Folic acid derivatives for PET imaging and therapy addressing folate receptor positive tumors

Folic acid, also known as vitamin B9, is the oxidized form of 5,6,7,8-tetrahydrofolate, which serves as methyl- or methylene donor (C1-building blocks) during DNA synthesis. Under physiological conditions the required amount of 5,6,7,8-tetrahydrofolate for survival of the cell is accomplished through the reduced folate carrier (RFC). In contrast, the supply of 5,6,7,8-tetrahydrofolate is insufficient under pathophysiological conditions of tumors due to an increased proliferation rate. Consequently, many tumor cells exhibit an (over)expression of the folate receptor. This phenomenon has been applied to diagnostics (PET, SPECT, MR) to image FR-positive tumors and on the other hand to treat malignancies related to a FR (over)expression. Based on this concept, a new 18F-labeled folate for PET imaging has been developed and was evaluated in vivo using tumor-bearing mice. The incorporation of oligoethylene spacers into the molecular structure led to a significant enhancement of the pharmacokinetics in comparison to previously developed 18F-folates. The liver uptake could be reduced by one sixth by remaining a tumor uptake of 3%ID/g leading to better contrast ratios. Encouraged by these results, a clickable 18F-labeled serine-based prosthetic group has been synthesized, again with the idea to improve the metabolic and pharmacokinetic profile of hydrophilic radiotracers. Therefore, an alkyne-carrying azido-functionalized serine derivative for coupling to biomolecules was synthesized and a chlorine leaving group for 18F-labeling, which could be accomplished using a microwave-assisted synthesis, a [K⊂2.2.2]+/carbonate system in DMSO. Radiochemical yields of 77±6% could be achieved.rnThe promising results obtained from the FR-targeting concept in the diagnostic field have been transferred to the boron neutron capture therapy. Therefore, a folate derivative was coupled to different boron clusters and cell uptake studies were conducted. The synthesis of the folate-boron clusters was straightforward. At first, a linker molecule based on maleic acid was synthesized, which was coupled to the boron cluster via Michael Addition of a thiol and alkene and subsequently coupled to the targeting moiety using CuAAC. The new conjugates of folate and boron clusters led to a significant increase of boron concentration in the cell of about 5-times compared to currently used and approved boron pharmaceuticals. rnMoreover, azido-folate derivatives were coupled to macromolecular carrier systems (pHPMA), which showed an enhanced and specific accumulation at target sites (up to 2.5-times) during in vivo experiments. A specific blockade could be observed up to 30% indicating an efficient targeting effect. A new kind of nanoparticles consisting of a PDLLA core and p((HPMA)-b-LMA)) as surfactants were developed and successfully radiolabeled via 18F-click chemistry in good RCYs of 8±3%rnThe nanoparticles were obtained via the miniemulsion technique in combination with solvent evaporation. The 18F-labeled nanoparticles were applied to in vivo testing using a mouse model. PET imaging showed a “mixed” biodistribution of low molecular weight as well as high molecular weight systems, indicating a partial loss of the 18F-labeled surfactant.rnIn conclusion, the presented work successfully utilized the FR-targeting concept in both, the diagnostic field (PET imaging) and for therapeutic approaches (BNCT, drug delivery systems). As a result, the high potential of FR-targeting in oncological applications has been shown and was confirmed by small animal PET imaging.rnFolsäure, Vitamin B9, ist die oxidierte Form der 5,6,7,8-Tetrahydrofolsäure, welche in der DNS Biosynthese als Lieferant u.a. von Methyl- und Methylenbausteinen (C1-Bausteine) fungiert. Unter physiologischen Bedingungen ist die Aufnahme der notwendigen Menge an 5,6,7,8-Tetrahydrofolat in die Zelle über den Reduced Folate Carrier (RFC) gewährleistet, wohingegen unter pathophysiologischen Bedingungen der erhöhte Bedarf an DNS Bausteinen aufgrund der gesteigerten Proliferationsrate, durch eine (Über)Expression des Folatrezeptors (FR) kompensiert wird. Diese (Über)Expression wird zum einen für die Diagnostik (PET, SPECT, MR) von FR-positiven Tumoren und zum anderen zur gezielten Therapie von malignen Tumoren eingesetzt. Auf dieser Grundlage wurde ein neues 18F-markiertes Radiofolate für die PET entwickelt und evaluiert. Der Einbau von Oligoethylenspacern bewirkte eine deutlich verbesserte Pharmakokinetik. Dies spiegelt sich durch eine um ein sechstel erniedrigte Anreicherung des Tracers in der Leber wieder, wobei die Tumoranreicherung bei einem ähnlichen Wert von 3%ID/g blieb im Vergleich zu ähnlichen Tracern. Dies führte zu einer deutlichen Verbesserung des Kontrasts. Auf diesen Erkenntnissen wurde eine serinbasierte prosthetische Gruppe entwickelt, die sich nahe am endogenen Serin orientiert und somit zu einer nochmaligen Verbesserung der Pharmakokinetik von Biomolekülen führen sollte. Dazu wurde ein Serinderivat dargestellt, welches eine Alkinfunktion für die Biomolekülanbindung und Chlor als Abgangsgruppe trug. Die Radiomarkierung konnte mittels einer mikrowellengestützten Synthese und eines Karbonat/Kryptofix Systems in DMSO durchgeführt werden. rnDie erfolgreiche Anwendung von Folatstrukturen in der Diagnostik konnte auch auf Therapieansätze übertragen werden. Hierbei wurden Folatborclusterderivate dargestellt, welche in der Bor Neuronen Einfangtherapie Anwendung finden. Dabei wurde zuerst das Borcluser über eine Michael Additionsreaktion eines Thiols mit einem Alken an das Linkermolekül angebunden. In einer zweiten CuAAC wurde nun das Folat gekoppelt. Darüber hinaus wurden die dargestellten Folsäurederivate an makromolekulare Trägersysteme (pHPMA) übertragen. Erste Zellaufnahmestudien zeigten eine deutliche Verbesserung der Anreicherung (Faktor 5) von Bor in den Zellen im Vergleich zu bisher verwendeten zugelassenen Borpharmazeutika. Die verwendeten Borcluster wurden über ein Linkermolekül mit der Folatstruktur gekoppelt. Durch die Verwendung des aktiven FR-targeting Konzepts konnte eine erhöhte Anreicherung (bis zu 2.5-fach) im Tumor während in vivo Experimenten gezeigt werden. Darüber hinaus konnte eine spezifische Blockade von 30% beobachtet werden, was nochmals die Effizienz des aktiven Targetings zeigte. Eine nochmalige Verbesserung der in vivo Eigenschaften wurde sich durch die Verwendung von nanopartikulären Systeme (PDLLA-Kern und p((HPMA)-b-(LMA)) Tenside), die mit Folsäure konjugiert sind, versprochen. Die über die Miniemulsion in Kombination mit der Solvent Evaporation Technik synthetisierten Systeme, noch ohne Folsäureanbindung, wurden hierbei erstmals mit Fluor-18 mit RCA von 8±3% markiert und im Mausmodell evaluiert. Die Systeme zeigten interessanterweise das Verhalten von sowohl niedermolekularen als auch hochmolekularen Systemen. Zusammenfassend konnte die erfolgreiche Anwendung des FR-targeting Konzepts in Diagnostik (PET)- und Therapieansätzen (BNCT, Drug Delivery) demonstriert werden.r

18F-Labeling Using Click Cycloadditions

Due to expanding applications of positron emission tomography (PET) there is a demand for developing new techniques to introduce fluorine-18 (t1/2=109.8 min). Considering that most novel PET tracers are sensitive biomolecules and that direct introduction of fluorine-18 often needs harsh conditions, the insertion of 18F in those molecules poses an exceeding challenge. Two major challenges during 18F-labeling are a regioselective introduction and a fast and high yielding way under mild conditions. Furthermore, attention has to be paid to functionalities, which are usually present in complex structures of the target molecule. The Cu-catalyzed azide-alkyne cycloaddition (CuAAC) and several copper-free click reactions represent such methods for radiolabeling of sensitive molecules under the above-mentioned criteria. This minireview will provide a quick overview about the development of novel 18F-labeled prosthetic groups for click cycloadditions and will summarize recent trends in copper-catalyzed and copper-free click 18F-cycloadditions

A Simple, Rapid Method for the Preparation of [11 C]Formaldehyde

A PET project: A powerful reagent for the synthesis of positron-emitting imaging molecules—[11C]formaldehyde—is accessible from [11C]methyl iodide and trimethylamine N-oxide (TMAO) in high yields and under mild conditions. Easy access to [11C]formaldehyde expands the scope of the carbon-11 toolbox and will lead to new reaction methodology and imaging compounds.Chemistry and Chemical Biolog

Radiolabeling of Nanoparticles and Polymers for PET Imaging

Nanomedicine has become an emerging field in imaging and therapy of malignancies. Nanodimensional drug delivery systems have already been used in the clinic, as carriers for sensitive chemotherapeutics or highly toxic substances. In addition, those nanodimensional structures are further able to carry and deliver radionuclides. In the development process, non-invasive imaging by means of positron emission tomography (PET) represents an ideal tool for investigations of pharmacological profiles and to find the optimal nanodimensional architecture of the aimed-at drug delivery system. Furthermore, in a personalized therapy approach, molecular imaging modalities are essential for patient screening/selection and monitoring. Hence, labeling methods for potential drug delivery systems are an indispensable need to provide the radiolabeled analog. In this review, we describe and discuss various approaches and methods for the labeling of potential drug delivery systems using positron emitters

18F-click labeling and preclinical evaluation of a new 18F-folate for PET imaging

Background The folate receptor (FR) is a well-established target for tumor imaging and therapy. To date, only a few 18 F-folate conjugates via 18 F-prosthetic group labeling for positron emission tomography (PET) imaging have been developed. To some extent, they all lack the optimal balance between efficient radiochemistry and favorable in vivo characteristics. Methods A new clickable olate precursor was synthesized by regioselective coupling of folic acid to 11-azido-3,6,9-trioxaundecan-1-amine at the γ-position of the glutamic acid residue. The non-radioactive reference compound was synthesized via copper-catalyzed azide-alkyne cycloaddition of 3-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)prop-1-yne and γ-(11-azido-3,6,9-trioxaundecanyl)folic acid amide. The radiosynthesis was accomplished in two steps: at first a 18 F-fluorination of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl-4-methylbenzenesulfonate, followed by a 18 F-click reaction with the γ-azido folate. The in vitro, ex vivo, and in vivo behaviors of the new 18 F-folate were investigated using FR-positive human KB cells in displacement assays and microPET studies using KB tumor-bearing mice. Results The new 18 F-folate with oligoethylene spacers showed reduced lipophilicity in respect to the previously developed 18 F-click folate with alkyl spacers and excellent affinity (Ki = 1.6 nM) to the FR. Combining the highly efficient 18 F-click chemistry and a polar oligoethylene-based 18 F-prosthetic group facilitated these results. The overall radiochemical yield of the isolated and formulated product averages 8.7%. In vivo PET imaging in KB tumor-bearing mice showed a tumor uptake of 3.4% ID/g tissue, which could be reduced by FR blockade with native folic acid. Although the new 18 F-oligoethyleneglycole (OEG)-folate showed reduced hepatobiliary excretion over time, a distinct unspecific abdominal background was still observed. Conclusions A new 18 F-folate was developed, being available in very high radiochemical yields via a fast and convenient two-step radiosynthesis. The new 18 F-OEG-folate showed good in vivo behavior and lines up with several recently evaluated 18 F-labeled folates

Comparison Study of Two Differently Clicked 18F-Folates—Lipophilicity Plays a Key Role

Within the last decade, several folate-based radiopharmaceuticals for Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) have been evaluated; however, there is still a lack of suitable 18F-folates for clinical PET imaging. Herein, we report the synthesis and evaluation of two novel 18F-folates employing strain-promoted and copper-catalyzed click chemistry. Furthermore, the influence of both click-methods on lipophilicity and pharmacokinetics of the 18F-folates was investigated. 18F-Ala-folate and 18F-DBCO-folate were both stable in human serum albumin. In vitro studies proved their high affinity to the folate receptor (FR). The lipophilic character of the strain-promoted clicked 18F-DBCO-folate (logD = 0.6) contributed to a higher non-specific binding in cell internalization studies. In the following in vivo PET imaging studies, FR-positive tumors could not be visualized in a maximum intensity projection images. Compared with 18F-DBCO-folate, 18F-Ala-folate (logD = −1.4), synthesized by the copper-catalyzed click reaction, exhibited reduced lipophilicity, and as a result an improved in vivo performance and a clear-cut visualization of FR-positive tumors. In view of high radiochemical yield, radiochemical purity and favorable pharmacokinetics, 18F-Ala-folate is expected to be a promising candidate for FR-PET imaging

Selective binding to monoamine oxidase a : in vitro and in vivo evaluation of f-18-labeled beta-carboline derivatives

In this study we synthesized four different F-18-labeling precursors for the visualization of the monoamino oxidase A using harmol derivatives. Whereas two are for prosthetic group labeling using [F-18]fluoro-d(2)-methyl tosylate and 2-[F-18]fluoroethyl-tosylate, the other three precursors are for direct nucleophilic F-18-labeling. Additionally the corresponding reference compounds were synthesized. The syntheses of [F-18]fluoro-d(2)-methyl-harmol and 2-[F-18] fluoroethyl-harmol were carried out using harmol as starting material. For direct nucleophilic F-18-labeling of the tracers carrying oligoethyled spacers (PEG), a toluenesulfonyl leaving group was employed. The radiolabeling, purification and formulation for each tracer was optimized and evaluated in vitro and in vivo. Stability tests in human serum showed that all tracers were stable over the observation period of 60 min. mu PET studies using of the synthesized tracers revealed that the tracers carrying PEG spacers showed no sufficient brain uptake. Consequently, the F-18-fuoro alkylated tracers [F-18] fluoro-d2-methyl-harmol and 2-[F-18]fluoroethyl-harmol were further evaluated showing SUVs in the brain of 1.0 +/- 0.2 g/mL and 3.4 +/- 0.5 g/mL after 45 min, respectively. In blockade studies the selectivity and specificity of both tracers were demonstrated. However, for [F-18]fluoro-d(2)-methyl-harmol a rapid washout from the brain was also observed. In vitro binding assays revealed that 2-[F-18] fluoroethyl-harmol (IC50 = 0.54 +/- 0.06 nM) has a higher affinity than the F-18-fluoro-d(2)-methylated ligand (IC50 = 12.2 +/- 0.6 nM), making 2-[F-18] fluoroethyl-harmol superior to the other evaluated compounds and a promising tracer for PET imaging of the MAO A

Considerations for Human ADME Strategy and Design Paradigm Shift(s) - An Industry White Paper.

The human absorption, distribution, metabolism, and excretion (hADME) study is the cornerstone of the clinical pharmacology package for small molecule drugs, providing comprehensive information on the rates and routes of disposition and elimination of drug-related material in humans through the use of 14 C-labeled drug. Significant changes have already been made in the design of the hADME study for many companies, but opportunity exists to continue to re-think both the design and timing of the hADME study in light of the potential offered by newer technologies, that enable flexibility in particular to reducing the magnitude of the radioactive dose used. This paper provides considerations on the variety of current strategies that exist across a number of pharmaceutical companies and on some of the ongoing debates around a potential move to the so called "human first/human only" approach, already adopted by at least one company. The paper also provides a framework for continuing the discussion in the application of further shifts in the paradigm

Non-labelled, Stable Labelled or Radiolabelled Approaches for Provision of Intravenous Pharmacokinetics in Humans: a Discussion Piece.

A review of the use of microdoses and isotopic microtracers for clinical intravenous pharmacokinetic (IV PK) data provision is presented. The extent of application of the varied approaches available and the relative merits of each are highlighted with the aim of assisting practitioners in making informed decisions on the most scientifically appropriate design to adopt for any given new drug in development. It is envisaged that significant efficiencies will be realized as IV PK data in humans becomes more routinely available for suitable assets in early development, than has been the case prior to the last decade