Quantification methods of 224Ra and 212Pb activity

applied to characterize therapeutic

radiopharmaceuticals

Thesis for the Degree of Philosophiæ Doctor by

Elisa Napoli


© Elisa Napoli, 2021

Series of dissertations submitted to the

Faculty of Medicine, University of Oslo

ISBN 978-82-8377-932-5

All rights reserved. No part of this publication may be

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Photo cover: Oscars Studio & Galleri, Lillestrøm
Print production: Reprosentralen, University of Oslo.
Acknowledgments
I would like to thank Oncoinvent, the University of Oslo, and the Norwegian Research
Council for having made this PhD work possible.

I want to express my deepest gratitude to Roy H. Larsen. I would not be here today living
in Norway and defending a thesis about Radium if not for him. He has taught me all I know
today about radioactivity and chemistry, and there are no words that can be written to
describe how grateful I am for this major opportunity that he has given me, together with
Tina B. Bønsdorff, my supervisor, manager, co-worker, and friend. It was Tina who first
took me home from the train station my very first day when I arrived in Norway. She has
always been by my side these years of my PhD studies, encouraging and guiding me.

I want to express my true appreciation for the excellent supervision by Øyvind S. Bruland
and Asta Juzeniene related to all my works and for always being available and for being
sources of inspiration during these past four years.

I would like to thank Gro Hjellum for introducing me to the quality control world, giving
me the opportunity to work at NIST, and always encouraging me. A special appreciation
goes to all my co-authors and co-workers at the Radiation Physics department of the National
Institute of Standards and Technology (NIST). Thanks to Denis Bergeron, Jeffrey Cessna,
Brian Zimmerman, Ryan Fitzgerald, and Marc Tyra for teaching me how to do radiation
metrology research and for welcoming me into their scientific community. I would
especially like to express my appreciation for the brilliant supervision of Denis Bergeron. I
really look up to you, and you have always been a source of inspiration. Thank you for being
so patient and for teaching me the secrets of the TDCR and LTAC.

I want to thank Jan Alfheim and all my co-authors and co-workers; in particular, Sara
Westrøm for always giving me the right advice. Ida Sofie Jorstad, Vilde Y. Stenberg and
Kim Lindland for being great lab partners and friends.

ii
I also want to acknowledge Ruth, Marion, Sandra T., Sandra K., Tore, Kristine, Idar,
Hong, Anne Cecilie, Kaja, Hans L., Kristin, Gerd and all my other former co-workers at
Oncoinvent, including Zimba, Tayco, and Luna. From Nordic Nanovector, I want to
acknowledge Roman, Ada, Tatiana, Astri, and Adam. From the Norwegian Radium
Hospital, Ma and Vladimir, who sadly passed away.

A big thank-you to all my current co-workers at Bayer; in particular, Iris and Nina.

Nor could I have reached this goal without my family and friends.

Voglio dedicare questa tesi di laurea a Mamma e Papà. Grazie per avermi sostenuto
moralmente, incoraggiato ed ascoltato. Grazie per essere i miei fan numeri uno e per avermi
dato tutto nella vita che mi ha permesso, oggi, di essere qui, a scrivere la mia tesi di
dottorato.

Grazie a Francesco e Valentina per il loro supporto morale in questi anni di crescita e per
tifare sempre per me. Grazie ai nonni e zia Ada che oggi sarebbero orgogliosi di questo
nuovo traguardo raggiunto. A "I compagni" (i compagni del liceo) e Ilaria B per esserci
sempre! Grazie a Flavia e Shoaib! Per avermi "sfamato" tutti questi anni con dolci
prelibatezze italiane a Oslo, ma sopratutto grazie per avermi sempre ascoltato e consigliato
Flavia, l'amica piu' amica del mondo.

I want to thank all my beloved friends and former roomies in Oslo, especially Betka,
Paulina, Aimeè, Anja, Martin, Kjersti, and the entire house of Katarinahjemmet in Oslo.

Last, but not least! I want to thank my boyfriend Sigurd for cooking delicious food and for
all his care while I was writing this thesis.

iii
Declaration of Interests

The work presented in this thesis was financially supported by the National Research
Council of Norway industrial PhD grants (n.259820 and 2862888) received by the privately
owned Norwegian company Oncoinvent AS, which has also funded the work.

Oncoinvent AS has an approved patent (US Patent 9539346 and European Patent 3111959)
IRUWKHXVHRISDUWLFOHVDQGDQĮ-emitting radionuclide. Publication III, included in this thesis,
is a collaboration between Oncoinvent AS and Nucligen AS, through the industrial PhD
grant n.260639 of the Research Council of Norway granted to Vilde Y. Stenberg. The
following authors of the publications included herein are/were employed at Oncoinvent AS
when the studies described took place: Elisa Napoli, Gro E. Hjellum, Tina B. Bønsdorff,
Sara Westrøm, and Ida Sofie Jorstad. Øyvind S. Bruland is working as part-time chief
medical officer at Oncoinvent. Elisa Napoli, Gro E. Hjellum, Tina B. Bønsdorff, Sara
Westrøm, Ida Sofie Jorstad, Øyvind S. Bruland, and Roy H. Larsen are also shareholders
and/or options holders in Oncoinvent AS. Roy H. Larsen is affiliated with Sciencons AS,
which is a shareholder of Oncoinvent AS. Øyvind S. Bruland is affiliated with Blaahaugen
AS, which is a shareholder and has a consultancy contract with Oncoinvent AS. Roy H.
Larsen is chairman of the board of Oncoinvent AS and Nucligen AS. Vilde Y. Stenberg,
Øyvind S. Bruland, and Roy H. Larsen own stocks in Nucligen AS.

iv

Table of Contents

Acknowledgments ................................................................................................................ ii
Declaration of Interests ...................................................................................................... iv
Abbreviations ..................................................................................................................... vii
List of Papers .................................................................................................................... viii
1 Introduction..................................................................................................................... 1
2 Background ..................................................................................................................... 2
2.1 Historical background .................................................................................................. 2
2.2 Radionuclide therapy ................................................................................................... 7
2.3 Therapeutic alpha emitters ........................................................................................... 9
2.3.1 Actinium-225/Bismuth-213 ........................................................................... 10
2.3.2 Astatine-211 ................................................................................................... 13
2.3.3 Lead-212 / Bismuth-212................................................................................. 14
2.3.4 Radium-223 .................................................................................................... 15
2.3.5 Radium-224 .................................................................................................... 18
2.3.6 Thorium-227................................................................................................... 20
2.4 Radionuclide standardization ..................................................................................... 20
2.4.1 Primary standardization methods ................................................................... 21
2.4.1.1 Liquid scintillation counting method—TDCR ..................................... 22
2.4.1.2 Live-timed anti-coincidence counting method—LTAC ...................... 23
2.4.1.3 CIEMAT/NIST..................................................................................... 24
2.4.1.4 The importance of the high-purity germanium detector (HPGe) ......... 24
2.4.2 Secondary standardization methods ............................................................... 25
3 Aims................................................................................................................................ 27
4 Research methods ......................................................................................................... 28
4.1 Radium-224 production ............................................................................................. 28
4.1.1 Radium-224 labeling of calcium carbonate microparticles............................ 29
4.2 Radium-224 activity determination ........................................................................... 29

v
 4.2.1 Primary methods ............................................................................................ 29
4.2.1.1 TDCR and CIEMAT/NIST efficiency tracing ..................................... 30
4.2.1.2 Anticoincidence counting LTAC ......................................................... 31
4.2.2 Secondary methods ........................................................................................ 32
4.2.2.1 Ionization chambers calibration ........................................................... 32
4.2.2.2 Sodium iodide detector calibration....................................................... 35
4.3 Lead-212 production .................................................................................................. 35
4.4 Lead-212 activity determination ................................................................................ 36
4.4.1 Calibrating ionization chamber detectors ....................................................... 37
4.4.2 Calibrating sodium iodide detectors ............................................................... 38
4.5 Lead-212 re-localization from 224Ra sources ............................................................. 40
4.5.1 Release of 220Rn in air from 224Ra sources ..................................................... 40
4.5.2 Re-adsorption of 212Pb on 224Ra CaCO3 labeled microparticles .................... 41
4.5.3 Pre-clinical study of therapeutic effect comparison between 224Ra CaCO3
microparticles labeling methods..................................................................... 42
4.5.4 Ethical considerations .................................................................................... 44
5 Summary of the papers ................................................................................................ 46
Paper I ............................................................................................................................... 46
Paper II.............................................................................................................................. 47
Paper III ............................................................................................................................ 48
Paper IV ............................................................................................................................ 49
6 Discussion ...................................................................................................................... 50
6.1 Radium-224 and 212Pb activity determination ........................................................... 50
6.1.1 Uncertainty perspectives for radioactivity determinations............................. 53
6.2 Rn-220 diffusion and re-adsorption of 212Pb: Implications for radiotherapeutic use of
224
Ra-CaCO3 microparticles ............................................................................................. 54
7 Conclusions & future perspectives .............................................................................. 60
References ............................................................................................................................ 2
Publications.........................................................................................................................60

vi
Abbreviations
BEGe broad-energy germanium
CaCO3 calcium carbonate
CRPC castrate-refractory prostate cancer
DOTA 1,4,7,10-tetraazacyclododecane tetraacetic acid
DS dial setting
FDA Food and Drug Administration (United States)
HPGe high purity germanium
IC ionization chamber
ip intraperitoneal
LET linear energy transfer
LTAC live-timed anti-coincidence counting
LS liquid scintillation
mAb monoclonal antibody
NaI sodium iodide
NIST National Institute of Standards and Technology
NNDC National Nuclear Data Center
NPL National Physical Laboratory
ORNL Oak Ridge National Laboratory
PAA polyacrylic acid
PC peritoneal carcinomatosis
PMT photomultiplier tube
PSMA prostate-specific membrane antigen
PSA peritoneal surface area
PTB Physikalisch Technische Bundesanstalt
QC quality control
TCMC 2-(4-Isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoyl methyl)-
cyclododecane
TDCR triple-to-double coincidence ratio
TAT targeted alpha therapy

vii
List of Papers

This thesis comprises four peer-reviewed publications:

Primary standardization of 224Ra activity by liquid scintillation counting.

Napoli, E., Cessna, J. T., Fitzgerald, R., Pibida, L., Collé, R., Laureano-Pérez, L.,
Zimmerman, B. E. and Bergeron, D. E. Appl Radiat Isot, 2020. 155, 108933.

224
Radionuclide calibrator responses for Ra in solution and adsorbed on calcium
carbonate microparticles.
Napoli, E., Cessna, J. T., Pibida, L., Fitzgerald, R., Hjellum, G. E., and Bergeron, D. E. Appl
Radiat Isot, 2020. 164, 109265.

212
Calibration of sodium iodide detectors and reentrant ionization chambers for Pb
activity in different geometries by HPGe activity determination.
Napoli, E., Stenberg, V. Y., Juzeniene, A., Hjellum, G. E., Bruland, Ø. S., Larsen and R. H.
Appl Radiat Isot, 2020. 166, 109362.

One manuscript has been submitted to PLOS One peer-reviewed journal:

224
Radon-220 diffusion from Ra-labeled calcium carbonate microparticles: Some
implications for radiotherapeutic use. 1
Napoli, E., Bønsdorff, T. B., Jorstad, I. S., Bruland, Ø. S., Larsen, R. H., Westrøm, S.
Plos one, 2021.16(3), e0248133.

1
The manuscript included in this thesis is an earlier draft of the published version in PLOS ONE journal.
Final full version available here: https://doi.org/10.1371/journal.pone.0248133.

viii
Apart from the work included in this thesis, I have contributed to the following published
papers during my PhD project period:

Ra-ODEHOLQJRIFDOFLXPFDUERQDWHPLFURSDUWLFOHVIRULQWHUQDOĮ-therapy:
Preparation, stability, and biodistribution in mice.
Westrøm, S., Malenge, M., Jorstad, I. S., Napoli, E., Bruland, Ø. S., Bønsdorff, T. B.,
Larsen, R. H. J Labelled Comp Radiopharm, 2018. 61(6): p. 472-486.

Determination of the internal pair production branching ratio of 90Y.

Pibida, L., Zimmerman, B. E., King, L., Fitzgerald, R., Bergeron, D. E., Napoli E., Cessna
J. T. Appl Radiat Isot, 2020. 156, 108943.

Calcium carbonate microparticles as carriers of 224Ra: Impact of specific activity in

mice with intraperitoneal ovarian cancer.
Li, R. G., Napoli, E., Jorstad, I. S., Bønsdorff, T. B., Juzeniene, A., Bruland, Ø. S., Larsen,
R. H., Westrøm, S. Current Radiopharmaceuticals, 2021. 14 (2)

Ra-224 activity, half-life, and 241keV gamma ray absolute emission intensity: A
NIST-NPL bilateral comparison.
Bergeron, D., Collins, S. M., Pibida, L., Cessna, J. T., Fitzgerald, R., Ivanov, P., Keightley,
J. D. and Napoli E. Appl Radiat Isot, 2021. 170, 109572.

ix

 Introduction

1 Introduction

This doctoral thesis relates to nuclear physics and chemistry applied to the therapeutic use of
radionuclides for cancer treatment. It is an industrial PhD work co-sponsored by the Norwegian
Research Council and the private company Oncoinvent AS in collaboration with the Oslo
University Hospital and National Institute of Standard and Technology (NIST), USA.
The data presented in this doctoral thesis were collected in the USA at NIST (papers I and II),
and in Norway at both Oncoinvent AS and the Oslo University Hospital (papers III and IV).

When dealing with radionuclide therapies, it is very important to determine the right dosage of
radioactivity that produce sufficient damage to tumor cells, while being tolerable to non-
targeted areas of the body. Standardization of the quantification methods for a radionuclide is,
from an international perspective, very important to secure a safe and consistent dosing (activity
level) of a radiopharmaceutical in in all countries where a product is being used. Radium-224
labeled CaCO3 microparticles are under clinical investigation by Oncoinvent AS as a potential
cancer therapeutic and in this regard, it was important to develop a primary standard for accurate
determination of the 224Ra radioactivity dosing given to each patient.

The main objectives of this work were to develop the first primary standard for activity
224
measurement of Ra; to describe and evaluate methods that can be used to quantify 224Ra and
212
Pb activity; to calibrate detectors commonly used for measurement in preclinical and clinical
trials and apply this to the evaluation of therapeutic 224Ra radiolabeled microparticles.
It was also a goal to use calibrated counters to study properties of multi-step decaying 224Ra-
220
labeled microparticles and the fate of radioactive progenies, including Rn and the longest
living progeny 212Pb.

1

 Background

2 Background

2.1 Historical background

Radioactivity relates to the process when unstable atomic nuclei lose energy by emitting
radiation. Particles of the same charge such as protons, and particles with no charge such as
neutrons, can co-exist close to each other (order of one femtometer distance) inside a nucleus.
The particles constituting a nucleus are subject to two forces: one repulsive force called
electromagnetic, and one attractive nuclear force called the strong force [1] which acts on both
protons and neutrons. The strong force is only effective at a femtometer distance, but is 100
times stronger than the electromagnetic one, ensuring that the nucleus is compact and stable.
A nucleus is considered stable when this remains unperturbed indefinitely unless an external
force interacts with it. The electromagnetic and the strong force are well balanced in a stable
nucleus because of a well-balanced ratio of protons and neutrons.
Lead-208 is considered the stable element with the highest N/Z ratio 1.51807 and the highest Z
= 83 [2], while H-1 and He-3 are the only stable nuclides with N/Z ratio below 1. Beyond these
values and nuclides, the imbalance between N and Z makes all isotopes "unstable" [3]. To
overcome this, the nucleus emits the "excess" particles through what is called "a radioactive
decay" until it reaches a state of stability.

In 1896-1902 Antoine Henri Becquerel (1852-1908) published his findings in the journal of
Comptes Rendus [4-10] claiming the discovery of what was later called “radioactivity” by his
doctoral student Maria 6NáRGRZVND(1867-1934) (also known as Marie Curie). At that time,
Becquerel, inspired by the discovery of X-rays from the physicist Wilhelm Conrad Röntgen in

2
 Background
1895, decided to perform an experiment of his own, to study the effects of sunlight exposure of
flourishing material. He placed a metal object (a Christian Maltese cross made of copper) on a
photographic plate, [11] hoping to prove that fluorescence was a visible form of X-rays. As
luck would have it, the fluorescence material that he wanted to use was an uranium-based
compound (potassium uranyl sulfate) and, as the sun was not shining on the day that he wanted
to prove his theory, he left the uranium compound wrapped in a black cloth on top of the copper
cross and the photographic plate in a drawer. Several days later when Becquerel reopened the
drawer, he observed a clear image of the Maltese cross on the photographic plate showing that
actual X-rays were "self-generated" by the uranium powder without contribution from the sun.

In 1897, a young Marie Curie began to study uranium compounds fascinated by its continuous
and spontaneous emission of X-rays, whereas until now those were only produced in vacuum
tubes and with the need of electrical power. Marie Curie discovered that uranium ore (called
pitchblende, sourced by St. Joachimstal mines in the now Czech Republic [12]) was more
radioactive than pure uranium, by testing it with an instrument similar to a nowadays ionization
chamber, that measure the electric charge generated by the interaction of the radioactivity with
the air (ionizing radiation). This observation lead Marie and her husband Pierre to understand
that there could be other radioactive elements other than uranium contained in pitchblende [13].
They were determined to isolate those substances and in 1898 they announced the discovery of
two new compounds: "radium" (226Ra) and "polonium" (210Po).
Marie Curie published her PhD thesis “Recherches sur les substances radioactives” (Research
on Radioactive Substances) on June 25th, 1903 [14], in terms of fame and impact possibly the
most important PhD thesis in the history of science.

Soon, other scientists and industrialists became interested in her work, particularly that on
radium properties and in how to produce radium for commercial purposes. Physical
measurements standards were therefore essential to be able to reproduce the same product
purity (free of Th-228 or what was called "mesothorium" at that time) [15].

Another important scientist in the field, Ernest Rutherford (1871-1937) won the Nobel Prize in
Chemistry in 1908 for his studies on "radiation and radioisotope chemistry” awarded for his
investigations into the disintegration of the elements, and the chemistry of radioactive
substances”. In 1910, a dedicated committee was constituted, later called "International
radiation measurements and standards commission", composed by Marie Curie, Ernest

3
 Background
Rutherford as president of the commission, Stefan Meyer, Frederick Soddy and other renowned
scientists. In 1910, at the congress of Radiology and Electricity in Brussels, it was decided by
the commission to produce reference standards to be used for comparisons in different
laboratories i.e., a 226Ra standard defined by weight. The first radium standard was prepared by
Curie in 1911 consisting it a glass ampoule containing 21.99 milligrams of radium chloride.
This standard was later stored, in 1913, in Paris at the International Bureau of Weights and
Measures as the first radium "primary standard" [15] [16].
Marie Curie was awarded two Nobel prizes. One prize in Physics was awarded in 1903 (together
with her husband Pierre and her professor Henri Becquerel) in recognition of Henri Becquerel’s
discovery of radioactivity and Pierre and Marie Curie studies on the same topic. The second
Nobel prize was awarded to Marie Curie alone in 1911 in chemistry for her discovery of radium
and polonium and her purification studies of radium.
In 1911, Curie and Rutherford met again in Brussel and decided to commission a new radium
primary standard, this time to Stefan Mayer from the "Institute of Radium research" in Vienna
[15]. This new primary standard was later compared with the Curie standard and found to be in
good agreement [17], by comparison performed with an instrument called "gold leaf
electroscope"[15]). Between 1912 and 1913, the international committee decided to produce
seven "secondary standards" (called "secondary" because they were assessed against the two
primary standards from Curie and Mayer), to be distributed internationally to the state members:
England, France, Germany, Japan, Portugal, Sweden and United States. All seven standards
were prepared by Otto Hönigschmid from Meyer’s laboratory in Vienna and certified by Curie,
Meyer and Rutherford [15]. Figure 1 shows the original certificate accompanying the secondary
standard source No.6 sent to the USA, received by the National Institute of Standards and
Technology (NIST) and signed by Marie Curie, Stephen Meyer and Ernest Rutherford.

4
 Background

Figure 1 International Radium Standards Commission certificate for the 226Ra secondary
standard No. 6 sent to the U.S.A. and received by the National Institute of Standards and
Technology (NIST), certified and signed by Stephan Meyer, Marie Curie and Ernest Rutherford
respectively in German, French and English [from the NIST archive, Gaithersburg MD, USA]
[15]

5
 Background

From 1920 onwards, products containing radium were commercialized including radium
chocolate and radium drinking water [18]. Those products were believed to be miraculous
against arthritis, impotence and aging. A popular skin care product was promising bright and
wrinkle free skin in 1933 [19]. But radium was also the main component of the patented U.S.
Radium Corporation luminous paint that between 1917 and 1926 employed hundreds of women
to paint clocks hands and faces with this new "light in the dark" paint, shaping their paintbrushes
with their mouths to maintain a fine point [20, 21].
These early practices would rather be found dangerous for human health and the cause of
harmful biological effect and death among several of the exposed individuals.

After the discovery of radium, numerous other radionuclides (or radioisotopes) of known
elements were discovered. Currently, several of those radionuclides with convenient half-lives
(in the order of hours or days) are utilized in research and in medicine for diagnostic or
therapeutic purposes.

Rutherford and the Curies were also responsible for the discovery of radon (222Rn, direct
226
daughter nuclide of Ra). Marie and Pierre Curie describe in their publications their studies
and measurements of this radioactive gas [22]. Thirty-nine radioactive isotopes have been
discovered of radon [23]. The most abundant radon isotopes and with longest half-lives are
222 220 224
Rn also just referred as "radon", Rn, the daughter of Ra, also known as "thoron" and
219 223
Rn, the daughter of Ra, also known in the past as "actinon". No stable isotopes of this
element exist [24].

Radon is a radioactive naturally occurring noble gas found in uranium ores, rocks such as
granite and limestone. Concentration of radon and progeny vary in different areas of the globe
according to the different concentration in soil of 226Ra, 224Ra, 223Ra and their generators [25].
The concentration of this gas is typically 20–2,000 Bq/m3 [26] in caves or poorly ventilated
houses and 1-100 Bq/m3 in open air [26]. Although, radon concentrations in uranium mines can
reach very high concentrations: for instance Gastein Healing Gallery averages 43 kBq/m3 (with
160 kBq/m3 as the maximum value registered for this specific mine) [27].

As early as in the fifteenth century, it was observed that miners where particularly prone to lung
diseases [28]. This were later attributed to high concentrations of radon exhalation [28].
6
 Background
Exposure to low concentrations of radon has been suggested to be curative against different
types of diseases [29]. In 1905, the chemist Frederick Soddy (1877-1956) suggested a treatment
for tuberculosis by internal radon emanation [30]. Radon spas and "health-mines" became
popular and still exists in Montana (USA), Austria, Poland, Czechoslovakia, Russia and Japan
where the public can be exposed to what is perceived to be therapeutic levels of radon [29, 31].
In later years the radium isotopes 224Ra, and in particular 223Ra, has attracted renewed interest
from scientific and medical communities as well as pharmaceutical companies for use in Į-
particle therapy against some forms of cancer.

2.2 Radionuclide therapy

Radionuclide therapy, as an integrated part of nuclear medicine, entails the use of radioactive
compounds as therapeutic agents against cancerous diseases. Radionuclide therapy differs from
external radiotherapy, which consists of irradiating a defined zone of the body where tumoral
areas are located with an external radiation beam generated by a linear accelerator.

Radionuclide therapy involves the internal administration of a radioactive compound.

Administration can be either systemic (unsealed radiopharmaceutical solution directly injected
into patients intravenously) or regional (applied to areas of the body, such as for intraperitoneal
(ip) infusion or brachytherapy, where sealed radioactive sources are placed close to or inside
the tumour).

A limited number of radionuclides have suitable radiotherapeutic properties. Their suitability

depends on the emission type (decay) of the radioisotope, the chemical property of the element,
the availability of the raw material, and the cost/feasibility of production, but most importantly,
the half-life. The half-life of a radioisotope is defined as the time that it takes for half of the
atoms of a specific radioactive material to decay.

For therapeutic purposes, radionuclides emitting Auger electrons, alpha- Į  DQG EHWD- ȕ
particles are often considered. The main characteristics of those types of decay modes are
summarized in Table 1. Alpha particles have the highest linear energy transfer (LET). The LET
defines the amount of energy deposited by the particle per unit of distance traveled. Alpha
particles consist of helium nuclei (two protons and two neutrons) of positive charge (+2), while
Auger electrons (negatrons) are negatively charged (í1) and ȕ-particles are negatrons or

7
 Background
positrons (positive charged electrons, +1). Alpha particles and Auger electrons are
monoenergetic, whereas ȕ-particles are emitted with a continuous spectrum of energies.

Table 1 Emission nature of radionuclides for therapeutic use [32, 33].

Particle *LET
Energy Range in tissue
decay mode (keV/—m)
Auger 1 eV–100 keV 4–26 2–500 nm
ȕ 50 keV–2.3 MeV 0.2–0.5 0.05–12 mm
Į 4–9 MeV 80–230 40–100 —m
*linear energy transfer

Alpha particles and Auger electrons short action range are compatible with micro-metastasis
targeting. While a single cancer cell can be inactivated at the 99% level, by typically as little as
19 Į-particle hits [33], several hundred to several thousand ȕ particle hits are required for a
similar degree of inactivation [34].

Apart from the radiation path length, the main differences among ȕ- DQG Į-emitting
radionuclides lie in the mechanism of action. Due to the high LET of Į-particles, the cells hit
by those are mainly subject to double strand DNA damage, whereas ȕ emitters mainly induce
single strand breaks that have a higher probability of cell auto-repair. Moreover, because of the
high energy of Į-particles per decay, less activity is required for patient treatment compared to
ȕ-emitting radionuclides. For radionuclides with some Ȗ- and X-ray emittance, this reduced
activity level would minimize external radiation exposure for hospital personnel, family
members, and other people interacting with the patient right after a treatment. On the other
KDQG Į-emitters are generally more harPIXO WKDQ ȕ-emitters when ingested or inhaled. It is
important to clarify that each type of decay is almost never 100% pure. $XJHUȕ, RUĮHPLVVLRQs
can therefore be accompanied by low amounts of Ȗ DQG ;-ray photons that are useful for
quantifying the activity of a specific nuclide using more affordable and commercially available
GHWHFWRUV0RUHRYHUWKHSUHVHQFHRIȖDQG;-ray photons makes it possible to image the actual
distribution of the radiopharmaceutical in patients after administration.

So far, clinical and preclinical research with radionuclides either under evaluation or already
approved for cancer therapy has involved several Auger electron emitting radionuclides,
111
including In, 67Ga, 195mPt, 125I, and 123I [35], VHYHUDO ȕ-emitting radionuclides, including

8
 Background
67
Cu, 77Br, 89Sr, 90Y, 105Rh, 131I, 149Pm, 153Sm, 166Ho, 177Lu, 186Re, 188Re, and 199Au, and several
Į-emitting radionuclides, including 213Bi, 223Ra, 224Ra, 225Ac, 211At, and 227Th [36, 37] as well
as RQHȕ-emitter nuclide, 212Pb, used as an in vivo JHQHUDWRURIWKHĮ-emitter 212Bi [38, 39].

A classic example of radionuclide therapy is the treatment of thyroid carcinoma with the
131
radioactive iodine isotope I. Iodine is the main component used to regulate gland function
and to produce the metabolically active hormones thyroxin and triiodothyronine. Upon
administration of the ȕ emitter 131I, this chemical element accumulates in high concentrations
in the thyroid, leading to the irradiation of the gland with ȕ particles and allowing targeted
radionuclide therapy.

Those types of radionuclides like 131I are considered “self-carriers,” as they can be delivered in
the area of interest without the need for a carrier component. When it comes to other
radionuclide types (e.g., lead, thorium), those need to be coupled to nano-/micro-sized particles,
peptides or monoclonal antibodies, through chemical linkers. In brachytherapy, medical devices
such as seeds or wires with encapsulated radionuclides are used. Ideally, the radiolabeled carrier
substance deposits the radiation dose in the targeted tumor cells, sparing healthy tissue and
therefore minimizing the radiation exposure and toxicity to non-targeted organs.
Immunoglobulin-based antibodies, which are polyclonal proteins normally present in the
human body, can also be made artificially as monoclonal antibodies (mAb). Monoclonal
antibodies are often studied as carriers in radionuclide therapy.

Unfortunately, not all radionuclides can be coupled to mAb with relevant stability. For instance,
radionuclides of radium lack adequate chemical characteristics to bind to linkers that permit a
stable conjunction between the radioisotope and the antibody. Targeted alpha therapy (TAT)
consists of Į-emitting radionuclides that selectively affect cancer cell aggregates.

2.3 Therapeutic alpha emitters

Among the numerous WKHUDSHXWLFĮ-emitters, section 2.2 lists those used for clinical purposes.

9
 Background
2.3.1 Actinium-225/Bismuth-213

Actinium-225 LVDQĮ-emitter, and its decay chain (Figure 2) involves four Į and three ȕ particles
[40]. The half-life of actinium-225 is 9.9179 ± 0.003 d [41], and it is the direct daughter of
225
Ra. The Actinium-225 daughter nuclide with the longest half-life, of 45.59 ± 0.06 min [42],
213 213
is Bi. Bismuth-213 is D PL[HG Įȕ-emitter that ȕ-decay to the short-lived Po
(t1/2 = 3.709 ± 0.012 —s) [41]. Both Ac and Bi nuclides can be attached to carrier molecules
using various chelator groups. The potential of both radionuclides, sometimes in combination
and sometimes in isolation, was demonstrated for leukemia, non-Hodgkin’s lymphoma,
melanoma, prostate cancer, bladder cancer, neuroendocrine tumors, and glioma. A
comprehensive overview is available in Morgenstern et al., 2018 [43].

There is a very limited amount of pure 225Ac in the world, and producing this nuclide by neutron
irradiation in a nuclear reactor is costly and challenging, as impurities from other long-lived
nuclides may complicate the release of the product when it comes to clinical use. Currently,
only three suppliers in the world [44] can extract 225Ac from old 229Th generators that were, in
233
turn, obtained by the decay of a U source (a fissile material used as reactor fuel and
225
investigated as a nuclear weapon component). The three current suppliers for pure Ac are
located at the U.S. Department of Energy, the Oak Ridge National Laboratory in Oak Ridge
(TN, USA) [45] at the Institute for Transuranium Elements (Karlsruhe, Germany) [46], and the
Leypunsky Institute for Physics and Power Engineering (in Obninsk, Russia) [47]. The listed
225
suppliers can, however, still produce limited quantities of pure Ac material every
year. Alternative large-scale production of 225Ac methods have been considered, such as proton
irradiation of 226Ra and spallation of 232Th. An overview of the various methods is described in
Morgenstern et al. [48].

The main issue with irradiating the target nuclide lies in the different long-lived impurities that
might occur during production. The 232Th spallation process, for instance, leads to the
coproduction of 227Ac (T1/2ௗ ௗ± 0.003 y), which it is currently not possible to separate
225 227
from Ac. In hospitals, the potential waste handling of the long-lived Ac, which is also a
radioactive radon gas generator (219Rn), would be a problem. However, it seems that the
impurity does not affect the dosimetry of patients [49].

10
 Background
The proton/deuterium or gamma photon irradiation of a 226Ra target seems to avoid the issue of
dealing with an 227Ac impurity, substituted by the possible presence of the short-lived 224Ac and
225
Ac (both with a half-life in the order of hours) when using protons. However, residuals of
the 226Ra (T1/2ௗ=1600 ± 7 y) mother nuclide may occur.

225
Several clinical trials involving Ac used in cancer therapy in combination with mAb are
currently ongoing or planned. Among them, a few examples are reported herein: the drug named
Lintuzumab-Ac-225™ from Actinium Pharmaceuticals (New York, USA) is currently running
225
a phase I and II clinical trial with Ac used as monotherapy or in combination with
chemotherapy. This radiopharmaceutical is intended as a therapeutic agent for refractory
multiple myeloma patients (NCT02998047, NCT03867682, NCT02575963 and
NCT03932318). Another product, initiated by the Xinhua Hospital and the Jiao Tong
University School of Medicine, both located in Shanghai, China, and featuring 225Ac, is using
225
a prostate-specific membrane antigen (PSMA) ligand as a targeting moiety for Ac. This
225
product is also planned for an early phase I clinical trial ( Ac-PSMA) intended for the
treatment of metastatic castrate-refractory prostate cancer (CRPC) patients (NCT04225910).
213
As for Bi, it has been used as intralesional TAT for patients with melanoma [50], myeloid
leukemia [51, 52], and prostate cancer [53].

11
 Background

233
U
3
ɲ *159.2(2) ·10 y
229
Th
ɲ *7932(25) y
225
22
Ra
ɴ 14.82(1
14.82(19) d

225
Ac
ɲ
*9.9179(30)
*9
*9.9
9 s
221
Fr
ɲ 4.79(2)
4
4.79(
79(( min
217
At
ɲ 32.3(4) ·10 s
3

45.59(6) m 2.09(3)% 45.59(6) m 97.91(3)%

213
2
209
Bi
ɲ ɴ- 213
Tl Po
209
2.161(7) m Pb 3.70(5) ђs
3
ɴ- ɲ
ɴ- 3.277(15
3.277(15) h
209
Bi
stable

Figure 2 Actinium-225 decay chain and parent nuclides. Half-lives are taken from The National
Nuclear Data Center (NNDC) [54] when they were not available from the Decay Data
Evaluation project (DDEP) database [55]. *Half-life for 225Ac is taken from [41].

12
 Background
2.3.2 Astatine-211

Astatine-211, with a half-life of 7.216 ± 0.007 h [42], decays (Figure 3) either directly by Į-
particle decay to 207Bi (41.78 ± 0.08%) [55] and followed by electron capture decay to
stable 207Pb, or directly by electron capture to 211Po (58.22 ± 0.08%) [55], IROORZHGE\RQHĮ-
particle emission to stable 207Pb [56].

Astatine-211 is produced by means of a cyclotron, by Į-irradiation of the naturally occurring

209 211
nuclide Bi. Because of the relatively short half-life of At, the logistical aspects are
challenging and the conjugation of the nuclide to targeting molecules, the quality control routine
assessments, and the shipping and administration of the product should take place at the same
site.

The first registered clinical study with 211At (NCT00003461) was performed at Duke University
in Durham (NC, USA) [57-60]. In this study, an 211At-labelled chimeric antibody (ch81C6) and
a MX35 ) DEƍ  antibody fragment were administered for the treatment of glioma tumor cells
remaining after surgical removal [59]. In 2009, the second phase I clinical trial [59] involving
the same product was implied in patients in remission from ovarian cancer with possible micro-
metastasis remaining. Patients were injected with 20–100 MBq of 211At activity, which resulted
in no evident toxicity to the bone marrow or other healthy organs, although no conclusions on
the therapeutic efficacy of this treatment are available yet [38]. At the Fred Hutchinson Cancer
211
Research Center in Seattle (WA, USA), At-BC8-B10 (CD45 mAb) was used in a clinical
phase I/II study (NCT03128034) for patients with acute myeloid leukemia and acute
lymphoblastic leukemia before an allogeneic stem cell transplant [61].

13
 Background

209 211
Bi(ɲ͕Ϯn) At

41.78(8)% 58.22(8)%
211 EC
ɲ At
207 211
2
Bi 7.216(7) h
Po
32.9(14) y 0.516(3) s
Žƌɴ
+
207 ɲ
Pb
stable

Figure 3 Astatine-211 production from 209%L ĮQ 211At nuclear reaction and 211At decay chain.
All radionuclides’ half-lives reported are taken from the Decay Data Evaluation Project
(DDEP) database [55].

2.3.3 Lead-212 / Bismuth-212

Lead-212 (10.64 ± 0.01 h) [42] LVDȕ-emitter that is considered an in vivo generator of WKHĮ-
212 212
emitter Bi (60.54 ± 0.06 min) [42], which decays either by emitting a ȕ-particle to Po
(64.07%) or by emitting an Į-particle to 208Tl (35.93%) (Figure 6).

Lead-212 belongs to the thorium natural radioactive series [62] and can be extracted from
228
Th/224Ra generators and is an indirect daughter of 224Ra, generated via the emanation of the
gas 220Rn and the decay of 216Po. Thorium-228 is a decay product of naturally occurring nuclide
228
Ra (t1/2 = 5.75 ± 0.04 y). Alternatively, 228Th can be obtained from spent nuclear fuel of 232U.

Lead-212 binds well via chelators to antibodies and small molecules, and it has been used by
the companies OranoMed and RadioMedix in clinical trials. A first phase I clinical trial was
212
completed in 2016 (NCT01384253) using Pb for treating patients with breast, peritoneal,
ovarian, pancreatic, and stomach neoplasms. Lead-212 was combined with a humanized mAb
trastuzumab conjugated with the bifunctional chelating agent TCMC ((1,4,7,10-Tetra-(2-
Carbamoyl Methyl)-Cyclododecane) [63, 64]). After promising pre-clinical studies [65], in

14
 Background
2018, a phase I clinical trial started with a tradename drug called AlphaMedix™ consisting of
²¹²Pb-DOTAMTATE, a somatostatin analog, intended for the treatment of neuroendocrine
tumors (NCT03466216). This clinical study [66] was performed to determine the safety,
pharmacokinetic, dosimetry, and initial effectiveness of the treatment during the dose escalation
stage. The results showed partial response to the treatment and low toxicity with common side
effects similar to those experienced by chemo-treated patients.

No clinical use of 212Bi alone has been registered until 2020. The main use of 212Bi in Į-therapy
is better understood when it comes to administration of 212Pb, which can be considered an in
vivo generator of the Į-emitter 212Bi.

2.3.4 Radium-223

Radium-223, with a half-life of 11.43 ± 0.03 days [42], releases four Į particles and two
ȕ-particles in its decay chain (Figure 4).

Radium-223 belongs to the uranium-actinium natural radioactive series [31]. Radium-223 is

the first Į-emitter to be commercially approved by the Food and Drug Administration (FDA)
in the USA (May 2013) and by the European Medicine Agency (EMA) (September 2013). The
223
Ra-based product (called Xofigo®, Bayer) was invented by Roy H. Larsen, Gjermund
Henriksen, and Øyvind S. Bruland in 1999 [67] under the product name Alpharadin®.
Alpharadin® was developed by the former Norwegian company Algeta ASA. Algeta ASA was
acquired in 2013 by Bayer HealthCare, who is now the sole producer of Xofigo®.

Radium is considered a “calcium mimetic” as it shares the same electronic configuration as

calcium. The calcium-mimetic property predisposes radium to form complexes with
hydroxyapatite in areas of increased bone turnover, such osteoblastic metastases [68, 69]. A
phase III clinical trial ALSYMPCA (ALpharadin in SYMptomatic Prostate CAncer patients)
using Xofigo® showed, for the first time, that Į-particles could improve survival in patients
with metastatic CRPC. A recent meta-analysis has concluded that a significant improvement of
overall survival and symptomatic skeletal event-free survival was obtained with bone-seeking
223 89
Į-emitting Ra, but not with bone-VHHNLQJ ȕ-emitting Sr [70]. Kratochwil et al. [71]
223 153
compared the effectiveness of the Į-emitter Ra with WKH ȕ-emitter Sm-EDTMP; this is

15
 Background
illustrated in Figure 5, which shows the normal tissue-sparing properties of Į-emitters and
ȕ-emitters in the targeting of skeletal metastases.

223
Numerous clinical trials and studies with Ra can be found in the literature. More than 20
clinical trials have been completed for 223Ra from phase I to III.

235
U
ɲ 6
704(1) ·10 y
231
Th
ɴ 25
25.522(10
25.5
5 )h
231
Pa
ɲ 3267
32670(260)
70(2 y
227
Ac
ɲ ɴ 21.772(3) y
21.
223 227
Fr Th
020(4)% ɲ
22.00 (7) min 0.020(4)% ɲ *18.697(7) d
*18
219 223
22
2
At ɴ Ra
56 (4) s 97% ɲ ɲ 11.4
11.43(3)
11 4 d
215 219
2
Bi ɴ Rn
ɲ 3 98(3 s
3.98(3)
ɴ 215
2
Po
ɲ 1.781(4) ·10 s
3

211
Pb
ɲ 36.1(2) min
2.15 (2) min 99.724(4)% 211 2.15 (2) min 0.276(4) %
ɴ Bi ɲ
207 211
Tl Po
4.774(12) min 207 0.516(3) s
ɴ Bi ɲ
stable

Figure 4 Actinium-227, 227Th and 223Ra decay chain. The half-lives nuclear data are taken from
the Decay Data Evaluation Project (DDEP) database [42].
*The half-life for 227Th is taken from Terrisse et al. [72].

16
 Background

Figure 5 Modified figure from Kratochwil et al.’s study [71]. An example of dose distribution
in a metastatic region of a bone trabeculae under the radiation effect of an Į- and ȕ-emitting
radiopharmaceutical. The small oval with magnified image of the treated area shows the
radiation uptake in healthy bone/off-target radiation; lower toxicity for healthy red marrow
(red circles) is registered for Į emitters (i.e., 223Ra) compared to ȕ-emitting bone seekers (i.e.,
153
Sm-EDTMP)[71]. The high energetic radiation dose RIĮHPLWWHUVis transferred to the bone
metastasis and irradiated to the surrounding bone (blue circle in Figure 5a) for 0.1 mm. In
contrast, ȕ-emitting radiopharmaceuticals deliver radiation doses to a wider range of 0.5-5 mm
in bones (blue circle in Figure 5b).

17
 Background
2.3.5 Radium-224

Radium-224, with a half-life of 3.631 ± 0.002 days [42], releases four Į and two ȕ particles in
its decay (Figure 6).

Radium-224 belongs to the thorium natural radioactive series [62], and it can be extracted from
a 228Th generator. Its convenient half-life permits ample timing for both production and quality
control before potentially shipping the 224Ra-based product to different countries.

Radium-224 has been used since the early 1900s in patients affected by leukemia, anemia [73],
and ankylosing spondylitis [74]. Radium-224 is the object of study in numerous clinical trials
both completed and in progress. The company Alpha Tau Medical (Jerusalem, Israel)
developed a medical device called “diffusing alpha emitter radiation therapy,” or Alpha
224
DaRT™ technology, consisting of different sizes of seeds where Ra is embedded in the
surface and is directly inserted into the tumor by means of an applicator [75]. This medical
device is intended for the treatment of skin-related tumors [76] and mucosal cavity/soft tissue
neoplasms (NCT03353077, NCT03015883, NCT03886181, NCT03889899, NCT03737734,
NCT04068155), pancreatic cancer (NCT04002479), and breast cancer (NCT03970967).

Another new 224Ra-based radiopharmaceutical, called Radspherin®, has been developed by the
224
Norwegian company Oncoinvent (Oslo, Norway). This consists of Ra-labeled calcium
carbonate (CaCO3) microparticles in suspension [77]. Radium calcium-mimetic properties are
exploited in this product for the easy adsorption of radionuclides on the carrier surface of
CaCO3 microparticles. Radspherin® is used in a radioactive treatment involving ip injection in
patients affected by peritoneal carcinomatosis (PC) from ovarian cancer (NCT03732768) and
colorectal carcinoma (NCT03732781). The treatment of colorectal carcinoma with
Radspherin® is conducted in combination with hyperthermic ip chemotherapy surgery
(HIPEC).

18
 Background

232
U
70.6(11) ɲ
228
Th
ɲ 1.9126(9)

224
Ra
ɲ 3.63
3.631(2)
3 63 d
220
Rn

ɲ 55.8
55.8(3)
55 8 s
216
Po
ɲ 300(
300(2) ns
212
Pb
ɴ- 10.64(1) h

212
Bi
208 ɲ ɴ 212
Tl Po

ɴ 208
Pb ɲ
stable

Figure 6 Thorium-228 decay chain. Half-lives for each nuclide are taken from the Decay Data
Evaluation Project (DDEP) database [42]. Uncertainty of the half-life is expressed as a
percentage in parentheses.

19
 Background
2.3.6 Thorium-227

Thorium-227 belongs to the actinium natural radioactive series [62]. It is the direct daughter of
227 223
Ac and direct parent of the recently discussed Ra. Thorium-227, with a half-life of
18.697 ± 0.007 days [72], decays by releasing five Į particles and two ȕ particles (Figure 4).

227 226
Thorium-227 is extracted from Ac, obtained by neutron irradiation of Ra at Oak Ridge
National Laboratory (ORNL). The production of 227Ac occurs by recycling 226Ra from medical
devices secured by the U.S. Department of Energy Isotope Program. After recovering 226Ra and
an extensive purification process, the radium is irradiated in the High Flux Isotope Reactor
227
(HFIR) at ORNL. This process ensures a sufficient supply of Ac for Bayer to fulfill the
worldwide demand for both Xofigo® and Targeted 227Thorium Conjugates (TTC) products.

Thorium-227 can be chelated using a macrocyclic ligand and attached using a chemical linker
to mAbs or small molecules. Several 227Th-based products used for cancer therapy have been
developed by Bayer: BAY1903149 (227Th chloride), with BAY1862864 being the TTC product,
227
based on Th-labeled conjugate (mAb/small molecules). Those investigated mAb/small
molecules are CD22-TTC, PSMA-TTC, HER2-TTC, and MSLN-TTC [78]. A phase I clinical
study with TTCs injected into patients suffering from relapsed or refractory CD22-positive non-
Hodgkin’s lymphoma has been completed (NCT02581878). Other ongoing clinical trials are
focusing on the treatment of patients affected by HER2-expression breast cancer
(NCT03724747), metastatic CRPC, advanced mesothelioma cancer (NCT03507452), and
serous ovarian cancer and pancreatic adenocarcinoma (NCT04147819).

2.4 Radionuclide standardization

As radionuclide therapy becomes an integral part of healthcare, there is an increased need for
accurate and consistent quantification of activities administered to patients.

The optimal activity (the dosage that maximizes damage to cancer cells but minimizes harm to
healthy organs and tissues) of a new radiopharmaceutical is mainly established during clinical
trials conducted in many countries at the same time. It is therefore necessary for every clinical
site to determine the activity to be administered to the patient using the same method of
quantification, such as using the same detector type calibrated against a common standard

20
 Background
reference source. To make this possible, radioactivity standards are developed and established
by national metrology institutes, including the National Institute of Standards and Technology
(NIST) in the United States, the National Physical Laboratory (NPL) in the United Kingdom,
the Physikalisch-Technische Bundesanstalt (PTB) in Germany, and the Laboratoire National
Henri Becquerel (LNHB) in France. Metrology institutions develop and disseminate national
standards of radioactivity; these primary standards are used to calibrate radioactivity detectors
and to generate secondary standards. Secondary standards are the calibration of this activity
against a primary standard for a specific analysis, i.e., for different clinically relevant
geometries of a specific radionuclide (syringes or dose vial geometry).

When radioactivity was discovered by Henri Becquerel in 1896, there was no instrument to
quantify radioactivity amounts. As mentioned in the historical background (section 2.1 of this
226
thesis), Marie Curie performed the first quantitative measurements of Ra activity by
226
extracting a pure 21.99 mg of RaCl2 source sealed in a glass tube and use it as a standard.
The unit of measure “Curie” (Ci) was later established as the amount of activity emitted by 1 g
of 226Ra, also equal to 3.7 × 1010 disintegrations (or nuclear transformations) per second.
However, with the establishment of the SI in 1960, the Ci unit could no longer be a derived
unit, and for practical reasons, the reciprocal of 1 second (s-1), later named “Becquerel” (Bq),
was adopted as the SI unit in 1975. The Bq is the unit of measurement corresponding to the
amount of disintegration per second of the radionuclide measured.

While in the United States, therapeutic procedures with radiopharmaceuticals are regulated by
the FDA authority, (requiring traceability of radioactivity measurements and a maximum of
10% deviation [79] between prescribed and administered dose), in Europe, there is no specific
standardization requirement other than having an appropriate quality control and quality
assurance system in place [79].

2.4.1 Primary standardization methods

The method of assessing a primary standard activity consists of a direct measurement of the
activity by determining the number of radioactive transformations per unit of time, without
recurring to the use of another radioactivity calibration or standard sources. There is a large
variety of different methods to achieve primary standardization, and an excellent summary can
be found in Pommé et al. [80].

21
 Background

The primary standardization methods adopted in paper I to be discussed in this dissertation were
based on the liquid scintillation (LS) counting triple-to-double coincidence ratio (TDCR)
method, the CIEMAT-NIST efficiency tracing with tritium, and the live-timed anticoincidence
counting (LTAC) method [81-83]. In paper III, a high-purity germanium detector (HPGe)
detector was used instead for a preliminary standard activity evaluation.

2.4.1.1 Liquid scintillation counting method—TDCR

The liquid scintillation counting technique consists of mixing a radioactive sample with a
cocktail of organic substances containing a fluorine molecule. The kinetic energy produced by
ĮDQGȕ-particles during the decay of the radionuclide is absorbed by the cocktail, bringing the
fluorine molecule to an excited energy state. When this de-excites, visible light is emitted and
therefore detected by the counter. The TDCR method was first put into place by Pochwalski in
1979 [84], and it consists of three photomultiplier tube (PMT) units used to determine the
specific efficiency of a radionuclide based on the statistical physical model of the photon
scintillation theory and the probability that the decay events can be detected by three PMTs.
This theory is based on two models considered equally valid: the binomial model and the
Poisson distribution model. Those models are used to calculate the theoretical efficiency of the
radionuclide, setting all the decay information. An example of a tool often used to calculate
efficiencies is the MICELLE2 code [85, 86].
The three PMTs are located 120 degrees apart from each other, so that the light generated in
the middle can be equally observed by each detector. The electronic system connected to the
three PMTs record for each event the triple coincidence counts, or ்ܰ (the number of events
detected by all three PMTs), and each double coincidence for each dual combination of PMTs
ܰ஽ (the number of events detected by two PMTs). The TDCR method is based upon the
variation of the parameter ‫( ܭ‬called the TDCR parameter), defined as:

்ܰ ‫் א‬
‫=ܭ‬ = (1)
ܰ஽ ‫א‬஽

The value of the parameter ‫ ܭ‬will always fall between 0 and 1, as ்ܰ is always lower than
ܰ஽ [81]. The high LET of Į-emitters, which makes those types of nuclides suitable candidates

22
 Background
for cancer therapy, makes it possible to obtain a very high counting efficiency in LS-based
counting techniques. In fact, we expect nearly 100% counting efficiency for ĮSDUWLFOHs.
When counting efficiency ‫ א‬for the triple and the double approaches 1, as in Į emitters, both
்ܰ and ܰ஽ get closer to the effective disintegration of the nuclide; therefore, ‫ ܭ‬will also
approach unity [81].

To verify that the experimental data are reliable, it is possible to vary the efficiency ‫א‬,
extrapolating it to 1, by applying a quenching factor. This will verify and test our model of the
relationship between ‫ ܭ‬and ܰ஽ (or ்ܰ ). There are three quenching techniques among the most-
used for TDCR: the defocusing method, operating on the electronic of the PMT and therefore
varying the parameter for the efficiency; chemical quenching, consisting of using substances
such as nitromethane that absorbs part of the energy of the photons so to alter the intensity of
the light; and gray filter application on the LS vial, which consists of surrounding the source
with different shades of gray filters [87].

2.4.1.2 Live-timed anti-coincidence counting method—LTAC

The live-timed anti-coincidence (LTAC) counting system involves the use of a proportional
counter or an LS counter for Į/ȕ detection and a Ȗ counter (for instance, a sodium iodide
thallium activated NaI(Tl) detector).
Knowing that ‫א‬ఉ is WKHȕ-efficiency, ܰ஺ is the anti-coincidence count rate, and ܰఊ is the Ȗ count
rate:

ܰ஺
‫א‬ఉ ൎ 1 െ (2)
ܰఊ

It is therefore possible to determine the source activity ܰ଴ as:

ܰ஺
ܰఉ ൎ ܰ଴ ቆ1 െ ݇௖.௙. ቇ (3)
ܰఊ
where
1 െ ߳ఉఊ
݇௖.௙. = ‫א‬
1 െ ቀ ஼ൗ‫ א‬ቁ (4)
ఊ

23
 Background
݇௖.௙. is the correction factor for Compton scattering and ‫א‬஼ is the efficiency of the additional
coincidence detected due to this phenomena [88]. This becomes

ܰఉ ܰఊ
ܰ଴ ൎ (5)
ܰఊ െ ܰ஺

By varying the low-level discriminator of the LS detector, it is possible to vary the LS detector
inefficiency. By extrapolating back to zero inefficiency, the activity becomes ܰ଴ for

ܰ஺
൘ܰ ՜ 0 (6)
ఊ

2.4.1.3 CIEMAT/NIST

The CIEMAT/NIST [89, 90] method with 3H consists of relating 3H counting efficiencies to
the efficiencies of the nuclide to be standardized by preparing paired samples of each nuclide
and measuring them on an LS counter. The reason that 3+LVXVHGLVEHFDXVHRILWVORZȕ-energy
emission, which makes it very sensitive to quenching effects. This way, it is possible to relate
the quenching of the 3H series to the quenching of the other nuclide series to be standardized to
calculate the efficiency of the unstandardized nuclide. This way, it is possible to determine an
efficiency curve for the unstandardized nuclide that is a function of the counting efficiencies of
3
H. At this point, the counting efficiency of 3H is measured as a function of the quenching
indicator: this means that known activity amounts of 3H are measured with different quenching
conditions.

2.4.1.4 The importance of the high-purity germanium detector (HPGe)

The HPGe detector is mainly applied in the primary standardization process for the
identification of radionuclidic impurities by Ȗ-spectrometry. The HPGe spectrometry technique
can be used to estimate activities in an effective manner when the detector is calibrated, and the
efficiency curve must be established with radioactive sources with known activity in a well-
defined geometry. The HPGe spectrometer is sensitive to both ȖDQG;-rays counted at energies
between 1 and ~1500 keV. Those types of system maintenance are quite demanding in terms
of daily checks and are expensive concerning the continuous cooling of the HPGe with liquid
nitrogen or electronically with the new models. It is very important that the system does not
24
 Background
experience any temperature variations, as the cooling system reduces thermal excitations from
the valence band.
The interaction of a Ȗ-ray in the crystal promotes electrons to the conduction band to create a
signal. The number of electrons promoted is read as a pulse height and is proportional to the
energy deposited by the Ȗ-ray. At temperatures higher than the liquid nitrogen (í195.79 °C),
electrons can easily cross the conduction band and therefore interact with the electric field and
generate electronic noise.

2.4.2 Secondary standardization methods

Measurements of the activity of radiopharmaceuticals at the end-user level, such as in a nuclear

medicine department in a hospital or at the radiopharmacy, are made under very different
conditions than those made at metrology institutes. It is very important to ensure traceability of
the measurement to a national standard. Secondary standards are often developed for re-entrant
well-type ionization chambers (ICs), commonly referred to as dose calibrators. Accurate
activity measurement by means of an IC depends on the application of calibration factor (dial
setting, or DS) for the specific instrument used, the radionuclide measured, and the geometry
of the sample (i.e., container used, activity in dry/liquid form, concentration mg/mL).

Dose calibrators generally consist of a pressurized gas ionization chamber that generates an
electrical signal when the gas is ionized by striking Ȗ-particles emitted by the radioactive source
placed in the chamber. The instruments therefore read out through an electrometer the amount
of current generated. The current is then converted into an activity using an internal factor,
which in most commercially available ionization chambers consists of a number that the user
inserts on a numeric keypad. This allows the instrument to display the amount of activity
measured in the MBq unit. The IC calibration factors in terms of current per unit activity
(pA/MBq) are generally determined by the instrument manufacturer [91, 92]. For Capintec
manufactured ionization chambers, the DS are obtained by the energy-dependent response
curve (or calibration curve), which is determined by measuring a minimum of two radionuclide
standards. Every other nuclide DS is subsequently predicted by interpolating along the
previously determined energy-dependent calibration curve and taking into consideration the
Ȗ-ray emission data for the nuclide. Detector responses are known to depend on the geometry
of the sample and the instrument used [93]. Moreover, different models from the same IC

25
 Background
manufacturer can differ with respect to chamber gas pressure, chamber wall thickness, and
material, resulting in a different output response.

Another important instrumentation often used to assess radioactivity in nuclear medicine is the
sodium iodide (tallium activated), or NaI(Tl) Ȗ-counter, which is a reliable yet affordable
instrument commercially available from various suppliers in automated versions.

This type of instrument is used in particular during preclinical trials, such as for product
characterization and stability studies, for measurements of tissue samples from animal studies
[94, 95], and for clinical applications described elsewhere [96]. Whereas measurements
performed with a dose calibrator are directly presented in units of activity (e.g., MBq), the NaI
counters’ results are expressed in counts per minute (CPM). Most automated Ȗ-counters (i.e.,
the Hidex Ȗ-counter used through the studies performed in papers III and IV) perform
measurements that are dead-time, decay corrected, and background corrected. Moreover, the
counter can be equipped with a balance unit that permits a user to directly determine the activity
per sample mass. This specific NaI counter also has the feature of applying a manufacturer-
determined calibration factor to translate the CPM value into activity units (Bq) for known
radionuclides used in nuclear medical imaging (such as 18F). It is also possible to apply custom-
determined calibration factors by measuring a known standard source.

Several clinically relevant Į emitters have been standardized. The first clinically important Į-
emitter, 223Ra, was standardized about a decade ago. The 223Ra activity primary standardization
was published in 2010 by NIST [97] and revised in 2015 by NPL [98] and by NIST [99]. The
first FDA-approved Į-therapeutic Xofigo is currently distributed internationally by Bayer with
224
NIST traceability to assure accurate activity administration to patients. The Ra activity
standardization was published in 2020 by NIST and Oncoinvent (paper I of this thesis [100]).
225
The activity standardization of Ac was published by the German Metrology Institute PTB
(Physikalisch-Technische Bundesanstalt) in 2020 [41]; the 227Th standardization was published
in 2019 by PTB and NPL [101-103].

26

 Aims

3 Aims

The main objectives of this doctoral thesis were:

1. To develop the first primary standard for activity measurement of 224Ra;
2. To describe and evaluate methods that can be used to quantify 224Ra and 212Pb activity
and calibrate detectors commonly used for measurement in preclinical and clinical trials
and apply this to the evaluation of therapeutic 224Ra radiolabeled microparticles.
220
3. To use calibrated detectors and counters to study and quantify Rn activity released
from 224Ra-based products and the fate of 212Pb generated from the released 220Rn.

For the first objective, the main key points were

- to accurately quantify 224Ra activity by developing a 224Ra primary activity standard and
calibrate detectors against the standard;
224
- to exploit the Ra primary activity standard for determining calibration factors for
different geometries;
- to calibrate commonly used Ȗ-detectors for measuring 212Pb activity.

For the second objective, the key points were

220 224
- to indirectly determine Rn activity released by several Ra based sources by
measuring 212Pb activity;
212 224
- to determine Pb activity re-localization in solution for different Ra labeling
methods of a CaCO3 microparticles carrier;

- to investigate the therapeutic effect of differently labeled 224

Ra-CaCO3 microparticles
in an animal model.

27

 Research methods

4 Research methods

It is important to note that all the measurements discussed in this dissertation were performed
for radionuclides in almost complete equilibrium with their progeny. For both 224Ra and 212Pb
sources, the equilibrium was considered reached after almost 4 days (224Ra/212Pb = 0.881;
224
Ra/212Bi = 0.872) to 6 days (224Ra/212Pb = 0.878; 224
Ra/212Bi = 0.868) from separation for
224
Ra sources, and 4 h for 212Pb sources (212Pb/212Bi = 0.905). This defines a limitation for our
results, as the activity determinations described herein are not valid for freshly produced pure
sources. The reason we chose to perform the studies at equilibrium was because it is more
practical when having a very complex decay chain such as the one of 224Ra and 212Pb. Another
reason is that the time between the production of the radiopharmaceutical, QC assays, and the
shipment of the product to hospitals, will permit the product to reach equilibrium anyway.

4.1 Radium-224 production

The 224Ra utilized in the experiments discussed in papers I and II were purchased from ORNL,
while the 224Ra used in papers III and IV was extracted at Oncoinvent laboratory from a 228Th
source (Eckert and Ziegler, Braunschweig, Germany) according to previously published
228
methods [94], U.S. patents n.9433690B1 and n.9433690B2 . In brief, Th (the decay chain
represented in Figure 6) was immobilized on a column containing an actinide resin. After
allowing time for ingrowth, 224Ra was eluted in 1 M HCl and evaporated to dryness. The 224Ra
activity used for CaCO3 particles labeling was later dissolved in a buffered solution to the
preferred volume (US patent 9539346B1) [104].

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 Research methods
4.1.1 Radium-224 labeling of calcium carbonate microparticles

Calcium carbonate microparticles were produced via 1–3 minutes of rapid mixing of two
dissolved substances in equal volume: 0.33M CaCl2 and 0.33M Na2CO3, either by magnetic
stirring or overhead stirring. The suspension of particles generated was then collected by
centrifugation process and dried to be subsequently stored as dry powder. This process is
described in detail by Westrøm et al. [94] as “second generation” CaCO3 microparticle
preparation.

The radiolabeling process of the CaCO3 microparticles used in this thesis was performed using
two methods: surface-labeling and inclusion-labeling. The surface-labeling method, used in
papers II and IV, consisted of a co-precipitation reaction of Ba2+, SO42-, and 224
Ra2+ cations
cations onto the microparticle surface, taking advantage of Ra2+ calcium mimetic properties
previously discussed in section 2.3.5. The labeling process is also discussed in detail elsewhere
[94]. The inclusion labeling method used in some studies and presented in paper IV consisted
224
of adding Ra activity in equilibrium with progeny during the CaCO3 microparticle
production.

The particles were afterwards washed and spun down twice with water to remove any unbound
224
Ra activity. The surface-labeled versus inclusion-labeled microparticles was tested to
evaluate potential differences in radon release and activity distribution in solution as well as the
therapeutic effects in an animal model.
Stability studies showed that the retained activity after 1 to 7 days, (when radiolabeling the
microparticles used in paper IV), was above 95% [94]. This was evaluated by using a Ȗ-counter
(i.e. NaI detector) to measure the activity contained in the particle portion and the liquid portion
of the final product after storing the product at room temperature.

4.2 Radium-224 activity determination

4.2.1 Primary methods

224
The primary Ra activity standardization is presented in paper I, and it was achieved using
two LS-based methods: TDCR and LTAC. The selection of these methods was entirely driven
by previous experience with activity standardization of RWKHUĮ-emitting radionuclides [98, 99,

29
 Research methods
102, 105]. Alpha spectroscopy was not performed since the presence of a radon gas in the decay
224
chain of Ra complicates the measurement process and would result in a possible
contamination of the detector.

224
All Ra sources for the primary standardization were prepared gravimetrically [106]. All
sources measured by ICs, HPGe, and NaI(Tl) detectors were diluted in 1 M HCl solution (the
reason for this choice is discussed in section 6.1 of this thesis). The sources counted in LS
counters were diluted in a liquid scintillation cocktail. Ultima Gold AB is a scintillant
specifically designed for Į/ȕ discrimination and is often used in the LS counting community.

4.2.1.1 TDCR and CIEMAT/NIST efficiency tracing

The TDCR detector used for the research discussed in this thesis was constructed at NIST. The
design of the NIST TDCR detector (showed in figure 7) is based on the one in use at the
National Laboratory Henri Becquerel (LNHB) (Gif-sur-Yvette, France) and the one at the
Radioisotope Centre POLATOM (Otwock, Poland).

For the CIEMAT/NIST method, the efficiency variation was achieved by chemical quenching
with nitromethane. All 224Ra sources were measured against matched 3H sources; therefore, it
cannot be considered a primary standardization method but, instead, more of a confirmatory
one.

The TDCR method involved instead measuring 224Ra LS vials by imposing a quench obtained
by surrounding the vial with different gray filters in order to vary the efficiency of detection.
LS Į-efficiencies were recorded as 100%. 7KHȕ-efficiencies were instead calculated using the
MICELLE2 code [107]. The LS sources were inserted into clear scintillation glass vials with
foiled-lined screw caps intended to decrease the risk of 220Rn gas escaping from the container.
An ad hoc experiment to determine any possible 220Rn leakage showed good retention of radon
in the vials used. For all methods, it was assumed that the short-lived (T1/2 = 300(2) ns) 212Po
212 212 212
daughter is detected with its Bi parent, yielding 100% efficiency for Po and Bi. This
assumption is consistent with previous treatment of the 213Po and 213Bi pair in the decay chain
of 229Th. Discussion of this phenomenon can be found in the following publications [108, 109]
relative to the same decay chain of 224Ra. In paper I, the coincidence resolving time was varied
to show this effect, but it was not observed. As reported in paper I, the expected response would

30
 Research methods
be too small to be detected in the range studied. The TDCR ratio determined depends on the
counting efficiency: equation 1 was used to determine the “true” activity of the 224Ra sample,
which was later used to calibrate the other detectors, such as ICs and NaI(Tl) counters for
measuring the same 224Ra activity.

Figure 7 The triple-to-double coincidence ratio (TDCR) liquid scintillation detector used for
data collection for papers I and II at the National Institute of Standards and Technology
(NIST) in Maryland, USA.

4.2.1.2 Anticoincidence counting LTAC

The LTAC detector used in paper I implements an anticoincidence counting method also used
224
for assessing the primary Ra activity standard. The LTAC unit used in this study was
224
assembled at NIST. The Ra activity mixed with the liquid scintillant was contained in a
hemispherical LS source placed on top of a photomultiplier tube sensitive to UV and visible
scintillation light from the source and sitting inside of a NaI(Tl) (sodium iodide thallium
activated) well detector sensitive to Ȗ-rays. As discussed in paper I, after setting the three energy
224
gates of interest for the NaI detector’s Ra activity determination, the LS count rate was
plotted against the LS detector Į-ȕ LQHI¿FLHQF\IRUHDFKJDWHVHW. When we increased/decreased
the efficiency by moving the lower level discriminator threshold and then extrapolated back to
zero inefficiency, the system read the true decay rate for all the particles in our decay chain.
Simulations in GEANT4 [110] were used to examine the gate sensitivity and weights, so as to
reach a sufficient linearity of the intercepts and to calculate correction factors.

31
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4.2.2 Secondary methods

4.2.2.1 Ionization chambers calibration

224
In both papers I and II, Ra activity contained in different geometries was measured on
reentrant ionization chambers (ICs) in order to establish the DS to be used for subsequent
calibrations of the instruments for 224Ra activity.

Those ICs were five different models of the Capintec CRC-15R, -55tR, -35R, -25PET,
and -55tPET, and a Vinten 671 ionization chamber (VIC), which is related to a sister chamber
at the National Physical Laboratory (NPL) in Teddington, UK, allowing for indirect comparison
of activity standards [111]. The Capintec chambers were selected since they are the commercial
brand mostly used in hospitals and at Oncoinvent AS.

The VIC chamber was also calibrated to allow a direct comparison of activity determination
with the NPL in the UK and avoid any inaccuracy and discrepancies discussed in section 6.1 of
this thesis. In paper I, the only geometry investigated was the 5 mL flame sealed NIST standard
glass ampoule containing 224Ra activity in 1 M HCl solution. This was measured in: the NIST
automated ionization chamber, the AutoIC, the VIC, and several Capintec chambers. In paper
II, a more in-depth assessment of several additional geometries was added for the Capintec ICs
measurements. These geometries are shown in Figure 8. DSs (or calibration factors) were
determined using the calibration curve method [93], which consisted of first assessing the
activity concentration of the 224Ra master solution on a calibrated ionization chamber (AutoIC)
[99] using calibration factors determined in paper I. The data acquisition process consisted of
using a LabVIEW-based interface to record multiple activity readings of a set of 15–10 different
DSs. The advantages of using the calibration curve method over a more traditional "dialing-in"
method (where the calibration factor is found by changing it until the correct activity is
displayed), lays in the possibility to adjust the calibration factor in the future if the "true activity"
is later revised, i.e. because of publication of revised and more accurate Ȗ-spectrometry data.
Moreover, generating a calibration curve and verifying the response of the detector for the
specific nuclide of interest makes it easier to detect how sensitive the response of the detector
is to the change of dial setting in the particular region of interest. Finally, the residuals on the
calibration curve fit can be used to estimate the uncertainty on the activity measured with the
ionization chamber at a given dial setting and/or the uncertainty on the dial setting for a given
activity.

32
 Research methods
The decay corrected value of the activity read (‫ )ݔ‬and the inverse of the dial setting value 1/DS
(‫ )ݕ‬were used as components to establish the following equation:

Ref. activity െ ܾ ିଵ
‫ = ܵܦ‬൤ ൨ (7)
݉

where “Ref.activity” stands for the reference activity based on the weight concentration of that
sample, in turn, based upon the AutoIC activity of the master solution; m represents the slope
of the regression line, and b represents the intercept, defined as:

σ(‫ ݔ‬െ ‫ݔ‬ҧ )(‫ ݕ‬െ ‫ݕ‬ത)

ܾ= (8)
σ(‫ ݔ‬െ ‫ݔ‬ҧ )ଶ

33
 Research methods

Figure 8 The calibrated geometries for 224Ra activities in solution discussed in paper II: 5 mL
glass ampoule with 5 mL solution, 20 mL 20R glass injection vials containing 2 mL to 20 mL
solution, and a 20 mL plastic syringe containing 12 mL solution.

34
 Research methods
4.2.2.2 Sodium iodide detector calibration

The sodium iodide counter measurements for 224Ra described in paper I were performed using
a Wallac Wizard 2480 automatic NaI(Tl) well-type Ȗ-counter at NIST laboratories modeled
with a GEANT4 Monte Carlo simulation made at NIST that can calculate activity based on
manufacturer settings.

224
The sodium iodide counter measurements for Ra described in paper IV were instead
performed using a Hidex ī-counter for convenience, described in section 4.4.2, along with 212Pb
activity determination assays.

4.3 Lead-212 production

Lead-212 was produced from 224Ra via 220Rn emanation adapted from Hassfjell [112] and using
a novel simplified single chamber diffusion system using two similar methods. In the first
method, 224RaCl2 solution, prepared as described by Westrøm et al. [113] and extracted [114]
from an equilibrated 228Th (Eckert and Ziegler, Germany) source, was distributed on the surface
of a paper strip. The strip (Figure 9) was previously attached to a syringe needle positioned on
a silicone septum of a micro reaction vessel glass v-vial lid. The sealed v-vial was left to rest
220
for 20-28 h in a fume hood, allowing Rn nuclides to build up and emanate in the vial.
220
Subsequently, Rn decayed into the short-lived 216Po, that, in turn, decayed into 212Pb, which
settled on the interior surfaces of the container.

After the 224Ra source was carefully removed, while avoiding any 224Ra contamination (verified
by HPGe measurement at 212Pb complete decay), the 212Pb was washed off the glass walls with
1M HCl and transferred into a new container.

212 224
The second method used to generate Pb was distributing Ra activity on quartz wool
(ProQuarz, Mainz, Germany), which was then fixed on the inside of the screw cap of a 100 mL
glass flask (Simax-Kavalierglass, Praha, Czech Republic). The sealed flask was left inverted in
a fume hood for 20–28 h to allow 220Rn/212Pb to build up, and then it was extracted, as in the
first method.
The two methods used to produce pure 212Pb activity, using the 220Rn emanation from a 224Ra
source, was an original idea of Roy H. Larsen, who co-authored paper IV.
35
 Research methods

Figure 9 The 212Pb/224Ra generator: 2–20 —L of a 224RaCl2 solution was distributed on a small
paper strip (15 × 5 mm) attached to a needle placed at the center of a 3 mL micro reaction
vessel glass v-vial (left). A few —L of 224Ra activity in HCl solution, distributed on quartz wool
(ProQuarz, Mainz, Germany), fixed on the inside of the screw cap of a 100 mL glass flask
(Simax-Kavalierglass, Praha, Czech Republic) (right). The vial/bottle was placed in a fume
hood for a period of 20–28 h while 220Rn emanated from the strip/wool. Lead-212 generated
from the emanated 220Rn which was attached to the inner walls of the vial was later dissolved
in 1M HCl.

4.4 Lead-212 activity determination

212
No primary activity standardization studies have so far been published for Pb, which
212
emphasizes the need to accurately estimate Pb activity for optimal dosages for preclinical
and clinical studies. During preclinical and clinical study phase, radionuclide activities are
usually assessed with sodium iodide (NaI) counters and ICs. The NaI counters are suitable for
activities of a few Bq to several kBq [115], while the ICs are employed for higher activities,
ranging from hundreds of kBq to several GBq [116].

Both IC and NaI instruments are used during radionuclide production for QC and for assessing
the total activity during preclinical studies. Most importantly, ICs are commercially available
and are commonly used in hospitals to assess the correct activity for administration to patients.
In contrast, NaI counters are mostly used for determining radionuclide dosages for animal
experiments and for biodistribution measurements at lower activity levels. Both counters have

36
 Research methods
some limitations. In fact, the low spectra resolution of the NaI counter makes it almost
224 212
impossible to resolve the Ra (240.986 keV) and the Pb (238.632 keV) peaks if the user
212
wants to measure Pb activity in a 224Ra sample, both for pre-equilibrium measurement of
224
Ra and for detecting possible breakthrough of 224Ra in a 212Pb sample.

212 224
Paper III describes the production of high purity Pb activity from a Ra-based diffusion
generator. The method used to calibrate those instruments consisted of using a 212Pb reference
activity inserted into a 5 mL flame sealed ampoule, determined by means of a high-purity broad
energy geranium (BEGe) detector. 7KH WZR Ȗ-lines used for assessing the reference activity
ZHUHȖ2,0(Bi) (238.63 keV, 43.6% ± 0.5% DQGȖ3,1(Bi) (300.09 keV, 3.18% ± 0.14%). Once the
212
Pb activity was determined, it was used as reference activity at a reference time.

A more accurate method of activity determination could have been used for this purpose to
reduce the uncertainty for the activity determined. An actual primary standardization could have
been performed, but the aim was to use commercially accessible instruments in a small lab
setting. Commercially available TDCR instrumentation might be the answer for performing a
more accurate activity assessment, although this should be paired with a good efficiency tracing
code calculator. Moreover, the efficiency curve of the HPGe detector was based upon an Eckert
& Ziegler multi-Ȗ SRM, but it could be of interest to perform a new efficiency calibration based
228
upon a combination of multi-Ȗ source and a Th standard for those missing peaks in the
224 212
specific region of interest for both Ra and Pb. This could have improved the activity
determination by minimizing the uncertainty of the measure. Another weakness of the study
concerns the activity level used, as the small lab setting, and the limited number of resources
prevented us from handling high activity levels. The advantage of using higher activities is to
have a much more complete overview of the calibration factors for the detector at comparable
activities used in clinical studies.

4.4.1 Calibrating ionization chamber detectors

As mentioned in section 2.4.2, ionization chambers measure a current (pA) directly displayed
in MBq units when it comes to Capintec ICs. The displayed activity changes based upon the
nuclide-specific DS on the instrument’s keyboard.

37
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The DS for 212Pb activity in paper III was determined using the calibration curve method [93]
of the Capintec CRC-55tR and the CRC-25R, which are the two models of Capintec available
212
at Oncoinvent. In this case, the method consisted of measuring an aliquot of a Pb master
212
solution that was three times stronger than the internal Pb standard measured on the HPGe
spectrometer.

Both sources were contained in 5 mL flame-sealed ampoules, as shown in Figure 10. Once the
IC ampoule was inserted in the chamber, three to five measurements each for 10 to 15 different
DSs were recorded. The data were analyzed as described in section 4.2.2.1.

The readings were decay corrected, normalized to the standard activity, and plotted against the
inverse of the DSs (1/DS). The fits of the resulting curves were then used to calculate the correct
DS for each chamber and for nuclides in equilibrium. The same measurements were also made
224 228
for two 5 mL ampoule certified sources: a Ra NIST activity standard and a Th NIST
traceable standard. This was done in order to have a complete overview of three nuclides related
to each other.

4.4.2 Calibrating sodium iodide detectors

Sodium iodide counters were used to assess both 224Ra and 212Pb activity for papers III (activity
determination assays) and IV (experiments involving both 224Ra and 212
Pb activity) using the
Hewlett Packard &REUD DXWR Ȗ-counter model #5002 and/or the Hidex Automatic Ȗ-counter
model #425-601. The samples that could be measured on the NaI counters have sample size
restrictions due to the automated system consisting of pre-set racks and sample holder sizes.

Paper III shows how to perform a NaI detector calibration for geometry-specific samples of
212
Pb activity using both the Hidex and the Cobra and an arbitrary glass container, size
12 mm × 75 mm, containing four different aliquots of 212Pb activity taken from the same master
solution used to prepare the reference activity (Figure 10).

The NaI calibration factors for each geometry were obtained by dividing the background and
decay-corrected CPM by the volume-corrected reference activity of the sample, determined via
HPGe measurements. The samples were measured multiple times and at different time points,
obtaining an average and normalized calibration factor from CPM to Bq.

38
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Figure 10 Overview of the different geometries of the samples used for calibrating ionization
chambers (ICs) and NaI(Tl) detectors.

In paper IV, the activity determination of 224Ra and 212Pb was achieved for the same gates set
for paper III. The efficiency factors specific for 224Ra nuclides and according to the geometry
212
used were previously assessed with the same method presented for Pb (Table 2). The
radioactivity background was measured before the experiments, and blanks were counted at the
same time as the sources to correct any possible Compton scattering effect, which was not
significantly observed in this study. It is important to discuss here that the main aim with paper
212 224
III was to describe indications on how to estimate Pb activities in Ra sources at
equilibrium. The reason we selected the detection gates presented in this publication
212 212
(exclusively including Pb emissions) was due to being able to precisely measure Pb to
calibrate the instruments without suffering any cross-contamination of the spectra coming from
228 224
other strong sources of Th and Ra placed on other counters at random occasions during
the data acquisition. This is because the data acquisition was mainly executed during a high
224 228
workload production at Oncoinvent’s laboratory of Ra sources using high doses of Th.
224 212
However, since paper IV measured both Ra and Pb sources with controlled and lower
background levels, for convenience, we decided to use one window of 65-345 keV, which is
212
also the most abundant for Pb. Moreover, by comparing the uncertainty and the
reproducibility of calibration factors between the Ȗ- and X-ray windows, they seemed
comparable.

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Table 2 Overview detection windows (keV) of the sodium iodide NaI(Tl) Ȗ-counter and
calibrated geometries used for determining activities in papers III and IV.

Instrument Detection gate (keV) Calibrated geometry

60–110 1.5 mL / 5mL plastic Eppendorf tube

Hidex
65–345 1 mL volume

70–80 12 mm × 75 mm glass tubes

Cobra
50–120 0.4 mL, 0.2 mL, 0.1 mL, 0.05 mL volume

4.5 Lead-212 re-localization from 224Ra sources

In paper IV, the release of 212Pb from its parent nuclide 224Ra through the intermediate progeny
220 224
Rn was studied for solutions of Ra when free and when adsorbed to CaCO3 particles. A
primary limitation of the study was not being able to directly measure 220Rn, but the amount of
radon gas produced had to be determined indirectly by measuring its daughter nuclide, 212Pb.
220
The main reason Rn cannot be measured directly is because it is an almost pure Į-emitter
and can mainly be measured with an Į-spectrometer. However, since radon is a gas, the detector
would be contaminated, making it a non-feasible option for assessing 220Rn activity.

4.5.1 Release of 220Rn in air from 224Ra sources

The release of 220Rn from 224Ra open sources was studied using two methods. The first method
involved the same setup used for the lead production explained in section 4.3 and shown in
Figure 9. This time, 224RaCl2 or 224Ra-CaCO3 microparticles in suspension were placed on the
paper strip and in the middle of the v-vial. The 224Ra-labeled CaCO3 microparticles used in this
study were either surface- or inclusion-labeled. After approximately 24 h, the paper strip (P)
and needle (N) were placed in two separate vials, and the lid was put back on the empty original
212
vial (V). By using the Bateman equation, the theoretical maximum Pb activity generated
220
(APb) through the Rn decay was calculated with the formula below to calculate for APb,t1 ,
assuming that APb,t0 = 0, where t1 is the time when the V, P, and N vials were measured and t0
is the time when the activity was applied to the paper strip.

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APb,t1 = APb,t0 × ݁ ିȜPb ×ǻt1

(9)
ȜPb
× ARa,t0 ൫݁ ିȜRa ×ǻt1 െ ݁ ିȜPb×ǻt1 ൯
ȜPb -ȜRa

where ȜPb = ln 2ΤPb(t½ ) and Pb(t½ ) = 10.64 h and the same is for ȜRa but with
224
Ra(t½ ) = 3.631 d; ARa,t0 is the total Ra activity applied on the paper strip and therefore
measured as the total activity (ܸ + ܲ + ܰ).
The decay was corrected at the time of preparation, t0.

212
At this point, knowing the theoretical expected Pb activity generated and the activity
220
measured in V, it was possible to calculate the percentage of released Rn by dividing the
measured activity by the expected 212Pb activity.
Two additional measurements on subsequent days were performed to ensure that there was no
224
Ra contamination in the original V.

220
The second method released Rn from liquid into air from distinct volumes of either free
cationic 224Ra2+ in solution (diluted in 0.9% NaCl or water) or a suspension of surface-labeled
(polyacrylic acid) PAA-coated CaCO3 microparticles in water.

224
This time, free cationic Ra2+ in solution or the 224
Ra-labeled particles in solution were
contained in an open tube (O1) that was, in turn, inserted in a bigger sealed tube (O2) and left
220 224
for 24 h. This was done to simulate the release of Rn from an open Ra solution source.
After 24 h, the two tubes (O1 and O2) were separated. O1 was transferred into a new tube, and
the 212Pb activity was assessed for both O1 and O2.

4.5.2 Re-adsorption of 212Pb on 224Ra CaCO3 labeled microparticles

220 212
To study the microparticle release of Rn and the microparticle re-adsorption of free Pb,
two methods were used.

In the first method, surface-labeled CaCO3 microparticles in sucrose solution were added to a
Slide-A-Lyzer MINI Dialysis Device (the inner compartment) and inserted into a conical 15 mL
centrifuge tube (the outer compartment), which was filled with a suspension of unlabeled

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CaCO3 particles suspended in Dulbecco’s PBS solution. The inner compartment had a
semipermeable membrane that allowed the diffusion of small chemical compounds such as
anions and cations (molecular weight cut-off of 20 kDa). The tube was shaken for 24 h at room
temperature by means of a table orbital shaker. After 24 h had passed, the dialysis device was
removed, and the activity present in both the dialysis device AD and the conical tube AS (which
comprised the cold-labeled particles AP placed in a separate tube upon measure) was measured.

212
The percentage of released Pb from the particles present in the dialysis device and the
adsorbed activity on the unlabeled particles present in the bottom of the conical tube was
calculated as follows:

CPM(AP+AS) (10)
% 212Pb released = × 100
CPM(AD+AS+AP)

CPM(AP )
% 212Pb adsorbed = × 100 (11)
CPM(AP +AS )

The second method used was a simplified version of what was just described, and it was more
focused on assessing whether the particles’ concentration would play an important role in the
212 212
percentage of Pb adsorption. This time, the setup consisted of inserting the Pb activity
directly into an Eppendorf tube containing various concentrations of unlabeled particles in
suspension, without the use of a dialysis device.
After the stirring process, the samples were centrifuged, to separate the microparticles from the
solution to individually measure them.

4.5.3 Pre-clinical study of therapeutic effect comparison between 224Ra CaCO3

microparticles labeling methods
224
The therapeutic effect of the Ra-labeled CaCO3 microparticles was evaluated in female
athymic nude mice (Athymic Nude-Foxn1nu) kept in a pathogen-free environment at the animal
facility of the Department of Comparative Medicine of The Norwegian Radium Hospital (Oslo
University Hospital, Norway). The mice were inoculated ip with a human ovarian epithelial
carcinoma cell line ES-2 (American Type Culture Collection, Wesel, Germany). Forty mice

42
 Research methods
were randomized and divided into six experimental groups (N = 4–8 animals per group),
224
summarized in Table 3, consisting of 0.9% NaCl only, Ra-CaCO3 microparticles (224Ra
activity dose range of 138–474 kBq/kg) surface-labeled with and without PAA coating, and
inclusion-labeled particles with and without PAA coating, respectively. One day following the
ES-2 cell inoculation, the different treatments were administered ip: 0.9% NaCl only, CaCO3
224
microparticles only, surface-labeled Ra-CaCO3 microparticles with and without PAA, and
inclusion-labeled 224Ra-CaCO3 microparticles with and without PAA.

The therapeutic effect of the injected radioactive product was evaluated by the time to reach
pre-determined humane endpoints. The activity levels used varied to some degree between the
groups; however, previous data indicate that the two low-activity concentration groups and the
two high-activity concentration groups received activity levels of similar efficiency, allowing
comparisons to be made [94].
A more formal animal toxicity determination was not included in the current study, but animals
were observed for signs like discomfort and weight loss often associated with acute and
subacute radiation radiotoxicity. The induction of radiotoxicity signals was not expected and
would, based on previous study with 224Ra-labeled CaCO3 microparticles [117], require much
higher activity levels than used herein.

43
 Research methods

Table 3 Overview of the groups in the animal therapy study. Group A was injected with 0.9%
NaCl only, while groups B–F were injected with microparticles suspended in sucrose solution
(94 mg/mL sucrose C12H22O11 and 2 mg/mL Na2SO4). *Injected with only 0.9% NaCl solution.
ഞ
mp: microparticles.

Injection solutions
Mice CaCO3 224 Labeling
PAA Ra
group mpᄾ method
A*
- - - -
x7
B
‫ض‬ ‫ض‬ - -
x4
C
‫ض‬ -
‫ض‬ surface
x5
D
‫ض‬ ‫ض‬ ‫ض‬ surface
x8
E
‫ض‬ ‫ض‬ inclusion
x8
F
‫ض‬ ‫ض‬ ‫ض‬ inclusion
x8

4.5.4 Ethical considerations

All procedures involving animals discussed in this dissertation and specifically in paper IV
were performed according to animal welfare needs and were approved by the National Animal
Research Authority (permit FOTS ID 7274). The experiments were performed in compliance
with regulations set by the same authority and by the EU Directive 2010/63/EU regarding the
protection of animals used for scientific purposes. The animals used in the experiments were
bred at the Department of Comparative Medicine of the Norwegian Radium Hospital (Oslo,
Norway). The mice were kept under pathogen-free conditions from birth to death, with food
and water supplied ad libitum. The tumor injection and the later treatment were administered
when the mice were older than 4 weeks. Changes in body weight, behavior, posture, and
appearance throughout the whole study were monitored, and human endpoints were reached
when predefined criteria were fulfilled. Among the criteria were rapid body weight loss (>10%
within one week), ascites build-up impairing the mobility of the mouse and/or cachexia, and
signs of discomfort/acute pain. All mice that reached the predetermined endpoints were
euthanized by cervical dislocation. The total number of animals used in the studies presented

44
 Research methods
in this dissertation was reduced to the absolute minimum (study in paper IV: N = 40, divided
into two control groups and four different therapy groups), and calculations were based upon
previous pilot experiments, which gave us the knowledge and experience of group type and size
to permit the collection of statistically relevant data.

45
 Summary of the papers

5 Summary of the papers

Paper I

Primary standardization of 224Ra activity by liquid scintillation counting

The purpose of this paper was to determine the first primary national standard for 224Ra activity
in equilibrium with progeny.

Main goals:
- to determine the 224Ra primary activity standard based on triple-to-double coincidence
ratio (TDCR) liquid scintillation counting and
- to confirm the standard using the live-timed anti-coincidence counting (LTAC) method,
CIEMAT/NIST efficiency tracing with 3H, Monte Carlo simulation and experimental
data from ionization chambers (ICs), and sodium iodide detectors (NaI).

Conclusions: For the first time, a primary standard for 224Ra has been determined. The standard
was developed at NIST and has been used to calibrate a NIST-automated IC for a 5 mL glass
ampoule geometry, allowing future calibrations at NIST, with a 0.62% (k = 2) calculated
uncertainty. Sufficient agreement between methods was recorded.

46
 Summary of the papers

Paper II

224
Radionuclide calibrator responses for Ra in solution and adsorbed on calcium
carbonate microparticles

New clinically relevant geometries for a radiopharmaceutical consisting of a suspension of

224
Ra surface-labeled CaCO3 microparticles are needed to be determined, consequently to the
primary standardization of 224Ra activity. Multiple ionization chambers (ICs), commonly used
in the hospital setting, were calibrated for different volumes and containers.

Main goals:
- to establish calibration factors for vial and syringe geometries for 224RaCl2 solution;
- to determine whether photon attenuation by CaCO3 (250mg/mL) would affect activity
responses for 224Ra when adsorbed onto microparticles; and
- to determine translation factors for different geometries, solution compositions, and
volumes.

Conclusions: Calibration factors for multiple ICs were established for 224RaCl2 solutions
and 224Ra surface-labeled CaCO3 microparticles for vials and syringe geometry. Calcium
carbonate microparticles attenuated the responses (> ı  Calibration factors for the container,
solution volume, and composition (water, acid solution) were determined, allowing an activity
determination below 0.5% for combined standard uncertainties.

47
 Summary of the papers

Paper III

212
Calibration of sodium iodide detectors and reentrant ionization chambers for Pb
activity in different geometries by HPGe activity determination

212
As no primary standardization has been published for Pb activity, there is a need to assess
212
activities for pre-clinical and clinical trials of Pb-based radiopharmaceuticals.

Main goals:
- to determine a suitable method to produce pure 212Pb;
- to establish geometry-specific calibration factors (or dial settings DS) for ionization
chambers (IC) and sodium iodide counters based on HPGe-determined 212Pb activities;
and
212
- to compare DSs for Pb activity with the one determined for known NIST/NIST
224 228
traceable Ra and Th standard.

212 220
Conclusions: High purity Pb could be produced using a Rn diffusion-based generator.
DSs for 212Pb activity in 5 mL glass ampoules were determined with a < 6% (k = 1) uncertainty,
using HPGe spectrometry. No significant differences among solution volumes (0.05 mL and
0.4 mL) of 212PbCl2 solution in glass test tubes were recorded for NaI counters. DSs determined
224 228
with NIST/NIST traceable Ra and Th standards seemed consistent with the theoretical
212
evaluation of the DS range of Pb activity.

48
 Summary of the papers

Paper IV

224
Radon-220 diffusion from Ra-labeled calcium carbonate microparticles: Some
implications for radiotherapeutic use

220 224
It is of interest to quantify Rn gas diffusion from its parent nuclide Ra, when free in
solution or when bound to different labeled CaCO3 microparticles, as this may influence the
therapeutic efficacy by extending the effective range of the Į-particles. The 212Pb re-adsorption
to CaCO3 microparticles may also enhance the retention of this nuclide in the injected area.

Main goals:
- to determine the percentage of 220Rn gas diffused from 224Ra sources;
220
- to compare the percentage of Rn diffusion from different solution volumes and
particle concentrations for 224Ra sources;
220
- to determine whether different particle concentrations affect the percentage of Rn
diffusion;
- to determine the re-associated activity ratio of 212Pb activity vs. the total 212Pb activity
released from surface- and inclusion-224Ra labeled CaCO3 microparticles; and
224
- to determine the therapeutic efficacy of CaCO3 microparticles, labeled with Ra,
through either surface adsorption or inclusion into the bulk of the microparticles, which
was hypothesized to impact 220Rn diffusion in an animal model.

Conclusions: The diffusion of 220Rn was not significantly different between the two labeling
methods, nor did the therapeutic index vary. Radon-220 diffusion was higher for low volumes
(1–10 ȝL) of 224Ra. The 212Pb re-association appeared to depend on particle concentrations; it
was lower for concentrations of < 1 mg/mL and 70–80% for 1 mg/mL to 50 mg/mL.

49
 Discussion

6 Discussion

6.1 Radium-224 and 212Pb activity determination

The activity standardization of radionuclides with complex decay, such as 224Ra, may require
access to advanced laboratory facilities with significant accuracy, equipment, and expertise.

224
The primary activity standard for Ra activity in equilibrium with its progeny (reported in
paper I) has been determined at NIST based on TDCR counting with efficiencies calculated
using the code MICELLE2. The activity standard determination was assessed experimentally
and confirmed by multiple other activity determination methods, as well as by Monte Carlo
simulations.

The primary standardization process for the first FDA- and EMA-approved Į-emitting
radionuclide,223Ra, was quite challenging for NIST and NPL [98, 118]. Based on the experience
with this radionuclide and the lessons learned, the methods were carefully chosen for the 224Ra
standardization. In 2010, NIST published results for the 223Ra primary and secondary standards.
Following some additional studies performed at NPL, researchers became aware of a
discrepancy that later resulted in D í9.5% deviation compared to the subsequent standard
released by NIST in 2015 [98, 118]. From the examination of the experimental part of the study
and the collection and analysis of the data, no errors were detected; therefore, the discrepancy
could not be attributed to dilutions or transfers of the activity to different containers from the
master solution. Afterward, it was noted that the actual 223Ra source used for the standardization
at NIST was the drug product solution directly sent from the manufacturer in a citrate

50
 Discussion
formulation, while the NPL standard was based upon 223Ra activity diluted in 1 M HCl [98]. In
further experiments presented in the publication relative to the new evaluated primary standard
223
for Ra activity, it was shown that the responses of the detector were decreased for citrate
solutions compared to the corresponding acid form by 2% [99]. It is common knowledge that
strong acid concentrations can increase the stability of lead solutions. As the radon escaping the
211
solution decays into Pb and is deposited on the neck of the flame-sealed ampoule, the
homogeneity of the activity in the sample can be re-established using acid solution. In fact, this
could be avoided by inverting the standard flame-sealed ampoule each time before every
measurement, as performed in paper I, in addition to stabilizing the solution using 1 M HCl
solution to dilute 224Ra.
Another concern was to find a high-quality sealed container to avoid any leakage of 220Rn from
the screw cap container. An initial pilot experiment was performed to rule out whether the
container used could be trusted to seal the sources completely. Finally, the 224Ra standardization
performed at NIST was also conducted in parallel at NPL. The data were found to be in
accordance between the two metrology institutes. The standardized activity samples for 224Ra
were used to calibrate the NIST AutoIC, thereby allowing future measurements of 224Ra in 5
mL ampoule geometry.

Dose calibrator DSs for 224Ra activity determination were later reported in paper II, also for a
set of different models of commercially used ICs (i.e., Capintec), using the NIST AutoIC
response as the reference activity, confirmed by additional TDCR measurements. The factors
were determined not only for the 5 mL ampoule geometry but also for 20 mL dose vials and
20 mL syringes as well as for water and acid solutions. The main goal of paper II was to assess
the possibility of using one DS for determining the 224Ra activity for a full (20 mL) or almost
empty (2 mL) dose vial. The 2 mL volume was also used to simulate the main location of
224
activity in a vial containing 20 mL of Ra CaCO3-labeled microparticles (particle
concentration of 250 mg/mL) suspended in the sediment at the bottom of the vial.

Another significant goal of paper II was to determine whether it was possible to measure the
224
Ra activity in a 20 mL 224RaCl2 solution in a 20R Shott vial and obtain the same response for
equal amount of activity contained in an identical geometry set up, but this time with 20 mL of
224
suspended Ra CaCO3-labeled microparticles. For the geometry used for the ampoules and
vials (for both the 2 mL and 20 mL solutions), the response of the Capintec IC was consistent
224
within < 1%. Moreover, repeated measurements of a 20 mL vial containing 20 mL of Ra

51
 Discussion
CaCO3-labeled microparticle suspension (250 mg/mL) did not register any significant response
attenuation (~0.3%) when the particles were agitated in the solution (the solution appeared
intensely white) compared to when the particles were still and deposited at the bottom of the
vial (with a transparent water solution on top).

224
In the syringe geometry, the attenuation from the particles when measuring 12 mL of Ra
CaCO3-labeled microparticle suspension (250 mg/mL) compared to 12 mL of 224RaCl2 solution
was ~1%. In the 20 mL vial geometry, the attenuation from the suspension of the particle was
higher, ~2.25%, but still acceptable for activity determination in the clinical setting (< 10%)
(59).

In Paper III, the calibration of the Capintec ICs CRC-55tR and CRC-25R at Oncoinvent for
measuring 212Pb, 224Ra, and 228Th activity was achieved for the 5 mL ampoule geometry using
the calibration curve method (59). The reference activities of the sources used for this purpose
were known for the NIST standard (224Ra)/NIST traceable standard sources (228Th) but not for
the 212Pb activity, which was determined using HPGe spectrometry.
Calibration factors for the NaI detectors (Hidex and Cobra) were determined for 212Pb activity
(with a test range between 2 kBq and 13 kBq) at equilibrium with its progenies contained in a
12 mm × 75 mm glass tube at different volume geometries (0.40, 0.20, and 0.05 mL). These
volume-specific factors (CPM/Bq) were not statistically different from each other.
Uncertainties of the factor were estimated by calculating the standard deviation of the
measurements made for triplicates for each geometry volume. The calibration factors were
determined for activities within the linearity range of the instruments, which was verified to
JLYHOLQHDUUHVSRQVHV ”r ” IRU the tested activity ranges of 0.07–13.00 kBq. A
proper primary standardization of 212Pb would have been optimal in order to more accurately
calibrate the instruments discussed in Paper III. Although in a small lab setting with limited
instruments and availability of 212Pb activity, an internal standard has been determined that can
be used during pre-clinical and clinical phases. A primary standard need to be assessed for any
commercialization purposes of 212Pb-based products intended for medical use.

It is important to remark that the activity estimation of 212Pb in paper IV was conducted mainly
with Ȗ-counters at a very low background level, and both the X-ray window of 60–110 keV and
the Ȗ-ray window of 65–345 keV (the window with the most abundant gammas for 212Pb) were
used. It was verified that it is possible to estimate 212Pb activity for both windows used, although

52
 Discussion
it is preferable to measure a blank sample for any possible background correction for Compton
scattering effects.

6.1.1 Uncertainty perspectives for radioactivity determinations

Uncertainty analysis has been performed for all studies included in this thesis. As for Paper I,
statistics played an important role in determining the uncertainty component of each activity
determination. For the TDCR and LTAC systems, the largest contributions to the uncertainty
components were given by the counting statistics defined as the standard deviation of repeated
measurements of a single source (N = 2 to 400) and repeated insertions of the single source in
the detector (N = 2 to 3). Moreover, other components were given by the uncertainty of the
model used (gray filters N = 4 for the TDCR and Monte Carlo models for the uncertainty of the
LTAC), the standard deviation of the activity concentration, the uncertainty due to the half-life
uncertainty propagation and the branching ratios of the progenies (nuclear data uncertainty
component), the uncertainty for the mass determination and for the TDC, and, finally, the
efficiency of the quenching model used to estimate the uncertainty of the Birks parameter
Kb = 0.0075 MeV/cm.

228 224 212

The DSs determined for Th, Ra, and Pb activities were evaluated using the same
procedure. The uncertainty for each DS was determined by combining multiple components
due to the uncertainty of the reference activity uA, the uncertainty of the half-life correction uH,
the calibration curve fit ufit, and the uncertainty due to experiment-to-experiment variance urpt,
which was determined as a standard deviation of the DSs in the various experiments. In Table
224 212
4, it is possible to compare the uncertainty of the Ra (paper II) and Pb (paper III) DS
determinations. As 212Pb has not been fully standardized yet, and since the activity was assessed
by HPGe spectrometry only, the largest contribution was due to the uncertainty of the activity
determination, and, consequently, experiment-to-experiment variation. Nonetheless, the
212
combined standard uncertainty (uC) for Pb activity IC calibration factor was below
6% (k = 1).

As for paper IV, all statistical analyses were performed in GraphPad Prism (version 8.2.1,
GraphPad Software, La Jolla, CA, USA) using a significance level of 0.05 using one-way
ANOVA. The Holm–Sidak method has been used to adjust p-values in survival curves.

53
 Discussion
Table 4 Comparison of DS determination uncertainty components among a NIST standardized
224
Ra source and a 212Pb non-standardized source measured with an HPGe detector.

Nuclide uA uH ufit urpt uC

224
Ra 0.3% <0.1% 0.4% 0.2% 0.7%
212
Pb 3.4% 0.1 <0.1% 4.4% 5. 6%

6.2 Rn-220 diffusion and re-adsorption of 212Pb: Implications for

radiotherapeutic use of 224Ra-CaCO3 microparticles

The release of 220Rn, discussed in section 4.5 for the solution volumes tested of open sources
of 224RaCl2 and 224Ra surface-labeled CaCO3 microparticles, was higher for small volumes (i.e.,
5–10 —L). However, the release of 220Rn activity from low volumes of 224Ra activity in an open
container could potentially be biased, as the 220Rn activity had to travel a greater distance in air
from the solution surface to the vial outlet as the 212Pb activity could potentially be deposited
224
on the inner walls of the tube containing the Ra activity before it could escape. This
confirmed the expectation that radon diffusion is more suppressed in water than air [119], as
shown in Figure 11.
To overcome this challenge, a different method was also developed, for a confirmatory
experiment, using the small paper strip placed inside the v-vial (Figure 9), a method also used
212
to produce Pb activity (section 4.3). Very small volumes of free radium or particle-bound
radium were applied on the paper strip, and the diffusion of 220Rn was indirectly determined by
measuring the empty vial after the strip had been carefully removed. When operated correctly,
no cross-contamination of the vial from the strip would be seen, as no radium activity could be
detected in the empty vial at 212Pb complete decay with HPGe spectrometry.

54
 Discussion

Figure 11 Schematic representation of the Brownian motion of a single 220Rn atom (yellow
SDWK 7KHILJXUHRQWKHOHIWWKHEOXHFLUFOHUHSUHVHQWVWKHDFWLRQUDQJHRIWKHĮUDGLDWLRQIURP
a single 224Ra particle (~100 —m), while the yellow area represents the extended action range
of the radiation due to 220Rn diffusion (~300–400 —m in water [75, 119-122] and ~28·103 —m
in air [119], represented by the yellow lined path). The figure on the right represents a single
event of 220Rn diffusing in water and air from a source of 224Ra-labeled CaCO3 microparticles
sedimented at the bottom of a water solution.

55
 Discussion
The results confirmed the outcome of the first method, showing a detected higher release of
220 224 224
Rn for RaCl2 solution compared to the Ra-labeled microparticles. An explanation for
this may be attributed to the porous structure of the CaCO3 microparticles and the Į recoil
method of diffusion of radon [123-125] into the porous cavities. The recoil range of <50 nm of
radon in most solids [119] suggests that if the recoil energy permits the diffusion for the whole
diameter of the pore, a re-adsorption into the CaCO3 microparticle matrix of the activity
emanated occur. An advantage in using micro-sized particles (ranging from 3 to 7—m), rather
than nanoparticles, is the prolonged retention of microparticles in cavitary regions such as the
peritoneal cavity [126], increasing the retention of radioactivity in the treated body cavity.
Moreover, the biocompatibility and the slow biodegradability of the calcium carbonate at pH
7.5 [127], typical pH values for the peritoneal fluid [128], make this carrier well suited as drug-
delivery method for patients.

220
No significant difference in Rn release between the different products from the two
microparticle labeling methods (p = 0.4131) was detected. This suggests that radon diffusion
224
could occur via the porous structure of the microparticles when Ra is embedded in the
particles [94, 129]. SEM images of the same type of CaCO3 microparticles that were used in
the experiment [94] show a degree of porosity with both labeling methods. The observed
220
diffusion of Rn from the microparticles are in agreement with the previously determined
radon diffusion coefficient for radon gas in limestone [130], a mineral composed of CaCO3.
While 220Rn diffuses for a few centimeters in air and a few hundred micrometers in water, the
diffusion in limestone is of a few millimeters [119]. This means that the diffusion length in
limestone is approximately a thousand times greater than the median diameter of the CaCO3
microparticles used in this study, suggesting that the retention of 220Rn in the microparticles in
expected to be low. However, water or air trapped in the pores and surrounding the
microparticles are likely to affect radon diffusion, as the diffusion coefficient is significantly
lower for radon in water or in air compared to calcium carbonate (i.e. limestone).
In the in vivo study performed with 224Ra-CaCO3 microparticles, discussed in section 4.5.3, no
statistically significant difference was observed between the surface- or inclusion-labeling
methods used (p-value • 0.1868). Because of variations in the radiolabeling yield, the
radioactivity dose of the different groups had a significant variation, although all were within
the window showing therapeutic effects previously in this model [131].
The survival curves of the saline control group and the group receiving non-radioactive
PAA-CaCO3 microparticles overlapped, showing that the microparticle carrier itself had no

56
 Discussion
effect in this cancer model. On the other hand, the mice survival was overall better in the 224Ra
labeled microparticles compared to the non-radioactive control treatment groups.
It is important to note that the slight differences in activity concentrations when comparing the
two low activity treatment groups and the two high activity treatment groups are not expected
to affect the therapeutic effect, since previous animal studies conducted with the same tumor
224
model and the same Ra-labeled CaCO3 microparticles treatment showed no significant
statistical difference in survival rates at different activity concentrations [131, 132]. Mice
receiving inclusion-labeled 224Ra-CaCO3 microparticles with a PAA coating were the one with
the highest activity dose. These particles appeared to be the less effective in terms of survival.
One may speculate that the reduced efficacy could be caused by a possible decrease of 220Rn
diffusion from the microparticles due to the presence of the polymer coating.

An interesting feature of the CaCO3 microparticles observed in this study was the ability of the
microparticles to re-adsorb 212Pb generated in the liquid phase from escaped 220Rn. This could
224
be a valuable feature in the clinical use of the Ra-labeled microparticles as it may reduce
212
leakage of Pb into systemic circulation, thereby reducing the risk of unwanted radiation
212
exposure of distant tissues, potentially allowing better control of the distribution of the Pb
daughters.
A dialysis unit with an inner and outer chamber separated by a semipermeable membrane
allowing the diffusion of cations but not the microparticle was used. The inner chamber
contained radiolabeled particles, and the outer chamber contained non-labeled particles. From
studies performed using this dialysis tool, it was found that when a fraction (6%) of the total
activity diffused from the radiolabeled particles inside the inner chamber to the outer chamber
solution, 70–80% of this fraction was later associated with cold-labeled CaCO3 microparticles
placed in the outer chamber. This adsorption effect was similar for the particle concentrations
220
studied (from 1 to 50 mg/mL), suggesting a potential feature of Rn diffusion that by first
approximation could lead to a "dose-smoothening" effect. This "dose-smoothening" effect
could overcome particle aggregation issues in the body, as previously observed during
biodistribution studies in rodents [131]. Heterogeneous distribution of microparticles was a
known problem with radioactive colloids (i.e., in Sullivan et al. [133]).

57
 Discussion

When the activity is stably bound to the microparticle, the Į-particle radiation range of the
particle would be less than 100 —m. By allowing the diffusion of 220Rn, the effective Į range
around a microparticle may be extended according to the 220Rn diffusion range in an aqueous
medium (Figure 11). Adsorption of the decay product of released 220Rn (i.e., 212Pb) onto nearby
microparticles can reduce the potential problem of distant re-localization of 212Pb into the blood,
among other places.

The microparticles release and re-adsorb radioactivity to the extent that the radiation zone
around a microparticle (made of ĮSDUWLFOHV coming from the microparticle-bound radionuclides
DQGĮSDUWLFOHVcoming from the released radon) is potentially extending the effective range of
the Į radiation zone, although being mostly confined in the peritoneal cavity, due to 220Rn and
its direct daughter nuclide 216Po relatively short half-life.

When it comes to radiotoxicity evaluation of 224Ra-labeled CaCO3 microparticles there are two
main features that can cause toxicity (1) Release of 224Ra and progenies due to biodegradation
of the particles and (2) 220Rn emanation from the particles causing release of 224Ra progenies.
Some of the 224Ra is released from microparticle biodegradation and is likely to reach systemic
circulation where it is rapidly taken up in the skeleton or released via the intestines (e.g.
223 224
Ra/Xofigo). As long as the amount of Ra radioactivity used, in a worst-case scenario,
would not cause a higher skeletal dose than 223Ra/Xofigo the risk for systemic toxicity from the
product is low.
When 220Rn is emanating from the particle most of the Į dose from the free radionuclides will
be produced locally in the cavity where the microparticles are applied. Lead-212, with its
substantial half-life, and its progenies has the potential to translocate into systemic circulation
and cause some systemic toxicity effects. However, it is important to point out that most of the
energy released from Į particles in the 224Ra decay chain (3 out of 4 alphas) comes before 212Pb
and its progenies. In the DaRT application it was shown that 30% and 50% of the 212Pb activity
is assumed to leave the targeted area and redistribute away from it [75, 134].
The results presented in paper IV indicate that the presence of CaCO3 microparticles reduces
the risk of translocation due to 220Rn emanation by re-adsorption of its daughter 212Pb. As seen
in animal therapy studies [131], even when the particles slowly dissolve and some release of
224
Ra and 212Pb has occurred, hematological analysis did not indicate any significant toxicity to

58
 Discussion
the bone marrow and, in general, the risk for radiotoxicity can be considered low compared to
off-target effects from many other cancer therapeutics. This is in agreement with the data from
224
ankylosing spondylitis patients who received a dose of 10 MBq of RaCl2 [135, 136] and
® 223
prostate cancer patient receiving Xofigo ( RaCl2) doses of 55 kBq per kg body weight
(3.85 MBq for a 70kg patient) [137], where both products have shown good safety profiles
compared with many cancer drugs in use today.
If for some reason very high radioactivity dosings with the 224Ra-labeled microparticles were
to be used, a substantial radiotoxicity might occur as indicated by published data in beagles and
human patients for intravenously administered 224Ra. Biodistribution studies with 224RaCaCO3
microparticles showed that the activity was predominantly located in the peritoneal cavity [94].
In adult beagles intravenously injected with 224RaCl2, red blood cells and liver were found to be
targeted by 212Pb and kidneys by 212Bi [138]. Also historical data of high cumulative doses of
224 224
Ra therapy, sometimes reaching a total dose of 140 MBq RaCl2 activity in ankylosing
spondylitis young patients [139], resulted in a long term toxicity and an increased risk of late
224
cancer. In conclusion, the risk for toxicity can be considered relatively low for Ra-labeled
CaCO3 microparticles when used at moderate activity levels, but treatment with very high
radioactivity doses is not recommended due to risk of bone marrow-, liver-, kidney toxicity etc
and the potential for carcinogenesis.

59
 Conclusions & future perspectives

7 Conclusions & future perspectives

224 212
The results presented in this doctoral thesis show how to assess Ra and Pb activity at
equilibrium with their progenies using both primary and secondary methods.

It is important to know that progenies are in equilibrium with the mother nuclides when using
the methods and calibration factors presented. The standardization work of 224Ra activity has
been important to assure correct radioactivity administration to patients during clinical trials
with the radiopharmaceutical Radspherin™. Radium-224 radiolabeled CaCO3 microparticles
may extend the survival of immunocompromised mice affected by ip ovarian cancer compared
to administration of free 224Ra. It has been hypothesized that the slight emanation of 220Rn from
CaCO3 microparticles may be beneficial in terms of microdistribution of the Į radiation in the
peritoneal cavity by extending the effective Į-particle range. Moreover, it was shown that the
decay product of 220Rn, 212Pb, has a high affinity for CaCO3 microparticles, determining a re-
association of the 220Rn-released activity onto the particles. This suggests the effective control
of activity localization and indicates that CaCO3 is a suitable carrier for 224Ra.

224 212 224

In clinical trials with Ra, monitoring Pb activity distribution compared to Ra activity
distribution may be important for human blood and urine samples. This can add information to
220 224
the biodistribution data from rodents on whether Rn released from a Ra-based
radiopharmaceutical can cause the re-localization of the activity outside the treated area in a
human system versus the tested animals.

Since both 224Ra and 212Pb are used as radiopharmaceuticals, there is a need to develop standard
methods for determining ingrowth status and activity levels for both radionuclides prior to

60
 Conclusions & future perspectives
equilibrium, i.e., by liquid scintillation and/or Ȗ-spectrometry methods. This is important if
recently produced products will be used for patient treatment. When the purification date and
time of free 224Ra activity is known and there is enough confidence in the radionuclidic purity
of the sample, the ingrowth of progenies of a nuclide can be calculated; factors for the different
ingrowth and decay ratios can be tabulated to better assess the 224Ra activities at a given time
after a known purification date. However, this would be valid only for sealed sources and,
therefore, for activities assessed prior to any chemical alteration of the product. An example of
this situation would be a patient vial released by the manufacturing site and measured with a
fixed DS specific for 224Ra in a calibrated ionization chamber. By knowing the time and date of
the product’s purification, it would be possible to determine the total activity contained in the
vial prior to opening the source by measuring it in the IC and multiplying the reading activity
by a decay factor, then dividing the same number by the ingrowth factor specific for the
measuring day.

224
The NPL in the UK has determined those factors for Ra activity in unpublished studies,
although the effect of volumes and vial types should be further evaluated for those non-
224
equilibrated solutions. The standardization of Ra is currently playing an important role for
224
ensuring that administered Ra activities to patients during clinical trials are accurately
measured and traceable to a primary standard.
212
A national primary activity standard for Pb has not yet been established; therefore, more
work with this nuclide is needed to further increase activity determination accuracy and to
212
ensure consistency among research institutes and hospitals using Pb in preclinical and
clinical settings.

61
 References

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suspension for intraperitoneal therapy. Radiology, 1983. 146(2): p. 539-541.
134. Arazi, L., et al., The treatment of solid tumors by alpha emitters released from (224)Ra-loaded
sources-internal dosimetry analysis. Phys Med Biol, 2010. 55(4): p. 1203-18.
135. Koch, W., Indication for 224 Ra-therapy in ankylosing spondylitis (Morbus Struempell-
Bechterew-Marie), in Biological Effects of 224Ra. 1978, Springer. p. 21-29.
136. Tiepolt, C., T. Grüning, and W.-G. Franke, Renaissance of 224Ra for the treatment of ankylosing
spondylitis: clinical experiences. Nuclear medicine communications, 2002. 23(1): p. 61-66.
137. Lassmann, M. and D. Nosske, Dosimetry of 223 Ra-chloride: dose to normal organs and tissues.
European Journal of Nuclear Medicine and Molecular Imaging, 2013. 40(2): p. 207-212.
138. Lloyd, R., et al., Radium-224 retention, distribution, and dosimetry in beagles. Radiation
research, 1982. 92(2): p. 280-295.
139. Wick, R., et al., Increased risk of myeloid leukaemia in patients with ankylosing spondylitis
following treatment with radium-224. Rheumatology, 2008. 47(6): p. 855-859.

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Publications

69
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Paper I

70
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Paper II

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Paper III

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Paper IV

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 224
Radon-220 diffusion from Ra-labeled calcium
carbonate microparticles: Some implications for
radiotherapeutic use

Elisa Napoli1,2,3, Tina B. Bønsdorff1, Ida Sofie Jorstad1, Øyvind S. Bruland2,4, Roy H.
Larsen1 and Sara Westrøm1*

1
Oncoinvent AS, Oslo, Norway
2
Institute of Clinical Medicine, University of Oslo, Oslo, Norway
3
Department of Radiation Biology, Institute for Cancer Research, Oslo University
Hospital, Oslo, Norway
4
Department of Oncology, Oslo University Hospital, Oslo, Norway

Submitted for publication in journal: PLOS ONE, November 2020.

1/28
Abstract

Alpha-particle emitting radionuclides continue to be the subject of medical research because

of their high energy and short range of action that facilitate effective cancer therapies.

Radium-224 (224Ra) is one such candidate that has been considered for use in combating
224
micrometastatic disease. In our prior studies, a suspension of Ra-labeled calcium

carbonate (CaCO3) microparticles was designed as a local therapy for disseminated cancers
224
in the peritoneal cavity. The progenies of Ra, of which radon-220 (220Rn) is the first,

together contribute three of the four alpha particles in the decay chain. The proximity of the
224
progenies to the delivery site at the time of decay of the Ra-CaCO3 microparticles can

impact its therapeutic efficacy. In this study, we show that the diffusion of 220Rn was reduced
224
in labeled CaCO3 suspensions as compared with cationic Ra solutions, both in air and

liquid volumes. Furthermore, free-floating lead-212 (212Pb), which is generated from

220
released Rn, had the potential to be re-adsorbed onto CaCO3 microparticles. Under
212
conditions mimicking an in vivo environment, more than 70% of the Pb was adsorbed

onto the CaCO3 at microparticle concentrations above 1 mg/mL. Further, the diffusion of
220
Rn seemed to occur whether the microparticles were labeled by the surface adsorption of
224 224
Ra or if the Ra was incorporated into the bulk of the microparticles. The therapeutic

benefit of differently labeled 224Ra-CaCO3 microparticles after intraperitoneal administration

was similar when examined in mice bearing intraperitoneal ovarian cancer xenografts. In
220 212
conclusion, both the release of Rn and re-adsorption of Pb are features that have

implications for the radiotherapeutic use of 224Ra-labeled CaCO3 microparticles. The release

of 220Rn through diffusion may extend the effective range of alpha-particle dose deposition,

and the re-adsorption of the longer lived 212Pb onto the CaCO3 microparticles may enhance

the retention of this nuclide in the peritoneal cavity.

2/28
Introduction

Cancer therapy with radionuclides has been the recipient of increased interest, and several

beta- and alpha-particle emitter-based therapeutic radiopharmaceuticals have either been

approved or are undergoing clinical investigation [1–7]. The radionuclides that are used

include the beta emitters 89Sr, 90Y, 131I, 153Sm, 177Lu and beta-emitting 212Pb, which generates

alpha-emitting progenies, as well as the alpha emitters 211At, 213Bi, 223Ra, 225Ac, 224
Ra and
227
Th. In general, long-range, low linear energy transfer (LET) beta emitters are believed to

be more suitable for the treatment of larger tumors than short-range, high-LET alpha

emitters, which are considered to be more effective for the treatment of micrometastases and

single-cell diseases [8].

From a logistical point of view, 224Ra has a convenient half-life of 3.63 days [9,10]. It decays

via several radioactive progenies, producing four alpha particles and two beta particles

(Table 1 and Fig 1). Recently, it has been subject of preclinical [11–15] and clinical [16–18]

research for its potential use in antitumor agents. While the properties related to high-LET
224
radiobiology [19] make Ra a potent cytotoxic agent, there are some concerns regarding

the fate of its progenies in vivo as daughter nuclides can distribute differently than a parent

because of differing biological affinities. For the brachytherapy application called diffusing
224
alpha-emitters radiation therapy (DaRT) in which Ra-loaded wires are implanted into

solid tumors, the distribution of progenies both within the tumor and in normal tissues have

been examined [13,20]. The release of progenies from one such 224Ra source has been shown

to have a therapeutic effect in a region of 5–7 mm in diameter.

3/28
Table 1. Details of the nuclear decay data for 224Ra and its daughters indicating x- and Ȗ-lines with 1%
or higher abundance and divided into two columns: one for energies in the 60-110 keV detection window
and the second for energies above 110 keV.

Daughter x- and Ȗ-lines, keV (Abundance)

Nuclide Half life
nuclide 60–110 keV > 110 keV
224
Ra 3.631 days (Rn) None 241.0 (4.12)%
220
Rn 55.8 s (Po) None None
216
Po 0.148 s (Pb) None None
212
Pb 10.64 h (Bi) 74.8 (10.1%) 238.6 (43.6%)
77.1 (16.9%) 300.1 (3.18%)
86.8 }
87.3 } (5.77%)
89.8 }
89.7 }
90.1 } (1.77%)
90.4 }
212
Bi 60.54 min (Po)/(Tl) None 727.3 (6.65%)
785.4 (1.11%)
1620.7 (1.51%)
212
Po 300 ns (Pb) None None
208
Tl 3.058 min (Pb) 72.8 (2.03%) 277.4 (6.6%)
75.0 (3.42%) 510.7 (22.5%)
84.5 } 583.2 (85.0%)
84.9 } (1.17%)
85.5 }
763.5 (1.80%)
860.5 (12.4%)
2614.5 (99.8%)
All data were taken from the Decay Data Evaluation Project [10]. To compare the x-ray and gamma incidences
between the radionuclides at equilibrium in the decay series, the branching factor (see Fig 1) for 212Bi, 212Po
and 208Tl must be considered.

4/28
Fig 1. Decay chain of 224Ra and progenies to stable 208
Pb. Half-life data are taken from the Decay Data
Evaluation Project [10].

We have previously described the use of a suspension of calcium carbonate (CaCO 3)

microparticles as carriers for 224Ra and its progenies [21]. This novel application is designed

to treat disseminated micrometastatic cancers, such as peritoneal carcinomatosis following

intraperitoneal (IP) administration. Radium-224 adsorbed on CaCO3 microparticles has

demonstrated antitumor activity against ovarian cancer xenografts in the peritoneal cavity of
224
mice [11,15]. Because of the multiple alpha-emitting daughters of Ra, it is important to

investigate the interaction of these progenies with the carrier compound. For example, 212Pb,

the progeny of 224Ra with the longest half-life in the decay chain (10.64 h [9,10], Fig 1), may

reach systemic circulation if it is prematurely released from the CaCO3 microparticles. A

5/28
release of 212Pb from the carrier compound can influence the dose delivered to the target area

and hence reduce the therapeutic effect of the product. Therefore, the behavior of the noble

gas 220Rn, the immediate daughter of 224Ra and the grandparent of 212Pb in the decay chain,
220
is of particular interest. Because it is gaseous, Rn may diffuse away from the CaCO3

microparticles and mediate a re-localization of the radioactivity.

In this study, we explored some fundamental product properties related to the two critical

progenies, 220Rn and 212Pb, when CaCO3 microparticles are used as a carrier compound for
224
Ra. The diffusion of 220Rn from the microparticles was investigated in both air and liquid
212 220
phases. The fate of Pb subsequent to its release due to the diffusion of Rn was also

studied under conditions mimicking an in vivo environment. Further, CaCO3 microparticles

224
labeled with Ra through either surface adsorption or inclusion into the bulk of the
220
microparticles were hypothesized to impact Rn diffusion and thus evaluated for their

therapeutic effect in mice following the IP inoculation of the human ovarian cancer cell line

ES-2.

6/28
Materials and Methods

Preparation of 224Ra-labeled CaCO3 microparticles

228
Radium-224 was extracted via a Th source from Eckert and Ziegler (Braunschweig,

Germany) or Oak Ridge National Laboratory (Oak Ridge, TN, USA) through previously
228
published methods [21,22]. In brief, the Th was immobilized on a column containing

DIPEX® (Eichrom Technologies LLC, Lisle, IL, USA) actinide resin. After allowing time
224
for ingrowth, the Ra was eluted in 1 M HCl and evaporated to dryness. For subsequent

use in radiolabeling, the residue was dissolved in 0.1 M HCl and pH adjusted to between 5

and 6 through the addition of NH4OAc (Merck, Darmstadt, Germany) to a final

concentration of 0.5 M.

The 224Ra-labeled CaCO3 microparticles were prepared by two different procedures: (1) the

adsorption of 224Ra onto the surfaces of pre-manufactured CaCO3 microparticles and (2) the

incorporation of 224Ra into the bulk during CaCO3 microparticle production.

The CaCO3 microparticles that were subsequently used for surface labeling with 224Ra were

prepared by a spontaneous precipitation process. In short, equal volumes of 0.33 M CaCl 2

(Merck) and 0.33 M Na2CO3 (Merck or VWR International, Radnor, PA, USA) were mixed

either by magnetic or overhead stirring. The microparticles were collected by centrifugation,

subsequently dried in an oven for 1 h at 180°C and stored as a dried powder. In addition, a

batch of CaCO3 microparticles was purchased from PlasmaChem GmbH (Berlin, Germany).

The microparticles had a mainly spherical geometry with diameters ranging from 3–7 —m

when representative batches were measured by laser diffraction (Mastersizer 3000, Malvern

Instruments Ltd, Worcestershire, UK).

7/28
For the surface radiolabeling, the microparticles were washed three times with water and

two times with 0.1 M Na2SO4 (Merck) before dispersion in either 0.9% NaCl or a sucrose

solution (composed of 94 mg/mL sucrose from Sigma-Aldrich, St. Louis, MO, USA and
224
2 mg/mL Na2SO4) as previously described [21]. Subsequently, Ra solution was added

along with 0.004–0.3 w/w% Ba2+ and 0.3–0.6 w/w% SO42í relative to the amount of CaCO3.

Microparticle suspensions were placed under orbital rotation for 1.5 h (HulaMixer,

Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) during the radiolabeling process.

The inclusion-labeled CaCO3 microparticles were prepared by rapidly mixing equal volumes
224
of 0.33 or 1 M CaCl2 solution containing Ra at the target radioactivity level and 0.004–

0.3 w/w% Ba2+ (relative to CaCO3) with 0.33 or 1 M Na2CO3 solution containing 0–0.7

w/w% SO42- (relative to CaCO3) with magnetic stirring or vortexing for 1–3 min. The mass

amount of the CaCO3 microparticles produced was determined by assuming the quantitative

yield of the precipitation process.

For both radiolabeling procedures, excess radiolabeling solution was removed prior to the

CaCO3 microparticles being washed twice with 0.9% saline, sucrose solution or water to

remove any 224Ra not bound to the particles.

In some experiments, the additive polyacrylic acid (PAA, average Mw ~250 000, 35% wt. in

H2O, Sigma-Aldrich) was used to coat the CaCO3 microparticle surface at a ratio of 1.3 —L

PAA solution per microparticle mg and added towards the end of the microparticle

crystallization process. Non-radioactive CaCO3 microparticles were also prepared for some

experiments through a mock labeling process following the same protocol as for surface-

labeling but without the addition of 224Ra.

8/28
Radioactivity measurements

Gamma-ray spectroscopy was performed using a Hidex Automatic Gamma Counter (Hidex,

Turku, Finland) equipped with a 3-inch diameter NaI crystal. The detector was shielded from

background radiation with a lead shield a minimum of 55 mm thick (80 mm on the conveyor

side). The counts per minute (CPM) was registered to the 60–110 or 65–345 keV detection

window. As can be seen in Table 1, the most abundant x and gamma radiation in these energy
212
ranges originate from Pb. For the analyses of the radioactive samples, it was therefore
212
assumed that the CPM in these detection windows originated only from the Pb as the

contribution from the other nuclides in the series was considered minimal. The activity of

the 212Pb was determined directly from the CPM in the 60–110 keV window [23], whereas
224
the Ra activity was determined indirectly based on counts in the 65–345 keV window

when the transient equilibrium between the 224Ra and 212Pb had been established. Transient
212
equilibrium can be assumed > 2 days after the initial Pb measurement when the sample
220
vial is left sealed. The data used for the Rn activity determination were acquired with

sources at secular equilibrium (> 6 h after separation and > 1 day from radiolabeling/transfer

to a new container) so that decay correction to a common reference time was achieved using

the half-life of 212Pb.

The limit of quantification (LOQ) was set to equal the average CPM plus 10 times the

standard deviation of the measurements of a series of blank samples. When the measured

CPM for a sample was below the LOQ value, the CPM was set as equal to the LOQ to

produce a theoretical maximum value.

9/28
Release of 220Rn from open 224Ra sources

220 224
The release of Rn from open Ra sources was evaluated with the two different

experimental setups as visualized in Fig 2.

Fig 2. Experimental setups to investigate 220Rn diffusion from open sources to air. A 3 mL glass micro
reaction vessel (A) and a sealed 5 mL Eppendorf tube (shown open for illustrative purposes) containing a
capless 1.5 mL Eppendorf tube (B) were used in separate experiments.

220
The first setup (Fig 2A) aimed at investigating the release of Rn through the air from a
224 224 224
Ra source. Two —L RaCl2 or 25 —g Ra-CaCO3 microparticles in 2 —L of water

suspension were applied on the surface of a small paper strip (1 × 1.5 cm, absorbent bench

paper) attached to a syringe needle that had previously been inserted through the silicone

septum of a 3 mL glass v-vial (Supelco Analytical, Merck) screw cap. Subsequently, the

screw cap with the radioactive sample was carefully inserted into the v-vial while avoiding

contact between the paper strip and the interior surfaces of the v-vial before the cap was

tightened. After approximately 24 h, the paper strip and needle were placed into two separate

10/28
vials (sample P and N). The cap was put back onto the empty original vial (V) and the

radioactivity in the now three vials was measured (time = t1). The total 224Ra activity applied

on the paper strip (ARa) at time of assembly (t0) was assumed to equal the decay corrected

sum of the activities in samples P, N and V:

CPM(V+P+N)t
ARa,t1 = 1
=ARa,t0 ×e-ȜRa ×ǻt1
EFRa

where EFRa is the efficiency factor (CPM/Bq) for the 65–345 keV window, ɉ= ln 2Τt½ and
220 212
ǻt1 = t1 െ t0 . The amount of Rn release into air was estimated by the measured Pb
212
activity in the empty original vial (V) divided by the theoretical maximum Pb activity

generated through 220Rn decay (APb) at the time of measurement (t1):

CPM(V)t ΤEFPb
% 220Rn release = 1
×100
APb,t1

where EFPb is the efficiency factor (CPM/Bq) for the 60–110 keV window [23] and

A„,t1 was calculated using the Bateman equation:

ȜPb
APb,t1 ൌ  „ǡ–Ͳ ൈ ݁ ିɉ„ ൈǻt1 ൈ ARa,t0 ൫݁ ିȜRa ൈǻt1 െ ݁ ିȜPb ൈǻt1 ൯
ȜPb -ȜRa

with „ǡ–Ͳ ൌ Ͳ. Although 212Pb was present in the samples applied to the paper strip, none

of this had the ability to translocalize from the paper strip to the inner surfaces of the vial,

and it can therefore be disregarded in the calculations above. Two additional measurements
224
on subsequent days were performed to ensure that there was no Ra contamination in the

original vial (V).

220
The second experimental setup (Fig 2B) sought to investigate the release of Rn from

solutions containing 224Ra. Distinct volumes from 5 to 1000 —L of either free cationic 224Ra2+

in solution (diluted in 0.9% NaCl or water) or a suspension of 4.3 mg surface-labeled

PAA-coated CaCO3 microparticles in water were added to 1.5 mL Eppendorf tubes with the

11/28
lids removed. Each sample tube (S) was inserted into a 5 mL Eppendorf tube (O1) and the

lid closed. After 1 day, the outer tube was opened, the inner sample tube transferred to a new

5 mL Eppendorf tube (O2) and the radioactivity in both tubes was measured. The amount of
220
Rn release from the liquid into the air was estimated by the measured 212Pb activity in the

empty outer tube (O1) divided by the total activity:

CPM(O1)
% 212Pb activity detected= × 100
CPM(O1) + CPMሺS in O2ሻ

The procedure was repeated after 3 and 7 days. The trapping efficiency of the 220Rn in the

Eppendorf tubes was verified in a separate experiment and found to be more than 99.8%. In
224
this experiment, a sample containing approximately 50 kBq Ra in a 1.5 mL Eppendorf

tube was contained in a sealed zip lock plastic bag for 1 or 7 days before the radioactivity in

the plastic bag was measured without the Eppendorf tube inside.

Adsorption of 212Pb onto CaCO3 microparticles

212 220
To investigate the chemical fate of Pb subsequent to its release caused by Rn gas
212
diffusion, a set of experiments was conducted to examine whether the Pb could be re-

adsorbed onto the CaCO3 microparticles.

In a pilot experiment, duplicate samples of 5 mg surface-labeled CaCO3 microparticles in

0.4 mL sucrose solution were added to a dialysis device (Slide-A-Lyzer MINI Dialysis

Device, 0.5 mL format, 20 kDa MWCO, Thermo Fisher Scientific). The device was placed

into a conical 15 mL centrifuge tube pre-filled with a suspension of 50 mg mock-labeled

CaCO3 microparticles in 14 mL Dulbecco’s PBS (pH 7, Gibco, Fisher Scientific), and the

tube was then capped with a screw lid. The tube was gently shaken at 150 rpm using a table

orbital shaker for 24 h at room temperature before the dialysis device was removed and the

tube centrifuged to collect the microparticles in the external solution. The radioactivity levels

12/28
in the dialysis device (AD), the supernatant of the external solution (AS) and the pelleted
212
microparticles from the 15 mL tube (AP) were measured. The percentage of released Pb

during the 24 h was estimated as follows:

CPM(AP+AS)
% 212Pb released ൌ  ൈ ͳͲͲ
CPM(AD+AS+AP)

The percentage of the released 212Pb activity that was adsorbed onto the CaCO3 particles was

estimated as:

CPM(AP )
% 212Pb adsorbed = × 100
CPM(AP +AS )

212
The dependency of the adsorption of Pb on the CaCO3 microparticle concentration was

examined in further experiments with a more simplified setup. In this case, 212Pb was used
212
directly and not as in the previous experiments where the source of Pb was the great-
224
grandparent nuclide Ra. Samples of mock-labeled CaCO3 microparticles in 75%

Dulbecco’s PBS and 25% fetal bovine serum solution (pH 7.5–8.5) with concentrations

ranging from 0.1–50 mg/mL were prepared in 1.5 or 5 mL Eppendorf tubes. Equal activities

of 212Pb were added to each sample before stirring with orbital motion at 450 rpm using an

Eppendorf C thermomixer or at 30 rpm using a HulaMixer at 37 °C. After 45–95 min, the

samples were centrifuged to separate the microparticles from the solution. The radioactivity

levels in the supernatant (AS) and the pelleted microparticles (AP) were measured, and the
212
percentage of Pb activity that had adsorbed onto the originally non-radioactive CaCO3

microparticles was determined as described above.

The 212Pb was produced and separated from the 224Ra via 220Rn emanation [24] using a single
224
chamber diffusion system [23]. A few —L of RaCl2 solution were distributed on quartz

wool (ProQuarz, Mainz, Germany) that was fixed on the inside of the screw cap of a 100 mL

glass flask (Simax-Kavalierglass, Prague, Czech Republic). The sealed flask was left

13/28
inverted overnight in a fume hood for the 220Rn to be released through the air inside the vial.
220 212
The Rn would then decay into Pb and become deposited on the interior walls of the

container. After 20 to 28 h, the cap with the 224Ra source was carefully removed, avoiding
224 212
the Ra contamination of the vial. The Pb was subsequently retrieved by washing the

glass walls with 1 M HCl solution.

Therapeutic effect of surface- and inclusion-labeled 224Ra-CaCO3

microparticles in mice

Female athymic nude mice (Hsd:Athymic Nude-Foxn1nu, bred at the Department of

Comparative Medicine, The Norwegian Radium Hospital, Oslo University Hospital, Oslo,

Norway) of 4–6 weeks of age at the start of the experiment were used. The animals were

maintained under pathogen-free conditions with food and water supplied ad libitum and

monitored for changes in body weight, behavior, posture and appearance throughout the

study. All procedures involving animals were approved by the Norwegian Food Safety

Authority (permit ID 7274) and performed in compliance with regulations set by the same

authority and EU Directive 2010/63/EU on the protection of animals used for scientific

purposes.

Human ovarian epithelial carcinoma cell line ES-2 (American Type Culture Collection,

Wesel, Germany) was cultured in McCoy’s 5A medium (Gibco, Fisher Scientific)

supplemented with 10% fetal bovine serum (Gibco, Fisher Scientific) and 1%

penicillin/streptomycin (Gibco, Fisher Scientific) at 37 °C in a humid atmosphere with 5%

CO2. The cells were harvested with TrypLE Express solution (Gibco, Fisher Scientific),

suspended in cold RPMI 1640 growth medium (Gibco, Fisher Scientific) and kept on ice

until inoculation.

14/28
 224
The therapeutic effects of four different variants of Ra-CaCO3 microparticles were

investigated: both surface- and inclusion-labeled microparticles each with and without PAA

coating. A total of 40 mice were randomized to the experimental groups and inoculated IP

with 1 × 106 ES-2 cells. One day later, the mice were given the different treatments as shown
224
in Table 2. All the animals that were treated with Ra-CaCO3 microparticles received a

single IP injection of 0.29–0.52 mL to achieve the same radioactivity dose based on their

body weight. The control animals received 0.9% NaCl (0.4 mL) or 5 mg CaCO3

microparticles (0.4 mL) dispersed in sucrose solution.

Table 2. Overview of the experimental groups included in the study investigating the therapeutic efficacy
of 224Ra-CaCO3 microparticles in mice.

224
Experimental group Ra- PAA Activity dose CaCO3 mass dose No. of
labeling coating (kBq/kg (mg per mouse) mice
method bodyweight)
0.9% NaCl n/a n/a n/a n/a 7
CaCO3 microparticles n/a Yes n/a 5.0 4
224
Ra-CaCO3 Surface No 350 4.6 ± 0.5 5
microparticles
224
Ra-CaCO3 Surface Yes 138 4.3 ± 0.4 8
microparticles
224
Ra-CaCO3 Inclusion No 179 5.5 ± 0.5 8
microparticles
224
Ra-CaCO3 Inclusion Yes 474 5.6 ± 0.5 8
microparticles
PAA: polyacrylic acid, n/a: not applicable

Therapeutic effect was evaluated by the time it took to reach the pre-determined humane

endpoints, which were defined as rapid body weight loss (> 10% within one week), ascites

build-up that severely impaired mobility and/or cachexia. Mice were euthanized by cervical

dislocation when they reached the predetermined endpoint and necropsied for gross

pathological examination.

15/28
Statistical Analysis

All statistical analyses were performed in GraphPad Prism (version 8.2.1, GraphPad

Software, La Jolla, CA, USA) using a significance level of 0.05. The release of 220Rn from
224
different Ra sources was analyzed by one-way ANOVA using the Tukey method to

correct for multiple comparisons. Survival curves were compared pairwise by log-rank tests

and the Holm-Sidak method to adjust the p-values for multiple comparisons.

16/28
Results

Release of 220Rn to air from open 224Ra sources

The release of 220Rn to air from open 224Ra microsources was measured indirectly through
212
the amount of daughter Pb that had re-localized. Radium-224, either in the form of free

cation or as surface- or inclusion-labeled CaCO3 microparticles, was applied on a paper strip

220
fixed on a needle suspended in a glass vial (Fig 2A). The percentage of Rn release was
212
estimated by dividing the Pb activity detected in the outer vial with the theoretical

maximum amount of 212Pb generated from 220Rn decay. The results displayed in Fig 3 show
220 224 224
significantly higher Rn release from Ra as a free cation than as Ra-labeled CaCO3

microparticles (one-way ANOVA, p ” 0.0039). No evident difference was seen between the

different 224Ra-labeling methods of the CaCO3 microparticles (p = 0.4862).

Fig 3. Release of 220Rn to air from various open 224Ra microsources. The 220Rn release was estimated
indirectly from measurements of the 212Pb activity that had re-localized due to 220Rn diffusion. Each
independent sample is indicated with a symbol, and a horizontal line represents the average of these three.

17/28
 220 224
The emanation of Rn was also evaluated for open liquid sources of Ra (Fig 2B).

Different volumes of free 224Ra or surface-labeled PAA-coated CaCO3 microparticles were

added to a tube without a cap that was contained inside a larger closed tube. After
212 212
approximately 1 day, the ratio of Pb activity detected in the outer vial to the total Pb

activity was used to indicate the 220Rn release from the open liquid sources. The results show
220 224
that the Rn release was at least 4 times lower when the Ra was adsorbed onto the

microparticles as compared with as a dissolved cation, which is in line with the findings
212 220
from the first experimental setup. The re-localization of Pb due to Rn diffusion also
220
appears to be dependent on the liquid volume of the sample, with higher Rn release at

lower volumes (Fig 4). The release at low volumes may be underestimated because of the
212
Pb activity deposited on the inner tube wall (Fig 2B). The experiment was repeated on

days 3 and 7, yielding similar results for the volume dependency (S1 Fig), which indicates

that a steady state was obtained after 1 day.

Fig 4. Detected 212Pb due to 220Rn release from open liquid sources of 224Ra approximately 1 day after
assembly. Sample volumes ranged from 5 to 1000 —L of either free cationic 224Ra or suspensions with 4.3 mg
PAA-coated CaCO3 microparticles surface labeled with 224Ra. Error bars represent standard deviation.

18/28
Adsorption of the 220Rn daughter 212Pb on CaCO3 microparticles

212 224
In order to investigate whether the Pb released from the surface-labeled Ra-CaCO3
212
microparticles could re-adsorb onto the microparticles, both the percentage of the Pb

activity released from the dialysis unit to the outer solution and the percentage of the 212Pb

adsorbed onto the originally non-radioactive microparticles were measured. Of the

212
approximately 6% Pb that had crossed the dialysis barrier, 75% was found to have re-

associated with the CaCO3 microparticles.

212
Subsequent experiments showed that the degree of Pb adsorption was high even at

relatively low CaCO3 microparticle concentrations (Fig 5). Adsorption decreased at CaCO3

microparticle concentrations below 1 mg/mL, whereas between 1 and 50 mg/mL, it appeared

to reach a plateau with adsorption of approximately 70–80%.

Fig 5. The percentage of 212Pb activity adsorbed onto CaCO3 microparticles at different CaCO3
microparticle concentrations.

19/28
Therapeutic effect of surface- and inclusion-labeled 224Ra-CaCO3

microparticles in mice

224
A single IP injection of Ra-CaCO3 microparticles significantly improved survival as

compared with both the saline and non-radioactive CaCO3 microparticle groups (p ” 0.023),

regardless of the different radiolabeling methods and PAA coating (Fig 6). The control

groups had no survivors beyond day 17, whereas all mice were alive at this time in the
224
different Ra-CaCO3 microparticle groups. No statistically significant difference was

found between the surface- and inclusion-labeled products (p • 0.1868), although the
224
survival curves indicate that treatment with the inclusion-labeled Ra-CaCO3

microparticles with a PAA coating had a slightly inferior effect as compared with the other
224
Ra-labeled microparticle treatments. The survival curves of the saline control group and

the group receiving PAA-CaCO3 microparticles overlap, showing that the microparticle

carrier itself had no effect in this cancer model.

Fig 6. Kaplan-Meier survival curves of athymic nude mice inoculated intraperitoneally with 1 × 106 ES-
2 cells and treated 1 day later with intraperitoneal injections of 0.9% NaCl, PAA-coated CaCO3
microparticles or 224Ra-CaCO3 microparticles both with and without PAA coating with an activity dose
ranging from 138–474 kBq/kg body weight. N = 4–8 animals per group.

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Discussion

220 224
The current study demonstrates that there is a significant diffusion of Rn from Ra-

CaCO3 microparticles. The degree of 220Rn diffusion seems to be relatively independent of

224
the radiolabeling method, that is, whether Ra was adsorbed onto the surfaces or

incorporated into the bulk of the microparticles during CaCO3 precipitation. This may be

explained by the porous structure of these CaCO3 microparticles [21,25] that allows 220Rn to

escape. However, both the air and liquid phase studies demonstrated that the diffusion of
220
Rn was significantly reduced when 224Ra was bound to microparticles as compared with

free cationic 224Ra.

224
All variants of the Ra-CaCO3 microparticles significantly extended the survival of the

mice with IP tumors, but the effect was relatively independent of the radiolabeling method.

One treatment, inclusion-labeled 224Ra-CaCO3 microparticles with a PAA coating, seemed

224
to be slightly less effective. Because of some variations in the Ra-labeling yield, the
224
activity dose was not directly comparable in the four different Ra-CaCO3 microparticle

groups. The highest activity dose (474 kBq/kg) was administered to the mice receiving

inclusion-labeled 224Ra-CaCO3 microparticles with a PAA coating. Therefore, this does not
224
explain why this variant of the Ra-CaCO3 microparticles appeared less effective. The
224
surface-labeled Ra-CaCO3 microparticles were given at an activity dose approximately

twice as high (350 kBq/kg) as the analog with the PAA coating (138 kBq/kg) and the

inclusion-labeled without (179 kBq/kg). Previous studies in the same tumor model with

activity doses of 150 and 300 kBq/kg showed a tendency for prolonged survival with the

highest dose; however, the difference was not statistically significant [11].

220
The release of Rn from CaCO3 microparticles affects the microdistribution of the alpha

particles from the 224Ra series. The mean distance 220Rn can travel subsequent to its escape

21/28
from the microparticles can be estimated by its mean diffusion length, which is dependent

upon half-life (55.8 s, Fig 1) and the diffusion coefficient of the material the radon atoms

traverse. The mean diffusion range is typically a few hundred micrometers in water and
224
centimeters in air (Table 3). In the case of using the Ra-CaCO3 microparticles as a

treatment for IP cancer when the intent is to irradiate liquid volumes and serosal surfaces in

the peritoneal cavity harboring micrometastases, the diffusion distance in water is probably

the most relevant to consider. As illustrated in Fig 7, 220Rn diffusion can result in an increase

in the irradiated volume from 224Ra-CaCO3 microparticles. The maximum distance an alpha

particle can travel in water is less than one third of the mean diffusion length of 220Rn in the

same medium. Thus, the irradiated volume can be increased by a factor of 27 through 220Rn
224
diffusion. This indicates that the alpha-particle related microdosimetry of Ra-labeled

microparticles may be significantly different from that of microparticles labeled with single-

step decaying alpha-emitting radionuclides. If only alpha-particle radiation is considered

224
significant for the biological activity of Ra-CaCO3 microparticles, then three out of four

alpha particles are produced by 220Rn and its progenies. Depending on the degree of radon

diffusion from the microparticles, a significant fraction of the therapeutic radiation dose can

be delivered beyond the alpha-particle range from a microparticle. Thus, the emanation of
220
Rn could be of benefit both for extending the effective alpha-particle range and in terms

of radiation “dose smoothening” as the microparticles may not be perfectly distributed in the

treated cavity. The brachytherapy application DaRT also exploits the daughter nuclides of
224
Ra for extending the effective alpha-particle range. Through modeling, it has been shown
224
that a point source of Ra (with approximately 100 kBq) placed in a solid tumor with

approximately 40% of the radon being released results in therapeutic alpha-particle dose

levels over a distance of 4–7 mm in diameter [20]. This distance is also in line with

22/28
preclinical studies in which necrotic regions of 5–7 mm in diameter have been observed after

the placement of a single 224Ra wire into squamous cell carcinoma tumors in mice [13].

Table 3. Overview of radon diffusion coefficients and resulting mean diffusion lengths in different
materials.

Material Temperature (°C) Diffusion coefficient (cm2/s) Mean diffusion length* (mm)
Water 37 1.9 × 10í5 [20,26] 0.4
Water 18 1.1 × 10í5 [27,28] 0.3
Water n/a 1 × 10 í5 [29] 0.3
Air n/a 0.1 [29] 28
 Žሺʹሻ
* The mean diffusion length is given by: ට , where D is the diffusion coefficient and Ȝ = is the decay constant of
ɉ – ͳΤ ʹ
220
Rn.

Fig 7. Illustration of the maximum distance alpha particles can travel in water (Lmax, Alpha = 100 —m) and
estimated mean diffusion length of 220Rn in water (Lmean, Radon = 300–400 —m) relative to the CaCO3
microparticle size (5 —m in diameter).

The diffusion of 220Rn from CaCO3 microparticles also raises questions about the fate of the

subsequent progenies. The immediate daughter of 220Rn, 216Po, has a half-life of only 0.15 s

23/28
and will decay essentially in the same location as the mother nuclide. However, the
212
subsequent progeny, Pb, has a sufficiently long half-life to allow it to be transported

further away from the parent nuclide and even redistribute from the peritoneal cavity.
224
Approximately 30% of the energy released from alpha particles in the Ra decay chain

originate from progenies of 212Pb. Hence, a significant fraction of the therapeutic radiation

dose can be lost if this radionuclide decays away from the target area. This is the case in the
212
DaRT application, where a considerable fraction of Pb (assumed to be between 30 and

50% [20,30]) leaves the tumor via systemic circulation and redistributes to distant organs
224
and tissues. With this in mind, a particularly interesting feature of Ra-CaCO3
212 220
microparticles is their ability to adsorb the free-floating Pb generated following Rn

escape from the microparticles. The data from liquid phase studies indicate that the
212
adsorption of Pb onto the microparticles occurs to a significant degree, even under

conditions mimicking an in vivo environment. The adsorption was also high over a wide

range of microparticle concentrations, indicating that this phenomenon can also occur in the

clinical treatment setting.

24/28
Conclusion

The 220Rn diffusion from 224

Ra-labeled CaCO3 microparticles is significant yet reduced as
224 220
compared with the release from cationic Ra. Furthermore, the diffusion of Rn from

microparticles and antitumor activity seem to be independent of the different radiolabeling

methods tested. There is a significant adsorption of 212Pb, the 220Rn daughter with the longest

half-life, onto CaCO3 microparticles even at microparticle concentrations of a few mg/mL.

Thus, the release of 220Rn and re-adsorption of 212Pb are features that may have implications
224 220
for the radiotherapeutic use of Ra-labeled CaCO3 microparticles. The diffusion of Rn

up to a few hundred micrometers can extend the effective range of the inherent short-range

alpha particles and may cause a “dose-smoothening effect” to counteract potential

heterogeneous distribution of microparticles in the treated cavity, while the re-adsorption of

212
Pb onto the CaCO3 microparticles can contribute to enhancing the retention of 212Pb in the

target area.

25/28
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