WO2011145957A1

WO2011145957A1 - Agents and methods for detection and/or imaging of hypoxia

Info

Publication number: WO2011145957A1
Application number: PCT/NZ2011/000083
Authority: WO
Inventors: Moana Tercel; Frederik Bastiaan Pruijn
Original assignee: Auckland Uniservices Limited
Priority date: 2010-05-20
Filing date: 2011-05-20
Publication date: 2011-11-24

Abstract

The invention relates to the detection and/or imaging or hypoxia. In particular, the invention relates to the use of 2- nitroimidazoles as agents for the detection and/or imaging of hypoxia. The invention also relates to a method of detecting and/or imaging hypoxic cells using a copper(I)-catalysed azide-alkyne 1,3-dipolar cycloaddition reaction between an alkyne-functionalised nitroimidazole and an azide-functionalised probe or an azide-functionalised nitroimidazole and an alkyne-functionalised probe.

Description

AGENTS AND METHODS FOR DETECTION AND/OR IMAGING OF HYPOXIA

TECHNICAL FIELD

The invention relates to the detection and/or imaging of hypoxia. In particular, the invention relates to the use of 2-nitroimidazoles as agents for the detection and/or imaging of hypoxia.

BACKGROUND TO THE INVENTION

The presence of hypoxia, i.e. oxygen concentrations below normal physiological levels, is a pathophysiological condition found in, for example, tumours/ ischemic tissue (e.g. cardiac ischemia, brain ischemia, etc.),^2,3 inflamed tissue (e.g. rheumatoid arthritis),⁴ and vascular disease (e.g. diabetes).⁵ In cancer, the presence of hypoxia has been associated with poor prognosis and treatment outcome.^6,7,8 Linkages have been postulated between cancer stem cells, hypoxia, and metastasis, and experimental evidence for these associations is growing,.^9,10,11,12 For these reasons, and particularly in the field of oncology, there is great interest in developing methods for the detection and imaging of hypoxia.^13,14

One established method for detecting hypoxic cells is based on the reduction of 2-nitroimidazoles.^13,14,15 It is well known that the reduction of 2-nitroimidazoles is an oxygen-sensitive process, and that reduction products derived from the 2-nitroimidazoles become covalently bound within the cells in which they are produced to form covalent adducts. For the purpose of this invention the covalent binding of the reduction products of nitroimidazoles to cell components (e.g. macromolecules) is defined as "labelling". Examples of 2-nitroimidazoles that react in this way include (but are not limited to) pimonidazole, EF5, fluoromisonidazo!e, FAZA, misonidazole, FETNIM, FETA, and CCI-103F.

pimonidazole EF5 fluoromisonidazole FAZA

misonidazole FETNIM FETA CCI-103F

A necessary step in the detection of hypoxic cells using 2-nitroimidazoles involves the detection and/or imaging of the covalent adducts (the 'hypoxia markers'). For the purpose of this invention "hypoxia markers" are defined as the covalent adducts resulting from oxygen-sensitive enzymatic bioreduction of nitroimidazoles. One widely used method for the detection of hypoxia markers employs antibodies which recognise the covalent adducts of 2-nitroimidazoles.¹⁶'^17,18 The antibody is further conjugated to a probe such as a fluorophore or a radiolabel, or a secondary antibody carrying such a probe, that allows detection by appropriate methods. For the purpose of this invention "probe" is defined as a molecule or moiety that allows detection by a suitable method, such as molecules or moieties that are fluorogenic, or molecules or moieties that are fluorophores, radiolabels or have luminescent or bioluminescent properties. For the purpose of this invention "fluorogenic" is defined as having fluorescent properties that are induced or enhanced after a chemical reaction. Antibodies that recognise reduction products derived from pimonidazole and EF5 are particularly well known. The combination of a 2-nitroimidazole and an appropriate antibody can be applied to single cells or tissue samples, which may be derived from in vitro or ex vivo experiments, and are employed in preclinical research and testing and in the analysis of clinical samples, for example, in immunostaining and immunohistochemistry methods. For the purpose of this invention "in vitro" is defined as cells or tissues or cell components outside a living body or organism; "in vivo" is defined as within a living organism; and "ex vivo" is defined as a sequence first involving the in vivo manipulation of a cell or tissue sample followed by an in vitro manipulation. For the purpose of this invention "immunostaining" is defined as the use of an antibody to detect a specific antigen in a sample and "immunohistochemistry" is defined as the use of an antibody for staining of tissue sections. Illustrative applications of immunostaining and immunohistochemistry methods using 2- nitroimidazoles in the oncology field include: to detect and study hypoxic cells in vitro (single cells, spheroids, and other multicellular structures),^19,20 in experimental tumour models,^19,21 and in tumours from cancer patients;²²'^23,24 to study the sensitivity of hypoxic cells to various anticancer therapies;²⁵ to study the effect of hypoxia on extravascular drug transport;²⁶ and to predict treatment outcome in preclinical tumour models,²⁷ and in human patients.²⁸

Despite the obvious usefulness of the techniques described above, the requirement for antibodies creates some difficulties. Many immunostaining and immunohistochemistry protocols start with preparation of the tissue or specimen by fixation in order to preserve cellular morphology and tissue architecture. However, fixation may diminish or abolish antibody binding (by loss of antigenicity), while antibody penetration into the specimen can be adversely affected by extensive protein cross-linking from prolonged aldehyde fixation. In the latter case antigen retrieval methods (e.g. by heat or enzymatic digestion) may be required to improve antibody binding by breaking protein cross-links.²⁹ The formation of protein cross-links during the fixation process is not compatible with technologies and methodologies that rely on protein mobility, for example, sodium dodecyl sulfate polyacryiamide gel electrophoresis (SDS-PAGE), Western blotting, liquid chromatography, and matrix-assisted laser desorption/ionization (MALDI). Further, since the 2-nitroimidazole-derived covalent adducts are intracellular, and antibodies cannot penetrate intact cell membranes, cells must also be permeabifised by treatment with a surfactant to partially dissolve the lipid membranes and allow the antibody to access the intracellular compartment.³⁰ Since antibodies have a low diffusion coefficient, prolonged contacting (incubation) times are often required with thicker specimens and even then full penetration may not be achieved.^31,32,33,34 While thicker specimens can be cut into smaller sections, such sectioning of specimens can lead to artefacts and loss of spatial and architectural information. Therefore, so-called whole mount sections can be advantageous but again may be limited by inefficient antibody penetration.³⁵

Immunostaining protocols therefore have a number of inherent disadvantages. These disadvantages can make the whole process difficult to establish, time-consuming, technically complicated, prone to artefacts from incorrect use of various reagents, unsuitable for thick specimens, and incompatible with other bioanalytical techniques. OBJECT OF THE INVENTION

It is an object of the invention to provide methods for the detection and/or imaging of hypoxia; it is an alternative object to provide compositions and 2-nitroimidazole agents for use in such methods; or alternatively to at least provide the public with useful choices.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a method for detection and/or imaging of hypoxic cells, the method including the in vitro or the ex vivo use of a copper(l)-catalysed azide-alkyne 1,3-dipolar cycloaddition reaction (herein referred to as 'click chemistry')_/ the alkyne being a terminal alkyne.

Preferably the method includes a click chemistry reaction between a hypoxia marker derived from an alkyne functionalised nitroimidazole and an azide functionalised probe, the alkyne being a terminal alkyne.

Alternatively the method includes a click chemistry reaction between a hypoxia marker derived from an azide functionalised nitroimidazole and an alkyne functionalised probe, the alkyne being a terminal alkyne.

Alternatively the method includes both a click chemistry reaction between a hypoxia marker derived from an alkyne functionalised nitroimidazole and an azide functionalised probe, and also a click chemistry reaction between a hypoxia marker derived from an azide functionalised nitroimidazole and an alkyne functionalised probe, the alkyne being a terminal alkyne in both reactions.

Preferably the hypoxia marker is derived from an alkyne functionalised 2-nitroimidazole and/or an azide functionalised 2-nitroimidazole

Preferably the 2-nitroimidazole is a compound of Formula (1):

N0₂

X

N N-X-Y

\=J ^■

Formula (I)

wherein:

X is a Ci-C₈ alkyl chain linker, optionally saturated or unsaturated, optionally straight or branched, optionally containing cyclic units and optionally further substituted with any one or more of:

N„ and/or O_n heteroatoms, which may be present as alcohol, ether, amine, amide, carbamate, azide and/or heterocyclic functional groups,

F_q atoms;

n is any number from 1 to 3;

q is any number from 1 to 6;

Y is a terminal alkyne or an azide; Z is

including enantiomers, pharmaceutically acceptable salts, and/or pharmaceutically acceptable solvates and/or chemical variants.

Alternatively the 2-nitroimidazo!e is a compound of Formula (II):

N0₂

^N-Z

X

Y

Formula (II)

wherein:

X may be absent or present,

N_n and/or 0„ heteroatoms, which may be present as alcohol, ether, amine, amide, carbamate, azide and/or heterocyclic functional groups,

F_q atoms;

n is any number from l to 3;

q is any number from 1 to 6;

Y is a terminal alkyne or an azide;

Z is

Alternatively the 2-nitroimidazole is a compound of Formula (III):

NO,

i x

Y .

Formula (III) wherein:

X may be absent or present,

X is a Cx-Ca alkyl chain linker, optionally saturated or unsaturated, optionally straight or branched, optionally containing cyclic units and optionally further substituted with any one or more of:

N_n and/or O_n heteroatoms, which may be present as alcohol, ether, amine, amide, carbamate, azide and/or heterocyclic functional groups,

F_q atoms;

n is any number from l to 3;

q is any number from 1 to 6;

Y is a terminal alkyne or an azide; ¹

Z is

Preferably in Formulae (I), (II), or (III) X is a Ci-C₈ alkyl chain linker, optionally saturated or unsaturated, optionally straight or branched, optionally containing cyclic units and optionally containing any one or more of:

N_n and/or O_n heteroatoms, which may be present as ether, amine, amide, carbamate, and/or heterocyclic functional groups,

n is any number from l to 3.

Preferably the 2-nitroimidazole is selected from the following:

Preferably the 2-nitroimidazole is selected from the following:

Preferably hypoxic cells are labelled with a 2-nitroimidazole in vivo or in vitro. Preferably the 2-nitroimidazole is administered in vivo.

Preferably the Cu(l) of the Cu(l) catalyst is added as a salt.

Preferably Cu(l) is added as CuBr or CuOAc. Alternatively the Cu(!) of the Cu(l) catalyst is formed from metallic Cu, a Cu(0) source, or a Cu(ll) source. Preferably the Cu(ll) souce is CuS0₄.

Preferably the Cu(l) source is formed from the Cu(ll) source and a reducing agent.

Preferably the reducing agent is sodium ascorbate or a phosphine.

Preferably the phosphine is tris(2-carboxyethyl)phosphine).

Preferably the method includes the use of a suitable metal chelating ligand.

Preferably the suitable chelating ligand is a tris(triazolyl)methylamine, such as tris(benzyltriazolyl)methylamine (TBTA) or a water soluble analogue such as tris(hydroxypropyltriazolyl)methylamine (THPTA).

Preferably when the 2-nitroimidazole is azide-functionalised the click chemistry is conducted under low oxygen conditions

Preferably the low oxygen conditions are less than or equal to about 20% by volume.

Preferably the method employs repeated exposure to the click chemistry reagents when a single exposure to the method does not result in a sufficient signal for detection and/or imaging purposes.

Preferably detection and/or imaging is carried out using fluorescence, radioactivity, or (bio)luminescent techniques and employs functionalised probes wherein the probe is either fiuorogenic, a fluorophore, a radiolabel or an N R active probe or a luminescent or bioluminescent probe.

Preferably the detection and/or imaging technique is fluorescence.

Preferably the probe is a fluorophore probe.

Preferably the probe is fiuorogenic. A fiuorogenic probe has latent fluorescent properties in that the fluorescent properties of the probe are induced or enhanced after the click chemistry reaction.

m the following:

including pharmaceutically acceptable salts, and/or pharmaceutically acceptable solvates and/or chemical variants.

In a third aspect the invention provides a method for detection and/or imaging of hypoxic cells, the method including the in vitro or the ex vivo use of a copper(l)-catalysed azide-alkyne 1,3-dipolar cycloaddition reaction between a hypoxia marker derived from a compound of the second aspect of the invention and a functiona lised probe.

Preferably the method further includes detection by antibody-based methods as well as by click chemistry methods. Preferably hypoxic cells are labelled with one or more 2-nitroimidazole compounds according to the second aspect in vivo or in vitro.

Preferably the 2-nitroimidazole according to the second aspect is administered in vivo.

Preferably the Cu(l) of the Cu(l) catalyst is added as a salt.

Preferably Cu(l) is added as CuBr or CuOAc. Alternatively the Cu(l) of the Cu(l) catalyst is formed from metallic Co, a Cu(0) source, or a Cu(ll) source. Preferably the Cu{ll) souce is CuS0 .

Preferably the Cu{l) is formed from the Cu(ll) source and a reducing agent.

Preferably the reducing agent is sodium ascorbate or a phosphine.

Preferably the phosphine is tris(2-carboxyethyl)phosphine). Preferably the method includes the use of a suitable metal chelating ligand.

Preferably when the 2-nitroimidazole is azide-fiinctionalised the click chemistry is conducted under low oxygen conditions.

Prefera bly the method employs repeated exposure to the click chemistry reagents when a single exposure to the method does not result in a sufficient signal for detection and/or imaging purposes.

Preferably detection and/or imaging is carried out using fluorescence, radioactivity, or (bio)luminescent techniques and employs functionalised probes wherein the probe is either fluorogenic, a fluorophore, a radiolabel or an NMR active probe or a luminescent or bioluminescent probe.

Preferably the detection and/or imaging technique is fluorescence.

Preferably the probe is a fluorophore probe.

Preferably the probe is fluorogenic. A fluorogenic probe has latent fluorescent properties in that the fluorescent properties of the probe are induced or enhanced after the click chemistry reaction.

In a fourth aspect the invention provides a method for identifying hypoxic cells, the method comprising the following steps:

a) in vivo administration of at least one alkyne functionalised 2-nitroimidazole and/or at least one azide functionalised 2-nitroimidazole , the alkyne being a terminal alkyne, to label hypoxic cells with an hypoxia marker; b) collecting labelled cells or tissue samples from the living organism; and

c) exposing the cells or tissue samples collected to an alkyne or azide functionalised probe, the alkyne being a terminal alkyne, and a Cu(l) source;

d) identifying hypoxic cells by detection of a signal generated by the probe by a method appropriate for the probe used.

Preferably the in vivo administration is by parenteral administration.

In a fifth aspect the invention provides a method for identifying hypoxic cells in tissue samples taken from a living organism to which at least one alkyne functionalised 2-nitroimidazole and/or at least one azide functionalised 2-nitroimidazole , the alkyne being a terminal alkyne, has been administered, the method comprising the use of click chemistry for the detection and/or imaging of hypoxic cells to identify the hypoxic cells.

in a sixth aspect the invention provides a composition for use in the detection and/or imaging of hypoxic cells, the composition including at least one nitroimidazole functionalised by a terminal alkyne and/or at least one nitroimidazole functionalised by an azide and a pharmaceutically acceptable carrier.

Preferably the composition is formulated for use as a parenteral composition.

Preferably the composition is administered in vivo using intraperitoneal or intravenous administration methods.

Preferably the nitroimidazoles are 2-nitroimidazoles selected from the 2-nitroimidazoles according to the first or second aspects of the present invention.

Preferably the concentration of 2-nitroimidazoles in the composition is sufficient to generate a detectable signal from hypoxic cells without causing solubility and/or toxicity complications.

Preferably the composition can be administered in vitro or in vivo.

Preferably the pharmaceutically acceptable carrier is saline solution.

In a seventh aspect the invention provides a two-part composition for use in the detection and/or imaging of hypoxic cells, the two-part composition including:

Part A, which is the composition according to the sixth aspect of the present invention; and

Part B, which comprises a probe functionalised by an azide or a probe functionalised by a terminal alkyne and a suitable carrier;

wherein when the nitroimidazole of Part A is functionalised by a terminal alkyne the probe cannot also be functionalised by a terminal alkyne and when the nitroimidazole of Part A is functionalised by an azide the probe cannot also be functionalised by an azide.

Preferably the concentration of 2-nitroimidazoles in the composition is sufficient to generate a detectable signal from hypoxic cells without causing solubility and/or toxicity complications. Preferably the furtctionalised probe is either fluorogenic, a fluorophore, an NMR active probe, or a radiolabel or a luminescent or bioluminescent probe.

Preferably the probe is a fluorophore probe.

Preferably Part A and Part B of the two-part composition are administered sequentially, wherein Part A is before Part B.

Preferably Part A can be administered in vitro or in vivo.

Preferably Part A is administered in vivo using intravenous or intraperitoneal methods.

Preferably Part B is administered in vitro or ex vivo together with a Cu(l) catalyst.

Preferably the Cu(l) of the Cu(l) catalyst is administered as a salt.

Preferably Cu(l) is administered as CuBr or CuOAc.

Alternatively the Cu(l) of the Cu(l) catalyst is formed from metallic Cu, a Cu(0) source, or a Cu(ll) source. Preferably the Cu(ll) souce is CuS0₄.

Preferably the Cu(l) source is formed from the Cu(li) source and a reducing agent.

Preferably the reducing agent issodium ascorbate or a phosphine.

Preferably the phosphine is tris(2-carboxyethyi)phosphine).

Preferably the suitable carrier of Part B includes aqueous buffer solutions, and organic or aqueous solvents.

In an eighth aspect the invention provides a three-part composition for use in the detection and or/imaging of hypoxia, the three-part composition including:

- Part A, which is the composition according to the sixth aspect of the present invention;

- Part B, which comprises a probe functionalised by an azide or a probe functionalised by a terminal alkyne and a suitable carrier; and

Part C, which comprises a Cu(l) source and a suitable carrier;

Preferably Part A and Part B are as described according to the sixth and seventh aspects of the present invention.

Preferably Part A is administered before Part B and Part C and preferably Part B and Part C are administered together.

Preferably Part B is administered in vitro or ex vivo together with Part C. Preferably Part C is a Cu(l) salt, a Cu(ll) salt or metallic copper.

Preferably the Cu(l) salt is CuBr or CuOAc.

Preferably the Cu(ll) salt is CuS0₄.

Preferably the reducing agent is sodium ascorbate or a phosphine.

Preferably the phosphine is tris(2-carboxyethyl)phosphine).

Preferably the suitable carrier of Part B and Part C includes aqueous buffer solutions, and organic or aqueous solvents.

In a ninth aspect the invention provides a four-part composition for use in the detection and/or imaging of hypoxic cells, the four-part composition including:

Part A, which is the composition according to the sixth aspect of the present invention;

Part B, which comprises a probe functionalised by an azide or a probe functionalised by a terminal a!kyne and a suitable carrier;

Part C, which comprises a Cu(l) source and a suitable carrier; and

- Part D, which comprises a chelating ligand capable of selectively chelating and stabilising Cu(l) and a suitable carrier;

Preferably Part A, Part B, and Part C are as described according to the sixth, seventh and eight aspects of the present invention.

Preferably Part A is administered before Part B, Part C and Part D and preferably Part B, Part C and Part D are administered together.

Preferably Part B is administered in vitro or ex vivo together with Part C and Part D.

Preferably Part D is a tris(triazolyl)methylamine or a water soluble analogue.

Preferably the tris(triazo!yl)methylamine is a tris(benzyltriazolyl)methylamine (TBTA).

Preferably the water soluble analogue is tris(hydroxypropyltriazolyl)methylamine (THPTA).

In a tenth aspect the invention provides novel compositions for use in the labelling of hypoxic cells, the compositions comprising any one or more of the following 2-nitroimidazoles:

and a carrier.

In an eleventh aspect the invention provides a use of a nitroimidazole functionalised by a terminal alkyne or a nitroimidazole functionalised by an azide, in the manufacture of a composition for the detection and/or imaging of hypoxic cells.

In a twelth aspect the invention provides a nitroimidazole functionalised by a terminal alkyne or a nitroimidazole functionalised by an azide for use in detection and/or imaging of hypoxic cells.

Preferably the nitroimidazole functionalised by a terminal alkyne or a nitroimidazole functionalised by an azide is a 2-nitroimidazole.

Preferably the 2-nitroimidazole is a compound of Formula (I), Formula (II), or Formula (II) according to the first aspect of the present invention.

Preferably the 2-nitroimidazole is selected from the following:

Preferably the 2-nitroimidazole is selected from the following:

In a thirteenth aspect the invention provides a kit of parts for use in the method of the first or third aspects of the present invention, the kit including either a 2-nitroimidazole functionalised by a terminal alkyne and a probe functionalised by an azide, or a 2-nitroimidazole functionalised by an azide and a probe functionalised by a terminal alkyne. The kit may also include a suitable copper salt, chelating ligand and reducing agent and other materials suitable for use in carrying out the method of the present invention. Preferably the kit also includes a suitable copper salt and chelating ligand and reducing agent.

Preferably the kit also includes aqueous buffer solution(s), or salts to make up buffer solutlon(s), organic solvent(s).

shows the fluorophore azides used in the click reactions;

shows HPLC chromatograms and UV-Visible spectra, which illustrate the progress of a reaction described in Example 7;

shows the progress of the reactions described in Example 7;

shows the results obtained when one reaction described in Example 7 was carried out in different aqueous buffers or in the presence of EDTA;

shows the fluorophore alkynes used in the click reactions;

shows HPLC chromatograms and UV-Visible spectra, which illustrate the progress of a reaction described in Example 8;

shows the progress of the reactions described in Example 9

shows the results obtained from the cytotoxicity study described in Example 10;

shows the results obtained from flow cytometry analysis of click reactions carried out in vitro, using nitroimidazole 2, as described in Example 11;

shows the results obtained from flow cytometry analysis of click reactions carried out in vitro, using nitroimidazoles 9 and 10, as described in Example 12;

shows the results obtained from flow cytometry analysis of click reactions carried out in vitro, using nitroimidazole 4, as described in Example 13;

shows the results obtained from flow cytometry analysis of click chemistry and immunostaining carried out in vitro using a combination of two 2-nitroimidazoles, as described in Example 14;

shows the results obtained from experiments comparing the extent of labelling of cells at variable oxygen concentrations, as assessed by immunostaining and by click chemistry protocols, as described in Example 14;

shows the results obtained from flow cytometry analysis of cells exposed to a single hybrid nitroimidazole 8 and detected by either click chemistry or immunostaining, as described in Example 15;

shows the results obtained from flow cytometry analysis of cells exposed to a single hybrid nitroimidazole 11 and detected by either click chemistry or immunostaining, as described in Example 15; Figure 16: shows results obtained from experiments described in Example 16 where hypoxic cells are detected by click chemistry with tetramethylrhodamine azide without the use of either fixation or permeabilisation;

Figure 17: shows results obtained from experiments described in Example 16 where hypoxic cells are detected by click chemistry with coumarin azide without the use of either fixation or permeabilisation;

Figure 18: shows the results obtained from flow cytometry analysis of click chemistry and immunostaining carried out ex vivo using a combination of two 2-nitroimidazoles, as described in Example 17;

Figure 19: shows fluorescent images of a fixed A431 tumour sample exposed to immunohistochemistry and click chemistry techniques;

Figure 20: shows fluorescent images of a fixed HCT116 tumour sample exposed to immunohistochemistry and click chemistry techniques;

Figure 21: shows results obtained from experiments described in Example 18 where hypoxic cells within multicellular layers are detected by click chemistry techniques;

Figure 22: shows results obtained from experiments described in Example 18 where click chemistry and immunostaining techniques are applied to multicellular layers;

Figure 23 shows Western blotting of samples from oxic and hypoxic cells after labelling by click chemistry techniques.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides the use of "click chemistry" as an alternative to known methods of detection and/or imaging of hypoxia.

The click chemistry technique is used, preferably with nitroimidazoles, in the process of detection and/or imaging of hypoxia. The most preferable compounds of use are 2-nitroimidazo!es.

Antibodies react in a highly specific manner and with high affinity to antigens (although cross-reactivity with multiple antigens is a common problem as well as non-specific background binding).

It would be highly advantageous to be able to detect and/or image markers of hypoxia, such as reduction products of nitroimidazoles, in the same way as with antibodies but by using a chemical reaction instead. Such reaction would preferably be fast, specific, and minimally or not at all interfere with the biological system or process of interest.

The copper(l)-catalysed Azide-Alkyne Huisgen Cycloaddition is a 1,3-dipolar cycloaddition between an azide and a terminal alkyne to give specifically 1,4-disubstituted regio-isomers of 1,2,3-triazoles as products. This reaction, which is also known as the copper(l)-catalysed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) or simply "click chemistry", is specific and fast. While click chemistry is, in general terms, a known technique, its use in a method for detection and/or imaging of hypoxia where the alkyne is a terminal alkyne is not known. The inventors are the first to have considered the use of click chemistry for this purpose and were surprised at the applicability and advantages offered. The invention can therefore be seen to include the use of "click chemistry" in the detection and/or imaging of hypoxia.

For the purposes of this specification, "click chemistry" may be defined as meaning "copper(l)-catalysed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) in which the alkyne is a terminal alkyne". The process is illustrated in Diagram 1 below showing a click reaction between molecule A bearing a terminal alkyne and molecule B bearing an azide, to form a triazole product linking A and B together.

Diagram 1:

The detection and imaging of hypoxia can be simplified to two steps.

The first step involves the labelling of the hypoxic cells with the nitroimidazole, preferably the 2- nitroimidazole. Nitroimidazoles other than 2-nitroimidazoles can be used if they have the right properties to be reduced by enzymes, for example, a reduction potential in the same range as 2- nitroimidazoles. The reducing enzymes generally require co-factors such as NADH and NADPH and are only active in live cells. Such matters would be known to the skilled person once in possession of this invention. This step requires enzymatic bio-reduction and is therefore carried out in living cells and/or organisms. The enzymatic bio-reduction of the 2-nitroimidazole in a hypoxic cell results in the formation of a covalent adduct which acts as a hypoxia marker that is capable of being detected via a subsequent click chemistry reaction with a detectable probe. The labelling step can be performed in vitro (provided the cells are alive) as well as in vivo. Preferably the 2-nitroimidazole is parentera!ly administered in vivo. In vitro labelling techniques include, but are not limited to, adding the 2-nitroimidazole to a culture or incubation medium containing tissue samples or cells to be tested. In vivo labelling techniques include administering the 2-nitroimidazole to the organism by intraperitoneal or intravenous administration (intraperitoneal administration is usually employed on laboratory animals while in humans the 2- nitroimidazo!e is usually administered via the intravenous route). The contacting time is usually in the order of a few hours but for in vivo applications these times can extend to 24 hours or longer after administration. The appropriate in vivo contacting time is dependent on factors such as the pharmacokinetics of the 2-nitroimidazole in use, and the method of administration, for example whether an intravenous administration is prolonged or given as a bolus.

The amount of 2-nitroimidazole administered can be varied depends on the nature and toxicity of the administered nitroimidazole, the route of administration, the dose rate, and the condition of the organism. Preferably the concentration or dose administered has minimal effect on biological processes, is non-toxic, and will result in a strong signal for detection of the probe once it has undergone click chemistry reaction with the hypoxia marker in the sample, which, as indicated above, forms upon the enzymatic bio-reduction of the administered nitroimidazole. A person skilled in the art will appreciate that toxicity, in its broadest sense, is determined by concentration or dose (as Paracelsus, the father of toxicology, wrote: "The dose makes the poison"). It is understood that toxicity and pharmacokinetics of nitroimidazoles may also vary considerably between species. The concentration of hypoxia marker in the sample correlates with the total contacting time with the nitroimidazole, the concentration of nitroimidazole during the contacting time (which may not be constant), and the level of hypoxia during the contacting time (which may not be constant). During the contacting time the labelling of live hypoxic cells with a hypoxia marker will take place dependent on these factors and this will be apparent to those skilled in the art. Generally, doses of 2-nitroimidazoles range between 0.1 g kg to 1 g/kg as a single dose or are divided into fractionated doses per day when the labelling is to take place in vivo. Preferably, the general dose is between 250 g/kg to 100 mg/kg. Variations in these dose levels can be adjusted using standard empirical routines for optimisation, which are well known in the art. Generally, concentrations of 2-nitroimidazole range between 0.1 pg/L to 1 g/L when the labelling is to take place in vitro or ex vivo. Preferably, the general concentration is between 250 g/L to 100 mg/L. Variations in these concentrations can be adjusted using standard empirical routines for optimisation, which are well known in the art.

Cells or tissue samples can be collected by standard techniques and methods. For example, cells may be harvested from an incubation or culture vessel or container (e.g. culture flask) by centrifugation ("spinning down") followed by one or more wash steps if required. Generally, cells need to be detached from the fixed substratum they grow in or on (e.g. the plastic of a culture flask) and this is generally done by treatment with enzymes (generally including trypsin) or by mechanical force (e.g. "scraping"). Cells may also need to be dissociated from each other and/or cells they are in contact with. Cells may also be grown directly on coverslips or spun (cytospin) or smeared onto coverslips for microscopy. Tissues (e.g. normal, diseased or malignant) are obtained by surgical removal (e.g. resection) or through needle suction (e.g. blood sample, tumour biopsy) or any other collection method to obtain tissues from living or deceased (e.g. a culled animal) organisms.

Any type of tissue sample can be collected. However, normal, diseased or malignant tissue is preferred for the purposes of this invention. Blood may also be collected because this, through circulation, provides both the input and output of any tissue. Once collected, a variety of methods can be employed for treating and/or storing cells and tissue samples to be tested. For example, tissues or cells may be fixed (e.g. in formalin), paraffin-embedded, or frozen fixed. Tissues may also be dissociated into single- cell suspensions using cocktails of proteolytic enzymes (e.g. trypsin) and then analysed by flow cytometry. Alternatively, tissues may be sectioned for microscopy or mounted as whole depending on the technique to be used. Permeabilisation may be performed by treating the sample with a surfactant.

The second step in the detection and imaging of hypoxia involves click chemistry. It is known that copper (I) is toxic to living systems (e.g. cells) and, therefore, click chemistry is restricted for use in the methods of the present invention to detection and/or imaging of hypoxia in non-living cells. Therefore, unlike the labelling step, the click chemistry step is done in vitro/ex vivo, i.e. outside the organism. Once the first step is done (i.e. the 2-nitrotmidazoie has been reduced by living hypoxic cells to form covalent adducts attached to the hypoxic cells) the method no longer requires live cells. It is understood that the detection and/or imaging of hypoxia according to the methods of the present invention is correlated with the presence of hypoxia during the labelling phase with the 2-nitroimidazole, i.e. when the cells are alive and before they are sampled, collected, harvested, or processed for subsequent click chemistry with a detectable probe. The click chemistry reaction is preferably between the covalent adduct of an alkyne functionalised nitroimidazole (i.e. the hypoxia marker) and an azide functionalised probe. Alternatively, the reaction is between the covalent adduct of an azide functionalised nitroimidazole and an alkyne functionalised probe. For the purpose of this invention "functionalised probe" is understood as either a probe carrying a terminal alkyne or a probe carrying an azide so that the terminal alkyne or the azide functionality enable the probe to undergo a click chemistry reaction, the "probe" being a molecule or moiety that allows detection by a suitable method. A dual approach involving both options is also possible, the dual approach involving the sequential administration of two different nitroimidazoles, one functionalised by a terminal alkyne and the other functionalised by an azide, for sequential labelling steps. The alkyne used in the click chemistry reaction is terminal and the probe used should be suitable for detection. Examples of useful probes include, and are not limited to, fluorescent, (bio)luminescent, radioactive, and NMR active probes. Fluorescent probes are particularly useful, and fluorophores bearing azide or terminal alkyne functional groups are known in the literature.

Surprisingly, the inventors have found that where click chemistry is utilised in the detection and/or imaging of hypoxic cells, certain methods of treating and/or storing tissue samples or cells may not be required. For example, fixing of tissues and/or cells and permeabilisation are not required. This provides an important advantage of the present invention over other methods of detecting and/or imaging hypoxic cells as the method of the present invention remains compatible with technologies and methodologies that rely on protein mobility (for example, SDS-PAGE, Western blotting, liquid chromatography, and MALDI). This is demonstrated in Example 19 which describes the use of the Western blotting technique with hypoxic cells labelled according to the method of the present invention. In contrast, where fixing and permeabilisation techniques are employed, technologies and methodologies that rely on protein mobility cannot be employed.

The two-step nature of the method of the present invention is illustrated in the Diagram 2 below. For illustrative purposes the diagram shows a nitroimidazole functionalised with a terminal alkyne and a probe functionalised with an azide, but the method can also be applied to the reverse orientation, i.e. a nitroimidazole functionalised with an azide and a probe functionalised with a terminal alkyne.

Diagram 2:

1) Labelling - living c

nitroimidazole

ypox a mar er

2) Click chemistry and detection

- in vitro or ex vivo

detection

signal

The invention therefore lies in the in vitro or ex vivo use of copper(l)-catalysed click chemistry reactions for use in the detection of hypoxic ceils, the reaction being between either a hypoxia marker derived from a nitroimidazole functionalised by a terminal alkyne and a probe functionalised by an azide, or alternatively between a hypoxia marker derived from a nitroimidazole functionaitsed by an azide and a probe functionalised by a terminal alkyne.

A variety of known nitroimidazole compounds could be used in the process of the invention.

Those involving terminal alkynes include:

Those involving azides include:

In a particularly preferred embodiment, the nitroimidazole used in accordance with the present invention is a compound of Formula (I):

N0₂

N^N-X-Y

Formula (I)

wherein:

N_n or 0„ heteroatoms (where n = any number from 1 to 3) which may be present as alcohol, ether, amine (primary, secondary or tertiary), amide (primary, secondary or tertiary), carbamate, azide or heterocyclic functional groups;

F_q atoms (where q = any number from 1 to 6);

Y represents a terminal alkyne (-GsC-H) or an azide (-N₃);

in an alternative preferred embodiment the nitroimidazole used in accordance with the present invention is a compound of Formula (II) or (III):

(III)

wherein:

X may be absent or present,

N„ or O_n heteroatoms (where n = any number from 1 to 3) which may be present as alcohol, ether, amine (primary, secondary or tertiary), amide (primary, secondary or tertiary), carbamate, azide or heterocyclic functional groups;

F_q atoms (where q = any number from 1 to 6);

Y is a terminal alkyne or an azide;

Z is

Preferably in Formulae (!), (II), or (III) X is a C_rC₈ alkyl chain linker, optionally saturated or unsaturated, optionally straight or branched, optionally containing cyclic units and optionally containing any one or more of:

N„ and/or O_n heteroatoms, which may be present as ether, amine, amide, carbamate, and/or heterocyclic functional groups,

n is any number from 1 to 3.

The inventors have also developed new 2-m^'troimidazoies which are useful in the process of the invention. These new compounds also form part of the invention in and of themselves as well as when used in the inventive process. Methods of synthesis for these new compounds are provided in Examples l to 6.

These include:

Examples 7 to 19 outline in detail how the click-chemistry based detection method can be carried out. In general terms the reaction can be described as follows. Given that the cells or tissue have been labelled with a hypoxia marker the hypoxia marker needs to be brought into contact with the appropriately matching functionalised probe in such a way that the click reaction can occur. This needs to be done in the presence of Cu(l). The Cu(l) can be added directly, for example in the form of CuBr or CuOAc. Alternatively, the Cu{l) can be generated in situ from metallic copper i.e. Cu(0), or from Cu(ll) salts such as CuS0₄. For example, reduction of Cu(ll) using suitable reductants, such as sodium ascorbate or a phosphine such as tris(2-carboxyethyl)phosphine, generates Cu(l). In the literature, click chemistry reaction times with biological samples vary from less than 5 minutes up to 60 minutes or more. Preferably, the click reaction takes about 30 minutes or less to carry out. These reaction times are much shorter than typically required for immunostaining and immunohistochemistry methods and thus represent an advantage (see Examples 14 and 17). The reaction can be performed at ambient temperature, or at elevated temperatures. Reactions can be optimised by the presence of a suitable metal chelating ligand. Examples of such iigands include TBTA and THPTA. These ensure that any side reactions are avoided or minimised by selectively chelating and stabilising the Cu(l) form. Chelating agents that compromise the Cu(l) catalytic activity, (e.g. EDTA, BSC) need to be avoided. If the probe contains a hapten then further steps may be required to bind a secondary probe in much the same way as a secondary immunostaining probe is used to bind (and detect) a primary immunostaining probe. The click reaction is very general and tolerant of a wide variety of structurally diverse azides and terminal alkynes, and this is one of the advantages of this technique. However, some requirements for optimal results have been suggested:³⁵

e Sodium ascorbate is the preferred reducing agent and should be present at concentrations of at least 2.5 mM

® Cu concentrations should fall in the range from 50-100 μΜ

® The ligand : Cu ratio should be at least 5 : 1

· Ascorbate should not be added to copper-containing solutions in the absence of the ligand

« Free thiols such as glutathione at more than two equivalents with respect to Cu are strong inhibitors of the CuAAC reaction and should be avoided

Surprisingly, the inventors have found that for the purposes of the method described in this patent the requirements pertaining to Cu concentrations, ligand to Cu ratios and compatible buffers are unnecessarily restrictive. In particular they have found that ^β Sodium ascorbate concenirations may range from 0.1 to 500 m ;

® Cu concentrations may fall in the range of between about 50 μΜ to about 1 mM;

Examples 7, 8, 11, 12 and 18 demonstrate that Cu concentrations within the range of between about 50 μΜ to about 1 mM are preferable for the purposes of the present invention. Example 11 also supports the finding that a ligand : Cu ratio of 1 : 1 is tolerated when employing click chemistry. With regard to the Tris buffer, the inventors found that a Tris buffer with a concentration of 50 mM at a pH 7.0 was tolerated. This is surprising given that the use of Tris buffer has previously been discouraged.³⁶

A. person skilled in the art will understand that a wider range of conditions will be compatible with the method described, in that a wider range of conditions will provide a sufficient concentration of cata!ytically active Cu(l) in solution to generate a detectable signal by click reaction in an appropriate time. In broad terms the copper concentration may range from 1 to 1000 μΜ, but preferably 50 to 200 μΜ, the ligand to copper ratio may range from 1 : 1 to 20 : 1, but preferably from 1 : 1 to 5 : 1; and the sodium ascorbate concentration may range from 0.1 to 500 mM, but preferably from 1 to 100 mM. A person skilled in the art will understand that the appropriate ranges will be influenced by factors such as the nature and solubility of the copper salt and ligand.

Subsequent imaging steps are dependent on the nature of the probe. Suitable imaging and/or detection techniques include fluorescence, radioactivity or (bio)luminescent techniques. The preferred imaging and/or detection technique is fluorescence. This may involve flow cytometry, fluorescence microscopy (wide field or laser confocal), a fluorescence plate reader, or any other detection device based on fluorescence signal. Preferably the probe employed is a fluorophore. Preferably the fluorophore probe is fluorogenfc, in that the fluorescent properties of the probe are induced or enhanced after the click chemistry reaction. A wide variety of suitable functionalised fluorophores are available, including azide- and alkyne-modified rhodamines, cyanines, coumarins, and fluoresceins, and their derivatives.³⁷'³⁸'³⁹'⁴⁰'^41,42,43 Many fluorophores are also available in reactive forms, such as activated esters or isocyanates or maleimides, which may be used to introduce the azide or alkyne functional group.

Alternatively, the probe can contain a radiolabel to be used with scintillation counting, autoradiography, or any other device designed to detect radioactivity. Non-fluorescent methods are typically based on enzymatic reactions such as peroxidase and alkaline phosphatase. These can be detected using light microscopy techniques. Luminescent or bioluminescent probes may also be employed. Typical equipment needed for detection of such probes include {bio)luminescence plate readers and microscopes. Where the probe contains a biotin moiety or other affinity tag these can be separated or isolated from the sample using an avidin-column or avidin-beads. Detection can then be carried out using standard analytical techniques such as HPLC, LC-MS, or other similar techniques.

Ideally, the samples are detected using a fully calibrated method with appropriate background correction and other corrections applied where necessary. This enables quantitative detection and imaging of 2-nitroimidazole adducts. It is to be noted that scales may be calibrated to read average p0₂ levels over the period of contact with the nitroimidazole. However, and similar to typical immunostaining protocols, full calibration need not necessarily be undertaken and alternatively detection signals may be compared within samples or across samples on a relative scale. In a typical case increased fluorescent staining of a hypoxia marker would indicate more hypoxia (i.e. lower oxygen concentration present at the time of contacting with the 2-nitroimidazole). Flow cytometry allows such measurements to be undertaken at the single-cell level, while microscopy provides an area or area measure (after appropriate background subtraction and thresholding) as output and thus allows analysis of the relative level and position of hypoxia within the sample at the time of contacting with the 2- nitroimidazo!e.

It is preferable that when the 2-nitroimidazole is azide-functionalised the click chemistry is conducted under low oxygen conditions. Preferably the low oxygen conditions are less than or equal to about 20% by volume. This is because low oxygen conditions helps to avoid or minimise non-specific binding of the alkyne-functionalised probe to other constitutes within the cell or tissue sample which are not related to hypoxia. Where such non-specific binding is allowed to occur, a high background noise can appear in the results of subsequent imaging and/or detection methods such that the desired signal resulting from the reaction between the alkyne-functionalised probe and hypoxia marker can be difficult to distinguish.

Where poor results are obtained from the imaging and/or detection methods, the second step of the method of the present invention can be repeated using fresh reagents until optimal results are obtained. Where the dual approach is employed, whereby two different nitroimidazoles, one functionatised by a terminal alkyne and the other by an azide, are sequentially administered during the labelling step, repeated exposure to functionalised probes by click chemistry methods is used to ensure that the probes do not react with each other in solution instead of the hypoxia markers in the cells.

The detection and/or imaging of hypoxic cells is also compatible with dual detection by antibody-based methods together with the click chemistry methods described above. For this reason, it is preferable that the detection and/or imaging of hypoxic cells includes such dual detection. This allows validation of click chemistry results with existing immunostaining methods which utilise hypoxia markers derived from EF5 or pimonidazole, for example, for detection and/or imaging of hypoxic cells. The level of validation can be regarded as high because the 2-nitroimidazo!e moiety of compounds of Formula (I) are identical to that of EF5 and pimonidazole and many other known and validated 2-nitroimidazoles for hypoxia imaging and detection, which increases the likelihood of similar enzymatic bioreduction and adduct formation in living cells (the "labelling" step)(Examples 14 and 17). Furthermore, compounds of Formula (II) and Formula (III) contain the same epitope as EF5 and pimonidazole and can, therefore, be detected using immunostaining methods with existing antibodies against these epitopes (Example 15). Preferably where dual detection by antibody-based methods is employed, the hypoxic cells are labelled with a 2-nitroimidazole in vivo or in vitro as previously described. Preferably, the 2-nitroimidazole is administered in vivo.

Comparison of click-chemistry and immunostaining detection methods reveals some similarities. Both are compatible techniques and can be done, sequentially, on the same sample. However, as indicated above, there are many technical advantages of click chemistry over immunostaining methods. The main differences are in the incubation times and temperature, the solvent and pH, the sample preparation required (fixation and permeabilisation, antigen retrieval, etc.), and the availability of suitable probe partners. In addition, immunostaining generally involves multiple demasking, blocking, and wash steps at various stages of the procedure.

Therefore, based on the above information, the inventive method for detecting and/or imaging hypoxic cells can include the following preparatory steps: Exposure of the tissues and/or cells to an alkyne or azide functionalised nitroimidazoie, allowing them to come into contact. Once in contact, oxygen-sensitive enzymatic bio-reduction of the nitroimidazoie takes place resulting in the formation of a covalent adduct ("hypoxia marker"). Such contact can be achieved by adding the nitroimidazoie to a culture or incubation medium containing the tissue samples or cells, or by administering the nitroimidazoie to the organism.

2) Collection of normal, diseased or malignant tissue samples using standard techniques, including and not limited to, surgical removal or biopsy. Cells may also be collected from in vitro samples using standard harvesting techniques.

3) Optional storage of tissue samples and cells prior to use in the second part of the method of the present invention.

Subsequent steps include the click-chemistry reaction, which should be carried out as described above, and the imaging of the hypoxic cells, preferably by fluorescence techniques. Consequently, it is preferable that the functionalised probe employed in the click-chemistry reaction contains:

1) A fluorophore to allow for imaging in devices designed to detect a fluorescent signal;

or alternatively either:

2) A radiolabel to allow for imaging in devices designed to detect radioactivity; or

3) Luminescent or bioluminescent probes to allow for imaging in (bio)luminescence plate readers and microscopes.

The invention may therefore also be seen as providing a method for identifying hypoxic cells in tissue samples taken from a living organism, and to which at least one alkyne functionalised 2-nitroimidazole (the alkyne being a terminal alkyne) and/or at least one azide functionalised 2-nitroimidazole has been administered, the method comprising the use of click chemistry for the detection and/or imaging of hypoxic cells.

The above description has focussed on the in vitro or ex vivo use of cells or tissue samples which have been obtained from a living organism. However, the method of the present invention may also be used in in vitro experiments on cell cultures which are propagated in a laboratory setting. However, before the labelling step and subsequent click chemistry reaction is carried out, it would be preferable to remove oxygen from the cell culture so that the cells within the culture mimic hypoxia conditions.

The present invention can also be seen as providing a composition for use in the detection and/or imaging of hypoxic cells, the composition including at least one nitroimidazoie functionalised by a terminal alkyne and/or at least one nitroimidazoie functionalised by an azide and a pharmaceutically acceptable carrier. Preferably the composition is formulated as a parenteral composition. Preferably, the methods of administration of the parenteral composition include, but are not limited to, intraperitoneal or intravenous administration.

The parenteral composition may also form part of a two-part composition for use in the detection and/or imaging of hypoxic cells, the two-part composition including:

Part A, which is the composition described above; and - Part B, which comprises a probe functionaiised by an azide or a probe functionaiised by a terminal alkyne and a suitable carrier;

wherein when the nitroimidazole of Part A is functionaiised by a terminal alkyne the probe cannot also be functionaiised by a terminal alkyne and when the nitroimidazole of Part A is functionaiised by an azide the probe cannot also be functionaiised by an azide.

Suitable nitroimidazoles for use in the above compositions are selected from any one or more of the nitroimidazoles discussed previously. In a preferred embodiment, the nitroimidazole is a compound of Formulae (I), (II) or (III).

As previously discussed, the amount of 2-nitroimidazoie for use in the present invention can be varied. Thus, the amount of 2-nitroimidazole for use in the compositions of the present invention can also be varied. The level of hypoxia marker that ends up being present within cells or a tissue sample is a function of concentration against time. That is, the higher the concentration of nitroimidazoles within the composition, the less contacting that may be required and vice versa. The concentration of nitroimidazoles within the composition should be sufficient to generate a detectable signal from hypoxic cells without causing solubility and/or toxicity complications. Likewise, the concentration of nitroimidazoles present within the composition should have minimal effect on biological processes. However, whether or not a particular concentration is sufficient is also dependent on a number of other factors, including but not limited to, the size of the mammal being administered the composition, whether the composition is administered via intraperitoneal or intravenous administration, the level of hypoxia present within the cell and/or tissue sample, the nature of the tissue sample (where tissue samples are analysed for the presence of hypoxia) and the metabolic capability of the tissue. As previously discussed, it is also understood that different nitroimidazoles may have different levels of toxicity. Generally, doses of 2-nitroimidazole range between 0.1 μg/kg to 1 g/kg as a single dose or can be divided into fractionated doses per day when the labelling is to take place in vivo. Preferably, the general dose is between 250 g/kg to 100 mg/kg. Variations in these dose levels can be adjusted using standard empirical routines for optimisation, which are well known in the art. Generally, concentrations of 2-nitroimidazole range between 0.1 g L to 1 g/L when the labelling is to take place in vitro or ex vivo. Preferably, the general concentration is between 250 μg/L to 100 mg/L. Variations in these concentrations can be adjusted using standard empirical routines for optimisation, which are well known in the art. For example, in in vitro experiments the inventors found that a nitroimidazole concentration of 100 μΜ (μιτιοΙ/L) gave an intense fluorescent signal during subsequent imaging steps, while during in vivo labelling of hypoxic cells and tissue samples taken from mice, the inventors found that a nitroimidazole concentration of 20 noM (resulting in a dose of 60 mg/kg) gave strong signals during subsequent imaging steps. EF5 (a known 2-nitroimidazole) is typically supplied in sterile vials at a concentration of 3 mg/mL. Before use it is dissolved in an aqueous solution of 5% dextrose and 2.4% ethanol and is administered intravenously at a rate of approximately 350 mL/h (resulting in a dose of 21 mg/kg)-⁴⁴ The skilled person would be able to determine suitable nitroimidazole concentrations for use in the compositions of the present invention without undue experimentation.

Suitable probes for use in Part B of the two-part composition include, but are not limited to, fluorescent, luminescent, radioactive, and NMR active probes. Fluorescent probes are particularly useful, and fluorophores bearing azide or terminal alkyne functional groups are known in the literature.

The composition inciuding at least one nitroimidazole functionaiised by a terminal alkyne and/or at least one nitroimidazole functionaiised by an azide and a pharmaceutically acceptable carrier can be administered to a living organism or to tissue samples or ceils in vitro, i.e. to a culture or incubation medium containing tissue samples or cells to be tested. Cells or tissue samples can be collected by standard techniques and methods as discussed previously. Preferably, where the composition is a parenteral composition it is administered to a living organism in vivo using intravenous or intraperitoneal methods, directly or in combination with any pharmaceutically acceptable carrier or salt known in the art. An example of a suitable carrier includes physiological saline. Other suitable carriers would be known to one skilled in the art and are described, for example in Remington: The Science and Practice of Pharmacy.*⁵ Examples of pharmaceutically acceptable salts include acetic or lactic acid. Examples of suitable vehicles include ethanol, propylene glycol and polyethylene glycol. Administering of the parenteral composition to the tissue samples or cells results in the labelling of hypoxic cells within a tissue sample or cells.

Where the composition is a two-part composition, Parts A and B are administered sequentially, wherein Part A is administered as described above and Part B is then administered together with a Cu{l) catalyst and suitable carriers as would be known to a person skilled in the art. The sequential administration of Part A and Part B of the two-part composition allows for the detection and/or imaging of hypoxic cells according to the method of the present invention.

As Cu(l) is toxic to living systems, Part B must be administered in vitro or ex vivo. Suitable sources of Cu(!) catalysts include Cu(l) salts such as CuBr or CuOAc, metallic copper (i.e. Cu(0)) or Cu(ll) salts such as CuS0₄. As previously stated, where Cu(ll) salts are employed, reduction of Cu(ll) using suitable reductants, such as sodium ascorbate or a phosphine such as tris(2-carboxyethyl)phosphine, is required. Suitable carriers for use with Part B of the two-part composition include, but are not limited to aqueous buffer solutions, and organic or aqueous solvents.

Therefore, the present invention could also be seen as providing a three-part composition for use in the detection and/or imaging of hypoxic cells, the three-part composition including:

- Part A, which is the composition including at least one nitroimidazole functionalised by a terminal alkyne and/or at least one nitroimidazole functionalised by an azide and a pharmaceutically acceptable carrier;

Part B, which comprises a probe functionalised by an azide or a probe functionalised by a terminal alkyne and a suitable carrier; and

- Part C, which comprises a Cu(l) catalyst and a suitable carrier;

Preferably Part A of the three-part composition is administered as described above for the two-part composition and preferably Part B and Part C are administered together and are administered in vitro or ex vivo for the reasons given above. Suitable carriers for use with Part B and Part C of the three-part composition include, but are not limited to aqueous buffer solutions, and organic or aqueous solvents. The sequential administration of Part A and Parts B and C together allows for the detection and/or imaging of hypoxic cells according to the method of the present invention. As discussed previously, the click chemistry aspect of the method of the present invention may be optimised by the presence of a suitable metal chelating ligand, which selectively chelates and stabilises the Cu(l) form. Examples of suitable chelating ligands include TBTA and THPTA. Therefore, the present invention could also be seen as providing a four-part composition for use in the detection and/or imaging of hypoxic cells, the four-part composition including:

Part A, which is the composition including at least one nitroimidazole functionalised by a terminal alkyne and/or at least one nitroimidazole functionalised by an azide and a pharmaceutically acceptable carrier;

Part C, which comprises a Cu(l) catalyst and a suitable carrier; and

Part D, which comprises a chelating ligand capable of selectively chelating and stabilising Cu(l); wherein when the nitroimidazole of Part A is functionalised by a terminal alkyne the probe cannot also be functionalised by a terminal alkyne and when the nitroimidazole of Part A is functionalised by an azide the probe cannot also be functionalised by an azide.

Preferably Part A is administered before Part B, Part C and Part D and preferably Part B, Part C and Part D are administered together and are administered in vitro or ex vivo. Preferably the methods of administration are as previously described.

Suitable chelating ligands for use in the four-part composition include TBTA and THPTA. Other suitable chelating ligands for use in the four-part composition are known^46,47 to those skilled in the art.

The invention also provides for the use of a nitroimidazole functionalised by a terminal alkyne or a nitroimidazole functionalised by an azide, in the manufacture of a composition for the detection and/or imaging of hypoxic cells. That medicament can also be the two part composition referred to above and includes the use of the probe.

Alternatively, the method provides a nitroimidazole functionalised by a terminal alkyne or a nitroimidazole functionalised by an azide for use in the detection and/of imaging of hypoxic cells. Suitable nitroimidazo!es are selected from any one or more of the nitroimidazoles discussed previously. In a preferred embodiment, the nitroimidazole is a compound of Formulae (I), (II) or (111).

The invention also provides novel compositions for use in the labelling of hypoxic cells, the compositions comprising any one of the five new 2-nitroimidazoles developed by the inventors together with suitable carriers as discussed above and as would be known to a person skilled in the art. These new 2- nitroimidazoles include:

The invention can also provide a kit of parts, the kit including either a 2-nitroimidazole functionalised by a terminal alkyne and a probe functionalised by an azide, or a 2-nitroimidazole functionalised by an azide and a probe functionalised by a terminal alkyne. The kit may also include a suitable copper salt, chelating ligand and reducing agent and other materials suitable for use in carrying out the method of the present invention.

The kit can be for:

1. In vitro use (for example, cell culture); or

2. Ex vivo use (for example, following administration of the nitroimidazole to mice and collection of the sample of interest).

The kit can also be for:

1. Single labelling (i.e. one nitroimidazole is functionalised by a terminal alkyne or an azide); or

2. Dual labelling (i.e. one nitroimidazole is functionalised by a terminal alkyne and another nitroimidazole is functionalised by an azide).

References to such uses are not intended to be restrictive.

Where the kit is used for single labelling for in vitro use it may contain at least one nitroimidazole functionalised by a terminal alkyne or an azide, a suitable probe, a source of Cu(l), and a metal chelating ligand. In addition, the kit may include aqueous buffer solution(s), or salts to make up buffer solutions(s), and organic solvent(s). The kit may further include cell fixation buffers and/or reagents, and cell permeabiiisation buffers and/or reagents. As already indicated, with cell permeable probes cell fixation and permeabiiisation can be avoided. The kit may also include protocols and instructions for using the kit.

Where the kit is used for single labelling for in vivo use it may contain at least one nitroimidazole functionalised by a terminal alkyne or an azide, a suitable probe, a source of Cu(l), and a metal chelating ligand. In addition, the kit may include aqueous buffer solution(s), or salts to make up buffer solutions(s), and organic solvent(s). The kit may contain sterile medium for injection into a living organism. The kit may further comprise one or more enzymes for dissociation of excised tissue and other necessary tools to prepare single-cell suspensions from excised/resected/co!lected tissue. Examples of suitable enzymes include pronase, collagenase and DNAase I. Use of enzymes would typically only apply to the use of single-cell suspensions for flow cytometry, for example. When using histochemical methods (for example, fluorescence microscopy), thin tissue sections are cut and labelled with a probe or probes. The kit may further include cell fixation buffers and/or reagents, and cell permeabilisation buffers and/or reagents. The kit may also include protocols and instructions for using the kit.

Employing click chemistry in methods for the detection and/or imaging of hypoxia is a novel technique which surprisingly offers many advantages. The technique allows the use of a range of click partners that can be either easily obtained commercially or synthesised by easy steps. This gives straightforward access to, for example, fiuorophores of any choice of excitation wavelength allowing for much easier and quicker access than immunostaining probes which typically only come with a limited range of (conjugated) fiuorophores. Large quantities of functionalised probes (such as functionalised fiuorophores) are easily obtained by chemical synthesis. Second, the technique is faster with high sensitivity. The click reaction can take 30 minutes or less to carry out, instead of overnight as with standard immunostaining probes. Third, the technique is compatible with existing immunostaining based methods and can therefore be employed for dual staining purposes. Fourth, the option of using either an alkyne functionalised nitroimidazole or an azide functionalised imidazole provides an orthogonal reporter pair for detecting or imaging changes in hypoxia. Fifth, the technique is simpler than known methods to carry out and requires fewer steps. This is especially the case when fixation and permeabilisation steps are optionally not employed. This in turn renders the technique compatible with other analytical techniques, such as Western blotting. Sixth, when used with fixed and permeabilised biological samples, antigen retrieval steps that are necessary for immunostaining may be optional and may not need to be employed. Finally, the technique is more suitable than immunostaining in applications requiring good tissue penetration because of the much smaller molecular size of the required probes and reaction components.

EXAMPLES

Examples 1-6, referring to Schemes 1-4, are representative of the invention, and provide detailed methods for preparing the compounds of the invention (nitroimidazoles 4, 8, 9, 10 and 11) and the known nitroimidazole 2. In these examples, elemental analyses were carried out in the Microchemical Laboratory, University of Otago, Dunedin, New Zealand. Melting points were determined on an Electrothermal 2300 Melting Point Apparatus. NMR spectra were obtained on a Bruker Avance-400 spectrometer at 400 MHz for H spectra referenced to Me₄Si. Column chromatography was carried out on silica gel unless otherwise stated.

Reagents:

a) ICH₂CONHCH₂CCH/Cs₂C0₃/D F/50 °C, 63%

b) /-Butyl chloroformate/NMM/propargylamine/CH₃CN/0 °C to r.t., 23% c) /-Butyl chloroformate/NMM/propargylamine/THF/O °C to r.t., 9% d) /-Butyl chloroformate/NM /H₂N(CH₂)₂N3/CH₃CN/0 °C to r.t., 55%

Scheme 1

Reagents:

a) NIS/D F/100°C,30%

b) NBS/CH₃CN, 100%

c) Cul/Nal/H₃CNH(CH₂)₂NHCH₃/dioxane/140 °C, 22%

d) i) TMSacetylene/Cul/PdCI₂(Ph₃P)₂/Et₃N/THF; ii) K₂C0₃/ eOH, 5%

Scheme 2

3 10 Reagents:

a) HC=C(CH₂)₂NH₂.HCI/PyBOP/DIPEA/CH₃CN/0⁰C, 51%

b) (HC≡CCH₂)₂NH.HCl/PyBOP/DIPEA/CH₃CN/0 ^oC, 58%

Scheme 3

Reagents:

a) i) HC≡C(CH₂)₄C≡CTIVlS/Cu!/PclCl2{Ph3p)₂/Et3N/THF; ii) K₂C0₃/MeOH, 15%

Scheme 4 Example 1. Synthesis of 2-(2-nitro-lW-imidazol-l-yl)-W-(2-propynyl)acetamide (2) (Scheme 1).

Method 1: Cs₂C0₃ (288 mg, 0.89 mmol) was added to a solution of azomycin (1) (100 mg, 0.89 mmol) in D F (5 mL) and the mixture was heated (50 °C) for 2 h under N₂. A/-Ethynyl-2-iodoacetamide⁴⁸ (197 mg, 0.89 mmol) was added and the mixture was heated (50 °C) for a further 3 h under N₂. EtOAc and water were added and the aqueous layer was extracted with EtOAc (x 5). The combined organic extracts were washed with brine and dried (Na₂S0₄) followed by flash chromatography (CH₂Cl2/MeOH; 100:0 to 47:3; gradient elution) to give nitroimidazole 2 (116 mg, 63%) as a white powder (HPLC purity 99.4%): mp 171- 173 °C; ^:H IM R [(CD₃)₂SO] 5 8.77 (t, J = 5.0 Hz, 1 H), 7.61 (d, J = 0.8 Hz, 1 H), 7.18 (d, J = 0.8 Hz, 1 H), 5.11 (s, 2 H), 3.91 (dd, J = 5.4, 2.5 Hz, 2 H), 3.15 (t, J = 2.5 Hz, 1 H).

Method 2: /V-Methylmorpholine {142 mg, 1.40 mmol) and isobutyl chloroformate (192 mg, 1.40 mmol) were added to a cooled (0 °C) solution of (2-nitro-ltf-imidazol-l-yl)acetic acid⁴⁹ (3) (200 mg, 1.17 mmol) in CH₃CN (100 mL). After 1 h propargyl amine (77 mg, 1.40 mmol) was added and the reaction was allowed to warm to room temperature over 48 h. EtOAc and water were added and the aqueous layer was extracted with EtOAc (x 5). The combined organic extracts were washed with saturated aqueous NaHC0₃, brine and dried (Na₂S0₄) followed by flash chromatography (CH₂CI₂/Me0H; 100:0 to 47:3; gradient elution) to give nitroimidazole 2 (57 mg, 23%) as a white powder (HPLC purity 99.8%).

The same procedure using THF as solvent gave nitroimidazole 2 (9%) (HPLC purity 98.9%).

Example 2. Synthesis of W-(2-azidoethy!)-2-(2-nitro-lW-!midazol-l-yl)acetamide (4) {Scheme 1).

/V-Methylmorpholine (210 mg, 2.11 mmol) and isobutyl chloroformate (240 mg, 1.75 mmol) were added to a cooled (0 °C) solution of 3 (300 mg, 1.75 mmol) in CH₃CN (50 mL). After 1 h 2-azidoethylamine⁵⁰ (180 mg, 2.11 mmol) was added and the reaction was allowed to warm to room temperature over 48 h. CH₃CN was removed and EtOAc and water were added and the aqueous layer was extracted with EtOAc (x 6). The combined organic extracts were dried (Na₂S0₄) followed by flash chromatography (CH₂Cl₂/MeOH; 100:0 to 47:3; gradient elution) to give nitroimidazole 4 (230 mg, 55%) as a white powder (HPLC purity 99.9%): mp 120-121 °C; *H NMR [(CD₃)₂SO] δ 8.54 (bs, 1 H), 7.61 (d, J = 1.0 Hz, 1 H), 7.18 (d, J = 1.0 Hz, 1 H), 5.10 (s, 2 H), 3.38 (m ,2 H), 3.28 (m, 2 H). Anal, calcd. for C₇H₉N₇0₃: C, 35.15; H, 3.79; N, 40.99. Found: C, 35.54; H, 3.81; N, 41.08.

Example 3. Synthesis of 2-(4-ethynyl-2-nitro-lH-imidazol-l-Yl)-/V-(2_/2,3,3,3- pentafluoropropyl)acetamide (8) (Scheme 2)

2-(4-lodo-2-nitro-lH-imidazol-l-vl)-/V-(2,2,3,3,3-pentafluoropropyl)acetamide (6).

Method 1: /V-lodosuccinimide (1.86 g, 8.28 mmol) was added to a heated (60 °C) solution of 2-(2-nitro- lH-imidazol-l-yl)-/V-(2,2,3,3,3-pentafluoropropyl)acetamide⁵¹ (5) (500 mg, 1.66 mmol) in DMF (5 mL). After 48 h a further portion of NIS (920 mg, 4.41 mmol) was added and the temperature was raised to 100 °C. After a further 56 h DMF was removed and the residue was extracted with EtOAc, which was washed with 1% sodium disulfite solution, brine and dried (Na₂S0₄). Flash chromatography (petroleum ether/EtOAc; 7:3 to 1:4; gradient elution) gave 6 (210 mg, 30%) as a light brown powder: mp 166-169 °C (/-Pr_zO); H NMR (CDCI₃) δ 7.25 (s, 1 H), 6.13 (bs, 1 H), 5.06 (s, 2 H), 4.05 (td, J = 14.8, 6.3 Hz, 2 H). Anal, calcd. for C₈H₆F₅I N₄0₃ + 0.1/-Pr₂O: C, 23.57; H, 1.70; N, 12.78. Found: C, 23.87; H, 1.50; N, 13.01.

Method 2:⁵² W-Bromosuccinimide (1.79 g, 9.93 mmol) was added to a solution of 5 (600 mg, 1.99 mrnol) in CH₃CN (5 mL). After 48 h a further portion of NBS (300 mg, 1.67 mmol) was added and a further portion of NBS (200 mg, 1.11 mmol) was added after a further 12 h. After a further 12 h CH₃CN was removed and EtOAc and water were added. The aqueous layer was extracted with EtOAc (x 3) and the combined organic extracts were washed with 1% sodium disulfite solution, brine and dried (Na₂S0₄) to give 2-(4-bromo-2-nitro-lW-imidazol-l-yl)-W-(2,2,3,3,3-pentafluoropropyl)acetamide (7) (760 mg, 100%) as a light brown powder: mp 164-166 °C (EtOAc); ^XH NMR (CDCI₃) δ 7.15 (s, 1 H), 6.03 (bs, 1 H), 5.07 (s, 2 H), 4.04 (td, J = 14.7, 6.4 Hz, 2 H). Anal, calcd. for C₈H₆BrF₅N403 + O.lEtOAc: C, 25.89; H, 1.76; N, 14.37. Found: C, 26.03; H, 1.53; N, 14.61.

Nal (315 mg, 2.10 mmol), Cul (10 mg, 0.05 mmol) and W, /v'-dimethylethylenediamine (10 mg, 0.11 mmol) were added to a nitrogen-purged solution of 7 (400 mg, 1.05 mmol) in dioxane (3 mL). The mixture was heated (130 °C) in a sealed tube for 15 h. Further portions of Nal (315 mg, 2.10 mmol), Cul (10 mg, 0.05 mmol) and N, W'-dimethylethylenediamine (10 mg, 0.11 mmol) were added and the mixture was heated (140 °C) for a further 48 h. The mixture was filtered through Celite (EtOAc/MeOH) and solvents were removed. EtOAc and water were added and the aqueous layer was extracted with EtOAc (x 3) and the combined organic extracts were washed with 1% sodium disulfite solution, brine and dried (Na₂S0₄). Flash chromatography (CH₂CI₂/MeOH; 100:0 to 49:1; gradient elution) gave 6 (100 mg, 22%).

2-(4-Ethynyl-2-nitro-lH-imidazol-l-yl)-/V-(2,2,3,3,3-pentafluoropropyl}acetamide (8).

Cul (6 mg, 0.03 mmol), PdCI₂(Ph₃P)₂ (10 mg, 0.015 mmol) and trimethylsilylacety!ene (115 mg, 1.17 mmol) were added to a nitrogen-purged solution of 6 (250 mg, 0.59 mmol) in THF (2 mL)/Et₃N (2 mL). After 18 h the reaction was filtered through silica gel/Celite (EtOAc) and solvents were removed. The residue was extracted with EtOAc which was washed with water, brine and dried (Na₂S0 ). EtOAc was removed and the crude product was used in the next step.

The crude product was added to a suspension of K₂C0₃ (80 mg) in MeOH (5 mL) and the reaction was stirred under nitrogen for 1 h. MeOH was removed followed by flash chromatography (CH^I^MeOH; 100:0 to 49:1; gradient elution) and pic chromatography (EtOAc) to give nitroimidazole 8 (10 mg, 5%) as a light brown powder (HPLC purity 98.6%). H N MR (CD₃OD) δ 7.66 (s, 1 H), 5.22 (s, 2 H), 4.02 (t, J = 15.3 Hz, 2 H), 3.65 (s, 1 H), 1 proton not observed. HRMS (ESI) for C₁₀H₈F₅N₄O₃ calcd. 327.0511, found 327.0508.

Example 4. Synthesis of / -(3-Butynyl)-2-{2-nitro-lW-lmida2ol-l-yl)acetam?de (9) (Scheme 3)

PyBOP (1.29 g, 2.48 mmol) and 3-butyn-l-amine hydrochloride (200 mg, 1.91 mmol) were added to a solution of 3 (330 mg, 1.91 mmol) and DIPEA (740 mg, 5.71 mmol) in CH₃CN (10 mL) at 0 °C. After 30 min the solvent was evaporated and EtOAc and H₂0 were added. The mixture was extracted with EtOAc (x 3) and the combined organic extracts were washed with brine a nd then dried (Na₂S0₄). Flash chromatography (CH₂CI₂/MeOH; 100:0 to 49:1; gradient elution) followed by trituration (MeOH/EtOAc//Pr₂0) gave nitroimidazole 9 (220 mg, 51%) as a cream powder (HPLC purity 99.2%): mp 142-144 °C; *H NMR (CD₃OD) 6 7.22 (d, J = 1.0 Hz, 1 H), 7.16 (d, J = 1.0 Hz, 1 H), 6.07 (s, 1 H), 5.04 (s, 2 H), 3.47 (q, J = 6.2 Hz, 2 H), 2.44 (td, J = 6.3, 2.6 Hz, 2 H), 2.03 (t, J = 2.6 Hz, 1 H). Anal, calcd. for

C, 48.75; H, 4.58; IM, 25.10. Found: C, 49.26; H, 4.73; N, 25.50.

Example 5. Synthesis of 2-(2-Nitro-lH-imidazol-l-yl)-A/,/V-di(2-propynyl)acetamide (10) (Scheme 3) PyBOP (1.19 g, 2.28 mmol) and W-(2-propynyl)-2-propyn-l-amine hydrochloride^53,54 (226 mg, 1.75 mmol) were added to a solution of 3 (300 mg, 1.75 mmol) and DIPEA (680 mg, 5.26 mmol) in CH₃CN (10 mL) at 0 °C. After 30 min the solvent was evaporated and EtOAc and H₂0 were added. The mixture was extracted with EtOAc (x 3) and the combined organic extracts were washed with brine and then dried (Na₂S0₄). Flash chromatography (CH₂Cl₂/MeOH; 100:0 to 49:1; gradient elution) followed by trituration {CH₂c iPr₂0) gave nitroimidazole 10 (250 mg, 58%) as a cream powder (HPLC purity 99.2%): mp 122- 124 °C; *H NMR (CD₃OD) 6 7.20 (s, d, J = 1.1 Hz, 1 H), 7.08 (d, J = 1.1 Hz, 1 H), 5.36 (s, 2 H), 4.35 (s, 2 H), 4.31 (s, 2 H), 2.47 (s, 1 H), 2.31 (s, 1 H). Anal, calcd. for C_uH₁₀N₄O₃ ·¼Η₂0: C, 52.70; H, 4.22; N, 22.35. Found: C, 52.82; H, 4.16; N, 22.29.

Example 6. Synthesis of 2-[2-Nitro-4-(l,7-octadiynyl)-lH-imidazol-l-yl3-/V"(2_f2,3,3,3- pentafiuoropropyljacetamide (11) (Scheme 4)

Cul (6 mg, 0.03 mmol), PdCI₂(Ph₃P)₂ (11 mg, 0.016 mmol) and trimethyl(l,7-octadiynyl)silane⁵⁵ (230 mg, 1.31 mmol) were added to a nitrogen-purged solution of 6 (280 mg, 0.65 mmol) in THF (3 mL) and Et₃N (3 mL). After 36 h further portions of Cul (6 mg, 0.03 mmol) and PdCl₂(Ph₃P)₂ (11 mg, 0.016 mmol) were added. After 48 h the reaction mixture was filtered through silica gel and Celite, eluting with EtOAc, and the solvents were evaporated. The residue was extracted with EtOAc and the extract was washed with water and then brine, and then dried (Na₂S0₄). The EtOAc was evaporated and the crude product was used in the next step.

The crude product was added to a suspension of K₂C0₃ (87 mg) in MeOH (10 mL) and the reaction was stirred under nitrogen for 48 h. MeOH was evaporated and the residue was purified by flash chromatography (petroleum ether/EtOAc; 9:1 to 3:2; gradient elution) and preparative layer chromatography (x 2) (petroleum ether/EtOAc; 1:1; CH^I^ eOH; 95:5). Trituration (EtOAc/CH₂CI₂/petroleum ether) gave nitroimidazole 11 (40 mg, 15%) as a light brown powder (HPLC purity 93.3%): mp 130-131 °C; H NMR (CD₃OD) δ 7.18 (s, 1 H), 6.19 (t, J = 5.9 Hz, 1 H), 5.06 (s, 2 H), 4.04 (td, J = 14.6, 6.4 Hz, 2 H), 2.43 (t, J = 6.7 Hz, 2 H), 2.24 (td, J = 6.8, 2.7 Hz, 2 H), 1.96 (t, J = 6.7 Hz, 1 H), 1.77-1.62 (m, 4 H). Anal, calcd. for C_l6Hi₅FsN₄0₃-/spetroieum ether: C, 48.78; H, 4.24; N, 13.23. Found: C, 49.01; H, 4.01; N, 13.23.

Example 7. Click reaction in solution with nitroimidazole 2

The ability of 2 to undergo a click reaction in aqueous solution with a variety of fluorophore azides was investigated by HPLC analysis. The fluorophore azides used (Figure 1) are commercially available (Alexa Fluor 488 azide, Alexa Fluor 647 azide), or prepared by reported methods (coumarin azide,⁵⁵ Nile Blue C3 azide"), or prepared by simple modification of reported methods (Nile Blue C6 azide,⁵⁷ tetramethylrhodamine azide⁵⁸). Each reaction mixture contained nitroimidazole 2 (10 μΜ), the fluorophore azide (20 μ ), CuS0₄ (100 μΜ), and TBTA ligand⁵⁹ (500 μΜ), in 100 mM phosphate buffer (pH 7.0) containing 5% DMSO. The reaction was initiated by the addition of sodium ascorbate (to a final concentration of 5 mM), and aliquots were withdrawn from the sealed reaction vessel (a brown glass HPLC vial held at room temperature) for analysis by HPLC (Alltima 2.1 x 150mm C8 5μιτι column and a 45 mM aqueous ammonium formate buffer (pH 4.5)/MeCN gradient) interfaced with a diode-array detector for UV/Vis- absorbance detection.

An example is illustrated in Figure 2 for the reaction between nitroimidazole 2 and Nile Blue C6 azide, showing chromatograms in Figure 2a and the UV-Vis spectra of the associated peaks in Figure 2b. The trace at t=0 min refers to the reaction mixture before the addition of sodium ascorbate. The trace at t=67 min shows consumption of 2 and of about half of the fluorophore azide (present in two-fold excess), accompanied by the formation of the click product, with a UV-Vis spectrum very similar to that of the fluorophore azide.

The progress of the reaction was monitored by comparison of peak areas over time with peak areas from the t=0 min samples, and is shown in Figure 3 for reaction with each of the fluorophore azides. For each fluorophore azide complete click reaction corresponds to consumption of half of the initially present material, and for the click product complete reaction corresponds to formation of half of the peak area of the initially present fluorophore azide (assuming equal extinction coefficients).

The data clearly show that under these conditions 2 undergoes a fast click reaction with each of the fluorophore azides illustrated. Before the first sampling point (ca. t=20 min) more than half of the reaction partners have been consumed, and more than half of the maximal amount of click reaction product has been produced.

The reaction between nitroimidazole 2 and coumarin azide was further investigated in a variety of aqueous buffers under otherwise identical conditions. The results are illustrated in Figure 4, and show that the click reaction is compatible with a variety of aqueous buffers (100 mM phosphate buffer (pH 7.0) or 50 mM Hepes buffer (pH 7.0) or 50 mM Tris buffer (pH 7.0)), but that the presence of an excess of the metal chelating agent EDTA (1 mM) completely suppresses the reaction. The inclusion of the Cu catalyst is clearly essential and this is consistent with literature reports that Cu(l) increases the reaction rate of the azide-alkyne cycloaddition reaction by about 10 million-fold compared to the thermal process.⁶⁰

Example 8. Click reaction in solution with nitroimidazole 4

The ability of nitroimidazole 4 to undergo a click reaction in aqueous solution with a variety of fluorophore alkynes was investigated by HPLC analysis. The fluorophore alkynes used (Figure 5) are commercially available (Alexa Fluor 488 alkyne) or prepared by reported methods (Nile Blue alkyne⁵⁷ and cyanine alkyne⁵⁷).

Each reaction mixture contained nitroimidazole 4 (10 μΜ), the fluorophore alkyne (20 μΜ), CuS0₄ (100 μΜ), and TBTA ligand (500 μΜ), in 100 mM phosphate buffer (pH 7.0) containing 5% DMSO. The reaction was initiated by the addition of sodium ascorbate (to a final concentration of 5 mM), and aliquots were withdrawn from the sealed reaction vessel (a brown glass HPLC vial held at room temperature) for analysis by HPLC (A!ltima 2.1 x 150mm C8 5μηη column and a 45 mM aqueous ammonium formate buffer (pH 4.5)/MeCN gradient) interfaced with a diode-array detector for UV-Vis- absorbance detection.

Figure 6 illustrates an example for the reaction between nitroimidazole 4 and Nile Blue alkyne, showing chromatograms in Figure 6a and the UV-Vis spectra of the associated peaks in Figure 6b. The trace at t=0 min refers to the reaction mixture before the addition of sodium ascorbate. The trace at t=42 min shows consumption of some of 4 and the fiuorophore alkyne (present in two-fold excess), accompanied by the formation of the click product, with a UV-Vis spectrum very similar to that of the fiuorophore alkyne. Similar results were observed for the reaction of 4 with Alexa Fluor 488 alkyne and cyanine alkyne.

Example 9. Click reaction in solution with nitroimidazoles 8, 9, 10, and 11

The ability of nitroimidazoles 8, 9, 10, and 11 to undergo click reactions in aqueous solution with fiuorophore azides was investigated by HPLC analysis. The reactions were performed as described in Example 7 using phosphate buffer, and the fiuorophore azide used was either Alexa Fluor 488 azide (for nitroimidazoles 8, 9, and 11) or Alexa Fluor 647 azide (for nitroimidazole 10). The progress of the reactions was monitored by comparison of peak areas over time with peak areas from the t=0 min samples, as described in Example 7, and the results are shown in Figure 7.

The data show that under these conditions all of the 2-nitroimidazoles undergo click reactions with fiuorophore azides. The reaction of nitroimidazole 10 is very fast and, compared to the other compounds, results in the consumption of more fiuorophore azide and the production of more click product, based on comparison of relative peak areas. 2-Nitroimidazole 10 contains 2 alkyne functional groups whereas nitroimidazoles 8, 9, and 11 contain one. The observed results are compatible with fast click reaction of both alkyne functional groups of nitroimidazole 0; This was confirmed by mass spectral analysis of the click product derived from 10. Compared to nitroimidazole 10 (m/z 246.1) and Alexa Fluor 647 azide (m/z 766.3) the click product had m/z 890.4, corresponding to the doubly charged ion of the product incorporating two Alexa Fluor 647 azide units. The product corresponding to click reaction of just one alkyne was not observed.

Example 10. Cytotoxicity of nitroimidazoles 2 and 4

The nitroimidazole alkyne 2 and nitroimidazole azide 4 were assessed in comparison to EF5 to see if any cytotoxicity is associated with the introduction of the alkyne or azide functional group. Clonogenic assays for cell sterilisation under aerobic and hypoxic conditions were performed using stirred and continuously gassed (5% C0₂ in air or N₂, respectively) suspensions of the human cervical carcinoma cell line SiHa (at 10⁵ cells/mL) as previously described.⁶¹ The drug exposure was 4 h. Colonies were grown for 14 days and stained with methylene blue, and those with >50 cells were counted to determine the plating efficiency (PE). Surviving fraction (SF) was calculated as PE(treated)/PE(controls). The results presented in Figure 8 show that no cell killing was associated with either nitroimidazoles 2 or 4 under aerobic (oxic) conditions up to the highest concentration tested (300 μΜ), and that under hypoxic conditions only, minimal cell killing was observed and only at the highest concentration used. This level of cytotoxicity is considerably less than the Ci₀ (concentration required for 1 log of cell kill, dotted horizontal line in Figure 8), and is not significantly different to that observed for EF5. Example 11. Click reaction in vitro using nitroimidazole 2

The nitroimidazole alkyne 2 was assessed for its ability to selectively label hypoxic cells in vitro using click chemistry with analysis by flow cytometry. Stirred suspensions of SiHa cells (at 10⁵ cells/mL) were continuously gassed at 37 °C under either oxic (5% C0₂ in 0₂) or hypoxic (5% C0₂ in N₂) conditions and treated with 2 (100 μΜ) for 2 h. At the end of the incubation period samples (1 x 10^s cells) were harvested by centrifugation, washed (1% BSA in PBS), fixed (4% paraformaldehyde in PBS at room temperature for 15 min, followed by washing), and permeabilised (0.3% Tween 20 in PBS at 4 °C for 30 min, followed by washing). A click reaction cocktail was prepared as follows: 10 μΜ Alexa Fluor 488 azide (from 2 mM stock in DMSO), 1 m CuS0₄ (from 100 mM stock in water), 1 m TBTA (from 200 mM stock in DMSO), and 100 m sodium ascorbate (from 2.5 M stock in water) in 10 mM Tris-saline at pH 7.4. The click reaction cocktail was used within 15 minutes of preparation, with the sodium ascorbate added last and immediately before addition to the cells. The click reaction cocktail (100 μΐ) was added to the prepared cell samples and incubated at room temperature for 30 min while protected from light. The cells were washed three times with PBS and resuspended in PBS for analysis by flow cytometry. The results are shown in Figure 9 where the histograms show relative fluorescence on a log scale on the x- axis and cell counts on the y-axis. Sample a) refers to hypoxic cells that were not exposed to 2, but otherwise treated identically to samples b) and c), including the click protocol. It is clear that there is minimal fluorescence associated with the oxic cells, but strong fluorescence associated with the hypoxic cells, clearly differentiating the two cell populations. If the median background staining from the blank sample a) is subtracted, the ratio between the hypoxic and oxic populations is 62-fold.

Similar results were observed when the fluorophore azide used was Alexa Fluor 647 azide or tetramethylrhodamine azide. A summary of these results is presented in Table 1. (Note, In Table 1 the values recorded for tetramethylrhodamine azide are absolute fluorescence readings since no blank was recorded for this experiment).

Table 1

This experiment was repeated for a variety of other human tumour cell lines, and in every case the nitroimidazole alkyne 2 was found to be capable of clearly distinguishing between oxic and hypoxic cells following click reaction with Alexa Fluor 488 azide. The average ratio between the fluorescence of hypoxic and oxic cells found in these experiments was as follows: HT29 (colon adenocarcinoma) 36 ± 6; A2780 (ovarian carcinoma) 38 ± 7; Panc-1 (pancreatic carcinoma) 48 ± 10; HCT116 (colon cancer) 55 ± 7; and H1299 (non-small cell lung carcinoma) 36 ± 3. Example 12. Click reaction in vitro using nitroimidazo!es 9 and 10

The nitroimidazole alkynes 9 and 10 were assessed for their ability to selectively label hypoxic cells in vitro using click chemistry with analysis by flow cytometry. SiHa cells were exposed to each nitroimidazole and then fixed and permeabilised as described in Example 11. The click reaction cocktail contained 10 μΜ Alexa Fluor 488 azide, 1 mM CuOAc, 5 m THPTA³⁶ and 100 mM sodium ascorbate in 10 mM Tris-saline at pH 7.4. The CuOAc stock was freshly prepared, and click reaction cocktail was used within 15 minutes of preparation, with the sodium ascorbate added last and immediately before addition to the cells. The cells were exposed to the click cocktail and analysed by flow cytometry as described in Example 11. The results are shown in Figure 10 where a) refers to cells exposed to nitroimidazole 9 and b) refers to cells exposed to nitroimidazole 10. Unstained samples were not exposed to nitroimidazole or click procedure, while blank samples were not exposed to nitroimidazole but did undergo the click procedure. In both Figure 10a and Figure 10b it is apparent that there is minimal fluorescence associated with oxic cells, but strong fluorescence associated with hypoxic cells, clearly differentiating the two cell populations. The ratio between the fluorescence of hypoxic and oxic cells is greater than 100 for both nitroimidazoles 9 and 10. The fluorescence of hypoxic cells treated with nitroimidazole 10 is approximately twice as strong as the fluorescence of hypoxic ceils treated with nitroimidazole 9. This is consistent with the observations of click reactions in solution described in Example 9, where nitroimidazole 10 undergoes a fast click reaction with two equivalents of fluorophore azide. The increase in fluorescent signal by the incorporation of, more than one clickable functional group in the nitroimidazole may be a useful and general way of increasing the sensitivity for click detection of hypoxic cells.

Example 13. Click reaction in vitro using nitroimidazole 4

The nitroimidazole azide 4 was assessed for its ability to selectively label hypoxic cells in vitro using click chemistry with analysis by flow cytometry. SiHa cells were exposed to nitroimidazole 4, then fixed, permeabilised, and clicked as described in Example 11, except that after the first 30 min clicking the cells were exposed to fresh click cocktail for a further 30 min. In addition, the click procedure was also conducted in an anaerobic chamber under conditions of severe anoxia (<0.001% 0₂). The results are shown in Figure 11 where panel a) refers to samples clicked under a normal atmosphere and panel b) refers to samples clicked in an anaerobic chamber. The blank sample clicked under a normal atmosphere display high background staining, despite the fact that the cells had not been exposed to nitroimidazole 4. This background signal was almost as high as that from oxic and hypoxic cells exposed to nitroimidazole 4 and only a small hypoxic/oxic ratio was observed. High background staining with other intracellular azide click targets has been reported and has precluded the use of some proposed azide click reagents.⁶² However we have found that if the click reaction is conducted in an anaerobic chamber, the blank sample displays only weak fluorescence and the hypoxic/oxic ratio is as large as that found for SiHa cells labelled using the nitroimidazole alkyne 2.

The ability to clearly distinguish between oxic and hypoxic cells using both a nitroimidazole alkyne and a nitroimidazole azide allows for complementary labelling by click chemistry using two different functionalised probes (for example a fluorophore azide and a fluorophore alkyne where the two fluorophores absorb and emit at different wavelengths). A particularly useful application would be in detecting temporal changes in hypoxia, which could be imaged, for example by the administration of a nitroimidazole alkyne followed at a later time by a nitroimidazole azide. Example 14. Staining of hypoxic cells by click reaction and by immunostaining using a combination of two nitroimidazoles

These experiments investigated whether the click reaction can be used in combination with immunostaining for the detection of hypoxic cells. For this purpose stirred suspensions of SiHa cells (at 10⁶ cells/mL), continuously gassed at 37 °C under either oxic (5% C0₂ in 0₂) or hypoxic (5% C0₂ in N₂) conditions were treated with a combination of 2 (100 μΜ) and EF5 (100 μ } for 2 h. At the end of the incubation period samples (1 x 10⁶ celis) were collected for click staining only, or for immunostaining only, or for immunostaining followed by click staining. The click staining was performed as described in Example 11 above using Alexa Fluor 647 azide. The immunostaining was performed according to the established protocol:¹⁶ cells were harvested by centrifugation, fixed (4% paraformaldehyde in PBS at 4 °C for 1 h followed by washing), permeabilised and blocked (PBS containing 0.3% Tween 20, 20% fat-free milk, 1.5% lipid-free albumin and 5% mouse serum, at 4 °C for 30 min, followed by washing), and immunostained (ELK3-51 Ab conjugated to Alexa Fluor 488 at 50 μ^ηηί in PBS containing 0.3% Tween 20 and 1.5% lipid-free albumin, 100 μΐ per sample, 4 "C overnight, followed by washing). When immunostaining was followed by click staining the same immunostaining procedure was used, and the samples were then treated with the click reaction cocktail as described above. All cell samples were suspended in PBS for analysis by flow cytometry. The results in Figure 12 show that whichever procedure is followed the oxic and hypoxic cells form two clearly distinguished populations, and that immunostaining first does not interfere with the ability of a subsequent click protocol to selectively detect hypoxic cells. The example also demonstrates that similar sensitivity of hypoxic cell detection can be achieved by the click stain procedure compared to the immunostain procedure, but the click procedure requires only 30 min incubation at ambient temperature as opposed to overnight at 4 °C for the immunostain method.

The above experiment established that the immunostaining and click staining procedures are compatible. A further experiment assessed whether the two procedures label the same cells when exposed to a gradient of oxygen concentrations, rather than to strictly oxic or hypoxic conditions. To establish a gradient of oxygen concentrations SiHa cells and HT29 cells were grown as multicellular layers (MCLs) by seeding onto collagen-coated Teflon support membranes. After 3 days MCLs containing 3-4 million cells and of approximate 10-20 cell diameters in thickness are formed.^53,64,55 Within these MCLs an oxygen gradient will be formed because of limited diffusion of oxygen from the surrounding media and metabolic consumption by the cells. This oxygen gradient can be manipulated by changing the gas phase to which the media is exposed. The MCLs were then incubated under an atmosphere of 5% 0₂ (balance 5% C0₂ in N₂) and exposed to 2 (100 μΜ) and EF5 (100 μΜ) for 2 h. At the end of the incubation period the MCLs were dissociated into single cell suspensions using trypsin, and samples from each were stained using click chemistry only, by immunostaining only, and by immunostaining followed by click staining as described above. The results are presented for representative MCLs from each cell line as scattergrams in Figure 13, where the x-axis represents fluorescence of cells stained with Alexa Fluor 647 azide (the click chemistry probe), and the y-axis represents the fluorescence of cells stained with Alexa Fluor 488 Ab conjugate (the immunostain probe). Each procedure independently illustrates a mixed population of cells derived from the MCLs that vary from low to high fluorescence (representing high to low oxygen concentration in the MCLs at the time of exposure to the 2- nitroimidazoles). Importantly, when the procedures are combined the scattergrams illustrate that each procedure stains the same cells to a similar extent irrespective of the oxygen concentration to which they were exposed.

Example 15. Staining of hypoxic cells with hybrid 2-nitroimidazoles 8 and 11.

The hybrid 2-nitroimidazoles of Formula II and Formula III have the potential to detect hypoxic cells either by a click reaction or by immunostaining. To examine this, stirred suspensions of SiHa cells (at 10⁶ cells/mL) were continuously gassed at 37 °C under either oxic (5% C0₂ in 0₂) or hypoxic (5% C0₂ in N₂) conditions and treated with nitroimidazole 8 (100 μΜ) for 2 h. At the end of the incubation period samples (1 x 10⁶ cells) were collected either for click staining (performed as described in Example 11 above using Alexa Fluor 488 azide) or for immunostaining (performed as described in Example 14 using EF5 Ab conjugated with Alexa Fluor 488) and analysed by flow cytometry. The results are shown in Figure 14, with click staining in Figure 14a and immunostaining in Figure 14b. In each case blank refers to cells that were not exposed to nitroimidazole 8 but incubated (under hypoxia) and stained under otherwise identical conditions. Figure 14a shows a small but distinct difference between the oxic and hypoxic cell populations, which are clearly well-differentiated by the immunostaining procedure in Figure 14b.

Another experiment was conducted in which SiHa cells were treated with nitroimidazole 11 under oxic or hypoxic conditions as described above. At the end of the incubation period samples were collected for click staining (performed as described in Example 12 but using Alexa Fluor 647 azide and 1 mM THPTA), or immunostaining (performed as described in Example 14 using EF5 Ab conjugated with Alexa Fluor 488), or for immunostaining followed by click staining. The cells were analysed by flow cytometry and the results are shown in Figure 15, where panel a) shows cells treated for immunostaining only, b) shows cells treated for click staining only, and c) and d) shows cells that underwent both immunostaining and click staining. Consistent with the examples described above, both immunostaining alone and click staining alone clearly distinguish between the oxic and hypoxic cells, with the ratio of fluorescence intensity between the hypoxic and oxic cells being 16 (immunostaining) and 98 (click staining) in these experiments. It is also clear that when click staining follows immunostaining this does not interfere with the immunostaining fluorescence signal, with panel c) showing approximately the same oxic and hypoxic fluorescence and ratio between them (16-fold) as panel a). When click staining follows immunostaining the click signal intensity is enhanced compared to click staining alone, but the oxic and hypoxic cell populations are still clearly distinguishable (18-fold ratio in panel d).

Taken together these results show that the EF5 antibody, raised specifically against EF5 adducts, can also recognise adducts derived from other 2-nitroimidazoles such as nitroimidazo!es 8 and 11 which share the same Nl substituent but possess other substituents on the nitroimidazole ring. It is also apparent that the presence of a 4-substituent on the 2-nitroimidazole (unlike all the known 2- nitroimidazole hypoxia markers as described in the background above) does not prevent the covalent binding of the nitroimidazole reduction product(s) to cellular macromo!ecules in hypoxic cells. It is further apparent that the click reaction procedure can be used to label hypoxic cells when the alkyne is attached at the 4-position of the nitroimidazole ring rather than attached as part of the Nl substituent. The click signal is stronger when the alkyne is attached via a short chain (as in 11) rather than directly to the nitroimidazole (as in 8). Thus the compounds of Formula II and Formula 111 allow for detection of hypoxic cells using a single hybrid agent that is compatible with either immunostaining or click staining. Example 16. Staining of hypoxic cells without fixation or permeabilisation.

All immunostaining techniques where the antigen is intracellular, as is the case for nitroimidazole- derived antigens, require some measure of cell permeabilisation to allow the large antibody to access intracellular sites. Since the click reaction reagents are considerably smaller and better able to penetrate intact cell membranes the click reaction has the potential to stain hypoxic cells without fixation and permeabilisation. A crucial requirement is for the fluorophore reaction partner (and all necessary reaction components) to be cell permeable. A first experiment assessed hypoxic cell staining without fixation or permeabilisation, using nitroimidazole 2 and two fluorophore azides - Atexa Fluor 488 azide and tetramethylrhodamine azide.

Stirred suspensions of SiHa cells (at lO⁶ cells/mL) were continuously gassed at 37 °C under either oxic (5% C0₂ in 0₂) or hypoxic (5% C0₂ in N₂) conditions and treated with 2 (100 μΜ) for 2 h. At the end of the incubation period samples (1 x 10⁶ cells) were harvested by centrifugation, washed (1% BSA in PBS), and directly incubated with the click reaction cocktail (containing either A!exa Fluor 488 azide or tetramethylrhodamine azide) as described in Example 11. The cells were suspended in PBS for analysis by flow cytometry. The results are shown in Figure 16 where a comparison is made between the fluorescence of eel! samples that are unstained (no exposure to click reaction cocktail), or blank {not exposed to 2 but subjected to the click reaction protocol), or incubated under oxic or hypoxic conditions and then subjected to the click reaction procedure. In Figure 16a where the fluorophore used is Alexa Fluor 488 azide, which is cell-impermeable, there is no useful distinction between the oxic and hypoxic cells, which in turn show insignificant staining beyond background (i.e. blank) levels. In Figure 16b where the only change is to use the cell-permeable tetramethylrhodamine azide as the fluorophore a clear distinction between the oxic and hypoxic cell populations is apparent. This distinction is made much stronger in Figure 16c, simply by repeating the click reaction protocol for a second time.

A second experiment was also performed using coumarin azide, another cell permeable fluorophore, and a double exposure to the click reaction cocktail. The results are shown in Figure 17, where there is once again a clear distinction between the oxic and hypoxic ceil populations, a distinction achieved without any ceil fixation or permeabilisation steps.

Taken together these experiments show:

1) where a suitable cell-permeable fluorophore partner is chosen the click reaction procedure is able to distinguish between oxic and hypoxic cells that have been neither fixed nor permeabi!ised;

2) tetramethylrhodamine azide and coumarin azide are two such suitable fluorophore partners;

3) when cells are neither fixed nor permeabilised the staining procedure is operationally very simple and fast; and

4) the staining of cells by the click reaction procedure can be improved (to provide, for example, a stronger signal and better population discrimination) by simple modifications such as repeat exposure to the click reaction cocktail. Further, the ability to distinguish hypoxic from oxic cells in a way that does not require fixation or permeabilisation allows for the analysis of such ceils by techniques {such as Western blotting) that are not compatible with fixation. One such application is illustrated in Example 19 below.

Example 17. Dual staining of hypoxic ceils ex vivo using EF5 with immunostaining and

immunohistochemistry and nitroimidazole 2 with click chemistry

A female NIH-III mouse (24 g) bearing an A431 human tumour xenograft (calliper measurements 16.4 x 15.8 mm) was injected intraperitoneal^ with EF5 and nitroimidazole 2 at a dose of 60 mg/kg (in saline) each. Two hours after drug administration the mouse was culled by cervical dislocation and the tumour excised. Approximately half of the tumour was fixed in 10% formalin for approximately 18 hours and subsequently transferred and stored in 70% EtOH for sectioning. The other half (500 mg) of the tumour was dissociated into a single-cell suspension using an enzyme cocktail.⁶⁶ Samples (1 x 10⁶ cells) were collected either for click staining (performed as described in Example 11 above using Alexa Fluor 647 azide) or for immunostaining (performed as described in Example 14 above using EF5 Ab conjugated with Alexa Fluor 488) and analysed by flow cytometry. The results are presented as scattergrams in Figure 18, where the y-axis represents fluorescence of cells stained with Alexa Fluor 647 (the click reaction probe), and the x-axis represents the fluorescence of cells stained with Alexa Fluor 488 (the immunostain probe). Each procedure independently illustrates a mixed population of cells derived from the tumour that vary from low to high fluorescence (representing high to low oxygen concentration in the tumour at the time of exposure to the 2-nitroimidazoles). Importantly, when the procedures are combined the scattergrams illustrate that each procedure stains the same cells to a similar extent, i.e. the signals from EF5 and nitroimidazole 2 are highly correlated. These results demonstrate that the click reaction approach and procedure with 2 gives identical results compared to the validated hypoxia marker EF5, in other words that EF5 and 2 label the same hypoxic tumour cells in vivo to the same extent.

The fixed tumour sample was sectioned and stained, with the immunohistochemistry protocol based on a reported procedure.⁶⁷ Briefly, sections were deparrafinised, treated for antigen retrieval (10 mM Citrate Buffer, pH 6.0, >100°C, 25 min then blocked with 10 % goat serum, 4°C, overnight), immunostained (EF5 Ab conjugated with Alexa Fluor 488, 100 μg/mL, 5 h, 4 °C), and click stained (as described in Example 11 but using Alexa Fluor 647 azide, 30 min, room temperature). Other sections were treated in the same manner but without the antigen retrieval step, followed by click staining. Fluorescence images were acquired at lOx magnification using a fluorescence microscope (Zeiss LSM 710 confocal) equipped with a digital camera. Representative images are presented in Figure 19a-c where in each case the scale bar is 1 mm long. Figure 19a and 19b depict a single section that was subjected to dual staining, with Alexa Fluor 488 signal (immunohistochemistry) shown in Figure 19a and Alexa Fluor 647 signal (click chemistry) shown in Figure 19b. The brightly stained areas represent regions of hypoxic cells within the tumour. Clearly the two procedures stain the same pattern of cells in the tumour sections, again demonstrating that the click reaction approach and procedure with 2 gives the same results compared to the validated hypoxia marker EF5. Figure 19c is an example of a section that was stained by click chemistry but without antigen retrieval, showing that click chemistry is able to detect hypoxic cells in tumour sections without the need for the time consuming and harsh conditions of the antigen retrieval procedure. The experiment was also performed using HCT116 tumour-bearing mice. The animals were dosed and the tumours were collected, sectioned, and stained as described above. Representative images are presented in Figure 20 where Figure 20a and 20b depict single sections from two different tumours that were subjected to dual staining, with Alexa Fluor 488 signal (immunohistochemistry) shown in the left panel and Alexa F!uor 647 signal (click chemistry) shown in the right panel. Clearly the two procedures stain the same pattern of cells in the tumour sections, again demonstrating that the click reaction approach and procedure with nitroimidazole 2 gives the same results compared to the validated hypoxia marker EF5.

Example 18. Click staining of hypoxic cells within multicellular structures

The distribution of hypoxic cells within a multicellular structure such as a solid tumour cannot be easily determined by immunohistochemical methods. The poor penetration properties of antibodies means that for intracellular antigens adequate staining of tissue sections requires an antigen retrieval step. Even with such a step staining is not anticipated to penetrate more than a few cell diameters into a tissue sample. In contrast, the small size of the reagents required for click staining means that they have the potential to penetrate into and stain hypoxic cells within much thicker tumour or tissue samples.

To investigate click staining of thick tissue samples multicellular layers of SiHa cells were grown for 2 days, as described in Example 14, to give MCLs approximately 150 μπι thick. The MCLs were then exposed to 2 (100 μΜ) for 2 h under either hypoxic (5% C0₂ in N₂) or oxic (5% 0₂ and 5% C0₂ in N₂) conditions. The MCLs were fixed (10% neutral buffered formalin, overnight in the dark) and permeabilised (0.3% Tween 20 in PBS at 4 °C for 30 min, followed by washing). A click reaction cocktail was prepared as follows: 10 μΜ Alexa Fluor 488 azide or Alexa Fluor 647 azide, 1 mM CuOAc (from a freshly prepared stock in water), 5 mM TBTA, and 100 mM sodium ascorbate in 10 mM Tris-saline at pH 7.4. The click reaction cocktail was used within 15 minutes of preparation, with the sodium ascorbate added last and immediately before addition to the MCLs. Each MCL was exposed to the click reaction cocktail (200 μΐ total, 100 μί added to either side of the MCL in a 24-well plate) at room temperature for 60 min, followed by refreshing the cocktail and incubating for a further 60 min. The MCLs were washed and mounted on slides and imaged by confocal microscopy. When the MCL was exposed to 2 under a gas phase of 5% C0₂ in N₂, so that all cells within the MCL were hypoxic at the time of exposure, subsequent click reaction with either Alexa Fluor 488 azide or Alexa Fluor 647 azide stained all cells with approximately equal intensity throughout the entire thickness of the MCL. This was confirmed by co- staining all cells with the nuclear marker Hoechst 33342. The experiment shows that the click reagents are capable of diffusing through solid tissue samples of at least 75 μηη in thickness, within 2 h, in sufficient quantities to provide a strong and easily detectable click signal. It is noteworthy that the prior permeabilisation step was very mild (only 30 min exposure to 0.3% Tween 20 in PBS at 4 °C) and that the MCLs were physically intact and could be handled and treated the same as untreated control MCLs.

When the MCL was exposed to nitroimidazole 2 under oxic conditions (5% 0₂ and 5% C0₂ in N₂) subsequent click reaction showed a bright fluorescent band only in the centre of the MCL. This is illustrated in Figure 21 for a representative MCL stained with Alexa Fluor 647 azide. The Figure represents a z-stack comprised of 120 scans of the MCL taken at 20x magnification, each scan 1.62 μιτι thick, then reconstructed and displayed in cut view. The xy plane (upper left quadrant) equates to 'looking down' on a selected scan of the MCL from above, and the xz and yz planes (lower left and upper right quadrants) to 'looking in from the sides' of the MCL at the positions marked on the xy projection. The top and bottom of the CL, as determined by Hoechst co-staining, are marked by the limits of the scale bar on the yz plane. The results described above in Example 14 showed that MCLs grown under these conditions and then incubated under 5% 0₂ contain a mixture of cells distributed across a range of oxygen concentrations. Figure 21 now shows that the cells at low oxygen concentrations are found at the centre of the MCL, consistent with limited diffusion of oxygen from the medium to the centre of the multicellular structure.

Attempts were made to illustrate the same spatial distribution of hypoxic cells in MCLs using EF5 and immunohistochemistry. MCLs were exposed to EF5 (100 μΜ) for 2 h under either hypoxic (5% CO_z in N₂) or oxic (5% 0₂ and 5% C0₂ in N₂) conditions, and fixed as above. Permeabilisation and immunostaining was either as described in Example 14 or following the established protocol for staining of tissue sections, as described in Example 17. MCLs exposed to EF5 under hypoxic conditions and immunostained gave extremely weak fluorescent signals following either of the permeabilisation and staining procedures. This is illustrated in Figure 22, where a) shows a representative MCL exposed to 2 under hypoxic conditions and stained using click chemistry with Alexa Fluor 488 azide, and b) shows a representative MCL exposed to EF5 under hypoxic conditions and stained using the tissue section protocol with antibody-Alexa Fluor 488 conjugate. The images in a) and b) were acquired at lOx magnification using identical microscope settings. Figure 22a illustrates click staining of hypoxic cells throughout the thickness of the multicellular layer, with staining of individual cells showing preservation of morphology during the sample preparation and staining procedure. Figure 22b shows that the immunostaining protocol, using the same fluorophore as for click staining, gives a much weaker fluorescent signal, to the point of being practically undetectable at the same settings for which a strong click signal is seen. This is true even after a harsh antigen retrieval step (>100 °C for 25 min) to expose intracellular antigens, a procedure that is unnecessary for click staining, and which makes the MCLs very fragile, difficult to mount, and creates artefacts such as holes in the MCLs. The very weak staining in EF5- treated MCLs is consistent with background staining, showing no difference between oxic and hypoxic MCLs and no spatial distribution within oxic MCLs.

In summary, click staining is suitable for imaging hypoxic cells within thick tissue samples, and intense fluorescent signals can be readily achieved under mild conditions which preserve sample morphology. In contrast immunostaining gives weak or no staining of hypoxic cells within thick tissue samples, and the recommended staining procedure causes considerable structural damage to the samples.

Example 19. Western blotting with click-labelled cells

Stirred suspensions of SiHa cells were incubated under either oxic or hypoxic conditions and treated with 2 followed by incubation with the click reaction cocktail containing tetramethylrhodamine azide as described in Example 16 but with addition of fresh click cocktail after 30 min and another 30 min of clicking. Samples of the aerobic and hypoxic cells were then pooled together in a 1:1 ratio in PBS containing 1 mM EDTA. The pooled population of cells was then sorted into two fractions on a fluorescence-activated cell sorter (FACS) based on the TAMRA fluorescence signal intensity; the dim cells were labelled "pooled-aerobic" and the bright cells "pooled-hypoxic".

Non-pooled and sorted cells were counted and 150,000 cells were spun down. The pellets were lysed in 15 μΐ lx LDS sample preparation buffer (106 mM Tris-HCl, 141 mM Tris-Base, 2 % lithium dodecyl sulfate (LDS), 10 % glycerol, 0.51 mM EDTA, 0.22 mM Coomassie Blue G250, 0.175 mM phenol red) in RIPA buffer (50 mM Tris-HCI pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCI, 1 tnM EDTA), plus 5% beta-mercaptoethanol. Samples were boiled for five minutes at 95°C.

The lysates were loaded onto precast 4-12 % Bis-Tris polyacrylamide gels. Proteins were separated at 150 V in lx MES running buffer pH 7.3 (50 mM 2-{N-morpholino)ethanesulfonic acid (MES), 50 mM Tris- Base, 0.1 % SDS, 1 mM EDTA, pH 7.3) for one hour, then transferred to nitrocellulose by blotting for one hour at 100 V using a tank blot system. The buffer contained 25 mM Tris-HCI pH 8.5, 0.2 M glycine and 20 % methanol. The membrane was cut along the 37 kDA band (using. prestained markers of known molecular size) to simultaneously probe for actin and BNIP3. Prior to that, membranes were agitated in blocking solution (5 % milk powder in Tris-buffered saline pH 7.5 (TBS; 3 g/L Tris-base, 8 g/L NaCI, 0.2 g/L KCI) containing 0.1½ Tween 20) for two hours at room temperature. Primary antibodies (diluted in 1% milk) were incubated overnight at 4°C. Before incubation with the secondary antibody (conjugated to horseradish peroxidise), as well as afterwards, the membrane was washed three times for 10 minutes in TBS-0.1% Tween 20. For detection, SuperSignal West Pico Chemiluminescent Substrate (Pierce) was used and chemiluminescence was measured with a LAS-3000 Imaging System. For band densitometry, ImageJ free software was employed. All calculations are normalised against actin.

The following antibodies were used: goat polyclonal anti-BNIP3 (1:1000), mouse monoclonal anti-actin (1:10,000), rabbit anti-goat (1:1000) and goat anti-mouse (1:1000).

Female CD-I nude mice (25-26 g) bearing SiHa human tumour xenografts (calliper measurements 16.4 x 15.8 mm) were injected intraperitoneal^ with nitroimidazole 2 at a dose of 60 mg/kg (in saline) as described in Example 17. Single-cell suspensions were prepared as describe in Example 17 and the cells underwent click staining with tetramethylrhodamine azide as described in Example 16. A portion of the cells were then sorted into two fractions by FACS as described above. Sorted and non-sorted cells from tumours were then lysed and used for Western blotting as described above.

SiHa cells under hypoxic culture conditions show the characteristic upregulation of BN1P3 as shown in Figure 23a. BNIP3 is known to be upregulated under hypoxic conditions through increased stabilisation of hypoxia-inducible factor-la (HIF-la).⁶⁸ A mixed population of aerobic and hypoxic SiHa cells can be separated sorted into two fractions using FACS that show increased BNIP3 signal by Western Blot in the "pooled-hypoxic" fraction as shown in Figure 23a. In SiHa tumours oxygen gradients exist, which affect BNIP3 expression. The more hypoxic cell fraction of SiHa tumours shows elevated levels of BNIP3 compared to the more aerobic cell fraction as shown in Figure 23b.

Collectively, the data show that labelling tumour cells with hypoxia marker nitroimidazole 2 and subsequent click reaction with the cel!-permeable tetramethylrhodamine azide without fixation and permeabilisation allows detection and isolation of hypoxic cells from aerobic cells that is compatible with Western blotting.

The foregoing describes the invention including preferred forms thereof. Modifications and alterations that would be readily apparent to the skilled person are intended to be included within the spirit and scope of the invention described and as defined in the attached claims.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of "including, but not limited to". The reference to any prior art in the specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge any country in the World.

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Claims

A method for detection and/or imaging of hypoxic cells, the method including the in vitro or the ex vivo use of a copper(l)-catalysed azide-alkyne 1,3-dipolar cycloaddition reaction, the alkyne being a terminal alkyne.

The method of claim 1 wherein the copper(l)-catalysed azide-alkyne 1,3-dipolar cycloaddition reaction is between a hypoxia marker derived from an alkyne functionalised nitroimidazole and an azide functionalised probe, the alkyne being a terminal alkyne.

The method of claim 1 wherein the copper{l)-catalysed azide-alkyne 1,3-dipolar cycloaddition reaction is between a hypoxia marker derived from an azide functionalised nitroimidazole and an alkyne functionalised probe, the alkyne being a terminal alkyne.

The method of claim 1 wherein copper(l)-catalysed azide-alkyne 1,3-dipolar cycloaddition reaction is between a hypoxia marker derived from an alkyne functionalised nitroimidazole and an azide functionalised probe, and also between a hypoxia marker derived from an azide functionalised nitroimidazole and an alkyne functionalised probe, the alkyne being a terminal alkyne in both reactions.

5. The method of any one of claims 2 to 4 wherein the hypoxia marker is derived from an alkyne functionalised 2-nitroimidazole and/or an azide functionalised 2-nitroimidazole

6. The method of claim 5 wherein the 2-nitroimidazole is a compound of Formula (I):

N0₂

N^N-X-Y

Formula (I)

wherein:

X is a Q-Cg alkyl chain linker, optionally saturated or unsaturated, optionally straight or branched, optionally containing cyclic units and optionally further substituted with any one or more of:

IM_n and/or O_n heteroatoms, which may be present as alcohol, ether, amine, amide, carbamate, azide and/or heterocyclic functional groups,

F_q atoms;

n is any number from l to 3;

q is any number from 1 to 6;

Y is a terminal alkyne or an azide;

Z is:

The method of claim 5 wherein the 2-nitroimidazole is a compound of Formula (II):

N0₂

^N-Z

X

Y

Formula (II)

wherein:

X may be absent or present,

X is a Ci-Cg alkyl chain linker, optionally saturated or unsaturated, optionally straight or branched, optionally containing cyclic units and optionally further substituted with any one or more of: i\l_n and/or O_n heteroatoms, which may be present as alcohol, ether, amine, amide, carbamate, azide and/or heterocyclic functional groups,

F_q atoms;

n is any number from l to 3;

q is any number from 1 to 6;

Y is a terminal alkyne or an azide;

Z is

The method of claim 5 wherein the 2-nitroimidazole is a compound of Formula (III):

N0₂ x

Y

Formula (ill)

wherein:

X may be absent or present,

X is a CrQ alkyl chain linker, optionally saturated or unsaturated, optionally straight or branched, optionally containing cyclic units and optionally further substituted with any one or more of: N_n and/or O_n heteroatoms, which may be present as alcohol, ether, amine, amide, carbamate, azide and/or heterocyclic functional groups,

F_q atoms;

n is any number from l to 3;

q is any number from 1 to 6;

Y is a terminal alkyne or an azide;

Z is

The method of any one of claims 6 to 8 the C C_g alkyl chain linker is optionally saturated or unsaturated, optionally straight or branched, optionally containing cyclic units and optionally containing any one or more of:

n is any number from 1 to 3.

The method of claim 5 wherein the 2-nitroimidazo!e is selected from the following:

The method of claim 5 wherein the 2-nitroimidazole is selected from the following:

12. The method of any one of claims 5 to wherein the hypoxic cells are labelled with a 2- nitroimidazole in vivo or in vitro.

13. The method of claim 12 wherein the 2-nitroimidazole is administered in vivo.

14. The method of any one of claims 1 to 13 wherein the Cu(l) of the copper (I) catalyst is added as a salt. The method of claim 14 wherein Cu(l) is added as CuBr or CuOAc.

16. The method of any one of claims 1 to 13 Cu(l) in the copper (I) catalyst is formed from metallic copper, a Cu(0) source, or a Cu(ll) source.

17. The method of claim 16 wherein the Cu(ll) source is CuS0₄.

18. The method of claim 16 wherein the Cu(l) is formed from the Cu(l!) source and a reducing agent. 19. The method of any one of claims 1 to 18 wherein the method includes the use of a suitable metal chelating ligand.

20. The method of any one of claims 1 to 19 wherein the detection and/or imaging is carried out using fluorescence, radioactivity, or (bio)Iuminescent techniques and employs functionalised probes wherein the probe is either fluorogenic, a fluorophore, or a radiolabel or a luminescent or bioluminescent probe.

21. A 2-nitroimidazole compound selected from the following:

22. A method for detection and/or imaging of hypoxic cells, the method including the in vitro or the ex vivo use of a copper(l)-catalysed azide-alkyne 1,3-dipolar cycloaddition reaction between a hypoxia marker derived from a compound of claim 21 and a functionalised probe.

23. The method of claim 22 wherein the method further includes detection by antibody-based methods.

24. The method of claim 22 wherein the hypoxic cells are labelled with any one or more of the 2- nitroimidazole compounds of claim 21.

25. The method of claim 24 wherein the 2-nitroimidazole compound(s) is administered in vivo.

26. The method of any one of claims 22 to 25 wherein the Cu(l) of the copper (I) catalyst is added as a salt.

27. The method of any one of claims 22 to 25 wherein Cu(l) of the copper (I) catalyst is formed from metallic copper, a Cu(0) source, or a Cu(ll) source.

28. The method of any one of claims 22 to 27 wherein the method includes the use of a suitable metal chelating ligand. The method of any one of claims 22 to 28 wherein the detection and/or imaging is carried out using fluorescence, radioactivity, or (bio)luminescent techniques and employs functionalised probes wherein the probe is either fluorogenic, a fluorophore, or a radiolabel or a luminescent or bioluminescent probe.

30. A method for identifying hypoxic cells, comprising the following steps:

a) in vivo administration of at least one alkyne functionalised 2-nitroimidazole and/or at least one azide functionalised 2-nitroimidazole, the alkyne being a terminal alkyne, to label hypoxic cells with a hypoxia marker;

b) collecting labelled cells or tissue samples from the living organism; and

31. The method of claim 30 wherein the in vivo administration is by parenteral administration.

32. The method of claim 30 or 31 wherein the alkyne functionalised 2-nitroimidazole and/or at least one azide functionalised 2-nitroimidazole is defined in any one of claims 6 to 11.

33. A method for identifying hypoxic cells in tissue samples taken from a living organism to which at least one alkyne functionalised 2-nitroimidazole and/or at least one azide functionalised 2- nitroimidazole, the alkyne being a terminal alkyne, has been administered, the method comprising the use of a copper(l)-cata!ysed azide-alkyne 1,3-dipolar cycloaddition reaction for the detection and/or imaging of hypoxic cells to identify the hypoxic cells.

34. A composition for use in the detection and/or imaging of hypoxic cells, the composition including at least one nitroimidazole functionalised by a terminal alkyne and/or at least one nitroimidazole functionalised by an azide and a pharmaceutically acceptable carrier.

35. The composition of claim 34 wherein the nitroimidazole is a 2-nitroimidazoles selected from any one or more of the 2-nitroimidazoles according to the any one of claims 6 to 11.

36. A two-part composition for use in the detection and/or imaging of hypoxic cells, the two-part composition including:

Part A, which is the composition according to claim 34 or 35; and

37. A three-part composition for use in the detection and or/imaging of hypoxia, the three-part composition including: Part A, which is the composition according to claim 34 or 35;

Part C, which comprises a Cu(l) source and a suitable carrier;

A four-part composition for use in the detection and/or imaging of hypoxic cells, the four-part composition including:

- Part B, which comprises a probe functionalised by an azide or a probe functionalised by a terminal alkyne and a suitable carrier;

- Part C, which comprises a Cu(l) source and a suitable carrier; and