceutical composition of Claims 19 or 20 or the kit of Claims 21 to 22 in the diagnostic imaging of apoptosis in vivo.