WO2009140743A2

WO2009140743A2 - Blood imaging

Info

Publication number: WO2009140743A2
Application number: PCT/BE2009/000027
Authority: WO
Inventors: Guy Bormans; Alfons Verbruggen
Original assignee: Katholieke Universiteit Leuven
Priority date: 2008-05-23
Filing date: 2009-05-25
Publication date: 2009-11-26
Also published as: WO2009140743A3

Abstract

The invention generally relates to the application of imaging techniques, in particular positron emission tomography (PET) or single photon emission tomography (SPECT), in the assessment of physiological parameters in a subject. The compounds of the present invention were shown to be cell membrane permeant and to have a specific binding affinity (or substrate affinity) for the carbonic anhydrases type I and II, and are accordingly useful for the in vivo and in vitro labeling of erythrocytes (RBC). It is accordingly an object of the present invention to provide said novel compounds, in particular the radio labeled variants thereof, and their use in imaging techniques to perform the assessment of physiological parameters, such as blood volume measurements, or blood flow measurements.

Description

BLOOD IMAGING

Field of the Invention

The invention generally relates to the application of imaging techniques, in particular positron emission tomography (PET) or single photon emission tomography (SPECT) or for ex vivo diagnostic purposes, in the assessment of physiological parameters in a subject. The radio labeled compounds of the present invention were shown to have a specific binding affinity for the carbonic anhydrases type I and/or II, and are accordingly useful for the in vivo and/or in vitro labeling of erythrocytes (RBC) in a manner sufficient to achieve blood imaging. It is accordingly an object of the present invention to provide said novel compounds, in particular the radio labeled variants thereof, and their use in imaging techniques to perform the assessment of physiological parameters, such as blood volume measurements, or blood flow measurements. It is accordingly also an object of the present invention to provide radio labeled compounds for use in a diagnostic imaging treatment to perform the assessment of physiological parameters, such as blood volume measurements, or blood flow measurements .

Background to the Invention

As already mentioned hereinbefore, Erythrocytes (RBC) labeled with a proper radionuclide that can be used for PET (positron emission tomography) or SPECT (single photon emission tomography) imaging allows performing the assessment of physiological parameters in a subject.

Blood volume measurements and blood flow measurements are for example used as physiological parameters in the diagnosis and/or disease monitoring of angiofibroma , hepatic hemangioma, renal cell carcinoma or the localization and confirmation of gastro intestinal bleeding (1, 2, 3, 4, 5, 6, 7). Scintigraphic blood flow measurement can be used to assess myocardial ejection fraction and regional wall motion (8) or to study the temporal changes of blood volume secondary to the administration of a pharmacologic active agent.

Heat altered radio labeled erythrocytes can be used for the diagnosis of hyposplenia (9), localization of splenosis (10), or to differentiate accessory spleen from tumor recurrence (11).

A first methodology, currently available for RBC labeling, consists of the in vivo labeling of RBC by inhalation of carbon-11 labeled carbonmonoxyde . Carbonmonoxide, which efficiently binds to hemoglobin in the RBC. However, handling of radioactive gasses carries serious contamination risks and the short half-life of carbon-11 may not be sufficient to enable visualization of gastrointestinal bleeding.

Also the alternative labeling with technetium-99m has certain drawbacks. Labeling with technetium-99m is typically performed by sequential pretinning of the erythrocytes by incubation with an anionic tin-II-complex (e.g. Sn2+-pyrophosphate) followed by incubation with [99mTc] -pertechnetate which will diffuse over the RBC membrane and be reduced to a lower valence by the Sn2+ followed by complexation of the reduced technetium to both heme and globine within the red blood cells. The resulting coπiplexation is clearly dependent on whether the pretinning and/or the incubation with pertechnetate are performed in vitro or in vivo. Full in vivo labeling generally results in low yields of RBC labeling due to competing complexation of technetium by plasma components. This in contrast to full in vitro labeling that is characterized by higher labeling yields but requires laborious blood manipulation with the associated risks of contamination of the manipulator or patients in case of accidental blood sample substitution.

Also radioactive halogen substituted RBC ligands, such as the longer-lived radionuclide fluorine-18, that provide extended time frames for PET scan data collection, are currently lacking. For this and the forementioned reasons, need for new and effective RBC labeling remains.

It is an object of the present invention to provide novel compounds that address one or more of these problems.

Brief Description of the Drawings

Figure 1 displayes the biodistribution in mice of [¹¹C] -1 showing a high and persistent concentration in blood.

Figure 2 demonstrates the binding of [¹¹C]-I to RBCs (% activity bound to RBCs) . 1 is incubation of RBCs during 10 min followed by separation from the supernatant and incubation (twice repeated) with PBS during 5 min and subsequent separation of supernatant and cells. 2 incubation of RBCs during 10 min with blood followed by separation of plasma and 2 subsequent washing. 3 incubation of RBCs during 20 min with blood followed by separation of plasma and 2 subsequent washing . 4 incubation of RBCs during 20 min in the presence of 0.77 mg/ml aminoethylbenzenesulphonamide

Figure 3 : Binding of ¹¹C- 1 to human red blood cells, Fraction A and B (Blank) incubated at RT for 10 and 20 min, Fraction C incubation at RT for 20 min in the presence of AEBS (5 μM)

Figure 4 : Binding of ¹¹C- 2 to human red blood cells, Fraction A and B (Blank) incubated at RT for 10 and 20 min, Fraction C incubation at RT for 20 min in the presence of AEBS (5 μM)

Figure 5: RBC binding inhibition of ¹⁸F-2 in the presence of increasing concentrations of acetazolamide; Fraction ^v A (Blank) , Fracitions B, C and D containing Acetazolamide (1, 10 and 100 μM respectively) incubated at room temperature for 1 h.

Figure 6 : Binding of ¹⁸F-I to human red blood cells, Fraction A (Blank) , Fraction B (¹⁸F-I and non radioactive reference compound 5μm) , Fraction C (¹⁸F-I and Acetazolamide lμm) incubated at RT for 1 or 5 min.

Figure 7: In vitro binding of ^99mTc- 5 to human red blood cells, Fraction A (^99mTc- 5 in plasma rich medium, incubated at RT 10 min) , Fraction B (^99mTc- 5 in plasma poor medium, incubated at RT, 10 min) , Fraction C (^99mTc- 5 in plasma poor medium, incubated at RT, 20 min) Figure 8 Total body microPET image (rat) at 60 min p.i. of ¹⁸F-2 showing most intense activity in the heart region and showing major blood vessels Figure 9 microPET image (60 min p.i.) zoom-in on the heart region showing the left and right atria/ventricles, aorta arc, descending aorta, vena cava

Summary of the invention

Generally this invention concerns a radio labeled compound, more particularly a radio labeled benzene sulfonamide compound, with a specific binding affinity for the carbonic anhydrases type I and/or II, for use in an imaging based diagnosis or an imaging based diagnostic procedure of a mammalian, including human, subject to diagnosing and/or monitoring blood volume and/or blood flow in said subject.

The present invention concerns a benzene sulfonamide compound of formula (I)

(I) wherein R comprises radiolabel connected to the phenyl ring directly or via an oxygen, sulfur, nitrogen, alkyl, aryl or any combination of these and whereby the radiolabel is from the group consisting of ¹¹C, ^99mTc, ¹³N, ⁶⁸Ga, ⁶⁷Cu and ⁶²Cu, or the radioactive is a halogen from the group consisting of ¹²²1, ¹²⁴I, ¹²³I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁸Br, ⁸²Br, and ¹⁸F. The phenyl ring of these compounds may be derivatized in other positions with additional substituents such as halogens, alkyl or aryl groups connected to the ring directly or via S, 0, N or other atoms. It also concerns any of these compounds for use in a diagnostic treatment for instance a diagnostic treatment for diagnosing and/or monitoring blood volume and/or blood flow in a subject.

Moreover the present invention concerns a benzene sulfonamide compound with the formula (II)

(D wherein;

- n represents an integer from 0 to 6;

- R is selected from [rll/C-] Ci_₆alkyloxy-, X-Ci_₆alkyl-, or X-Ci- 6alkyloxy-;

- X represents a radioactive halogen selected from the group consisting of 1^±2-"2-I, 1¹2^Z3^J-I,

131- I, 7'5⁰Br, 7'6⁰ _rBr, 7"7τBr,

including solvates and pharmaceutically acceptable addition salts thereof. It also concerns any of these compounds for use in a diagnostic treatment for instance a diagnostic treatment for diagnosing and/or monitoring blood volume and/or blood flow in a subject.

Furthermore the present invention concerns a compound with the formula (II)

(D wherein;

- n represents an integer from 1 to 6; - R is selected from [¹¹C] Ci_₆alkyloxy-, X-Ci-₆alkyl-, or X-Ci- 6alkyloxy-;

- X represents a radioactive halogen selected from the group consisting of ¹²²I, ¹²³I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, 7⁸Br, ⁸²Br, and ¹⁸I; including solvates and pharmaceutically acceptable addition salts thereof. Another embodiment of present invention is such a benzene sulfonamide compound but wherein; n represents an integer from 1 to 6, in particular n represents 2; R represents [¹¹C] Ci_₆alkyloxy-, or X-Ci- βalkyloxy-; and X represents a radioactive halogen selected from the group consisting of ¹²³I, and ¹⁸I. It also concerns any of these compounds for use in a diagnostic treatment for instance a diagnostic treatment for diagnosing and/or monitoring blood volume and/or blood flow in a subject.

Furthermore present invention concerns a compound that consists of those compounds selected from the group consisting of; 4- (2- (4-

[¹¹C] Methoxybenzamido) ethyl) benzenesulphonamide (hereinafter also referred to as (¹¹C-I) ) and other derivatives thereof with a sulphonamide group for use in a diagnostic treatment for instance a diagnostic treatment for diagnosing and/or monitoring blood volume and/or blood flow in a subject.

A particular embodiment of present invention is benzenesulfonamide compound, with the structure

or with the structure

or with the structure

(Tc = ""¹Tc) for use in a diagnostic treatment for instance a diagnostic treatment for diagnosing and/or monitoring blood volume and/or blood flow in a subject.

Furthermore the present invention concerns a composition comprising any of the here above in this application described benzene sulfonamide compounds The composition can be an aqueous injection further comprising one or more of a stabilizer (e.g., ascorbic acid), a solubilizer (e.g., - cyclodextrins and the like), a dispersing agent (e.g., Tween 80, carboxymethyl-cellulose, sodium alginate and the like) , a preservative (e.g., methylparaben, propylparaben, benzyl alcohol, chlorobutanol and the like) , an isotonicity agent (e.g., sodium chloride, glycerol, sorbitol, glucose and the like) and the like. Such composition can be used in a diagnostic treatment for instance a diagnostic treatment for diagnosing and/or monitoring blood volume and/or blood flow in a subject, for instance in PET or SPECT imaging techniques or for ex vivo diagnostic purposes

Furthermore the present invention comprises a pack for blood imaging for monitoring t blood volume and/or blood flow in a mammalian, including human, subject, the pack comprising a radio labeled benzene sulfonamide have a specific binding affinity for the carbonic anhydrases type I and/or II.

Detailed Description of the Invention

This invention relates to methods and compounds for diagnosing and/or monitoring physiological parameters in a subject, and is based on the finding that 4- (2- (4- Methoxybenzamido) ethyl) benzenesulphonamide (MEMBEBSA) and derivatives thereof, hereinafter represented by the compounds of formula (I) , have a specific binding affinity for carbonic anhydrase types I and II.

Carbonic anhydrase (CA) is an ubiquitous metalloenzyme of which 16 mammal isoforms have been described. Carbonic anhydrase type I and II are abundant in erythrocytes (12) (resp 20 μM and 160 μM (13) ) where it serves to increase the transport capacity for CO₂. Interaction with CA I and II in the RBCs has been found to be responsible for extensive but saturable binding to RBCs of several drugs that have affinity for carbonic anhydrase such as the anticancer agent indisulam (14) and the antiepileptic drug topiramate (15). The former shows a RBC concentration of about 50% of the plasma concentration whereas the latter shows a threefold higher concentration in RBCs than in plasma.

As used herein, and in a first aspect, the present invention provides MEMBEBSA and MEMBEBSA derivatives represented by those compounds of formula (I)

( D wherein;

- n represents an integer from 1 to 6;

- R is selected from [¹¹C] Ci-βalkyloxy-, X-Ci_₆alkyl-, or X-Ci- εalkyloxy-;

- X represents a radioactive halogen selected from the group consisting of ¹²²1, ¹²⁴I, ¹²³I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, 7⁷Br, ⁷⁸Br, ⁸²Br, and ¹⁸F; including solvates and pharmaceutically acceptable addition salts thereof.

In a particular embodiment the MEMBEBSA derivatives consists of those compounds of formula (I) hereinbefore wherein n represents an integer from 1 to 6, in particular¹ n represents 2; R represents [¹¹C] Ci_₆alkyloxy-, or X-Ci- ₆alkyloxy-; and X represents a radioactive halogen selected from the group consisting of ¹²³I, and ¹⁸F.

In a more particular embodiment the MEMBEBSA derivatives consists of those compounds selected from the group consisting of; 4- (2- (4-

[¹¹C] Methoxybenzamido) ethyl) benzenesulphonamide (hereinafter also referred to as (¹¹C-I) ) and including stereo-isomeric forms, solvates and pharmaceutically acceptable addition salts thereof.

As the Cl-6 alkoxy group, alkoxy group having 1 to 6 carbon atoms such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, pentyloxy, hexyloxy and the like can be mentioned.

As the Cl-6 alkyl group, alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like can be mentioned.

As the salt of compound (I), a pharmaceutically acceptable salt is preferable and, for example, salt with inorganic base, salt with organic base, salt with inorganic acid, salt with organic acid, salt with basic or acidic amino acid and the like can be mentioned. As the inorganic base that forms a salt, for example, alkali metal such as sodium, potassium and the like; alkaline earth metal such as calcium, magnesium and the like; aluminum, ammonium and the like can be mentioned. As the organic base that forms a salt, for example, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N',N^?- dibenzylethylenediamine and the like can be mentioned. As the inorganic acid that forms a salt, for example, hydrochloric acid, hydrobromide acid, nitric acid, sulfuric acid, phosphoric acid and the like can be mentioned. As the organic acid that forms a salt, for example, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like can be mentioned. As the basic amino acid that forms a salt, for example, arginine, lysin, ornithine and the like can be mentioned, and as the acidic amino acid that forms a salt, for example, aspartic acid, glutamic acid and the like can be mentioned.

Compound (I) can be converted to a pharmaceutically acceptable salt thereof by a method known in the art.

The non radioactive reference compound 4- (2- (4-

Methoxybenzamido) ethyl) benzenesulphonamide (1) was obtained by reacting of a 4-methoxybenzoylchloriede with 4- (2- aminoethyl) benzenesulphonamide in pyridine

The precursor for labeling is synthesized by reaction of a 4-Acetoxyzoylchloride with 4- (2- aminoethyl)benzenesulphonamide in pyridine. The acetylgroup is then removed by treatment with sodiummethanolate to obtain 4- (2- (4-hydroxybenzamido) ethyl) benzenesulphonamide. Radiolabelling with carbon- 11 is performed by reaction of 4- (2- (4-hydroxybenzamido) ethyl) benzenesulphnamide with methyliodide . As provided in more detail in the examples hereinafter, the compounds of formula (I) , are in particular useful for PET

(positron emission tomography) or SPECT (single photon emission tomography) imaging of physiological parameters in a subject.

As used herein, and in another aspect the present invention also concerns labeled CAI and/or CAII inhibitors, substrates or ligands of CAI and/or CAII for use in a diagnostic treatment of in vivo erythrocytes (RBC) labeling for medical imaging. Such medical imaging can be ET (positron emission tomography) or SPECT (single photon emission tomography) imaging to assess physiological parameters in a subject. Such physiological parameters can be blood volume measurements, or blood flow measurements. Suitable CAI and/or CAII inhibitor are compounds with a sulphonamide group. These inhibitors can be labelled or conjugated to a radioisotope. The ligands or substrates can be labeled or conjugated to a radioisotope. These compounds of the present invention were shown to be cell membrane permeant and to have a specific binding affinity (or substrate affinity) for the carbonic anhydrases type I and II, and are accordingly useful for the in vivo and in vitro labeling of erythrocytes (RBC) .

As used herein, and in another aspect the present invention also concerns labelled CAI and/or CAII inhibitors substrates or ligands of CAI and/or CAII for use in a diagnostic treatment of in vivo erythrocytes (RBC) labelling for ex vivo diagnostics. Suitable CAI and/or CAII inhibitor are compounds with a sulphonamide group. These inhibitors can be labelled or conjugated to a radioisotope. The ligands or substrates can be labelled or conjugated to a radioisotope. The compounds of the present invention were shown to be cell membrane permeant and to have a specific binding affinity (or substrate affinity) for the carbonic anhydrases type I and II, and are accordingly useful for the in vivo and in vitro labeling of erythrocytes (RBC) . Such compounds of the present invention need to be cell membrane permeant and to have a specific binding affinity (or substrate affinity) for the carbonic anhydrases type I and II.

Erythrocytes (RBC) labelled with a proper radionuclide that can be used for PET (positron emission tomography) or SPECT (single photon emission tomography) imaging allows performing the assessment of physiological parameters in a subject .

The physiological parameters to be assessed using the compounds of the present invention, are blood volume measurements and blood flow measurements. Said parameters are for example used as physiological parameters in the diagnosis and/or disease monitoring of angiofibroma , hepatic hemangioma, renal cell carcinoma, the localization and confirmation of gastro intestinal bleeding, to assess myocardial ejection fraction and regional wall motion or to study the temporal changes of blood volume secondary to the administration of a pharmacologic active agent.

Heat altered radio labeled erythrocytes can be used for the diagnosis of hyposplenia, localization of splenosis, or to differentiate accessory spleen from tumor recurrence . It is accordingly an object of the present invention to provide the use of compounds as defined hereinbefore in a method of diagnosing and/or monitoring physiological parameter (s) in a subject, in particular in any one of the aforementioned indications, and more in particular in PET or SPECT imaging techniques.

In view of the utility of the compounds according to the invention, there is provided a method for diagnosing and/or monitoring physiological parameters in a subject, which comprises administering an amount of a compound according to the present invention, to said subject. As used herein, an effective amount is an amount sufficient to enable in vivo localization of the labeled compound and detection thereof. The effective amount will of course, vary with the particular compound, the purpose, the organ of interest, the radioactivity of the radionuclide atom and the like, but will be below pharmacologically significant levels.

Is it uselful to try t specify this? The amount of marker injected may vary from about 0.1 μCi to 20 mCi . For intraorgan regional flow determinations the amount will generally be from about 2 to 25 μCi, while for whole blood imaging the amount will be from about 50 μCi to 1 mCi, more usually from about 100-500 μCi . For instance a suitable injection is 1-20 mCi in 1-20 ml IV injection

The marker is typically administered intravenously in any convenient physiological medium, such as saline, phosphate- buffered saline, saline having physiological amounts of other metals, e.g., K, Ca, Mg, glucose, insulin, or the like, in physiologically acceptable amounts. Generally, it will be formulated as an aqueous injection together with a stabilizer (e.g., ascorbic acid), a solubilizer (e.g., - cyclodextrins and the like), a dispersing agent (e.g., Tween 80, carboxymethyl-cellulose, sodium alginate and the like) , a preservative (e.g., methylparaben, propylparaben, benzyl alcohol, chlorobutanol and the like) , an isotonicity agent (e.g., sodium chloride, glycerol, sorbitol, glucose and the like) and the like, according to a conventional methods known in the art .

It is accordingly a further object of the present invention to provide a composition useful for diagnosing and/or monitoring physiological parameters in a subject, which comprises a compound according to the invention. In particular an aqueous injection comprising said compounds together with one or more of a stabilizer (e.g., ascorbic acid), a solubilizer (e.g., -cyclodextrins and the like), a dispersing agent (e.g., Tween 80, carboxymethyl-cellulose, sodium alginate and the like), a preservative (e.g., methylparaben, propylparaben, benzyl alcohol, chlorobutanol and the like), an isotonicity agent (e.g., sodium chloride, glycerol, sorbitol, glucose and the like) and the like

This invention will be better understood by reference to the Experimental Details that follow, but those skilled in the art will readily appreciate that these are only illustrative of the invention as described more fully in the claims that follow thereafter. Additionally, throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.

EXAMPLES The following examples illustrate the invention. Other embodiments will occur to the person skilled in the art in light of these examples.

MATERIALS AND METHODS

Synthesis of 4- (2- (4-

[HC]Methoxybenzamido) ethyl)benzenesulphonamide (llC-1)

4- (2- (4-Methoxybenzamido) ethyl) benzenesulphonamide (1)

4-Methoxybenzoylchloride (1.71 g, 10 mmol) was added slowly at room temperature to a stirred solution of 4- (2- aminoethyl) benzenesulphonamide (2 g, 10 mmol) in dry pyridine (60 ml). The clear solution was stirred overnight. Pyridine was removed under reduced pressure and absolute ethanol (40 ml) was added to the residue and the precipitate was filtered off and recrystallized from methanol. Yield: 880 mg white needles (26.3 %) .

4-Acetoxybenzoylchloride (2)

The compound was synthesized following a literature procedure (16) 4- (2- (4-Acetoxybenzamido) ethyl)benzenesulphonam±de (3)

4-Acetoxybenzoylchloride (1.09 g, 5.5 mmol) was dissolve in CH₂Cl₂ (20 ml) and added drop by drop to a stirred solution of 4- (2-aminoethyl) benzenesulphonamide (1.1 g, 5.5 mmol) in dry pyridine (40 ml) . The solution was stirred for 72 h, evaporated and treated with ethanol (40 ml) . The precipitate was filtered off and recrystallized from water/ethanol . The white crystals were collected and dried under vacuum. Yield: 1.5 g (75 %) .

4- (2- (4-hydroxybenzam±do) ethyl)benzenesulphonam±de (4) Sodium (46 mg, 2 mmol) was dissolved in dry methanol (30 ml). 4- (2- (4-Acetoxybenzamido) ethyl) benzenesulphonamide (720 mg, 2 mmol) was added and the mixture was stirred for 4 h at room temperature. The mixture was poured into ice water and acidified with 1 N HCl to pH 2. A white solid was formed, which was isolated, washed water (50 ml) and recrystallized from water/ethanol. Yield: 512 mg, (80 %) .

4- (2- (4- [¹¹C]Methoxybβnzamido) ethyl)benzenesulphonamide (¹¹C-I)

To 200 μg of the phenol precursor 4 dissolved in 0.2 mL dimethyl formamide (DMF) about 1 mg of cesium carbonate was added and ^i:LC-methyl iodide was bubbled through the solution which was heated at 90 °C for 15 min.

The reaction mixture was purified using semi prep HPLC Column in a C18 XBridge column (Waters) 4.6 mm x 150mm (5μm) eluted with a mixture of 20% EtOH and 80% NaOAC 0.05M pH5.5 at a flow rate of lml/min. The eluate was monitored by a UV detector (254 nm) and a radiometric detector. The fraction eluting at 11.0 min was isolated and analyzed by HPLC on a C18 XBridge (Waters) column (3 x 100mm, 3.5μm) eluted with a mixture consisting of 20% ACN and 80% NaOAC 0 . 05M pH5. 5 At a flow rate of 0 . 8ml/min. The retention time of ¹¹C-I was found to be identical to that of 1 ( 5 . 0 min) . The radiochemical yield was found to be about 30% (relative to starting [¹¹C] methyliodide and the radiochemical purity was found to be higher than 95% .

Synthesis of 4-["C]methoxy-benzenesulphonamide (^uC-2) nC-2 was synthesized by addition of commercially available precursor p-hydroxybenzenesulfonamide (0.8 mg) in DMF (0.2 ml), NaOH IN (2.5μL) in the presence of [¹¹C]CH₃I, heated at 90 °C for 15 min. After cooling down to RT, the reaction mixture was diluted with 1 ml water and purified by HPLC on an XTerra Ci₈ column (5um, 7.8 mm^χl50 mm, Waters). The mobile phase consisted of mixtures of solution (NH₄OAc 0.05 M/ EtOH; 90:10 v/v) at a flow rate of 2 mL/min. The radio labeled product [¹¹C] -2 was collected at 11 min with a good yield 90%. The identity of [¹¹C] -2 was confirmed by coelution with authentic non-radioactive 4-methoxybenzenesulfonamide (Alfa Aesar GmbH & Co. KG, Karlsruhe, Germany) using analytical RP-HPLC on an XTerra Ci₈ column (5μm, 4.6 mm^χ250 mm, Waters) eluted with (NH₄OAc 0.05 M/ EtOH; 80:20 v/v) at a flow rate of 1.0 mL/min, detected at UV 236 nm, Rt = 8 min, radio chemical purity >98%.

Synthesis of precursor and radiolabelling of ^99mTc-5

Since technetium is a metal, chelating agent or ligand is required to label with technetium. -Therefore we used S-BAT (S,S-bis-trityl-Λ7-BOC-l,2-ethylenedicysteamine) and effectively conjugated with the active moiety AEBS and labeled with technetium. The S- BAT ligand was synthesized from our earlier published procedure and conjugated with AEBS with a yield of 66%. The ^99mTc-5 was synthesized in one- pot, two-step procedure. The S-BAT lignad was deprotected from trityl and BOC protecting groups by hydrolysis and this deprotected conjugate was subsequently labelled with Na "¹¹¹TcO₄ ^" by an exchange labelling method.

In a test vial, (precursor 200 μg in ACN) and HCL 0.5N (60μL) was added and heated at 100⁰C for 20 min. After cooling down to RT, to the vial 200 μL buffer mixture consisting of phosphate buffer 0.5 M pH 7.0 (5mL) , Na₂EDTA 0.1 M (2.5mL), sodium potassium tartrate (0.14mmol, 2.5 ml) was added along with 15 μL SnCl₂.2H₂O (0.09 mmol in 5ml of 0.05M HCL), Na "¹¹¹TcO₄ (370-740 MBq/0.5 ml) and heated at 100 °C for 15 min. After cooling down, the reaction mixture was purified by HPLC on an XTerra Ci₈ column (5μm, 4.6 mm^χ250 mm, Waters). The mobile phase consisted of 60:40 v/v; 0.05 M ammonium acetate and ethanol at flow rate of 1.0 mL/min. The radiolabeled ^99mTc-5 obtained with a radio chemical yield 49% and purity > 98%.

The understanding compounds were tested on the blood imaging capacity:

18 F-I

18 F-2

99m Tc-5

Synthesis/radiosynthesis of the Fluoride labelled compounds compounds Radiosynthesis of 4-(2-(4-

F]fluoroethoxybenzamido)ethyl)benzenesulphonamide ([ F]-I)

Production of ¹⁸F-Fluoride

[¹⁸F] Fluoride was obtained using an [¹⁸O (p,n)] nuclear reaction by irradiation of 0.5 mL of 97% enriched [¹⁸O]H₂O (Rotem HYOX¹⁸, Rotem industries, Beer Sheva, Israel) in a niobium target with 18-MeV protons from a Cyclone 18/9 cyclotron (Ion Beam Applications, Louvain-la-Neuve, Belgium). After irradiation for about 30 min [¹⁸F]F^" was transferred from the target to semi-automated synthesis module and separated from [¹⁸O] H₂O using a SepPak Light Accell plus QMA^® anion exchange catridge (Waters) , which was preconditioned by successive treatments with 0.5 M potassium carbonate solution (10 mL) and water (2 x 1OmL) . The trapped [¹⁸F] F^" fluoride ion was then eluted into a reaction vial with a solution of 2.47 mg potassium carbonate and 27.92 mg Kryptofix 222 dissolved in 0.75mL of H₂O/CH₃CN (5:95 v/v) . A stream of helium was passed to evaporate the solvent from reaction vial while heating at 110⁰C for 5 min. After evaporation, [¹⁸F]F^" was further dried by azeotropic distillation using 1 ml of anhydrous acetonitrile added in two portions to remove traces of water.

Radiosynthesis of [¹⁸F] fluoroethylbromide

2-Bromoethyl triflate (BrCH₂CH₂OTf) (5 μL) in o- dichlorobenzene (0.7 ml) was added to a reaction vessel containing dried [¹⁸F]F^" and potassium-kryptofix complex. The reaction vial was then heated to 110°C, and the resulting [¹⁸F] fluoroethylbromide ([¹⁸F] FEtBr) was distilled with a stream of helium (4-5 ml/min) and trapped into a reaction vessel containing the suitable phenol precursor.

Radiosynthesis of ¹⁸F-I [¹⁸F] fluoroethylbromide was trapped into a reaction vessel containing 1 mg of 4- (2- (4-hydroxybenzamido) ethyl) - benzenesulphonamide (see 3.4) in DMF (0.2 ml), NaOH IN (2.5 μl) . When the maximum radioactivity was distilled (about 10- 15 min) the reaction mixture was heated at 90⁰C for 15 min, to evaporate the residual ( [¹⁸F] FEtBr) the heating was continued at 70⁰C for 10 min, after cooling down to room temperature (RT) . The reaction mixture was diluted with 1 ml water and purified by HPLC on an XTerra Ci₈ column (5μm, 7.8 mm^χl50 mm, Waters) . The mobile phase consisted of mixtures of solution (NH₄OAc 0.05 M/ EtOH; 90:10 v/v) at a flow rate of 2 mL/min.

The same labeling precursor of was used to synthesize the reference compound, the precursor was dissolved in DMF and reacted with FCH₂CH₂Br, K₂CO₃ (equivalent ratios) at 70°C for 16 h, the reference compound was obtained with an chemical yield of 65% The radio labeled product ¹⁸F-I was collected at 5 min, yielding 65%. The identity of the tracer ¹⁸F-I was confirmed by coelution with non-radioactive compound using analytical RP-HPLC on an XTerra Ci₈ column (5μm, 4.6 mm^χ250 mm, Waters) eluted with (NH₄OAc 0.05 M/ ACN; 75:25 v/v) at a flow rate of 1 mL/min, detected at UV 254nm, Rt = 5 min, radio chemical purity >99% .

Radiosynthesis of 4- [¹⁸F] fluoroethoxy-benzenesulphonamide (¹⁸F-2) [¹⁸F] fluoroethylbromide (see 1.2) was trapped into a reaction vessel containing 0.8 mg p-hydroxybenzenesulfonamide (TCI Europe NV, Belgium) in DMF (0.2 ml), NaOH IN (2.5μL). When the maximum radioactivity was distilled (about 10-15 min) the reaction mixture was heated at 90⁰C for 15 min, to evaporate the residual ([¹⁸F]FEtBr) the heating was continued at 70⁰C for 10 min, after cooling down to room temperature (RT) . The reaction mixture was diluted with 1 ml water and purified by HPLC on an XTerra Ciβ column (5μm, 7.8 mm><150 mm, Waters) . The mobile phase consisted of mixtures of solution (NH₄OAc 0.05 M/ EtOH; 90:10 v/v) at a flow rate of 2 mL/min.

The radio labeled product 18F-2 was collected at 16 min, with a radiochemical yield of 65%. The identity of the tracer ¹⁸F- 2 was confirmed by coelution with non-radioactive compound F-2 (synthesized by published procedure with a yield of 60%) using analytical RP-HPLC on an XTerra Ci₈ column (5μm, 4.6 mm^χ250 mm, Waters) eluted with (NH₄OAc 0.05 M/ EtOH; 80:20 v/v) at a flow rate of 0.8 mL/min, detected at UV 236nm, Rt = 11 min, radio chemical purity >99% (Figure. 2) .

Biodistribution study in normal mice

A solutions of [HC]-I obtained after RP-HPLC purification was diluted using 0.9 % NaCl for injection to a concentration of 3.7 MBq/mL and 37 MBq/mL, respectively. Biodistribution of both tracers was studied in male NMRI mice (body mass 30-40 g) . The mice were anesthetized by inhalation of isoflurane. A volume of 0.1 mL of the diluted tracer solution was then injected via a tail vein and the mice were sacrificed by decapitation at 2 or 60 min post injection (p.i., n=4 per time point). All organs and body parts were dissected. Blood was collected in tared tubes and the mass of all organs and body parts was determined. The radioactivity in each organ was counted using a 3-in. NaI (Tl) well counter, corrected for background radioactivity and expressed as percentage of the injected dose (% ID) or as percentage of the injected dose per gram tissue (% ID/g) . For the calculation of total radioactivity in blood, blood mass was assumed to be 7 % of the body mass.

In vitro labelling of human RBCs

From a human whole blood samples (4ml) , 1 mL of whole blood is transferred into preweighed tube. lμCi (lOμL) of HPLC purified radio tracer (¹¹C-I) was added to the 1 ml whole blood and incubated at room temperature (RT) for 10 minutes.

After incubation, the cells and plasma were separated by centrifugation for 5 min at 3000 rpm.

Total CPMs are counted by the γ-counter after which the (supernatant) plasma is transferred into a preweighed tube.

The (sediment) cells are rinsed by adding 400μL of phosphate buffer saline 0.05 M pH 7.4 (PBS) and incubating for 5 minutes at RT , later PBS was separated by centrifugation for 5 min at 3000 rpm. The radioactivity in blood cells, plasma and rinsings of PBS are counted in gamma counter separately and the activity in blood cells and plasma per gram was calculated as follows.

CPMs in the cells/g %Activity in blood cells^~ x 100

Total CPM/g (cells + plasma + PBS rinsings)

CPMs in the plasma/g % Activity in plasma "= x 100

Total counts/g (cells + plasma + PBS rinsings)

Procedure for Cell fraction 1, 2 and 3

The collected whole blood sample (4ml) was centrifuged for 5 min at 3000 rpm to separate the blood cells and plasma. The blood cells are then divided into three fractions 1 mL each and transferred to 5 mL tubes. Processing of cell fractions 1 and 2

To the cell fractions 1 and 2, lOμCi (lOOμL) of HPLC purified radio tracer (¹¹C-I) and 500μL of PBS was added and incubated at room temperature (RT) . For cell fraction 1 the incubation time is 10 minutes and for cell fraction 2 the incubation time is 20 minutes

After incubation 500 μL of PBS is added, the cells are then subjected to centrifugation for 5 min at 3000 rpm to remove the unbound tracer followed by the rinsing procedure as follows.

- The cells are rinsed by adding ImL PBS, incubating for 5 minutes at RT, later the PBS was separated by centrifugation for 5 min at 3000 rpm.

- The rinsing procedure is carried out twice to remove the unbound tracer and traces of plasma.

- The radioactivity in blood cells and PBS rinsing fractions are counted in gamma counter separately.

- The percentage of retained tracer in the cells is calculated as follows

CPMs in the cells X 100

Total CPM (cells + PBS rinsing solution)

Processing of cell fraction 3

To the cell fraction 3, aminoethylbenzosulfonamide (1 mg dissolved in 200μL of PBS), lOμCi (lOOμL) of HPLC purified radio tracer (¹¹C-I) was added consecutively and incubated at room temperature (RT) for 20 minutes. After incubation, the cells are centrifuged for 5 min at 3000 rpm to remove the unbound tracer followed by rinsing procedure as follows . - The cells are rinsed by adding ImL PBS, incubating at RT for 5 minutes, later the rinsing solution (PBS) was separated by centrifugation for 5 min at 3000 rpm.

- The rinsing procedure is carried out thrice to remove the unbound tracer and traces of plasma. - The radioactivity in blood cells and rinsing fractions, are counted in gamma counter separately.

- The percentage of retained radio tracer in the cells is calculated as described above

In vitro binding inhibition study with acetazolamide and 1⁸F-2 in human red blood cells

From a healthy human volunteer about 4 mL of blood was collected into a BD vacutainer ™ 4.5 ml (containing lithium heparin; BD, Franklin lakes, USA) . The whole blood was divided into 4 fractions (A, B, C and D) each containing 0.5 mL in 1.5 ml eppendorf tubes. Tracer ¹⁸F-2 about 37 KBq/0.25 mL (<5% of EtoH) was added to the fractions (A, B, C and D) followed by addition of 1, 10 and 100 μM acetazolamide (AAZ) to the fractions B, C and D using fraction A as blank. The fractions B, C and D were treated with the acetazolamide due to its inhibitory properties to the most isoforms of CAs for example, Ki of CA I, 250nm; CA II, 12 nM; CA IV 70 nM; CA IX, 25 nM ^[16] . All the fractions were incubated at room temperature for 1 h. After incubation all the fractions were centrifuged for 5 min at 3000 rpm to separate the plasma and erythrocytes. The supernatant layer (plasma) was pipetted off carefully and transferred to other eppendorf, the unbound tracer from erythrocyte fraction was rinsed by the addition of phosphate buffer pH 7.4 (0.5 itiL) and incubated for 5 min at RT followed by the centrifugation for 5 min at 3000 rpra, the supernatant layer was pipetted off and transferred to other eppindorf. The rinsing procedure was repeated twice to remove the unbound tracer from the erythrocyte fractions. The radioactivity of all fractions, plasma and subsequent washings was quantified on automated gamma counter equipped with a 3-in. NaI(Tl) well crystal coupled to a multi-channel analyzer (Wallac 1480 Wizard 3'', Wallac, Turku, Finland).

Xn vitro binding inhibition study with AEBS in human red blood cells The in vitro blood cell analysis was carried out as mentioned earlier (II.1) with some modifications. The cells were incubated at RT for 10 or 20 min, the binding of the tracers ¹¹C-I and ^uC-2 to RBCs in the presence of [4- (2- aminoethyl-benzene) sulfonamide (AEBS) was quantified.

μPET imaging studies with ¹⁸F-2

To assess the in vivo applicability of ¹⁸F-2, dynamic micro- PET imaging was performed on a healthy male wistar rat using a Siemens Focus 200, micro-PET scanner with spatial resolution of 2 mm. The rat was anesthetized with isoflurane and injected with 40 MBq of ¹⁸F-2 via lateral tail vein. Immediately after injection, the rat underwent scanning for 60 min while being kept sedated with 1.5% to 2% isoflurane. Results and discussion

Radiolabelling

Radiolabelling was performed by alkylation of compound 4 with methyliodide and subsequent purification of [HC]-I with RP-HPLC. HPLC analysis of the purified compound showed a sufficient radiochemical purity (>95%) and identical retention time of the radio labeled compound [¹¹C] -1 with authentic 1.

RBC Binding inhibition with 4-aminoethγlbenzenesulphonamide (AEBS)

The tracers ¹¹C-I or ^i:LC-2 show specific binding to RBCs (>90%) when incubated with human blood to be >90% within 10 minutes after incubation, incubation for an additional 10 minutes did not seem to influence the fraction of radiotracer that was bound to the RBC fraction. After addition of 5 μmol of 4-aminoethylbenzenesulphonamide (AEBS), which has a Ki of 2.4mM or 160 nM for CAI or CA II respectively, a 20% reduction of acticivity bound to RBCs was observed for ¹¹C-I and 55% reduction for [¹¹C] -2 (Figures

3 and 4) . This indicates that the tracers are retained in blood cells by binding to the CA I/II expressed in erythrocytes.

RBC Binding inhibition with acetazolamide

The binding of ¹⁸F-2 is inhibited progressively by increasing concentrations of acetazolamide, a compound for which the affinity for CAI and CAII has been published. From the results it can be depicted with the blanc (fraction A) there was a retention of >90% of ¹⁸F-2 in RBCs, whereas fractions B, C and D treated with increasing concentrations of acetazolamide (1, 10 and 100 μmol shown a progressive reduction in binding of ¹⁸F-2 to the erythrocytes. When compared to the blanc (Fraction A) 11%, 65% and 80% reduction in RBC binding was observed, indicating the retention in erythrocytes is due to binding to CA I/II which are expressed in erythrocytes. The results confirm that ¹⁸F-2 efficiently binds to the erythrocytes and is retained selectively by binding to the carbonic anhydrases (CA I/II) .

¹⁸F-I was also tested in vitro showing fast uptake in RBCs as values as high as 90% RBC labeling were observed after an incubation time of only 1 minute. Increase of the incubation time did not result in an increased fraction of ¹⁸F-I associated with RBCs. Incubation in the presence of acetazolamide resulted in a decrease of the fractions of ¹⁸F- 1 associated with RBCs indicating that ¹⁸F-I binds to CAI/II within the RBCs.

RBC binding of ^99mTc-5 Incubation experiments with whole human blood showed that the fraction of ^99mTc-5 associated with RBC is relatively low compared to the tested ¹¹C and ¹⁸F compounds. Additional tests using plasma poor RBCs (isolated RBCs resuspended in NaCl 0.9%) are still able to diffuse through the RBC membrane and bind to CAI/II. In view of the larger size of the technetium complex the rate of diffusion is however slower as compared to the ¹¹C and ¹⁸F labeled compounds so that the uptake in RBCs significantly increases from an incubation time of 10 min (45%, fraction B) compared to an incubation time of 20 min (70%, fraction C) indicating that the compound can be used for in vitro labeling of RBCs. Biodistribution in mice

The biodistribution in mice (FIG. 1) shows that [HC]-I is predominatly retained in blood (79.3% at 2 min pi, 67% at 60 min p.i.). It appears that a small fraction of the injected activity is excreted to the intestines (12% of ID) and the urine (5% of ID) at 60 min p.i. As the radiotracer is injected intravenously it appears that it is quickly extracted from plasma to the RBCs

In order to confirm whether the tracer agent binds to RBCs and not to plasma proteins further tests were carried out in vitro using human blood.

Figure 2 shows that more than 95% of the activity is associated with RBCs even after an incubation period as short as 10 min (2) which is identical to an incubation period of 20 min (3) . Incubation of the labeled red blood cells with PBS for 2 subsequent periods (1) does not result in a decrease of the RBC associated activity indicating that the tracer is tightly bound within the RBCs. Coincubation with a product with known affinity for CA (aminoethylbenzenesulphonamide) decreases the RBC associated fraction of tracer to about 75% indicating that the binding of the tracer within the RBC indeed occurs through interaction with CA II/I.

These results indicate that the studied compound [¹¹C]-I can be used as an efficient in vivo or in vitro labeling agent for RBCs. μPET imaging study with ¹⁸F-2

To assess the in vivo applicability of ¹⁸F-2, dynamic micro- PET imaging was performed on a healthy male wistar rat using a Siemens Focus 200, microPET scanner with spatial resolution of 1.2 mm. The rat was anesthetized with isoflurane and injected with 40 MBq of ¹⁸F-2 via lateral tail vein. Immediately after injection, the rat underwent scanning for 60 min while being kept sedated with 1.5% to 2% isoflurane. Images were reconstructed from the raw data and were analyzed with acquisition sinogram and image processing software that accompanies the Siemens micro-PET.

From the Figure 8 and 9 , it can be seen that IV injection of ¹⁸F-2 provides excellent images of the heart and major blood vessels despite the relatively limited resolution of the microPET camera. The microPET image provides a proof of concept that small organic molecules with affinity for

CAI/II such as [¹⁸F] -2 efficiently label the erythrocytes in vivo allowing wide application for blood pool imaging.

Absence of activity in the bladder and the liver suggests that ¹⁸F-2 does not leak out of the blood compartment.

References

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9. Gotthardt M, Broker S, Schlieck A, Bauhofer A, Herbst B, Behe M, Corstens FH, Behr TM, Gorg C. Scintigraphy with 99mTc-labeled heat-altered erythrocytes in diagnosing hyposplenia: prospective comparison to 99mTc-labeled colloids and colour-coded duplex ultrasonography. Nuklearmedizin. 2007; 46 (4) : 135-40

10. Alvarez R, Diehl PGVI, Avram A, Brown R, Piert M. Localization of splenosis using 99mTc-damaged red blood cell SPECT/CT and intraoperative gamma probe measurements. Eur J Nucl Med MoI Imaging. 2007 Jun;34 (6) :969. Epub 2007 Apr 25.

11. Valdes Olmos RA, Horenblas S, Kartachova M, Hoefnagel CA, Sivro F, Baars PC. 99mTc-labelled heat- denatured erythrocyte SPET-CT matching to differentiate accessory spleen from tumour recurrence. Eur J Nucl Med MoI Imaging. 2004 Jan; 31 (1) : 150. Epub 2003 Dec 2

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Claims

1. A benzene sulfonamide compound of formula (I)

(D wherein R comprises radiolabel connected to the phenyl ring directly or via an oxygen, sulfur, nitrogen, alkyl, aryl or any combination of these and whereby the radiolabel is from the group consisting of ¹¹C, ^99mTc, ¹³N, ⁶⁸Ga, ⁶⁷Cu and ⁶²Cu, or the radiolabel is a halogen from the group consisting of ¹²²I₇ ¹²⁴I, ¹²³I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁸Br, ⁸²Br, and ¹⁸F;

2. The benzene sulfonamide compound of claim 1, wherein the phenyl ring may be derivatized in other positions with additional substituents such as halogens, alkyl or aryl groups connected to the ring directly or via S, 0, N or other atoms.

3. A benzene sulfonamide compound of claim 1, with the formula (II)

( I I ) wherein ;

- n represents an integer from 0 to 6;

- R is selected from [¹¹C] Ci_₆alkyloxy-, X-Ci_₆alkyl-, or X-C_1-. βalkyloxy-;

- X represents a radioactive halogen selected from the group consisting of ¹²²I, ¹²³I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, 7⁸Br, ⁸²Br, and ¹⁸I; including solvates and pharmaceutically acceptable addition salts thereof.

4. A benzene sulfonamide compound of claim 1, with the formula (II)

( II ) wherein;

- n represents an integer from 1 to 6;

- R is selected from [¹¹C] Ci_₆alkyloxy-, X-Ci_₆alkyl-, or X-Ci- βalkyloxy-;

5. A benzene sulfonamide compound according to claim 1 wherein; n represents an integer from 1 to 6, in particular n represents 2; R represents [¹¹C]Ci-_GaIkYlOXy-, or X-C_x- βalkyloxy-; and X represents a radioactive halogen selected from the group consisting of ¹²³I, and ¹⁸I.

6. A benzene sulfonamide compound according to claim 1 that consists of those compounds selected from the group consisting of; 4- (2- (4- [¹¹C] Methoxybenzamido) ethyl) benzenesulphonamide (hereinafter also referred to as (¹¹C-I) ) and other derivatives thereof with a sulphonamide group.

7. A benzene sulfonamide compound as in claim 1, with the structure

8. A benzene sulfonamide compound as in claim 1, with the structure

9. A benzene sulfonamide compound as in claim 1, with the structure

10. A benzene sulfonamide compound as in claim 1, with the structure

11. A composition comprising a benzene sulfonamide compound as defined in any one of claims 1 to 10.

12. A composition according to claim 11 wherein said composition is an aqueous injection further comprising one or more of a stabilizer (e.g., ascorbic acid), a solubilizer (e.g., -cyclodextrins and the like), a dispersing agent (e.g., Tween 80, carboxymethyl-cellulose, sodium alginate and the like), a preservative (e.g., methylparaben, propylparaben, benzyl alcohol, chlorobutanol and the like) , an isotonicity agent (e.g., sodium chloride, glycerol, sorbitol, glucose and the like) and the like.

13. Use of a benzene sulfonamide compound as defined in any one of claims 1 to 10, or of compositions as defined in claims 11 and 12, in a method of diagnosing and/or monitoring physiological parameter (s) in a subject.

14. Use of benzene sulfonamide compounds as defined in any one of claims 1 to 10, or compositions as defined in claims 11 and 12 in PET or SPECT imaging techniques or for ex vivo diagnostic purposes

15. Use according to claims 13 or 14 in the measurement of blood volume and/or blood flow.

16. A radiolabeled benzene sulfonamide compound with a specific binding affinity for the carbonic anhydrases type I and/or II, for use in a diagnostic treatment

17. A radiolabeled benzene sulfonamide compound with a specific binding affinity for the carbonic anhydrases type I and/or II, for use in an imaging based diagnosis of a mammalian, including human subject, to diagnosing and/or monitoring blood volume and/or blood flow.

18. A benzene sulfonamide compound as defined in any one of claims 1 to 10, or a composition as defined in any one of the claims 11 and 12, for use in an imaging based diagnosis of a mammalian, including human subject, for diagnosing and/or monitoring blood volume and/or blood flow.

19. A pack for blood imaging for monitoring blood volume and/or blood flow in a mammalian, including human, subject, the pack comprising a radio labeled benzene sulfonamide compound with a specific binding affinity for the carbonic anhydrases type I and/or II.

20. A pack for blood imaging for monitoring blood volume and/or blood flow in a mammalian, including human, subject, the pack comprising a benzene sulfonamide compound of any of the claims 1 to 10.