**Binary Radiotherapy of Melanoma – Russian Research Results**

Victor Kulakov3, Elena Grigirjeva2, Elena Koldaeva2, Alisa Arnopolskaya3,1 and Alexey Lipengolts3,1 *1Federal Medical Biophysical A.I. Burnasyan's Centre of Federal Biomedical Agency of the Russian Federation 2Russian N.N. Blochin's Cancer Research Centre of Russian Academy of Medical Science 3Moscow Engineering & Physics Institute (State University) Russian Federation* 

### **1. Introduction**

122 Advances in Cancer Therapy

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> Radiotherapy (RT) in its different forms –distance, contact, intraoperative - currently is a powerful means of damaging cancer cells. RT is used both, as a radical monotherapy and in combination with different radiosensitizers or antitumor chemotherapeutic agents. The main problem of radiotherapy is the inevitable effect of ionizing radiation on normal tissues that results in unwanted side effects. Solving this problem was achieved practically by improvement of technical capacities of the ionizing radiation source and planning systems of irradiation procedure. The up-to-date therapeutic devices combine capacities of a medical imaging system (computed tomography) with a radiation therapeutic device. The combination according to the "two-in-one" principle ensures not only an ideal guiding of radiation on the target, but also constantly corrects the guiding, taking into consideration displacement of the target in the process of the patient's breathing, etc. The up-to-date planning systems help to increase reliability of precise guiding of ionizing radiation. Thus, unwanted action of ionizing radiation on normal tissues is reduced to the minimum, but is not excluded completely. In the last 100 years RT has transformed into a separate powerful trend in medicine. RT is equipped with sophisticated therapeutic devices widely used in oncological clinical practice - about 70% of oncological patients need these or those variants of RT. What are the prospects of the further improvement of RT? Obviously, the further technical improvement of therapeutic devices cannot be considered as a single way of RT development.

#### **1.1 Binary radio therapy: What is it?**

The most real trend of RT development is the development and introduction into clinical practice of Binary radiotherapy (BRT). Currently it is possible to speak about two types of BRT. They are neutron capture therapy (NCT), the idea of which was proposed in 1936 (Locher, 1936) and photon capture therapy (PCT). In English scientific literature is used the term "photon activation therapy". The principles of NCT and PCT (Khokhlov, 2004) are very similar. A malignant tumor is saturated with a preliminarily administered agent containing

Binary Radiotherapy of Melanoma - Russian Research Results 125

After many years of purposeful searches, two compounds were selected for BNCT mercaptoundecahydrododecaborate (Sivaev, 2002) - Na2B12H11SH (BSH) and *p*- (dihydroxybor)-L-phenylalanine (BPA) (Snyder, 1958). As the clinical experience has shown, these compounds are not optimal for BNCT, but nevertheless it is possible to use BSH rather successfully at the combined treatment of brain tumors with BNCT (Japan, USA, countries of EU), and BPA - at the treatment of skin melanoma and its metastases (Japan, USA). Clinical trials are being conducted to study effectiveness of BNCT with BPA in treating brain tumors (Japan, USA). A parallel intensive scientifically grounded search of new, more

An important aspect of the effective conduction of BNCT is also the existence of the methods which are capable to provide the optimal individual planning and control of its conduction. First of all, it is necessary to know the absolute concentration of 10B, its microdistribution in the process of irradiation, that will allow to choose correctly the time of irradiation, to enhance accuracy of dosimetry and microdosimetry, thereby determining success of BNCT. In this connection of particular value is the development and mastering in clinical setting of the neutron radiation method of determining boron in tumor and normal tissues in real time by (n,)-reaction to 10B, and also a number of other analytical methods allowing to determine intracellular concentration and location of boron, such as quantitative autoradiography of high performance, electron spectroscopy and others. Up to the present the evaluation of boron concentration in tumor at BNCT is conducted as a rule on the basis of preliminarily obtained data, for example, during surgical operation for removal of brain tumor, or indirectly - by the blood level of boron taking into account also preliminarily established

Interaction of photon radiation with different elements is characterized by dominating in a particular energy range (Fig. 1) of photon absorption by heavy elements (with Z≥53) on absorption by light elements composing biological tissues (H, O, N, C, Na, K, Cl etc.). This difference can be used for local increasing of energy absorption in the target (tumor) by administration or accumulating some heavy element in necessary region of irradiation.

Fig. 1. Energy dependence of specific photon kerma for the basic elements of biological

perfect, boron-containing compounds for BNCT is going on.

ratio of its concentrations tumor/blood.

tissue and some heavy elements (Sheino, 2006).

an element which is highly capable of interacting with the particular external ionizing radiation. Then tumor distant irradiation is carried out and local release of energy directly in the target tumor takes place. The main task is to make the total dose in the tumor to be lethal for its cells, and radiation exposure of normal tissues not to exceed the limits of their radiotolerance. Ideally, this permits to replace sophisticated technical devices for guiding ionizing radiation into the tumor with self targeting system based on biological or pharmacokinetic principles, and to increase considerably specificity and therapeutic effectiveness of radiation treatment technology. It is this circumstance that is the main impetus initiating research in this direction. For implementation of BRT two components are needed - a source of ionizing radiation (flow of neutrons or photons of certain energy) and pharmaceutical which composition includes the elements being highly capable of interaction with ionizing radiation.

#### **1.2 Physic principals of binary radio therapy**

The basis of BRT are the physical processes occurring as a result of interaction of certain elements, being constituents of composition of special pharmaceuticals, with external ionizing radiation. Let us consider in more details peculiarities of BRT taking as an example boron neutron capture therapy (BNCT), gadolinium neutron capture therapy (GdNCT) and PCT. 10B-Neutron Capture Therapy (BNCT) is based on nuclear reaction 10В(n,,)7Li occurs as the result of interaction between stable isotope 10B with thermal neutron (Еn=0.025 eV). That interaction has very high probability (σ=3890 barn). Also as the result of the following nuclear reaction high energy short range charged particles – nucleus of helium (α-particle) and nucleus of lithium are emitted. These particles has high linear energy transfer (LET) to tissue (200 keV for α-particle and 350 keV for lithium nucleus) and short path length (about 14 μm) comparable to the diameter of a single cell. Such charged particles are equally lethal for oxygenated cells, hypoxic cells and cells in G0-stage. Sub lethal and potentially lethal damages caused by this kind of particles are nonreparable unlike the damages induced by the photon radiation. That is why BNCT is the most effective in treating tumors with cells are highly capable for DNA reparation. i.e. melanoma and glioblastoma. And if selective accumulation of 10B in tumor cells is provided then selective radiation exposure could be achieved on the cell level: only tumor cells with 10B inside would be destroyed leaving healthy tissues undamaged, thus killing all indefinitely small metastasis. In theory BNCT could overcome major limitations of photon radiotherapy: too high radioresistance of some tumor cells and to low radiotolerance of normal tissues.

For successful implementation of BNCT in clinical practice a complex of complicated chemical, medical, biological, physical and engineering problems must be solved. The most important task of them is development of 10B-containing drug capable to deliver necessary therapeutical amount of 10B into malignant tumor cells providing 10B optimum intracellular distribution for a time necessary for neutron irradiation. It was calculated that for a fluence of 1013 n/cm2 concentration of 10B should be about 20-35 μg/g or 109 atoms of 10B per cell. To prevent damage of healthy tissues in the irradiated volume there must be at least 3 times less concentration of 10B in them than in the tumor tissue. The requirement of the 10B amount in the tumor cell depends greatly on it's intracellular localization. It's assumed that 2 μg/g of 10B is enough for successful BNCT in case of it's localization inside the cells nuclei. Therefore the radiobiological efficacy of 10В(n,,)7Li reaction greatly depends on so called "factor of compound" provided by peculiarities of chemical structure of boron with main substance, it's metabolism and it's distribution among the most important organelles of the tumor cell.

an element which is highly capable of interacting with the particular external ionizing radiation. Then tumor distant irradiation is carried out and local release of energy directly in the target tumor takes place. The main task is to make the total dose in the tumor to be lethal for its cells, and radiation exposure of normal tissues not to exceed the limits of their radiotolerance. Ideally, this permits to replace sophisticated technical devices for guiding ionizing radiation into the tumor with self targeting system based on biological or pharmacokinetic principles, and to increase considerably specificity and therapeutic effectiveness of radiation treatment technology. It is this circumstance that is the main impetus initiating research in this direction. For implementation of BRT two components are needed - a source of ionizing radiation (flow of neutrons or photons of certain energy) and pharmaceutical which composition includes the elements being highly capable of interaction

The basis of BRT are the physical processes occurring as a result of interaction of certain elements, being constituents of composition of special pharmaceuticals, with external ionizing radiation. Let us consider in more details peculiarities of BRT taking as an example boron neutron capture therapy (BNCT), gadolinium neutron capture therapy (GdNCT) and PCT. 10B-Neutron Capture Therapy (BNCT) is based on nuclear reaction 10В(n,,)7Li occurs as the result of interaction between stable isotope 10B with thermal neutron (Еn=0.025 eV). That interaction has very high probability (σ=3890 barn). Also as the result of the following nuclear reaction high energy short range charged particles – nucleus of helium (α-particle) and nucleus of lithium are emitted. These particles has high linear energy transfer (LET) to tissue (200 keV for α-particle and 350 keV for lithium nucleus) and short path length (about 14 μm) comparable to the diameter of a single cell. Such charged particles are equally lethal for oxygenated cells, hypoxic cells and cells in G0-stage. Sub lethal and potentially lethal damages caused by this kind of particles are nonreparable unlike the damages induced by the photon radiation. That is why BNCT is the most effective in treating tumors with cells are highly capable for DNA reparation. i.e. melanoma and glioblastoma. And if selective accumulation of 10B in tumor cells is provided then selective radiation exposure could be achieved on the cell level: only tumor cells with 10B inside would be destroyed leaving healthy tissues undamaged, thus killing all indefinitely small metastasis. In theory BNCT could overcome major limitations of photon radiotherapy: too high radioresistance of some

For successful implementation of BNCT in clinical practice a complex of complicated chemical, medical, biological, physical and engineering problems must be solved. The most important task of them is development of 10B-containing drug capable to deliver necessary therapeutical amount of 10B into malignant tumor cells providing 10B optimum intracellular distribution for a time necessary for neutron irradiation. It was calculated that for a fluence of 1013 n/cm2 concentration of 10B should be about 20-35 μg/g or 109 atoms of 10B per cell. To prevent damage of healthy tissues in the irradiated volume there must be at least 3 times less concentration of 10B in them than in the tumor tissue. The requirement of the 10B amount in the tumor cell depends greatly on it's intracellular localization. It's assumed that 2 μg/g of 10B is enough for successful BNCT in case of it's localization inside the cells nuclei. Therefore the radiobiological efficacy of 10В(n,,)7Li reaction greatly depends on so called "factor of compound" provided by peculiarities of chemical structure of boron with main substance, it's metabolism and it's distribution among the most important organelles of the tumor cell.

with ionizing radiation.

**1.2 Physic principals of binary radio therapy** 

tumor cells and to low radiotolerance of normal tissues.

After many years of purposeful searches, two compounds were selected for BNCT mercaptoundecahydrododecaborate (Sivaev, 2002) - Na2B12H11SH (BSH) and *p*- (dihydroxybor)-L-phenylalanine (BPA) (Snyder, 1958). As the clinical experience has shown, these compounds are not optimal for BNCT, but nevertheless it is possible to use BSH rather successfully at the combined treatment of brain tumors with BNCT (Japan, USA, countries of EU), and BPA - at the treatment of skin melanoma and its metastases (Japan, USA). Clinical trials are being conducted to study effectiveness of BNCT with BPA in treating brain tumors (Japan, USA). A parallel intensive scientifically grounded search of new, more perfect, boron-containing compounds for BNCT is going on.

An important aspect of the effective conduction of BNCT is also the existence of the methods which are capable to provide the optimal individual planning and control of its conduction. First of all, it is necessary to know the absolute concentration of 10B, its microdistribution in the process of irradiation, that will allow to choose correctly the time of irradiation, to enhance accuracy of dosimetry and microdosimetry, thereby determining success of BNCT. In this connection of particular value is the development and mastering in clinical setting of the neutron radiation method of determining boron in tumor and normal tissues in real time by (n,)-reaction to 10B, and also a number of other analytical methods allowing to determine intracellular concentration and location of boron, such as quantitative autoradiography of high performance, electron spectroscopy and others. Up to the present the evaluation of boron concentration in tumor at BNCT is conducted as a rule on the basis of preliminarily obtained data, for example, during surgical operation for removal of brain tumor, or indirectly - by the blood level of boron taking into account also preliminarily established ratio of its concentrations tumor/blood.

Interaction of photon radiation with different elements is characterized by dominating in a particular energy range (Fig. 1) of photon absorption by heavy elements (with Z≥53) on absorption by light elements composing biological tissues (H, O, N, C, Na, K, Cl etc.). This difference can be used for local increasing of energy absorption in the target (tumor) by administration or accumulating some heavy element in necessary region of irradiation.

Fig. 1. Energy dependence of specific photon kerma for the basic elements of biological tissue and some heavy elements (Sheino, 2006).

Binary Radiotherapy of Melanoma - Russian Research Results 127

model of canine spontaneous melanoma. The chapter presents the used neutron and photon bundles. It presents the main characteristics of the used pharmaceutical products with 10В ([10В]-boron phenylalanine in the pharmaceutical form borate ether with D-fructose; [10В]- BSH - mercapto-closo-undecaborateas a sodium salt (Sivaev, 2002) and gadopentetat in the pharmaceutical form ensuring delayed elimination of the substance from the injection site). The results of remote consequences of BRT and traditional methods of melanoma treatment - surgical intervention, immune therapy, action of ionizing radiation (neutrons and gammaradiation) are given. The comparison of therapeutic effectiveness of NCT with 10В and Gd used as both monotherapy and in combination with adjuvant immune therapy with interleukin-2 (Roncoleukin) is presented. Separately there are presented the results of the micropharmacokinetic studies - distribution of BSH and a number of new boron-containing agents across the main organelles of melanoma cell. For the results beyond the scope of the

Neutron irradiation was performed on the irradiation facilities of Moscow Engineering and Physics Institute research reactor IRT and research reactor RR-8 of RRC "Kurchatov Institute". The IRT facility includes the irradiation room for positioning cell cultures and laboratory animals including dogs in the neutron beam. The neutron beam delivered to the irradiation room has the following characteristics: thermal neutron flux – 1.1×109 n/cm2s, fast neutron flux – 5.8×107 n/cm2s, photon dose rate - 1.8×10-4 Gy/s. Irradiation room is equipped with video surveillance to observe the state of irradiation object and with the physiological parameters monitoring system (heart and breath frequency, blood pressure, body temperature ) as well as with drug delivery system. IR-8 reactor facility is designed for irradiation small object only - cell cultures and small animals. Thermal neutron flux on IR-8 facility was 1.2×108 n/cm2s, fast neutron flux – less than 0.96×107 n/cm2s, photon dose rate - 8.3×10-7 Gy/s. Small animals were not anesthetized prior the irradiation. At the IR-8 animals were irradiated in Teflon® cages, at IRT animals were situated in lead boxes, which were limiting animal's movements but not interfering feeding and defecations. Local irradiation of transplanted into the rear paw tumor immobilized in advance was performed with dose of 2.5 Gy-Eq. Considering that poor fluence power thermal neutron irradiation was

prolonged for time individual group as control for each animal group was used.

X-rays irradiations were performed using radiobiology autoprotective X-rays facility with anode voltage 220 kV and dose rate at the position of cell culture monolayer 2.0 Gy/min for cell culture irradiation and X-rays irradiation of mice bearing B-16 melanoma was performed with an X-ray unit with anode voltage of 150 kV and dose rate of 0.7 Gy/min at

Thermal neutron absorbed dose at NCT was measured with prompt gamma neutron activation analysis (PGNAA). Average dose in tissue without drug was 0.25 Gy/h. Total absorbed dose was determined by 3 nuclear reactions, which provide the major part of absorbed dose (95-97%) during interaction of thermal neutrons with nuclides of biological

preclinical studies, the references to the published works are given.

**2. Materials and methods** 

treated tumor volume.

tissue - 1H (n,γ)2H; 14N (n,p)14C; 10B (n,α)7Li.

**2.2 Dosimetry** 

**2.1 Irradiation of biological objects** 

In terms of Radiotherapy such approach can be implemented by administration or accumulation in a tumor volume the necessary amount of a pharmaceutical containing some heavy element with subsequent irradiation of the tumor region with X-rays of certain energy spectrum. As the result of such irradiation local increase of absorbed dose in the tumor occurs. In case of necessary amount of heavy element in the target is provided the increase of absorbed dose can be 2-3 times higher than for the same irradiation but with no drug administered (Karnas, 1999; Sheino, 2006). Emission of short range radiation caused by photoabsorption on heavy elements directly in the irradiated target is effective factor of tumor cell growth suppression. Significant that in PCT absorbed dose in healthy tissues could be lower than it's tolerant dose. Thus radiation influence on healthy tissues is decreasing and selectivity of tumor tissue damage is increasing during irradiation procedure. Such binary technology was called Photon Capture Therapy. Combination of biological self targeting of radiation and it's main localization in target volume makes PCT prospective Radiotherapy technology.

Fig. 2. Relative increase of a dose in a biological tissue for various elements with Z>53 at their concentration of 1% in dependence on energy of photons. (Sheino, 2006).

Calculated estimation show (Sheino, 2006) that proper therapeutic efficacy of PCT could be achieved if the concentration of heavy element is around ~ 10 mg/g. At present there are no drugs with i.v. administration capable to accumulate in tumor tissues in such amount. That is why gadolinium containing MRI-contrast pharmaceutical Dipentast® (Russia) and intratumoral way of administration were used in our primary studies of PCT.

#### **1.3 Tasks of research**

The chapter presents the results of the preclinical studies on BRT conducted in Russia. Melanoma was chosen as the main object of the research. The studies were carried out in conformity with the effective RF requirements for three main directions: 1. the studies on the В-16 mouse melanoma cell culture; 2. the studies of mice with the transplanted B-16 melanoma; the studies on dogs with spontaneous B-16 melanoma. The similarity of canine and human melanomas permitted to replace the transplanted nude melanomas with the model of canine spontaneous melanoma. The chapter presents the used neutron and photon bundles. It presents the main characteristics of the used pharmaceutical products with 10В ([10В]-boron phenylalanine in the pharmaceutical form borate ether with D-fructose; [10В]- BSH - mercapto-closo-undecaborateas a sodium salt (Sivaev, 2002) and gadopentetat in the pharmaceutical form ensuring delayed elimination of the substance from the injection site). The results of remote consequences of BRT and traditional methods of melanoma treatment - surgical intervention, immune therapy, action of ionizing radiation (neutrons and gammaradiation) are given. The comparison of therapeutic effectiveness of NCT with 10В and Gd used as both monotherapy and in combination with adjuvant immune therapy with interleukin-2 (Roncoleukin) is presented. Separately there are presented the results of the micropharmacokinetic studies - distribution of BSH and a number of new boron-containing agents across the main organelles of melanoma cell. For the results beyond the scope of the preclinical studies, the references to the published works are given.
