WO2004071505A2

WO2004071505A2 - Radiolabelled d-amino acids for use in diagnosis with spect and pet and systemic radionuclide therapy

Info

Publication number: WO2004071505A2
Application number: PCT/US2004/003829
Authority: WO
Inventors: John R. Mertens
Original assignee: Mallinckrodt Inc.
Priority date: 2003-02-11
Filing date: 2004-02-11
Publication date: 2004-08-26
Also published as: WO2004071505A3

Abstract

The present invention relates to D-amino acid of the general formula NH2-CHR-COOH, wherein R is a variable side chain, for use in diagnosis with SPECT and PET or in systemic radionuclide radiotherapy, wherein the D-amino acid is labelled in the side chain R, which is preferably benzyl, methyl benzyl, ethyl benzyl, 3-hydroxybenzyl or 4-hydroxybenzyl, either of which may be optionally substituted in a free ortho, meta, or para position with (CH2)n-X, wherein n is 0, 1 or 2 and X is a radioactive halogen selected from F, Cl, Br, I or a polamino polycarboxylic (PAPC) chelator. The invention further relates to pharmaceutical compositions for diagnosis or therapy of cancer.

Description

RADIOLABELLED D-AMINO ACIDS FOR USE IN DIAGNOSIS WITH SPECT AND PET AND SYSTEMIC RADIONUCLIDE THERAPY

Field of the Invention

The present invention relates to new radiolabelled D-amino acids, to pharmaceutical compositions comprising them and to their use in diagnosis, in particular with SPECT and PET, and in therapy, such as systemic radionuclide therapy.

Background of the invention

Positron emission tomography (PET) is a scanning technique used in conjunction with small amounts of radiolabelled compounds. A very small amount of a radiolabelled compound is inhaled by or injected into the patient. The injected or inhaled compound accumulates in the tissue to be studied. As the radioactive atoms in the compound decay, they release smaller particles called positrons, which are positively charged. When a positron collides with an electron (negatively charged), they are both annihilated, and two photons (light particles) are emitted. The photons move in opposite directions and are picked up by the detector ring of the PET scanner. A computer uses this information to generate three-dimensional, cross-sectional images that represent the biological activity where the radiolabeled compound has accumulated.

PET is often performed with the radioactive tracer ¹⁸F-deoxyglucose (FDG). All living cells utilize glucose. Some cells metabolize glucose faster than others. Cancer cells are hyperactive and divide quickly, and therefore metabolize the injection of the radioactive tracer FDG faster than normal cells. On a PET scan, cancer cells appear "hot" and significantly more prominent than normal cells.

A related technique is called single photon emission computed tomography (SPECT). SPECT is similar to PET, but the compounds used contain heavier, longer-lived radioactive atoms that emit high-energy photons, called gamma rays, instead of positrons. SPECT is used for many of the same applications as PET, and is less expensive than PET.

Systemic radionuclide therapy is a form of radiotherapy that involves administering the source of the radiation into the patient. With systemic radionuclide therapy the physiology of the disease provides a major contribution to the therapy ultimately resulting in the delivery of the radionuclide to the tumor. By using a radioactive material that will be delivered to the tumor by the patient's own physiologic processes, it is possible to deliver a large dose of radiation to certain tumors with a minimal amount of patient manipulation.

As explained above, the subtle changes in the metabolic phenotype such as higher uptake and metabolism of amino acids permit the in vivo study of tumours using radioactive labelled L-amino acids coupled to SPECT (Single Photon Emission Computed Tomography) and PET (Positron Emission Tomography). Presently, some L-amino acids are already used in clinical studies, such as 3- I-alpha-methyl-L-tyrosine (L- IMT) for SPECT (Biersack et al. (1989) J. Nucl. Med. 30:110; Jager et al. (2001) Nucl. Med. Comm. 22(1):87) and 2-¹⁸F-L-tyrosine (Coenen et al. (1989) J. Nucl. Med. 30:1376), 0-(2-^I8F- ethyl-L-tyrosine (L-¹⁸FET)(Heiss et al. (1999) J. Nucl. Med. 40:1367) and ¹⁸F-alpha- methyl-L-tyrosine (L-¹⁸FMT) for PET. The uptake of 2-¹⁸F-L-tyrosine in inflammatory lesions is lower than that of ¹⁸F-FDG, used for routine PET diagnosis of tumours. A drawback limiting the applicability of L- IMT, L- FET and L- FMT is the high renal accumulation.

Summary of the invention

Whatever in the future the approaches for therapy of cancers will be, an accurate and specific non-invasive diagnosis on biomolecular level of tumours and metastases remains of primary importance. SPECT and PET are techniques that can play an important role therein. However, new and better tracer molecules for use in these techniques will always be necessary.

In the current state of the art and in the next future also specific target directed vector molecules labelled with radioactive isotopes emitting beta-particles, alpha-particles, Auger electrons as well as low energy gamma photons with high electron conversion are needed for systemic radiotherapy.

It is thus the object of the invention to provide new tracers and vector molecules for the diagnosis and treatment of cancer not showing the drawbacks of the currently known tracers.

It is a further object of the invention to provide the use of such novel tracers and vector molecules in diagnosis and therapy. Detailed description of the invention

In the research that led to the invention two new potential tumour tracers for SPECT were developed: 2-¹²³I-L-tyrosine (2-¹²³I-L-Tyr) and 2-¹²³I-L-phenylalanine (2- ^I23I-L-Phe). These tracers were evaluated in vitro using a RIM (rat rhabdomyosarcoma cells) and WiDr (human colonadenocarcinoma cells) tumour cell model. RIM tumour bearing rats were used as in vivo rodent model. It was thus shown that the uptake of ¹²³I- methyl-L-tyrosine and the new tracers 2-¹²³I-L-tyrosine and 2-¹²³I-L-phenylalanine in tumour cells occurred for the larger part (-80%) by the system L amino acid transport system (LAT). The increased accumulation is mainly determined by strongly increased amino acid transport activity rather than by incorporation into proteins. It was demonstrated by Langen et al. ((2002) Nucl. Med. Biol. 29:625-631) that the expression of L transporters and related reversible uptake depended on the proliferation rate of human glioma cells.

The L transporter is a major nutrient transport system responsible for Na⁺- independent transport of large neutral amino acids including synthetic amino acids by an obligatory exchange mechanism coupled to an anti-port system. The heterodimeric L transporter contains subunits named LAT1 and LAT2. Recently it was shown that in tumour cells within the neutral amino acid transporter system L, the hLATl transporter (hLATl/h4F2hc heterodimeric membrane glycoproteins) is related to tumour growth and progression of malignant tumours. LAT1 expression was scarcely detected in non-tumour areas. LAT1 is highly expressed (up-regulated) in proliferating tissues, in particular malignant tumours, as it plays a critical role in cell growth and proliferation.

A remarkable characteristic of system L and the hLAT-1 amino acid uptake is its broad substrate selectivity, which enables the transporter to accept amino acid related compounds, such as D-amino acids and some cancer drugs. LAT2 transports all of the isomers of neutral alpha-amino acids by facilitated diffusion. LAT2 has a high level of expression in small intestine, kidney, placenta, brain and in epithelia and blood-tissue barriers. LAT2 does not transport D-amino acids.

Although 2-¹²³I-L-Tyr and 2-¹²³I-L-Phe are in "in vitro conditions" not significantly incorporated in the cell proteins (they are non-proteinogenic) they showed a retarded efflux in MEM buffer (a buffer containing essential and non essential amino acids mimiclάng in vivo conditions) as compared to the efflux of the intracellular free fraction of ;H-L-Phe and;H-L-Tyr. This could be explained by the fact that the efflux is ruled by both the specific activity of the radioactive tracers, which is very high representing very low mass, and the difference in affinity for the internal transporter part between these radio iodinated analogues, which is slightly smaller, and all the natural L-amino acids present inside the cells.

When evaluated in vivo in RIM tumour bearing rats, the tracers show high uptake in the tumour (comparable with the uptake of 3-¹²³I-methyl~L-Tyr) while no renal accumulation or significant uptake in induced acute inflammatory tissue was observed. The uptake and washout kinetics in vitro and in vivo were almost identical for both compounds.

2-¹²³I-L-Tyr is currently used in human studies. High uptake in tumours is observed while a fast clearance of the tracer by the kidneys to the bladder is observed. High selectivity is proven by the fact that the uptake in inflammatory lesions such as tuberculosis was negligible.

All the development of and studies with radioactively labelled amino acids for tumour diagnosis with PET and SPECT were focussed on the L-enantiomeric form as it was assumed that the amino acid tracers had to be incorporated into the tumour cell proteins.

Recently, it was shown that the increased accumulation is mainly determined by strongly increased amino acid transport activity rather than incorporation into proteins and that the expression of L transporters and related reversible uptake depended on the proliferation rate of human glioma cells.

The present inventors have also demonstrated that I.V. injection of 2-^!31I-L-Tyr (¹³¹I is a β-emitter) in RIM tumour bearing rats revealed significant tumour growth inhibition as compared with the control group. Although the uptake of the radio iodinated tyrosine analogue is reversible, the retarded efflux allows to initiate radiolysis causing the growth inhibition.

Furthermore, the inventors found that in the cancer cell lines rat RIM rhabdomyosarcoma, human HT29 and WiDr colon adenocarcinoma cells, A2058 melanin producing melanoma, C36 non-melanin producing melanoma and C6 glioma, used in the in vitro evaluation model, the uptake of both the enantiomeric L form and D form of neutral amino acids such as L-tyrosine (L-Tyr), D-tyrosine (D-Tyr), L-phenylalanine (L- Phe), D-Phe, 2-Br-L-Phe, 2-Br-D-Phe, 2-I-L-Phe, 2-I-D-Phe, 2-methyl-L-Phe, 2-methyl-D- Phe, L-Leucine,;H-L-Phe, ^I4C-D-Phe,;H-L-Tyr, 2-^{123 125}I-L-Tyr, 2-^{,23 125}I-L-Phe, 2-^{123 125}I- D-Phe, 4-¹²⁵I-L-Phe occurred for at least 80 % through a Na⁺ independent obligatory exchange of the anti-port type which can be defined as a LAT transport system.

For amino acid analogues, such as ¹²³I-methyl-L-Tyr, 2-¹²³I-L-Tyr, 2-¹²³I-L-

Phe, F-O-ethyl-L-Tyr among others, which are not involved in the biochemical incorporation pathways, the uptake [*AAt,in], as a result from influx and efflux, reaches an equilibrium where [*AAeq]₀ut/SKi.mAA_0Ut = [*AAeq]i„/SKi.mAA_in (equation 1)(*AA refers to the radioactive amino acid, mAA to the amount in moles of the natural amino acids in the outer (out) compartment and in (in) the cancer cells, respectively. Ki is the relative Km value in the two compartments.

It was shown in RIM cells that the incorporation rate of [³H]-L-Phe in vitro is very much slower (0.03 %/min) than the short time reversible uptake Idnetics (2.6 %/min) and that it follows a pseudo zero order reaction pattern. Both linear parts of the time related curves, representing the total uptake (upper) and the incorporation fraction (lower), respectively, show the same slope up from 20 minutes. This means that the uptake in the growing cells by a reversible exchanging mechanism remains constant and that the LAT coupled influx of radioactivity is directly related to the requirement of amino acids for protein synthesis or the cell metabolism. Related to the equation mentioned above the uptake at equilibrium of 2-¹²⁵I-L-Phe, even not incorporated into the proteins, reflects the total "amino acid turn-over" of the tumour cells.

It is known that D-amino acids are only accidentally taken up from some nutrients and that the uptake of D-amino acids in normal tissue is low to negligible.

This invention shows that the LAT1 transport system present in cancer cells, is a suitable transport system for the influx of neutral synthetic amino acids showing the D- enantiomeric form (D-amino acids) like the amino acids mentioned above.

It was also surprisingly shown that, due to the LAT1 anti-port system and the competition of the neutral L-amino acids present inside the cancer cells, the efflux of the radioactively labelled D-amino acids, with a high specific activity, and thus very low concentration as compared to the L-amino acids present in the cells (equation 1) showed significantly slower kinetics than observed for the radioactive L-analogues, resulting in a longer retention in the cells, which makes them candidates for radionuclide therapeutic purposes. Moreover as the D-amino acids are not significantly transported trough the blood-brain barrier (BBB), radiolabeled D-amino acids compared to the uptake of the L- analogue can show if the BBB is damaged by the tumour growth.

The background observed in vivo in rat and human for 3-¹²³I-methyl-L-Tyr, 2- ¹⁸F-L-Tyr, 2-¹²³I-L-Tyr and 2-¹²³I-L-Phe can be due partially to LAT2 induced uptake. As it was shown that LAT2 does not transport D-amino acids, the non-specific background will be reduced when using radioactive D-amino acid analogues.

The present invention is thus based on the finding that lipophilic neutral radioactively labelled synthetic amino acids showing the D enantiomeric form show a low, almost non-significant uptake in normal tissue and inflammatory tissue, show an uptake in tumour cells by the LAT amino acid transport system almost comparable with that of their L enantiomeric form analogues, which linked to a lower background uptake results in a better tumour to background ratio, and show a longer retention (slower efflux) in tumour cells than the L analogues due to their high specific activity and extreme low mass linked to a lower affinity than their L analogues vis-a-vis the LAT transport system (competition- retention model), making them appropriate candidates for systemic radionuclide tumour therapy.

The invention thus provides novel D-amino acid of the general formula NH₂- CHR-COOH, wherein R is a variable side chain, for use in diagnosis with SPECT and PET or in systemic radionuclide radiotherapy, wherein the D-amino acid is labelled in the side chain R. D-amino acids that are radioactively labelled in their R side chain have not been described before, in particular not for use in diagnosis and therapy.

In a specific embodiment R is a bidentate or tridentate bifunctional chelating molecule. Depending on the radionuclide that is used for labelling of the D-amino acid R can be varied. For labelling with a radioactive halogen R is selected from the group consisting of benzyl, methyl benzyl, ethyl benzyl, 3-hydroxybenzyl or 4-hydroxybenzyl, either of which may be optionally substituted in a free ortho, meta, or para position with (CH₂)_n-X, wherein n is 0, 1 or 2 and X is a radioactive halogen selected from F, CI, Br, I.

For labelling with radioactive Tc and Re, R is a polyamino polycarboxylic (PAPC) chelator that is optionally coupled to the D-amino acid via a spacer, and is in particular selected from the group consisting of histidine, DOTA, DTPA and EDTA. When use is made of radioactive Pd, In, Y and Lu, as the radioactive element, R is a is a polyamino polycarboxylic (PAPC) chelator that is optionally coupled to the D- amino acid via a spacer, and is in particular selected from the group consisting of DOTA, DTPA and EDTA, and the radioactive label is complexed with the chelator. The spacer is for example an alkyl group of the formula (CH₂)_n- wherein n is 1-7, preferably 1-5.

Of the above mentioned radioactive isotopes ¹²³1, ^99mTc, ^mIn, and ¹⁸F are particularly useful as diagnostic radioisotopes. ¹²³1, ^99mTc, ^mIn emit gamma rays suitable for detection with SPECT, whereas ¹⁸F emits beta plus particles resulting in annihilation gamma rays of 51 IKeV detectable with PET. For systemic radionuclide therapy, for example by I.V. injection, the above mentioned D-amino acids are preferably labelled with ^,31I, ^18θ/188Re, ⁹⁰Y, ¹⁰³Pd, ¹⁷⁷Lu and ^mIn because these isotopes emit radiation that causes cell death.

For diagnosis 2- I-D-Tyr, 2- ID-Phe are particularly suitable and for therapy 2- I-D-Tyr, 2- I-D-Phe are preferred. Both tracers can be obtained by Cu assisted substitution in reducing conditions (Mertens et al. "Cu¹⁺ Assisted Nucleophilic Exchange Radiohalogenation: Application and Mechanistic Approach" in New Trends in Radiopharmaceutical Synthesis, Quality Assurance, and Regulatory Control, Editor: A.M .Emran, Plenum Press, NY, 1990, pp. 53-65) with a yield of 85-90% for non-isotopic exchange on 2-Br-D-Tyr or 2-Br-D-Phe yielding carrier free tracer after HPLC separation or an almost quantitative yield for isotopic exchange yielding carrier added tracer.

D-4-N,N-diethylamine-aminophenylalanine or D-β-4-EDTA-phenylalanine are custom synthesized D-analogues labelled with ^mIn for both diagnosis and therapy or with ¹⁰³Pd, ⁹⁰Y, ¹⁷⁷Lu for therapy.

2-¹⁸F-alkyl-D-phenylalanine can be obtained by nucleophilic aliphatic substitution of ¹⁸F^" on for example the tosylated precursor followed by mini-column purification.

The invention further relates to pharmaceutical compositions for use in diagnosis or therapy, comprising one or more radiolabelled D-amino acids of the invention and one or more suitable diluents, carriers, excipients or additives. Suitable diluents, carriers and excipients are for example 91 sodium chloride solution, 5% glucose solution, etc. As additives appropriate supporting complexing agents and stabilizing agents well known in formulating radiopharmaceuticals are used. The formulation of radiopharmaceuticals is well known to the person skilled in the art.

For example, for diagnostic applications with SPECT advantageously sterile kit formulations are used that are brought to the appropriate pH (4.5-7) and isotonicity as mentioned in Example 1 regarding labelling of 2-I-D-Phe. For PET non-carrier added

I purified (HPLC or multi mini-column system) ¹⁸F-labelled compounds are used in isotonic saline. A typical composition of the invention for use of ¹³¹I-D-Phe in systemic radionuclide therapy is used in a kit formulation as mentioned in Example 1 diluted in isotonic saline and is used for both bolus injection and infusion.

The invention further relates to the use of the novel compounds in diagnosis with PET or SPECT or in systemic radionuclide therapy. These techniques and the way the novel compounds of the invention are used therein are well-known to the skilled person.

The present invention will be further illustrated in the non-limiting examples that follow and in which reference is made to the following figures:

Figure 1: Uptake of 2-¹²⁵I-D-Phe/2-I-D-Phe as function of time in different cancer cells.

Figure 2: Uptake of-2-¹²⁵I-D-Phe/2-I-D-Phe showing a reversible first order reaction fit. Data: mean ± SD (n=3)

Figure 3: "Slow" MEM stimulated efflux of 2-¹²⁵I-D-Phe as compared to L- [³H]-Phe.

Figure 4: Uptake of 2-¹²⁵I-D-Phe/2-I-D-Phe in HEPES+ according to a Michaelis-Menten plot.

Figure 5: Lineweaver-Burk plot showing different lines for different concentrations of inhibiting compound with different slopes but the same intercept on the 1/mass-uptake axis.

Figure 6: Ex vivo bio-distribution of 2-¹²³I-D-Phe in RIM tumour bearing rat.

Figure 7: Activity biodistribution in vivo as function of time. Planar camera acquisition.

Figure 8: Integral picture after 60 minutes of acquisition.

Figure 9: Planar acquisition RIM tumour bearing NuNu mouse.

Figure 10: Planar acquisition of A2058 melanoma bearing NuNu mouse. EXAMPLE

EXAMPLE 1

MATERIALS AND METHODS

Synthesis and Radiosynthesis of respectively 2-I-D-Phenylalanine (2-I-D-Phe) and radioactive 2-'^{23 125 131}I-D-Phe

1. Synthesis of the precursor 2-I-D-Phe

2-Iodo-D-phenylalanine (2-I-D-Phe) was prepared using the Cu¹⁺non-isotopic exchange method (Mertens et al. Eur. J. Nucl Med (2002) 29(1):722; Lahoutte et al. JNucl Med. (2003) 44(9): 1489-94). Briefly, in a reaction vial, a 10 ml aqueous solution containing 30.3 mM 2-Br-L-phenylalanine (Peptech Corp., Burlington, Ma, USA), 4.46 mM CuS0₄ (Merck), 8.9 mM citric acid (Merck), 9.0 mM SnS0₄ (Merck), 10.7 mM gentisic acid (Merck) and 44.5 mM Nal (Merck) was added. The solution is flushed with N₂ for 10 minutes and heated at 160EC for 16h under N₂ atmosphere. After centrifugation the solution containing the product was transferred to a new flask and the water was evaporated. After re-uptake in an appropriate volume of mobile phase, purification was performed by HPLC using a 7 μm Hibar Lichrosorb RP-select column (250 x 25 μm) and 20/80 MeOH (Merck)/H₂0 containing 1 mM NH₄Ac (Merck) (pH 5.5, λ = 261 nm) at 13 ml/min. Further purification was performed by repeated dissolving of the remaining precipitate in MeOH coupled to evaporation of the MeOH fraction.

2. Radiochemistry: labelling procedure

2-[¹²⁵I]-D-phenylalanine (2-[¹²⁵I]-D-Phe) was prepared using the Cu¹⁺ assisted Kit preparation method (Mertens et al. Eur. J. Nucl Med (2002) 29(1):722; Lahoutte et al. J Nucl Med. (2003) 44(9): 1489-94). In a 1 mL septum-closed vial, 28 μl of a CuSO₄.5FI₂0 (Merck) solution (1.3xl0^"8 mol/μl) was added to a mixture of 2-I-D-Phe (1 mg), SnS0₄ (0.5 mg), gentisic acid (Merck) (1.25 mg) and citric acid (Merck) (2.5 mg) in 500 μl H₂0. This solution was flushed with N₂ for 10 minutes. 37 MBq Carrier-free sodium [¹²⁵I] iodide in 10^"2M NaOH (Bristol-Meyers Squibb Pharma, Belgium) was added. The reaction vial placed in a septum-closed safety container was heated at 100EC during 60 minutes. The reaction mixture followed by 500 μl of a 71 mM Na₃-citrate solution was passed through a sterile 0.22 μm Ag-membrane filter (Millipore). Quality control of 2-I-D-Phe and the radioiodinated analogue was achieved by HPLC, using a C8-column (LichrosPher 100RP8 (5 μm), Lichrocart 125-4) and 10/90 MeOH/ H₂0 v/v containing 1 mM NH Ac as mobile phase at 1.0 ml/minute while monitoring UV absorption (Shimadsu UV detector, 280 nm) and radioactivity (Nal- detector).

Chiral chromatography was performed on a 5μm Chirobiotic T (Astec) column (150 mm x 4.6 mm) using 80/20 v/v Methanol/H₂0 containing 20 mM ammonium acetate at a flow of 1 mL/minute. In these conditions a complete separation of the chiral isomers was obtained. The capacity values (K=) of 2-I-L-Phe and 2-I-D-Phe were 2.7 minutes and 3.8 minutes, respectively.

3. In Vitro Evaluation of 2-r^{123 125/131}η-D-Phe .

The non radioactive amino acids used were: L-Tyr, D-Tyr, L-Phe, 2-Br-L-Phe, 2-Br-D-Phe, 2-I-L-Phe, 2-I-D-Phe, 2-L-methyl-Phe, 2-D-methyl-Phe.

The radioactively labelled amino acid analogues were: ; H-L-Phe, ¹⁴C-D- Phe, ; H-L-Tyr, 2- ' I-L-Tyr, 2- " I-L-Phe as references and the new compound 2- ^123/125I-D-Phe.

4. Cell model

4.1. Cell cultures

RIM rhabdomyosarcoma cells, A2058 melanin producing melanoma cells, C32 non-melanin producing melanoma cells, HT29 colon carcinoma cells, WiDr colon carcinoma cells among other types were cultivated in cell culture flasks (NUNC) at 37EC and a 5% C0₂ atmosphere in MEM (Minimum Essential Medium with Earl=s salts and with L-glutamine) (Invitrogen) in the presence of 10% (v/v) Foetal Bovine Serum (FBS) (Invitrogen), 100 IU/mL penicillin (Invitrogen) and 100 μg/mL streptomycin (Invitrogen). For in vitro experiments cells were cultivated in 6-well-plates (NUNC) for 2 days until adhesive mono-layers, containing about 4 (± 0.2) million cells per well were obtained.

4.2. In vitro evaluation

All in vitro experiments were earned out in 6-well-plates, using three wells for each data-point. Influx and efflux were studied both in a Na⁺ containing buffer (HEPES+: 100 mM NaCl, 2 mM KC1, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM Hepes, 5 mM Tris, 1 g/L glucose and 1 g/L Bovine Serum Albumine, pH = 7,4), a Na⁺ free buffer (HEPES-: 100 mM Choline-Cl, 2 mM KC1, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM Hepes, 5 mM Tris, 1 g/L glucose and 1 g/L Bovine Serum Albumin, pH = 7,4) and MEM buffer (pH 7.2, containing essential and non essential amino acids of which 1.2 mM of amino acids known to be transported by the L transport system). The process was terminated by physical withdrawal of the buffer and washing three times with ice-cold phosphate-buffered saline (PBS). Subsequently, the cells were detached from the well with 2 mL of 0.1 M NaOH. The radioactivity of the samples was counted using a gamma-counting-system (Cobra-inspector 5003, Canberra Packard, Meriden, CT, USA).

This cell model was already used for the evaluation of several radioiodinated amino acids, which were later on tested in vivo in tumour bearing rats. It was shown that the in vitro uptake results could accurately predict the uptake obtained later in vivo in rodents (Tony Lahoutte, John Mertens, Vicky Caveliers et al., J Nucl Med (2003) 44, 1489-1494 and J. Mertens, T. Lahoutte, C. Joos, A. Bossuyt, European Journal of Nuclear Medicine and molecular imaging, (2002) 29, Supplement 1).

4.2.1. Time and concentration dependency

The cells were incubated for times ranging from 1 to 20 minutes in 1 ml of 0.1 mM 2-I-L-Phe in HEPES⁺ and HEPES^" or MEM containing 37 KBq 2-[¹²⁵I]-D-Phe.['⁴C]- D-phenylalanine (Amersham Biosciences) was used as reference product. Saturation of the uptake was measured at 15 min with concentrations of 2-I-D-Phe varying from 0.01 to 0.2 mM. The data were fit to the Michaelis-Menten relation and the KiΗ_apParent , Vmax and Ki values were calculated from Eady-Hofstee and Hanes-Woolf and Lineweaver-Burk (LWB) plots. "Km_apPaιe_nt" is the combination of the Km values related to the transport system(s) involved.

4.2.2. Inhibition of 2-¹²⁵I-D-Phe influx

In these inhibition experiments the cells were incubated with 37 KBq 2-¹²⁵I-D- Phe for 15 minutes in HEPES⁺ and HEPES^" buffer supplemented with 5 mM L-Phe or BCH (2-amino-2-norbomane-carboxylic acid). LWB plots were used for the calculation of Ki. 4.2.3. Trans-stimulation of 2-^I25I-D-Phe efflux

The cells were incubated with 37 KBq 2-¹²⁵I-L-Phe for 15 minutes in HEPES+ and HEPES- buffer. The incubation medium was removed and the cells were washed tliree times with ice-cold PBS. Subsequently HEPES buffer containing 5 mM L-Phe or 5mM BCH or MEM buffer was added. The efflux medium was removed after 20 minutes, the cells were washed tliree times with ice-cold PBS, detached with 0.1 M NaOH, suspended and counted.

5. Animals: Wagrij Rats and NuNu Mice

Wag/Rij rats and NuNu mice were subcutaneously injected in the right flank with 1 million RIM rhabdomyosarcoma cells (Harlan, Netherlands). Tumours were grown for 4 weeks.

The animals had free access to water and food until 4 hours before tracer injection. The animals were anaesthetised (halothane orNembutal). Afterwards the animals were sacrificed by intravenous injection of KC1. The study protocol was approved by the ethical committee for animal studies and the National Institutes of Health principles of laboratory animal care (NIH publication 86-23, revised 1985) were followed.

5.1. Biodistribution: ex vivo

The animals were injected with 5MBq 2-¹²⁵I-D-Phe and sacrificed 10 minutes post injection. The organs and tissues of interest were removed rapidly, washed and weighed. The radioactivity of the samples was counted by use of a gamma ray counting system. The amount of radioactivity in the samples is expressed as differential absorption ratio (activity per gram of sample divided by the activity injected per gram rat)

5.2. Dynamic planar imaging.

Dynamic imaging of the rat injected in the penis vein was performed with a gamma camera equipped with a medium energy collimator (resolution 11 mm at full width at half maximum). Imaging was started immediately after I.V. injection of 18.5 Mbq 2-¹²³I- D-Phe. A total of 240 images of 10 s each were acquired in 128X128 matrices with a zoom factor 302 (pixel size 1.5 mm) and a photo peak window set around 159 KeV.

Regions of interest were drawn around the tumour, the contra-lateral background area, left ventricle, kidneys and total body. The tracer uptake was calculated as DUR (differential uptake ratio).

RESULTS

In vitro

1. Uptake- and Efflux-time kinetics

The uptake of 2-¹²⁵I-D-Phe/2-I-D-Phe in different cell types increases with time following a hyperbole to reach an apparent equilibrium value of 0.35% per million cells around 15 minutes. (Fig. 1) followed by a slight increase. After 12 hours the uptake amounted to 1.4 %.

The uptake kinetics of 2-¹²⁵I-D-Phe are the same as the short time kinetics (up to 15 minutes) obtained for ¹⁴C-D-phenylalanine ([^*AAj_n]_eq = 0.4 %) in the same conditions. The uptake in MEM mimics in vivo conditions in blood as MEM contains about 1.2 mM amino acids, which are known to be transported by the L transport system.

Contrary to 2-¹²⁵I-L-Phe/2-I-L-Phe which uptake pattern follows a first order reversible reaction, this is not the case for the D-analogue (Fig. 2). The deviation of linearity points to an accumulation effect caused by a hindered efflux due to competitive inhibition by the natural L-amino acids inside the cells. This accumulation effect is proven by the slow MEM stimulated efflux Idnetics of 2-¹²⁵I-D-Phe as compared to L-Phe (Fig. 3). 75% of 2-¹²⁵I-D-Phe remains in the cells while this only amounts 8% for natural L-Phe.

2. Concentration depending kinetics

The uptake at the 15 minutes time point (exchange rate at equilibrium) of-2- ¹²⁵I-D-Phe/2-I-D-Phe for concentrations ranging from 0.1 to 2 mM in HEPES+ was measured. The uptake as a function of concentration is saturable and follows the Michaelis-Menten relation (Fig. 4). The related Eady-Hofstee shows a single substrateBtransport system interaction. The resulting apparent Km value is 46 μM and is comparable to these of ¹⁴C- D-Phenylalanine (Km: 55μM) in the same conditions.

3. Transport Type Characteristics

The uptake of 2-¹²⁵I-D-Phe was reduced to 15% of the original value by the presence of 5mM 2-aminobicyclo-[2,2,l]heptane-2-carboxylic acid (BCH), a specific inhibitor of L transport system, and to 3 % by 5 mM L-phenylalanine in both HEPES- and HEPES+. This points to the fact that the major part of the uptake is ruled by the same transport system belonging to the L type. The inhibition of 2-¹²⁵I-D-Phe uptake by BCH, phenylalanine and tyrosine was competitive as revealed from a double-reciprocal Lineweaver-Burk plot showing different lines for different concentrations of inhibiting compound with different slopes but the same intercept on the 1/mass-uptake axis. Inhibition of ³H-L-Phe/L-Phe concentration dependent uptake by 2-I-D-Phe related Lineweaver-Burk plots also showed a typical competitive inhibition pattern (Fig. 5) resulting in an apparent Ki value of 57 μM for 2-I-D-Phe. This proves that radioiodinated 2-I-D-Phe shows a high affinity for the transport system involved.

In vivo

1. Biodistribution

As shown in Fig. 6 after 30 minutes the uptake of the 2-I-D-Phe is the same as the L form in most of the organs. High uptake in the pancreas is typical for rodent and it shows that 2-I-D-Phe is still recognized as an amino acid.

In the tumour it is somewhat lower for the D form but still a good tumour/blood ratio is obtained.

2. Planar Imaging

Fig. 7 shows the bio-distribution as a function of time measured with planar camera acquisition.

An accumulation in the tumour with increasing time is observed. Injection of phenylalanine and methionine into the rat displaced the activity for a short time, showing that the reversible exchange occurred by the LAT system, followed by a slow but continuous accumulation.

This observation proves that in case of therapy a infusion is preferred over a bolus injection.

The accumulated picture after 60 minutes shows a clear uptake in the tumour and a fast clearance through the lddneys to the bladder.

3. Nunu Mice

Planar acquisition of 2-¹²³I-D-Phe distribution respectively RIM tumour and A2058 melanine producing melanoma bearing NuNu mice.

Fig. 9 shows that the uptake (counts per pixel) in a RIM tumour bearing NuNu mice of 2-¹²³I-D-Phe measured by means of SPECT acquisition of ~5%ID/pixel. A high uptake in the tumour is observed while the clearance of the tracer occurs mostly through the lddneys to the bladder. No significant uptake in the thyroid shows that the deiodination is negligible. This distribution is comparable with the uptake of 2-¹²³I-L-Phe in the same mice.

Fig. 10 shows that in vivo in NuNu mice the uptake in human A2058 melanoma cells the tumour/backgiOund ratio amounts up to 5 allowing SPECT acquisition of these rumours. This SPECT acquisition, represented in Fig. 10, shows a high uptake in the tumour and a large clearance through the lddneys to the bladder. Here also no significant uptake in the thyroid is observed. Stability and lack of deiodination is of prime importance for radioiodinated (¹³¹I) compounds used for therapeutic purposes.

EXAMPLE 2

Alternative labelling procedures

Derivatization of the amino acid with Histidine is performed according to the reaction scheme shown below.

The preparation of [^99mTc(H₂0)₃(CO)₃]⁺ is carried out according to Alberto=s procedure (J. Am. Chem. Soc. 123, 3135-3136 (2001), ASynthesis and Properties of Boranocarbonate : A Convenient in Situ CO Source for the Aqueous Preparation of [^99mTc(H₂0)₃(CO)₃]⁺@) General Labeling Procedures with Tc(CO)₃ ⁺ are as follows. 100 μl of the amino acid derivative solution (10^"3 M) is added to a 10 ml vial, which has been previously sealed under nitrogen atmosphere. To this vial, 900 μl of the [^99mTc(H₂0)₃(CO)₃]⁺ solution are added and the vial is placed in a boiling water bath for 30 minutes.

The preparation of the [Re(H₂0)₃(CO)₃] precursor is done according to Schibli et al. (Bioconjugate Chem. 13, 750-756, 2002).

The amino acid derivatization procedure can also be applied to conjugate different kinds of chelator e.g. DTP A, DOTA, etc, useful for coordination to other radioisotopes like In-I l l, Y-90, Lu-177.

H₂

-Derivative HOOC^/

oxidation

BOC protection

lodination

Claims

1. D-amino acid of the general formula NH₂-CHR-COOH, wherein R is a variable side chain, for use in diagnosis with SPECT and PET or in systemic radionuclide radiotherapy, characterized in that the D-amino acid is labelled in the side chain R.

2. D-amino acid as claimed in claim 1, wherein R is a bidentate or tridentate bifunctional chelating molecule.

3. D-amino acid as claimed in claim 1, wherein R is selected from the group consisting of: benzyl, methyl benzyl, ethyl benzyl, 3-hydroxybenzyl or 4-hydroxybenzyl, either of which may be optionally substituted in a free ortho, meta, or para position with (CH₂)„-X, wherein n is 0, 1 or 2 and X is a radioactive halogen selected from F, CI, Br, I.

4. D-amino acid as claimed in claim 1, wherein R is a polamino polycarboxylic (PAPC) chelator optionally coupled to the D-amino acid via a spacer of the formula (CH₂)_n, wherein n is 1-7, preferably 1-5, and the chelator is in particular selected from the group consisting of histidine, DOTA, DTPA, EDTA, and the radioactive label is complexed with the chelator and selected from the group consisting of radioactive Tc, Re.

5. D-amino acid as claimed in claim 1, wherein R is a polamino polycarboxylic (PAPC) chelator that is optionally coupled to the D-amino acid via a spacer of the fomiula (CH₂)_n, wherein n is 1-7, preferably 1-5, and the chelator is in particular selected from the group consisting of DOTA, DTPA and EDTA, and the radioactive label is complexed with the chelator and selected from the group consisting of radioactive Pd, In, Y, Lu.

6. D-amino acid as claimed claim 1, which D-amino acid is selected from the group consisting of 2-I*-D-phenylalanine, 2-I*-D-tyrosine, 2-Br*-D-phenylalanine, 2-Br*- D-tyrosine, 2-Cl*-D-phenylalanine, 2-Cl*-D-tyrosine, 2-F*-D-phenylalanine, 2-F*-D- phenylalanine, wherein I*, Br* and F* are radioactive isotopes of the respective elements.

7. D-amino acids as claimed in claim 6, wherein I* is selected from ^{1 1}I, ¹²²I,

123τ 124τ 125τ 131τ

D-amino acids as claimed in claim 6, wherein Br* is selected from, ⁷⁵Br,

⁷⁷Br, ⁸²Br.

9. D-amino acids as claimed in claim 6, wherein F* is

10. D-amino acid as claimed in claim 1 labelled with a radioactive element selected from the group consisting ^99mTc, ¹⁸⁶Re, ¹⁸⁸Re, ^mIn, ⁸⁵Y, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ¹⁷⁷Lu, ^{, 03}Pd.

11. Pharmaceutical composition, comprising one or more D-amino acids as claimed in claim 1 and one or more diluents and/or carriers and/or excipients and/or additives.

12. Use of D-amino acids as claimed in claim 1 for the preparation of pharmaceutical composition for diagnosis of cancer.

13. Use as claimed in claim 12, wherein the diagnosis is performed by means of Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET).

14. Use of D-amino acids as claimed in claim 1 for the preparation of a pharmaceutical composition for the treatment of cancer.

15. Use as claimed in claim 14, wherein the treatment comprises systemic radionuclide therapy.