[ 90 Y]Yttria Alumino Silicate Glass Microspheres: A Biosimilar Formulation to “TheraSphere” for Cost-Effective Treatment of Liver Cancer

Abstract

Background:

Selective internal radiation therapy (SIRT) using a suitable β⁻-emitting radionuclide is a promising treatment modality for unresectable liver carcinoma. Yttrium-90 (⁹⁰Y) [T _1/2 = 64.2 h, E _β(max) = 2.28 MeV, no detectable γ-photon] is the most preferred radioisotope for SIRT owing to its favorable decay characteristics.

Objective:

The present study describes indigenous development and evaluation of intrinsically radiolabeled [⁹⁰Y]yttria alumino silicate ([⁹⁰Y]YAS) glass microsphere, a formulation biosimilar to “TheraSphere” (commercially available, U.S. FDA-approved formulation), for SIRT of unresectable liver carcinoma in human patients.

Methods:

YAS glass microspheres of composition 40Y₂O₃-20Al₂O₃-40SiO₂ (w/w) and diameter ranging between 20 and 36 μm were synthesized with almost 100% conversion efficiency and >99% sphericity. Intrinsically labeled [⁹⁰Y]YAS glass microspheres were produced by thermal neutron irradiation of cold YAS glass microspheres in a research reactor. Subsequent to in vitro evaluations and in vivo studies in healthy Wistar rats, customized doses of [⁹⁰Y]YAS glass microspheres were administered in human patients.

Results:

[⁹⁰Y]YAS glass microspheres were produced with 137.7 ± 8.6 MBq/mg YAS glass (∼6800 Bq per microsphere) specific activity and 99.94% ± 0.02% radionuclidic purity at the end of irradiation. The formulation exhibited excellent in vitro stability in human serum and showed >97% retention in the liver up to 7 d post-administration when biodistribution studies were carried out in healthy Wistar rats. Yttrium-90 positron emission tomography scans recorded at different time points post-administration of customized dose of [⁹⁰Y]YAS glass microspheres in human patients showed near-quantitative retention of the formulation in the injected lobe.

Conclusions:

The study confirmed the suitability of indigenously prepared [⁹⁰Y]YAS glass microspheres for clinical use in the treatment of unresectable hepatocellular carcinoma.

Introduction

Primary as well as metastatic liver malignancies have emerged as serious heath challenge and, currently, one of the most prevalent causes of cancer-related deaths worldwide.^1

–4 Among the different types of liver malignancies, hepatocellular carcinoma is the most prevalent and accounts for around 90% of liver cancer cases globally.^2,4,5 Selective internal radiation therapy (SIRT) using a suitable β⁻-emitting radionuclide is one of the most promising treatment modalities of unresectable liver carcinoma.^6

–11 Intrinsically [⁹⁰Y]Y-labeled glass microsphere formulation is the most widely used radiotherapeutic agent for SIRT.^6,10,11

Yttrium-90 (⁹⁰Y) is a pure β⁻-emitter with a physical half-life of 64.2 h and decays to stable ⁹⁰Zr by near exclusive β⁻-particle emission with maximum and average energy of 2.28 MeV and 937 keV, respectively.^10,11 The particulate emission from ⁹⁰Y has mean tissue penetration of 2.5 mm and a maximum of 11 mm,¹¹ thereby effective in delivering cytotoxic dose to a large tumor mass. The [⁹⁰Y]Yttria glass microsphere formulation that is most extensively used in SIRT across the globe is sourced from a commercial manufacturer in the name of TheraSphere^®.¹¹ It is a radiochemical formulation consisting of millions of nonbiodegradable glass microspheres, having diameter in the range of 20–35 μm, in sterile physiological saline, and in which ⁹⁰Y is an integral constituent of the glass.¹²

For SIRT, [⁹⁰Y]Yttria glass microspheres are administered via intra-arterial route through hepatic artery. Malignant liver tumors are generally highly vascular and receive the majority of their blood supply from the hepatic artery (80%–100%), compared with liver parenchyma, which receives its blood supply primarily from the portal vein. Therefore, the intra-arterial injection of [⁹⁰Y]Yttria glass microspheres would primarily deliver ionizing radiation dose to the tumor in local or regional manner, whereas the dose to liver parenchyma would be considerably lower as hepatic artery provides only ∼25% of total hepatic blood flow.^13,14 Once administered in the hepatic artery, the microspheres preferentially lodge in the vasculature of the malignant hepatic cells and deliver cytotoxic doses of ionizing radiation from ⁹⁰Y, normally around 50–1000 Gy in the treated liver tumor depending on size with median of ∼200 Gy.¹⁵

One of the major constraints in the broader utility of [⁹⁰Y]Yttria glass microsphere in the treatment of liver cancer is the prohibitively high cost of the dose of commercially available formulation for individual patient care. In India, the cost of treatment using ready-to-use formulation of TheraSphere imported from commercial manufacturer is $10,000–12,000, depending on the dose size. Taking it into consideration, it was felt pertinent to develop a new [⁹⁰Y]Yttria glass microsphere formulation, which will be biosimilar to TheraSphere and can be made available at an affordable cost (within $1000 for each dose).

In this direction, yttria alumino silicate (YAS) glass microspheres having 20Al₂O₃-40SiO₂-40Y₂O₃ (w/w) chemical composition and particle size in the range of 20–36 μm have been synthesized following the procedure developed indigenously and characterized. Intrinsically ⁹⁰Y-labeled [⁹⁰Y]YAS glass microspheres were produced by thermal neutron irradiation of cold microspheres at a suitable thermal neutron flux in a research reactor. In the present study, the authors report in detail the systematic approach following which intrinsically radiolabeled [⁹⁰Y]YAS glass microsphere formulation, suitable for human clinical applications, was developed and evaluated as a biosimilar formulation to TheraSphere.

Experimental

Materials and equipment

Yttrium oxide, alumina, and silica used for the preparation of YAS glass were of spectroscopic grade and procured from Merck, GmbH. All chemicals and reagents used were of analytical grade. Medical grade sterile saline and water for injection were used. High purity irradiation grade quartz tubes were used for encapsulation of YAS glass microspheres for irradiation.

Powder X-ray diffractometer (XRD; Bruker D8 Discover Instrument) with collimated CuKα radiation source was performed to confirm the glassy nature of the prepared glass and glass microspheres. Scanning electron microscope (SEM) images of the glass microspheres were acquired using SEM model: Stereoscan S-240 (Cambridge, United Kingdom). Chemical analysis was carried out by energy dispersive X-ray fluorescence (EDXRF) technique (EDXRF spectrometer EX 3600M; Xenemetrix, Israel), having Rh-anode X-ray tube and in-built acquisition and analysis software. Radioactivity of [⁹⁰Y]Y-YAS glass microspheres produced was measured using a precalibrated isotope dose calibrator (Curiementor 3; PTW-Freiburg, Germany).

Radionuclidic purity of [⁹⁰Y]YAS glass microsphere formulation was ascertained by γ-ray spectrometry using a HPGe detector (Canberra Eurisys, France) coupled to a PC-based 4K channel analyzer (MCA). A ¹⁵²Eu reference source, obtained from Amersham, Inc., was used for energy and efficiency calibration of the HPGe detector. All the nuclear data used were taken from Table of Isotopes.¹⁶ All other radioactivity measurements were carried out using a well-type NaI(Tl) scintillation counter (Mucha, Raytest, Germany), unless mentioned otherwise.

Synthesis of YAS glass microspheres

Glass preparation

Yttrium alumino silicate glass having composition 40Y₂O₃-20Al₂O₃-40SiO₂ (wt%) was prepared by melt-quench process. High purity chemicals were used to ensure that chemical impurities which can be activated under neutron irradiation are avoided in raw material. Each batch of 100 g charge was prepared by weighing calculated amounts of initial constituents in the form of oxides. The batch was mixed thoroughly and heated at 110°C overnight to remove any moisture present. The mixture was melted in a Pt-Rh crucible in electrically heated R-L furnace at around 1550°C–1650°C and held for sufficient time to obtain a clear melt free from any air bubbles.

The melt was stirred and held for sufficient time to obtain a clear melt free from any air bubbles. The melt was removed from the furnace and quenched by pressing in between two metal plates to obtain small pieces of cracked glasses. The glassy nature of the prepared material was confirmed by XRD. Furthermore, the glass frits were slowly ground and sieved through 20 and 36 μm sieves to collect the feed particles in between 20 and 36 μm size range. The crushing and sieving processes were carried out repeatedly to collect sufficient amount of the feed particle of required sizes for spheroidization. At final stage, the feed particles are cleaned using air jet to remove any dust particle present with the feed particles.

Flame spheroidization

The glass feed particles are converted into glass microspheres by introducing them into oxy-hydrogen (O₂-H₂) flame. Before exposure to flame, glass particles were heated to remove moisture and agglomeration. The glass particles are directed into the flame through a feeder vibrated manually. The flame was conditioned by passing H₂:O₂ at a ratio of 2:1. The glass particles were exposed to the flame for a short time for melting and allowed to spheroidize due to surface tension followed by rapid cooling. The flame is directed into a quartz tube/container that collects the glass microspheres after being expelled from the flame. The schematic of flame spheroidization process is shown in Figure 1.

FIG. 1.

Schematic diagram of flame spheroidization process for synthesis of YAS glass microspheres. YAS, yttria alumino silicate.

Post-synthesis treatment

After the spheroidization of glass particles, the glass microspheres were sorted by sieving to obtain the particles of desired size range of 20–36 μm. The separated glass microspheres of 20–36 μm size range were further screened to remove the particles with air bubbles and other defects. Glass microspheres with microbubbles were screened by sedimentation technique using heavy liquids of suitable density. After the removal of defective particles, the glass microspheres were subsequently cleaned with acetone, dried, and finally heated in a furnace to remove any organic impurities.

Physicochemical characterization

YAS glass microspheres prepared were observed under SEM to check for sphericity, size, and visible defects. XRD and small-angle X-ray spectra (SAXS) were recorded before and after neutron irradiation to observe the glassy nature of the microspheres and evaluate the surface changes of the glass microsphere after irradiation. Analysis of chemical composition of various batches of YAS glass microspheres was carried out using EDXRF and inductively coupled plasma optical emission spectroscopy (ICP-OES) techniques.

Formulation of [⁹⁰Y]YAS glass microspheres

Irradiation

Depending on dose requirement, 20–60 mg of YAS glass microspheres were taken in a quartz ampoule [5 mm (ϕ) × 12 mm (l)], and the ampoule was flame sealed (Fig. 2a). The sealed quartz ampoule containing the glass microspheres was irradiated at a thermal neutron flux of ∼1.4 × 10¹⁴ n·cm⁻²·s⁻¹ for 7 d, after placing inside a cold-pressure-weld type cylindrical 1S aluminum container of dimension 22 mm and length 44 mm (Fig. 2b).

FIG. 2.

(a) YAS glass microspheres sealed in a quartz ampoule, (b) 1S aluminum irradiation container, and (c) sealed quartz container with [⁹⁰Y]YAS glass microspheres dose formulation. YAS, yttria alumino silicate.

Dose formulation

The irradiated glass microspheres contained in sealed quartz ampoules were allowed to cool for 24 h after the end of irradiation, and the ampoules were retrieved from containers after cutting open the Al irradiation container by remote operation inside a 50 mm lead-shielded chamber. Subsequent handling of irradiated microspheres was carried out in a leak-tight glove box of the dimension 1.8 × 1.8 × 0.9 m³ equipped with neoprene gloves and other associated accessories. The accessories include remotely operated pipette, vortex mixer, standard glass vial station, semi-automatic glass vial sealing unit, and so on. High efficiency particulate air filters were provided in the exhaust line, and aseptic environment was maintained inside the glove box.

The quartz ampoule was broken carefully inside the glove box, and the irradiated glass microspheres were transferred into a sterile tapered-bottom quartz container, as shown in Figure 2c. The glass microspheres were washed twice using 1 mL of sterile physiological saline in each occasion by remote operation. Finally, 0.6 mL of sterile physiological saline was added to the [⁹⁰Y]YAS glass microspheres, sealed, and autoclaved (20 min at ∼121°C and 15 psi pressure).

Quality control of [⁹⁰Y]YAS glass microspheres

[⁹⁰Y]YAS glass microsphere formulation prepared was subjected to the following quality control tests.

Activity assay

The radioactivity content of [⁹⁰Y]YAS glass microsphere formulation was measured by a precalibrated isotope dose calibrator. The ⁹⁰Y activity was recorded in gigabecquerel from the measurement.

Specific activity

The specific activity of the formulation was calculated as ⁹⁰Y activity (GBq) produced per milligram of YAS irradiated for dose formulation.

Radionuclidic purity

The radionuclidic purity was determined by recording γ-ray spectra of an appropriate aliquot withdrawn from [⁹⁰Y]YAS glass microsphere formulation using an HPGe detector coupled to a 4 K MCA system. A ¹⁵²Eu reference source (Amersham, Inc.) was used for both energy and efficiency calibration of the detector. All the nuclear data used were taken from Table of Isotopes.¹⁶ Gamma ray spectra of aliquots of [⁹⁰Y]YAS glass microsphere formulation were recorded for 1 h at regular time intervals. Aliquots were preserved for more than 10 half-lives of ⁹⁰Y, and spectra were recorded for 24 h to determine the activity of long-lived radionuclide impurities produced.

Radiochemical purity

The radiochemical purity of [⁹⁰Y]YAS glass microsphere formulation was determined in the following way. The radioactive glass microspheres in 0.6 mL physiological saline were allowed to settle completely by incubating for ∼30 min. Subsequently, an aliquot (typically, 0.1 mL) was withdrawn from the supernatant and ⁹⁰Y activity of the aliquot was measured. The percent radiochemical purity was determined from the activity data using the following equation: $% R a d i o c h e m i c a l p u r i t y = (100 - A_{s} \times 6 ∕ A_{t})$ (1)

where A _s and A _t are the background corrected ⁹⁰Y activities associated with 0.1 mL of supernatant withdrawn and total activity of the [⁹⁰Y]YAS dose formulation, respectively.

Sterility

Sterility of the [⁹⁰Y]YAS glass microsphere formulation was tested by the direct inoculation method following the approved Indian Pharmacopeia (IP) procedure. The test was carried out in “Fluid Thioglycolate Medium” and “Soybean–Casein Digest Medium” as per IP. The sterility of the formulation is confirmed by incubating the test solution with a portion of the media at the specified incubation temperature (as per IP) for 14 d. No growth of microorganisms confirmed sterility of the formulation.

Apyrogenicity

Apyrogenicity of [⁹⁰Y]YAS glass microsphere formulation was tested by gel clot-bacterial endotoxin test (BET) assay method as per approved IP procedure, described previously.¹⁷ The endpoint-turbidimetric assay, which is based on the quantitative relationship between the concentration of endotoxins and the turbidity (measured by absorbance or transmission) of the reaction mixture at the end of an incubation period, is used to determine endotoxin concentration.

In vitro stability assay

In vitro stabilities of [⁹⁰Y]YAS glass microsphere formulation with regard to release of ⁹⁰Y radioactivity was studied both in physiological saline and in human serum. Aliquot [⁹⁰Y]YAS glass microsphere formulation was withdrawn, diluted to 2.0 mL by the addition of physiological saline, and stored at 37°C up to 7 d. The percentage of ⁹⁰Y activity leached out into normal saline from the formulation at different time intervals post-preparation was determined following the same procedure described for the determination of radiochemical purity of the formulation. From this, the percent radioactivity remained associated with YAS glass microspheres as function of time post-preparation, which ascertains the in vitro stability of the formulations in normal saline, was determined.

To determine in vitro stability in human serum, aliquot of the formulation was withdrawn and mixed with 1.0 mL of freshly isolated human serum and mixed thoroughly. The mixtures were stored at 37°C up to 7 d. The percent radioactivity remained associated with YAS glass microsphere as function of time post-preparation was determined as in the previous case.

Furthermore, it was pertinent to study the effect of radioactivity on the shape, size, and morphology of [⁹⁰Y]YAS glass microspheres upon its storage in vitro. These parameters could influence the biological properties of [⁹⁰Y]YAS glass microspheres produced by neutron irradiation of cold particles. For this, a portion of [⁹⁰Y]YAS glass microsphere formulation prepared was stored at room temperature for 30 d for ⁹⁰Y radioactivity to decay (>10 half-lives of ⁹⁰Y), and subsequently, SAXS and SEM of the sample were recorded.

Biodistribution studies

Biodistribution studies of [⁹⁰Y]YAS glass microsphere formulation were carried out in a group of healthy male Wistar rats each weighing 200–250 g. Each animal was anaesthetized using a combination dose of xylazine hydrochloride and ketamine hydrochloride. Anaesthetized animals were prepared for aseptic laparotomy, and subsequently, the hepatic artery is exposed surgically. Appropriately diluted aliquots of freshly prepared [⁹⁰Y]YAS glass microsphere formulation (0.1 mL, ∼5 MBq radioactivity) were injected through the portal vein of each animal. The total counts corresponding to the activity of [⁹⁰Y]YAS glass microsphere aliquot taken in the syringe in a flat-type NaI(Tl) scintillation counter were recorded just before injection, and the counts in empty syringe were subtracted to obtain total counts injected in each animal (typically 400,000–500,000/30 s).

Abdominal organs were placed in situ, and surgical wound was stitched immediately. After the completion of the surgical procedures, the animals were kept under observation until they completely recovered from the anesthetized conditions. To obtain the biodistribution pattern of the injected preparation, randomly selected group of four animals each were sacrificed at 3, 24, 72, and 144 h post-injection (p.i.) by CO₂ asphyxiation. The tissue and the organs were excised, washed with physiological saline, and dried, and the activity associated with each organ and tissue was measured in terms of counts in the same NaI(Tl) scintillation counter. For determination of activity in the blood, sample was withdrawn using syringe and counted directly. The uptake of [⁹⁰Y]YAS glass microsphere in different organs and tissue is calculated from these data and expressed as percent injected activity (dose, %ID) per organ.

The total uptake in the blood, skeleton, and muscles was determined by considering that the respective organ/tissue constitute 7%, 10%, and 40% of the total body weight of the animals, respectively.^18,19 While calculating the total skeletal uptake in the animals, tibia is considered as the representative of the skeleton. For comparative evaluation, biodistribution pattern of TheraSphere (Boston Scientific) was also obtained at 3 h post-administration of the formulation in another set of healthy Wistar rats following exactly the same protocol as described above. All the animal experiments are carried out in compliance with the relevant national laws for conducting animal experimentations in India with prior approval of Institutional Animal Ethics Committee of Bhabha Atomic Research Centre.

Human clinical study

Preliminary human clinical investigation of [⁹⁰Y]YAS glass microsphere formulation was carried out in a male patient (56 years) with right lobe hepatocellular carcinoma. Triphasic computed tomography (CT) of the thorax, abdomen, and pelvis was performed (on PET/CT Gemini TF 64; Philips Medical System, Best, the Netherlands) before the TARE procedure, which revealed segment VII live lesion in the patient. Dedicated 3D contouring software (EBW; Philips Medical System) was used to estimate tumor and liver volumes from CT. Tumor and liver mass, required to calculate the activity [⁹⁰Y]YAS glass microspheres to be administered, was determined by considering the density of the liver to be 1.07 g/mL.²⁰

Activity or dose of [⁹⁰Y]YAS glass microspheres required for the therapy was calculated as per the protocol reported earlier²¹ and was found to be 3.4 GBq for the patient selected for TARE. Customized dose of 3.4 GBq of [⁹⁰Y]YAS glass microsphere formulation was prepared and administered through right hepatic artery with superselective approach. Positron emission tomography (PET)/CT images were recorded at 24 h post-administration to ascertain the localization of the glass particles. The human clinical studies reported herein were carried out after getting regulatory clearance from the institution ethical committee of Tata Memorial Centre, Mumbai, India, and with informed written consent from the patients concerned.

Results

Synthesis and characterization of YAS glass microspheres

YAS glass microspheres were synthesized using flame spheroidization process with conversion efficiency of almost 100% and >99% sphericity. SEM images of the glass microspheres (Fig. 3a) confirmed the high degree of sphericity and uniformity of the microspheres prepared and absence of any visible defects. The absence of any characteristic peaks in the XRD pattern of synthesized glass microspheres (Fig. 3b) confirmed typical glassy nature of the material. Chemical composition of YAS glass microspheres synthesized in different batches was analyzed by a combination of EDXRF and ICP-OES techniques, and the results are summarized in Table 1. The data showed that chemical composition remained unchanged after synthesis of glass microsphere using high temperature process. A representative EDXRF spectrum YAS glass microsphere is shown in Figure 3c. Particle size distribution analysis of glass microspheres carried out using laser diffraction particle size analyzer (Fig. 3d) showed that more than 90% of particles were within the range of 20–36 μm.

FIG. 3.

Characterization of YAS glass microspheres: (a) SEM image, (b) XRD pattern, (c) EDXRF spectrum, and (d) particle size distribution. EDXRF, energy dispersive X-ray fluorescence; SEM, scanning electron microscope; XRD, X-ray diffractometer; YAS, yttria alumino silicate.

Table 1.

Compositional Analysis of Yttria Alumino Silicate Glass Microspheres

Batch	Wt% of components
Batch	Al₂O₃	SiO₂	Y₂O₃
Batch I	19.4 ± 1.3	40.4 ± 1.1	40.1 ± 2.6
Batch II	18.0 ± 1.6	39.3 ± 1.4	43.2 ± 3.0
Batch III	20.5 ± 1.3	40.8 ± 1.5	38.1 ± 2.0
Batch IV	21.5 ± 1.3	41.0 ± 1.6	40.3 ± 1.6
Batch V	19.1 ± 1.2	40.5 ± 2.3	42.1 ± 2.0
Batch VI	19.5 ± 1.2	39.7 ± 2.3	38.5 ± 1.5

Formulation and quality control of [⁹⁰Y]YAS glass microspheres

Intrinsically [⁹⁰Y]Y-labeled glass microsphere formulation was produced in six different batches by thermal neutron irradiation of 20–60 mg of YAS glass microspheres in a research reactor. During this process, ⁹⁰Y is produced by ⁸⁹Y(n,γ)⁹⁰Y route [thermal neutron capture cross section of ⁸⁹Y (σ = 1.28 b)]. Since natural yttrium is mononuclidic (100% in ⁸⁹Y), the radioisotope other than ⁹⁰Y that is expected to form on neutron irradiation of yttrium would be ^90mY, which decays to ⁹⁰Y by isomeric transition with a short half-life of ∼3.2 h and emission of γ-photons. However, due to its short half-life, it almost completely decays in the time required for dose formulation and transportation for its clinical use, and hence not taken into consideration.

The data on batch yield, specific activity, and radionuclidic impurity analysis are given in Table 2, which shows that [⁹⁰Y]YAS glass microspheres were produced with a specific activity of 137.7 ± 8.6 MBq/mg of microspheres, which corresponds to ∼6800 Bq of ⁹⁰Y radioactivity per microsphere. Radionuclidic purity of the formulations was >99.9%, desirable for human clinical applications. The production of radionuclidic impurities specified in Table 2 was due to neutron activation of chemical impurities present in the constituents YAS glass.

Table 2.

Yield, Specific Activity, and Radionuclidic Purity Data of Six Batches of [⁹⁰Y]Yttria Alumino Silicate Glass Microspheres at the End of Irradiation

Batch No.	YAS glass microspheres irradiated, mg	Yield, GBq	Specific activity, MBq/mg	Radionuclidic purity, %	Impurity burden of individual radioisotopes, %
1	35	4.83	138	99.92	⁴⁶Sc: 3 × 10⁻³, ⁵¹Cr: 3 × 10⁻², ⁵⁹Fe: 1.4 × 10⁻², ⁶⁵Zn: 2 × 10⁻³, ⁹⁵Nb: 3 × 10⁻³, ¹³⁴Cs: 8 × 10⁻³, ¹⁸¹Hf: 2.1 × 10⁻²
2	25	3.18	127	99.94	⁴⁶Sc: 4 × 10⁻³, ⁵¹Cr: 2.5 × 10⁻², ⁵⁹Fe: 1.1 × 10⁻², ¹³⁴Cs: 6 × 10⁻³, ¹⁸¹Hf: 2.1 × 10⁻²
3	40	5.72	143	99.93	⁴⁶Sc: 2 × 10⁻³, ⁵¹Cr: 2.8 × 10⁻², ⁵⁹Fe: 1 × 10⁻², ¹³⁴Cs: 8 × 10⁻³, ¹⁸¹Hf: 1.8 × 10⁻²
4	60	7.50	125	99.93	⁴⁶Sc: 2.5 × 10⁻³, ⁵¹Cr: 2 × 10⁻², ⁵⁹Fe: 1.5 × 10⁻², ⁶⁵Zn: 2 × 10⁻³, ¹³⁴Cs: 8.8 × 10⁻³, ¹⁸¹Hf: 2 × 10⁻²
5	50	7.10	142	99.93	⁴⁶Sc: 3 × 10⁻³, ⁵¹Cr: 2.5 × 10⁻², ⁵⁹Fe: 1 × 10⁻², ¹³⁴Cs: 7.4 × 10⁻³, ¹⁸¹Hf: 2.3 × 10⁻²
6	45	6.53	145	99.93	⁴⁶Sc: 2 × 10⁻³, ⁵¹Cr: 2.8 × 10⁻², ⁵⁹Fe: 1.5 × 10⁻², ¹³⁴Cs: 8 × 10⁻³, ¹⁸¹Hf: 2 × 10⁻²

Decay properties of radionuclidic impurities are as follows:

Sc: T _1/2 = 83.83 d, E _β(max) = 357 keV, E _γ = 889 keV (99.9%).

Cr: T _1/2 = 27.7 d, no β, E _γ = 320 keV (9.83%).

Fe: T _1/2 = 44.5 d, E _β(max) = 1.57 MeV, E _γ = 1.29 MeV (43.2%).

Zn: T _1/2 = 244.1 d, E _β(max) = 324 keV, E _γ = 1.11 MeV (50.8%).

Nb: T _1/2 = 34.97 d, E _β(max) = 924 keV, E _γ = 766 keV (99.8%).

134

Cs: T _1/2 = 2.06 y, E _β(max) = 658 keV, E _γ = 605 keV (97.6%), 796 keV (85.4%).

181

Hf: T _1/2 = 42 d, E _β(max) = 407 keV, E _γ = 482 keV (82.9%).

YAS, yttria alumino silicate.

Radiochemical purity of all the six batches of [⁹⁰Y]YAS glass microsphere formulations determined by following the procedure was found to be >99.0%. Sterility and BET revealed that all the batches of [⁹⁰Y]YAS glass microsphere formulations were sterile and with <175 EU bacterial endotoxin content. The results of all the quality control tests are summarized in Table 3.

Table 3.

Results of Quality Control Tests of [⁹⁰Y]Yttria Alumino Silicate Glass Microspheres Produced

Test	Acceptance criteria	Test result
Specific activity (activity per unit microsphere)	Should be least 2500 Bq	Not <6000 Bq
Radionuclide purity	Should be >99.9% (the total impurity burden from all radionuclide should not exceed 0.1% of the total activity of ⁹⁰Y)	99.93% ± 0.01%
Radiochemical purity	Should be >98%	99.6% ± 0.1%
Sterility	Should be sterile	Sterile
BET	<175 EU in total volume of the formulation	<175 EU

BET, bacterial endotoxin test.

In vitro stability assay

[⁹⁰Y]YAS glass microspheres exhibited excellent in vitro stability with regard to the release of ⁹⁰Y activity from the formulation when stored at 37°C in physiological saline and in human serum. Leaching of ⁹⁰Y activity from the radiolabeled microspheres was found to be negligible (<0.2%) even after it was stored for 7 d in both the media.

Figure 4a shows the SAXS profiles of YAS glass microspheres (black) and [⁹⁰Y]YAS glass microsphere recorded 30 d (>10 half-lives of ⁹⁰Y) after formulation of the radiolabeled product allowing ⁹⁰Y activity to decay almost completely (red). It is evident that there is almost no change on the surface of the glass microspheres after neutron irradiation to produce ⁹⁰Y-labeled formulation. Similarly, SEM image of decayed [⁹⁰Y]YAS glass microsphere (Fig. 4b) showed very uniform and spherical monodispersed glass microsphere, which indicated that both the size and shape of the [⁹⁰Y]YAS glass microspheres remained almost intact on its storage up to 30 d.

FIG. 4.

SAXS profile (a) and SEM (b) of [⁹⁰Y]YAS glass microspheres showing particle size, surface morphology, and shape of YAS glass microspheres remain intact on thermal neutron irradiation. SAXS, small-angle X-ray spectra; SEM, scanning electron microscope; YAS, yttria alumino silicate.

Biodistribution studies

Biodistribution pattern of [⁹⁰Y]YAS glass microspheres expressed as the percentage of injected activity (dose) at different time points after intra-articular administration of the formulation in healthy Wistar rats is summarized in Table 4. The study revealed excellent retention of administered microparticles in the liver (97.62 ± 0.68%ID at 24 h and 94.23 ± 0.57%ID at 144 h p.i.). The uptake in any other major organ/tissue, particularly, the lungs, gastro intestinal tract, and spleen, that might have resulted due to leakage of the radioactive microspheres from the liver, was found to be insignificant. Almost no accumulation of activity in the skeleton up to 144 h post-administration indicated that there was no leaching of ⁹⁰Y activity from the microspheres lodged in the liver. Furthermore, a comparison of biodistribution patterns of [⁹⁰Y]YAS glass microspheres with commercially available TheraSphere in healthy Wistar rats at 3 h post-administration through portal vein, as shown in Figure 5, revealed near-identical pattern of the developed formulation with TheraSphere. Overall, the biodistribution pattern revealed all the favorable characteristics of the developed formulation desirable for human clinical use.

FIG. 5.

Comparison of biodistribution patterns of [⁹⁰Y]YAS glass microspheres with commercially available TheraSphere^® in healthy Wistar rats at 3 h post-administration through portal vein. YAS, yttria alumino silicate.

Table 4.

Biodistribution Pattern of [⁹⁰Y]Yttria Alumino Silicate Glass Microspheres in Healthy Wistar Rats

Organ	%ID
Organ	3 h	24 h	72 h	144 h
Blood	0.33 ± 0.08	0.13 ± 0.02	0.08 ± 0.02	0.00 ± 0.00
Liver	97.62 ± 0.68	97.06 ± 0.52	96.63 ± 1.08	94.23 ± 0.57
GIT	0.68 ± 0.11	0.43 ± 0.06	0.48 ± 0.11	0.53 ± 0.11
Kidneys	0.08 ± 0.03	0.13 ± 0.04	0.06 ± 0.02	0.03 ± 0.01
Stomach	0.05 ± 0.02	0.06 ± 0.02	0.07 ± 0.03	0.03 ± 0.02
Heart	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Lungs	0.32 ± 0.04	0.25 ± 0.04	0.29 ± 0.07	0.31 ± 0.05
Skeleton	0.10 ± 0.03	0.18 ± 0.04	0.12 ± 0.03	0.16 ± 0.04
Muscles	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Spleen	0.33 ± 0.03	0.26 ± 0.05	0.19 ± 0.04	0.20 ± 0.05

Data are presented as mean ± standard deviation. At each time point, three animals were taken.

GIT, gastro intestinal tract; %ID, %injected dose.

Human clinical study

Figure 6a shows representative post-therapy PET/CT image of a 56-year male patient with right lobe hepatocellular carcinoma 24 h after the administration of 3.4 GBq of [⁹⁰Y]YAS glass microspheres, whereas Figure 6b and c represents transaxial CT and transaxial PET/CT of the liver of the same patient. Target-specific localization and near-complete retention of the formulation in the cancerous site are quite evident from these images. Furthermore, preliminary investigation revealed that the administration of 3.4 GBq therapeutic dose of the developed [⁹⁰Y]YAS glass microsphere formulation injected into the patient was well tolerated by the patient, as no adverse side-effect of the therapeutic procedure was reported.

FIG. 6.

Post-therapy PET/CT image of a 56-year male patient with right lobe hepatocellular carcinoma 24 h after administration of 3.4 GBq of [⁹⁰Y]YAS glass microspheres showing (a) biodistribution, (b) transaxial CT of the liver and (c) transaxial PET/CT of the liver. PET, positron emission tomography; YAS, yttria alumino silicate.

Discussion

Intrahepatic administration of customized doses of [⁹⁰Y]Y-glass microsphere is one of the most effective therapeutic modalities for patients with inoperable liver malignancies.^6

–10 The radioactive glass microspheres are guided to the artery that supplies blood to the liver using a catheter inserted through an incision in the groin. The nonbiodegradable glass microspheres lodge in the hepatic arterioles (branches of the artery) and embolize the blood vessels feeding the tumor. These lodged microspheres exert a radiotherapeutic effect by β⁻-particles emitting from ⁹⁰Y, which destroys the local malignant tissue with little damage to the surrounding normal tissue. The primary objective of the present investigation was in-house development of a robust methodology for preparation of therapeutic doses of [⁹⁰Y]Yttrium glass microsphere ([⁹⁰Y]YAS glass microsphere) biosimilar to the commercially available product (TheraSphere), which will ensure its wider availability to a larger population of patients at an affordable cost. This was achievable in India due to the availability of a research reactor with medium thermal neutron flux.

European Association of Nuclear Medicine procedure guideline for the treatment of liver cancer specifies the use of [⁹⁰Y]Yttrium glass microspheres (TheraSphere) that consists of insoluble glass microparticles in which ⁹⁰Y is an integral constituent of the glass.²² The diameter of glass microspheres (specific gravity 3.7 g/dL) ranges from 20 to 35 μm with mean sphere diameter 25 μm. Each of these microspheres contain >2500 Bq of ⁹⁰Y radioactivity at calibration time.¹² Yttrium alumino silicate glass particles with chemical composition 20Al₂O₃-40SiO₂-40Y₂O₃ (w/w) were synthesized by melt-quench technique and converted to glass microspheres by flame spheroidization. The chemical composition of the microspheres was same as that of TheraSphere.

Particles with diameter within the range of 20–36 μm were segregated to produce [⁹⁰Y]YAS glass microsphere formulation. Considering the mean diameter of the glass particles to be 30 μm and specific gravity 3.7 g/dL (reported for TheraSphere), each milligram of the material synthesized will have ∼20,000 microspheres. On irradiation at a thermal neutron flux of ∼1.4 × 10¹⁴ n·cm⁻²·s⁻¹ for 7 d in a research reactor, cold YAS glass microsphere was converted to [⁹⁰Y]YAS glass microsphere with specific activity of 137.7 ± 8.6 MBq/mg at the end of irradiation. This roughly translates to ∼6800 Bq of ⁹⁰Y activity associated with each microsphere at the end of irradiation. Taking into consideration 24 h of decay loss of activity from the end of irradiation to human clinical application at hospital, each glass microsphere will carry ∼4000 Bq ⁹⁰Y activity at the time of its clinical utilization.

This value is higher compared with that mentioned in commercial TheraSphere specification.¹² Higher specific activity or activity per unit microsphere means that fewer particles need to be administered to achieve the desired dose. As a result, the microparticles will be less embolic with reduced concern of vascular stasis. Overall, a comparison of technical specifications of [⁹⁰Y]YAS glass microsphere formulation prepared vis-a-vis TheraSphere is given in Table 5, which shows that the in-house-developed formulation compares well with the commercially available TheraSphere in terms of chemical composition, particle size range, radioactivity content, purity, and so on. Recently, Pham et al. have reported the synthesis and preclinical evaluation of [⁹⁰Y]Y-glass microspheres of 18–30 μm diameter following the similar melt-quench process of glass formulation, although the specific activity of the formulation was reported to be significantly lower (∼23 MBq/mg).²³

Table 5.

Technical Specifications of Developed [⁹⁰Y]Yttria Alumino Silicate Glass Microsphere Formulation Vis-a-Vis TheraSphere Confirming the Biosimilarity

Parameter	⁹⁰ Y[YAS] glass microsphere	TheraSphere^®12
Chemical composition	20Al₂O₃-40SiO₂-40Y₂O₃ (w/w)	20Al₂O₃-40SiO₂-40Y₂O₃ (w/w)
Particle size range	20–35 μm	20–36 μm
Available doses	3 GBq or custom dose size	Standard (3, 5, 7, 10, 15, 20 GBq) or custom dose size
Dose composition	Radioactive microspheres in 0.6 mL water for injection	Radioactive microspheres in 0.6 mL water for injection
Specific activity (activity per unit microsphere)	∼4000 Bq/microsphere	>2500 Bq/microsphere
Product purity	RN purity: >99.9% RC purity: >99.0%	RN purity: Not specified ⁹⁰Y is not surface bound

RC, radiochemical; RN, radionuclidic; YAS, yttria alumino silicate.

Conclusions

Formulation of ready-to-use therapeutic doses of [⁹⁰Y]YAS glass microsphere suitable for human clinical use in the treatment of unresectable liver cancer has been achieved. YAS glass microspheres of composition 40Y₂O₃-20Al₂O₃-40SiO₂ (w/w) and diameter ranging between 20 and 36 μm were prepared by flame spheroidization process. [⁹⁰Y]YAS glass microspheres with adequate radionuclidic and radiochemical purity for human clinical use were produced by thermal neutron irradiation of YAS glass microspheres. Leaching of ⁹⁰Y from the microspheres was negligible (<0.2%) both in physiological saline and in human serum. In vivo evaluation in healthy Wistar rats showed near-complete retention of [⁹⁰Y]YAS glass microspheres in the liver of the test animals when administered through portal vein. Preliminary human clinical evaluation of the formulation in a patient with right lobe hepatocellular carcinoma also showed retention of ∼98% of the administered activity of [⁹⁰Y]YAS glass microspheres in the injected lobe by post-therapy ⁹⁰Y-PET scans.

Footnotes

Acknowledgments

The authors from Bhabha Atomic Research Centre are grateful to the Department of Atomic Energy, Government of India, for the financial support. The valuable service of the staff members of the animal house facility of Bhabha Atomic Research Centre in carrying out animal biodistribution studies is also acknowledged.

Authors' Contributions

K.V.V.: Data curation, formal analysis, writing—original draft. A.R.: Data curation, formal analysis. A.D.: Data curation, formal analysis. R.C.: Data curation, formal analysis, writing—review and editing. H.D.S., S.K.: Data curation, formal analysis. A.J.: Data curation, formal analysis. A.P.: Data curation, formal analysis. V.R.: Formal analysis, supervision. M.G.: Conceptualization, data curation, formal analysis, writing—review and editing. S.C.: Conceptualization, data curation, formal analysis, writing—original draft, review and editing, supervision.

Disclosure Statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Funding Information

The work is funded by Department of Atomic Energy, Government of India.

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[ 90 Y]Yttria Alumino Silicate Glass Microspheres: A Biosimilar Formulation to “TheraSphere” for Cost-Effective Treatment of Liver Cancer

Abstract

Background:

Objective:

Methods:

Results:

Conclusions:

Introduction

Experimental

Materials and equipment

Synthesis of YAS glass microspheres

Glass preparation

Flame spheroidization

Post-synthesis treatment

Physicochemical characterization

Formulation of [90Y]YAS glass microspheres

Irradiation

Dose formulation

Quality control of [90Y]YAS glass microspheres

Activity assay

Specific activity

Radionuclidic purity

Radiochemical purity

Sterility

Apyrogenicity

In vitro stability assay

Biodistribution studies

Human clinical study

Results

Synthesis and characterization of YAS glass microspheres

Formulation and quality control of [90Y]YAS glass microspheres

In vitro stability assay

Biodistribution studies

Human clinical study

Discussion

Conclusions

Footnotes

Acknowledgments

Authors' Contributions

Disclosure Statement

Funding Information

References

Formulation of [⁹⁰Y]YAS glass microspheres

Quality control of [⁹⁰Y]YAS glass microspheres

Formulation and quality control of [⁹⁰Y]YAS glass microspheres