Mikhailov V. A., Scientific Medical Laser Centre, Moscow, Russia

Skobelkin O. K., Andreeva C. P.,  Petuchov M. I., State Centre of Science of Laser Medicine, Moscow, Russia

 

Abstract

Multiple studies done by us to investigate the effects of various laser irradiations on tumoral growth. both in experiments and in clinics (4, 5, 6) have shown that tumoral-static effect of low-level laser radiation (LLR) may be explained by the stimulation of the immune system (7) and by the dirrect changes in the metabolism in tumoral cells after LLR, In our work we made an attempt to study changes taking place in tumoral cells,

 

Introduction

The selectivity of accumulation or irradiation of different chemotherapeutic drugs in tumoral tissue is one of the main factors determining their curative-diagnostic efficiency. Pharmacokinetics of hematoporphyrine derivatives (HpD) in intact and diseased organism have been studied by some researchers (1, 2, 3). However, changes in HpD pharrnacokinetics under some side-effects (for example, the tumoral process at different stages, the inflammatory process in the organism, etc.) have not been studied in fact. The tumoral specificity of these preparations depends on the chemical structure, changes in their transport and in the organism damaged by the disease.

Various preparations are bound in a different way by normal and tumoral cells. We preferred HpD synthetized in Russia as it has the greatest possibility to accumulate in tumoral cells (3).

The purpose of our article is to present an experimental research on the effect of different schemes of LLR on HpD accumulation in tumoral tissue.

 

Materials and Methods

The experiments have been done on white female rats weighing 120-150 gr divided in 4 groups, 47 animals in each. Walker's carcinosarcoma N 256 from USA bank was inoculated subcutaneously in the left lateral part of the body in dosage 0.5 ml. The peculiarity of this tumor lies in a rapid growth with the period of duplication in 2-3 days. The experimentation was done on the 4-5th/day after innoculation when possible spontaneous necrosis and peripheric hemorrhages are absent. The preparation, a derivative of hematoporphyrine (HpD) synthetized in Institute of Chemical Technology (prof. Mironov A.) was injected in the dosage 10 mg/kg under the ether narcosis intravenously in the jugular vena. The accumulation of HpD in the tumor was studied in each animal decapitated in 1/2, 3, 6, 12, 24 and 48 hours. Cryostatic species 18-20 microns thick were prepared from the tumour; then they were studied with microspectroflowmetric methods. The fluorescence intensity was measured qualitatively without the fluorescence background and was calculated in relative units. laser irradiation was performed by a semiconductor laser (GaAs) with the wavelength 890 nm and max. pulse power 5 W.

 

Research purpose

The general strategy of this work lies in the following:

1. To study the accumulation of HpD in the tumor under different schemes of LLR and to determine the best regime which effects the accumulation most strong.
2. To determine terms of significant changes in HpD accumulation and discharge from the tumoral tissue under LLR.

 

Results

LLR was applied one time as well as many times; and it was done both before HpD injection and after it. Each seria of animals has been divided into two groups; they got different dosages of irradiation - minimal (0,03067 J/cm²) and maximal (0,3067 J/cm²).

In the first seria animals were injected only with HpD. They were controls.

In the second seria animals were irradiated once immediately after HpD injection.

In the third seria animals were irradiated twice: first time -immediately after HpD injection, second time - in 30 min after the first one.

In the fourth seria animals were irradiated once 30 min befor HpD injection.

In the fifth seria animals were irradiated twice: the first time -1 day befor HpD injection, the second time - 30 min befor HpD injection (Fig. 1).

 

Fig.1. Accummulation of hematoporphyrin derivative (HpD) in the tumoral tissue of experimental animals of different parameters of laser therapy.

                Fluorescent intensity in relative units

 

                                                            Time after the HpD injection (hours)

          I. Injection of only HpD (controls)

II. Direct single (max. dosage) irradiation after HpD injection

III. Double irradiation (minimal dosage) immediately and 30 min after HpD injection

IV. Single irradiation (max. dosage) 30 min before HpD injection

V. Double irradiation (minimal dosage) 1 day and 30 min before HpD injection.

In the second and third serias the accumulation of the preparation in the tumor practically does not differ of that in controls. In the third seria the accumulation was somewhat lower than in the controls; however the difference was not statistically significant. Different dosages of irradiation practically did not influence the preparation's accumulation. In the fourth and fifth seria a significant reduction in this accumulation in the tumor has been found out, and this phenomenon has been watched in all periods of investigation and the fluorescence intensity was almost similar. In these serias of investigations the quantity of the absorbed sensitizer depended little on the dosage of irradiation. However, in the fifth and fourth serias it was found out that under the minimal dosage of irradiation (0.03067 J/cm² ) the photosensitizer was accumulated less than under the maximal one. The most interesting results are presented in Table 1.

 

Table 1. Statistical analysis of the data obtained revealed the following results expressed in relative units.

Time after HpD	1 Series	2 Series	3 Series	4 Series	5 Series
30 min.	180 ± 15.5	164 ± 18.5	159 ± 13.6	21.4 ± 6.2	20 ± 6.2
3 hours	127 ± 8.8	110 ± 14.1	99.8 ± 14.3	27.2 ± 2.3	24.4 ± 4.8
6 hours	96.4 ± 17.0	91 ± 19.3	89 ± 8.5	11.1 ± 2.3	6.4 ± 2.1

 

Discussion

This research has been made by us on the basis of our data obtained for the analysis of LLR effect on inoculated tumors. At first, taking into consideration the research made by T. Ohshiro (1989) we expected that LLR would increase the accumulation of HpD and thus it could be used for the improvement of photodynamic therapy effect. According to the theory LLR had to effect the tumoral blood circulation through changes in the vascular wall and cellular membranes permeability. However, in the II and III series of investigations when LLR was used after HpD injection, we did not observe any significant changes in the preparation accumulation and discharge comparing to the controls. We suggested that those changes which had to take place after LLR had not developed yet. In the fourth series when LLR was made once before HpD injection the insignificant reduction of the preparation accumulation in the tumor has been noted. In the fifth seria, when LLR was made 24 hours before HpD injection a sharp reduction in the quantity of photosenthyzer in the tissue was found out; moreover during all periods of the investigation it was similar (Fig.1), The preparation accumulation depended little on the irradiation dosage. However, in the fourth and fifth serias under minimal dosages the preparation accumulated less than under maximal ones.

Thus, the investigation made presented a clear picture of HpD accumulation in tumoral tissue. The effect of laser therapy takes place 30 min after beginning and is characterised by a sharp reduction of the preparation in tumoral tissue. It could be explained both by the impairments in the preparation's transport through the vascular floe (to be more exact, through the capillary wall) and by the impairments of that transport through the cellular membraine). The reduction of the preparation's discharge out of cells irradiated by laser in comparison to controls also supports this suggestion.

Thus, application of low-level lasers in the experiment for studying the accumulation of hematoperphyrines in tumors has shown that laser irradiation is the most effective when being performed 30 min befor the HpD injection. HpD accumulation in tumoral tissues is reduced by 111% in comparison to controls laser therapy after HpD injection does not influence the preparation accumulation.

 

Conclusions

1. The effect of laser therapy takes place in 30 minutes after the irradiation and is accompanied by a sharp reduction of the preparation's accumulation in the tumoral cells.

2. Changes in HpD accumulation and impairments in its discharge seem to be caused by the changes in the permeability both in the capillary wall and in the cellular membraine after LLR.

 

References

1. Gaiduk M.I,, Grigorjantz V.V., Mininkov V.D., Mironov A.P., Sukhin T.M. Fiberoptic laser spectroflowmetry of medico-biological objects. Proceedings of XIII Inter. Conf. on Gogerent and honlinear optics. Minsk, 1988, vol. 1, section I-VIII. p. 454-455.

2. Andrioni A., Cubeddu R., De Silvistris S., Laporta P., Ambesi-Impiombato P.S., Esposito M., Mastrocinque M., Tramontano D. Effect of laser irradiation on hematoporphyrine -treated normal and transformed thyroid cells in culture. Cancer Res., 43:2076, H983.

3. Barabash R., Skobelkin O.K., Petuchov M.I., Normansky V.E., Rjashsky O.K., Mironova M.I., Andreeva K.P., Kolobanov A.S. Pharmakokinetics of hematoporphyrines, its derivative and fluorescein in carcinocarcoma growth. Fannakologia i Toksikologia, 1990, 53.- N3 p. 24-26.

4. Mikhailov V.A., Skobelkin O.K., Denisov I.M. Application of low-level lasers in preoperative preparation of oncologic patients, Proc. of Inter. Conf., Tashkent, p. Ill, 1989, pu40-41

5. Mikhailov V.A., Skobelkin O.K., Denisov I.M. Study of the effect of different dosages of laser irradiation on the tumoral growth and the determination of the most effective combination with different groups of chemiopreperation in the experiment, Inter. Conference "New in laser Medicine", Moscow, part II, 1990, p. 60.

6. Mikhailov V.A., Skobelkin O.K., Eliseenko V.I., Denisov I.M. The effect of different groups of chemiopreparations in the combination with laser light on the growth of experimental tumours.All - Union Conference "Actual problems of application of magnit and. electro-magnit fields in medicine", Abstracts, Leningrad, 1990, p. 110.

7. Mikhailov V.JL., Zaharov S.D., Skobelkin O.K., Eliseenko V.I., Denisov I.M., Tchaitzev R.G., Silkina G.I. Activation of the immune system in oncopatients by low-level laser light in preoperative period. All-Union Conf. "Low-level laser light in medicine". Chabarovsk, 1990, p. 62-63.