Synthesis, Radiolabeling, and Characterization of Plasma Protein

Sep 12, 2017 - Synthesis, Radiolabeling, and Characterization of Plasma Protein-Binding Ligands: Potential Tools for Modulation of the Pharmacokinetic...
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Synthesis, Radiolabeling, and Characterization of Plasma ProteinBinding Ligands: Potential Tools for Modulation of the Pharmacokinetic Properties of (Radio)Pharmaceuticals Cristina Müller,*,†,‡ Renáta Farkas,† Francesca Borgna,§ Raffaella M. Schmid,† Martina Benešová,†,‡ and Roger Schibli*,†,‡ †

Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institut, 5232 Villigen-PSI, Switzerland Department of Chemistry and Applied Biosciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland § Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padova, Italy ‡

S Supporting Information *

ABSTRACT: The development of (radio)pharmaceuticals with favorable pharmacokinetic profiles is crucial for allowing the optimization of the imaging or therapeutic potential and the minimization of undesired side effects. The aim of this study was, therefore, to evaluate and compare three different plasma protein binders (PPB-01, PPB-02, and PPB-03) that are potentially useful in combination with (radio)pharmaceuticals to enhance their half-life in the blood. The entities were functionalized with a 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator via a L-lysine and β-alanine linker moiety using solid-phase peptide chemistry and labeled with 177Lu (T1/2 = 6.65 days), a clinically established radiometal. The binding capacities of these radioligands and 177Lu−DOTA were evaluated using human plasma and solutions of human serum albumin (HSA), human α1-acid glycoprotein (α1-AGP), and human transthyretin (hTTR) by applying an ultrafiltration assay. 177Lu−DOTA−PPB-01 and 177Lu−DOTA−PPB-02 bound to a high and moderate extent to human plasma proteins (>90% and ∼70%, respectively), whereas the binding to hTTR was considered negligible (90%) with a high fraction bound to hTTR (∼50%). Plasma protein binding of the 177Lu−DOTA complex, which was used as a control, was not observed (80%) from HSA by ibuprofen, specific for Sudlow’s binding site II and coherent with the aromatic structures, and >80% by their respective binding entities. 177Lu−DOTA−PPB-03 was displaced from hTTR by the site-marker L-thyroxine (>60%) and by its binding entity PPB-03* (>80%). All three radioligands were investigated with regard to the in vivo blood clearance in normal mice. 177Lu−DOTA−PPB-01 showed the slowest blood clearance (T1/2,β: >15 h) followed by 177Lu−DOTA−PPB-03 (T1/2,β: ∼2.33 h) and 177Lu−DOTA−PPB-02 (T1/2,β: ∼1.14 h), which was excreted relatively fast. Our results confirmed the high affinity of the 4-(4-iodophenyl)-butyric acid entity (PPB-01) to plasma proteins, while replacement of the halogen by an ethynyl entity (PPB-02) reduced the plasma protein binding significantly. An attractive approach is the application of the transthyretin binder (PPB-03), which shows high affinity to hTTR. Future studies in our laboratory will be focused on the application of these binding entities in combination with clinically relevant targeting agents for diagnostic and therapeutic purposes in nuclear medicine.



INTRODUCTION The development and selection of drug candidates with favorable pharmacokinetic and pharmacodynamic profiles is crucial for successful drug discovery because it allows the optimization of the biological activity and the minimization of undesired side effects.1 Drug candidates that are cleared too quickly from blood circulation require large doses or repeated administration, which may lead to unwanted toxicity to healthy organs and tissue.2 Increasing the half-life of therapeutics in the blood has, therefore, been regarded as a valuable means to reduce the number of injections and improve the efficacy of the drug. A wide variety of techniques, meant to enhance the blood © XXXX American Chemical Society

circulation time of drug candidates, have been proposed and studied.3,4 Conjugation of drug molecules with high-molecularweight polyethylene glycol (PEG) or carbohydrate polymers has been used to increase the blood permanence by reducing glomerular filtration.4 In the case of peptide-based molecules, chemical modification with, for instance, unnatural amino acids have been employed to reduce unwanted enzymatic degradation.5 Another strategy to increase the blood circulation time of Received: July 1, 2017 Revised: August 10, 2017

A

DOI: 10.1021/acs.bioconjchem.7b00378 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

The aim of this study was to evaluate and compare three different plasma protein binders potentially useful in combination with radiopharmaceuticals. Among those was a 4-(4iodophenyl)butyric acid derivative (PPB-01) previously discovered from a DNA-encoded chemical library by Dumelin et al.20 This was the entity used in combination with antibody fragments,21 folic acid radioconjugates,16,22 a DOTA-based bisphosphonate,17 and PSMA-targeting radioligands,18,19 in which the desired effect of enhanced blood circulation of the biomolecule was successfully demonstrated. As the currently most investigated plasma protein binder in combination with radiopharmaceuticals, PPB-01 served for comparison with other potentially powerful plasma protein-binding entities. The second compound was based on an ethynyl benzene entity (PPB-02). The ethynyl moiety served as a bioisosteric replacement of the iodine of compound PPB-01 based on a study in which the ethynyl group was evaluated as a halogen bioisostere.23 The third entity (PPB-03) was recently reported to effectively bind to transthyretin (hTTR).12 The three entities were functionalized with a DOTA chelator via a L-lysine and βalanine linker moiety using solid-phase peptide chemistry (Figure 1). Radiolabeling was performed with 177Lu (177Lu: T1/2 = 6.65 days; Eβ−average = 134 keV; Eγ = 113 and 208 keV), a clinically established radiometal for targeted radionuclide therapy. The three radioligands were investigated extensively in vitro and compared with regard to their blood clearance in mice.

drugs is to enhance their binding capability to blood components with long circulation half-lives, such as the Fc region of IgG isotype antibodies, lipoproteins, or blood plasma proteins.1,6 Indeed, the extent of binding to plasma proteins influences the pharmacokinetic profile of drugs that show reduced blood clearance with high binding capability.1 Plasmabinding properties may reduce the therapeutic effect because only the free fraction can penetrate biological barriers and reach the therapeutic target.2 It is, however, considered as a valuable strategy with which to avoid undesired side effects of drugs with a narrow therapeutic window, such as anticancer therapeutics.7 The most important proteins responsible for drug binding are human serum albumin (HSA) and human α1-acid glycoprotein (α1-AGP).1 HSA accounts for 55% of the total plasma protein pool (blood plasma concentration of ∼700 μM) and is, thus, a major protein component of blood plasma.8,9 It is composed of a single, nonglycosylated amino acid chain with a molecular weight of ∼66 kDa and a long blood plasma half-life of ∼19 days.1,7 HSA plays an important role in the transport of various endogenous and exogenous compounds because it has an extraordinary ligand-binding capacity and can reversibly bind acidic and neutral compounds.1 It is composed of three main domains (I, II, and III) that are each divided into two helical subdomains (A and B).8 A pair of main drug-binding sites have been identified: (i) Sudlow’s site I is located on the subdomain IIA and binds bulky heterocyclic anions such as the anticoagulant warfarin, whereas (ii) Sudlow’s site II is located in the subdomain IIIA and binds preferentially to extended aromatic carboxylates molecules such as ibuprofen, a nonsteroidal anti-inflammatory drug.8 Human α1-AGP is smaller (38−48 kDa) than HSA, highly glycosylated and present at lower blood plasma levels (10− 30 μM).1 Its blood plasma half-life of ∼2−3 days is dependent on the extent of glycosylation.10 α1-AGP is responsible for the transport of lipophospholipids and biliverdin but also for exogenous ligands such as antibiotics and anticancer drugs. The binding capacity of human α1-AGP is lower than the binding capacity of HSA, but the binding affinity of its ligands, mostly basic compounds, is commonly higher.1 Human transthyretin (hTTR), originally referred to as prealbumin, is a protein (55 kDa) present in blood plasma at much lower levels (∼5 μM) than HSA. It acts as a transport molecule for thyroxine hormones and holo-retinol-binding protein.11 hTTR is a homotetramer and has an in vivo half-life of ∼2 days.12 Recently, drug binding to hTTR has been demonstrated as a novel strategy to prevent fast clearance of therapeutic drugs.12 Targeted radionuclide therapy makes use of a tumortargeting agent to deliver the therapeutic radionuclide to the tumor site, where it acts as a source of radiation to damage cancer cells.13 These radiotherapeutic drugs should ideally accumulate specifically at diseased sites while sparing healthy tissue from radiotoxic effects.14 It has been demonstrated preclinically that plasma protein binding may be of value for the development of folate-based radiopharmaceuticals enabling high tumor uptake and reducing renal clearance and related toxicities.15,16 More recently, a DOTA-based bisphosphonate as well as PSMA-targeting ligands have been modified with an albumin-binding entity to improve their tissue-distribution profiles.17−19 It is, thus, of interest to identify and compare different types of plasma protein-binding entities to be conjugated with targeting ligands to optimize the pharmacokinetics of these (radio)therapeutics.



RESULTS Synthesis. The syntheses of the individual plasma proteinbinding entities (PPB-01, PPB-02, and MePPB-03) are described in detail in the Supporting Information. 4-(4Iodophenyl)butanoic acid (PPB-01) was obtained from a

Figure 1. Chemical structures of the investigated plasma proteinbinding ligands (A) DOTA−PPB-01, (B) DOTA−PPB-02, and (C) DOTA−PPB-03. The plasma protein-binding entities are colored in blue (PPB-01), yellow (PPB-02), and pink (PPB-03). B

DOI: 10.1021/acs.bioconjchem.7b00378 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry Scheme 1. Synthesis of DOTA−PPB-01, DOTA−PPB-02, and DOTA−PPB-03

Figure 2. HPLC chromatograms of (A) retention times.

177

Lu−DOTA−PPB-01, (B)

177

Lu−DOTA−PPB-02, and (C)

177

Lu−DOTA−PPB-03 with corresponding

Figure 3. (A) Fraction of radioligands bound to proteins in human plasma as well as to specific plasma proteins determined by a filter assay. These values were calculated based on the measurement of the filtered fraction (i.e., free fraction) and expressed as the percentage of the corresponding loading solutions, which were set to 100%. (B) The half-maximum binding (50%) was obtained at [HSA]-to-[radioligand] ratios of 234, 3270, and 396 for 177Lu−DOTA−PPB-01, 177Lu−DOTA−PPB-02, and 177Lu−DOTA−PPB-03, respectively. C

DOI: 10.1021/acs.bioconjchem.7b00378 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

(80% and >65%, respectively. The binding of 177 Lu−DOTA−PPB-02 was, however, decreased to