**2. Materials and methods**

126 Advances in Cancer Therapy

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

Fig. 2. Relative increase of a dose in a biological tissue for various elements with Z>53 at

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

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

their concentration of 1% in dependence on energy of photons. (Sheino, 2006).

intratumoral way of administration were used in our primary studies of PCT.

prospective Radiotherapy technology.

**1.3 Tasks of research** 

### **2.1 Irradiation of biological objects**

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 treated tumor volume.

#### **2.2 Dosimetry**

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

Binary Radiotherapy of Melanoma - Russian Research Results 129

centrifuged at 1200 rev/min during 7-10 min. Supernatant was carefully removed. 200 μl of DMSO were added into each hole for formazane solution. Optical density of homogenous solution was determined in "Picon" photometer at 530 nm. Optical density of solution was demonstration of redox-intensivity in cell culture. For clonogenic test cells of both groups were sewed in 6-holes plates with full medium and in 3, 5, 7 days cell colonies were count

Fig. 3. Preclinical trials of BNCT in dogs with spontaneous malignant melanoma. Infusion of BPA drugs using a feeding pump (left); X-ray control of the compound administration

Boron tumor concentration was measured in vivo with PGNAA (Khokhlov, 2008; Khokhlov, 2009). During irradiation this concentration in tumor varied about 7-8 μg/g for [10B]-BSH,

Gd tumor concentration varied about 10-100 mg/сm3, Gd concentration in biological samples was measured with neutron-activation analysis (Zaitsev, 2004 а) or using roentgen

In 5 min., 1 and 24 h after BSH, KUG-1 administration experimental tumors were excised, rinsed, weighed and homogenized. The cell organelles were prepared using routine

Subcellular fractions (nuclei, mitochondria, lysosomes, cytosol) were received for further

Kinetics of B-16 melanoma cell population was studied at determination of melanocytes and

For this purpose tumors of exponential growth phase were used. On 7-th day after subcutaneous inoculation experimental С57BL/6 mice (body weight of 17-20 g; tumor size

fluorescent analysis in X-Art-М concentration analyzer (Comita Ltd, Russia).

using LOMO microscope.

(richt).

**2.5 Quantification of boron and gadolinium** 

10-20 μg/g for ВРА and 10-11 μg/g for KUG-1.

techniques of differential centrifugation (De Duve, 1967).

**2.7 Study of kinetics of B-16 melanoma cell population** 

DNA-synthesizing cells percentage in total studied tumor cells.

**2.6 Subcellular boron distribution** 

boron determination with PGNAA method.

Dose rate of X-rays in irradiation position was performed using DKS-AT5350/1 dosimeter (Russia) and dosimetric films Gafchromic® EBT. Optical density of dosimetric films was measured on Varian Cary-50 spectrophotometer.

#### **2.3 Animals and cell culture**

B-16 melanoma cell culture for experiments was received from Blockin's Cancer Research Center (Russia) storage. These cells were grown as monolayer in glass flasks containing mixture of RPM6-1640 and Eagle's nutrient media with 10% calf serum and gentamicyne. Grown cells were suspended in saline. Cell concentration in suspension was 1000 per 1 ml.

Cultural flasks in PCT in vitro studies were divided to experimental and control groups. Both groups were irradiated using X-rays with 10 Gy, 20 Gy, 30 Gy and 40 Gy. Before irradiation Dipentast® was added into experimental flasks for 0.2 mg/ml Gd concentration.

Experiments in vivo were conducted on С57Bl/6 male and female mice bearing subcutaneously transplanted melanoma В-16. All mice were of veterinary certification.

The mice were subcutaneously inoculated into rear paw with (3.5-4)106 melanoma cells. All NCT and PCT experiments were conducted 10-11 days after transplantation (tumor volume was about 0.9-1.0 сm3).

For NCT studying the mice were injected intraperitoneally with saline solutions of KUG-1 and BSH in amount of 0.2 ml containing 150 mg of boron per kg body weight for BSH and 25 mg of B per kg body weight for KUG-1. After injection of studied compounds ( in 6 hours for BSH and in 24 hours for KUG-1) tumor was locally irradiated.

For PCT the animals were divided into four groups: 1- experimental group irradiated with X-rays and administered with Gd-DTPA before irradiation, 2 – mice only locally irradiated with X-rays but not injected with Gd-DTPA, 3 – nonirradiated mice but only administered with Gd-DTPA with the same amount that the experimental group, 4 – untreated mice. When tumor volume was achieved 1 сm3 1 and 3 animal groups were intratumorally administrated with 0.175 ml Dipentast®. After administration mice from 1 group were immediately irradiated with X-rays in 20 Gy dose.

For NCT studies Dogs with spontaneous oral cavity melanoma were selected in LLC "Biocontrol" based on the results of clinical examination. The owners of the dogs were agreed on experimental treatment.

60 dogs with oral cavity melanoma were selected for the study . The dogs were examined before and after treatment using clinical and histological methods. The dogs were divided on the following groups: I group – without treatment; II group – surgical treatment; III – distant gamma-therapy, IV group – neutron therapy; V – BNCT; VI - GdNCT ; VII – Complex treatment (NCT with adjuvant immunotherapy with Ronkoleukine); VIII group – NCT without immunotherapy. BNCT was performed with [10В]-BPA in pharmaceutical form of boron ether solution with D-fructose. GdNCT was performed with Dipentast®.

In V group BPA were administered by two routes: in the artery which nourish tumor and intravenously (Fig. 3). Administration of the medicine was controlled by X-ray examination. Irradiation was performed 2.0-2.5 hours after BPA administration

#### **2.4 Cell vitality assessment**

For cell vitality assessment after irradiation MTT-test (Mosman, T., 1983) and clonogenic test were used. For MTT-test cells were sewed in 96-holes flat-bottomed plates and incubated during 3, 5, 7 days. 2-3 hours before termination 10 μl of (4,5-dimethylthiazoline-2)-2,5 diphenyltetrazoly bromide (final concentration was 1 mg/ml). After incubation cells were

Dose rate of X-rays in irradiation position was performed using DKS-AT5350/1 dosimeter (Russia) and dosimetric films Gafchromic® EBT. Optical density of dosimetric films was

B-16 melanoma cell culture for experiments was received from Blockin's Cancer Research Center (Russia) storage. These cells were grown as monolayer in glass flasks containing mixture of RPM6-1640 and Eagle's nutrient media with 10% calf serum and gentamicyne. Grown cells were suspended in saline. Cell concentration in suspension was 1000 per 1 ml. Cultural flasks in PCT in vitro studies were divided to experimental and control groups. Both groups were irradiated using X-rays with 10 Gy, 20 Gy, 30 Gy and 40 Gy. Before irradiation Dipentast® was added into experimental flasks for 0.2 mg/ml Gd concentration. Experiments in vivo were conducted on С57Bl/6 male and female mice bearing subcutaneously transplanted melanoma В-16. All mice were of veterinary certification. The mice were subcutaneously inoculated into rear paw with (3.5-4)106 melanoma cells. All NCT and PCT experiments were conducted 10-11 days after transplantation (tumor volume

For NCT studying the mice were injected intraperitoneally with saline solutions of KUG-1 and BSH in amount of 0.2 ml containing 150 mg of boron per kg body weight for BSH and 25 mg of B per kg body weight for KUG-1. After injection of studied compounds ( in 6 hours

For PCT the animals were divided into four groups: 1- experimental group irradiated with X-rays and administered with Gd-DTPA before irradiation, 2 – mice only locally irradiated with X-rays but not injected with Gd-DTPA, 3 – nonirradiated mice but only administered with Gd-DTPA with the same amount that the experimental group, 4 – untreated mice. When tumor volume was achieved 1 сm3 1 and 3 animal groups were intratumorally administrated with 0.175 ml Dipentast®. After administration mice from 1 group were

For NCT studies Dogs with spontaneous oral cavity melanoma were selected in LLC "Biocontrol" based on the results of clinical examination. The owners of the dogs were

60 dogs with oral cavity melanoma were selected for the study . The dogs were examined before and after treatment using clinical and histological methods. The dogs were divided on the following groups: I group – without treatment; II group – surgical treatment; III – distant gamma-therapy, IV group – neutron therapy; V – BNCT; VI - GdNCT ; VII – Complex treatment (NCT with adjuvant immunotherapy with Ronkoleukine); VIII group – NCT without immunotherapy. BNCT was performed with [10В]-BPA in pharmaceutical form of boron ether solution with D-fructose. GdNCT was performed with Dipentast®. In V group BPA were administered by two routes: in the artery which nourish tumor and intravenously (Fig. 3). Administration of the medicine was controlled by X-ray examination.

For cell vitality assessment after irradiation MTT-test (Mosman, T., 1983) and clonogenic test were used. For MTT-test cells were sewed in 96-holes flat-bottomed plates and incubated during 3, 5, 7 days. 2-3 hours before termination 10 μl of (4,5-dimethylthiazoline-2)-2,5 diphenyltetrazoly bromide (final concentration was 1 mg/ml). After incubation cells were

for BSH and in 24 hours for KUG-1) tumor was locally irradiated.

Irradiation was performed 2.0-2.5 hours after BPA administration

immediately irradiated with X-rays in 20 Gy dose.

agreed on experimental treatment.

**2.4 Cell vitality assessment** 

measured on Varian Cary-50 spectrophotometer.

**2.3 Animals and cell culture** 

was about 0.9-1.0 сm3).

centrifuged at 1200 rev/min during 7-10 min. Supernatant was carefully removed. 200 μl of DMSO were added into each hole for formazane solution. Optical density of homogenous solution was determined in "Picon" photometer at 530 nm. Optical density of solution was demonstration of redox-intensivity in cell culture. For clonogenic test cells of both groups were sewed in 6-holes plates with full medium and in 3, 5, 7 days cell colonies were count using LOMO microscope.

Fig. 3. Preclinical trials of BNCT in dogs with spontaneous malignant melanoma. Infusion of BPA drugs using a feeding pump (left); X-ray control of the compound administration (richt).

#### **2.5 Quantification of boron and gadolinium**

Boron tumor concentration was measured in vivo with PGNAA (Khokhlov, 2008; Khokhlov, 2009). During irradiation this concentration in tumor varied about 7-8 μg/g for [10B]-BSH, 10-20 μg/g for ВРА and 10-11 μg/g for KUG-1.

Gd tumor concentration varied about 10-100 mg/сm3, Gd concentration in biological samples was measured with neutron-activation analysis (Zaitsev, 2004 а) or using roentgen fluorescent analysis in X-Art-М concentration analyzer (Comita Ltd, Russia).
