WO2006117218A2 - Serotonin-transport-protein binding radiopharmaceutical for the production of a diagnostic medicament for the diagnosis of headache - Google Patents

Abstract

The present invention relates to the use of radiopharmaceuticals which specifically bind the serotonin-transport-protein (SERT) for the production of a diagnostic drug for the diagnosis of headache. The radiopharmaceuticals are tracers, which have the capacity to specifically bind the Serotonin-Transport-Protein in the context of a Positron-Emission-Tomography (PET) or a Single Photon Emission Tomography (SPECT). The present invention further relates to a screening method for identifying substances, which selectively modify the binding of these radiopharmaceuticals to the serotonin transporter. These substances are, e.g. precursors of putative therapeutics for the treatment of headache.

Description

Serotonin-transport-protein binding radiopharmaceutical for the production of a diagnostic medicament for the diagnosis of headache.

Introduction

This invention relates to the use of a Serotonin-Transport-Protein (SERT) binding radiopharmaceutical for the production of a diagnostic drug for the diagnosis of headache. Radiophar- maceuticals herein are so called tracers, which in the context of an examination with Positron- Emission-Tomography (PET) or Single Photon Emission Tomography (SPECT) are able to specifically bind SERT by which it is possible to detect the presence of SERT. The present invention further relates to a method of screening for substances which are able to selectively modify the binding of these radiopharmaceuticals. These substances may be, e.g. precursors of putative therapeutics for the treatment of headache.

Around 12-18% of the female population and 5-8% of the male population suffers from migraine. (Rasmussen BK. Migraine and tension-type headache in a general population: precipitating factors, female hormones, sleep pattern and relation to lifestyle. Pain 1993; 53. 65- 72; Russell MB, Rasmussen BK, Thornvaldesen P, Grasso J. Prevalence and sex ratio of the subtypes of migraine. International Journal of Epidemiology 1995; 24:612-618.) In contrast to migraine, cluster headache is a rare primary headache disorder with a prevalence of 0,01 - 0,1% of the population(Bahra A, May A, Goadsby PJ. Cluster headache: a prospective clinical study with diagnostic implications. Neurology 2002; 58: 354-361). Both primary headache disorders are characterized by unilateral headache attacks of high pain intensitiy as well as by headache specific autonomous symptoms. Migraine attacks may occur throughout a life span with a high individual variation of attack frequency. In contrast, around 90% of cluster headache patients suffer from the episodic type, i. e. they cycle between headache phases and long remission phases. Irrespective of the individual burden of the disease, migraine is a cost-intensive disorder because of high direct (drugs) and indirect costs. Thus, migraine has a great impact on our socioeconomic system (Davies GM, Santanello N, Garth W, Lerner D, Block GA. Validation of a migraine work and productivity loss questionnaire for use in migraine studies. Cephalalgia 1999; 19: 497-502). The monaminergic neurotransmitter serotonin (5-HT) has been assumed to play an important role in migraine pathophysiology. 5-HT is known to be involved in a wide range of CNS functions like sleep, mood, anxiety and cognition. In respect to migraine, the most prominent role of serotonin is pain modulation. Around 80% of the brain serotonergic neurons reside within the raphe nuclei in the brainstem. The raphe nuclei form two distinct groups, the rostral and the caudal raphe complex. The rostral raphe complex, which is located in mesencephalon and pons of the brainstem, projects rostrally to virtually all regions of the central nervous system. The caudal serotonergic system in the medulla oblongata sends serotonergic projections to the spinal cord. The raphe nuclei belong to the "supraspinal nocifensive system", i.e. they are responsible for nocifension of nociceptive signals from the periphery.

So far, evidence for the involvement of serotonin in the pathophysiology of headache disorders mainly derives from pharmacological treatment strategies (Ferrari MD & Saxena PR. On serotonin and migraine: a clinical and pharmacological review. Cephalalgia 1993; 13: 151- 165).

The unselective serotonin agonists called "ergotamines" have been widely used for the treatment of migraine and cluster headache (Jost W. Selbach O. Medikamentδse Akuttherapie. Aus: Therapie der Migrane (ed. Host W & Selbach O), Uni-Med Verlag Bremen- London- Boston. 2001). The "ergotamines" have recently been replaced by highly selective serotonin receptor agonists, which act at the 5HTl _B and 5HTl_D-receptor-subtype (Diener HC & Eiker- mann A. Behandlung der Migraneattacke. Aus: Kopfschmerzen (ed: Diener HC), Georg Thieme Verlag Stuttgart, New York 2003; Ferrari MD, Roon KI, Lipton RB, Goadsby PJ. Oral triptans (serotonin 5HT IB/ID agonists) in acute migraine treatment: a meta-analysis of 53 trials. Lancet 2001, 358: 1668-1675; Ekbom K, Krabbe A, Micelli G, Prusinski A, Cole JA, Noronha D for the Stumatriptan Cluster Headache Long-term study group. Cluster headache attacks treated for up three months with subcutaneous sumatriptan (6mg). Cephalalgia 1995; 15: 230-236; Schuh-Hofer S, Reuter U, Kinze S, Einhaupl KM, Arnold G. Treatment of acute cluster headache attacks with 20 mg sumatriptan nasal spray-an open pilot study. J Neu- rol 2002; 249: 94-99). Serotonergic drugs are not only used for acute therapy but also for prophylactic treatment. Methysergide, a 5HT-2-receptor antagonist with agonistic effects on 5HTl B/ ID-receptors is regarded as the most potent prophylactic drug in the treatment of migraine and cluster headache ( Silberstein SD. Methysergide. Cephalalgia 1998; 18: 421- 435). Finally, tricyclic antidepressants are used in the United States as first-line drugs for prophylactic migraine treatment. (Silberstein SD. Practice parameter: Evidence-based guidelines for migraine headache (an evidence-based review)- Neurology 2000; 55: 754-763).

Attempts towards an assessment of the brain serotonergic system of migraineurs in vivo were hampered due to methodologic limitations. For many decades, migraine research thus was forced to focus on biochemical analyses of 5-HT and its metabolites in various body fluids (blood, plasma, urine) and in platelets of patients. Such investigations were performed primarily in migraine patients but also in patients with medication overuse headache. Despite of an extensive research effort, the results often were inconsistent, partly for methodological rea- sons, partly because of confounding factors (Ferrari MD & Saxena PR. On serotonin and migraine: a clinical and pharmacological review. Cephalalgia 1993; 13: 151-165; D'Andrea, G., Cananzi, AR., Perini, F., Hasselmark, L. Platelet models and their possible usefulness in the study of migraine pathogenesis. Cephalalgia 1995; 15: 265-271)

The first pilot analysis of the brain serotonin metabolism of migraine patients was performed by Chugani in 1999. Chugani chose a method where the cerebral capacity of the brain serotonin synthesis may be ascertained using PET- and the radiopharmacon ^uC-α-methyl-L- tryptophan (Chugani DC, Niimura K Caturvedi S, Muzik O, Fakhouri M, Lee ML, Chugani HT. Increased brain serotonin synthesis in migraine. Neurology 1999; 53 (7): 1473-9.). This analysis performed on 11 patients showed a global increase in brain serotonin synthesis in 8 patients with migraine without aura. Local differences in anatomic regions were however not found in this examination. Due to the small size of patients this examination may be consider to be a pilot-study.

A brain-imaging study performed by Weiller et al. in 1995 pointed to the pontomesencephalic brainstem being an "anatomical hot spot" for migraine pathophysiology. (Weiller C, May A, Limmroth V, Jϋptner M, Kaube H, Schayck RV, Coenen HH. Diener HC. Brain stem activation in spontaneous human migraine attacks. Nature medicine 1995; 1: 658-660). In this 150- H2O-PET-study, Weiller demonstrated a significant increase of cerebral blood flow (rCBF) in the dorsal aspects of mesencephalon and pons of migraineurs during spontaneous migraine attacks which even persisted after successful treatment. Though the resolution of 15O-H2O- PET does not allow the localization of specific brainstem nuclei, the region of increased rCBF comprised the anatomic region of the rostral raphe nuclei. The raphe nuclei are the origin of serotonergic neurons: around 80% of the brain serotonergic neurons reside within the raphe nuclei in the brainstem. The raphe nuclei form two distinct groups, the rostral and the caudal raphe complex. The rostral raphe complex, which is located in mesencephalon and pons, projects rostrally to virtually all regions of the central nervous system. The caudal serotonergic system in the medulla oblongata sends serotonergic projections to the spinal cord. The raphe nuclei belong to the "supraspinal nocifensive system", i.e. they are responsible for nocifen- sion of nociceptive signals from the periphery. Respecting the functional role of the brainstem raphe nuclei, the study by Weiller supported the idea of an involvement of the brainstem nocifensive system, embodied by the serotonergic raphe nuclei, in migraine pathophysiology. However, Weiller' s study did not implicate a functional dysregulation of the brainstem sero- tonergic system in migraineurs since the method he used was not able to measure the brain serotonin metabolism.

The role of serotonin in cluster headache and medication overuse headache is described as follows. As outlined above, there is an assumption according to which the serotonergic sys- tern plays a pathophysiological role also in cluster headache. In [¹⁵O]H₂O-PET-examinations in cluster headache patients, it was possible to prove a specific regional enhancement of blood flow in the ipsilateral hypothalamus during acute cluster headache attacks (May A, Barra A; Buchel C, Frackowiak RSJ, Goadsby PJ. Hypothalamus activation in cluster headache attacks. Lancet 1998;352: 275-278). The hypothalamus belongs to the important projection ar- eas of the serotonergic raphe nuclei. Chronobiological aspects such as the circadian and cir- canual occurrence of cluster headache attacks which play an important role for the pathophysiology of cluster headache could be explainable with a serotonergic dysfunction in the region of the hypothalamus (in particular in the region of the nucleus suprachiasmaticus) (Glass JD. Grossman GH, Farnbauch L, DiNardo L. Midbrain raphe modulation of nonphotic circadian clock resetting and 5-HT release in the mammalian suprachiasmatic nucleus. J Neu- rosci 2003; 23: 7451-7460). Thus it is conceivable that one may find a specific regional dysregulation of the serotonin transport protein in the region of the hypothalamus in cluster patients in contrast to migraine patients. This would expand the applications of specific SERT- radiopharmaceuticals to the differential diagnosis of cluster headache.

There is evidence for the involvement of the serotonergic system not just in the "primary" headache diseases migraine and cluster headache, but also in the most common secondary headache disease, the medication overuse headache. The prevalence of the medication overuse headache in the population is about 1 % (Castillo J, Munoz P, Guitera V, Pascual J. Epi- demiology of chronic daily headache in the general population. Headache 1999; 39: 190-196). The medication overuse headache develops based on a long time abuse of pain killers and clinically leads to constant head pain. In a study relating to the serotonin concentration in platelets of patients with medication overuse headache a significant lowering of the serotonin concentration was observed when compared to migraine patients (Srikiatkhachorn A, Anthony M. Platelet serotonin in patients with analgesic-induced headache. Cephalalgia 1996; 16: 423-6). Due to the outstanding role of serotonin in pain pathophysiology an extensive serotonin deficiency may be the cause of medication overuse headache.

Only very recently has it become possible by means of nuclear medical methods (SPECT and PET) to display the serontonin transport protein in vivo (Brϋcke T, Kornhuber J, Angelberger P, Asenbaum S, Frassine H, Podreka I. SPECT imaging of dopamine and serotonin transporters with [¹²³I]-beta-CIT Binding kinetics in the human brain. J Neural Transm Gen Sect 1993; 94:137-46, Okada T, Fujita M, Shimada S, Sato K, Schloss P, Watanabe Y, Itoh Y, Tohyama M, Nishimura T: Assessment of affinities of beta-CIT, beta-CIT-FE, and beta-CIT-FP for monoamine transporters permanently expressed in cell lines. Nucl Med Biol 1998; 25:53-8, Hesse, S., Barthel, H., Schwarz, J., Sabri, O., Mϋller, U. Advances in in vivo imaging of serotonergic neurons in neuropsychiatric disorders. Neurosci. Biobehav. Rev. 2004; 28: 547-63). The first radiopharmaceutical utilized for this was the tropan derivative beta-CIT (Hill J, Ak- erman KK, Kuikka JT, Bergstrom KA, Halldin C, Nikola T, Rasanen P, Tiihonen J, Vauhkonen M, Karhu J, Kupila J, Lansimies E, Farce L: Iodine- 123 labeled nor-beta-CIT as a potential tracer for serotonin transporter imaging in the human brain with single-photon emission tomography. Eur J Nucl Med 1998, 25:19-23). The disadvantage of this radiopharmaceutical is the relatively poor serotonergic specificity due to the fact that apart from the serotonin transporter (SERT) also the dopamintransporter (DAT) is radioactively marked and thus an exact analysis of the central serotonin system is only possible in a restricted manner (Bergstrom KA, Kuikka JT, Ahonen A, Vanninen E: [¹²³I]-beta-CIT, a tracer for dopamine and serotonin re-uptake sites: preparation and preliminary SPECT studies in humans. J Nucl Biol Med 1994;38(St): 128-31, Pirker W, Asenbaum S, Kasper S, Walter H, Angelberger P, Koch G, Pozzera A, Deecke L, Podreka I, Brocke T: beta-CIT SPELT demonstrates blockade of 5HT-uptake sites by citalopram in the human brain in vivo. J Neural Transm Gen Sect 1995; 100:247-56, Hiltunen J, Akerman KK, Kuikka JT, Bergstrom KA, Halldin C, Nikula T, Rasanen P, Tiihonen J, Vauhkonen M, Karhu J, Kupila J, Lansimies E, Farde L: Iodine-123 labeled nor-beta-CIT as a potential tracer for serotonin transporter imaging in the human brain with single-photon emission tomography. Eur J Nucl Med 1998;25: 19-23, Tauscher J, Pirker W, de Zwaan M, Asenbaum S, Brucke T, Kasper S: In vivo visualization of serotonin transporters in the human brain during fluoxetine treatment. Eur Neuropsychopharmacol 1999; 9: 177-9, Jones DW, Gorey JIG, Zajicek K, Des S, Urbina R, Lee KS, Heinz A. Knable MB, Higley DR, Weinberger D, Linnoila M Depletion-restoration studies reveal the impact of endogenous dopamine and serotonin on [I-I23]b-C1T SPECT imaging in primate brain. J Nucl Med 1998; 39 (Suppl):42). Recently 3 radiopharmaceuticals were developed for the visualization of the serotoneric transport system with SPECT: ¹²³I-IDAM, ¹²³I-ODAM (Zhuang et al. 2000) and ¹²³IADAM (Oya S, Choi SR, Hou C, Mu M, Kung MP, Acton PD, Sicifano M, Kung HF. 2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine (ADAM): an improved serotonin transporter ligand. Nucl Med Biol 2000;27:249-54). All three radiopharmaceuticals are characterized by a high selectivity (Kung MP, Hou C, Oya S, Mu M, Acton PD, Kung HF. Characterization of [¹²³I]-ADAM as a novel single-photon emission tomography tracer for serotonin transporters. Eur J Nucl Mad 1999; 26:844-53, Choi SR, Hou C, Oya S, Mu M, Kung MP, Siciliano M, Acton PD, Kung HF. Selective in vitro and in vivo binding of [¹²³I]-ADAM to serotonin transporters in rat brain. Synapse 2000; 38:403-12) and their excellent suitability for the imaging with SPECT (Acton PD, Kung MP, Mu M, Plossl K, Hou C, Siciliano M, Oya S, Kung HF. Single-photon emission tomography imaging of serotonin transporter; in the non-human primate brain with the selective radioligand [¹²³I]-ADAM. Eur J Nucl Med 1999, 26:854-61). Comparative analysis seems to suggest that ¹²³I-ADAM is superior to the other 2 radiopharmaceuticals due to its higher uptake, its higher selectivity and specificity which is based on a low unspecific binding (Acton PD, Choi SR, Hou C, Plossl K, Kung HF Quantification of serotonin transporter; in nonhuman primates using [¹²³I]-ADAM and SPECT. J Nucl Med. 2001 ;42: 1556-62). Its largest advantage over the tropan derivative β-CIT which was previously used is its 10.000 fold higher affinity to the serotonin transporter when compared to the dopamine- or norepinephrintransporter (Oya S, Choi SR, Hou C, Mu M, Kung MP, Acton PD, Sicifano M, Kung HF. 2-((2-((dimethylamino)methyl)phenyl)thio)- 5-iodophenylamine (ADAM): an improved serotonin transporter ligand. Nucl Med Biol 2000;27:249-54). By means of ¹²³I-ADAM-SPECT it is possible to visualize the serotonin transporter in vivo in a highly selective fashion and to semiquantitatively analyse the serotonin transporter and thereby examine disorders which previously have only been attributed by means of indirect evidence to a pathogenetic involvement of the serotonergic system. Apart from the SPECT-radiopharmaceuticals also the PET-radiopharmaceuticals ^{1 1}C-McN 5652, (S)-([^l8F]flouromethyl)-(x)-McN5652 and ^{1 1}C-DASB are held to be very specific and suited for visualizing the Serotonin-Transport-Protein in vivo.

As already outlined above the diagnosis of "migraine" and "cluster headache" presently is only based on an extensive headache anamnesis. The International Headache Society has developed standardized criteria for the diagnosis of headache disorders which were only recently revised and published (Cephalalgia 2004, 24: Suppl 1). Up until now, globally and apart from the anamnesis criteria there is no diagnostic procedure which allows the diagnosis of migraine in headache patients. In spite of the fact that in the context of long lasting clinical experience it has been shown in a highly specialized headache centre that it is very problematic for many doctors working in an out-patient centre to diagnose migraine and cluster headache as well as to differentially diagnose the two diseases and in spite of the absence of epidemiological studies in this context one must assume a high number of false diagnoses. This does not only affect the individual patient but also the healthcare system from an economic view point due to insufficient false therapeutic approaches. In particular due to this aspect there is an essential application for the use of SERT-radiopharmaceuticals for the diagnosis and differential diagnosis of migraine, in particular because migraine apart from i.e. diabetes mellitus or back pain may be counted to one of the largest wide spread diseases.

Summary of the Invention

In one embodiment of the present invention methods and corresponding components are provided for which allow for the examination of the dysregulation of the serotonin transport protein in patients with primary and secondary headache disorders like migraine, cluster headache and medication-overuse headache. A further embodiment of the present invention relates to diagnosis and clinical monitoring of patients with migraine, cluster headache or medication overuse headache as well as the use of the invention in the context of the development of medicaments.

According to a first embodiment of the present invention the problem according to the present invention is solved by the use of a serotonin-transport-protein (SERT) binding radiopharmaceutical for the production of a diagnostic medicament for the diagnosis of headache. The radiopharmaceutical ¹²³I-ADAM for brain-SPECT- is particularly preferred however, the invention is not limited to ¹²³I-ADAM.

In one embodiment the radiopharmaceutical carries an isotope or a radioactive moiety chosen from the group consisting of a PET isotope, a SPECT isotope, a radioactive isotope, selected from the group consisting of ¹²³1, ¹³¹1, ¹²⁵1, ⁷⁷Br, ¹⁸F, ¹¹C and ⁷⁶Br.

In a preferred embodiment the radiopharmaceutical is a compound according to Formula I: Formula (I)

or a pharmaceutically acceptable salt thereof; wherein,

X is hydrogen, F, Cl, Br, I, NO₂, NR³R⁴ or -L-Ch, CH3(CH2)nO-, X(CH₂)_nO-, CH₃(CH₂)_nS-, X(CH₂)_nS-, CH₃(CH₂)_nOCH₂,

X(CH₂)_nOCH₂-, CH₃(CH₂)_n(O)-, X(CH₂)_n(O)-, CH₃(CH₂)_n(O)O-, X(CH₂)_n(O)O-, (CH₂)_nCH₃, or (CH₂)_nCH₂X, wherein n = 0, 1 , 2, or 3;

Y is hydrogen, -CH₂OR⁵, -CH₂NCH₃R⁶, NO₂, -NR³R⁴, -L-Ch, hydroxy(Ci-

C₄)alkyl, halo(Ci-C₄)alkyl, nitro, azido or halo;

R³ and R⁴ are independently

hydrogen, hydroxy, C₁-C₅ alkyl, halo(Cι-C₄)alkyl, halobenzoyl, C₃-C₈ cycloalkyl, Ci_-5 alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, phenyl, benzyl, naphthyl, naphthylmethyl or -L-Ch;

R⁵ is hydrogen, Ci-C₅ alkyl, C₃-C₈ cycloalkyl, Ci_-5 alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, phenyl, benzyl, naphthyl, naphthylmethyl or -L- Ch; W is Y' or alkyl-Y'

Y' is hydrogen, -CH₂OR⁵, -CH₂NCH₃R⁶, NO₂, -NR³R⁴, -L-Ch, hydroxy(C,-

C₄)alkyl, halo(Ci-C₄)alkyl, azido or halo;

R⁶ is hydrogen, Cj-C₅ alkyl, C₃ -C₈ cycloalkyl, Ci_-5 alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, halo(Ci-C₃)alkyl, phenyl, benzyl, naphthyl, naphthylmethyl or L-Ch;

Z is S, O, NR⁷, CR⁸R⁹, C(O) or -C(=CR⁸R⁹)-;

R⁷ is hydrogen, C₁-C5 alkyl, C₃-C₈ cycloalkyl, Ci_-5 alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, phenyl, benzyl, naphthyl, naphthylmethyl or -L- Ch;

R⁸ and R⁹ are independently

hydrogen, Ci-C₅ alkyl, or chloro;

A, R¹ and R² are independently

Hydrogen, halogen, cyano, Ci-C₄ alkyl, halo(Ci-C₄)alkyl, halo(Ci- C₄)alkanoyl or (Ci-C₄)alkoxy or L-Ch;

L is a covalent bond or a linking group selected from the group consisting of -(CH₂)_n -and -(CH₂)_n -C(O)-, where n is 1-5;

Ch is a tetradentate ligand capable of chelating a metal;

and

with the proviso that at least one of A, X, Y, R³, R⁴, R⁵, R⁶, or R⁷ is -L-Ch and/or, one or both of X or A is Br or I and/or of Y, Y', R¹, R² or A contains ¹⁸F and wherein any halogen within the compound may by a radioisotope and/or wherein a carbon atom in R may be ¹¹C. Detailed Description of the Invention

By applying the preferred ¹²³I-ADAM-SPECT the inventors were able to detect a significant elevation of availability of the Serotonin-Transport-Proteins ("SERT") in the pontomesen- cephalic region, the anatomic region of the rostral raphe complex (see fig. 1) in migraine patients. The serotonin-transport-protein is responsible for transporting the biogenic amine serotonin back from the synaptic gap into the neuron, thereby regulating the concentration of available serotonin in the synaptic gap. Based on examinations performed in the context of this invention it was surprisingly found that migraine patients differ significantly from con- trol patients, which do not have migraine, with respect to the availability of the serotonin- transport-protein. The results presented herein are the first examination results concerning the representation of the availability of the serotonin-transport-protein in the brain of migraine patients. The inventors are the first ever to specifically apply a radiopharmaceutical (¹²³I- ADAM) which specifically binds SERT to the visualization of the serotonin-transporters in the brain of migraine patients. In the context of the present invention, surprisingly in migraine patients (see fig. 1), the ratio of the SERT-availability in the pontomesencephalic brainstem (i.e. target region) compared to the occipital lobe (i.e. reference region) was 0.88 +/- 0.16 (mean +/- standard deviation). Compared to the ratio measured in healthy subjects, which was 0.58 +/- 0.20, this ratio was significantly elevated in in migraine patients. Based on the specific teaching herein, one skilled in the art will be able to identify other statistically relevant ratios as well as their displacement which may vary according to pain, applied radiopharmaceutical, number of patients analyzed and method of examination applied. This however, is encompassed by the general principal of the present invention. The method developed by the inventors may find access to clinical routine as a diagnostic non-invasive method. Ac- cording to the present invention a preferred radiopharmaceutical may be a standard PET- radiopharmaceutical chosen from the group consisting of ¹⁵O-H₂O-ligands, ¹¹C-McN 5652, (S)-([¹⁸F]fluoromethyl)-(+)-McN5652; ¹¹C-DASB and ¹¹C-DAPP. Alternatively, a radiopharmaceutical also preferred, may be a common SPECT-radiopharmaceutical selected from the group of ¹²³I-ADAM ¹²³I-IDAM, ¹²³I-ODAM, ¹²³IB-CIT and ¹²³I-nor-β-CIT. Preferred radiopharmaceuticals are described below in the section "detailed description of the invention". Surprisingly the inventors found that, using a radiopharmaceutical for measuring the availability of SERT in the pontomesencephalic region of the brain which comprises the rostral raphe complex that there is a significant elevation of the availability of the serotonin- transport-proteins ("SERT") in the pontomesencephalic region, the anatomic region of the rostral raphe complex.

Scientifically there is no reason not to assume, that these results are not reproducible with any radiopharmaceutical which specifically binds the serotonin-transporter. This means that according to the invention a preferred radiopharmaceutical may be applied in clinical therapeu- tic monitoring of migraine of cluster headache or of medication overuse headache.

A further embodiment of the present invention relates to an in vitro method for screening for substances which modulate the binding of a radiopharmaceutical which specifically binds the Serotonin-Transport-Protein (SERT) comprising the steps of: a) providing for an in vitro sys- tern, which comprises the SERT and the radiopharmaceutical which specifically binds SERT, b) providing for a candidate substance, c) both bringing in contact SERT and the radiopharmaceutical which binds SERT specifically, with and without the candidate substance, and d) measuring the binding between SERT and the radiopharmaceutical which specifically binds SERT wherein, a substance is identified by the difference in binding both with and without the candidate substance. According to the invention a modulation may be an inhibition or an elevation of the binding of the radiopharmaceutical to SERT. It is expected that substances that modify the specific binding of the radiopharmaceutical to the Serotonin-Transport- Protein (SERT) are useful therapeutics or at least lead compounds for the treatment of headache, in particular migraine, cluster headache and/or medication overuse- headaches. Pre- ferred are in particular such substances identified which modulate selectively the SERT in vitro system from the pontomesencephalic region of the brain and in particular from the serotonergic raphenuclei because these, based on the results of the present invention, are presumed to be particularly suited for the treatment of headache in particular migraine. An "in vitro-system" in accordance with the present invention is an array or device for the above- mentioned screening method which allows for measuring the binding of a radiopharmaceutical which specifically binds to the Serotonin-Transport-Protein (SERT). The system comprises at least the SERT and the radiopharmaceutical which is SERT specific and alternatively SERT may be present, in for example, a recombinant cell model by which the modulation may be determined. It is possible, to measure, e.g. an amount of the radiopharmaceutical which is bound, which may be selected from the group consisting of the common PET- or SPECT-radiopharmaceuticals (see above). Further, an indirect detection may be performed using SERT, e.g. by determining uptake of serotonin (or derivatives thereof) or by means of labelled antibodies which may either bind SERT or the radiopharmaceutical. The candidate substance may also be labelled. All components of the in vitro system of the present invention may be present in a kit which may be used to perform the screening method according to the invention. Candidate substances may by selected from all substances which are used in the context of screening experiments in particular substances such as low molecular weight substances ("small molecules") which are present in, e.g. commercially available compound Ii- braries. Possible substances are also beta blockers metoprolol, propranolol, flunarizin or valproic acid of which the application to the specific treatment of headache or migraine therapy has previously been determined.

A further embodiment of the present invention relates to a method for the production of a pharmaceutical for the treatment of headache comprising a screening method as defined above and the mixing of the substance identified with a suitable pharmaceutical excipient. A further embodiment of the present invention relates to a pharmaceutical composition (pharmaceutical) comprising the substance identified by the invention and adjuvants as well as accessory substances this pharmaceutical composition may be characterized in that the sub- stance is present as a depot substance together with a suitable, compatible excipient or a diluent.

It is also preferred according to present invention that the pharmaceutical composition comprises a further therapeutic substance. This therapeutic substance may be any substance known to the persons skilled in the art which is therapeutically applied for pain therapy comprising such substances as for example beta blockers such as metoprolol, propranolol, flunarizin or valproic acid.

According to the present invention the above-mentioned pharmaceutical composition may be present in form of tablets, dragees, capsules, drops, suppositories, injections or infusions, for the per oral, rectal or parenteral use. Such pharmaceutical forms as well as their production are known to those skilled in the art. A further aspect of the present invention relates to the use of at least one of the above-mentioned identified substances for the treatment of diseases such as headache, in particular migraine, cluster headache and/or the medication overuse headache. This use may be applied for example in the form of a depot substance together with a suited pharmaceutically diluent excipient.

A further aspect of the invention is the use of one or more of the substances according to the invention for the production of a medicament for the treatment of diseases such as headache, in particular migraine, cluster headache and/or medication overuse headache. The production may be performed analogously to the procedure outlined above.

The present invention further relates to a method of treatment wherein the disease may be selected from the group of headache, in particular migraine, cluster headache and/or medication overuse headache wherein, the method in encompasses the step of administering a substance identified according to the invention and wherein, the substance is a pharmaceutical composition according to the invention. The administration may be performed, e.g. together with a depot substance a pharmaceutically acceptable diluent or excipient. The examination results described by the inventors further open the possibility of applying the SERT radiopharmaceuticals not only to the diagnosis and differential diagnosis of headache but also of applying radiopharmaceuticals to field of therapeutic monitoring. As outlined above, serotonergic substances such as amitriptyline are already being used for migraine prophylaxis. There are further migraine prophylactics available apart from amitriptyline such as for example the beta blockers metoprolol and propranolol the calcium antagonist flunarizin or the anticonvul- sivum valproic acid. The mode of action of these prophylactics remains unclear up until now. In general the prophylactic therapy is complicated by the fact that the patient reaction to a given prophylactic chosen by the doctor is not predictable. This means, that often times a number of medicaments need to be "tried" before a suitable and active substance is identified. The use of radiopharmaceuticals may help in examining the mode of action of migraine prophylactic treatment with respect to their influence on the serotonergic system thereby clarifying the reason for so called "non-responding" patients. In addition, SERT- radiopharmaceuticals also appear to be useful for therapy monitoring.

A further application of SERT-radiopharmaceuticals is the diagnosis and differential diagnosis of medication overuse headache. According to epidemiological data the most common primary headache disorder underlying medication overuse headache is migraine. The second most common primary headache disease underlying medication overuse headache is chronic tension type headache (Evers S, Suhr B, Bauer B, Grotemeyer KH, Husstedt W. A retrospec- tive long-term analysis of the epidemiology and features of drug-induced headache. J Neurol. 1999; 246: 802-9). Medication overuse headache is of considerable economic relevance. First of all it leads - and this is due to the suffering of the patient - to frequent none productive times. Medication overuse headache causes high therapeutic costs due to the fact that patients go through a five to ten days stationary detoxification or the patients are on sick leave during detoxification. If the medication overuse headache is not recognized in due time this may lead to irreversible kidney damage resulting in the necessity of life long dialysis or kidney transplantation. Due to the fact that the diagnosis is based solely on the reliable information provided for by the patient relating to the monthly uptake of pain killers the diagnosis of medication overuse headache is often difficult. Apart from the medicament anamnesis there are no further criteria for the differential diagnosis of chronic tension type headache or chronic migraine without medication overuse on the one hand and and medication overuse headache on the other hand. As outlined above there is indirect evidence for serotonergic dys- regulation in patients with medication overuse headache. Thus, also here, SERT- radiopharmaceuticals could provide for diagnostic and differential diagnostic applications.

The method of diagnosis described by the inventors is thus suited for the application in clinical routine. In summary the present invention provides for the following applications:

According to a first embodiment of the present invention the problem according to the present invention is solved by the use of a serotonin-transport-protein (SERT) binding radiopharmaceutical for the production of a diagnostic medicament for the diagnosis of headache.

Diphenylsulfide compounds of the present invention can be synthesized using methods analogous to those described in Ferris, R. M. et al, J. Pharm. Pharmacol. 47:775-781 (1995); Brieaddy, L. E., "Substituted diphenylsul fides as serotonin uptake inhibitors, " published International Patent Appl. No. WO 93/12080 (1993); and Mehta, N. B. et al, "Halogen substituted diphenylsulfides, " published European Patent Application EP 402,097 Al (1990). It is important that these are SERT specific. Radiopharmaceuticals ac- cording to the invention are also disclosed in Hesse S, Barthel H, Schwarz J, Sabri O, Mϋller U: "Advances in in vivo imaging of serotonergic neurons in neuropsychiatric disorders". Neurosci Biobehav Rev 2004; 28: 547 - 63, which is herewith incorporated by reference. Synthesis of Serotonin-Transport-Protein (SERT) binding radiopharmaceuticals according to the invention may be performed as described in WO 03/078393 (Inventors Kung and Shiue), incorporated by reference herewith. Synthesis of Serotonin-Transport-Protein (SERT) binding radiopharmaceuticals according to the invention may be performed as described in WO 00/66537 (Inventor Kung), incorporated by reference herewith. Synthesis of Serotonin- Transport-Protein (SERT) binding radiopharmaceuticals according to the invention may be performed as described in WO 03/096978 (Inventors Huang and Laruelle) incorporated by reference herewith.

The radiopharmaceutical ¹²³I-ADAM for brain-SPECTis particularly preferred however; the invention is not limited to ¹²³I-ADAM.

Herein, the following abbreviations are used the meaning of which is disclosed as follows:

McN 5652: (+)-6β-(4-methylthiophenyl)-l,2,3,5,6α,10β- hexahydropyrrolo[2,l- a]isoquinoline ADAM: 2-{2-[(dimethylamino)methyl]phenylthio}-5-iodophenylamine

DASB: 3-amino-4-{2[(dimethylamino)methyl]phenylthio) benzonitrile

PAPA: 5-bromo-2-{2 [(dimethylamino)methyl]phenylthio} phenylamine

AFM: 2-{2-[(dimethylamino)methyl]phenylthio}-5-fluoromethylphenylamine

¹²³I-ADAM: [2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine] F-ADAM: N,N-dimethyl-2-(2-amino-5-[18F]fluorophenylthio)benzylamine (5-[18F]-

ADAM)

C-AFM: [( 11 )C]2-[2-(dimethylaminomethyl)phenylthio]-5-fluoromethylphenylamine

([(1 I)C] AFM)

I-ODAM: [(5-iodo-2-(2-dimethylaminomethylphenoxy)-benzyl alcohol, 9)]

In one embodiment the radiopharmaceutical carries an isotope or a radioactive moiety chosen from the group consisting of a PET isotope, a SPECT isotope, a radioactive isotope, selected from the group consisting of ¹²³1, ¹³¹1, ¹²⁵1, ⁷⁷Br, ¹⁸F, ^{1 1}C and ⁷⁶Br.

In a preferred embodiment the radiopharmaceutical is a compound according to formula I: Formula (I)

or a pharmaceutically acceptable salt thereof; wherein,

X is hydrogen, F, Cl, Br, I, NO₂, NR³R⁴ or -L-Ch, CH3(CH2)nO-, X(CH₂)_nO-, CH₃(CH₂)_nS-, X(CH₂)_nS-, CH₃(CH₂)_nOCH₂,

X(CH₂)_nOCH₂-, CH₃(CH₂)_n(O)-, X(CH₂)_n(0)-, CH₃(CH₂)_n(O)O-, X(CHz)_n(O)O-, (CH₂)_nCH₃, or (CH₂)_nCH₂X, wherein n = 0, 1, 2, or 3;

Y is hydrogen, -CH₂OR⁵, -CH₂NCH₃R⁶, NO₂, -NR³R⁴, -L-Ch, hydroxy(C,-

C₄)alkyl, halo(Ci-C₄)alkyl, nitro, azido or halo;

R³ and R⁴ are independently

hydrogen, hydroxy, Ci-C₅ alkyl, halo(Ci-C₄)alkyl, halobenzoyl, C₃-C₈ cycloalkyl, Ci_-5 alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, phenyl, benzyl, naphthyl, naphthylmethyl or -L-Ch;

R⁵ is hydrogen, Ci-C₅ alkyl, C₃-C₈ cycloalkyl, Ci_-5 alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, phenyl, benzyl, naphthyl, naphthylmethyl or -L-

Ch;

W is Y' or alkyl-Y'

Y' is hydrogen, -CH₂OR⁵, -CH₂NCH₃R⁶, NO₂, -NR³R⁴, -L-Ch, hydroxy(C,-

C₄)alkyl, halo(Ci-C₄)alkyl, azido or halo;

R⁶ is hydrogen, Ci-C₅ alkyl, C₃ -C₈ cycloalkyl, Ci_-5 alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, halo(Ci-C₃)alkyl, phenyl, benzyl, naphthyl, naphthylmethyl or L-Ch;

Z is S, O, NR⁷, CR⁸R⁹, C(O) or -C(=CR⁸R⁹)-; R⁷ is hydrogen, Ci-C₅ alkyl, C₃-C₈ cycloalkyl, Ci_-5 alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, phenyl, benzyl, naphthyl, naphthylmethyl or -L- Ch;

R and R are independently

hydrogen, C₁-C₅ alkyl, or chloro;

A, R¹ and R² are independently

Hydrogen, halogen, cyano, Ci-C₄ alkyl, halo(Ci-C₄)alkyl, halo(Ci- C₄)alkanoyl or (Ci-C₄)alkoxy or L-Ch;

L is a covalent bond or a linking group selected from the group consisting of -(CH₂),, -and -(CH₂)_n -C(O)-, where n is 1-5;

Ch is a tetradentate ligand capable of chelating a metal;

and

with the proviso that at least one of A, X, Y, R³, R⁴, R⁵, R⁶, or R⁷ is -L-Ch and/or, one or both of X or A is Br or I and/or of Y, Y', R¹, R² or A con- tains ¹⁸F and wherein any halogen within the compound may by a radioisotope and/or wherein a carbon atom in R⁶ may be ^{1 1}C.

In a preferred embodiment Ch, the tetradentate ligand capable of chelating a metal may be selected form the group of:

and

wherein, R⁹ is hydrogen, a sulphydryl protecting group, methoxymethyl, methoxyethoxy- methyl,p-methoxybenzyl or benzyl. Synthesis thereof is as described in WO 00/66537 (In- ventor Kung), incorporated by reference herewith

Ch may further be a technetium chelate, wherein Ch is selected from the group consisting of

Synthesis thereof may be performed as described in WO 00/66537 (Inventor Kung), incorporated by reference herewith.

The term "alkyl" as employed herein by itself or as part of another group refers to both straight and branched chain radicals of up to 8 carbons, preferably 6 carbons, more preferably 4 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, and isobutyl.

The term "alkoxy" is used herein to mean a straight or branched chain alkyl radical, as defined above, unless the chain length is limited thereto, bonded to an oxygen atom, including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the like. Preferable chain lengths are disclosed above.

The term "halo" employed herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine. The term "haloalkyl" as employed herein refers to any of the above alkyl groups substituted by one or more chlorine, bromine, fluorine or iodine with fluorine and chlorine being preferred, such as chloromethyl, iodomethyl, trifluoromethyl, 2,2,2- trifluoroethyl, and 2- chloroethyl.

The term "hydroxyalkyl" as employed herein refers to a group containing a hydroxyl group attached to a carbon chain.

It is preferred that W is (Ci -C₄)- Y' and in particular W is -(CH₂)-Y\

It is further preferred that Z is S.

It is preferred that Y is NH_2.

The following radiopharmaceuticals are particularly preferred. Here the radiopharmaceutical is a compound according to Formula II, III, IV, V or VI (below), wherein R⁶ in Formula II, III, IV, V or VI is hydrogen, C_rC₅ alkyl, C₃ -C₈ cycloalkyl, Ci_-5 alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, halo(Ci-C₃)alkyl, phenyl, benzyl, naphthyl, naphthylmethyl or L-Ch and wherein, I in Formula II, V and VI is a radioisotop selected from the group consisting of ' ³I, ¹³¹I and ¹²⁵I:

Formula II (I-ADAM):

Formula III (F-ADAM):

Formula IV (F-AFM):

Formula V (I-OB-ADAM):

Formula VI (I-ODAM):

In a preferred embodiment the serotonin-transport-protein and binding radiopharmaceutical is selected from the group of radiolabeled 5-methyl-6-nitroquipazine and 3-(3[¹⁸F]fluoro- propyl)-6-nitroquipazine. These preferred serotonin-transport-protein binding radiopharmaceuticals are disclosed in Sandell et al., Bioorganic and Medicinal Chemistry Letters 12 (2002) 3611-3613, as well as Lee et al, Bioorganic and Medicinal Chemistry 11 (2003) 4949- 4958.

The results of the examination performed with the radiopharmaceutical ¹²³I-ADAM have shown a significant elevation of availability of this radiopharmaceutical in the pontomesencephalic region in migraine patients. Therefore, SERT specific radiopharmaceuticals will be suited for diagnostic imaging of migraine. The most common false diagnosis of migraine is tension type headache. The pathophysiology of tension type headache is so far, very unknown. The medical treatment for migraine and tension type headache differs significantly. Although amitriptyline is effective both in the preventive treatment of migraine and tension type headache, selective serotonin-agonists are solely effective in the acute therapy of migraine. This in turn means, that the serotonergic system in the case of tension type headache possibly only plays a secondary unspecific however, not causal pathophysiological role.

According to the invention in one embodiment the radiopharmaceutical is used to measure the availability of SERT in the pontomesencephalic region of the brain, which comprises the ros- tral raphe complex.

According to present knowledge concerning the efficacy of serotonergic substances in cluster headache, serotonin plays an essential role in the pathophysiology of cluster headache. Nuclear medical examinations have shown an increase of regional cerebral blood flow in the hypothalamus of patients suffering from acute cluster headache attacks. The hypothalamus belongs to one of the specific projection areas of serotonergic neurons. , Thus, with the application of radiopharmaceuticals which specifically bind SERT, a diagnosis of cluster headache appears to be possible by detecting specific serotonergic dysregulation in the hypothalamus.

The most common secondary headache is the medication overuse headache. In many cases up until today, this leads to kidney damage which intern leads to the necessity for dialysis or kidney transplantation due to the toxicity of medicaments. Medication overuse headache is thus a serious economic problem for the health care system. The diagnosis of medication overuse headache presently relies solely on clinical statements. An examination of platelets in patients with medication overuse headache has provided for evidence of a massive lack of serotonin in affected individuals. The method of diagnosis described by the inventors shall thus initially be applied in order to verify the hypothesis of a dysregulation of the Serotonin- Transport-Protein in patients with medication overuse headache. Later the method may also be applied to the diagnosis and monitoring of patients with pain killer abuse. Apart from this the application of radiopharmaceuticals which specifically bind SERT seem to make sense for the differential diagnostic differentiation between a primary headache (chronic tension type headache, chronic migraine) and the secondary medication overuse headache. Migraine patients profit both from acute therapy as well as prophylactic therapy of serotonergic substances. The method of examination described by the inventors may in future be relevant for drug-monitoring and for identifying patients which do not react to standard medicaments.

According to the invention in one embodiment the radiopharmaceutical is used in therapy monitoring in migraine patients, cluster headache or medication overuse headache.

The invention relates to a method for positron emission tomography (PET) imaging or single photon emission computed tomography (SPECT) of serotonin transporter sites in a mammal comprising the steps of (i) labelling serotonin transporter sites (SERT) with an image- generating amount of a PET or SPECT radiopharmaceuticals, which is able to bind SERT specifically or, with the radiolabeled compounds according to the compounds according to the invention (see above) and (ii) measuring the spatial distribution of the compound in the mammal by PET or SPECT (iii) so as to thereby image the serotonin transporter sites, wherein, (iv) radiopharmaceutical uptake is determined in the pontomesencephalic brainstem.

In a preferred embodiment of the method, additionally, radiopharmaceutical uptake is determined in a reference region. It is particularly preferred that the reference region is devoid of SERT. In a particularly preferred embodiment of the invention the reference regions is located in the occipital lobe. The final target parameter which is equivalent to the SERT availability is calculated from the activity of the radiopharmaceutical binding to SERT according to the following formula: Target region (pontomesencephalic region/brainstem) - reference region (occipital lobe)/ reference region

In a preferred embodiment the invention relates to a method of identifying migraine attacks in SPECT or PET images by calculating an activity quotient from SPECT or PET images of brain using serotonin-transport-protein binding radiopharmaceuticals according to the invention from regions of the pontomesencephal and/or rostral raphe nucleor complex, wherein this activity quotient is higher than 0.55. In a particularly preferred embodiment it is higher than 0.8 even more preferred higher than 0.88. It is further preferred that the mammal is human and the human is a headache patient. In preferred embodiment the headache patient is selected from the group comprising migraine, medication overuse headache and tension type headache.

The invention relates to an in vitro screening method for substances which specifically modify the binding of a radiopharmaceutical which specifically bind the Serotonin-Transport- Protein (SERT) comprising the steps of, a) providing for an in-vitro system, which comprises the SERT and the SERT specific radiopharmaceutical, b) providing for a candidate substance, c) bringing together the SERT specific radiopharma- ceutical and the SERT with and without the candidate substance, and d) measuring the binding between SERT and the SERT specific radiopharmaceutical, wherein, a difference in binding between with and without the candidate substance identifies a substance which specifically modifies the binding of a radiopharmaceutical which specifically binds the Serotonin-Transport-Protein (SERT).

It is preferred that the SERT of the in-vitro system stems from the pontomesencephalic region of the brain and in particular from the serotonergic raphe nuclei.

It is further preferred that the radiopharmaceutical is chosen from the conventional SERT spe- cific PET-radiopharmaceuticals, SERT specific SPECT radiopharmaceuticals or the compounds according to the invention as outlined above.

In this method the modification of binding is an increase or decrease in binding of the radiopharmaceutical to the SERT.

The invention also relates to a method for the production of a pharmaceutical for the treatment of headache, comprising a method of in vitro screening as detailed above further comprising mixing the identified substance with a suited pharmaceutical carrier.

The invention in the following shall be described in more detail with the aid of the figures and examples however, without limiting the invention to the specific examples.

Examples The following hypotheses was checked in the context of the examination: when comparing migraine patients to controls that are healthy, sex identical and similar in age (age-matching), migraine patients will show a significant difference of the serotonin-transporter-binding (SERT) in the brainstem which is a highly and densely innervated serotonergic pontomesen- cephalic region (the region of the rostral raphe complex). Serotonin-Transport-Binding was determined semiquantitativly with the aid of ¹²³I-ADAM-SPECT. Written informed consent was obtained from all subjects after the purpose and methods of this study had been fully explained. The parameter goal was the semiquantitative determination of the ¹²³I-ADAM-uptake in ROI-technique in the region of the pontomesencephalic brainstem (rostral raphe complex) by means of ¹²³I- AD AM-brain SPECT-technique 4 hours after an intravenous injection.

Radiopharmaceutical I- AD AM was used for the nuclear medical functional imaging of the serotonergic system in the central nervous system (Oya S, Choi SR, Hou C, Mu M, Kung MP, Acton PD, Sicifano M, Kung HF. 2-((2-((dimethylamino)methyl)phenyl)thio)-5- iodophenylamine (ADAM): an improved serotonin transporter ligand. Nucl Med Biol 2000;27:249-54). The radiopharmaceutical ¹²³I-ADAM is commercially available however not medically approved. The radiopharmaceutical has been extensively, pharmacologically and toxically tested. It has been shown that the use of the substance is safe in human beings (Oya S, Choi SR, Hou C, Mu M, Kung MP, Acton PD, Sicifano M, Kung HF. 2-((2- ((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine (ADAM): an improved serotonin transporter ligand. Nucl Med Biol 2000;27:249-54; Choi SR, Hou C, Oya S, Mu M, Kung MP, Siciliano M, Acton PD, Kung HF. Selective in vitro and in vivo binding of [¹²³I]-ADAM to serotonin transporters in rat brain. Synapse 2000; 38:403-12; Acton PD, Choi SR, Hou C, Plossl K, Kung HF Quantification of serotonin transporter; in nonhuman primates using [¹²³I]- ADAM and SPECT. J Nucl Med. 2001 ;42: 1556-62; Ahonen AK, Kauppinen, TA, Heikman, P, Bergstrδm KA, Launes, J, Nikkinen, P, Hiltunen, J. [¹²³I]-labeled ADAM -a selective novel radioligand for imaging of serotonin transporters in the human brain. J Nucl Med 2002a; 43:232 P (Poster auf der 49. SNM-Tagung 2002); Ahonen AK, Kauppinen, TA, Heikman, P, Bergstrom, KA, Nikkinen, P, Kylkinen, T, Kamarainen, EL, Puronto, O, Wtu- han, J. Imaging of serotonin transporters in human beings using [¹²³I]-ADAM. Eur J Nucl Med 2002b; 29 Suppl 1: 8120 (abstract 269, Poster EANM-Tagung 2002); Kauppinen, TA, Bergstrδm, KA, Heikman, P, Hiltunen, J, Ahonen, AK. Biodistribution and radiation dosimetry of [¹²³I]-ADAM in healthy human subjects: preliminary results. Eur J Nucl Med 2003; 30: 132-136). Prior to the application of the radiopharmaceutical ¹²³I-ADAM to humans the manufacture (MAP of medical technologies Oy, Elementtitie 27, 41160 Tikkakoski, Finnland) performs tests relating to radiochemical and radionuclide pureness relating to pH as well as to sterility. Altogether up until now, no side effects or other unwanted occurrences have been seen when applying the radiopharmaceutical ¹²³I-ADAM to humans.

19 patients with migraine, diagnosed on the basis of the criteria of the International Headache Society, were examined (17 patients with migraine without aura, 2 patients with migraine with aura). A control group of 10 healthy patients was examined. Patients and control individuals were chosen in an age and sex adjusted manor (migraine: w: m = 17:3; age: 44 +/- 10.9 ys, (mean +/- S.D); controls: w:m= 8:2, age: 43.6+/- 11.1 ys)). There was no significant difference between the mean age of the control and the study population (p = 0.93). Due to the fact that recent imaging studies suggest a change in the SERT-availability of patients suffering from a major depression (Malison RT, Price LH, Berman R, VanDyck CH, Pelton GH, Carpenter L, Sanaccra G, Owens MJ, Nemeroff CB, Rajeevan N, Baldwin RM, Selby[ JP, Innis RB, Charney DS. Reduced brain serotonin transporter availability in major depression as measured by [123I]-2B-carbomethoxy-3B-(4-iodophenyl)tropane and Single Photon Emission Tomography. Biol Psychiatry 1998; 44: 1090-1098.; Pirker W, Asenbaum S, Kasper S, Walter H, Angelberger P, Koch G, Pozzera A, Deecke L, Podreka I, Brocke T: beta-CIT SPELT demonstrates blockade of 5HT-uptake sites by citalopram in the human brain in vivo. J Neural Transm Gen Sect 1995;100:247-56.; Willeit M, Praschak-Rieder N, Neumeister A, Pirker W, Asenbaum S, Vitouch O et al., [123I]-β-SPECT imaging shows reduced brain serotonin transporter availability in drug-free depressed patients with seasonal affective disorders. Biol Psychiatry 2000; 47: 482-489.), an unstructured psychiatric interview was performed in patients as well as control individuals in order to exclude a psychiatric disease as a possible confounding factor . Further, standardized questionnaires were performed in order to exclude depression or anxiety conditions.

Inclusion criteria were defined as follows:

Patients with migraine headache

- patient (m o. f) age between 18 and 65 years

- documented clinical diagnosis of a migraine without aura or a migraine with aura or cluster headaches being of episodic type - presents of diagnosis criteria according to ICD-IO: migraine with aura: G 43.1 migraine without aura: G. 43.0

- no prophylactic treatment of the headache syndrome within the last eight weeks prior to the inclusion in the study

- patient must be able to understand the patient information, the necessary examinations, must be willing to meet appointments and must be willing to give a declaration of consent.

Healthy test persons

- voluntary test persons (m o. f) in the age of between 18 and 65 years - good physical health in accordance with age, documented in the context of the clinical examinations during screening

- no history of neurological or psychiatric diseases

- test person must be able to understand the study information, willing to meet the appointments and provide a written declaration of consent.

Exclusion criteria for patients were defined as follows:

General exclusion criteria:

- use of medicaments which may interfere with the application of the radiopharmaceutical ¹²³I-ADAM (un-permitted medicaments)

- impairment of the kidney or liver function which would result in an influence on the pharmacokinetics of ¹²³I-ADAM: liver diseases with a serum-creatinin-level of over threefold of the normal level, liver diseases with a transaminase level elevation of threefold more than the normal level and / or γ-GT-elevation of more than fivefold - exposition to radiation due to profession or otherwise > 15 mSv per year

- abuse of drugs or alcohol prior to or at the time of the study

- participation in an other nuclear medical examination within a period of 5 halftimes of the substance used for screening - pregnant women or women during lactation.

Special exclusion criteria:

- Parkinson-syndrome (M. Parkinson, Multisystem atrophy, progressive supranuclear palsy) - cerebrovascular diseases, ZNS-tumors, hydrocephalus with normal pressure

- dementia (DSM IV/ICD 10 criteria)

- repeated cerebral ischemia with stepwise deterioration

- a past encephalitis

- evidence for structural damage of the basal ganglion or the brainstem - presence of a depressive disease (ICD 10 F 32, F 33)

- presence of a general anxiety condition (ICD 10 F 41.1 )

- comorbidity with a further primary headache: tension type headache (ICD 10 G. 44.2), cluster headache (ICD 10 G 44.0) in migraine patients, comorbidity with facial pains (for example atypical facial pain, ICD 10 G 50.1), neuralgy of the trigeminus (ICD 10 G. 50)

No-go criteria were defined as follows:

The doctor treating the patient in the study could end the study based on important grounds at any given time. Important grounds are for example unforeseeable non-tolerable side effects. Patients were taken out of the study if a severe or medically intolerable occurrence happened, symptoms of a disease occurred which fell within the exclusion criteria or the patient wished to end the study or the compliance of the patient was not sufficient.

Un-allowed medicaments (known interaction with ¹²³I-ADAM) were defined as follows:

- CNS-stimulants: cocaine, amphetamines, methylamphetamines, methylphenidats

- medicaments for the therapy of anorexia / obesity: phentermines, mazindols, dextroamphetamines - symphatomimetica: norephedrines, phenylpropanolamines

- antidepressives in particular substance class SSRI, SNRI, NASSA, NARI

- anti-parkinson medicaments (L-dopa, dopamine-agonists, MAO-/COMT-inhibitors, amantadines, budipines) - 5HT lB/lD-receptoragonists ("triptanes") up until 48 hours prior to the examination

Screening was performed as follows: The inclusion of patients/test persons into the study took place in the context of a screening in a clinic for neurology whereby an anamnesis and a neurological/psychiatric examination was performed as well as a screening regarding the inclu- sion and exclusion criteria.

The patients were informed of the planned examinations both orally and in writing. The declaration of consent was a prerequisite prior to the inclusion to the study. Visiting plan for test persons and migraine patients: First visit: The earliest on the day after the screening (1 day to 2 weeks) the patient received an intravenous injection of the radiopharmaceutical ¹²³I-ADAM at the clinic for nuclear medicine. Upon injection the patients were observed for adverse events (blood pressure, bleeding at the injection site).

¹²³I-ADAM-SPECT: 4 hours upon injection of the radiopharmaceutical ¹²³I-ADAM-SPECT examination was performed in the nuclear medical clinic. An examination took approximately 45 minutes. Upon injection the patient/test persons were consulted with respect to possible side effects/undesirable effects.

Visit 2: A follow-up was performed one day upon the nuclear medical examination, this means that the patient/test person was evaluated with respect to undesirable effects/happenings. This questioning took place in a telephone conversation in an outpatient status.

For the SPECT-examination the patient received an intravenous injection of 130 MBq ¹²³I- ADAM (after prior blockade of the thyroid gland with perchlorat). Four hours upon injection the ADAM-brain-SPECT was performed. The ¹²³I-ADAM-brain-SPECT-examination was performed with a double-headed camera (MultiSpect2, Fa. Siemens) with a Low Energy High Resolution Collimator (LEHR) with a matrix of 128 x 128 with 120 projections (60 per camera head) with an angle step of 30 degrees and a time of 40 seconds per projection without zoom. Thereupon, the SPECT-raw data was iteratively reconstructed using a commercially available reconstruction software ReSPECT (.Company: SCIVIS).

In order to precisely define regions of interests (ROIs) on the SPECT images, detailed ana- tomical information was obtained by MR-Imaging. Using a 1.5-T Siemens Magnetom Vision scanner (Siemens, Erlangen, Germany) equipped with a standard head coil we collected a high resolution Tl -weighted 3D-MP RAGE dataset (TR = 9.7 ms, TE = 4 ms, flip angle = 90°, field of view = 256 x 256 mm, voxel size l x l x l mm, 190 slices) in every.subject. To facilitate the precise coregistration of the two imaging modalities, four round-shaped external markers (φ 1,5 cm, height 0,5 cm) containing a thin column (φ 2 mm) of tocopherol inside were fixed on the head at predefined positions (according to the 10-20 scheme for EEG- registration nasion, inion, Cz and a fourth marker at half of the length between the preauricular fossa and the lateral margin of the eyebrow).

Thereupon a fusion of the three-dimensional magnetic resonance images and the iteratively rreeccoonnssttrruucctteedd ¹¹²²³³II--AADDAAMM--bbrraaiinn--SSPPEECCTT--ddaattaa wwaass ppeerrffoormed with the aid of a commercially available software MPI-tool by Advanced Tomo Vision.

The pontomesencephalic region, which comprises the rostral raphe complex, was chosen as target region. The "Raphe-nuclei" are the nuclei of origin of serotonergic neurons. (Tδrk I.

Anatomy of the Serotonergic System. Annals of the New York Academy of Science 1990;

600: 9-34.). The caudal Raphecomplex is not of interest for the examination of the serotonin metabolism in the brain because this nuclear complex projects "downwards" to the spinal cord but not orally to the brain. For the statistical analysis therefore the entire anatomic region of mesencephalon and pons was selected.

The data was analyzed as follows: Regions of interest (ROI) were drawn on co-registered images along their anatomic border. The brainstem ROI comprised the mesencephalon and pons (including rostral raphe complex, substantia nigra, ventral tegmental area). Using the software program MPI tools, we assessed counts/mm³ in the volume of the mesopontine brainstem and the occipital lobe.

The entire uptake in the target region was determined as follows: In a first step all counts per pixel were multiplied with the entire amount of pixels in one particular "region of interest" (ROI) (total impulses x total pixels). The total impulses of one ROI were then added for all slices (total impulses of one target region). In parallel the total volume of the target region was determined. Thereupon the total impulses of the target region was divided by the total volume of the target region. The quotient determined thereby (AQ in counts/mm³) is the AQ which enters in the total calculation.

Choice of reference region: In the past the cerebellum has been chosen in nuclear medical examinations for the determination of cert concentration as reference region (Oya S, Choi SR, Hou C, Mu M, Kung MP, Acton PD, Sicifano M, Kung HF. 2-((2-((dimethyl- amino)methyl)phenyl)thio)-5-iodophenylamine (ADAM): an improved serotonin transporter ligand. Nucl Med Biol 2000;27:249-54.; Shioe K, Ichimiya T, Sahara T, Takano A, Suite Y, Yasuno F, Hirano M, Shinohara M, Kagami M, Okubo Y, Nankai M, Kanba S. No association between genotype of the promoter region of serotonin transporter gene and serotonin transporter binding in human brain measured by PET. Synapse 2003; 48:184-188.; Buchert R, Thomasius R, Nebeling B, Petersen K, Obrocki J, Jenicke L, Wilke F, Wartenberg L, Zapleta- lova P, Clausen M. Long-term effects of "Ecstasy" use on serotonin transporters of the brain investigated by PET: J Nucl Med 2003; 44: 375-384.). However in visual analysis of the ¹²³I- ADAM-brain-SPECT-examination according to the invention in migraine patients one could find a relevant accumulation of the radiopharmaceutical in the cerebellum which was clearly higher than baseline. Thus, due to the fact that the cerebellum did not seem suited as reference region an alternative anatomical region was chosen in which the uptake was in the region of the baseline. Since the occipital lobe is regarded devoid of SERT, we selected this region as the reference region. In analogy to the target region (see above) an activity quotient was calculated for the reference region. The final target parameter which is equivalent to the SERT availability was calculated from the activity quotients according to the following formula:

Target region (pontomesencephalic region) - Reference region (occipital lobe)/ reference region

In a preferred embodiment the invention relates to a method of identifying migraine attacks in SPECT or PET images by calculating an activity quotient from SPECT or PET images of brain using serotonin-transport-protein binding radiopharmaceuticals according to the invention from regions of the pontomesencephal and/or rostral raphe nucleor complex, wherein when this activity quotient is higher than 0.55 migraine may be diagnosed. In a parcularly preferred embodiment it is higher than 0.8 even more preferred higher than 0.88 when migraine is diagnosed.

The activity quotient obtained in the ¹²³I-ADAM-examination was subjected to a statistical analysis (two-sided t-test for unbound random sampling).

¹²³I-ADAM uptake in the mesopontine brainstem: For the healthy test persons an activity quotient of 0,58 +/- 0,20 (target region - reference region/reference region) was determined. In contrast, the activity quotient was elevated to 0,88 +/- 0,16 in migraine patients. This differ- ence was significant in the context of a two-sided t-test for unbound random samples (p < 0,01).

Correlation between migraine frequency and mesopontine SERT-availability.

The monthly migraine days of our study population ranged from 2 to 17 days (see Table 1). In order to test a possible correlation between the severity of migraine, represented by the individual monthly migraine days, and the mesopontine SERT-availability, a linear regression analysis was performed. The statistical analysis revealed a positive correlation between mean monthly migraine days and '²³I-ADAM uptake in the rostral brainstem (r = 0.5; p = 0.027, see Fig. 6).

Clinical data of migraine patients and epidemiological data of healthy subjects.

Of the 19 migraine patients enrolled in our study, 17 patients suffered from migraine without aura (moa) and 2 patients suffered from migraine with aura (mwa). The migraine days refer to a mean of monthly migraine days during the last three months before study enrolment. Clinical data of migraine patients is shown in Table 1. BDI refers to the Beck Depression Inventory (BDI) according to: Beck AT, Ward CH, Mendelson M, Mock J, Erbaugh J: "An inventory for measuring depression" in Arch Gen Psychiatry 1961; 4: 561 - 71. Table 1

Epidemiological data of healthy subjects is shown in Table 2. 10 healthy subjects were enrolled in our study. The age of the healthy subjects matched to the age of the migraine patients (p = 0.93). One healthy subject (no. 3) was a smoker. There was no evidence for a current or a past psychiatric disorder. Table 2

Description of the Figures

The results of the examination are depicted in figures 1 to 6 as follows:

Figure 1: Depiction of the activity quotient of healthy controls and migraine patients in a box-plot-analysis. The difference between the two groups was highly significant (p < 0,001) in the present case, in the context of the present invention in elevation of the ratio of SERT in migraine patients between pontomesencephalic and occipital was about 0,8 to 1,0 and in particular 0,88. Such changes in ratio are to be expected in other headache forms and with other radiopharmaceuticals as well as with other procedures of examination.

The figures 2 to 5 show examples of results of healthy patients (controls) and patients from various regions of the brain. In the top row the cerebral magnetic resonance image is shown; the middle row shows the respective result for the SPECT-analysis. For Region-of interest analysis, an image fusion of SPECT and MRI was performed (see lower pannel) . The brain- slices are depicted three-dimensionally: in the left column the transversal pictures are depicted, in the middle column the coronae layers are depicted and in the right column the sagittal-images are depicted. The arrows mark the target region (pontomesencephalic region). Already upon visual examination in elevated availability of SERT due to an elevated activity storage in the target region of patients when compared to control patients is detectable.

Figure 2: In the bottom row of figure 2 the fusion image (SPECT and MRT) of a healthy control is shown. The arrow in the left and right bottom picture marks the "pons". In the fusion image of the lower picture row one may observe an in comparison low activity storage in control persons.

Figure 3 shows the fusion pictures of a patient with migraine without aura. In the bottom row of pictures, the arrows again point to the pontomesencephalic region. In comparison to the control person already upon visual inspection (in particular in the sagittal-picture) a significant elevation of activity storage may be observed.

Figure 4: In order to make the difference between control persons and migraine patients particularly clear the results of a control person and a migraine patient are directly compared. The transverse image shown here show the region of the mesencephalon (red arrow). The difference in activity storage between control person and patient may be clearly observed. In figure 5 the region of the "pons" is shown in transversal pictures (red arrow). Also here a clear difference between control person and migraine patient becomes apparent in the marked regions.

Figure 6: Correlation of SERT-availability and migraine frequency

We studied the relationship between migraine severity and SERT-availability in the mesopon- tine brainstem. Linear regression analysis revealed a significant correlation between mean monthly migraine days and ¹²³I-ADAM binding in the mesopontine brainstem (r = 0.5; p = 0.027).

Claims

Claims

1. Use of a serotonin-transport-protein binding radiopharmaceutical for the production of a diagnostic medicament for the diagnosis of headache.

2. Use according to claim 1 wherein the radiopharmaceutical carries an isotope or a radioactive moiety chosen from the group consisting of a PET isotope, a SPECT isotope, a radioactive isotope, selected from the group consisting of ¹²³I, ¹³¹I, ¹²⁵I, ⁷⁷Br, ¹⁸F, ¹¹C and

7₆ Br.

3. Use according to claims 1 or 2, wherein, the radiopharmaceutical is a compound according to Formula I:

Formula (I):

or a pharmaceutically acceptable salt thereof; wherein,

X is hydrogen, F, Cl, Br, I, NO₂, NR³R⁴ or -L-Ch, CH3(CH2)nO-, X(CH₂)_nO-, CH₃(CH₂)_nS-, X(CH₂)_nS-, CH₃(CH₂)_nOCH₂, X(CH₂)_nOCH₂-, CH₃(CH₂)_n(O)-, X(CH₂)_n(O)-, CH₃(CH₂)_n(O)O-, X(CH₂)_n(O)O-, (CH₂)_nCH₃, or (CH₂)_nCH₂X, wherein n = 0, 1, 2, or 3;

Y is hydrogen, -CH₂OR⁵, -CH₂NCH₃R⁶, NO₂, -NR³R⁴, -L-Ch, hydroxy(Ci- C₄)alkyl, halo(Ci-C₄)alkyl, nitro, azido or halo;

R » 3 and J r R>4 are independently hydrogen, hydroxy, C1-C₅ alkyl, halo(Ci-C₄)alkyl, halobenzoyl, C₃-C₈ cycloalkyl, Ci_-5 alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, phenyl, benzyl, naphthyl, naphthylmethyl or -L-Ch;

R⁵ is hydrogen, Ci-C₅ alkyl, C₃-C₈ cycloalkyl, Cu₅ alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, phenyl, benzyl, naphthyl, naphthylmethyl or -L-

Ch;

W is Y' or alkyl-Y'

Y' is hydrogen, -CH₂OR⁵, -CH₂NCH₃R⁶, NO₂, -NR³R⁴, -L-Ch, hydroxy(C_r

C₄)alkyl, halo(Ci-C₄)alkyl, azido or halo;

R⁶ is hydrogen, C_rC₅ alkyl, C₃ -C₈ cycloalkyl, C_)-5 alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, halo(Ci-C₃)alkyl, phenyl, benzyl, naphthyl, naphthylmethyl or L-Ch;

Z is S, O, NR⁷, CR⁸R⁹, C(O) or -C(=CR⁸R⁹)-;

R⁷ is hydrogen, Ci-C₅ alkyl, C₃-C₈ cycloalkyl, Ci_-5 alkylcarbonyl, (C₃-C₈ cycloalkyl) carbonyl, phenyl, benzyl, naphthyl, naphthylmethyl or -L-

Ch;

R⁸ and R⁹ are independently

hydrogen, Cj-C₅ alkyl, or chloro;

A, R¹ and R² are independently

Hydrogen, halogen, cyano, Ci-C₄ alkyl, halo(Ci-C₄)alkyl, halo(Ci-

C₄)alkanoyl or (Ci-C₄)alkoxy or L-Ch;

L is a covalent bond or a linking group selected from the group consisting of -(CH₂),, -and -(CH₂)_n -C(O)-, where n is 1 -5;

Ch is a tetradentate ligand capable of chelating a metal;

and with the proviso that at least one of A, X, Y, R³, R⁴, R⁵, R⁶, or R⁷ is -L-Ch and/or, one or both of X or A is Br or I and/or of Y, Y', R 1₅ rR> 2 or A contains ¹⁸F and wherein any halogen within the compound may by a radioisotope and/or wherein a carbon atom in R⁶ may be ^{1 1}C.

4. Use according to claim 3, wherein, W is (Ci-C₄)-Y' and in particular -(CH₂)-Y\

5. Use according to claims 3 and 4, wherein, Z is S.

6. Use according to claims 3 to 5, wherein, Y is NH₂.

7. Use according to claims 3 to 6, wherein, the radiopharmaceutical is a compound according to Formula II, III, IV, V or VI, wherein R⁶ in Formula II, III, IV, V or VI is hydrogen, Ci-C₅ alkyl, C₃ -C₈ cycloalkyl, Ci_-5 alkylcarbonyl, (C₃-C₈ cycloalkyl)carbonyl, halo(Ci-C₃)alkyl, phenyl, benzyl, naphthyl, naphthylmethyl or L- Ch and wherein, I in Formula II, V and VI is a radioisotop selected from the group consisting of ¹²³I, ¹³¹I and ¹²⁵I:

Formula II (I-ADAM):

Formula III (F-ADAM):

Formula IV (F-AFM):

Formula V (I-OB-ADAM):

Formula VI (I-ODAM):

8. Use according to claims 1 or 2 wherein the serotonin-transport-protein binding radiopharmaceutical is selected from the group of 5-methly-6-nitroquipazine and 3-(3[¹⁸F]- fluoropropyl)-6-nitroquipazine.

9.Use according to claims 1 to 8 wherein, the radiopharmaceutical is used to measure the availability of SERT in the pontomesencephal region of the brain, which comprises the rostral raphe nucleor complex.

10. Use according to one of the claims 1 to 8 wherein, the radiopharmaceutical is used in therapy monitoring in migraine patients, cluster headache or medicament induced headache.

11. In vitro screening method for substances which specifically modify the binding of a radiopharmaceutical which specifically bind the Serotonin-Transport-Protein (SERT) comprising the steps of: a) providing for an in-vitro system, which comprises the SERT and the SERT specific radiopharmaceutical, b) providing for a candidate substance, c) bringing together the SERT specific radiopharmaceutical and the SERT with and without the candidate substance, and d) measuring the binding between SERT and the SERT specific radiopharmaceutical, wherein, a difference in binding between with and without the candidate substance identifies a substance which specifically modifies the binding of a radiopharmaceutical which specifically binds the Serotonin-Transport-Protein (SERT).

12. Method according to claim 11, wherein the SERT of the in-vitro system stems from the pontomesencephal region of the brain and in particular from the serotonergic raphe nuclei.

13. Method according to claims 11 or 12, wherein the radiopharmaceutical is chosen form the conventional SERT specific PET-radiopharmaceuticals, SERT specific SPECT radiopharmaceuticals or the compounds according to claims three to seven.

14. Method according to one of the claims 11 to 13, wherein the modification of binding is an increase or decrease in binding of the radiopharmaceutical to the SERT.

15. Method for the production of a pharmaceutical for the treatment of headache, comprising a method according to one of the claims 11 to 14 and mixing the identified substance with a suited pharmaceutical carrier.

16. Method for positron emission tomography (PET) imaging or single photon emission computed tomography SPECT of serotonin transporter sites in a mammal comprising the steps of

(i) labelling serotonin transporter sites (SERT) with an image- generating amount of a PET or SPECT radiopharmaceuticals, which is able to bind SERT specifically or, with the radiolabeled compounds according to claims three to seven and (ii) measuring the spatial distribution of the compound in the mammal by PET or

SPECT

(iii) so as to thereby image the serotonin transporter sites, wherein, (iv) radiopharmaceutical uptake is determined in the pontomesencephal brainstem.

17. Method according to claim 16, wherein, additionally radiopharmaceutical uptake is determined in a reference region.

18. Method according to claims 16 to 17, wherein, the mammal is human and the human is a headache patient.