Synthesis of n.c.a. [188, 189, 191pt] Cisplatin From a Cyclotron-produced n.c.a. 188, 189, 191PtCl42-Complex

We developed a novel method for production of no-carrier-added (n.c.a.) [ 188, 189, 191 Pt]Pt (cid:0) Cl 42- from an Ir target material, and then synthesized n.c.a. [*Pt]cis-[Pt (cid:0) Cl 2 (NH 3 ) 2 ] ([*Pt]cisplatin) from [*Pt]Pt (cid:0) Cl 42- . [*Pt]Pt (cid:0) Cl 42- was prepared as a synthetic precursor of n.c.a. *Pt complex by a combination of resin extraction and anion-exchange chromatography after the selective reduction of Ir (cid:0) Cl 62- with ascorbic acid. The ligand-substitution reaction of Cl with NH 3 was promoted by treating n.c.a. [*Pt]Pt (cid:0) Cl 42- with excess NH 3 and heating the reaction mixture, and n.c.a [*Pt]cisplatin was successfully produced without employing precipitation routes. After this treatment, [*Pt]cisplatin was isolated through preparative HPLC with a radiochemical purity of 99+% at the end of synthesis (EOS).


Introduction
Targeted radionuclide therapy (TRT) is a type of radiation therapy in which malignant tissues are internally irradiated with radiopharmaceuticals emitting with β --ray, α-ray, or Auger electron (Auger e -). β -rays are the most commonly used in the clinic. Recently, α-rays have attracted a great deal of interest because of their high therapeutic e cacy, e.g., 225 Ac-PSMA-617 for metastatic castration-resistant prostate cancer [1]. Auger e -, the third candidate, are also expected to be used in TRT, and many radiopharmaceuticals labeled with Auger eemitters (e.g., 123, 125 I and 111 In) have been developed [2]. However, the therapeutic e cacy has been modest or low in clinical trials performed to date [3][4][5][6][7], and the causes and potential solutions remain unexplored.
An Auger eis a low-energy electron released following inner-shell excitation, and each excited atom emits multiple Auger e -. The range of Auger eis extremely short, 2-500 nm, yielding a high linear energy transfer (LET) of 4-26 keV/µm in the limited nano-scale range [8]. For example, the locally absorbed radiation dose around an 125 I decay site was estimated to be 1.6 MGy within a radius of 2 nm [9]. The effective range of Auger eis smaller than a single cell, suggesting that it is necessary to transport radiopharmaceuticals to intracellular regions that are sensitive to radiation. DNA is expected to be a prime target of Auger etherapy [2,[10][11][12][13]. More double-strand breaks can be induced when an Auger eemitter is closer to the DNA [14][15][16], suggesting that radiopharmaceuticals must be brought as close as possible to DNA to ensure an e cient interaction between Auger eand DNA in the nano-scale range.
Therefore, in many radiopharmaceuticals developed to date, Auger-emitting radionuclides of 123, 125 I and 111 In were labeled to DNA-targeting molecules, e.g., a nucleic acid derivative such as deoxyuridine (UdR) [3,17], a nuclear localization signal (NLS) [12,18], or a DNA-binding molecule [19][20][21], to ensure their transport to DNA. Although antimetabolites based on nucleic acid derivatives are incorporated into DNA, they are limited for use in TRT treatment due to their unavoidable distribution in the intestinal tract, which is radiosensitive. In almost all drugs, however, Auger eemitters labeled to DNA-targeting molecules are either not combined with DNA, or are combined indirectly through an intermediary molecule; consequently, there is expected to be a distance between the DNA and the Auger eemitter. Auger e -emitters directly combined with DNA may induce DNA damage most e ciently, but most radioelements are not combined with DNA by themselves. Radioelements should be labeled to intermediary DNAtargeting molecules when being transported to DNA, and such drug design is unalterable.
Platinum has a natural property that is useful in this context. Many platinum complexes (e.g., cisplatin, carboplatin, and oxaliplatin) have been used as platinum-based antineoplastic drugs, and platinum complexes with appropriate leaving groups can form direct DNA adducts between Pt and nucleobases [22]. 191 Pt (T 1/2 = 2.80 d, EC = 100%), 193m Pt (T 1/2 = 4.33 d, IT = 100%), and 195m Pt (T 1/2 = 4.01 d, IT = 100%), summarized in Table 1 [23, 24], are promising candidate radionuclides [25] that have a suitable half-life and a very high Auger eyield, e.g., an average of 32.8 electrons emitted per decay for 195m Pt vs. 14.7 electrons for 111 In [26]. Therefore, platinum complexes labeled with radio-Pt as the center metal allow many Auger eto be released very close to DNA, and are therefore appropriate for detailed studies to make sure of the degree of the therapeutic effect by Auger e -. In this work, we focus on cis-[Pt Cl 2 (NH 3 ) 2 ] (cis-diamminedichloroplatinum (II)), commonly called cisplatin, which can form direct DNA adducts between Pt and nucleobase as an intra-stand cross-link [22]. Cisplatin is a widely used chemotherapeutic agent, and its value is supported by a large number of basic and clinical studies over the years. In the clinic, cisplatin is also used in combination with external radiation because it can increase therapeutic e cacy by causing DNA damage via different routes [27]. Because radio-Pt-labeled cisplatin acts as both an anticancer agent that can target and chemically damage DNA and an Auger eemitter, it is expected to provide a superior therapeutic effect as an in vivo radio-chemotherapy agent.
Contrary to these expectations, however, the production method of no-carrier-added (n.c.a.) radio-Pt remains to be established at a practical level. Although the degree of therapeutic e cacy was reported in previous studies using carrier-added radio-cisplatin with low speci c activity (~MBq/mg) [28,29], it was doubtful whether the fundamental potential of Auger eitself could be detected without being masked by the chemotherapeutic effects of non-radioactive cisplatin carriers. To reveal the therapeutic potential of Auger e -, the DNA-damaging effect of radio-cisplatin needs to be investigated using n.c.a. radio-Pt.
Available radio-Pt is commonly produced by a reactor via the nat Pt(n,x) 191, 193m, 195m Pt reaction, resulting in carrier-added radio-Pt derived from a non-radioactive Pt target material. Although n.c.a. 191, 193m Pt can be produced by a cyclotron from a target material of iridium (Ir) or osmium (Os), several issues related to the chemical properties of both Ir and Os make it di cult to produce 191, 193m Pt with high yield and high purity [30][31][32][33]. Therefore, we demonstrated the production of n.c.a. 191 Pt from an Ir target using a cyclotron [34,35]. In this work, we established a procedure for producing n.c.a. *Pt Cl 4  before separation.
The and non-radioactive cisplatin in saline solution (0.5 mg/mL) were also analyzed to identify respective retention times.
Preparation of n.c.a. *Pt Cl 4

2-
The preparation scheme is shown in Fig. 1; the details of this scheme are as follows. greenish-yellow, as Ir Cl 6 2was selectively reduced to Ir Cl 6 3whereas *Pt Cl 6 2remained intact. After the reduction, the mixed solution was loaded into a TBP-resin column made by connecting three TBP-resin cartridges (2 mL cartridge, TrisKem International, Rennes, France), as *Pt Cl 6 2was selectively extracted into the resin. The column was rinsed with 3 mol/L HCl (5 mL), and then water (6 mL) was used as an eluting agent. To reduce *Pt to *Pt , an ascorbic acid solution (water + ascorbic acid injection = 2 + 8 mL) was added to the collected elution (6 mL

Synthesis of n.c.a. [*Pt]cisplatin
The synthesis scheme is shown in Fig. 1. In this work, the solid-phase extraction was applied in place of the liquid-liquid extraction because column separation is more suitable for expansion into a remote automatic device for further development.
*Pt Cl 6 2was extracted onto the TBP-resin column in the presence of HCl, and then quickly eluted with The extraction e ciencies for the recovery of *Pt were above 90% (n = 3, Table 2).
While only Ir Cl 6 2was reduced by ascorbic acid in 6 mol/L HCl, we found that ascorbic acid is also applicable to reduce *Pt Cl 6 2to *Pt Cl 4 2in dil. HCl (<1 mol/L). After elution from the TBP-resin column, *Pt Cl 6 2was reduced by ascorbic acid rapidly in <1 mol/L HCl. Then, the crude *Pt Cl 4 2solution was puri ed by anion exchange chromatography (AEC) with a QMA column. The radiochromatogram obtained during QMA-AEC is shown in Fig. 2. Although *Pt Cl 4 2was predominantly observed (f2-12), some *Pt Cl 4 2changed to other complexes, which passed through the column without any interaction (f0) or were strongly retained and remained on the column (C), as shown in Fig. 2. Additionally, the early eluted fractions (f1-4) were removed from the product because they contained impurities derived from ascorbic acid. As a result of these losses, a pure fraction of n.c.a. *Pt Cl 4 2was isolated at an e ciency of 60-70% (n = 3, Table2). Overall, as summarized in Table 2, the e ciency of the preparation of *Pt Cl 4 2was nearly constant, and the n.c.a. *Pt Cl 4 2product was obtained. Furthermore, no organic solvents were used in our method for separation of *Pt Cl 6 2from a bulk Ir target, which contributes the green chemistry and reduces the workloads on the quality control.

Synthesis of n.c.a. [*Pt]cisplatin
Bulk cisplatin is commonly produced by forming a crystal precipitate [36], but this approach is di cult to apply to n.c.a. radionuclides. Therefore, we synthesized n.c.a. [*Pt]cisplatin from *Pt Cl 4 2in solution, and then separated it by preparative HPLC. The radiochromatogram obtained during preparative isolation is shown in Fig. 3. [*Pt]cisplatin was detected at a retention time of 28-30 min, in good agreement with the value for non-radioactive cisplatin. The radiochemical yield for [*Pt]cisplatin, de ned as the ratio of 191 Pt radioactivity of isolated [ 191 Pt]cisplatin to the total 191 Pt collected after evaporation, was 5-15%. The low e ciency was due to a decrease in *Pt Cl 4 2purity during evaporation, in addition to the low synthetic yield of the ligand-substitution reaction between Cl and NH 3 . In HPLC analyses using an anion-exchange column, the peak intensity of *Pt Cl 4 2decreased with time, whereas an unknown peak that was not retained on the column and a peak of *Pt Cl 6 2appeared. The purity of *Pt Cl 4 2in the evaporated solution was 60% or less, suggesting that n.c.a. *Pt Cl 4 2is unstable. In support of this observation, the synthesis yield was increased to 30-40% when the collected elution (1 mol/L HCl and 0.5 mol/L KCl) from the QMA column was used immediately without evaporation.
Even when the purity of *Pt Cl 4 2was not so reduced, the synthetic yield was less than 50%. In the conventional synthetic method for bulk cisplatin, the synthetic yield is around 60% when K 2 [Pt C1 4 ] is treated directly with NH 3 , and an accurate two equivalents of NH 3 to Pt should be added in order to prevent excess ligand substitutions [36,37]. In this study, we used n.c.a. *Pt Cl 4 2-  E ciency contains an uncertainty of 5% in the radioactivity measurement.