**3.2 Influence on cell cycle and kinetics of cell population of B-16 melanoma**

This research was devoted to study the influence of BSH – boron carrier at anticancer neutron capture therapy on cell population of murine melanoma B-16. It was proved that prevalent accumulation of tumor cells in synthetic and premitotic phases (S and G2/M) of cell cycle was caused by BSH effect. Probably this phenomenon of cell proliferation may be linked to compensatory population response on damaging factor of BSH (Ado, 1993).

 The detected activity of the latter coupled with its ability to penetrate into the nucleus and mitochondria may be the reason for its efficacy in NCT of melanoma and opens new perspectives of the well known drug. These data are shown in tables 3, 4.


Table 3. Quantity of melanocytes and labeled cells in peripheral and central zones of of В-16 melanoma after BSH administration (100 mg/kg of body weight)


Table 4. Influence of ВSH on cell cycle of B-16 melanoma after BSH administration

So, BSH is the drug of threefold action and this action allows to realize full potential of BSH.


The results of investigations demonstrated most efficacy of NCT during the first 24 h after BSH administration due to both subcellular distribution and influence on cell cycle.

In further searching of new boron compounds for NCT it would be remembered that any chemical agent is probably active agent effecting on cell biology.

#### **3.3 In vitro studies of photon capture therapy**

132 Advances in Cancer Therapy

This research was devoted to study the influence of BSH – boron carrier at anticancer neutron capture therapy on cell population of murine melanoma B-16. It was proved that prevalent accumulation of tumor cells in synthetic and premitotic phases (S and G2/M) of cell cycle was caused by BSH effect. Probably this phenomenon of cell proliferation may be linked to compensatory population response on damaging factor of BSH (Ado, 1993). The detected activity of the latter coupled with its ability to penetrate into the nucleus and mitochondria may be the reason for its efficacy in NCT of melanoma and opens new

**Peripheria Centre** 

50 19.52.0 58.45.7 13.71.4 8.40.9 22.1 150 20.12.1 57.24.5 14.31.5 7.90.8 22

50 16.71.5 56.45.4 8.91.1 18.72.0 27.6 150 16.21.3 54.65.1 16.61.3 12.71.3 29.3

50 17.01.8 57.95.6 10.11.1 15.91.6 25.1 150 16.01.4 57.05.5 14.31.3 12.71.3 27.0

(saline) 20.11.9 59.25.7 14.01.5 6.70.7 20.7

1 4.0±0.9 10.9±1.1 9.2±1.3 12.0±1.2 2 13.1±1.2 15.3±1.3 16.7±1.3 8.8±1.0 3 22.0±1.5 18.9±1.3 17.6±1.4 12.3±1.2

Table 3. Quantity of melanocytes and labeled cells in peripheral and central zones of of В-16

Table 4. Influence of ВSH on cell cycle of B-16 melanoma after BSH administration

1. BSH is boron carrier to deliver boron into tumor and it's vital structures.

So, BSH is the drug of threefold action and this action allows to realize full potential of BSH.

2. BSH stimulates proliferating cells (S+G2/M), increasing sensitivity for all types of

3. BSH influences on cell differentiation ( synthesis of melanin is index of cell

The results of investigations demonstrated most efficacy of NCT during the first 24 h after

In further searching of new boron compounds for NCT it would be remembered that any

BSH administration due to both subcellular distribution and influence on cell cycle.

chemical agent is probably active agent effecting on cell biology.

Control Experiment Control Experiment

Phase of cell cycle IG1 IIG1 S G2/M S+

G2/M

**3.2 Influence on cell cycle and kinetics of cell population of B-16 melanoma** 

perspectives of the well known drug. These data are shown in tables 3, 4.

melanoma after BSH administration (100 mg/kg of body weight)

BSH, <sup>10</sup>В µg /kg BW

**Days after administration** 

Time after administration, h

3

12

24

irradiation.

<sup>24</sup>Control

differentiation for melanoma).

On the base of obtained results of МТТ-test and clonogenic test curves of dependence " part of survived cells – exposition dose" were constructed. (Fig. 4 and Fig. 5).

MTT-test data had shown equal parts of survived cells in both groups on 3-rd day. On 5-th day part survived cells in experimental group was essentially low and was retained at this level to 7-th day. RGG-test data had shown (Fig. 5) practically complete suppression of cell growth in experimental group even at 10 Gy. In control group significant quantity of cell colonies were observed.

Binary Radiotherapy of Melanoma - Russian Research Results 135

Absorbed dose in tumor from reaction on В-10,Gy 1.76 1.23 Absorbed dose in tumor from reaction on N, Gy 0.42 0.79 Absorbed dose in tumor from reaction on H, Gy 0.45 0.36 Total absorbed dose in tumor 2.63 2.38 Concentration of В-10 in tumor, initial, μg/g 11.3 8.3 Concentration of В-10 in tumor, initial, μg/g 9.9 7

Fig. 6. Growth of transplanted B-16 melanoma in C57bl/6 after thermal neutron irradiation

On the base of obtained results of irradiation curves of tumor growth were constructed.

Criterion Group 1 Group 2 Group 3

(Fig. 7). Quantitative assessment of efficacy of each force was determined. (Table 6).

Tumor growth delay time, days 15±1 8±1 -

Tumor volume doubling time, days 16±2 11±1 -

Tumor growth index (IG) 0.1±0.03 0.25±0.05 0.75±0.05

Obtained data demonstrate that local irradiation of melanoma containing Dipentast® resulted in significant inhibition of tumor growth in comparison with irradiation only. Administration of Dipentast® without consecutive irradiation didn't result in expressed

at presence of BSH and KUG-1

**3.4.2 Photon capture therapy** 

anticancer effect.

Table 6. Quantitative parameters of effect assessment

Table 5. Results of thermal neutron irradiation mice bearing B-16 melanoma

В-10-carrier for NCT KUG-1 BSH

Fig. 5. Cell survival for control and experimental groups (clonogenic test data) , 3-rd and 5-th days.

#### **3.4 Radiobiological studies in vivo**

Studies of binary radio therapy in animals with potentially suitable for clinical application pharmaceuticals

#### **3.4.1 Boron neutron capture therapy**

The therapeutic efficacy of NCT was studied using two compounds: BSH and KUG-1 containing 55% and 20% boron relatively. PGNAA data demonstrated that maximal boron content in tumor (12 μg/g of tissue) was achieved in 1 hour after BSH injection decreasing by 6 hours. Then this level was not varying for 48 hours. On the same time the compound rapidly eliminated from all tissues adjacent to tumor excluding skin. As result in 12-48 hours interval there was sufficient for NCT ratio tumor/adjacent tissue including blood. The boron content suitable for NCT in the case of introduction of KUG-1 was accumulated in 24 hours and was constant not less then 15 hours. It should be noted that the total absorbed dose in tumor was determined from three major nuclear reactions - (n,γ)2H; 14N (n,p)14C; 10B (n,ά)7Li - contributing essential part in to absorbed dose at interactions between thermal neutrons and nuclides of living tissue and was equal to 95-97%. The time of tumor volume duplication (Тd) at NCT with BSH injection was increased to 14-15 days comparatively with 3-4 days for intact animals. In the case of Ph1 injection this time was prolonged to 8-10 days. The injection of Ph2 did not make this parameter differ from control meanings.

 The increase of survival of treated mice was equal to 57% in the case of BSH administration and only 14% at KUG-1 administration in spite of KUG-1 accumulation coefficient was more than the same for BSH. It is caused with toxic side effect of KUG-1 (hepatic- and neurotoxicity).

KUG1 is perspective for purposes of NCT but needs a development for eliminations of toxic side effects. Results of irradiation are shown in table 5 and in figure 6.

Studies of binary radio therapy in animals with potentially suitable for clinical application

The therapeutic efficacy of NCT was studied using two compounds: BSH and KUG-1 containing 55% and 20% boron relatively. PGNAA data demonstrated that maximal boron content in tumor (12 μg/g of tissue) was achieved in 1 hour after BSH injection decreasing by 6 hours. Then this level was not varying for 48 hours. On the same time the compound rapidly eliminated from all tissues adjacent to tumor excluding skin. As result in 12-48 hours interval there was sufficient for NCT ratio tumor/adjacent tissue including blood. The boron content suitable for NCT in the case of introduction of KUG-1 was accumulated in 24 hours and was constant not less then 15 hours. It should be noted that the total absorbed dose in tumor was determined from three major nuclear reactions - (n,γ)2H; 14N (n,p)14C; 10B (n,ά)7Li - contributing essential part in to absorbed dose at interactions between thermal neutrons and nuclides of living tissue and was equal to 95-97%. The time of tumor volume duplication (Тd) at NCT with BSH injection was increased to 14-15 days comparatively with 3-4 days for intact animals. In the case of Ph1 injection this time was prolonged to 8-10 days. The injection of Ph2 did not make this parameter differ from

 The increase of survival of treated mice was equal to 57% in the case of BSH administration and only 14% at KUG-1 administration in spite of KUG-1 accumulation coefficient was more than the same for BSH. It is caused with toxic side effect of KUG-1 (hepatic- and

KUG1 is perspective for purposes of NCT but needs a development for eliminations of toxic

side effects. Results of irradiation are shown in table 5 and in figure 6.

Fig. 5. Cell survival for control and experimental groups (clonogenic test data) , 3-rd and

**5 days**

**Colonies fraction,%**

**5 10 15 20 25 30 35 40**

 **X-rays**

**X-rays dose,Gy**

 **X-rays +0.00125M Gd-DTPA**

**5 10 15 20 25 30 35 40**

**3.4 Radiobiological studies in vivo** 

**3.4.1 Boron neutron capture therapy** 

 **X-rays**

**X-ray dose,Gy**

**X-rays + 0.00125M Gd-DTPA**

5-th days.

pharmaceuticals

control meanings.

neurotoxicity).

**Colonies fraction,%**

**3 days**


Table 5. Results of thermal neutron irradiation mice bearing B-16 melanoma

Fig. 6. Growth of transplanted B-16 melanoma in C57bl/6 after thermal neutron irradiation at presence of BSH and KUG-1

### **3.4.2 Photon capture therapy**

On the base of obtained results of irradiation curves of tumor growth were constructed. (Fig. 7). Quantitative assessment of efficacy of each force was determined. (Table 6).


Table 6. Quantitative parameters of effect assessment

Obtained data demonstrate that local irradiation of melanoma containing Dipentast® resulted in significant inhibition of tumor growth in comparison with irradiation only. Administration of Dipentast® without consecutive irradiation didn't result in expressed anticancer effect.

Binary Radiotherapy of Melanoma - Russian Research Results 137

radical removal of the tumor. At these stages of tumor process the tumor size appears to be

To evaluate effectiveness of neutron capture radiotherapy, that is, NCT, the results obtained were compared to the animals subject to the action of neutron bundle alone. In this group (IV, n=5) partial regression was attained in 80% of cases, and complete regression - in 20% of cases. In 100% of cases the continued growth of the tumor within 101±28.8 days was also

In group V, where irradiation with neutron bundle was performed with a preliminary administration of boron agents, that is, BNCT, complete tumor regression was possible to be attained in 78% of cases. (Fig. 8) At BNCT, in cases of complete tumor regression no recurrence was observed. The absence of recurrence in complete tumor regression proves

In this study the boron product was administered by two routes: intravenously and regionally into the artery feeding the tumor. Having analyzed the results obtained, we got sufficiently significant data on that the route of the product administration exerts no effect on NCT effectiveness. Thus, during further studies of BNCT it is possible to administer 10В

During conduction of NCT with Dipentast®, that is, GdNCT (group VI), somewhat other results were obtained as compared to BNCT. First, tumor regression took place more slowly in at GdNCT comparing to BNCT, and complete tumor regression was noted only in 46% of cases (Fig.9). In 66.7% of animals recurrence of the tumor was seen 1067.5 days after the procedure of GdNCT. Nevertheless, the effect of irradiation at GdNCT was sufficiently high that suggested a possibility of development of an effective technology of melanoma

Since so far a small number of studies on GdNCT was conducted and there are no data on the optimal concentration of gadolinium inside the tumor which is necessary to achieve the tumoricide dose, we studied the GdNCT effect at different doses of gadolinium in tumor. The studied range of concentrations was from 10 mg/cm3 to 100 mg/cm3. During the clinical trial the regularity was determined at which the best clinical effect of GdNCT was

We also conducted the study on investigating distribution of the power of total dose depending on the gadolinium concentration inside tumor. Such study was performed on the

The study of total dose rate distribution during GdNCT at different concentration showed that at the gadolinium concentration over 12 mg/cm3 the total dose in the tissue sharply decreases by several times. So, at concentrations of 6-12 mg/cm3 the total dose power at the depth of 0.5 cm of tissue is 150-200 cGy/min and does not increase with the growth of the gadolinium concentration in target (Fig. 10). Such effect is due to the fact that isotopes of gadolinium 157Gd (the content in the natural element 15.68%) have high sections of capture of thermal neutrons. That causes an increase of absorption of thermal neutrons during GdNCT at high concentrations of gadolinium products. The so-called "shielding effect"

Thus, the analysis of distribution of the total dose, by using the solving of the equation of transfer of neutrons and photons by the method of discrete ordinates according to the RADUGA-5 program, showed that the optimal concentration of 157Gd is within the range of 6-12 mg of Gd in 1 cm3 of tumor. These conclusions help to optimize subsequently the

complete destruction of melanoma cells in the primary tumor focus.

BFA intravenously that facilitates conduction of the BNCT procedure.

observed at the gadolinium concentration in tumor of up to 12 mg/cm3.

sufficiently large.

treatment on the basis of GdNCT.

basis of the developed model of GdNCT (1).

process of GdNCT for achieving the maximum result.

diagnosed.

occurs.

Fig. 7. Tumor volume growth kinetics for irradiated with Gd-DTPA injection group (▲), only irradiated group (■), only administered with Gd-DTPA group (∆), untreated group (□)

#### **3.5 Results of neutron capture therapy studies in dogs with spontaneous melanoma of the oral mucosa. Development of combined radiotherapy scheme on the basis of NCT and adjuvant immunotherapy**

The improvement of the technology of neutron capture therapy under the condition of nuclear reactors, conduction of detailed studies with the purpose of comparative evaluation of effectiveness of different variants of neutron capture technology and traditional methods of treatment is an important and topical clinical task. The conduction of preclinical studies will permit to choose the most efficient scheme of treatment of melanoma, since melanoma of the oral mucosa in dogs may be regarded as a correct model of human melanoma.

The studies on effectiveness of BNC technology in treatment of spontaneous melanoma of canine oral mucosa have been conducted for several years. The preclinical study of neutron capture therapy on dogs with the diagnosis of oral mucosa melanoma has yielded the following results.

In group I of the animals which did not receive any treatment, the average animal life expectancy was 38±13.6 days. 100% of the animals were euthanized due to their severe condition. The reason for euthanasia of such animals was local propagation of the tumor and systemic metastasis.

In the group of animals who underwent surgical removal of the oral mucosa tumor, 100% recurrence occurred within the period of 26 ±12.5 days. In group III where the animals received traditional radiotherapy, stabilization of tumor growth was seen in 75% of cases, partial regression in 12.5% of animals and complete regression - also in 12.5% of animals. In 100% of cases resumption of tumor growth within 30±5.5 days was observed.

Thus, both at surgical treatment and at radiotherapy recurrence occurred in 100% of cases. The statistical analysis showed that the duration of the recurrence-free period at the use of these methods of treatment was in fact equal.

This indicates that surgical treatment and radiotherapy are virtually low-effective methods of treating melanoma of oral mucosa in dogs at stage II-III of btumor process. This probably can be explained by the specific location of the tumor when it is impossible to perform

**Gd-DTPA + X-rays**

**Control Gd-DTPA X-rays**

**Irradiation**

**5 10 15 20 25 30 35 40 4**

**Days after tumor inoculation**

Fig. 7. Tumor volume growth kinetics for irradiated with Gd-DTPA injection group (▲), only irradiated group (■), only administered with Gd-DTPA group (∆), untreated group (□)

**3.5 Results of neutron capture therapy studies in dogs with spontaneous melanoma of the oral mucosa. Development of combined radiotherapy scheme on the basis of** 

The improvement of the technology of neutron capture therapy under the condition of nuclear reactors, conduction of detailed studies with the purpose of comparative evaluation of effectiveness of different variants of neutron capture technology and traditional methods of treatment is an important and topical clinical task. The conduction of preclinical studies will permit to choose the most efficient scheme of treatment of melanoma, since melanoma

The studies on effectiveness of BNC technology in treatment of spontaneous melanoma of canine oral mucosa have been conducted for several years. The preclinical study of neutron capture therapy on dogs with the diagnosis of oral mucosa melanoma has yielded the

In group I of the animals which did not receive any treatment, the average animal life expectancy was 38±13.6 days. 100% of the animals were euthanized due to their severe condition. The reason for euthanasia of such animals was local propagation of the tumor

In the group of animals who underwent surgical removal of the oral mucosa tumor, 100% recurrence occurred within the period of 26 ±12.5 days. In group III where the animals received traditional radiotherapy, stabilization of tumor growth was seen in 75% of cases, partial regression in 12.5% of animals and complete regression - also in 12.5% of animals. In

Thus, both at surgical treatment and at radiotherapy recurrence occurred in 100% of cases. The statistical analysis showed that the duration of the recurrence-free period at the use of

This indicates that surgical treatment and radiotherapy are virtually low-effective methods of treating melanoma of oral mucosa in dogs at stage II-III of btumor process. This probably can be explained by the specific location of the tumor when it is impossible to perform

100% of cases resumption of tumor growth within 30±5.5 days was observed.

of the oral mucosa in dogs may be regarded as a correct model of human melanoma.

**Tumor volume, cm**

**NCT and adjuvant immunotherapy** 

following results.

and systemic metastasis.

these methods of treatment was in fact equal.

**3**

radical removal of the tumor. At these stages of tumor process the tumor size appears to be sufficiently large.

To evaluate effectiveness of neutron capture radiotherapy, that is, NCT, the results obtained were compared to the animals subject to the action of neutron bundle alone. In this group (IV, n=5) partial regression was attained in 80% of cases, and complete regression - in 20% of cases. In 100% of cases the continued growth of the tumor within 101±28.8 days was also diagnosed.

In group V, where irradiation with neutron bundle was performed with a preliminary administration of boron agents, that is, BNCT, complete tumor regression was possible to be attained in 78% of cases. (Fig. 8) At BNCT, in cases of complete tumor regression no recurrence was observed. The absence of recurrence in complete tumor regression proves complete destruction of melanoma cells in the primary tumor focus.

In this study the boron product was administered by two routes: intravenously and regionally into the artery feeding the tumor. Having analyzed the results obtained, we got sufficiently significant data on that the route of the product administration exerts no effect on NCT effectiveness. Thus, during further studies of BNCT it is possible to administer 10В BFA intravenously that facilitates conduction of the BNCT procedure.

During conduction of NCT with Dipentast®, that is, GdNCT (group VI), somewhat other results were obtained as compared to BNCT. First, tumor regression took place more slowly in at GdNCT comparing to BNCT, and complete tumor regression was noted only in 46% of cases (Fig.9). In 66.7% of animals recurrence of the tumor was seen 1067.5 days after the procedure of GdNCT. Nevertheless, the effect of irradiation at GdNCT was sufficiently high that suggested a possibility of development of an effective technology of melanoma treatment on the basis of GdNCT.

Since so far a small number of studies on GdNCT was conducted and there are no data on the optimal concentration of gadolinium inside the tumor which is necessary to achieve the tumoricide dose, we studied the GdNCT effect at different doses of gadolinium in tumor. The studied range of concentrations was from 10 mg/cm3 to 100 mg/cm3. During the clinical trial the regularity was determined at which the best clinical effect of GdNCT was observed at the gadolinium concentration in tumor of up to 12 mg/cm3.

We also conducted the study on investigating distribution of the power of total dose depending on the gadolinium concentration inside tumor. Such study was performed on the basis of the developed model of GdNCT (1).

The study of total dose rate distribution during GdNCT at different concentration showed that at the gadolinium concentration over 12 mg/cm3 the total dose in the tissue sharply decreases by several times. So, at concentrations of 6-12 mg/cm3 the total dose power at the depth of 0.5 cm of tissue is 150-200 cGy/min and does not increase with the growth of the gadolinium concentration in target (Fig. 10). Such effect is due to the fact that isotopes of gadolinium 157Gd (the content in the natural element 15.68%) have high sections of capture of thermal neutrons. That causes an increase of absorption of thermal neutrons during GdNCT at high concentrations of gadolinium products. The so-called "shielding effect" occurs.

Thus, the analysis of distribution of the total dose, by using the solving of the equation of transfer of neutrons and photons by the method of discrete ordinates according to the RADUGA-5 program, showed that the optimal concentration of 157Gd is within the range of 6-12 mg of Gd in 1 cm3 of tumor. These conclusions help to optimize subsequently the process of GdNCT for achieving the maximum result.

Binary Radiotherapy of Melanoma - Russian Research Results 139

Thus, the clinical results agree entirely with the further studies on investigating distribution

Comparing the time of recurrence in control and at GdNCT we found that the time of recurrence at GdNCT (1067.5 days) significantly exceeds the time of recurrence at surgery (2612.5 days) and at traditional gamma-therapy (305.5 days). Such advantage of the GdNCT method comparing to traditional therapy is explained by a high local dose of

The differences in the results of BNCT and GdNCT, in our opinion, may be explained by

the differences in the pharmacokinetic properties of the used products - 10В-boron phenylalanine during BNCT and the complex of gadolinium with DTPA during GNCT; different products of nuclear and physical reactions of nuclei of 10В and Gd with thermal

During BNCT there is a selective accumulation of 10В-boron phenylalanine inside the tumor cells. The ratio of 10В concentrations in the tumor and in normal tissues is not less than 2.5. The run of alpha-particles which are formed during irradiation is commensurable with the cell diameter, and the nuclei of 6Li – less than the cell diameter. Therefore secondary radiation affects predominantly tumor cells, by which a high effect of BNCT effect is achieved. And in the given study during BNCT we observed degree 1-2 of local radiation

During GdNCT the used product with gadolinium - Dipentast® is mainly in the intercellular space, and accumulation of the product inside tumor is achieved only by intratumoral administration of the product. As a result of absorption of thermal neutrons by the nuclei of 157Gd there are formed high-energy gamma-quanta, low-energy electrons and roentgen radiation. It is secondary radiation that causes death of tumor cells. However, the character of secondary radiation leads to affection of healthy tissues as well. At the use of GdNCT degree 2 of local radiation reactions was diagnosed in all animals, and in one case - stage 4.Such difference in the mechanism clinically should manifest itself in an increase of side effects on healthy tissues during GdNCT. Indeed, during GdNCT the degree of radiation lesions of healthy tissues was somewhat higher (degrees 2 and 4 of local radiation reactions) than at irradiation with neutrons and during BNCT (degree 1-2

An increase of recurrence rate at GdNCT even in complete regression of tumor shows that at GdNCT it is not always possible to achieve death of all tumor cells. One of the reasons of this result may be the excessive concentration of 157Gd inside tumor. As natural gadolinium has the section of neutron capture two orders higher than boron has, at its excessive concentration in tumor the effect of screening on the tumor surface may occur that does not allow to achieve destruction of cells in a deeper layer. During further studies, at improvement of GNCT technology, it is necessary to administer gadolinium product in such way so that the gadolinium concentration in tumor tissue would not

Despite the conducted treatment, in all animals there was recorded systemic metastasis into the lungs, that is, despite local cure of oral mucosa melanoma, the further generalization of tumor process could not be avoided. Thus, NCT is effective comparing to other methods for local treatment of the primary tumor focus but influences in no way the process of formation of metastases. Therefore the task of this study was not only to evaluate NCT effectiveness during treatment of the primary tumor focus, also development of the

of the total dose power depending on the gadolinium concentration inside tumor.

irradiation of 80-100 Gy per the irradiated target.

two reasons:

neutrons.

reactions.

of local radiation reactions).

exceed 12 mg/cm3.

Fig. 8. BNCT Results of spontaneous melanoma of the oral mucosa: A - Melanoma before BNCT, B - 2 weeks after procedure BNCT. Full Tumor regression

Fig. 9. GdNCT Full regression of melanoma. A – Oral Melanoma, B - 1,5 months after GdNCT

Fig. 10. Tumor dose distribution in different 157Gd concentration.

A. B.

A. B.

Fig. 10. Tumor dose distribution in different 157Gd concentration.

GdNCT

Fig. 9. GdNCT Full regression of melanoma. A – Oral Melanoma, B - 1,5 months after

BNCT, B - 2 weeks after procedure BNCT. Full Tumor regression

Fig. 8. BNCT Results of spontaneous melanoma of the oral mucosa: A - Melanoma before

Thus, the clinical results agree entirely with the further studies on investigating distribution of the total dose power depending on the gadolinium concentration inside tumor.

Comparing the time of recurrence in control and at GdNCT we found that the time of recurrence at GdNCT (1067.5 days) significantly exceeds the time of recurrence at surgery (2612.5 days) and at traditional gamma-therapy (305.5 days). Such advantage of the GdNCT method comparing to traditional therapy is explained by a high local dose of irradiation of 80-100 Gy per the irradiated target.

The differences in the results of BNCT and GdNCT, in our opinion, may be explained by two reasons:

the differences in the pharmacokinetic properties of the used products - 10В-boron phenylalanine during BNCT and the complex of gadolinium with DTPA during GNCT;

different products of nuclear and physical reactions of nuclei of 10В and Gd with thermal neutrons.

During BNCT there is a selective accumulation of 10В-boron phenylalanine inside the tumor cells. The ratio of 10В concentrations in the tumor and in normal tissues is not less than 2.5. The run of alpha-particles which are formed during irradiation is commensurable with the cell diameter, and the nuclei of 6Li – less than the cell diameter. Therefore secondary radiation affects predominantly tumor cells, by which a high effect of BNCT effect is achieved. And in the given study during BNCT we observed degree 1-2 of local radiation reactions.

During GdNCT the used product with gadolinium - Dipentast® is mainly in the intercellular space, and accumulation of the product inside tumor is achieved only by intratumoral administration of the product. As a result of absorption of thermal neutrons by the nuclei of 157Gd there are formed high-energy gamma-quanta, low-energy electrons and roentgen radiation. It is secondary radiation that causes death of tumor cells. However, the character of secondary radiation leads to affection of healthy tissues as well. At the use of GdNCT degree 2 of local radiation reactions was diagnosed in all animals, and in one case - stage 4.Such difference in the mechanism clinically should manifest itself in an increase of side effects on healthy tissues during GdNCT. Indeed, during GdNCT the degree of radiation lesions of healthy tissues was somewhat higher (degrees 2 and 4 of local radiation reactions) than at irradiation with neutrons and during BNCT (degree 1-2 of local radiation reactions).

An increase of recurrence rate at GdNCT even in complete regression of tumor shows that at GdNCT it is not always possible to achieve death of all tumor cells. One of the reasons of this result may be the excessive concentration of 157Gd inside tumor. As natural gadolinium has the section of neutron capture two orders higher than boron has, at its excessive concentration in tumor the effect of screening on the tumor surface may occur that does not allow to achieve destruction of cells in a deeper layer. During further studies, at improvement of GNCT technology, it is necessary to administer gadolinium product in such way so that the gadolinium concentration in tumor tissue would not exceed 12 mg/cm3.

Despite the conducted treatment, in all animals there was recorded systemic metastasis into the lungs, that is, despite local cure of oral mucosa melanoma, the further generalization of tumor process could not be avoided. Thus, NCT is effective comparing to other methods for local treatment of the primary tumor focus but influences in no way the process of formation of metastases. Therefore the task of this study was not only to evaluate NCT effectiveness during treatment of the primary tumor focus, also development of the

Binary Radiotherapy of Melanoma - Russian Research Results 141

A. B.

C. D.

Fig. 11. BNCT of primary osteosarcoma IIb stage. A, B – Roentgenoscopy before operation;

C, D - Roentgenoscopy 2.5 months after surgery.

multimodality treatment aimed at both the primary tumor focus and prevention of quick metastasis of tumor. We formed two groups of animals - groups VII and VIII in which we determined life expectancy. These groups embraced the animals at one stage of tumor process in which complete regression was achieved as a result of NCT. In this study the adjuvant therapy influence on life expectancy of dogs with oral cavity melanoma (group VII) was investigated by using Ronkoleukin®. For inclusion into the scheme of treatment of melanoma on the basis of BNCT and GdNCT as the means hampering the process of metastasis, the Russian agent Roncoleukin® was chosen which is an immunostimulator, it enhances the antibacterial, antiviral, antifungal and antitumor immune response.

We managed to carry out the study on a small group of animals, but the results obtained show significantly that life expectancy at conduction of immunotherapy increase on the average by three times. So, in the study group of the animals which received successful BNCT at stage II of tumor process and also receive adjuvant immunotherapy, the average life expectancy was about 305 days. In group VIII where BNCT was conducted without immunotherapy, the average life expectancy was 113 days.

Thus, in cases of successful NCT at concomitant conduction of immune therapy we observed the maximum life expectancies, and most such animals were long-living ones.

The studies performed by us have shown that treatment of oral mucosa melanoma should be of multimodality character and be aimed at both combating the primary tumor focus and remote metastases.

During conduction of NCT it is necessary to attain complete regression of tumor, it is in these cases that the maximum life expectancies may be reached.

This approach to the diseases, namely the combination of NCT and adjuvant immune therapy permitted us to gain the maximum results in treating melanoma.

In this chapter the results of complex method of oral canine melanoma are shown, but also we studied the possibility of NCT combination with other methods for canine osteosarcoma treatment. Induction chemotherapy, intraarterious administration of BPhA, bone resection, extracorporal BNCT of bone fragment, radiated bone implantation and adjuvant chemotherapy were included in treatment scheme (Fig. 11). 24 hours after operation dog can lean on the leg. 2.5 months after operation, results of biopsy showed absence of tumor cells in replanted bone. X-ray imaging showed full consolidation of replanted bone with maternal bone, without carcinogenesis signs (Fig. 11, 12), Total life span of the dog was 2.5 years without recurrence.

Results of highly malignant tumors treatment show that, instead of high metastatic activity and low traditional methods efficacy , it is possible to achieve high therapeutic efficacy with application of complex treatment scheme on the basis of NCT.

#### **4. Conclusion**

There is a large world positive experience of BNCT clinical application in combined treatment of more than 300 patients with brain tumors (Japan, USA, EU countries) and more than 30 patients with melanoma, including metastatic melanoma (Japan, USA), which shows the prospects for future development and optimization of this type of binary therapy. According to Japanese clinical data for 1968-1996, average life span of patients with brain tumors was from 640 till 1811 days, depending on histological type of tumor, without strong mental disorders. 6 patients after BNCT live more than 10 years. Average life span of such

multimodality treatment aimed at both the primary tumor focus and prevention of quick metastasis of tumor. We formed two groups of animals - groups VII and VIII in which we determined life expectancy. These groups embraced the animals at one stage of tumor process in which complete regression was achieved as a result of NCT. In this study the adjuvant therapy influence on life expectancy of dogs with oral cavity melanoma (group VII) was investigated by using Ronkoleukin®. For inclusion into the scheme of treatment of melanoma on the basis of BNCT and GdNCT as the means hampering the process of metastasis, the Russian agent Roncoleukin® was chosen which is an immunostimulator, it

We managed to carry out the study on a small group of animals, but the results obtained show significantly that life expectancy at conduction of immunotherapy increase on the average by three times. So, in the study group of the animals which received successful BNCT at stage II of tumor process and also receive adjuvant immunotherapy, the average life expectancy was about 305 days. In group VIII where BNCT was conducted without

Thus, in cases of successful NCT at concomitant conduction of immune therapy we observed the maximum life expectancies, and most such animals were long-living ones. The studies performed by us have shown that treatment of oral mucosa melanoma should be of multimodality character and be aimed at both combating the primary tumor focus and

During conduction of NCT it is necessary to attain complete regression of tumor, it is in

This approach to the diseases, namely the combination of NCT and adjuvant immune

In this chapter the results of complex method of oral canine melanoma are shown, but also we studied the possibility of NCT combination with other methods for canine osteosarcoma treatment. Induction chemotherapy, intraarterious administration of BPhA, bone resection, extracorporal BNCT of bone fragment, radiated bone implantation and adjuvant chemotherapy were included in treatment scheme (Fig. 11). 24 hours after operation dog can lean on the leg. 2.5 months after operation, results of biopsy showed absence of tumor cells in replanted bone. X-ray imaging showed full consolidation of replanted bone with maternal bone, without carcinogenesis signs (Fig. 11, 12), Total life span of the dog was 2.5 years

Results of highly malignant tumors treatment show that, instead of high metastatic activity and low traditional methods efficacy , it is possible to achieve high therapeutic efficacy with

There is a large world positive experience of BNCT clinical application in combined treatment of more than 300 patients with brain tumors (Japan, USA, EU countries) and more than 30 patients with melanoma, including metastatic melanoma (Japan, USA), which shows the prospects for future development and optimization of this type of binary therapy. According to Japanese clinical data for 1968-1996, average life span of patients with brain tumors was from 640 till 1811 days, depending on histological type of tumor, without strong mental disorders. 6 patients after BNCT live more than 10 years. Average life span of such

enhances the antibacterial, antiviral, antifungal and antitumor immune response.

immunotherapy, the average life expectancy was 113 days.

these cases that the maximum life expectancies may be reached.

application of complex treatment scheme on the basis of NCT.

therapy permitted us to gain the maximum results in treating melanoma.

remote metastases.

without recurrence.

**4. Conclusion** 

Fig. 11. BNCT of primary osteosarcoma IIb stage. A, B – Roentgenoscopy before operation; C, D - Roentgenoscopy 2.5 months after surgery.

C. D.

Binary Radiotherapy of Melanoma - Russian Research Results 143

BNCT. Complex treatment scheme, which combines traditional methods of treatment with

Therapeutic possibilities of BRT are note finally defined, but this method of treatment has low cost and it can be easily installed and transported to other hospital. It makes us to intensify the studies for effective BRT technologies creation, putting more attention for creation of specific pharmaceuticals, which can accumulate in the tumor with tumor/normal concentration gradient more than 3. To minimize resources for new pharmaceuticals

Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J.D. (1989). Molecular Biology of the Cell (second edition). Garland Publishing Inc., ISBN 0-8240-3695-6, London Gabel D., (1989). tumor-seeking compounds for boron neutron capture therapy: synthesis

Grigorieva E.Yu., Nikolaeva T.G., Koldaeva E.Yu., Naidenov M.G., Sprishkova RA., (2005).

De Duve C., 1967. General principles in enzyme Cytology. Ed. by Roodyn, D.B. Academic

Karnas, S.J., Yu, E., McGarry, R.C., Battista, J.J. (1999). Optimal photon energies for IUdR K-

Khokhlov, V.F., Kulakov V.N., Zaitsev K.N., Riaboukhine Yu.S., Yarmonenko S.P., Deineko

Khokhlov V.F., Kulakov V,N., SheinoI.N., Nasonova T.A. & Mitin V.N. (2004). Patent RF

Khoknlov V.F., Zaitsev K.N. , Beliaev V.N., Kulakov V.N., Kvasov V.I., Lipengolts A.A.,

Khoknlov V.F., Zaitsev K.N., Beliaev V.N., Kulakov V.N., Lipengolts A.A., Portnov A.A.

Koldaeva E.Yu., Grigorieva E.Yu., Kulakov V.N., Sauerwein W. (2010). BSH for BNCT of B-

Kulakov V.N., Bregadze V.I., Sivaev I.B., Nikitin C.M., Gol'tyapin Yu.V., Khokhlov V.F.

and biodistribution, *Clin Aspects Neutron capture Ther*. pp.233-241. Upton. – New-

Na210B12H11SH (BSH) – pharmaceuticall for neutron capture therapy and it's influence on melanoma B-16 cell cycle, *Rossiyskiy Bioterapevtitcheskiy Zhurnal (in* 

edge radiosensitization with filtered x-ray and radioisotope sources. Physics in Medicine and Biology, Vol. 44, No. 10, (October 1999), pp. 2537-2549, ISSN 0031-9155.

O.N., Kornienko V.N. (2001). The Rushian Project on Neutron Capture Therapy for Cancer. Therapy *Frontiers in Neutron Capture, ed. by Hawthorne et al. Kluwer* 

Portnov A.A. (2008). Prompt Gamma Neutron Activation Analysis of 10B and Gd in Biological Samples at the MEPhI Reactor. Proceedings of 13th International Congress on Neutron Capture Therapy "A new option against cancer". Italy,

(2009). Prompt gamma neutron activation analysis of 10B and Gd in biological samples at the MEPhI reactor. Applied Radiation and Isotopes. Vol. 67.- Issue 7-8S.-

16 Melanoma in a Murine Model. *New Challenges in Neutron Capture Therapy 2010*, *Proceedings of 14th International Congress on Neutron Capture Therapy*, October 25-29,

(2001). Design of Boron- and Gadolinium- Containig Agents for NCT *Frontiers in Neutron Capture, ed. by Hawthorne et al. Kluwer Academic/Plenum Publishers, N-Y,* 

BRT can significantly improve malignancy treatment.

**5. References** 

York-London.

Press, London.

#2270045.

S251–S253.

2001, p.p. 843-846.

*rus.)*. № 3, pp. 30-33.

selection for further studies we apply developed screening scheme.

*Academic/Plenum Publishers, N-Y*, 2001, p.p. 425-432.

Florence, November 2-7. ENEA. pp. 415-417.

2010, Buenos Aires, Argentina, p. 144-146.

Ado, A.D., (1993). *Physiopathology (in rus), Triada-X, Moscow /* 

Fig. 12. A dog on walk 2.5 months after BNCT. Surgery region is marked in yellow

patients after traditional methods of treatments was 8-10 months. Doctor Nakagawa Y., taking into consideration BNCT opportunities and analyzing his own clinical experience, supposes, that BNCT is the best method for malignant brain tumors treatment, which needs further development of neutron beams and 10B-containing drugs (Nakagawa & Hatanaka, 1997; Nakagava et. al., 2003). Preliminary analysis of melanoma treatment clinical results shows, that more than 2 years life span for patients with melanoma was 78%, treatment can be succeed in T3-4 N0M0 melanoma in more than 90% of patients. (Mishima 1996; Busse et al., 1997; Barth et al., 2005).

Achieved by Russian scientists results prove the advantages of BRT in comparison with traditional methods of treatment. ВРА is more effective substance for BNCT than BSH and KUG-1. Full tumor regression was reached in 80% of cases with application of BNCT, and in the group of GdNCT full tumor regressions was in 50% of dogs with oral cavity melanoma, while the application of traditional gamma-therapy provides only 12,5 % of full tumor regression. Complex therapy, which combines BNCT with adjuvant immunotherapy with Ronkoleukin® (Interleukin-2) can increase the total life span more than 3 times. To compare with human life span we can speak about possibility of 5-10 years recurrence-free period for humans. As canine melanoma is the correct model of human melanoma, such complex method of treatment significantly more effective than traditional methods of treatment One of the reason of BNCT efficacy with BSH is the capability of BSH to penetrate into the cell nucleus and mitochondrions. This fact opens new prospects for future application of BSH in BNCT. Complex treatment scheme, which combines traditional methods of treatment with BRT can significantly improve malignancy treatment.

Therapeutic possibilities of BRT are note finally defined, but this method of treatment has low cost and it can be easily installed and transported to other hospital. It makes us to intensify the studies for effective BRT technologies creation, putting more attention for creation of specific pharmaceuticals, which can accumulate in the tumor with tumor/normal concentration gradient more than 3. To minimize resources for new pharmaceuticals selection for further studies we apply developed screening scheme.

#### **5. References**

142 Advances in Cancer Therapy

Fig. 12. A dog on walk 2.5 months after BNCT. Surgery region is marked in yellow

1997; Barth et al., 2005).

patients after traditional methods of treatments was 8-10 months. Doctor Nakagawa Y., taking into consideration BNCT opportunities and analyzing his own clinical experience, supposes, that BNCT is the best method for malignant brain tumors treatment, which needs further development of neutron beams and 10B-containing drugs (Nakagawa & Hatanaka, 1997; Nakagava et. al., 2003). Preliminary analysis of melanoma treatment clinical results shows, that more than 2 years life span for patients with melanoma was 78%, treatment can be succeed in T3-4 N0M0 melanoma in more than 90% of patients. (Mishima 1996; Busse et al.,

Achieved by Russian scientists results prove the advantages of BRT in comparison with traditional methods of treatment. ВРА is more effective substance for BNCT than BSH and KUG-1. Full tumor regression was reached in 80% of cases with application of BNCT, and in the group of GdNCT full tumor regressions was in 50% of dogs with oral cavity melanoma, while the application of traditional gamma-therapy provides only 12,5 % of full tumor regression. Complex therapy, which combines BNCT with adjuvant immunotherapy with Ronkoleukin® (Interleukin-2) can increase the total life span more than 3 times. To compare with human life span we can speak about possibility of 5-10 years recurrence-free period for humans. As canine melanoma is the correct model of human melanoma, such complex method of treatment significantly more effective than traditional methods of treatment One of the reason of BNCT efficacy with BSH is the capability of BSH to penetrate into the cell nucleus and mitochondrions. This fact opens new prospects for future application of BSH in Ado, A.D., (1993). *Physiopathology (in rus), Triada-X, Moscow /* 


**7**

*Cuba* 

**Clinical Development Paradigms for Cancer** 

Scientific progress is inhabited by paradigm shifts. In 1962, Tomas Kuhn popularized the concept of ¨paradigm shift¨ arguing that scientific advancement is not evolutionary, but rather is a "series of peaceful interludes punctuated by intellectually violent revolutions", and in those revolutions "one conceptual world view is replaced by another" (Kuhn, 1962). Again according to Kuhn, ¨paradigm shifts occur when anomalies in the old paradigm accumulate and cannot be overlooked anymore¨. This is probably the situation today in the

For many years, chemotherapy has been the gold standard of cancer therapy. The era of cancer chemotherapy began in the 1940s with the first use of nitrogen mustards and folic acid antagonist drugs (De Vita et al, 2005) and a major break-through in 1965, when James Holland, Emil Freireich, and Emil Frei hypothesized that cancer chemotherapy should follow the strategy of antibiotic therapy for tuberculosis with combinations of drugs, each with different mechanisms of action (Frei et al, 1965). Cytotoxic chemotherapy in fact succeeded in producing major therapeutic effects, including cures, in hematological malignancies and some solid tumors such as testicular and ovarian cancers. However its contribution to the treatment of most solid tumors has been much less. The success history of chemotherapy in leukemia and lymphoma simply did not repeat in most solid tumors. Chemotherapy has the disadvantage of its high toxicity, because is an unspecific treatment

The paradigm of selective killing of cancer cells in a way alike to what antibiotics do for infections, created in turn a standardized procedure for stepwise drug development through conventional Phase I, II and III trials, which was soon adopted and translated into regulations by many drug regulatory authorities. The designs of these clinical trials respond

However, although retaining the main concept and adapting to the old paradigm malfunctioning, variants have been introduced for cancer drugs approval: i.e. approvals

From January 1973 through December 2006, 68 new drugs were approved for cancer therapy from which 31 were approved without 2 arms randomized clinical trials including a

to the need of demonstrating the drug efficacy at its maximal tolerable dose (MTD).

without randomized trials and approvals based in accelerated approval regulations.

control arm with different therapy, supportive care or placebo (Tsimberidou, 2009).

**1. Introduction** 

treatment of advanced cancer.

However, changes are difficult. Human beings resist changes.

which effect does not distinguish between normal and tumor cells.

**Vaccines: The Case of CIMAvax EGF®**

Gisela González, Tania Crombet and Agustín Lage

*Center of Molecular Immunology* 

