The importance of tyrosines in multimers of cyclic RGD nonapeptides: towards αvß6-integrin targeted radiotherapeutics.

In a recent paper in this journal (RSC Med. Chem., 2023, 14, 2429), we described an unusually strong impact of regiospecific exchange of phenylalanines by tyrosines in 10 gallium-68-labeled trimers of certain cyclic RGD peptides, c[XRGDLAXp(NMe)K] (X = F or Y), on non-specific organ uptakes. We found that there was, in part, no correlation of liver uptake with established polarity proxies, such as the octanol-water distribution coefficient (log D). Since this observation could not be explained straightforwardly, we suggested that the symmetry of the compounds had resulted in a synergistic interaction of certain components of the macromolecules. In the present work, we investigated whether a comparable effect also occurred for a series of 5 tetramers labeled with lutetium-177. We found that in contrast to the trimers, liver uptake of the tetramers was well correlated to their polarity, indicating that the unusual observations along the trimer series indeed was a unique feature, probably related to their particular symmetry. Since the Lu-177 labeled tetramers are also potential agents for treatment of a variety of αvß6-integrin expressing cancers, these were evaluated in mice bearing human lung adenocarcinoma xenografts. Due to their tumor-specific uptake and retention in biodistribution and SPECT imaging experiments, these compounds are considered a step forward on the way to αvß6-integrin-targeted anticancer agents. Furthermore, we noticed that the presence of tyrosines in general had a positive impact on the in vivo performance of our peptide multimers. In view of the fact that a corresponding rule was already proposed in the context of protein engineering, we argue in favor of considering peptide multimers as a special class of small or medium-sized proteins. In summary, we contend that the performance of peptide multimers is less determined by the in vitro characteristics (particularly, affinity and selectivity) of monomers, but rather by the peptides' suitability for the overall macromolecular design concept, and peptides containing tyrosines are preferred.

Combining Cisplatin with Different Radiation Qualities-Interpretation of Cytotoxic Effects In Vitro by Isobolographic Analysis.

BACKGROUND: The combination of platinum-containing cytostatic drugs with different radiation qualities has been studied for years. Despite their massive side effects, these drugs still belong to the therapeutic portfolio in cancer treatment. To overcome the disadvantages of cisplatin, our study investigated the cytotoxic effects of combining radionuclides with cisplatin. METHODS: FaDu cells were treated with cisplatin (concentration ≈ 2 µM) and additionally irradiated after two hours with the alpha-emitter 223Ra, the beta-emitter 188Re as well as external X-rays using dose ranges of 2-6 Gy. Cell survival was followed by colony formation assays and plotted against cisplatin concentration and radiation dose. The results were interpreted by isobolograms. RESULTS: Isobolographic analyses revealed a supra-additive cytotoxic effect for the combination of cisplatin and 223Ra. A sub-additive effect was observed for the combination of cisplatin and 188Re, whereas a protective effect was found for the combination with X-rays. CONCLUSIONS: The combination of cisplatin and 223Ra may have the potential to create a successfully working therapy scheme for various therapy approaches, whereas the combination with 188Re as well as single-dose X-ray treatment did not lead to a detectable radiosensitizing effect. Thus, the combination with alpha-emitters might be advantageous and, therefore, should be followed in future studies when combined with cytostatic drugs.

Equilibrium Thermodynamics of Macropa Complexes with Selected Metal Isotopes of Radiopharmaceutical Interest.

To pursue the design of in vivo stable chelating systems for radiometals, a concise and straightforward method toolbox was developed combining NMR, isothermal titration calorimetry (ITC), and europium time-resolved laser-induced fluorescence spectroscopy (Eu-TRLFS). For this purpose, the macropa chelator was chosen, and Lu3+, La3+, Pb2+, Ra2+, and Ba2+ were chosen as radiopharmaceutically relevant metal ions. They differ in charge (2+ and 3+) and coordination properties (main group vs lanthanides). 1H NMR was used to determine four pKa values (±0.15; carboxylate functions, 2.40 and 3.13; amino functions, 6.80 and 7.73). Eu-TRLFS was used to validate the exclusive existence of the 1:1 Mn+/ligand complex in the chosen pH range at tracer level concentrations. ITC measurements were accomplished to determine the resulting stability constants of the desired complexes, with log K values ranging from 18.5 for the Pb-mcp complex to 7.3 for the Lu-mcp complex. Density-functional-theory-calculated structures nicely mirror the complexes' order of stabilities by bonding features. Radiolabeling with macropa using ligand concentrations from 10-3 to 10-6 M was accomplished by pointing out the complex formation and stability (212Pb > 133La > 131Ba ≈ 224Ra > 177Lu) by means of normal-phase thin-layer chromatography analyses.

Cyclotron-Based Production of 67Cu for Radionuclide Theranostics via the 70Zn(p,α)67Cu Reaction.

Theranostic matched pairs of radionuclides have aroused interest during the last couple of years, and in that sense, copper is one element that has a lot to offer, and although 61Cu and 64Cu are slowly being established as diagnostic radionuclides for PET, the availability of the therapeutic counterpart 67Cu plays a key role for further radiopharmaceutical development in the future. Until now, the 67Cu shortage has not been solved; however, different production routes are being explored. This project aims at the production of no-carrier-added 67Cu with high radionuclidic purity with a medical 30MeV compact cyclotron via the 70Zn(p,α)67Cu reaction. With this purpose, proton irradiation of electrodeposited 70Zn targets was performed followed by two-step radiochemical separation based on solid-phase extraction. Activities of up to 600MBq 67Cu at end of bombardment, with radionuclidic purities over 99.5% and apparent molar activities of up to 80MBq/nmol, were quantified.

Modulating the pharmacokinetic profile of Actinium-225-labeled macropa-derived radioconjugates by dual targeting of PSMA and albumin.

Rationale: Small 225Ac-labeled prostate-specific membrane antigen (PSMA)-targeted radioconjugates have been described for targeted alpha therapy of metastatic castration-resistant prostate cancer. Transient binding to serum albumin as a highly abundant, inherent transport protein represents a commonly applied strategy to modulate the tissue distribution profile of such low-molecular-weight radiotherapeutics and to enhance radioactivity uptake into tumor lesions with the ultimate objective of improved therapeutic outcome. Methods: Two ligands mcp-M-alb-PSMA and mcp-D-alb-PSMA were synthesized by combining a macropa-derived chelator with either one or two lysine-ureido-glutamate-based PSMA- and 4-(p-iodophenyl)butyrate albumin-binding entities using multistep peptide-coupling chemistry. Both compounds were labeled with [225Ac]Ac3+ under mild conditions and their reversible binding to serum albumin was analyzed by an ultrafiltration assay as well as microscale thermophoresis measurements. Saturation binding studies and clonogenic survival assays using PSMA-expressing LNCaP cells were performed to evaluate PSMA-mediated cell binding and to assess the cytotoxic potency of the novel radioconjugates [225Ac]Ac-mcp-M-alb-PSMA and [225Ac]Ac-mcp-D-alb-PSMA, respectively. Biodistributions of both 225Ac-radioconjugates were investigated using LNCaP tumor-bearing SCID mice. Histological examinations of selected organs were performed to analyze the occurrence of necrosis using H&E staining, DNA damage via γH2AX staining and proliferation via Ki67 expression in the tissue samples. Results: Enhanced binding to serum components in general and to human serum albumin in particular was revealed for [225Ac]Ac-mcp-M-alb-PSMA and [225Ac]Ac-mcp-D-alb-PSMA, respectively. Moreover, the novel derivatives are highly potent PSMA ligands as their KD values in the nanomolar range (23.38 and 11.56 nM) are comparable to the reference radioconjugates [225Ac]Ac-mcp-M-PSMA (30.83 nM) and [225Ac]Ac-mcp-D-PSMA (10.20 nM) without albumin binders. The clonogenic activity of LNCaP cells after treatment with the 225Ac-labeled ligands was affected in a dose- and time-dependent manner, whereas the bivalent radioconjugate [225Ac]Ac-mcp-D-alb-PSMA has a stronger impact on the clonogenic cell survival than its monovalent counterpart [225Ac]Ac-mcp-M-alb-PSMA. Biodistribution studies performed in LNCaP tumor xenografts showed prolonged blood circulation times for both albumin-binding radioconjugates and a substantially increased tumor uptake (46.04 ± 7.77 %ID/g for [225Ac]Ac-mcp-M-alb-PSMA at 128 h p.i. and 153.48 ± 37.76 %ID/g at 168 h p.i. for [225Ac]Ac-mcp-D-alb-PSMA) with favorable tumor-to-background ratios. Consequently, a clear histological indication of DNA damage was discovered in the tumor tissues, whereas DNA double-strand break formation in kidney and liver sections was less pronounced. Conclusion: The modification of the PSMA-based 225Ac-radioconjugates with one or two albumin-binding entities resulted in an improved radiopharmacological behavior including a greatly enhanced tumor accumulation combined with a rather low uptake in most non-targeted organs combined with a high excretion via the kidneys.

Efficient Production of the PET Radionuclide 133La for Theranostic Purposes in Targeted Alpha Therapy Using the 134Ba(p,2n)133La Reaction.

Targeted Alpha Therapy is a research field of highest interest in specialized radionuclide therapy. Over the last decades, several alpha-emitting radionuclides have entered and left research topics towards their clinical translation. Especially, 225Ac provides all necessary physical and chemical properties for a successful clinical application, which has already been shown by [225Ac]Ac-PSMA-617. While PSMA-617 carries the DOTA moiety as the complexing agent, the chelator macropa as a macrocyclic alternative provides even more beneficial properties regarding labeling and complex stability in vivo. Lanthanum-133 is an excellent positron-emitting diagnostic lanthanide to radiolabel macropa-functionalized therapeutics since 133La forms a perfectly matched theranostic pair of radionuclides with the therapeutic radionuclide 225Ac, which itself can optimally be complexed by macropa as well. 133La was thus produced by cyclotron-based proton irradiation of an enriched 134Ba target. The target (30 mg of [134Ba]BaCO3) was irradiated for 60 min at 22 MeV and 10−15 µA beam current. Irradiation side products in the raw target solution were identified and quantified: 135La (0.4%), 135mBa (0.03%), 133mBa (0.01%), and 133Ba (0.0004%). The subsequent workup and anion-exchange-based product purification process took approx. 30 min and led to a total amount of (1.2−1.8) GBq (decay-corrected to end of bombardment) of 133La, formulated as [133La]LaCl3. After the complete decay of 133La, a remainder of ca. 4 kBq of long-lived 133Ba per 100 MBq of 133La was detected and rated as uncritical regarding personal dose and waste management. Subsequent radiolabeling was successfully performed with previously published macropa-derived PSMA inhibitors at a micromolar range (quantitative labeling at 1 µM) and evaluated by radio-TLC and radio-HPLC analyses. The scale-up to radioactivity amounts that are needed for clinical application purposes would be easy to achieve by increasing target mass, beam current, and irradiation time to produce 133La of high radionuclide purity (>99.5%) regarding labeling properties and side products.

Strained Ammonium Precursors for Radiofluorinations.

The increasing application of positron emission tomography (PET) in nuclear medicine has stimulated the extensive development of a multitude of novel and versatile techniques to introduce fluorine-18, especially for the radiolabelling of biologically or pharmacologically active molecules. Taking into consideration that the introduction of fluorine-18 (t1/2 =109.8 min) mostly proceeds under harsh conditions, radiolabelling of such molecules represents a challenge and is of enormous interest. Ideally, it should proceed in a regioselective manner under mild physiological conditions, in an acceptable time span, with high yields and high specific activities. Special attention has been drawn to 2-fluoroethyl and 3-fluoropropyl groups, which are often the active sites of radiofluorinated compounds. Precursors containing an ammonium leaving group - such as a strained azetidinium or aziridinium moiety - can help to overcome these obstacles leading to a convenient and mild introduction of [18 F]fluoride with high radiochemical yields.

Cisplatin - A more Efficient Drug in Combination with Radionuclides?

AIM: The combination of conventional chemotherapeutic drugs with radionuclides or external radiation is discussed for a long period of time. The major advantage of a successful combination therapy is the reduction of severe side effects by decreasing the needed dose and simultaneously increasing therapeutic efficiency. METHODS: In this study, pUC19 plasmid DNA was incubated with the cytostatic drug cisplatin and additionally irradiated with 99mTc, 188Re and 223Ra. To verify the contribution of possibly excited platinum atoms to the emission of Auger electrons we determined DNA damages, such as single- and double strand breaks. RESULTS: The threshold concentration value of cisplatin, which was tolerated by pUC19 plasmid DNA was determined to be 18-24 nM. Nevertheless, even at higher dose values (>100 Gy) and simultaneous incubation of cisplatin to 200 ng plasmid DNA, no significant increase in the number of induced single- and double-strand breaks was obtained, compared to the damage solely caused by the radionuclides. CONCLUSION: We thereby conclude that there is no direct dependence of the mechanism of strand break induction to the absence or presence of platinum atoms attached to the DNA. Reported increasing DNA damages in therapy approaches on a cellular level strongly depend on the study design and are mainly influenced by repair mechanisms in living cells. Nevertheless, the use of radioactive cisplatin, containing the Auger electron emitter 191Pt, 193mPt or 195mPt, is a bright prospect for future therapy by killing tumor cells combining two operating principles: a cytostatic drug and a radiopharmaceutical at the same time.

The impact of barium isotopes in radiopharmacy and nuclear medicine - From past to presence.

With the exception of beryllium, divalent cations of every alkaline earth metal are characterized by their calcimimetic behavior. Thus, in vivo biodistribution of these cations mostly occurs in form of a massive accumulation in bone tissues, consisting of hydroxyapatite to a major extent. Apart from the lightest elements beryllium and magnesium, animal studies and human studies regarding the overall in vivo behavior were carried out by using radioisotopes of the elements calcium, strontium, barium and radium. To date, only strontium with its radioisotopes and radium gained importance for applications in nuclear medicine, mainly for pain-reducing and palliative treatment of bone metastases. In contrast, barium radioisotopes can be ascertained as useful imaging agents and possible diagnostic analogues for theranostic approaches. This review focuses on the characteristic and chemical behavior of barium compounds, possible radioactive barium isotopes for future applications in nuclear medicine and radiopharmacy as well as recent results regarding barium-131 as diagnostic match for radium isotopes used in targeted alpha therapy.

Towards Targeted Alpha Therapy with Actinium-225: Chelators for Mild Condition Radiolabeling and Targeting PSMA-A Proof of Concept Study.

Currently, targeted alpha therapy is one of the most investigated topics in radiopharmaceutical cancer management. Especially, the alpha emitter 225Ac has excellent nuclear properties and is gaining increasing popularity for the treatment of various tumor entities. We herein report on the synthesis of two universal 225Ac-chelators for mild condition radiolabeling and binding to conjugate molecules of pharmacological interest via the copper-mediated click chemistry. A convenient radiolabeling procedure was investigated as well as the complex stability proved for both chelators and two PSMA (prostate-specific membrane antigen)-targeting model radioconjugates. Studies regarding affinity and cell survival were performed on LNCaP cells followed by biodistribution studies, which were performed using LNCaP tumor-bearing mice. High efficiency radiolabeling for all conjugates was demonstrated. Cell binding studies revealed a fourfold lower cell affinity for the PSMA radioconjugate with one targeting motif compared to the radioconjugate owing two targeting motifs. Additionally, these differences were verified by in vitro cell survival evaluation and biodistribution studies, both showing a higher cell killing efficiency for the same dose, a higher tumor uptake (15%ID/g) and a rapid whole body clearance after 24 h. The synthesized chelators will overcome obstacles of lacking stability and worse labeling needs regarding 225Ac complexation using the DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid) chelator. Moreover, the universal functionalization expands the coverage of these chelators in combination with any sensitive bio(macro)molecule, thus improving treatment of any addressable tumor target.

Recent Insights in Barium-131 as a Diagnostic Match for Radium-223: Cyclotron Production, Separation, Radiolabeling, and Imaging.

Barium-131 is a single photon emission computed tomography (SPECT)-compatible radionuclide for nuclear medicine and a promising diagnostic match for radium-223/-224. Herein, we report on the sufficient production route 133Cs(p,3n)131Ba by using 27.5 MeV proton beams. An average of 190 MBq barium-131 per irradiation was obtained. The SR Resin-based purification process led to barium-131 in high radiochemical purity. An isotopic impurity of 0.01% barium-133 was detectable. For the first time, radiolabeling of the ligand macropa with barium-131 was performed. Radiolabeling methods under mild conditions and reaction controls based on TLC systems were successfully applied. Small animal SPECT/ computed tomography (CT) measurements and biodistribution studies were performed using [131Ba]Ba(NO3)2 as reference and 131Ba-labeled macropa in healthy mice for the first time. Biodistribution studies revealed the expected rapid bone uptake of [131Ba]Ba2+, whereas 131Ba-labeled macropa showed a fast clearance from the blood, thereby showing a significantly (p < 0.001) lower accumulation in the bone. We conclude that barium-131 is a promising SPECT radionuclide and delivers appropriate imaging qualities in small animals. Furthermore, the relative stability of the 131Ba-labeled macropa complex in vivo forms the basis for the development of sufficient new chelators, especially for radium isotopes. Thereby, barium-131 will attain its goal as a diagnostic match to the alpha emitters radium-223 and radium-224.

Sub-10 nm Radiolabeled Barium Sulfate Nanoparticles as Carriers for Theranostic Applications and Targeted Alpha Therapy.

Invited for this month's cover is the group of Kristof Zarschler at the University of Dresden, Germany. Read the full text of their Full Paper at 10.1002/open.202000126.

Sub-10†nm Radiolabeled Barium Sulfate Nanoparticles as Carriers for Theranostic Applications and Targeted Alpha Therapy.

The treatment of cancer patients with α-particle-emitting therapeutics continues to gain in importance and relevance. The range of radiopharmaceutically relevant α-emitters is limited to a few radionuclides, as stable chelators or carrier systems for safe transport of the radioactive cargo are often lacking. Encapsulation of α-emitters into solid inorganic systems can help to diversify the portfolio of candidate radionuclides, provided, that these nanomaterials effectively retain both the parent and the recoil daughters. We therefore focus on designing stable and defined nanocarrier-based systems for various clinically relevant radionuclides, including the promising α-emitting radionuclide 224Ra. Hence, sub-10 nm barium sulfate nanocontainers were prepared and different radiometals like 89Zr, 111In, 131Ba, 177Lu or 224Ra were incorporated. Our system shows stabilities of >90 % regarding the radiometal release from the BaSO4 matrix. Furthermore, we confirm the presence of surface-exposed amine functionalities as well as the formation of a biomolecular corona.

The effect of hypoxia on the induction of strand breaks in plasmid DNA by alpha-, beta- and Auger electron-emitters 223Ra, 188Re, 99mTc and DNA-binding 99mTc-labeled pyrene.

INTRODUCTION: Radiation-induced DNA damage occurs from direct and indirect effects. The induction is influenced by the physical characteristics of the radionuclide, especially its linear energy transfer. Hypoxia reduces the effect of irradiation treatment in tumor cells and leads to poor patient outcomes. High linear energy transfer emitters can overcome this obstacle. Our aim is to demonstrate the influence of hypoxia on the interaction of different radiation qualities with isolated DNA. METHODS: PuC19 Plasmid DNA was irradiated with 223Ra, 188Re, 99mTc and 99mTc-labeled pyrene with and without DMSO under hypoxia or normoxic conditions. DNA damages in form of single-(SSB) and double-strand breaks (DSB) were analyzed by gel electrophoresis. RESULTS: Radiation doses up to 200 Gy of 223Ra, 188Re and 99mTc led to maximal yields of 80% SSB and 30%, 28% and 32% DSB, respectively. Hypoxia had minor effects on damages from 223Ra, but caused a small enhancement in DSB for 188Re and 99mTc. DMSO prevented DSB completely and reduced SSB from the "free" radionuclides to comparable levels. DNA-binding 99mTc-labeled pyrene induced less SSB and DSB compared to [99mTc]TcO4-. However, the incubation with DMSO could prevent the SSB and DSB induction only to a minor extent. CONCLUSIONS: Hypoxia does not limit DNA damage induced by 223Ra, 188Re, 99mTc and 99mTc-labeled pyrene. Dose-dependent radiation effects were comparable for alpha-emitters and both high- and low-energy electron emitters. The radioprotection by DMSO was not influenced by hypoxia. The results indicate the contribution of mainly indirect radiation effects for 99mTc, 188Re and 223Ra. 99mTc-labeled pyrene caused direct DNA damages and Auger-electrons from 99mTc-labeled pyrene are more effective than high-energy electrons or alpha particles. ADVANCES IN KNOWLEDGE: Without the consideration of DNA repair mechanisms, oxygen has no direct influence in radiation-induced DNA damages by different radiation qualities. IMPLICATIONS FOR PATIENT CARE: The short-time stimulation with oxygen during patient radiation could have minor influence compared to constant oxygen flooding to overcome hypoxic barriers.

Modified Calix[4]crowns as Molecular Receptors for Barium.

Invited for this month's cover picture is the group around Dr. Constantin Mamat at the Institute of Radiopharmaceutical Cancer Research at the Helmholtz-Zentrum Dresden-Rossendorf (Germany) together with Prof. Martin Köckerling from the University of Rostock (Germany). The cover picture shows the ability of special functionalized calix[4]crown-6 derivatives to stably bind group 2 metals like barium. This binding mode is highly important for radiopharmaceutical applications not to lose the respective radiometal in vivo to avoid high background signals and/or false positive results and damages in other tissues. For this purpose, different calix[4]crowns were tested, based upon their potential to stably bind barium as surrogate for radium. Radium nuclides are known to be good candidates for usage in α-targeted therapies. Currently, radium-223 is used for α-therapy of bone metastases because of its calcium mimetics. Our aim is to apply the radium to treat other cancer tissues. That's why we need novel chelators to stably fix groups 2 metals like barium and radium. Read the full text of their Full Paper at https://doi.org/10.1002/open.201800019.

Modified Calix[4]crowns as Molecular Receptors for Barium.

A series of modified calix[4]crown-6 derivatives was synthesized to chelate the heavy group 2 metal barium, which serves as a non-radioactive surrogate for radium-223/-224; radionuclides with promising properties for radiopharmaceutical use. These calixcrowns were functionalized with either cyclic amide moieties or with deprotonizable groups, and the corresponding barium complexes were synthesized. Stability constants of these complexes were measured by using NMR and UV/Vis titration techniques to determine logK values of >4.1. Further extraction studies were performed to characterize the binding affinity of calixcrowns to radioactive barium-133. Additionally, the ligands containing cyclic amides were investigated regarding their rotational barriers by using temperature-dependent NMR measurements.

Direct and Auger Electron-Induced, Single- and Double-Strand Breaks on Plasmid DNA Caused by 99mTc-Labeled Pyrene Derivatives and the Effect of Bonding Distance.

It is evident that 99mTc causes radical-mediated DNA damage due to Auger electrons, which were emitted simultaneously with the known γ-emission of 99mTc. We have synthesized a series of new 99mTc-labeled pyrene derivatives with varied distances between the pyrene moiety and the radionuclide. The pyrene motif is a common DNA intercalator and allowed us to test the influence of the radionuclide distance on damages of the DNA helix. In general, pUC 19 plasmid DNA enables the investigation of the unprotected interactions between the radiotracers and DNA that results in single-strand breaks (SSB) or double-strand breaks (DSB). The resulting DNA fragments were separated by gel electrophoresis and quantified by fluorescent staining. Direct DNA damage and radical-induced indirect DNA damage by radiolysis products of water were evaluated in the presence or absence of the radical scavenger DMSO. We demonstrated that Auger electrons directly induced both SSB and DSB in high efficiency when 99mTc was tightly bound to the plasmid DNA and this damage could not be completely prevented by DMSO, a free radical scavenger. For the first time, we were able to minimize this effect by increasing the carbon chain lengths between the pyrene moiety and the 99mTc nuclide. However, a critical distance between the 99mTc atom and the DNA helix could not be determined due to the significantly lowered DSB generation resulting from the interaction which is dependent on the type of the 99mTc binding motif. The effect of variable DNA damage caused by the different chain length between the pyrene residue and the Tc-core as well as the possible conformations of the applied Tc-complexes was supplemented with molecular dynamics (MD) calculations. The effectiveness of the DNA-binding 99mTc-labeled pyrene derivatives was demonstrated by comparison to non-DNA-binding 99mTcO4-, since nearly all DNA damage caused by 99mTcO4- was prevented by incubating with DMSO.