Development of Semiautomated Module for Preparation of 131 I Labeled Lipiodol for Liver Cancer Therapy

Abstract

Intra-arterial injection of ¹³¹I Lipiodol is an effective treatment option for primary hepatocellular carcinoma as it delivers high radiation dose to liver tumor tissue with minimal accumulation in adjacent normal tissue. The present article demonstrates design, fabrication, and utilization of a semiautomated radiosynthesis module for preparation of ¹³¹I labeled Lipiodol. The radiolabeling method was standardized for preparation of patient dose of ¹³¹I labeled Lipiodol radiochemical yield (RCY); radiochemical purity (RCP) and pharmaceutical purity of the product were determined using optimized procedures. Sterile and apyrogenic ¹³¹I labeled Lipiodol in >60% RCY could be prepared with >95% RCP. Preclinical evaluation in animals indicated retention of more than 90% of activity at 24 hours postportal vein injection. This is the first report demonstrating potential application of simple user friendly and safe semiautomated system for routine production of ¹³¹I labeled Lipiodol, which is adaptable at centralized hospital radiopharmacies. The described prototype module can be modified as per demand for preparation of other therapeutic radiopharmaceuticals.

Introduction

Hepatocellular carcinoma (HCC) is a major form of malignant disease^1
–3 especially in the Asian and African regions, where more than 95% of patients do not survive 5 years past the initial diagnosis.^4,5

Transarterial chemoembolization (TACE) has been successfully practiced for treating HCC for more than three decades. While the majority of normal hepatic blood supply is through the portal vein, neoangiogenic vessels primarily connect to the hepatic artery; hence therapeutic agents administered intra-arterially get preferentially localized in regions of tumor. This is the underlying principle of TACE. Drugs like cisplatin, doxorubicin, methotrexate, and paclitaxel have been used as single agents or in different combinations, in conjunction with Lipiodol for locoregional therapy.^6,7 In this study, Lipiodol serves as both drug carrier and embolizing agent. Intra-vassal retention of Lipiodol leads to starvation of tumor cells of nutrient and oxygen supply and delivers high doses of the drug(s) locoregionally, which provides greater chemotherapeutic effect than by the systemic route. Embolization in conjunction with a radiotherapy agent is called radioembolization. There are two main categories of radioembolic agents approved for clinical use.^8,9 First category is based on micron-range particulates that encapsulate or adsorb therapeutic radionuclides like ⁹⁰Y-bearing glass spheres (TheraSphere^®) and polymeric SIR-Spheres^® and another is Lipiodol or related embolic substances tagged with therapeutic radioactivity. Lipiodol or ethiodized oil is a naturally iodinated fatty acid ethyl ester of poppy seed oil (37%w/w of iodine). It is used as a magnetic resonance imaging contrast agent for the liver and has also been labeled with therapeutic radionuclides such as ¹³¹I and ¹⁸⁸Re for HCC treatment.¹⁰ ¹³¹I is easily produced by irradiation of Tellurium target in nuclear reactors, and the dry distillation process for ¹³¹I production leads to availability of large quantities of ¹³¹I on regular basis. Suitable half-life of ¹³¹I permits convenient logistics of production and quality control checks before product release.

Standard activity of 2.22 GBq (60mCi)¹³¹I labeled Lipiodol in 2–10 mL volume is administered by slow intra-arterial injection under fluoroscopic guidance as per European Association of Nuclear Medicine (EANM) guidelines for Lipiodol therapy.¹¹ ¹³¹I labeled Lipiodol would be best adapted for patients with small tumors dispersed in the liver to deliver high-dose radiation to the tumor and little radiation to the surrounding liver. Lipiocis^® is licensed in France for the treatment of patients with HCC and portal vein thrombosis. Low cost of ¹³¹ I labeled Lipiodol compared to other agents makes it a promising therapeutic agent especially for cancer patients in developing countries.¹² The high-energy gamma radiations (0.364 MeV, 81% abundance) of ¹³¹I pose considerable safety related limitations for preparation of patient doses of ¹³¹I labeled Lipiodol with high amounts of initial activity (>3.7 GBq) of ¹³¹I. Although automated modules for radiosynthesis are widely reported for preparation of positron emission tomography agents, comparatively fewer reports are available on preparation of therapeutic radiopharmaceuticals using automated or semiautomated modules.^13,14 Hence, a semiautomated modular system was designed and fabricated to ensure operator safety, as well as pharmaceutical purity and safety of the product. Quality control tests were carried out to estimate radiochemical yield (RCY), radiochemical purity (RCP), and to determine sterility and apyrogenicity of the preparation. Preclinical evaluation was done in Wistar rats to ascertain liver retention of ¹³¹I labeled Lipiodol.

Materials and Methods

Lipiodol^® Ultra Fluid 4.8 g Iodine per 10 mL (38% w/w) was procured from Guerbet (Asia Pacific). No carrier added ¹³¹I-Sodium iodide solution of >99.99% chemical purity, specific activity ∼18.5 GBq/mL radioactive concentration, and >99.9% radionuclidic purity were produced as reported.¹⁵ Analytical grade absolute ethanol (Assay: >98.5%) was from Brampton, Canada. Whatman 3MM paper was used for paper chromatography. All glassware and accessories used for production were cleaned and processed to render them sterile.

Radioactivity assay of ¹³¹I activity was carried out by high-resolution gamma ray spectrometry using an HPGe detector (EGG Ortec/Canberra detector) coupled to a 4K multichannel analyzer system. All other radioactivity measurements were carried out using ion chamber or NaI (Tl) scintillation counter (Mucha, Raytest, Germany). Sterility Test Kits containing fluid thioglycollate media (FTG) and soybean casein digest media (SCD) were obtained from HiMedia Laboratories, India, and Endotoxin standards and LAL reagent pyrogen-free water for endotoxin detection were procured from Charles River Laboratories India Pvt. Ltd.

Design and fabrication of semiautomated module

An independent stand alone modular system was designed suitable to be fitted in a mini hot cell. The module holds two lead pots, one containing Na¹³¹I vial and the other housing the reaction vessel. The reaction vessel assembly was enclosed in silicon glycerin bath. Facility was made for remote addition of reagents to vial containing Na¹³¹I activity, as well as to reaction vessel. The heating of reaction vessel was precisely carried out using thermocouple. All transfers were carried out by tubing connected to solenoid valves, which are mounted on plate and connected to junction box; inert nitrogen gas of high purity was used for pressurizing vessels. The module was placed inside indigenously designed and fabricated shielded facility similar to commercially available mini hot cell with adequate lead shielding having negative pressure and charcoal filters fitted at release duct. Electrical valve operations and heating controls were placed separately on control panel. Valve operation sequence was standardized based on optimized reaction parameters. Radiation field, air activity, and activity released through charcoal trap were monitored during all operations. Several batches of ¹³¹I labeled Lipiodol were prepared using standardized operating protocol.

Preparation of ¹³¹I labeled lipiodol

Isotope exchange reaction between organic iodine of Lipiodol with ionic ¹³¹I was carried out with slight modifications in the reported procedure.¹⁶ The initial standardization of optimum time and temperature was carried out using trace amounts of radioiodine keeping same volume of aqueous ethanol and Lipiodol estimated for patient dose preparation.

Typically 2 × 2 mL of absolute ethanol was injected to the ¹³¹I activity vial (∼3.7 GBq in 0.1 mL) followed by transfer of complete activity into the reaction vessel reactor and heating at 55°C for 15 minutes under nitrogen. Two milliliters Lipiodol was injected directly into the reaction vessel, and the exchange reaction was carried out by heating Lipiodol with ¹³¹I in ethanol at 80°C for 20 minutes and, subsequently, at 100°C for 30 minutes.

The reaction mixture was allowed to cool and additional 1 mL Lipiodol was added to the reaction vial and mixed, and the product was transferred to product vial through 0.22 μ sterile filter. Five hundred microliters of sample was transferred to two other similar vials for quality control analyses. The reaction was monitored for ¹³¹I activity measurement at all the stages of production. The percentage reaction yield was calculated by measuring activity in product vials compared to starting activity by decay correction.

Test for radionuclide identification

The gamma-ray spectrum of the preparation was taken as per standard procedure of gamma ray spectrometry using a high-purity germanium crystal or Ge (Li) crystal assembly. The most prominent gamma photon of 0.364 MeV energy was examined to estimate radionuclide purity of ¹³¹I.

Test for RCP

The RCP of ¹³¹I labeled Lipiodol was determined by paper chromatography. Two microliter aliquots of carrier solution (1.5% KI) and sterile product were spotted on Whatman 3MM paper chromatography strips, which were then developed in two different solvent systems, 85% methanol and ether/petroleum ether (1:2), respectively, by ascending chromatography. The movement of ionic I-131 and the radiolabeled product was measured using NaI (Tl) detector with suitable energy window.

Stability

The product was stored at 4°C protected from light and tested repeatedly for RCP using paper chromatography technique for up to 3 weeks.

Sterility and bacterial endotoxin testing

To ensure pharmaceutical purity of the product, sterility and bacterial endotoxin testing (BET) was carried out as per Indian Pharmacopeia procedures listed in General Chapter on Radiopharmaceuticals.¹⁷ 0.1 mL of ¹³¹I labeled Lipiodol was diluted with 1.9 mL Lipiodol Ultra Fluid injection under aseptic conditions and used for sterility and BET.

Biological evaluation

In vivo distribution studies of ¹³¹I labeled Lipiodol were carried out in normal adult Wistar rats (male, ∼200–225 g). The animals were fasted for 6 hours before the procedure. Approximately 100 μL of ¹³¹I labeled Lipiodol was administered through portal vein following a previously reported protocol of viable surgery under anesthesia.¹⁸ Animals were sacrificed at different time points, and percentage of injected activity associated with different organs/tissue was determined.

Results and Discussion

Design and fabrication of semiautomated module

The simple remote operating procedures standardized for production of ¹³¹I labeled Lipiodol considerably reduce dose exposure to the radiation worker and also assure pharmaceutical purity and safety of product. Figure 1 depicts the schematics of the semiautomated synthesis module.

FIG. 1.

Schematics of the semiautomated synthesis module for production of ¹³¹I labeled Lipiodol. B, bubbler; F, sterile membrane filter; H, heater; N, Nitrogen gas inlet; P, sterile apyrogenic product vial; R1, vial containing Na¹³¹I placed inside a lead pot; R2, reaction vessel; S1 and S2, syringe ports; T, thermocouple; V, vent.

Actual photograph of the module with two lead pots, valve mounting plate, junction box, etc. is shown in Figure 2. The safety features that were included while designing the module resulted in significantly reducing the radiation exposure to the operator. The cleaning and drying procedures of module carried out before synthesis and collection of final product in sterile pyrogen-free vial through sterile membrane filter ensured compliance with good manufacturing practices. The optimized cleaning protocol after batch preparations removes most of the residual activity, which aids in maintenance of module parts.

FIG. 2.

Actual photograph of the semiautomated synthesis module inside shielded facility for production of ¹³¹I labeled Lipiodol.

Preparation of ¹³¹I labeled lipiodol

It was observed that under controlled nitrogen flow, complete drying of aqueous alkaline solution of Na ¹³¹I in ethanol occurs within 10 minutes of heating at 55°C. The radiolabeling yields varied between 50% and 70% when 2 mL of Lipiodol was used. The yield was found to be consistent when temperature was increased from 80°C to 100°C, however longer time heating at 100°C did not result in substantial increase in yields. Diluting reaction mixture with additional 1 mL Lipiodol resulted in >80% recovery of the product from the reaction vessel without compromising quality.

The activity distribution determined as in-process quality control (QC) parameter showed low retention (<3% to 5%) of Na¹³¹I in activity vial, indicating almost complete transfer to reaction vessel. The activity recovered in ethanol during distillation was ∼5% to 7%, and ∼10% to 15% of the activity was trapped in charcoal. The sterile syringe filter used for terminal filtration showed <2% retention of the product.

Evaluation of ¹³¹I labeled Lipiodol

The principal gamma photon energy peaks of 364 and 637 keV (±5 keV) were observed for all the samples of ¹³¹I labeled Lipiodol in gamma ray spectrum fulfilling the test of radionuclide identification. Table 1 depicts RCYs (60%–70%) obtained when production of ¹³¹I labeled Lipiodol was carried out by varying ¹³¹I activity from 222 to 5180 MBq. RCP of the product was >95% when estimated by paper chromatography in two different solvent systems. In 85% methanol as solvent, ¹³¹I labeled Lipiodol shows R_f of 0–0.2, while R_f of free Iodide is 0.8–0.9. Another solvent system, ethyl ether/petroleum ether, wherein ¹³¹I labeled Lipiodol shows R_f of 0.9 and R_f of free Iodide as 0.0–0.1, was used for comparison. RCP values of different batches of ¹³¹I labeled Lipiodol estimated on next day and 2 weeks after preparation are tabulated in Table 2. The final specifications and QC acceptance criteria are given in Table 3. The stability of the product evaluated over a period of 3 weeks ascertains the logistics of supply to other nuclear medicine centers.

Table 1.

Scaling Up of Radiosynthesis of ¹³¹I Labeled Lipiodol Under Optimized Conditions with Semiautomated Module

Batch no.	Na¹³¹I activity in MBq (before batch preparation)	¹³¹ I-labeled Lipiodol produced in MBq	% RCY
1	222	141	63.3
2	600	410	68.5
3	1685	1147	68
4	2812	1776	63
5	3053	2138	70
6	3774	2571	68
7	4810	3248	67.4
8	5180	3675	71

RCY, radiochemical yield.

Table 2.

Radiochemical Purity of ¹³¹I Labeled Lipiodol

	% ¹³¹I labeled Lipiodol
Batch. no.	Next day of preparation	After 2 weeks
1	95.1 ± 1.2	94.6 ± 1.1
2	97.2 ± 1.6	96.3 ± 2.6
3	97.3 ± 0.9	98.2 ± 1.9
4	97.1 ± 1.2	96.6 ± 2.1
5	97.0 ± 1.6	97.9 ± 3.3
6	95.6 ± 3.8	96.3 ± 2.9
7	96.2 ± 2.9	97.7 ± 1.5
8	95.6 ± 3.2	96.2 ± 2.7

The figures are rounded off to the nearest integers to one decimal point.

RCP, radiochemical purity.

Table 3.

Specifications/Acceptance Criteria of ¹³¹I Labeled Lipiodol Injection

Product	¹³¹ I labeled Lipiodol
Description	¹³¹I labeled Lipiodol is a ready to use sterile, pyrogen-free injectable preparation. The preparation contains ¹³¹I iodinated ethyl esters of fatty acids of poppy seed oil (¹³¹I labeled Lipiodol)
Appearance	Clear yellow to light brown liquid
Radionuclide identification	Principal energy peaks 364 keV(γ)
RNP	>99.9%
RCP	>95%
Radioactive concentration	740–925 MBq/mL
Sterility test	Complies with i.p. (no microbial growth till 14 days in FTG and SCD growth mediums)
BET	Complies with i.p. (<175 EU per total volume or 25 EU per mL)
Biodistribution/imaging	∼90% retention of activity in the liver up to 24 hours
Expiry 7 days from the date of reference (preparation)

BET, bacterial endotoxin testing; FTG, fluid thioglycollate medium; RNP, radionuclide purity; SCD, soybean casein digest medium.

There are not many reports on preclinical animal biodistribution studies of ¹³¹I labeled Lipiodol. Hence our pharmacokinetic studies extended till 5 day postinjection gives more insight to the in vivo fate of the product. The results of in vivo distribution studies are given in Table 4. At 24 hours, more than 90% of injected ¹³¹I activity was associated with the liver with negligible activity in the nontarget tissues and organs. Good localization and retention of the labeled preparation in the hepatic tissue were observed. At 3 days postsurgery, around 80% of ¹³¹ I labeled Lipiodol was retained in the liver, while after 5 days liver activity decreased to 70% compared to >90% at 24 hours. Normal liver contains specific macrophages (Kupffer cells), which are known to metabolize Lipiodol and will cause its release from the liver over a period of days.¹⁹ It is expected that in HCC tissue, which lacks macrophages, such loss of radiolabeled Lipiodol will not be observed ensuring retention and in vivo stability of the product after locoregional intravascular application. Other organs did not show any appreciable accumulation of the activity, and most of the activity was excreted through urine. It is notable that the thyroid, known to absorb iodine from the bloodstream, did not harbor any mentionable radioactivity from the preparation, indicating that ¹³¹I-iodine remains stably associated with Lipiodol.

Table 4.

In Vivo Distribution Data (%ID/Organ) of ¹³¹I Labeled Lipiodol in Normal Rats (n = 4)

Organ/tissue	1 Day Avg ± SD	3 Days Avg ± SD	5 Days Avg ± SD
Liver	93.87 ± 1.66	77.81 ± 7.61	69.99 ± 6.56
Intestine	0.62 ± 0.04	2.81 ± 1.02	1.31 ± 0.51
Stomach	0.41 ± 0.04	1.41 ± 0.19	1.27 ± 0.99
Kidney	0.10 ± 0.02	0.40 ± 0.22	0.17 ± 0.19
Heart	0.02 ± 0	0.15 ± 0.05	0.00 ± 0.00
Lungs	0.04 ± 0.02	0.80 ± 0.49	0.73 ± 1.01
Spleen	0.03 ± 0.01	0.06 ± 0.05	0.17 ± 0.29
Muscle	1.73 ± 0.76	1.09 ± 0.39	1.69 ± 0.40
Blood	0.35 ± 0.16	1.15 ± 0.94	0.22 ± 0.29
Bone	0.60 ± 0.13	0.59 ± 1.03	0.00 ± 0.00
Thyroid	0.13 ± 0.01	0.45 ± 0.04	0.19 ± 0.05
Urine	2.13 ± 0.62	13.72 ± 7.21	28.04 ± 5.89

SD, standard deviation.

Conclusions

To the best of our knowledge this is the first report on successful radiosynthesis of an ¹³¹I labeled Lipiodol utilizing semiautomated synthesis module useful in nuclear medicine setups where access to a ¹⁸⁸W-¹⁸⁸Re generator is economically or logistically inconvenient.¹³¹I labeled Lipiodol can be easily and safely synthesized by installing such modules inside mini hot cells in centralized or hospital radiopharmacy. Preparation of other therapeutic radiopharmaceuticals can also be envisaged using similar modules for radiosynthesis.

Footnotes

Acknowledgments

Research at the Bhabha Atomic Research Centre (BARC) is part of the ongoing activities of the Department of Atomic Energy, India, and is fully supported by government funding. The authors express their sincere thanks and gratitude to Dr. M. R. A. Pillai, former Head, Radiopharmaceuticals Division, and Dr. Grace Samuel, former Head, RPES, Radiopharmaceutical Division for their encouragement during this work. The authors also gratefully acknowledge officers and staff of irradiation section and health physicists at BARC for their help during the course of the work.

Disclosure Statement

Research at Bhabha Atomic Research Centre is fully supported by Government of India funding. The authors do not have any conflict of interest.

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Development of Semiautomated Module for Preparation of 131 I Labeled Lipiodol for Liver Cancer Therapy

Abstract

Introduction

Materials and Methods

Design and fabrication of semiautomated module

Preparation of 131I labeled lipiodol

Test for radionuclide identification

Test for RCP

Stability

Sterility and bacterial endotoxin testing

Biological evaluation

Results and Discussion

Design and fabrication of semiautomated module

Preparation of 131I labeled lipiodol

Evaluation of 131I labeled Lipiodol

Conclusions

Footnotes

Acknowledgments

Disclosure Statement

References

Preparation of ¹³¹I labeled lipiodol

Preparation of ¹³¹I labeled lipiodol

Evaluation of ¹³¹I labeled Lipiodol