University of Groningen
Metallodrugs for therapy and imaging: investigation of their mechanism of action
Spreckelmeyer, Sarah
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Propositions
for the thesis
Metallodrugs for Therapy and Imaging: Investigation of Their Mechanism of Action
Sarah Spreckelmeyer
1) Bifunctional chelators as radiopharmaceuticals for imaging or therapy are useful tools in personalized medicine.
2) The milestones in cancer research could not have been achieved without the interdisciplinary work of multiple research facilities.
3) The combination of PET or SPECT with CT or MRI is an enormous progress in medicine.
4) The pharmacy study is an excellent preparation for working in an interdisciplinary environment.
5) Inorganic Chemistry offers a great toolbox for the design of molecules for therapy and diagnosis.
6) Although cisplatin is used in the clinic as anticancer agent, not much is known about its mechanism of transport.
7) Work hard, play hard in order to be successful in life. – adapted from James de Koven
8) “Um zu wissen, was im Leben wichtig ist, muss man die Welt gesehen haben.” – Marteria (“You need to have seen the world to know what is important in life.”)
9) The statement “Life isn’t about waiting for the storm to pass. It’s about learning to dance in the rain” (Vivian Greene) applies to the process of a PhD project.
Paranimphs
Gerian Prins
Malte Schulze
Cover design: Carlos Niermeier
Layout design: Sarah Spreckelmeyer
Printed by: Ipskamp Printing
The research presented in this thesis was financially supported
by CANDA. Printing of this thesis was supported by the University
of Groningen, Faculty of Science and Engineering and the
University Library.
ISBN (printed version): 978-94-034-0442-4
ISBN (digital version): 978-94-034-0441-7
No parts of this thesis may be reproduced or transmitted in any
form or any means, electronical or mechanical, including
photocopying, recording or any information storage and retrieval
system, without permission of the author
INVITATION
You are cordially invited to attend the public defence of the
doctoral thesis of
SARAH SPRECKELMEYER
entitled
Metallodrugs for Therapy and Imaging – Investigation of Their
Mechanism of Action
Friday, 23 February 2018
at 16:15 hours
Reception immediately after.
sarah.spr@gmx.de
Address of the defence:
Bestuursgebouw
Oude Boteringestraat 44
Groningen
Address of the reception:
Academiegebouw
Broerstraat 5
Groningen
Metallodrugs for Therapy and Imaging:
Investigation of Their Mechanism of
Action
PhD thesis
to obtain the joint degree of PhD at the
University of Groningen and the University of British Columbia
on the authority of
the Rector Magnificus of the University of Groningen, Prof. E. Sterken,
the Faculty of Graduate and Postdoctoral Studies (Chemistry) of the University of British Columbia
and in accordance with
the decision by the College of Deans of the University of Groningen
This thesis will be defended in public on Friday, 23 February 2018 at 16.15 hours
by
Sarah Spreckelmeyer
born on 8 May 1989
in Osnabrück, Germany
Supervisors
Prof. G. M. M. Groothuis
Prof. C. Orvig
Co-supervisor
Prof. A. Casini
Assessment Committee
Prof. F. J. Dekker
Prof. M. Wolf
Prof. J. Reedijk
Für Opa Hubert
Table of contents
Introduction………...……….………...…………...13
1. Metallodrugs for therapy……….…..……….………14
1.1. Pharmacology of metallodrugs for therapy……….……..….……16
1.1.1. Pharmacokinetics (PK) and toxicity of cisplatin……….……18
1.2. Radiopharmaceuticals for therapy……….………..………19
1.2.1. Bifunctional chelator……….………...21
1.2.1.1. Biological targets discussed in this work…………...…….22
1.3. Mechanism of targeting of radiopharmaceuticals for therapy…-………23
2. Metallodrugs for imaging……….……….………..……….……23
2.1. Radiopharmaceuticals………..………23
2.1.1. PET and SPECT technique……….……….………….………24
2.1.2. Mechanism of accumulation of radiopharmaceuticals for cancer imaging………26
2.2. Fluorescence imaging………27
3. References………..………..…..………28
The aim of the thesis………..……...…….…………31
Part A: Vancouver………..………36
A1: p-NO2-Bn-H4neunpa and H4neunpa-Trastuzumab: Bifunctional Chelator for Radiopharmaceuticals and 111In Immuno-SPECT Imaging……….37
1. Abstract………38
2. Introduction………...………..………..………39
3. Results and Discussion………..……….………41
3.1. Synthesis and characterization of the ligand……….…………41
3.2. Synthesis and characterization of non-radioactive metal complexes……….………44
3.2.1. NMR……….…….……….…44
3.2.2. IR………...………45
3.2.3. Thermodynamic stability……….…..……..…………46
3.3. Radiolabeling experiments with unmodified chelator………..……51
3.4. Stability studies with unmodified chelator………53
3.5. Initial biodistribution studies……….………..….………55
3.6. Preparation of Bioconjugates and in vitro characterization……...………59
3.7. Biodistribution and SPECT/CT imaging studies………..………61
6. References………..………..…..………82
7. Supporting Information………..……….…………88
A2: H4neunpa: A Bifunctional Acyclic Chelator with Many Faces……….………..98
1. Abstract……….………..………99
2. Introduction………..………100
2.1. Subchapter 1………..……….……….……….101
2.1.1. Results and Discussion………102
2.1.1.1. Synthesis………..………..………102
2.1.1.2. Radiolabeling with 111In……….………103
2.1.1.3. Stability of H4neunpa-PSMA-L in human serum………104
2.2. Subchapter 2……….………..…..……104
2.2.1. Results and Discussion……….……….……….…………106
2.2.1.1. Metallacage exo-functionalization……….………106
2.2.1.2. La-complexation reaction………109
2.2.1.3. Fluorescence spectroscopy……….………110
2.3. Subchapter 3………111
2.3.1. Results and Discussion………..……….……113
2.3.1.1. Radiolabeling with 225Ac……….……….…113
2.3.1.2. 225Ac/213Bi iTLC chromatograms………..114
2.3.1.3. Sb-complexation……….……….118
3. Summary………..………119
4. Experimental……….………121
5. References………..…..…………124
6. Supporting Information……….………127
A3: Tetrahydroxamic Acid Bearing Ligands: EDTA and DTPA Analogues…….134
1. Abstract……….………..………135
2. Introduction………..………136
3. Results and Discussion……….…………141
3.1. Synthesis and characterization………141
3.2. Metal complexation reactions……….………..………143
3.3. Infrared (IR) spectroscopy………..………..………144
3.4. In vitro cell experiments………..………146
3.5. Stability determination of Fe-EDT(M)HA and Fe-EDT(B)HA by UV-VIS spectroscopy……….……….148
3.6. 89Zr-radiolabeling……….……….…….150
3.7. Density Functional Theory (DFT) ………151
5. Experimental………..………..………….………158
6. References………..……….………166
7. Supporting Information……….………169
A4: Overcoming the Limitations in Thrombosis Treatment: A Bifunctional Chelator as Positron Emission Tomography-Imaging Probe for Detecting Blood Clots……….….176
1. Abstract……….………..………..………..………..…………..177
2. Introduction………..………..………..………..………..178
3. Results and Discussion………..………..…..……….………..183
3.1. Synthesis………..……….………..……….……..183
3.1.1. Thiol bioconjugation of compound 3……….…...187
3.2. DTNB assay………..……..……….…..190 4. Conclusions………..………..……….192 5. Experimental……….……..………..……….192 6. References………..….………..……….197 7. Supporting Information………..………..………199
Part B: Groningen………..…………..……….204
B1: Cellular Transport Mechanisms of Cytotoxic Metallodrugs: An Overview Beyond Cisplatin……….……….…………..205
1. Abstract……….………...………..…206
2. Introduction………..………..………..………..………..207
3. Transport processes of metal-base compounds………..……..211
3.1. Anticancer Pt drugs………..211
3.1.1. Cu transporters……….………..……….……..212
3.1.2. Organic cations transporter (OCTs) and toxin extrusion proteins (MATEs)………..………..….220
3.2. Experimental anticancer metal compounds……….…..225
3.2.1. Ruthenium complexes………..225
3.2.2. Gold complexes……….…..………..……..229
3.2.3. Iridium complexes……….………..234
3.3. Transporter-targeted anticancer metal compounds………..…….235
4. Conclusion and Perspectives………..……..237
B2: Exploring the Potential of Gold(III) Cyclometallated Compounds as
Cytotoxic Agents: Variations on the C^N Theme………..250
1. Abstract………..………..………..…………251
2. Introduction………..………..………..………..………..…………252
3. Results and Discussion……….………253
3.1. Synthesis and structural characterization………..………….…………254
3.2. Antiproliferative activity………..………....………..…..………258 3.3. PARP-1 inhibition………..………..………..………..…………261 4. Conclusions………..……….…..…………..………..….………261 5. Experimental section………..………..……..………..…………262 6. References………..………..………..………..………..…….……268 7. Supporting Information………..………..………..…………271
B3: On the Toxicity and Transport Mechanisms of Cisplatin in Kidney Tissues in Comparison to a Gold-based Cytotoxic Agent………..285
1. Abstract………..………..……..…..286
2. Introduction……….…..………..……….…….287
3. Results and discussion……….………..……….…..292
3.1. Toxicity evaluation……….….….…..……….…..292
3.1.1. ATP content determination……….…….…..292
3.1.2. Histomorphology……….………..………..294
3.1.3. Expression of kidney-injury molecule 1 (KIM-1), villin, p53 and BAX……….……….298
3.2. Uptake studies……….……….…..301
3.2.1. Metal content determination by ICP-MS……….…301
3.2.2. Effect of temperature on uptake in PCKS……….…….…302
4. Conclusions……….……….……….…..304
5. Experimental methods……….…..………..………….…..307
6. References……….…..………..………..……….……….…..312
B4: Investigation of the Molecular Accumulation Mechanisms of an Au(III) Cyclometallated Compound Compared to Cisplatin in vitro: Are OCT2 and CTR1 involved?...318
1. Abstract………..……….…..……….……..….…..319
2. Introduction……….…..………..………...…..320
3. Results and Discussion………..…………..…..323
3.1. Synthesis and characterization……….……..…….…..………..…323
3.2. Fluorescence……….….…..………..……….…...324
3.3. Antiproliferative effects……….………..….…….325
3.4.1. Competition experiments………..………326
3.4.2. Metal content determination………..………..………….…..331
3.4.3. Passive/active mechanisms………..……….…..338 3.5. Copper accumulation……….……….…….…..……….….…..340 3.6. Fluorescence microscopy………..………..……….…..344 4. Conclusions……….………..…….…….…..346 5. Experimental section………..……….…..……….…..348 6. References……….….………..……….…..……….….…..354
General Discussion and Future Perspectives……….…………..………...356
Samenvatting……….……...367
Acknowledgements……….………...………....377
Curriculum Vitae………..……….….378
Introduction
Metallodrugs are organic compounds that contain one or more metal ions or metal bindings either with a coordinative or covalent bond.1 Although most of the FDA and EMA approved drugs are based on organic molecules, the use of metallodrugs in medicinal inorganic chemistry offers a great toolbox for therapeutic and/or diagnostic purposes, as will be shown in this chapter. After reading this introduction, the reader may understand the reasons for the increased interest in metallodrugs for therapy and imaging as well as may agree to the fact, that the periodic table offers us a great toolbox to perform research in the field of medicinal inorganic chemistry. The role of metallodrugs in therapy and imaging2-5 in general was extensively reviewed before and in the following, we will focus on the work that is related specifically to our research.
1 Metallodrugs for therapy
One of the first therapeutic metallodrug was Salvarsan, an arsenic based antimicrobial agent developed by Paul Ehrlich in 1912 for the treatment of syphillis.6 Its structure has long been assumed to have an As=As double bond, but in 2005, a mass spectral analysis showed that Salvarsan has an As-As single bond.7 A few years later, in 1965, Barnett Rosenberg and Loretta VanCamp accidentally discovered cisplatin, which possesses antitumor activity (Figure 1).8 Nowadays, therapeutic metallodrugs find applications in many diseases, like arthritis (auranofin), cancer (cisplatin), depression (Li2CO3), bacterial infections (Flamazine, thiomersal) and ulcer (Pepto-Bismol) (Figure 1).
Figure 1. Approved metallodrugs for therapy.
Due to their great success, interest in the research and development of more specific and effective metallodrugs was raised. As a consequence, numerous metallodrugs were developed and studied for effects in different diseases, but also for diagnostic purposes. Metallodrugs currently in clinical trials are BBR3464,9 Satraplatin (JM216),10 Picoplatin (AMD-473),11 NAMI-A,12,13 BMOV14 and Fosrenol15 (Figure 2). Au S P O O O O O O O O O Auranofin Pt NH3 Cl NH3 Cl Cisplatin H2N S N O O N N Ag+ -Flamazine S Hg ONa O Thiomersal O O O Bi OH Pepto-Bismol
Figure 2. Metallodrugs for therapy currently in clinical trials.
1.1 Pharmacology of metallodrugs for therapy
The pharmacology of drugs has two main areas: pharmacodynamics (PD) and pharmacokinetics (PK).16 The difference between both is that pharmacodynamics studies the effect of a drug on the biological system, and pharmacokinetics studies the effects of the biological system on the drug (Figure 3).
Figure 3. Pharmacodynamics and pharmacokinetics. Pt H3N Cl NH2(CH2)6 NH3 Pt H2N NH3 NH2 Pt H3N (H2C)6H2N Cl NH3 H3N BBR3464 Pt Cl Cl H3N O O O O Satraplatin 4+ Ru Cl Cl Cl Cl S N NH O -N H H N + NAMI-A V O O O O O O O BMOV La2(CO3)3 Fosrenol Pt Cl Cl H3N Picoplatin
Pharmacodynamics describes the interaction of the drug with its target, which then determines its application, e.g. cisplatin alkylates DNA (target), which leads eventually to apoptosis and is thus used as anticancer agent (application).17 Common targets of therapeutic drugs are, among others, the cellular membranes (penicillin), enzymes (aspirin), transporter proteins (selective serotonin reuptake inhibitors, SSRIs), nucleic acids (cisplatin, doxorubicin), or receptors (agonist or antagonist). In general, there is a direct relation between the concentration of the drug at the target and its effect, which translates into a dose-response relationship.
Pharmacokinetics, which focuses on the Absorption, Distribution, Metabolism and Excretion (ADME), describes the different processes a drug has to undergo until it reaches its target and which metabolites are formed and how those are excreted.16 The dose-response relationship is particularly important in the relation between PK and PD, and between PK and safety, which enables predicting at which dose a drug is effective and safe, and if undesired side-effects and toxic effects are dose-limiting. The window of concentrations that shows sufficient efficacy and acceptable side-effects is also called the therapeutic window.18 The route of administration (orally, intravenous, subcutaneous, etc.) plays a crucial role for the exposure of the body to the drug and its metabolites. Before a new compound reaches market approval, it has to pass several pre- and clinical trials to demonstrate its effectiveness and safe application (in vitro, in vivo studies in animals and humans and PK/PD models) as well as non-inferiority with drugs with the same application.
Concerning metallodrugs for therapy, the pharmacodynamics is not the critical part, since metallodrugs can be successfully designed for specific targets and in pre-clinical experiments, it is tested, whether the drug reaches its target and is effective or not. These studies are mostly performed in vitro, ex vivo or in animal studies in vivo. It is possible to already get useful information about the pharmacokinetics, efficacy and safety of the new drug in these preclinical studies, but the
main parameters for clinical approval need to be studied in clinical phases I-III studies.
1.1.1 Pharmacokinetics (PK) and toxicity of cisplatin
In this section the PK and toxic effects of cisplatin are briefly described. Cisplatin is usually administered to patients via intravenous injection and 90 % of the injected dose is known to bind to plasma proteins such as serum albumin. The drug is distributed to the tissues, in addition to the tumor tissue it distributes especially in the kidneys, liver and prostate, where cisplatin accumulates intracellularly via various transporter proteins and/or by passive diffusion. Beside cisplatin’s great success in the treatment of various kinds of cancer, its treatment is also hampered by severe side-effects like ototoxicity, myelosuppression and nephrotoxicity. Nephrotoxicity is by far the most dominant dose-limiting side-effect and there is strong evidence that the tubular cells are the main targets of cisplatin’s toxic metabolites.19
Activation of cisplatin to its highly reactive and toxic metabolites includes spontaneous intracellular aquation reactions, which involve the substitution of the chloride ligands with water/hydroxide molecules.20,21 Cisplatin is metabolized via a formation of conjugates between glutathione (GSH) and cisplatin through the action of glutathione-S-transferase. The metabolisms can take place in the liver and kidney. These cisplatin-GSH conjugates can be excreted via the kidneys, but can also be metabolized to toxic metabolites.22 Many of the mechanisms of cell uptake and efflux are not yet fully understood at a molecular level.23 Specifically, several drug transporters play an important role in drug accumulation as well as drug efflux, as greatly reviewed by Petzinger et al.24 For cisplatin, the organic cation transporter 2 (OCT2) and copper transporter 1 (CTR1) seem to be involved in the uptake of cisplatin in kidneys, and, therefore, responsible for the observed nephrotoxicity during chemotherapy. Other studies indicate that multi-extrusion proteins (MATEs) and ATP7A/B might be responsible for cisplatin excretion and thus
also play a role in the accumulation in kidney tissue is still largely unknown.
1.2 Radiopharmaceuticals for therapy
Another important area of metallodrugs for therapy is the field of radiopharmaceuticals. Radiopharmaceuticals are molecules that incorporate a radioactive metal, also called radiometals. Radiometals for therapy are beta-emitters, alpha-emitters or Auger electron emitters.25 As shown in Figure 4, the travel path in tissue/cells highly depends on the kind of emission as well as the energy. Auger electrons have a very low energy; therefore they can only travel through one or two cells. Alpha emitters are highly energetic particles that can reach a few cell diameters. Beta particles on the other hand, have a medium energy and travel the longest, up to a hundred of cells.26 Alpha particles, which are helium nuclei, have the main advantage that they, if targeted to a receptor on cancer cells, only destroy cancer cells instead of surrounding healthy tissue, in contrast to beta emitters. The decay scheme of those is shown in Figure 5.
Figure 4. Track length in tissues of alpha particles, beta-particles, and Auger electrons relative to their energy; reproduced with permission from reference.27
Alpha: AZX à A − 4Z − 2Y + 42α
Beta: AZX à AZ + 1Y + 0−1β
Figure 5. Alpha and beta decay scheme.
Radiopharmaceuticals for therapy currently on the market are 177Lu-DOTATOC (neuroendocrine tumor) and 177Lu-PSMA-617 (prostate cancer), 131I-MIBG (thyroid cancer), 153Sm-EDTMP (bone metastases), 89SrCl2 (bone metastases) and 90Y-silicate (synovitis in the knees) as beta emitters (Figure 6), and 223RaCl2 (prostate cancer osteoblastic bone metastases) as the only alpha emitter to date.28
Figure 6. Therapeutic Radiopharmaceuticals.
There is only one alpha emitter currently clinically approved, but the interest in various radiometals for targeted alpha therapy (TAT) is increasing due to the advantages of alpha emitters discussed above.
N N N N O OH O HO O OH O N H O H N O N H O H N O HN O NH2 HN O OH N H O S S HO OH NH OH 177Lu 177Lu-DOTATOC N N N N O OH O HO O OH O N H 177Lu 177Lu-PSMA-617 O HN O NH NH O NH O OH O OH O HO 131I N H NH NH2 131I-MIBG 153Sm N N PO2 PO2 O O PO2 PO2 O O 153Sm-EDTMP
5-From the radiochemistry point of view it is part of the uranium decay chain. Uranium decays via four alpha decays and three beta decays to non-radioactive 209Bi (Figure 7). Current problems in that field of research include the development of an efficient production of 225Ac and purification 29 as well as its recoil energy and radiolysis properties. The development of a stable chelator for 225Ac is challenging due to its multiple daughter atoms, which need to be stably bound as well. Only a few publications have appeared on the chelator design for 225Ac.30,31
Figure 7. Decay chain of 233U via 225Ac to stable 209Bi (adapted with permission from reference32).
1.2.1 Bifunctional chelator
In order to apply these radiometals to specific biological targets, the “free” radiometal ions must be sequestered from aqueous solution using chelators to obviate trans-chelation. Chelators used for this application are typically covalently linked to a biologically active targeting molecule, making an active targeting radiopharmaceutical.
The goal for this approach is to tightly bind a radiometal ion so that when injected into a patient, the targeting molecule can deliver the isotope without any radiometal loss from the radiopharmaceutical. Acyclic and macrocyclic chelators are currently under investigation for this purpose. Acyclic chelators possess faster radiolabeling properties than macrocyclic ligands; however, the stability inertness of acyclic chelators tends to be inferior to macrocycles in vivo.25
1.2.1.1 Biological targets discussed in this work
Clinically relevant targets for diagnostic as well as therapeutic radiopharmaceuticals are prostate specific membrane antigen (PSMA), HER2/neu positive tumors and blood clots.
Prostate cancer is one of the most common cancer types in men. This disease is still difficult to diagnose in an early stage, since the prostate-specific antigen (PSA) screening is not sensitive enough. Other biomarkers are currently investigated, like urinary PCA3 and TMPRSS2-ERG.33 177Lu-PSMA-617 and 68Ga-PSMA-11 are organic molecules targeting PSMA, and are used for therapy or diagnosis of prostate cancer, respectively. PSMA is a 750 amino acid type II transmembrane glycoprotein. It is overexpressed (up to 1000 times higher than normal prostate cells) in virtually all prostate cancers (5-10% of prostate cancers appear not to express the PSMA glycoprotein). The treatment and diagnosis with these PSMA targeting peptides is fairly new. Consequently, no long term toxicity evaluations were performed yet. Nevertheless, these two new radiopharmaceuticals seem to have a huge impact on prostate cancer patients, as they are new therapeutic options in addition to external beam radiotherapy and androgen blockage.34
HER2/neu is a member of the human epidermal growth factor receptor family. This receptor is overexpressed in 30% of all breast cancers35 and most recently also discovered in gastric cancer.36 Trastuzumab is a humanized monoclonal antibody that binds
pathways.37 Due to its high specificity, it can be used as targeting vector for radiopharmaceuticals.
Beside the diagnosis and therapy of different cancer types, the early detection of blood clots by targeted radiopharmaceuticals plays a major role in the successful treatment of thrombosis. The coagulation cascades and fibrolysis process consist of many peptides that might be useful as target for radiopharmaceuticals. Factor Xa seems to be a favorable targeting vector, as it accumulates highly localized at the side of injury.38
1.2.2 Mechanisms of targeting of radiopharmaceuticals for therapy 177Lu-DOTATOC consists of the macrocyclic chelator DOTA that is attached to a peptide that targets neuroendrocine tumors. 177 Lu-PSMA-617 also consists of the macrocyclic chelator DOTA, which is attached to a peptide that targets PSMA, specific for prostate cancer. 131I-MIBG is a noradrenaline analogue and so-called “false” neurotransmitter. Thus, this radiopharmaceutical accumulates in tissues with rich adrenergic innervation, essentially neuroectodermal tissue, including tumors of neuroectodermal origin. 153Sm-EDTMP consists of the acyclic chelator ethylenediamine tetra(methylene phosphonic acid) (EDTMP) and the beta emitter 153Sm. Phosphonate functional groups preferably accumulate in bone, thus it is used in treating osteosarcoma. The Na+/I- symporter uses the sodium gradient, built up by Na+/K+ ATPase, in the cell membrane for the co-transport of iodide and sodium into the cell. 123I and 131I as well as 99m Tc-pertechnetates use this mechanism to accumulate in specific tissues. 2 Metallodrugs for imaging
2.1 Radiopharmaceuticals
In the field of metallodrugs for diagnosis of certain diseases, radiopharmaceuticals featuring metal ions as positron emitters (ß+) or gamma emitters (γ), are standardly used in the clinic in nuclear imaging techniques.39 Contrast agents for magnet resonance imaging (MRI), such as Gd3+, or for computed tomography (CT), such as gold
nanoparticles, will not be discussed here. The nuclear imaging techniques frequently applied, CT, MRI, single photon emission computed tomography (SPECT) and positron emission tomography (PET) have different spatial resolutions and sensitivities (Figure 8). CT and MRI have a good resolution, below 1mm. However, their sensitivity is quite low. In contrast, PET and SPECT have a lower resolution compared to CT and MRI, but their sensitivity is in the nmolar range. Thus, a combination of both groups is standardly used in the clinic (eg. PET/CT or PET/MRI).
Figure 8. Sensitivity and spatial resolution of nuclear imaging techniques. Reproduced with permission from reference 40
2.1.1 PET and SPECT technique
Positron emission tomography (PET) is a functional imaging technique that provides sensitive, quantitative and non-invasive three-dimensional images of a variety of molecular processes and targets. PET tracers emit positrons that annihilate with electrons nearby, generating two gamma (151 MeV) photons that are emitted in 180° from each other. The detection of these emissions coincident in time supplies then information about the localization of the radiation event (Figure 9A). The current workhorse for PET imaging is 18 F-fluorodeoxyglucose (18F-FDG, Figure 10)41, which is an excellent marker for tissue metabolism and widely used to explore cancer metastasis;
Figure 10), 64Cu, 86Y, 89Zr and 44Sc have aroused increasing interest for PET imaging as well.42
Figure 9. PET and SPECT technique (adapted with permission from reference).43 Figure 10. Tracers used for PET imaging.
Single photon emission computed tomography (SPECT) is an older and less resolving imaging technique, which measures – in contrast to PET imaging - the gamma rays emitted by radio tracers
N N N N O OH O HO O OH O N H O H N O N H O H N O HN O NH2 HN O OH N H O S S HO OH NH OH 68Ga 68Ga-DOTATOC NH O NH O OH O OH O HO 68Ga-PSMA-11 O HO HO HO 18F OH [18F]FDG NH O HN O N N O HO O HO O OH HO HO 68Ga
directly (Figure 9B). The most common isotope used in SPECT is still 99mTc, but the radiometals 67Ga, 111In and 177Lu find use in chelator-based radiopharmaceuticals as well.44,45
The workhorse of modern clinical imaging is, with about 80% of world’s use of radioactive isotopes in nuclear medicine, 99mTc, a metastable nuclear technetium isomer that emits readily detectable γ rays (140 keV). Despite its numerous and favourable use in SPECT imaging, 67/68Ga has attracted a lot of attention throughout the years and is considered as valuable alternative to 99mTc.25 Beside its ability to form stable complexes with a variety of multidentate ligands, 68Ga3+ shows suitable properties for PET imaging, for instance a short half-life time with 68 min, decay via positron emission by 89% and a maximum positron energy of 1.899 keV.
Another important metal used in nuclear medicine for SPECT imaging and therapeutic applications, is the post-transition metal Indium.25 Out of the 39 known indium isotopes, the cyclotron-produced isotope 111In is the most stable artificial isotope, which decays via electron capture with a half-life of 67.2 h. Since it emits both γ rays (245 and 172 keV) that can be used for imaging, and Auger electrons that can be used for therapy, it shows desirable properties for a variety of applications in nuclear medicine.
2.1.2 Mechanism of accumulation of radiopharmaceuticals for cancer imaging
The imaging agent 18F-FDG is taken up by tumor cells via the glucose transporters GLUT1 and GLUT4.46 Once intracellular, FDG is phosphorylated to FDG-6-phosphate and is not metabolized further. Importantly it is accumulating more in tumors than in normal surrounding tissues, probably because many tumor cells express more GLUT1 than normal cells.47 However, despite the FDG success, there is urgent need for novel radiotracers, since GLUT1 expression largely varies among tumor types affecting the efficiency of FDG uptake. The SPECT radiotracers 99mTc-sestamibi and 99mTc-tetrofosmin are known
glycoprotein-expressing multidrug-resistant tumor cells extrude both compounds out of the cell whereas P-glycoprotein-negative, drug sensitive tumor cells accumulate the substances. These two drugs are used for imaging of the heart muscle, and are studied for brain tumor imaging as well.49
2.2 Fluorescence imaging
Fluorescence imaging is the visualization of fluorescent dyes or proteins as labels for molecular processes or structures. It enables a wide range of experimental observations including the location and dynamics of gene expression, protein expression and molecular interactions in cells and tissues. Fluorescence results from a process that occurs when certain molecules absorb light. The absorption of light raises their energy level to an excited state. As they decay from this excited state, they emit fluorescent light. Examples of fluorescent probes for biological imaging in in vitro or in vivo experiments50 are Cy5.5 or 4’,6-diamidino-2-phenylindole (DAPI) (Figure 11). Metal containing fluorescent probes are for example ruthenium incorporating
bis(2,2’-bipyridine)-(5-aminophenanthroline)rutheniumbis(hexafluorophosphate) (here: Ru-1) or lanthanide probes like 1,4,7,10-tetraazacyclodecane-1,4,7-triacetate (DO3A) with Eu3+ or Tb3+ (Figure 11).
Figure 11. Examples of fluorescent metal-complexes and ligands used for fluorescent probes. 3 References
(1) Gasser, G.; Ott, I.; Metzler-Nolte, N. J. Med. Chem. 2011, 54, 3-25. (2) Mjos, K. D.; Orvig, C. Chem. Rev. 2014, 114, 4540-4563.
(3) Medici, S.; Peana, M.; Nurchi, V. M.; Lachowicz, J. I.; Crisponi, G.; Zoroddu, M. A. Coord. Chem. Rev. 2015, 284, 329-350.
(4) Abrams, M.; Murrer, B. Science 1993, 261, 725-730.
(5) Barry, N. P. E.; Sadler, P. J. Chem. Commun. 2013, 49, 5106-5131.
(6) Ehrlich, P.; Bertheim, A. Berichte der deutschen chemischen Gesellschaft 1912, 45, 756-766.
(7) Lloyd, N. C.; Morgan, H. W.; Nicholson, B. K.; Ronimus, R. S. Angewandte Chemie Internat. Ed. 2005, 44, 941-944.
(8) Rosenberg, B.; Vancamp, L.; Krigas, T. Nature 1965, 205, 698-699. (9) Jodrell, D. I.; Evans, T. R. J.; Steward, W.; Cameron, D.; Prendiville, J.; Aschele, C.; Noberasco, C.; Lind, M.; Carmichael, J.; Dobbs, N.; Camboni, G.; Gatti, B.; De Braud, F. Eur. J. Canc., 40, 1872-1877.
(10) Bhargava, A.; Vaishampayan, U. N. Expert Opin. Investig. Drugs 2009, 18, 10.1517/13543780903362437.
(11) Hamilton, G.; Olszewski, U. Expert Opin. Drug Metab. Toxicol. 2013, 9,
N N O OH Cy5.5 N H HN H2N NH2 NH DAPI N N H2N Ru N N N N [PF6]2 Ru-1 NH N N N OH O OH O HO O DO3A
(12) Leijen, S.; Burgers, S. A.; Baas, P.; Pluim, D.; Tibben, M.; van Werkhoven, E.; Alessio, E.; Sava, G.; Beijnen, J. H.; Schellens, J. H. M. Investig. New Drugs 2015, 33, 201-214.
(13) Trondl, R.; Heffeter, P.; Kowol, C. R.; Jakupec, M. A.; Berger, W.; Keppler, B. K. Chem. Sci. 2014, 5, 2925-2932.
(14) Almac, E.; Bezemer, R.; Kandil, A.; Aksu, U.; Milstein, D. M.; Bakker, J.; Demirci-Tansel, C.; Ince, C. Intens. Care Med. Exper. 2014, 2, 3.
(15) D'Haese, P. C.; Spasovski, G. B.; Sikole, A.; Hutchison, A.; Freemont, T. J.; Sulkova, S.; Swanepoel, C.; Pejanovic, S.; Djukanovic, L.; Balducci, A.; Coen, G.; Sulowicz, W.; Ferreira, A.; Torres, A.; Curic, S.; Popovic, M.; Dimkovic, N.; De Broe, M. E. Kidney Internat., 63, S73-S78.
(16) Tripathi, K. D. Essentials of Medical Pharmacology; Jaypee Brothers Medical Publishers, 2013; Vol. 7.
(17) Hurley, L. H. Nat. Rev. Cancer 2002, 2, 188-200.
(18) Blix, H. S.; Viktil, K. K.; Moger, T. A.; Reikvam, A. Pharmacy Practice 2010, 8, 50-55.
(19) Miller, R. P.; Tadagavadi, R. K.; Ramesh, G.; Reeves, W. B. Toxins 2010, 2, 2490-2518.
(20) Kelland, L. 2007, 7, 573.
(21) Hall, M. D.; Okabe, M.; Shen, D.-W.; Liang, X.-J.; Gottesman, M. M. Annual Rev. Pharmacol. Toxicol. 2008, 48, 495-535.
(22) Andersson, A.; Fagerberg, J.; Lewensohn, R.; Ehrsson, H. J. Pharm. Sci., 85, 824-827.
(23) Spreckelmeyer, S.; Orvig, C.; Casini, A. Molecules 2014, 19, 15584-15610. (24) Petzinger, E.; Geyer, J. Naunyn-Schmiedeberg's Archives of Pharmacology 2006, 372, 465-475.
(25) Price, E. W.; Orvig, C. Chem. Soc. Rev. 2014, 43, 260-290. (26) Kassis, A. I. Sem. Nucl. Med. 2008, 38, 358-366.
(27) Pouget, J.-P.; Navarro-Teulon, I.; Bardies, M.; Chouin, N.; Cartron, G.; Pelegrin, A.; Azria, D. Nat. Rev. Clin. Oncol. 2011, 8, 720-734.
(28) Liepe, K.; Shinto, A. Therapeut. Adv. Med. Oncol. 2016, 8, 294-304. (29) Robertson, A. K. H.; Ramogida, C. F.; Rodriguez-Rodriguez, C.; Blinder, S.; Kunz, P.; Sossi, V.; Schaffer, P. Phys. Med. Biol. 2017, 62, 4406-4420.
(30) Comba, P.; Jermilova, U.; Orvig, C.; Patrick, B. O.; Ramogida, C. F.; Ruck, K.; Schneider, C.; Starke, M. Chemistry 2017.
(31) Wilson, J. J.; Ferrier, M.; Radchenko, V.; Maassen, J. R.; Engle, J. W.; Batista, E. R.; Martin, R. L.; Nortier, F. M.; Fassbender, M. E.; John, K. D.; Birnbaum, E. R. Nucl. Med. Biol., 42, 428-438.
(32) Wilson, J. J.; Ferrier, M.; Radchenko, V.; Maassen, J. R.; Engle, J. W.; Batista, E. R.; Martin, R. L.; Nortier, F. M.; Fassbender, M. E.; John, K. D.; Birnbaum, E. R. Nucl. Med. Biol. 2015, 42, 428-438.
(33) Cuzick, J.; Thorat, M. A.; Andriole, G.; Brawley, O. W.; Brown, P. H.; Culig, Z.; Eeles, R. A.; Ford, L. G.; Hamdy, F. C.; Holmberg, L.; Ilic, D.; Key, T. J.; La
Vecchia, C.; Lilja, H.; Marberger, M.; Meyskens, F. L.; Minasian, L. M.; Parker, C.; Parnes, H. L.; Perner, S.; Rittenhouse, H.; Schalken, J.; Schmid, H.-P.; Schmitz-Dräger, B. J.; Schröder, F. H.; Stenzl, A.; Tombal, B.; Wilt, T. J.; Wolk, A. The Lancet. Oncol. 2014, 15, e484-e492.
(34) Emmett, L.; Willowson, K.; Violet, J.; Shin, J.; Blanksby, A.; Lee, J. J. Med. Rad. Sci. 2017, 64, 52-60.
(35) Mitri, Z.; Constantine, T.; O'Regan, R. Chemotherap. Res. Pract. 2012, 2012, 7.
(36) Sanford, M. Drugs 2013, 73, 1605-1615. (37) Nat Rev Clin Oncol 2015, 12, 126-126.
(38) Pryzdial, E. L. G.; Meixner, S. C.; Talbot, K.; Eltringham-Smith, L. J.; Baylis, J. R.; Lee, F. M. H.; Kastrup, C. J.; Sheffield, W. P. J. Thomb. Haem. 2016, 14, 1844-1854.
(39) Anderson, C. J.; Welch, M. J. Chem. Rev. 1999, 99, 2219-2234. (40)
https://lh3.googleusercontent.com/- lPj9XnIej08/T9TSwuKcxJI/AAAAAAAAEtM/Ldh9-f0vouk/w402-h318-n/F7scaleSensImage.jpg.
(41) Almuhaideb, A.; Papathanasiou, N.; Bomanji, J. Annals of Saudi Med. 2011, 31, 3-13.
(42) Conti, M.; Eriksson, L. EJNMMI Physics 2016, 3, 8.
(43) Xu, C.; Mu, L.; Roes, I.; Miranda-Nieves, D.; Nahrendorf, M.; Ankrum, J. A.; Zhao, W.; Karp, J. M. Nanotechnol. 2011, 22, 494001-494001.
(44) Uribe, C. F.; Esquinas, P. L.; Tanguay, J.; Gonzalez, M.; Gaudin, E.; Beauregard, J.-M.; Celler, A. EJNMMI Physics 2017, 4, 2.
(45) Reitblat, T.; Ben-Horin, C. L.; Reitblat, A. Annals of the Rheumatic Diseases 2003, 62, 257-260.
(46) Kaiser, F.; Pelzer, T.; Kreissl, M.; Higuchi, T.; Arias-Loza, P. J. Nucl. Med. 2013, 54, 24.
(47) Carvalho, K. C.; Cunha, I. W.; Rocha, R. M.; Ayala, F. R.; Cajaíba, M. M.; Begnami, M. D.; Vilela, R. S.; Paiva, G. R.; Andrade, R. G.; Soares, F. A. Clinics 2011, 66, 965-972.
(48) Alexiou, G. A.; Xourgia, X.; Vartholomatos, E.; Tsiouris, S.; Kalef-Ezra, J. A.; Fotopoulos, A. D.; Kyritsis, A. P. Internat. J. Mol. Imaging 2014, 2014, 5.
(49) Kelly, J. D.; Forster, A. M.; Higley, B.; Archer, C. M.; Booker, F. S.; Canning, L. R.; Chiu, K. W.; Edwards, B.; Gill, H. K.; McPartlin, M.; Nagle, K. R.; Latham, I. A.; Pickett, R. D.; Storey, A. E.; Webbon, P. M. J. Nucl. Med. 1993, 34, 222-227. (50) Rao, J.; Dragulescu-Andrasi, A.; Yao, H. Current Opin. Biiotechn. 2007, 18, 17-25.
The aim of the studies described in this thesis is the design of new metallodrugs and radiopharmaceuticals for diagnosis and cancer therapy as well as elucidating the mechanism of cellular uptake and excretion and the anti-cancer activity as well as organ toxicity of some new Au containing metallodrugs in comparison to cisplatin. Specifically,
Part A deals with the synthesis and characterization of acyclic chelators and their coupling to peptides as targeting moieties for therapy or diagnosis and
Part B deals with the investigation of mechanism of uptake and excretion and the toxicity of several Au(III) compounds and cisplatin. Part A was carried out at the University of British Columbia, Vancouver, BC, in Canada under the supervision of Prof. Chris Orvig. The Orvig Group investigated a novel family of acyclic chelators which is based on the picolinic acid scaffold, the “pa” family of chelators (Figure 1).
Figure 1. The „pa“-family – Acyclic chelators of the Orvig group.
The first identified member of this group is called H2dedpa and a promising 67/68Ga chelator. The hexadentate acyclic chelator binds gallium isotopes quantitatively (RCY > 99%) within 30 min and under
NH HN N N OH O HO O N N N OH O N HO O N OH OH HO O O O N N N OH O N HO O N OH HO O O NCS H2CHXdedpa H5decapa p-SCN-Bn-H4neunpa N N N N OH O HO O H4CHXoctapa O HO O OH N N N N OH O HO O H4octapa O HO O OH NH HN N N OH O HO O H2dedpa N N N N OH O HO O H2azapa N N N N N N N N N N OH O HO O H6phospa P P O O OH OH HO OH
mild conditions with high specific activities. Unfortunately, the chelator-64Cu complex showed decreased serum stability in vitro within 24h, which makes H2dedpa unsuitable for in vivo applications. Both, radiolabeling kinetics and kinetic inertness could be significantly improved by modifying the H2dedpa scaffold with an 1R-2R-(-)-trans-cyclohexane diamine backbone. This pre-organization of donor atoms created a chiral dedpa2- analogue called H2CHXdedpa which could be successfully used for in vivo imaging of gallium isotopes.
The promising results for the chelator H2dedpa led to the investigation of larger frameworks based on the H2dedpa scaffold and generated several chelators for larger radiometal ions like 111In, 90Y, 177Lu, 89Zr, 225Ac namely, H4octapa, H2azapa, H6phospha and H5decapa.
H4octapa is an octadentate acyclic chelator (N4O4) derived from H2dedpa with two additional carboxylic arms. It is capable of quantitatively radiolabeling 111In at ambient temperature in 10 min with high specific activities (97.5% RCY) and shows comparable results for 177Lu as well. Indium complexes with H
4octapa show an extraordinary high in vivo stability over 24h and compared with DOTA an improved clearance and stability, which is demonstrated by a lower uptake in kidney, liver and spleen. The formation of a single isomer and the lack of fluxional behavior as well as the higher pM value (pM = -log [Mn+]free) (logKML = 26.8) makes H4octapa a very strong and promising alternative to the current “gold standard” DOTA.
Two other interesting derivatives are H2azapa and H6phospha. H2azapa is an analogue of H2dedpa containing two triazole rings which shows quantitative radiolabeling with 64Cu but low in vivo stability and H6phospha is a methylene-phosphonate derivative of H4octapa, which is the first reported chelator that shows any binding with 89Zr.
The expansion of this unique scaffold led to the discovery of H5decapa (N5O5), a hexadentate analogue of H4octapa, which shows promising radiolabeling results for 177Lu due to its higher denticity. To enable bioconjugation, an additional functionalization was introduced into
H5decapa resulting in the novel ligand H4neunpa, which will be the subject of this work.
Chapter A1 deals with the synthesis and characterization of an acyclic chelator for 111In radiolabeling. First, the chelator H4neunpa was synthesized and characterized with analytical methods and preliminary radiolabeling experiments in vitro showed promising results, which then led to in vivo experiments in mice for SPECT imaging and biodistribution studies.
In A2, the same chelator was used to link to various therapeutic molecules such as trastuzumab or a PSMA targeting peptide, and a metallacage was linked to H4neunpa in order to synthesize a fluorescent probe for in vitro imaging. Another aim was to change the radiometal from 111In to 225Ac, 213Bi or 177Lu in order to develop radiopharmaceuticals for not only SPECT imaging, but also targeted alpha therapy or therapy with beta emitters, respectively.
The synthesis of a chelator for 89Zr radiolabeling for therapy was investigated in chapter A3, since up to now, there is no stable chelator for the positron emitter 89Zr approved for clinical use. 89Zr does have promising properties. Its physical half-life of 3.2 days matches the biological half-life of antibodies. Consequently, it could be used for the development of a novel bifunctional chelator The well-known chelator EDTA was used as a lead structure which was modified with hydroxamic acid arms.
The use of radiopharmaceuticals for cancer diagnosis and treatment is well established, whereas the field of thrombosis is still lacking non-invasive methods to detect blood clots at low concentrations. Blood coagulation factor FXa seems to be a promising targeting vector, since it highly accumulates at the side of blood clots. Thus, in A4 we developed a bifunctional chelator for 68Ga radiolabeling for the diagnosis of low concentrations of blood clots.
under the supervision of Prof. Angela Casini (now at the University of Cardiff) and Prof. Geny Groothuis.
In chapter B1, a review is presented on the transport mechanisms of cisplatin compared to other metallodrugs, as this topic is fundamental for understanding the mechanism of action of metallodrugs used in the clinic and in preclinical studies. There is still a lack of knowledge about the transporter proteins involved in the uptake and efflux of metallodrugs on a molecular level, both in cancer cells and in the organs. Additionally, many experiments were performed in cells in vitro and rare studies are published using ex vivo or in vivo models.
Chapter B2 introduces a new class of C^N Au(III) cyclometallated compounds synthesized by B. Bertrand. The synthesis and characterization of these compounds is described as well as their biological evaluation via IC50 determination on several cancer cell lines and PARP-1-inhibition studies.
The most cytotoxic compound from chapter B2 was then chosen to be further evaluated in chapter B3 in an ex vivo model of precision cut tissue slices in an attempt to characterize the uptake and efflux mechanisms as well as the toxicity, compared to cisplatin. Moreover, ICP-MS was used to detect the metal content in the cells and tissues and histomorphology was applied for an additional toxicity evaluation. The expression level of KIM-1, villin, p53 and Bax as markers for toxicity was taken into account as well.
Chapter B4 deals with another Au(III) compound from the family evaluated in chapter B2, that contains a fluorescent coumarin moiety. This compound was evaluated in vitro for specific uptake and efflux mechanisms (OCT2, MATE, CTR1) as well as by fluorescent microscopy for determination of subcellular distribution.
In the final chapter, the results are summarized and discussed and future perspectives for the development of metallodrugs for therapy and imaging are presented.
Chapter A1
p-NO
2-Bn-H
4neunpa
and H
4neunpa-Trastuzumab:
Bifunctional Chelator for
Radiometalpharmaceuti-cals and
111In Immuno-SPECT Imaging
Sarah Spreckelmeyer,a,b Caterina F. Ramogida,c Julie Rousseau,d Karen Arane,c Ivica Bratanovic,c Nadine Colpo,d Una Jermilova,d Gemma M. Dias,d Iulia Dude,d Maria de Guadalupe Jaraquemada-Peláez,a François Bénard,d Paul Schaffer,d Chris Orviga
a Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British
Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T 1Z1, Canada
b Dept. Pharmacokinetics, Toxicology and Targeting, Research Institute of Pharmacy,
University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands
c Life Sciences Division, TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia,
V6T 2A3, Canada
d BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, V5Z 1L3,
Can-ada
1 Abstract
Potentially nonadentate (N5O4) bifunctional chelator p-SCN-Bn-H4neunpa and its immunoconjugate p-SCN-Bn-H4neunpa-Trastuzumab for 111In radiolabeling are synthesized. The ability of p-SCN-Bn-H4neunpa and H4neunpa-Trastuzumab to radiolabel quantitatively 111InCl3 at ambient temperature within 15 min or 30 min, respectively, is presented. Ther-modynamic stability determination with In3+, Bi3+ and La3+ resulted in high pM values. In vitro human serum stability assays have demon-strated both 111In complexes to have high stability over 5 days. Mouse biodistribution of [111In][In(p-NO2-Bn-neunpa)]-, compared to that of [111In][In(p-NH
2-Bn-CHX-A"-DTPA)]2-, at 1 h, 4 h and 24 h shows fast clearance of both complexes from the mice within 24 h. In a second mouse biodistribution study, the immunoconjugates 111 In-neunpa-Trastuzumab and 111In-CHX-A”-DTPA-Trastuzumab demonstrate a simi-lar distribution profile, but with slightly lower tumor uptake of 111 In-neunpa-Trastuzumab compared to 111In-CHX-A”-DTPA-Trastuzumab. These results were also confirmed by Immuno-SPECT imaging in vivo. These initial investigations reveal the acyclic bifunctional chelator p-SCN-Bn-H4neunpa to be a promising chelator for 111In (and other radio-metals) with high in vitro stability, and also show H4neunpa-Trastuzumab to be an excellent 111In chelator, with promising biodistri-bution in mice.
2 Introduction
Early detection and specific therapy are the key factors for the successful treatment of cancer. 111In (t 1/2 = 2.8 days) and/or 177Lu (t 1/2 = 6.6 days) are important radioisotopes in nuclear medicine that match either the requirements for single photon emission tomography (SPECT) and performing dosimetry, or for therapeutic purposes, respec-tively. 111In being a cyclotron–produced radiometal (via the 111Cd(p,n)111In reaction) emits gamma rays (245 and 171 keV) and Au-ger electrons. 177Lu being a reactor-produced radiometal (176Lu(n,gamma)177Lu) emits primarily beta particles (490 keV) that can be used for therapy.1
A common method to incorporate metallic radioisotopes (i.e. radiometals) into radiopharmaceuticals is via chelation of the desired radioisotope using a bifunctional chelator (BFC). As implied by the name, BFCs possess two properties – they must chelate the radiometal of interest in a tight and stable metal-ligand complex, and the BFC must incorporate a point of attachment for conjugation to a targeting vector (e.g. biomolecule of interest in disease progression such as a peptide or antibody). Both macrocyclic and acyclic chelators are used in the clinic, and are also of interest in the field of medicinal inorganic chem-istry research. The pros and cons of cyclic vs acyclic chelators are widely known and beyond debate.2 Relevant to 111In and 177Lu, macro-cyclic DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) is the gold-standard chelator, while acyclic chelator DTPA (diethylene-triamine pentaacetic acid) and chiral analogue CHX-A”-DTPA are ubiq-uitous in 111In radiopharmaceutical development (Figure 1). Recent studies developed bifunctional somatostatin analogues of DOTA with increased stability in vivo.3 As an acyclic gold-standard, the commer-cially available radiopharmaceutical OctreoScan (111In-DTPA octeotride) reached approval in 1994 (Figure 1). Since the success of OctreoScan, several more bifunctional acyclic 111In chelators that contain different biomolecules have been developed, hoping to overcome the limitations of OctreoScan. These include an increased physiological uptake which
restricts the detection of small lesions, prolonged imaging protocol and relatively high radiation dose to the patients, as well as low image qual-ity.4
Our group has developed several promising acyclic chelators for 111In and/or 177Lu, based on picolinic acid binding motifs, which we have since dubbed the “pa”-family of chelators.5-8 Of note, octadentate H4octapa (N4O4) and its bifunctional analogue p-SCN-Bn-H4octapa showed exceptional complexation properties (quantitative 111In or 177Lu radiolabeling in 10-30 minutes at ambient temperature) and favorable
in vivo stability of resulting complexes.9,10 Furthermore, chiral ligands
H2CHXdedpa (N4O2) and H4CHXoctapa (N4O4) showed promising 68Ga and 111In radiolabeling properties, respectively, and subsequently im-pressive stability in human serum.8
Our group continues to design ligands that may incorporate large metal ions (such as radioactive actinides/lanthanides for imag-ing/therapy), which possess ideal properties for radiopharmaceutical incorporation, e.g. fast, mild, and quantitative complexation of radio-metals at low ligand concentrations; formation of resultant thermody-namically stable and kinetically inert metal-complexes; and a conven-ient point of attachment to targeting vectors. Herein, we report the syn-thesis and characterization of a novel nonadentate (CN = 9) acyclic chelator H4neunpa (N5O4, referred to herein as either p-NO2-Bn-H4neunpa or p-NO2-Bn-H4neunpa) and bifunctional analogue p-SCN-Bn-p-NO2-Bn-H4neunpa that was designed as a bifunctional analogue of H5decapa (N5O5), re-ported by our group in 2012.5 The carboxylic acid group on the middle nitrogen atom has been replaced by p-nitrobenzene-ethylene to keep its symmetry, and act as the bifunctional arm to attach the ligand to a bi-omolecule through a thiourea bond (Figure 1). We hypothesized that the extended diethylenetriamine backbone and nine coordinating at-oms of H4neunpa may favorably form complexes with large metal ions such as In3+ (92 pm, CN = 8)11, Lu3+ (103 pm, CN = 9), or Bi3+ (117 pm, CN = 8). Radiolabeling of 111In and 177Lu to H4neunpa was assessed and compared to gold-standards DOTA and CHX-A”-DTPA, and an in vivo
neunpa complexes were also determined. Moreover, coupling of the HER2/neu targeting monoclonal antibody (mAb) Trastuzumab was per-formed via the reaction between the antibody’s primary-amine(s) with the isothiocyanate functional group of p-SCN-Bn-H4neunpa. The bio-conjugate was labeled with 111In, and in vivo biodistribution and SPECT/CT imaging studies were conducted and compared directly to a 111In-CHX-A”-DTPA-Trastuzumab conjugate.
Figure 1. Structures of cyclic (DOTA) and acyclic (OctreoScan, CHX-A"-DTPA)
com-mercial chelators, and acyclic “pa”-ligands H2CHXdedpa, H4CHXoctapa, H4octapa,
H5decapa, and novel nonadentate chelator p-SCN-Bn-H4neunpa discussed in this work.
3 Results and Discussion
3.1 Synthesis and characterization of the ligand
The synthesis of the previously reported analogue H5decapa used N-benzyl protection, N-alkylation with an alkyl halide, benzyl deprotection via hydrogenation, a second alkyl halide N-alkylation, and finally deprotection in refluxing HCl (6M).10 The N-benzyl protection was found to be the yield-limiting step because the deprotection always resulted in partly eliminating the picolinic acid moieties. The use of O-nitrobenzenesulfonyl (nosyl) was found to give better cumulative yields compared to N-benzyl protection. Based on that, the bifunctional
ana-NH HN N N OH O HO O N N N OH O N HO O N OH OH HO O O O N N N OH O N HO O N OH HO O O NCS H2CHXdedpa H5decapa p-SCN-Bn-H4neunpa N N N N OH O HO O H4CHXoctapa O HO O OH N N N N OH O HO O H4octapa O HO O OH N N N N DOTA N N N NH HO O O O OH O HO O OH OctreoScan somatostatin O OH O HO O OH O HO N N N OH HO O O O OH O HO O HO p-SCN-Bn-CHX-A''-DTPA NCS
logue H4neunpa, was synthesized with a general reaction scheme that follows N-nosyl-protection, bifunctionalization on the middle nitrogen atom via N-alkylation, N-alkylation with picolinic acid, nosyl-deprotection with thiophenol, a second alkyl halide N-alkylation and ester-deprotection with LiOH to yield p-NO2-Bn-H4neunpa 6 (Scheme 1). The isothiocyanate (NCS) analogue for mAb conjugation, p-SCN-Bn-H4neunpa 9, was synthesized from the intermediate 5 followed by nitro-reduction, ester-deprotection with LiOH and isothiocyanate formation with thiophosgene (Scheme 1).
Starting from the diethylenetriamine backbone, the two primary amines were protected with the 2-nitrobenzenesulfonyl groups to yield compound 1. Compound 1 is highly polar due to the two nosyl groups, thus a highly polar solvent like methanol is needed to separate it from the column. The second step is N-alkylation with 4-(2-bromoethyl)nitrobenzene. In order to maintain symmetry of the ligand, the ideal spot for bifunctionalization is the middle nitrogen. After that,
N-alkylation with methyl-6-bromomethyl picolinate5 was performed to
yield compound 3. The most challenging step was the nosyl-deprotection, constantly resulting in low yields of compound 4. The deprotected product is unfortunately highly polar and likely adsorbs on the surface of potassium carbonate, as seen by the red color of the salt. It was not possible to remove the large fractions of the deprotect-ed product completely from the salt, which explains the low yield re-ported in the Experimental Section. Subsequently, alkyl halide N-alkylation was performed to yield product 5 with 71 % yield. p-NO2-Bn-H4neunpa 6 was synthesized in a final step of ester deprotection with LiOH. This compound was further used for radiolabeling experiments as well as potentiometric stability titrations. The 1H NMR spectrum of the final product is shown in Figure 2.
p-SCN-Bn-H4neunpa 9 was synthesized starting from the
inter-mediate 5 of the previous reaction route. Reduction of the nitro group with palladium on carbon yielded the amine-functionalized product 7. The hydrolysis of the two tert-butyl esters and two methyl esters was
43 10 eq. of lithium hydroxide to the reaction mixture at room temperature to yield the product, with a 50 % yield. The final step is the synthesis of the isothiocyanate-functionalized product 9. This was achieved by the reaction of excess thiophosgene with the aromatic primary amine to yield the final product with a 59 % yield. Overall, the synthesis of p-SCN-Bn-H4neunpa from diethylenetriamine has a cumulative yield of 2.3 %, comparable to the overall synthesis yield of H5decapa (2.5 %).
Scheme 1. Synthetic scheme for p-SCN-Bn-H4neunpa and p-NO2-Bn-H4neunpa.
NH2 N H NH2 SO2Cl NO2 NO2 Br SH Na2CO3, THF K2CO3, DMF Na2CO3, DMF N O O Br K2CO3, THF Na2CO3, CH3CN O O Br Pd/C 10% glacial CH3COOH LiOH THF/H2O 3:1 SCCl2 H2O, DCM N N N OH O HO O N N N N N O O O O N N O O O O NO2 NH N HN N N O O O O NO2 N N N N N O O O O NO2 O2 S OS2 NO2 O2N N H N NH NO2 O2 S O2 S NO2 O2N N H NH NH O2 S O2 S NO2 O2N N N N OH O HO O N N HO OH O O NO2 LiOH THF/H2O 3:1 N N N O O O O N N O O O O NH2 N N N OH O HO O N N HO OH O O NH2 6 p-NO2-Bn-H4neunpa 1 2 3 4 5 7 8
3.2 Synthesis and characterization of non-radioactive metal com-plexes
3.2.1 NMR
Three complexation experiments were performed with La3+, In3+ and Bi3+. 1H NMR spectra of the p-NO
2-Bn-H4neunpa ligand precursor, and corresponding La and In complexes can be found in Figure 2. The [La(p-NO2-Bn-neunpa)]- complex shows 1H NMR upfield shifts of the alkyl-region; this effect has been previously observed in our group.13 The aromatic region is more resolved and shows a splitting of the peaks. Integration of all peaks gives the same number of protons com-pared to the uncomplexed ligand. Furthermore, the HSQC spectra of this complex (Figure S2) shows the same number of carbons compared to the bare ligand, suggesting that there is only one isomer in solution. In contrast, the 1H NMR spectrum of [In(p-NO2-Bn-neunpa)]- shows more splitting in the aromatic and alkyl regions. The aromatic peaks are sharp and well resolved and integrating the peaks suggests one major static isomer. In addition, the COSY spectrum of this complex shows clear coupling of several peaks in the complex alkyl region (Fig-ure S12), leading to the assumption there are fluxional isomers in solu-tion. Comparing these results to those with [In(decapa)]2-, which gave a complex 1H NMR spectrum with multiple isomers presumably due to several unbound carboxylates10, we can see an improvement in terms of isomerization by replacing one carboxylate group with the function-alization arm on the middle nitrogen atom of the diethylenetriamine backbone. Due to insolubility of the Bi complex, the 1H NMR spectrum cannot be used for proper assignments (Figure S1).
Figure 2. 1H NMR spectra of A: p-NO
2-Bn-H4neunpa (400 MHz, CDCl3, 25 °C); B:
[La(p-NO2-Bn-neunpa)]- (400 MHz, CDCl3, 25 °C); C: [In(p-NO2-Bn-neunpa)]- (400 MHz,
DMSO-d6, 25 °C).
3.2.2 IR
Due to the insolubility of [Bi(p-NO2-Bn-neunpa)]-, an IR experi-ment on the solid was performed (Figure 3). Shifts of various peaks of the ligand itself compared to the Bi complex can be observed. The OH stretch at 2500 cm-1 disappeared after complexation, suggesting that the carboxylic acids are bound to the metal ion; the carboxyl stretch at 1700 cm-1 disappeared as well, supporting this assumption. The two stretches of the nitro functional group (1500 cm-1 and 1400 cm-1) stayed the same. The stretch at 1200 cm-1 in the ligand spectra can be assigned as a C-N stretch that shifts to lower energies (1000 cm-1) when bound to the metal ion.
3. 3 C D 3O D 3. 3 C D 3O D A: ligand B: La complex 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 f1 (ppm) 2. 5 D M S O -d 6 C: In complex
Figure 3. IR spectra of p-NO2-Bn-H4neunpa and [Bi(p-NO2-Bn-neunpa)]-.
3.2.3 Thermodynamic Stability
The extended diethylenetriamine backbone, along with the non-adentate N5O4 binding motif of H4neunpa, were specifically designed to accommodate binding of larger metal ions. As such, the protonation constants of H4neunpa as well as the stability constants of the respec-tive La3+, Bi3+ and In3+ complexes were determined at 25 ºC in 0.16 M NaCl aqueous solution. The stepwise protonation constants (log K) obtained are presented in Table 1 together with protonation and stabil-ity constants reported for the related ligands H5decapa, H4octapa, DTPA and CHX-A”-DTPA. A straightforward comparison of the ability of different ligands to coordinate a specific metal ion (rather than the thermodynamic stability constants alone) is the conditional stability constant or pM value. pM is defined as (-log [Mn+]free) and is calculated at specific conditions ( [Mn+] = 1 µM, [Lx-] = 10 µM, pH 7.4 and 25 ºC), taking into consideration both metal-ligand association and ligand ba-sicity. The protonation constants of the new synthesized ligand H4neunpa were determined by potentiometric titrations at pH 1.8-11.5 and by combined potentiometric-spectrophotometric titrations16,17 over the pH range 2.5-11.5. 800 1200 1600 2000 2400 2800 3200 3600 4000 50 60 70 80 90 100 cm-1 Tr an sm it ta n ce [ % ] p-NO2-Bn-H4neunpa [Bi(p-NO2-Bn-neunpa)]
