¹⁸⁸Re Labeled liver therapeutic drugs for hepatic carcinoma (HCC)

Abstract

$^{188}Re$ is one of the most readily available generator derived and useful radionuclides for therapy emitting ${\beta}^-$ particles (2.12 MeV, 71.1% and 1.965 MeV, 25.6%) and imageable gammas (155 keV, 15.1%). The $^{188}W/^{188}Re$ generator is an ideal source for the long term (4-6 months) continuous availability of no carrier added (NCA) $^{188}Re$ suitable for the preparation of radiopharmaceuticals for radionuclide therapy. Rhenium-188 has been used for the preparation of therapeutic radiopharmaceuticals for the management of diseases such as bone metastasis, rheumatoid arthritis and primary cancers. Several early phase clinical studies using radiopharmaceuticals based on $^{188}Re$ -labeled phosphonates, antibodies, peptides, lipiodol and particulates have been reported. In this review, we addressed the current development status of $^{188}Re$ radiopharmaceuticals for liver cancer therapy and their applications.

Introduction

Various radioisotopes have been used for trans-arterial radiotherapy of hepatocellular carcinoma (HCC) (Table 1), among them rhenium-188 (¹⁸⁸Re) is most commonly used. Rhenium-188 is a high energy β-emitting radioisotope obtained from the ¹⁸⁸W/¹⁸⁸Re- generator (Figure 1), which has shown utility for a variety of therapeutic applications in nuclear medicine, oncology, and interventional radiology/cardiology. The decay of ¹⁸⁸Re is accompanied by a 155 keV predominant energy β-emission, which could be detected by γ-cameras, for imaging, biodistribution, or absorbed radiation dose studies. It has an attractive physical properties (Table 1) and its potential low cost associated with a long-lived parent make it an interesting option for clinical use.

Figure 1. Decay scheme for ¹⁸⁸Re production. 

Table 1. Examples of radioisotopes used for HCC transarterial metabolic radiotherapy.

The clinical efficacy, for several therapeutic applications, of a variety of ¹⁸⁸Re-labeled agents is demonstrated. The high energy of the β-emission of ¹⁸⁸Re is particularly well suited for effective penetration in solid tumors. Its total radiation dose delivered to tissues is comparable to other radionuclides used in therapy. Furthermore, radiation safety and shielding requirements are an important subject of matter. In the case of bone metastases treatment, therapeutic ratios are presented in order to describe the efficacy of ¹⁸⁸Re usage.

Lipiodol

Lipiodol is an iodinated and esterified lipid of poppy seed oil that has been used as a contrast agent for the detection of liver cancer (1-4). When injected through the hepatic artery it accumulates in the liver cancer because of its high viscosity. This property of lipiodol has encouraged many researchers to use it as a radioisotope carrier. Other carriers such as microspheres can also be used. However, lipiodol is the most effective and convenient carrier because of its excellent targeting ability as well as its capacity to be accurately monitored by X-ray.

There have been many attempts to label lipiodol with therapeutic radioisotopes, including ¹⁶⁶Ho, ¹³¹I, ⁹⁰Y, ¹⁸⁶Re and ¹⁸⁸Re (5-9).

¹³¹I-labelled lipiodol is commercially available and is currently used in many countries. However, its high cost and high external radiation dose due to its high energy gamma radiation limits its general use (6, 1012). Among the metallic beta emitters, ¹⁸⁸Re became the most important candidate for labelling lipiodol because of its convenience and economy. Early attempts to label lipiodol with ¹⁸⁸Re were not very successful, since ¹⁸⁸Re is obtained as an aqueous solution and lipiodol is available as an oil. Direct chemical reaction is precluded because they are not miscible. If a bichelating agent were to be linked with lipiodol in order to facilitate labelling with ¹⁸⁸Re, the chemical properties of the resulting conjugate would be changed. In addition, its chemistry would be difficult to characterize since lipiodol is not composed of a single substance.

Bifunctional chelating agents for ¹⁸⁸Re Labeling

Several N₂S₂ based bifunctional chelating agents, 2,2,9,9-tetramethyl-4,7-diaza-1,10-decanedithiol (TDD) (1),  4-Octyl-2,2,9,9-tetramethyl-4,7-diaza-1,10decanedithiol (ODD) (13), 4-Dodecyl-2,2,9,9-tetramethyl4,7-diaza-1,10-decanedithiol (DDD) (13), 4-hexadecyl2,2,9,9-tetramethyl-4,7-diaza-1,10-decanedithiol (HTDD) (14), 4-hexadecyl-2,2,9,9-tetramethyl-4,7-diaza1,10-decanedithioacetate (HTDD-A) (2) and 4-hexadecyl4,7-diaza-1,10-decanedithiol (HDD) (15), have been developed for ¹⁸⁸Re labeling (Figure 2).

Figure 2. Chemical structures of ¹⁸⁸Re labeled N₂S₂ based bifunctional chelating agents. 

¹⁸⁸Re-lipiodol for Hepatic Carcinoma (HCC)

Hepatic carcinoma is a most prevalent cancer worldwide and although most cases still occur in developing countries in South-East Asia and Africa (16).  Almost 700,000 new cases are diagnosed yearly throughout the world and unfortunately the prognosis remains poor since more than 500,000 deaths are ascribed to HCC each year (16). Thus the development of therapeutic radiopharmaceuticals for its management if intercepted early, is an active field of radiopharmaceuticals research for many researchers. ¹³¹I labelled lipiodol is a commercially available radiopharmaceutical for the treatment of hepatocarcinoma (17). Lipiodol or ethiodized oil, contains iodine combined with ethyl esters of fatty acids and is used as a contrast agent in myelography. The inactive iodine of lipiodol are exchanged with ¹³¹I, followed by solvent extraction of the labeled product for preparation of the radiopharmaceutical.

There are several reports describing the preparation of ¹⁸⁸Re-lipiodol starting with Kim et al. suspending ¹⁸⁸Re sulphur colloid in lipiodol (18). A N₂S₂ (diaminedithiol) based chelating agent 2,2,9,9-tetramethyl-4,7-diaza1,10-decane dithiol (TDD, Figure 2) was synthesized and labeled with technetium (^99mTc-TDD) or rhenium (¹⁸⁸Re-TDD) in high yields (72.0 ± 5.0 %), and found to be stable in room temperature and in human serum at 37ºC for up to 48 h (13). ¹⁸⁸Re-TDD showed high accumulation in the hepatoma (5 min: 33.1±24.1; 60 min: 13.5±10.6; 24 h: 3.02 ±2.78) was observed in hepatoma-bearing Sprague-Dawley (SD) rats after injection through the hepatic artery (13). However, ¹⁸⁸Re-TDD tumor retention is not enough to treat liver cancer. Therefore a new form of TDD, 4-hexadecyl2,2,9,9-tetramethyl-4,7-diaza-1,10-decanedithiol (HTDD)  was developed to improve tumor retention by introducing a long alkyl chain and it is labeled with ¹⁸⁸Re in high yields (65.0 ± 7.0 %).  A comparative study between ¹⁸⁸Re-TDD and ¹⁸⁸Re-HTDD were performed in VX2 carcinoma (liver) bearing rabbits. The residences times of radioactivity in the liver were 10.2 ± 1.0 h in the ¹⁸⁸Re-TDD and 17.6 ± 0.8 h in the ¹⁸⁸Re-HTDD (p = 0.034) (1). A comparative study between ¹⁸⁸Re-HTDD and ¹³¹I-lipodol was conducted in patients (Ghent University hospital, Belgium). This study showed that ¹⁸⁸Re-HDD/lipiodol yielded smaller cytotoxic effect and a lower radiation exposure for an expected higher tumor-killing effect. Dosimetryguided transarterial radionuclide therapy with ¹⁸⁸Re-HTDD/lipiodol in a patient with unresectable hepatocellular carcinoma was evaluated. This study showed that transarterial radionuclide therapy with ¹⁸⁸Re-HTDD/lipiodol appears to be a safe, effective and promising therapeutic option in cases of unresectable hepatocellular carcinoma with portal vein thrombosis (19, 20). The maximum tolerated activity to be safely injected in the patient was calculated to be about 8.325 GBq (225 mCi) with the lungs being the dose limiting organ. Two doses of the radiopharmaceutical resulted in the complete disappearance of a large volume tumor and the patient was disease free for 18 months. A large scale clinical trial involving 93 patients in India and Vietnam using ¹⁸⁸Re-HDD-lipiodol were conducted (21). Similarly, 35 treatments in 28 patients with ¹⁸⁸Re-HDD-lipiodol activity ranging from 4.81-7.03 GBq(130-190 mCi) has been reported (22, 23). The studies confirmed that the patients tolerated the dose and no severe complications were reported. Response assessment showed partial response in 1, stable disease in 28 and disease progression in 2 treatments. There was a significant reduction in AFP (An alpha-fetoprotein) levels measured in patients six weeks after treatment. Radiolabeling procedure and radiolabeling conditions of ¹⁸⁸Re-N₂S₂ based derivatives were presented in Table 3.

Table 2. Physical properties of ¹⁸⁸Re.

Table 3. Reported conditions of Rhenium chelating agents for optimal radiolabeling efficiencies.

¹⁸⁸Re-EDD/Lipiodol

^99mTc-ECD (Ethyl cyteinate dimer) is a known brain perfusion imaging agents and it is an approved drug.  Ethyl cyteinate dimer (ECD) was labelled with ¹⁸⁸Re instead of ^99mTc and then extracted by Lipiodol to form a new agent ¹⁸⁸Re-ECD/Lipiodol (79.77±3.0), and it found to be stable in human serum at 37ºC for at least 2 days (Figure 2) (24). Radiolabeling conditions of ¹⁸⁸ReECD/lipiodol were presented in Table 3. ¹⁸⁸Re-ECD/ Lipiodol showed high accumulation in the hepatoma (1 h: 11.19±4.11; 24 h: 7.30±2.20; 48 h: 3.55 ±1.03) was observed in hepatoma-bearing Sprague-Dawley (SD) rats after injection through the hepatic artery.

¹⁸⁸Re(III)-SSS-lipiodol

SSS-lipiodol [SSS = (S2CPh)(S3CPh)2] is an another N₂S₂ based bifunctional chelating agent developed and labeled with ¹⁸⁸Re with a very high radiochemical yields (87 ± 9.1%), showed a clear advantage over the previously mentioned radiolabeling techniques for  ¹⁸⁸Re (Figure 3). Radiolabeling conditions of ¹⁸⁸Re-SSS lipiodol were presented in Table 4. The radiochemical purity (93 ± 3.4 %), is satisfactory and the radiolabeling is stable for at least 48 h in-vitro. ¹⁸⁸Re-SSS lipiodol was injected to the hepatic artery of healthy pigs  and they were sacrificed at 1, 24 and 48 h post-injection, for ex-vivo γ-counting.  Ex-vivo γ-counting confirmed the predominantly hepatic uptake and revealed weak lung and intestinal uptake. There was very weak urinary elimination (2.3 ± 0.5% at 48 h) and a slightly higher level of intestinal elimination (4.8%±1.9% at 48 h). The autoradiographic studies showed ¹⁸⁸Re-SSS lipiodol to be located mainly in sinu-soids, like ¹³¹I-lipiodol. The main difference is represented by very weak urinary elimination and more marked intestinal elimination (25).

Table 4. Reported conditions of ¹⁸⁸Re-SSS/lipiodol for optimal radiolabeling efficiencies.

A comparative evaluation of ¹⁸⁸Re-SSS-lipodol with ¹³¹I-lipiodol in rats bearing HCC, was performed. This study shows that, in HCC-bearing rats, treatment by ¹³¹I-lipiodol is more effective compared with the ¹⁸⁸Re-SSS lipiodol/¹³¹I-lipiodol mixture or ¹⁸⁸Re-SSS lipiodol alone, because it is the only treatment that makes it possible to obtain a prolonged improvement of survival. This study also shows that treatment by ¹⁸⁸ReSSS lipiodol alone is ineffective with the HCC-tumour model used here, which is built up of a single, small tumour (26).

However, this may not be extendable to HCC in humans as the tumors are fairly large. The International Atomic Energy Agency (IAEA) conducted a multicountry Phase I/II clinical trial involving 185 patients in eight countries using ¹⁸⁸Re-HTDD/Lipiodol in which three complete responses and 19 partial responses were reported (27, 28). The overall results of the clinical studies with ¹⁸⁸Re-HTDD/Lipiodol demonstrated that it is a clinically useful agent (27, 28)  

However, potential liver leakage of activity has been a concern and there are more developments to improve the radiolabeling yields and the in vivo stability of the ¹⁸⁸Re-SSS lipiodol radiopharmaceutical. 

Figure 3. Procedure for the preparation of a lipiodol solution of ¹⁸⁸Re-labeled agents

¹⁸⁸Re-DEDC-lipiodol

Radiolabeling yields (> 95%) have been considerably improved by labeling lipiodol through rhenium(V)Nitride-bis(diethylthiocarbamate) (DEDC) complex (29). This method involves the reaction of [¹⁸⁸Re] [ReO₄]¯ with N-methyl S-methyl dithiocarbazate(DTCZ), as donor of nitrido nitrogen atoms, sodium oxalate and SnCl2 to afford a mixture of two intermediate compounds. When this mixture is reacted with the sodium salt of a dithiocarbamate ligand (L) of the type Na[R₂N-C(=S)S] (R = CH₃, CH₃CH₂, CH₃CH₂CH₂), the formation of the bis-substituted, neutral complexes [¹⁸⁸Re][Re(N)(L)2] is easily obtained in high yield (> 95%). The Re(V) nitrido precursor was prepared in high yields using a lyophilized kit formulation and the resultant complex being highly lipophilic is quantitatively extracted into lipiodol. It was found that the ¹⁸⁸Re-N-DEDC-lipiodol was highly stable in physiological solution and in rat’s blood. It selectively gets accumulated in the tumor with high target to nontarget ratios (30). Furthermore, results of transchelation experiments showed that these compounds were inert toward transchelation by cysteine and glutathione (29).

The results of the initial clinical trials showed that this could be a useful radiopharmaceutical for the therapy of unresectable hepatocellular carcinoma. 

Automated preparation of ¹⁸⁸Re-DEDC-lipodol

An automated synthesis of ¹⁸⁸Re-lipodol was first reported by Uccelli et al. (Figure 4) (31). Automated modular preparation of ¹⁸⁸Re-DEDC-lipodol allows easy preparation of sterile and pyrogen-free samples of ¹⁸⁸Re-lipiodol ready to be administered to the patient. Important advantages include the possibility to incorporate high ¹⁸⁸Re activity into the lipiodol hydrophobic phase and a sharp reduction of radiation exposure of the operator assisting the labelling procedure. The flowchart of the automated synthesis is described in Fig. 6. Briefly, Generator-eluted [¹⁸⁸Re] [ReO₄]Na was first collected from the ¹⁸⁸W/¹⁸⁸Re generator onto a vial (collecting vial), and initial activity (5.1±1.2 GBq; n=10) counted in a β-counter. This vial was, then, inserted into the automated system. Activity was transferred from the collecting vial to a tandem cation-anion concentration system by mean of a peristaltic pump (P). This system was made by three alumina cationic exchange cartridges placed in series (SCE) followed by one anionic exchange cartridge (QMA). After the [¹⁸⁸Re]perrhenate solution passed through the tandem concentration system, activity remained quantitatively trapped into the anionic column (32-34). The accumulation of [¹⁸⁸Re][ReO₄] Na onto the anionic column was monitored by a small radiation-sensitive photodiode detector (Det1). This concentration system was used only once and discarded after use. The [¹⁸⁸Re]perrhenate adsorbed onto the QMA cartridge was then subsequently eluted with 3.0 ml of saline (V2) and transferred to vial A. Before insertion of vial A into the automated system, 0.1 ml of glacial acetic acid were added under sterile conditions using an insulin-type syringe. The resulting solution was heated at 80°C for 5 min and then transferred to the conicbottom glass reactor vial (R). The content of vial B was dissolved by transferring 1.5 ml of sterile water (V3). The resulting solution was, then, introduced into the reactor vial (R), and the mixture heated at 80°C for 20 min to afford the final complex ¹⁸⁸ReN-DEDC. After cooling, the reaction solution was passed through a SepPak C18 cartridge onto which the radioactive complex was retained. Accumulation of the radioactive product on the C18 column was monitored using another small radiation-sensitive photodiode detector (Det2). The cartridge was first washed with 20 ml of water (V4) followed by 1.5 ml ethanol/water (50:50 v/v) (V5). The product was recovered by washing the cartridge with 2.0 ml of ethanol (V6), and the resulting solution passed through a sterile 0.22-μm membrane filter and collected in vial C. Residual ethanol was removed by heating vial C at 100°C, under a nitrogen stream. Lipiodol (2.0 ml) was finally added to vial C to dissolve the radioactive compound. The total time for completing the whole procedure was 80 min. Application of this modular reaction system could be also extended to the preparation of other ¹⁸⁸Re- radiopharmaceuticals and to compound labelled with different β-emitting therapeutic radionuclides.

¹⁸⁸Re(N)(cys) (PNP)

Synthesis, characterization and biological evaluation of a ¹⁸⁸Re(N)(cys) (PNP) mixed ligand complex for the preparation of ¹⁸⁸Re-lipiodol have been reported (Figure 5) (35). Radiolabeling conditions of ¹⁸⁸Re-PPP derivatives were presented in Table 5. [¹⁸⁸Re(N)(cys ~)(PNP)]+/0 mixed-ligand compounds were efficiently prepared in aqueous solution from perrhenate using a multistep procedure based on the preliminary formation of the labile ¹⁸⁸ReIII–EDTA species, which easily undergo oxidation/ligand exchange reaction to afford the [¹⁸⁸Re^V≡N]²⁺ core in the presence of dithiocarbazate. The final mixed-ligand compounds were obtained, at 100°C, by adding the two bidentate ligands to the buffered [¹⁸⁸Re^V≡N]²⁺ solution (pH 3.2– 3.6). However, a relatively high amount of cys~ ligand was required to obtain a quantitative radiochemical yield. The complexes were stable toward reoxidation to perrhenate and ligand exchange reactions. In vivo studies showed rapid distribution and elimination of the complexes from the body. No specific uptakes in sensitive tissues/organs were detected (35). The continued developments in the preparation and production of ¹⁸⁸Re-lipiodol show the interest in this product as it addresses the management of a widely prevalent cancer.

Table 5. Reported conditions of various ¹⁸⁸Re-PNP derivatives for optimal radiolabeling efficiencies.

Conclusion

Rhenium-188 is produced by an ¹⁸⁸W/¹⁸⁸Re onsite generator in a convenient and inexpensive way in the majority of hospitals. Usage of Re-188 radiopharmaceuticals could also enhance therapeutic efficacy to other malignancies (e.g., nonresepctable liver cancer, nonmelanoma skin cancer, and breast cancer) as well as treatment of arthritis and inhibition of arterial restenosis. Therapeutic ratios in these cases could provide a powerful and reliable tool for the estimation of treatment benefits. Therefore, ¹⁸⁸Re has a great potential for radionuclide therapy in field of nuclear medicine and will be needed researchers great efforts.