Division Radiochemistry and Radiopharmacology (E0300 / E030) Head: Prof

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Research Program E Division E0300 Radiological Diagnostics and Therapy Radiochemistry and Radiopharmacology

Division Radiochemistry and Radiopharmacology (E0300 / E030) Head: Prof. Dr. Michael Eisenhut (temporary) Scientists tion and processing of no-carrier-added positron emitting Dr. Joseph Eisenbarth radionuclides. Dr. Matthias Gilbert Thereafter, the Radiopharmaceutical Chemistry Group Dr. Jochen Schuhmacher (PET-Tracers, E0301) was transformed and reorganized Dr. Hannsjörg Sinn to the Radiopharmacy Group, now almost entirely de- Klaus Weber voted to GMP conform production of PET-Tracers. E0302 Priv. Doz. Dr. Andreas Wunder was renamed in Immunodiagnostics and left unchanged regarding the research goals. E0303 dealt with macromo- Graduate Students lecular carriers (mainly albumin) for cytotoxic drugs and Marcus Wolf fluorescent probes. This research is now replaced by Qin Wang other carrier systems which comprise tumor specific sub- Visiting Scientists stances usefull as carriers for anticancer agents. The Dr. Claudia Bauer name was shortened to Drug Transporters (E0303). The Dr. Stefan Martin former Targetry Group (E0304) was closed in favour of a Dr. Thomas Fietz group called New Radiopharmaceuticals (E0304). This decision was necessary because of several changes in Technicians research activities. All scientific aspects of radiopharma- Ulrike Bauder-Wüst ceutical research are dealt now within E0304. Nicole DiGallo (trainee) In summary, the Division of Radiochemistry and Radio- Hans Gasper pharmacology is now organized and briefly described as Harald Hauser follows: Bettina Helfert (trainee) E0301 Radiopharmacy J. Eisenbarth und K. Weber Ronald Matys The Radiopharmacy group is producing radiopharma- Heike Marx ceuticals for clinical applications. The agents are used by Stefan Rahn (trainee) the Clinical Cooperation Unit Nuclear Medicine (E0600) Dorina Rauch for clinical phase I and II studies. In addition, the devel- Armin Runz opment and validation of production procedures for new Ursula Schierbaum 250 compounds are performed for the approval by pharma- Hans-Hermann Schrenk ceutical inspection authorities. Ulrike Wagner-Utermann E0302 Immunodiagnostics J. Schuhmacher Germanium-68/Gallium-68 radionuclide generators are Research at the Division of Radiochemistry and Radio- produced and preclinical investigations of new tumor se- pharmacology is aiming at the development of new imag- lective peptides with animal models are performed. In ing agents and cancer targeted drugs, which are of poten- addition, several clinical studies involving PET-imaging of tial value for the diagnosis and therapy of tumors. neuroendocrine tumors with Gallium-68 labeled soma- Complementary to these investigations, the department tostatin analogs (DOTA-TOC) are performed. is also providing radiopharmaceuticals externally devel- E0303 Drug Transporters M. Eisenhut oped to support ongoing clinical studies at the Clinical Drug transporters derived from radiopharmaceutical re- Cooperation Unit Nuclear Medicine (E0600). The produc- search projects of this group are: Selective receptor medi- tion technologies necessary to provide new agents have ated transport of cytotoxic agents into tumor cells (cur- to be adapted to the local GMP-production facility and the rently somatostatin receptor binding peptides conjugated cyclotron Scanditronix MC32NI. If necessary, the precur- to antisense oligodeoxynucleotides like thioates, PNAs). sor chemistry and labeling techniques are improved. All E0304 New Radiopharmaceuticals M. Eisenhut preclinical studies needed for the evaluation of new The development and preclinical evaluation of radiola- agents and the clinical trials are performed in close col- belled compounds as potentially useful imaging agents laboration with the Clinical Cooperation Unit Nuclear for PET and SPECT: Medicine (E0600). Radiolabeled peptides for apoptosis imaging with PET/ In May 2001 the direction of the Division was taken over SPECT; from Dr. H. Sinn. At that time the Division was structured Radioiodinated Benzamides and 99mTc Benzamide in four research groups. E0301 Radiopharmaceutical Complexes for melanoma imaging; Chemistry (PET-Tracers), dealing with oncological topics New PET-Tracers for metabolic (functional) imaging of of PET-Chemistry; E0302 Radiopharmaceuticals for cancer. Immunoscintigraphy, development of pretargeting techniques with bispecific antitumor/anti-Gallium chelate antibodies; E0303 Macromolecular Compounds for Clinical Application in Oncology, albumin as carrier of fluorescence tags and cytotoxic drugs; E0304 Targetry, produc-

DKFZ 2003: Research Report 2001/2002 Research Program E Division E0300 Radiological Diagnostics and Therapy Radiochemistry and Radiopharmacology

Radiopharmaceutical Production The metabolism of 3'-fluoro-3'-deoxythymidine (FLT) fol- (Radiopharmacy E0301) lows a similar pathway as thymidine prior to integration into DNA. Due to the lack of a 3'-hydroxy group the DNA J. Eisenbarth, K. Weber, S. Martin, H. Marx, polymerization terminates after incorporation of FLT. D. Rauch, A. Runz, U. Wagner-Utermann, [18F]FLT is phosphorylated by TK I forming [18F]FLT-5'- M. Eisenhut monophosphate and captured within the cell resembling Cooperations (internal): Prof. Dr. U. Haberkorn, Prof. Dr. L. the metabolic trapping of [18F]FDG. Consequently, the ra- Strauss, Prof. Dr. J. Clorius, Clinical Cooperation Unit Nuclear diolabelled FLT derivative [18F]FLT (see Figure) was intro- Medicine (E0600); Priv.Doz. Dr. Dr. J. Debus, Clinical Cooperation duced as a proliferation marker for positron emission to- Unit Oncological Radiation Therapy (E0500); Dr. W.-E. Hull, Prof. Dr. W. Lehmann, Central Spectroscopy (R0400). mography [1]. Cooperations (external): Priv.Doz. Dr. W. Mier, Dr. A. Mohammed, The syntheses and 18F-labelling of a series of thymidine Department of Nuclear Medicine, Ruprecht-Karls-University, precursors exhibiting an alternative protection group Heidelberg; Prof. Dr. F. Rösch, Institute for Nuclear Chemistry, strategy have been developed. The major advantage to Johannes-Gutenberg-University; Mainz. Prof. Dr. B. Johannsen, Dr. B. Füchtner, Institute of Bioanorganic and Radiopharmaceuti- previous accomplishments rests upon the Boc-protection cal Chemistry, Research Center Rossendorf, Dresden; Prof. Dr. of the 3-N-position allowing an one-step hydrolysis of the R. Baum, Zentralklinik Bad Berka. radiolabelled compound in homogenous solution. The 18 The Radiopharmacy Group is providing radiopharmaceu- total number of six [ F]FLT-labelling precursors were in- ticals for clinical investigations. J. Eisenbarth is in charge vestigated by an automated radiosynthesis in order to op- of the manufacturing und K. Weber is responsible for the timize this clinically interesting radiopharmaceutical. The 18 quality control of radiopharmaceuticals. The agents used radiopharmaceutical [ F]FLT is currently investigated in in the first place are [18F]FDG, [15O]Water and [18F]FLT. two clinical studies to prove its efficacy as a proliferation Other tracers like [18F]FDOPA can be provided for experi- marker. mental purposes. For clinical studies, however, produc- Several other projects have been started to promote im- tion approvals are needed which are granted by the local provements on the targetry site: [121I]Iodide: Radioiodine governmental agency. For three years now, all 11C and 13N is a versatile radioisotope which can conveniently be labeling activities are abandoned due to the shut-down of used for the labeling of peptides, proteins and othe low the nuclear research reactor positioned between the cy- molecular imaging agents. Therefore, target related work clotron and the hot-cell laboratory. In addition, this lab was initiated to provide [121I]iodine for future animal PET was not accepted for GMP conform production of radio- and clinical PET applications. Although 124I is commer- pharmaceuticals. As a substitute a small clean room was cially available effords to start in-house production were 251 set up in the cyclotron hall housing all 18F production fa- launched because the setup of a vertival beam line of the cilities. One major obstacle is however associated with MC32NI was open for special applications. Rigid new ra- this situation: Cyclotron and production cannot be used at diation protection laws and the unresolved situation of the same time. the nuclear reactor, however, brought the project to a halt. Owing to the above mentioned restrictions, all develop- [18F]Fluoride: [18F]fluoride production is commonly per- ments which have been performed during the last three formed via the 18O(p,n)18F nuclear reaction using isotope years were exclusively focussed on 18F labeled com- enriched [18O]water. The expense for this target material pounds. At the beginning, the development for alternative is high because recovery of the enriched water leads to labeling methods for [18F]FLT (3´-deoxy-3´-[18F]fluorthymi- contamination with [16O]water. The development of a tita- dine) were initiated. nium gas target using [18O]oxygen prevents this effect be- O cause it can be removed from the target by condensation H The achievements ob- without [16O]oxygen dilution. The recovery rate amounted N 4 tained are briefly de- 3 5 CH3 scribed below: to 98% which implies a loss of about 10 €/charge. This 2 H O compares well with a loss of 100 €/charge by using H 1 6 Thymidine is utilized HO N 18 5' [ O]water. The technology is optimized by an automated H H H by proliferating cells O e system currently under construction. 4' 1' for DNA replication. 3' 2' 1 H H The recycling of Spherical Nb-Target: The conventional target technology 18F H x nucleosides from de- with isotope enriched [18O]water was always a problem 18 [ F]FLT graded DNA, the sal- with the silver and titanium targets available in the depart- vage pathway, is a ma- ment. Therefore, a high flux beam target for [18F]fluoride jor source for this process, because less energy is production was designed by S. Zeisler, D. Becker and H. needed in comparison to the de novo synthesis. Cata- Gasper. The layout of the sperical Nb-containment pro- lyzing the formation of thymidine-5'-monophosphate vided water cooling around the sphere. Niob was chosen (dTMP) the enzyme thymidine kinase I (TK I) is necessary because of the chemical inertness of the metal during for the salvage pathway in proliferating tissue. In cells en- the nuclear reaction with up to 35 µA beam current and tering the S-phase TK I is increased 15-fold as compared 1.2 GBq/µAh. This target was modified and is now in rou- to cells in other phases of the cell cycle. Therefore, TK I tine application for e.g. [18F]FDG synthesis. upregulation may be exploited for the development of Purification of [18O]Water: The work up of used [18O]water markers for cell proliferation. which is routinely performed by destillation in presence of

DKFZ 2003: Research Report 2001/2002 Research Program E Division E0300 Radiological Diagnostics and Therapy Radiochemistry and Radiopharmacology

β+ 68 68 (t1/2 68 min, 88%) is available from a Ge/ Ga radionuclide generator recently developed at the DKFZ. For imaging, specific molecular carriers showing high target affinities and fast nontarget tissue clearances are of pivotal interest. Monoclonal antibodies (MAbs) and small regulatory peptides can specifically target malignant cells expressing the corresponding tumor associated antigens and receptors. Consequently, MAbs and Fig. 1: [18O]-water before eletrolysis regulatory peptides labeled with a positron emitter should enable a specific and sensitive scintigraphic tumor localization with PET. Pretargeted Immunoscintigraphy Immunoscintigraphic tumor localization with labeled MAbs often resulted in false negative findings due to the low radioactivity contrasts between the tumor and the surrounding normal tissues. As a promising technique to improve sensitivity of immunoscintigraphic tumor localization multistep targeting methods have been developed using antitumor MAbs additionally conjugated with a high-affinity noncovalent binding site for a small, tissue Fig. 2: [18O]-water after eletrolysis diffusible, radiolabeled hapten, which is given after the localization of the MAbs on the tumor. These methods cir- cumvent the limitations of high bloodpool and liver back- KMnO4/NaOH does not sufficiently remove organic con- ground activity as well as of macromolecule targeting of taminations like CH3CN and EtOH. The invention of K. Weber provides an electrolytic device which breaks water solid tumors related to the administration of MAbs la- 18 beled prior to injection and allow optimization of tumor-to- down to its elements H2 and [ O]O2 and burns the gas- eous components back to [18O]water. Figures 1 and 2 normal tissue contrasts by reducing background activity. demonstrate the purification efficiency of this method. An To combine the specificity of antitumor MAbs with the im- automated supply of the used target water was designed proved tumor contrast of pretargeting and the sensitivity 252 and the setup is currently tested for routine performance. of PET we developed the reagents and treatment sched- ule for a three-step targeting method. For the complex- Publications (* = external co-author) ation of Ga3+ we synthetized N,N´-[2 hydroxy 5-(ethylene β- [1] Mier W*; Haberkorn U; Eisenhut M: 18F FLT; Portrait of a prolif- carboxy) benzyl]ethylene diamine N,N´diacetic acid eration marker. European Journal of Nuclear Medicine 29 (2001) 165-169. (HBED-CC) by a Mannich reaction starting with 3-(4 hy- [2] Martin S; Eisenbarth J; Wagner-Utermann U; Mier W*; Henze droxy phenyl) propionic acid, N,N´ ethylene diamine M; Pritzkow H*; Haberkorn U, Eisenhut M: A new precursor for diacetic acid and formaldehyde. The Ga-HBED-CC che- the radiosynthesis of [18]FFLT. Nuclear Medicine and Biology 29 late is of high kinetic and thermodynamic stability and is (2002) 263-273. excreted rapidly via the kidneys. Immunization of mice resulted in the production of high affinity anti-Ga HBED-CC 68Ga-Radiopharmacuticals for PET MAbs (K = 1.4 x 1010 M-1 ). The first step is the administra- (Immunodiagnostics E0302) tion of a bispecific monoclonal antibody (BS-MAb) pre-

pared from the F(ab´)2 fragments of an antitumor MAb J. Schuhmacher, H. Hauser, R. Matys and the F(ab´) fragments of an anti-Ga chelate MAb via a Cooperations (internal): Prof. Dr. U. Haberkorn, Dr. M. Henze, mixed functional chemical linker. Clinical Cooperation Unit Nuclear Medicine (E0600). After a localization period of 18 - 24 h a blocker was given Cooperations (external): Dr. S. Kaul, Prof. G. Bastert, Department of Gynecological Oncology, University Hospital Heidelberg; Prof. which consisted of the nonradioactive Ga chelate cova- H. Mäcke, Department of Radiological Chemistry, Institute of lently coupled to human apotransferrin serving as a high Nuclear Medicine, University Hospital Basel; Dr. S. Froidevaux, molecular weight carrier with slow extravasation. This Prof. A.N. Eberle, Department of Endocrinological Research, Uni- blocker saturated the anti-Ga chelate binding sites of the versity Hospital Basel; Dr. F. Le Gall, Affimed Therapeutics AG, BS-MAb still present in the circulation. Shortly after the Heidelberg. blocker injection 68Ga HBED-CC complex was injected The general objective of this group is the application of which rapidly penetrated into the tumor tissue binding to the short lived positron emitter 68Ga for the development the prelocalized BS-MAb. Excess 68Ga chelate was rapidly of new imaging agents. The main group 3 element Ga cleared from the circulation via the kindneys. Activity distri- shows in the M3+ state excellent complex stabilities with a bution was examined 1 h postinjection of the Ga chelate. series of chelating agents. These chelates can either be Using three different antitumor MAbs and the same anti- used as haptens for tumor targeting in the pretargeted Ga chelate MAb for BS-MAb preparation multistep target- immunoscintigraphy technology or covalently bound to ing resulted in tumor-to-blood ratios of 3.6, 4.7 and 2.6 in peptides or proteins. The short-lived positron emitter 68Ga rat pancreas carcinoma, human colon carcinoma and

DKFZ 2003: Research Report 2001/2002 Research Program E Division E0300 Radiological Diagnostics and Therapy Radiochemistry and Radiopharmacology human mammary carcinoma bearing nude mice respec- dimer (scFv)2 (diabody) with 68Ga, which is based on an tively. This compares to ratios of 0.6 - 0.8 24 h postin- isothio cyanate derivatized HBED as the bifunctional ch- jection obtained with the corresponding native MAbs la- elator. The second step will be the in vitro and in vivo beled with Iodine-131 prior to injection. characterization of the labeled diabody to evaluate its po- In a preliminary clinical trial we attempted to assess the tential for colon carcinoma localization. efficacy of pretargeting and PET for breast cancer localization in patients. For this reason the antimucin MAb 12 Receptor imaging of neuroendocrine tumors with H 12 was chosen as the antitumor part in BS-MAb prepa- PET ration. MAb 12 H 12 is a mouse IgG1, which recognizes Most tumors of endocrine origin like gastric carcinoids, the carbohydrate side chains of the tumor associated gly- pancreatic tumors, meningiomas and small cell lung coprotein TAG 12, which differs in glycosylation from the cancers show a highly increased expression of soma- MUC1 mucin on normal epithelial cells. TAG 12 is over- tostatin receptors. Somatostatin is a small, cyclic peptide expressed by the vast majority of epithelial cell adenocar- hormone consisting of 14 aminoacids which is synthe- cinomas and is shed into the circulation due to proteolytic sized and released from endocrine cells. It exerts a wide cleavage from the cell membrane. variety of antisecretory actions on the central nervous sys- Ten patients with biopsy proven primary breast carcino- tem, the hypothalamus and the gastrointestinal tract. Es- ma were infused with 10 mg of the BS-MAb. Eighteen pecially, the inhibitory action of somatostatin on the re- hours later 10,7 mg of the blocker were injected intra- lease of growth hormone (Somatotropin) from receptor veneously, followed 15 min later by 9.6 µg of the Ga che- positive tumor cells, resulting in an antiproliferative effect, late with ~ 260 MBq of 68Ga. PET imaging was started 60 suggested its potential as a therapeutic agent. Due to the - 90 min after Ga chelate injection. Fourteen of 17 known rapid proteolytic degradation of somatostatin in blood lesions, averaging 25 ± 16 mm in size were clearly visu- synthetic analoga were developed. The most efficient alized as foci of increased activity with PET. Uptake of the analogon, an octapeptide designated Octreotide, is high- Ga chelate by intraductal tumor sites was much higher ly resistant to degradation and retained an affinity of 9 -1 than by infiltrating tumor sites suggesting a potential of > 10 M to type II and V receptors similar to that of the na- the antimucin BS-MAb for detecting breast carcinoma in tive somatostatin. Conjugated with the chelator DTPA and situ. Nevertheless, PET was able to detect infiltrating tu- labeled with Indium-111 Octreotide proved to be of high mor sites 10 mm in size and contrasting only by a factor specificity for the localization of neuroendocrine tumors of two from surrounding normal breast tissue (Fig. 1). [10]. This demonstrates the better sensitivity of PET for detec- Sensitivity of the 111In Octreotide is restricted, however, to a 253 tumor size of ≥ 25 mm, due to the biokinetics of the compound and due to single photon detection with conventional gamma cameras. Derivatization with the macrocyclic ligand DOTA enables peptide labeling with metallic radionuclides other than 111In [11]. We have developed a fast and efficient technique for labeling DOTA-Octreotide with the short-lived positron emitter 68Ga resulting in a ≥ 85% labeling yield and with a recovery of ~ 50% of the ini- tial radioactivity. 68Ga-DOTA-Octreotide proved to be of Fig. 1 Patient with bilateral disease examined in the supine po- high in vivo stability and was cleared from the circulation sition. The transaxial slices show a tumor recurrence with intra- more rapidly because of its increased hydrophilicity com- dermal disease 2 years after resection of the primary tumor pared to 111In-DTPA-Octreotide. In a first preliminary clini- (left). Primary IDC branching into the mammillary region(right). cal trial with meningioma and gastric cancer patients PET imaging with 68Ga-DOTA-Octreotide resulted in high tion of breast cancer at low tumor contrasts compared to contrast images of le- conventional immunoscintigraphy. Since the low affinities sions less than 7 mm of antimucin MAbs and antigen shedding proved to be not in size 1 - 2 h postin- optimal for increasing tumor-to-tissue ratios, pretargeting jection (Fig. 2). 68Ga- with anti-CEA and anti-EpCAM BS-MAbs which are avail- DOTA-Octreotide is now able with much higher affinities and the absence of anti- produced routinely. gen shedding might further increase the sensitivity for As promising carriers to breast cancer detection. target malignant mela- The recent development of genetically engineered single- noma analogues of the chain fragments (sc Fv), which consist of only the antibody variable regions held together by a flexible linker, Fig. 2 Coronal slice of a now provides small sized (~ 26 kD) immuno reactive 55 year old patient with a molecules which more rapidly penetrate into solid tu- neuro-endocrine tumor mors than F(ab)´and F(ab)2 fragments and are cleared (carcinoid) showing tho- from the circulation within hours. Thus we are developing racic, hepatic, pelvic, ab- a rapid technique for labeling an anti-CEA scFv and its dominal and skeletal metastases.

DKFZ 2003: Research Report 2001/2002 Research Program E Division E0300 Radiological Diagnostics and Therapy Radiochemistry and Radiopharmacology

Fig. 3 PET im- [7] Schuhmacher J; Klivenyi G; Kaul S; Matys R; Hauser H; age (coronal and Clorius JH: Pretargeting of human mammary carcinoma xe- transaxial slices) nografts with bispecific anti-MUC1/anti-Ga chelate antibodies of melanoma and immunoscintigraphy with PET. Journal of Nuclear Medicine bearing mice 1 h and Biology, 28 (2001) 821-828. post injection of 2 [8] Henze M; Schuhmacher J; Hipp R*; Kowalski J*; Becker DW; 68 MBq Ga-DOTA Doll J; Mäcke HR*; Debus J; Haberkorn U: Pet imaging of MSH. Tumors sonatostatin receptors using 68Ga-DOTA-DPhe1-Tyr3-octreotide were inoculated (DOTA DOC). Journal of Nuclear Medicine, 42 (2001) 1053-1056. in the right lateral [9] Brix G*; Bellemann M; Hauser H; Doll J: Ermittlung von Recov- thoracic wall. ery-Koeffizienten für die Bestimmung der Arteriellen-Input- Funktion aus dynamischen PET-Messungen. Nuklearmedizin 41 (2002) 184-190. alpha-melano- cyte-stimulat- Somatostatin Receptor Mediated Transport α ing hormon ( (Drug Transporters E0303) - MSH) have emerged. Such M. Eisenhut, W. Mier* an analogue, Cooperations (internal): Prof. Dr. U. Haberkorn (E0600) , S. derivatized with Sieger Clinical Cooperation Unit Nuclear Medicine (E0600); Dr. the macrocyclic W.-E. Hull, Prof. Dr. W. Lehmann, Department of Spectroscopy chelator DOTA (R0400); Dr. H. Spring, Strukturelle Genanalyse (A0601). * Cooperations (external): Priv.Doz. Dr. W. Mier, Dr. A. and showing a high receptor affinity of 1 x 109 M-1, has Mohammed, Department of Nuclear Medicine, Ruprecht-Karls- been developed at the University of Basel (S. Froidevaux). University, Heidelberg. We have modified our labeling technique for labeling the DOTA - MSH with 68Ga. Preliminary results in melanoma Antisense oligonucleotides (ODNs) are on the brink of bearing mice are shown in Fig. 3. Optimization of the bio- extensive clinical application. They represent ideal tools kinetics should further improve tumor uptake and thus for the treatment of gene-related disorders like hereditary, enable the clinical use of this tracer for melanoma detec- viral or cancer diseases. Despite the excellent specificity tion with PET. of antisense ODNs, their efficiency is limited by rapid degradation by exo- and endonucleases. These prob- Publications (* = external co-author) lems have, to a great extent, been solved by using modi- 254 [1] Hofmann M*; Maecke H*; Börner AR*; Weckesser E*; fied backbones, mixed backbones and non-standard Schöffski P*; Oei ML*; Schuhmacher J; Henze M; Heppeler A; bases. However, these modifications do not significantly Meyer GJ*; Knapp WH*: Biokinetics and imaging with the soma- improve the poor cellular uptake. Site specific delivery of tostatin receptor PET radioligand 68Ga-DOTATOC: preliminary data. European Journal of Nuclear Medicine, 28 (2001) 1751- ODNs is still one of the major obstacles for clinical appli- 1757. cations. Consequently, several attempts have been made [2] Kissel M; Peschke P; Subr V*; Ulbrich K*; Schuhmacher J; to increase the intracellular bioavailability of ODNs. How- Debus J; Friedrich E*: Synthetic macromolecular drug carriers: ever, no tumor specific drug targeting systems has been biodistribution of poly[(N-2-hydroxypropyl)methacrylamide] and developed for ODNs. its accumulation in solid rat tumors. Journal of Pharmaceutical Science and Technology, 55 (2001) 191-201. Octreotide (OC) has found widespread application as a transporter for radioisotopes in nuclear medicine. OC la- [3] Doll J; Werling A; Bublitz O; Hauser H; Semmler W; Brix G*: 68 99m 111 123 Auswirkung von Aktivitäts- und Dichteverteilungen außerhalb beled with Ga, Tc, In or I are used for in vivo im- des Gesichtsfeldes auf die Genauigkeit von 3D-PET-Messungen. aging of SSTR-expressing tumors and metastases. Par- In: Medizinische Physik 2001. Hrsg.: Welker K, Zink K. Berlin: ticle emitting isotopes linked to OC have been shown to DGMP, (2001) 345-346. enable internal radiotherapy. For example, the β-emitting [4] Kissel M; Peschke P; Subr V*; Ulbrich K*; Schuhmacher J; [90Y]DOTA-D-Phe1-Tyr3-octreotide (DOTATOC) was suc- Debus J; Friedrich E*: Biodistribution of charged poly(N-2- cessfully used in a clinical trial for the radiotherapy of (hydroxypropyl)methacrylamide in tumor bearing rats. In: Proc. Int’l. Symp. Control. Rel. Bioact. Mater. Hrsg.: . San Diego, USA: , SSTR-expressing tumors. In summary, the preclinical (2001) 1346-1347. and clinical results indicate the potential of OC to direct [5] Kowalski J*; Henze M; Schuhmacher J; Mäcke HR*; Haberkorn U: Evaluation of PET imaging using 68Ga-DOTA-D phe1-tyr3-octreotide (DOTATOC) in comparison to (111In)- DTPAOC (octreoscan) SPECT. First results in patients with neuroendocrine tumors. Journal of Nuclear Medicine (2002) submit- ted. [6] Schuhmacher J; Kaul S*; Klivenyi G; Junkermann H*; MagenerM*; Henze M; Doll J; Haberkorn U; Amelung F; Bastert G*: Immunscintigraphy with positron emission tomography: Gal- lium-68 chelate imaging of breast cancer pretargeted with bispecific anti MUC1/anti-Ga chelate antibodies. Cancer Re- search, 61 (2001) 3712-3717.

Chemical structure of PNA-conjugate (Base sequence = Tyr- AGC GTG CGC CAT CCC-peptide).

DKFZ 2003: Research Report 2001/2002 Research Program E Division E0300 Radiological Diagnostics and Therapy Radiochemistry and Radiopharmacology large substituents linked to the N-terminal site of the cy- 15-mer PNA. The receptor affinities revealed little effect clic peptide. for the PNA-moiety on specific SSTRs binding, melting In this context the question was raised whether the studies proved the integrity of the hybridizing characteris- SSTR-selective peptide OC exhibits carrier capacity to tics. In vivo the PNA-octreotate conjugate showed a ten- transport drugs with high molecular weight like ODNs fold accumulation in tumor tissue as compared to the into tumor cells. In a recently published report we de- unconjugated PNA-control. This SSTR-selective binding scribed the synthesis of an anti-bcl-2 phosphorothioate, was inhibited by coinjection of excess octreotide. The conjugated to the SSTR-affine cyclic octapeptide endosomal accumulation of a rhodamine tagged PNA- octreotate (TATE) H-D-Phe-cyclo[Cys-Phe-D-Trp-Lys-Thr- octreotate conjugate into SSTR positive AR42J cells indi- Cys]-Thr-OH (Mier, W. et al. Bioconjug. Chem. 2000; 11: cated successful receptor mediated internalization. This 855). The receptor binding and hybridisation characteris- study demonstrates for the first time that selective accu- tics were found almost unchanged when compared to mulation of PNAs in tumor tissue is feasible by SSTR- the free peptide or ODN moiety. Now, we investigated the mediated transport indicating potential for tumor directed tumor targeting of this phosphorothioate-octreotate con- ODN therapy. jugate (PS-TATE) and compared the results with the anti- Publications (* = external co-author) bcl-2 peptide-nucleic-acid oligomer octreotate conjugate [1] W. Mier*, R. Eritja*, A. Mohammed*, U. Haberkorn, M. Eisenhut: (PNA-TATE). PNAs are synthetic DNA analogs in which Peptid-PNA-Konjugate: gezielter Transport von Antisense- the sugar phosphate backbone is substituted by neutral Oligonucleotiden in Tumoren. Angewandte Chemie, 2003 in amide backbone linkages. This modification has been press. chosen because PNA has attracted major attention [2] Haberkorn U; Altmann A; Eisenhut M: Functional genomics and caused by its potential for noninvasive diagnostic, proteomics - the role of nuclear medicine. European Journal of pretargeting, as well as pharmaceutical applications. The Nuclear Medicine 29 (2002) 115-132. synthesis of PNA-TATE conjugates, labeling, binding and [3] S. Wagner, R. Eritja*, M. Zuhayra*, F. Oberdorfer*, A. Mohammed*, W. Mier*, U. Haberkorn and M. Eisenhut: Synthesis hybridisation characteristics and the subsequent and Properties of Radiolabeled CPTA-Oligonucleotides. J labelled biodistribution in SSTR-positive tumor bearing rats are Comp Radiopharm 46 (2003) 175-186. described. In addition, cellular internalization of [4] J. Hoffend*, G. Linke*, A. Mohammed*, C.P. Tiefenbacher*, M. rhodamine tagged PNA-TATE conjugate using confocal Eisenhut, U. Haberkorn. 99mTc-BAT-2NI, a novel nitroimidazole microscopy was investigated. tracer – in vivo uptake studies in ischemic myocardium. Euro- pean Journal of Nuclear Medicine (2003) in press. Using rats bearing the SSTR-positive cell line CA20948 the anti-bcl-2 phosphorothioate ODNs (PS) conjugated to 255 the SSTR-selective peptide octreotate showed a tumor Human Serum Albumin Conjugates uptake of 0.57 ± 0.25 %ID/g 1 h after injection. The (Drug Transporters E0303) unconjugated, control PS yielded a similar result indicat- H. Sinn, A. Wunder, H.-H. Schrenk, U. Bauder-Wuest ing that octreotate did not increase the tumor uptake. To avoid strong unspecific tissue binding observed with the Cooperations (internal): Dr. E. Frei, Abt. Molekulare Toxikologie C0300; Dr. P. Peschke, Klinische Koopperationseinheit Strahlen- PS moiety, the ODN part was replaced by peptide nucleic therapie E0500, Prof.Dr. J. Clorius, Klinische Koopperationsein- acids (PNA, see Figure). Octreotate was assembled by heit Nuklearmedizin E0600, Dr. U. Zillmann Zentrales Tierlabor solid phase synthesis and extended with an anti-bcl-2 R0200, Dr. T. Haase Medizinische Physik E0400, Dr. M. Reinwald, Dr. F. Kießling Abt. Onkologische Diagnostik und Therapie E0100. Cooperations (external): Dr.G. Stehle, Prof. Dr. D.L Heene, Univ. A B Klinik Mannheim; Dr. G. Hartung, Prof. Dr. W. Queißer, III Med. Clinic, Univ. Klinik Mannheim; Dr. T. Egelhof, Neuroradiologie, Univ. Essen; Dr. C. Fiehn, T. Möhler, R. Max, Dr. M. Becker, Eye-Clinic, Univ. Heidelberg; Dr. J. Gahlen, Surgery, Univ. Heidelberg; Dr. P. Kremer, Neurosurgery, Univ. Heidelberg; Dr. E.H.K. Stelzer, EMBL Heidelberg; Dr. Dr. A. Kübler, Head & Neck Surgery, Univ. Heidel- berg; Prof. Dr. H.H. Fiebig, Clinics for Tumor Biology, Freiburg; Prof. Dr. V. Sturm, Dr. J. Voges, Neurosurgery, Univ. Köln; Dr. B. Werner, Impfstoffwerke Dessau – Tornau; Dr. F. Reiter, Klinge C D Corp., München. Human serum albumin (HSA) is taken up by solid tumors because it serves as a nitrogen and energy reserve. This finding was exploited for the tumor targeted transport of cytotoxic and fluorescence agents. From the uptake studies perfomed with radiolabelled HSA it was found that during the labelling procedure the native character of the protein must be maintained, otherwise the bioavailability Confocal laser scanning fluorescence microscopy images of of the protein was markedly reduced and a rapid accu- AR42J pancreatic cancer cells obtained with sulforhodamine B labeled PNA-Octreotate conjugate for 1 h at 37 °C (A), and with mulation in the liver was observed. These observations Lysotracker® yellow , a stain for lysosomal compartments (B). C were transfered to Methotrexate-albumin conjugates shows merged A and B. D shows the differential interference (MTX-HSA). The results of therapy experiments with tu- contrast image of the cell cluster in transmitted light.

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mor bearing rats revealed improved efficacy of the drugs, [2] Reuther T*; Kübler AC; Zillmann U; Flechtenmacher C*; Sinn H- accompanied with a considerable reduction of side ef- J: Comparison of the In Vivo Efficiency of Photofrin II-, mTHPC-, mTHPC-PEG-and mTHPCnPEG-Mediated PDT in a Human fects. Xenografted Head and Neck Carcinoma. Lasers in Surgery and Motivated by the positive preclinical results a phase-I Medicine, 29 (2001) 314-322. clinical trial was started using MTX-albumin. After ap- [3] Roelofs TA*;Grashew G*; Schneider M*; Rakowsky M*; Sinn proval from the local ethic commission this study was H; Schlag PM*: Multiphoton versus single photon excitation of performed under the control of the Arbeitsgemeinschaft photosensitizers for laser-induced fluorescence diagnosis and photodynamic therapy of cancer cells. Proccedings of SPIE. Internistischer Onkologen (AIO) in collaboration with the 2001; 259-262. oncologic division of the Klinikum Mannheim. The MTX- [4] Kiessling F*; Fink C; Hansen M*; Bock M; Sinn H*; Schrenk HH; albumin was produced under GMP conditions by the Krix M; Egelhof T*; Fusenig NE*; Delorme S: Magnetic resonance Impfstoffwerk Dessau. In 1998 the patent was licenced to imaging of nude mice with heterotransplanted high-grade squa- Klinge Pharma, München. Afterwards, this company con- mous cell carcinomas: Use of a low-loaded, covalently bound tinued the ongoing clinical studies. Gd-HSA conjugate as contrast agent with high tumor affinity. In- vestigative Radiology, 2002; 37: 193-198. Apart from MTX-HSA activities the scope of the albumin [5] Vajkoczy P*, Farhadi M*, Gaumann A*, Heidenreich R*, Erber conjugation technology was extended to other anticancer R*, Wunder A, Tonn JC*, Menger MD*, Breier G*. Microtumor drugs. Today, a great variety of biologically active com- growth initiates angiogenic sprouting with simultaneous expres- pounds have been conjugated to albumin as there are sion of VEGF, VEGF receptor-2, and angiopoietin-2. J Clin Invest. Aminopterin, Aureomycine, Doxorubicin, Ellipticine, Mito- 2002; 109: 777-85. xantrone, Quercetine, a new cis-Platinum complex and [6] Kremer P*, Hartung G, Bauder-Wüst U, Schrenk H-H, Wunder Tetracyclines. In addition, a series of fluorescent dyes A, Heckel S, Zillmann U, Sinn H. Efficacy and tolerability af an aminopterin-albumin conjugate in tumor-bearing rats. Anti-Cancer were conjugated to albumin. The applications of these Drugs 2002; 13: 1-10. conjugates ranged from the imaging of solid tumor, in- [7] Weigand M, Hartung G, Roboz J, Sieger S, Wolf M, Sinn H, flamed tissue to lymphnodes (see Fig. 1) in intraopera- Schrenk H.-H., Wiessler M, Frei E. Anti-Cancer Drug Design tive laser induced fluorescence diagnosis (LIFD) and for 2002; 16: 227-237. photodynamic therapy (PDT). The wave lengths ranges from 526 nm of fluorescein (AFlc) to about 660 nm of a Radiolabeled peptides for apoptosis tetracarboxyphenylchlorine. AFlc-HSA has a bright fluores- imaging (New Radiopharmaceuticals E0304) cence without any photoactivity, therefore, preferred by neurosurgeons, whereas the chlorine conjugate is pre- M. Eisenhut, C. Bauer, M. Gilbert 256 ferred in PDT, due to its high photoactivity and the high Cooperations (internal): Prof. Dr. U. Haberkorn, Clinical Coopera- penetration depth of the red light. tion Unit Nuclear Medicine (E0600); Prof. Dr. P. Krammer, Immungenetik (G0300); Dr. W.-E. Hull, Prof. Dr. W. Lehmann, Cen- Publications (* = external co-author) tral Spectroscopy (R0400). [1] Burger AM*; Hartung G*; Stehle G*; Sinn H; Fiebig HH*: Pre- Cooperations (external): Priv. Doz. Dr. W. Mier, Dr. A. Mohammed, clinical evalution of a methotrexate-albumi conjugate (MTX-HSA) Department of Nuclear Medicine, University Heidelberg; Priv. Doz. in human tumor xenografsin vivo. International Journal of Cancer, Dr. R. Kinscherf Department of Anatomy, University Heidelberg; 92 (2001) 718-724. Prof. M. Little, Dr. S. Knackmuß, Affimed Heidelberg. The imaging of apoptosis is attractive for the clinician because 1. induction of apoptosis in tumors treated with various modalities may be assessed, 2. the extent of apoptosis may be visualized in degenerative disease involving the severity of the estimated disease and 3. the rejection of transplanted organs may be visualized at early stages. Furthermore, measurements before and after therapeutic intervention could be used for therapy monitoring allowing early changes of ineffective treatment schedules. For the in vivo detection of apoptosis mainly two targets in the apoptotic pathway are of interest: The presentation of phosphatidylserine residues at the outer side of the plasma membrane and the appearance of activated caspases. Phosphatidylserine is maintained at the inner site of the plasma membrane by the ATP-dependent enzymes floppase and translocase. Apoptosis induces in- activation of these enzymes and activation of scramblase leads to the appearance of phosphatidylserine on the outer side of the membrane. This effect has been recently used to develop an imaging agent for apoptosis. Annexin V, a 35 kD human protein with high affinity for cell Fig. 1. Lymph node image after injection of TCPP/TCPC-HSA. Op. membrane bound phosphatidylserine, was labeled with situs (rat) was illuminated with UV-light.

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99mTc and investigated for its uptake in apoptotic cells. An [3] Haberkorn U; Henze M; Altmann A; Jiang S; Morr I; Mahmut M; increased accumulation was found in Jurkat cells where Peschke P; Debus J; Eisenhut M: Transfer of the human sodium iodide symporter gene enhances iodide uptake in hepatoma cells. the programmed cell death was initiated by growth factor Journal of Nuclear Medicine, 42 (2001) 317-325. deprivation, anti-CD95 antibody and doxorubicin treat- [4] Henze M; Mohammed A*; Mier W*; Nollert J*; Rudat J*; Dietz ment. Also anti-CD95 treated mice showed a 3-fold rise A*; Eisenhut M; Haberkorn U: Pretreatment evaluation of the hy- in hepatic 99mTc-Annexin V accumulation in response to popharynx and larynx carcinoma with 18F-fluorodeoxyglucose severe liver damage with histologic evidence of apop- (FDG), 123(alpha)-methyl-typosine (IMT) and 99mTc-sestamibi tosis. Finally, increased uptake was detected in animal (MIBI). European Journal of Nuclear Medicine 29 (2001) 324-330. models using the acute rejection of transplanted hetero- topic cardiac allografts or transplanted murine B cell lym- 99mTc Benzamide Complexes phomas treated with cyclophosphamide. (New Radiopharmaceuticals E0304) Since caspases play a key role during the early period of M. Eisenhut the intracellular signal cascade of cells undergoing apoptosis we considered benzyloxycarbonyl-Val-Ala-DL- Cooperations (internal): Prof. Dr. U. Haberkorn Clinical Coopera- tion Unit Nuclear Medicine (E0600); Dr. W.-E. Hull, Prof. Dr. W. Asp(O-methyl)-fluoromethyl ketone [Z-VAD-fmk], a pan- Lehmann, Central Spectroscopy (R0400). caspase inhibitor, as a potential apoptosis imaging Cooperations (external): Priv. Doz. Dr. W. Mier, Dr. A. Mohammed, 131 agent. Applying the Tl(TFA)3/[ I]iodide method Z-VAD-fmk Department of Nuclear Medicine, University Heidelberg; Prof. Dr. was labelled at the benzyloxycarbonyl protecting group. A. Jones, Prof. Dr. A. Mahmood, Harvard Medical School, Boston, The success of radioiodination, however, depended on USA. the presence of carrier iodide resulting in specific radio- The development of melanoma imaging agents has activities of 2.6 GBq/µmol and the formation of a mixture been pursued for more than twenty years. These efforts of the 2- and 4-iodophenyl derivative (61%) which could have been spurred by the need for selective radiophar- not be separated by HPLC. Uptake measurements were maceuticals for the early detection of melanoma me- performed with Morris hepatoma cells (MH3924Atk8) tastases which would enhance the success of therapy which showed expression of the Herpes Simplex Virus and of follow-up of patients suffering from this disease. thymidine kinase (HSVtk) gene. Apoptosis was induced Although visible at by treatment of the cells with 25 µM ganciclovir. The an early stage, the TUNEL assay revealed 1.3 ±0.3 and 23 ±1.1% apoptotic primary tumor is cells immediately and 24 h after therapy, respectively. A often underesti- two-fold increase of [131I]IZ-VAD-fmk uptake was found at mated. It is known 257 the end of treatment with the HSVtk/suicide system which that cutaneous tu- constantly remained elevated for the following 4 hours. mor thicknesses The slow cellular influx and lack of uptake saturation of as small as 1 mm [131I]IZ-VAD-fmk are evidence for simple diffusion as involve dissemi- transport nation of daughter mechanism. cells into local In addition, lymph nodes be- the absolute fore general inva- cellular up- sion into other or- take of gans occurs. The 131 [131I]IZ-VAD-fmk [ I]IZ-VAD- ability of mela- fmk was noma cells to take found to be low. This quality was related to the rather high up tyrosine (like a lipophilicity of [131I]IZ-VAD-fmk causing unspecific binding sympathetic neu- to macromolecules in the medium. Instead of using an ron) and to oxidize inhibitor, synthetic caspase substrates are currently in- it to dopa, and finally to form polymeric melanin, has vestigated which may accumulate in the apoptotic cell by been exploited by several approaches for the develop- metabolic trapping thereby enhancing the imaging sig- ment of melanoma targeting compounds. Most recently nal. Current work deals with syntheses of caspase sub- radioiodinated benzamides, for example, BZA [N-(2-di- strates and the formation of anti apoptosis single-chain ethylaminoethyl)-4-[123I]iodobenzamide] and IMBA [N-(2- antibodies. diethylaminoethyl)-3-[123I]iodo-4-methoxy-benzamide] have been studied in patients, and both compounds dis- Publications (* = external co-author) play considerable melanoma uptake and excellent con- [1] Haberkorn U; Kinscherf R*; Krammer PH; Mier W*; Eisenhut M: Investigation of a potential scintigraphic marker of apoptosis: trast in scintigraphic images, with a specificity of approxi- radioiodinated Z-Val-Ala-DL-Asp(O-methyl)-fluoromethyl ketone. mately 100%. Nuclear Medicine and Biology, Oct;28(7) (2001) 793-798. Based on the melanoma-affinity characteristics of radio- [2] Haberkorn U; Altmann A; Jiang S; Morr I; Mahmut M; Eisenhut iodinated benzamides, the development of 99mTc com- M: Iodide uptake in human anaplastic thyroid carcinoma cells af- plexes containing structural elements of N-(dialkyl- ter transfer of the human thyroid peroxidase gene. European Journal of Nuclear Medicine, 28 (2001) 633-638. aminoalkyl)benzamide as pharmacophores has been pursued. Thus, a series of ‘3+1’ mixed-ligand [99mTc]oxo-

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technetium complexes [TcO(SN(R)S)(SNX2)] were synthe- and followed the rank order 19 > 15 >> 7. Comparison of sized and studied in B16 murine melanoma cells as well the 1-h melanoma uptake values with the corresponding as in the C57Bl6/B16 mouse melanoma model. In bio- blood counts indicates an interdependence, thus sug- distribution studies with the compound in which R=Me gesting that the degree of melanoma uptake is propor- and X=Bu, up to 5% ID/g and an average melanoma/non- tional to the concentration of the 99mTc complex circulating target tissue ratio (M/NTT) of 12.6 was obtained at one in the blood (bioavailability). hour post-injection. When the [99mTc]oxotechnetium(V) complexes of tetradentate amine-amide-dithiol (AADT) Publications (* = external co-author) chelates containing only tertiary amine substituents, [1] Friebe M*; Mahmood A*; Bolzati C*; Drews A*; Johannsen B*; Eisenhut M; Kraemer D*; Davison A*; Jones AG*: 99mTc oxotech- AADT-CH -[CH ] -NR (n = 1, 2; R = Et, n-Bu), were tested, 2 2 n 2 netium(V) complexes amine-amide-dithiol chelates with dialkyl- melanoma uptake of 7.6% ID/g (M/NTT 9.5) was obtained aminoalkyl substituents as potential diagnostic probes for malig- at one hour after administration. In other approaches the nant melanoma. Journal of Medical Chemistry, 2001; 44: 3132- complete benzamide pharmacophore N-(2- 3140. diethylaminoethyl)benzamide was connected to bis(ami- [2] Mahmood A*; Friebe M*; Eisenhut M; Bolzati C*; Drews A*; nothiol) ligands (BAT), resulting [99mTc]oxotechnetium Johannsen B*; Davidson A*; Jones AG*: Amine-amide-di-thiol (AADT)99mTc-complexes with dialkylaminoalkyl substituents as [TcO(BAT)] and [99mTc]nitridotechnetium [TcN(BAT)] com- potential diagnostic probes for malignant melanoma. Journal of plexes that displayed melanoma uptake between 0.43 labelled Comp. Radiopharm, 2001; 44: 51-53. and 1.51 %ID/g (M/NTT 4.5 and 1.6) one hour post injec- [3] M. Eisenhut, A. Mohammed*, W. Mier*, M. Friebe*, A. tion in the C57Bl6/B16 mouse model. The synthesis and Mahmood*, A.G. Jones*, U. Haberkorn. Melanoma Affine 99mTc in-vivo evaluation in the C57Bl6/B16 mouse melanoma Complexes Comprising the N-(2-diethylaminoethyl)benzamide model of 99mTc complexes has been performed with Structure Element. Journal of Medicinal Chemistry 2002; 45: ligands containing the N-(2-diethyl-aminoethyl)benz- 5802-5805. amide structural element using (1) a chelate design that integrates the phenyl ring of the pharmacophore within Novel octreotide derivatives the complex, (2) a bis(aminothiol) ligand connected via a (New Radiopharmaceuticals E0304) butylene spacer, and (3) a hydrazinonicotinamide ligand Q. Wang, M. Eisenhut with tricine as ancillary co-ligand. Cooperations (internal): Prof. Dr. U. Haberkorn Clinical Coopera- The ligands 3,4-bis(S-benzoyl-2-thioacetylamido)-N-(2- tion Unit Nuclear Medicine (E0600); Dr. W.-E. Hull, Prof. Dr. W. diethylaminoethyl)benzamide and 4-(S-benzoyl-2-thio- Lehmann, Department of Spectroscopy (R0400). acetyl-glycyl-glycylamido)-N-(2-diethylaminoethyl)benz- Cooperations (external): Priv. Doz. Dr. W. Mier, Dr. K. Graham, 258 amide were synthesized with an integrated ligand-phar- Department of Nuclear Medicine, University Heidelberg. macophore design, while N-(2-diethylaminoethyl)-4-[4- The cyclic tetradecapeptide somatostatin is a potent in- (2,10-dimercapto-2,10-dimethyl-4,8-diazaundec-6-yl)bu- hibitor in the secretion of growth hormones. In addition, a tyloxy]-benzamide, a tetradentate bis(aminothiol) ligand, large number of carcinoid tumours have been shown to and 6-hydrazinopyridine-3-[N-(2-diethylaminoethyl)]carb- express receptors for somatostatin. Due to an in vivo oxamide, a hydrazinonicotinamide, were based on a pen- half-life of 2-3 mins, smaller cyclic peptides have been dent approach. After complexation with 99mTc the biodistri- developed. One of these discoveries is octreotide - a cy- bution of the HPLC-purified compounds was evaluated in clic eight-membered peptide with a disulphide bridge. It the C57Bl6/B16 mouse melanoma model. The 99mTc has been shown to have an in vivo half-life in humans of complex formed with ligand 11, Tc-12, displayed the high- 60-90 mins and to be 2000 more times effective in inhib- est melanoma uptake at 1 and 6 h post injection (3.42 iting growth hormone secretions. Today, derivatives of and 4.35 %ID/g). The melanoma uptake of 99mTc com- octreotide represent the most important peptides for re- plexes of the remaining ligands was considerably lower ceptor targeting in oncological applications. Conse-

DKFZ 2003: Research Report 2001/2002 Research Program E Division E0300 Radiological Diagnostics and Therapy Radiochemistry and Radiopharmacology quently, these peptides ofer an enormous potential as carrier molecules for drug targeting applications. The figure shows the importance of the different amino acids of octreotide: Constituting the pharmacophor in numerous somatostatin receptor-affine substances, the sequence Phe(Tyr)-Trp-Lys-Thr has been thoroughly investigated. In contrast, systematic investigations of hydrophobic and hydrophilic modifications of octreotide have not been performed. The properties of hydrophilic and lipophilic derivatives of Tyr3-octreotate were also fully investigated. As the potential of conjugates formed at the C-terminal end is unknown, a large series of C-terminaly drivatised compounds were investigated. The receptor affinities were determined by competition experiments with 125I-Tyr3- octreotide on rat cortex membranes. After labeling with 125I, the organ distribution of the derivatives was investigated in a rat model bearing the pancreas tumor CA20948. The C-terminal modification resulted in conjugates with high affinity to SST-receptors but unfavourable pharmacokinetics. The ethyleneglycol modified derivatives and the short chain fatty acid-modified derivatives revealed high receptor affinities. In vivo, these compounds have a substantially decreased uptake in the liver, the fatty acid modified derivatives showed very low kidney uptake. In addition, the organ distribution of several conjugates revealed strong accumulation in the pancreas. This might be the base for the development of pancreas-specific tracers.

Publications (* = external co-author) [1] Qin Wang, K. Graham*, T. Schauer*, T. Fietz, A. Mohammed*, 259 U. Haberkorn, M. Eisenhut, W. Mier*. Pharmacological properties of hydrophilic and lipophilic derivatives of octreotate.Nucl Med Biol. in press. [2] Graham K.A.N.*, Wang Q., Eisenhut M., Haberkorn U., *Mier W. A general method for functionalising both the C- and N-termi- nals of Tyr3-octreotate. Tetrahedron Letters 43 (2002) 5021- 5024. [3] Mier W*; Eritja R*; Mohammed A*; Haberkorn U; Eisenhut M: Synthesis and labeling of peptide nucleic acid oligomers conjugated to octreotate. Journal of Labelled Compounds and Radiopharmaceuticals, Suppl. 1, 44 (2001) 954-956. [4] Mier W*; Haberkorn U; Eisenhut M: Synthesis and isolation of active esters of DOTA. Journal of Labelled Compounds and Radiopharmaceuticals, 44 (2001) 814-816. [5] Mier W*; Rossmann M*; Mohammed A*; Haberkorn U; Eisenhut M: 3-End-labeling procedure for phosphorothioate oligonucleotides and oligonucleotideconjugates. Journal of Labelled Com- pounds and Radiopharmaceuticals, 44 (2001) 163-166.

DKFZ 2003: Research Report 2001/2002