PLESNYS, Albinas (Girinio g. 8, Vilnius, LT-08401, LT)
PLESNYS, Albinas (Girinio g. 8, Vilnius, LT-08401, LT)
CLAIMS
1. Radiation source for activation of hematoporphyrines (HpD) in gamma dynamic therapy characterised in that it emits 0,511 MeV energy quanta.
2. Radiation source according to claim 1 characterized in that it is the source of radioactive krypton.
3. Radiation source according to claim 1 characterized in that it is the source of radioactive gallium.
4. Radiation source according to claim 1 characterized in that it is the source of radioactive sodium.
5. Radiation source according to claim 1 characterized in that it is the source of radioactive aluminum.
6. Use of gamma rays 0,511 MeV energy gamma quanta for HpD activation.
7. Gammadynamic tumour treatment method that includes HpD administering into tumour and tumour irradiating characterized in that for tumour irradiating it ijses a radiation source according to any of claims 1-4.
8. Gammadynamic tumour treatment method according to claim 6, characteriz e d in that the the single absorbed ionizing radiation dose is 0,02-0,2 Gy.
9. Use of the radiation source according to any of the claims 1-4 for production of device for tumour treatment and prevention/prophylaxis. |
Porphyrin activation by different spectrum electromagnetical waves - new malignant tumour treatment possibilities
Technical field
The invention is connected to biomedicine, radiomedicine, radiosensibilisation, electromagnetical waves medicine.
Background art Photosensitized tumour therapy, otherwise called photodynamic therapy (PDT) is one of the malignant tumour treatment methods, based on light sensitive substance — photosensibilizers - selective accumulation in tumorous or other quickly proliferating tissues. The most often used sensibilisers are porphyrins and their derivatives. After absorbing the appropriate length light quantum, porphyrins become activated, interact with by-existent oxygen and activate it (turns from the triplet condition into the single condition). Activated oxygen eagerly reacts with the substances in cell's cytoplasm and organelle membrane. Because of the photochemical reactions tumorous cells die, but the abreast tissues are little harmed.
Porphyrins are the main breathing pigments and ferment components, therefore essential to each cell. They all are tetrapyrole porphyrin derivatives and differ among themselves in 1-8 positions adhering radicals. Porphyrins belong to amphoteric compounds group. They possess features of both acids and alkalis. In acid environment they adhere to a proton, compose a positive charge bearing ions, and after melting in strong alkalis become negative charge possessing compounds. Junctured bounds* in a porphyrin molecule determine certain equal typical their features: red light fluorescence; porphyrin crystal, illuminated with ultraviolet rays, shine with dark red light.
Hematoporphyrin derivatives (HpD) are mixtures of dicarboxyline porphyrines, which possess different radicals. From those polymers and dimmers are considered to be active. Especially plenty of porphyrins, that are more apt to aggregate, accumulate in tumorous tissues.
PDT era started in 1960 since the works by R.L. Lipson in the USA appeared (R.L. Lipson, EJ. Baldes. The photodynamic properties of a hematoporhyrin derivative. Arch Dermatol 1960; 82: 508-516). In 1976 J.F. Kelly's publication about HpD and light
interaction application in human bladder tumour diagnosis and treatment appeared. (J.F.
Kelly, M.E. Snell. Hematoporphyrin derivative: A possible aid in the diagnosis and therapy of carcinoma of the bladder. J Urol 1976; 115: 150-151). However, the greatest contribution to PDT development and wide application in clinic was made by TJ. Dougherty with his co-authors (TJ. Dougherty, J.E. Kaufman, A. Goldfarb, K.R.
Weishaupt, D. G. Boyle, and A.Mittleman. Photo radiation therapy for the treatment of malignant tumors. Cancer Res 1978; 38: 2628-2635).
Unfortunately, visible light penetrates into tissues just for some centimeters. This narrows PDT application indications. Therefore, new more penetrative porphyrin activation ways are being looked for. However, most of the current works are intended for searching light sources that emit light of a greater wave length (are more penetrative) in the visible part of spectrum.
In 1986 we suggested new sensitized tumour therapy methodology — radiosensitized tumour therapy (RST), which we called gammadynamic treatment (GDT). It is such a way of treatment, when accumulated endogenic and exogenic porphyrins in tumorous tissues are activated by an appropriate length wave (of appropriate power) gamma rays of little intensity. (Bloznelyte L., Slekys G., B: Thesises of speeches in conference "Development and integration of contemporary ray diagnostics methods" (CH. 2). Vilnius 1987: 289-90).
During gammadynamic treatment, a small dose (2Gy single and 6Gy sum doses) of gamma rays is used to activate a sensitizer — porphyrin. This sensitized tumour therapy methodics significantly expands malignant tumour treatment possibilities in oncology and we have successfully applied it since 1989 in our clinic. (L. Bloznelyte. About photodynamic and gammadynamic treatment in oncology. Doctoral thesis. Vilnius 1990.
L. Bloznelyte. Photodynamic and gammadynamic therapy - some recent developments. In: Dougherty TJ, editor. Optical Methods for Tumor Treatment and Detection:
Mechanisms and Techniques in PDT π, SPIE Proceedings 1993; 1881: 48-51. L.
Bloznelyte, A. Stancius. Sensitized tumour therapy. Acta medica Lithuanica 1994; 1: 51-
3).
In biomedicine literature there happens to find scientific works with researches on porphyrin as a radiosensitizer.
In 5 th decade of the previous age F.H J. Figge was the first to get interested into ionizing radiation and porphyrin interaction (F.HJ. Figge and R. Wichterman. Effect of hematoporphyrin on X-radiation sensitivity in Paramecium. Science 1955; 122: 468-469).
After having accomplished experiments with Strong A strain mice that were vaccinated
with sarcoma 180, the scholars noted that while applying conventional radial treatment for a complete sarcoma regression 8000 r dose is required. Having injected the mice with hematoporphyrin (Hp), to achieve a full sarcoma regression 4000 r dose is enough (H.P. Mack, W.K. Diehl, G.C. Peck, F.HJ. Figge. Evaluation of the combined effects of hematopoφhyrin and radiation. Cancer 1957; 10 (3): 529-539). This work is also the first clinical study on hematoporphyrin and radiation interaction. 25 patients with cervical cancer a conventional radial treatment was applied alongside with prescribing i/v hematoporphyrin. Those patients showed more rapid than usual tumour regression and they tolerated radial treatment better. In 1966 L. Cohen and S. Schwartz article appeared which confirmed Figge's achieved results. They noticed porphyrins' dual features - both radio protective and radiosensitizational. Their features depend on the porphyrin type and dose. This Israel and USA scholars' work declares that mice rhabdomyosarcome tumours disappeared after affecting them with small hematopoφhyrin doses (0,05 mb) and x-rays (2750 r). While increasing the Hp dose to 0,25 and 1,25 mg, the radiosensitizing effect decreased much. (L. Cohen, S. Schwartz. Modification of radiosensitivity by porphyrins H Transplanted rhabdomyosarcome in mice. Cancer Res 1966; 26: 1769-73).
In 1986 F. Y. Zhao and co-authors work was published about hematoporphyrin derivative's (HpD) radiosensitizing effect while treating tumours in head and neck location. They, after injecting HpD, irradiated the tumour with 40 Gy (used 60 Co) and noticed that this improved radial treatment effect more than 30%. (F. Y. Zhao, K.H. Zhang, H.N. Huang, K.H. Sun, Q.B. Ling, B. Xu. Use of hematopoφhyrin derivative as a sensitizer for radiotherapy of oral and maxillofacial tumours: a preliminary report. Lasers in Medical Science 1986; 1: 253-256). On the basis of the latter work we posed a scientific hypothesis, that ionizing radiation rays of certain power are able to activate poφhyrins themselves. Therefore, to activate a sensitizer that accumulated in a tumour, a small gamma ray amount could be sufficient (and this would enable the treatment to be repeated plenty of times and apgly it, even when tumorous process is most outspread). To verify that assumption in 1986 we carried out an experiment with mice, which were vaccinated with stomach pylorus tumour OZ. After 24, 48 and 72 h after HpD injection, mice tumours were irradiated by single 2, 4 or 8 Gy gamma ray dose. During the week, all tumours necrotized. We continued the experiments, while investigating small γ ray dose (1-3 Gy) impact on radiosensitization of different hystogenesis tumours. Sum γ ray doses were 3-9 Gy. We carried out the
experiments with tumours of mice and rat from 8 more different strains: Voker carcinosarcoma 256 and sarcoma 45 were vaccinated into Vistar line rats subcutaneously, and glioma C6 and glioblastoma A.101.8 -into Vistar line rats head brains. Stomach pylorus tumour OZ-5 and hepatoma 22 were vaccinated into C3HA line mice subcutaneosly, lung epidermoid carcinoma Liuis - to hybrid mice CDF 1 subcutaneously, mammary gland adenocarcinoma Ca755 - to C57Blline mice, and sarcoma S- 180 to BALB/c mice subcutaneously. Brain tumours (glioma C6 and glioblastoma A.101.) were vaccinated stereotaxically: the skull between the ear and eye, 2mm right from the sagittal line trephined by 2mm diameter stomatologist drill. After that tumorous cells suspension (5xl0 5 /50 μl) was injected stereotaxically with a special needle.
In the case of tumour implanting subcutaneously, after the tumour has reached 1 cm size, 5mg/kg sensitizer- hematoporphyrine derivative (HpD) - was injected into eye angle vein sinus or abdomen, hi rats' subcutaneous tumour case, HpD was injected into abdomen, with tumour of 0,5-2 cm 3 size. For rats with implanted brain tumours HpD was injected into tail vein: for rats with glioma C6 it was done 12 th day after implanting, and in glioblastoma A. 101.8 case - on the 15 th day after implanting. In 24, 48 and 72 h after HpD injection, the tumour was irradiated by γ rays radiated by 60 Co in single 1-3 greys (Gy) dose. Total dose was 3-9 Gy.
During all preclinical researches the experimental animals were divided into groups. The first group contained investigative experimental animals that after implanting the tumour received gammadynamic treatment: radiosensitiser HpD was injected and, in 24, 48 and 72 h after HpD injection the tumours were irradiated with single γ ray dose - 2Gy. Total dose was 6 Gy.
Alongside the investigative group there were three control animals groups: 1. Pure control - the group consisted of experimental animals, that, after tumour implantation, we did not apply any treatment.
2. Sensitilizators control group, consisting of experimental animals, that, after implanting a tumour, were just injected with radiosensibiliser and no further treatment was applied. 3. Radiotherapy control group, consisting of experimental animals to which, after tumour implanting, just radio therapy without radiosensitizer was applied.
During entire preclinical researches, the main treatment effectiveness criteria were: animals' survival and tumour growth inhibition. In control groups, animals that received
just radiotherapy without radiosensibiliser, both - survival and tumor growth was similar to the pure control animals (untreated ones).
During some experimental researches we additionally researched different single and total γ ray doses impact on GDT effectiveness. During these experiments single 1; 1,5; 2 and 3 Gy and total 3; 4; 5; 6 and 9 Gy doses were researched. During entire experiments, alongside there were additional radiotherapy control groups, consisting of experimental animals, which, after tumour implantation received only radiotherapy without radiosensitizer, and got respective single 1; 1,5; 2 and 3 Gy, and sum 3; 4; 5; 6 and 9 Gy doses. Having accomplished 12 experimental researches with more than 800 mice and rats, we noticed that not all strains tumours are sensitive to GDT. The best results were achieved with rats' carcinosarcoma W-256, sarcoma 45 and mice stomach pylorus tumour. It is important to note, that while irradiating a sensitized tumour with hematoporphyrine derivatives with a single γ ray dose, bigger than 2GY, and sum doze bigger than 6Gy, gammadynamic treatment effect does not increase. The results that we received proved that GDT effectiveness depends much on the histological tumour type. GDT did not have any significant impact on healthy tissues. This is also verified by M.A. Biel and other's works, researching photosensitizers and ionizing radiation impact on healthy tissues (M.A. Biel, T. Kim, MJ. Trump. Effect of radiation therapy and photofrin on tissue response in a rat. Lasers Surg Med 1993; 13 (6): 672-6). According to their data, usual sensitizator doses do not change radioactive ray effect on healthy tissues. We started researching gammadynamic treatment in an experiment in 1986 and in clinic in 1989. (L. Bloznelyte. About photodynamic and gammadynamic treatment in oncology. Doctoral thesis. Vilnius 1990). In 1989 GDT was applied in a clinic - Lithuanian Institute of Oncology - for the first time. Since 1989, GDT was applied to 305 symptomatic oncological patients, to whom, because of the tumour expansion, a conventional treatment (surgical, ray, chemotherapy) could not be applied or their possibilities were exhausted. Out of these 305 patients 112 were diagnosed with primary or metastatic malignant brain tumours, the rest had melanoma, sarcoma or adenocarcinoma of other localization.
After one or several GDT courses, tumours completely disappeared in 24 patients. These patients were clinically healthy after 1-8 years after the treatment. 83 symptomatic patients showed improvement after GDT. After applying GDT in clinics, it was established that GDT is especially effective while treating adenocystic cancer, part of
melanomas (more seivine cells type), sarcomas (osteogenic sarcomas, leomyosarcomas, lymph sarcomas), adenocarcinomas (breast, prostate) and is hardly effective while treating squamos cell and basal cell cancer. (L. Bloznelyte-Plesniene. Gammadynamic therapy. Universal Lithuanian Encyclopedia. 2004) While researching both in experiment and clinic, it was determined that optimal sum gamma ray dose for hematoporphyrine derivative (HpD) activation depends on tumour hystogenesis and is 4-6 Gy. Increasing the dose treatment effect does not change.
In 2001 corporate Munchen and Padova universities scholars' experimental work was published, which verified the results that we received. Having carried out several repeated experiments with mice that were implanted with human bladder tumours, a radiosensitizing photofrin effect on tumorous tissue was observed (M. Schaffer, P.M. Schaffer, L. Corti, G. Sotti, A. Hofstetter, G. Jori, and E. Duhmke. Photofrin II as an efficient radio sensitizing agent in an experimental tumor. Onkologie 2001; 24: 482-485). Most of these scholars generalized their works in USA in patent No 9,843,977. The first patent to verify porphyrine as radiosensibiliser features was A. Richter, J.G. Levy and D. Dolphin's patent No WO 94/04147 (USA patent Nr. 5,945,439). During the latter decade more publications devoted for different radiosensitizers, enhancing radial treatment effectiveness while applying it to tumours of different hystogenesis have appeared.
There is no exhaustive physical and biochemical explanation for radiosensitizers' operation mechanism yet. F. Bistolfi presents one of the possible hypotheses. According to him, in radiosensitized tumour therapy, the main role falls on peroxidation of biomembrane lipids, oxygen of single condition and active metabolites that outcom&* after water radiolysis (F. Bistolfi. Red radio luminescence and radiochemiluminescence: premises for a photodynamic tumour therapy with X-rays and haematoporphyrin derivatives. Panminerva Medica 2000; 42 (1): 69-75).
Both, while researching GDT in the experiment, and applying the method in clinic, we noticed that for treatment of different hystogenesis tumours differently prepared (with different modifications) hematoporphyrine derivatives are most effective. While applying photodynamic treatment, this phenomenon was not noticed. It is not described by other scholars who researched photodynamic therapy. Neither had we observed it, though we have carried out the works researching suitability of different photosensitizers, while treating tumours of different histological types. (Scientific program "Laser sensibilization in tumour therapy" report 1998-2000; http://www.ff.vu.lt/lsnt/ataskaita.html).
As HpD solutions are unstable, therefore, they should be prepared during each experiment, which is time consuming and does not ensure identity of solutions (hematoporphyrine derivatives are dicarboxyde porphyrin, but possessing different radicals, mixtures). The first who synthesized oligomeric porphyrin mixture in a dualstage method, called hematoporphyrin derivative, were S. Schwartz and R.L. Lipson. TJ. Dougherty was the first to describe HpD preparation in an article (CJ. Gomer, TJ. Dougherty. Determination of [ 3 H]- and [ 14 C]-hematoporphyrin derivative distribution in malignant and normal tissue. Cancer Res. 1979; 39: 146-151). In 1987 and 1989 TJ. Daugherty and co- authors' USA patents No 4,649,151 and 4,866,168 about hematoporphyrine derivatives and their preparation appeared. In the first phase, while hematoporphyrin reacts in sulphur and vinegar acid mixture, hematoporphyrin acetate forms, hi the second phase unpurifϊed hematoporphyrin acetate is affected with 0,1 n soda hydroxide solution and turns into a mixture, called hematoporphyrin derivative (HpD or more rarely used name Photofrin I). Bi-ring hematoporphyrine isomers, possessing ethereal junction, are attributed to the most active components that are in HpD and which selectively accumulate in cancerous cells. Sterilized, purified by diafiltration method, HpD derivative form is called Photofrin H It is a complex oligomeric porphyrin mixture from several tens of individual compounds, which whole structure is not explored. Most often in oligomers poφhyrin rings are joined by ethereal juncture, and the biggest oligomers in the mixture possess up to 9 hematoporphyrin fragments. Scholars that researched HpD derivatives debated for a long time about etheric and esteric juncture relation in these oligomeric derivatives. D.Kessel's prepared HpD derivatives from purified hematoporphyrin diacetate, posses more esteric junctions than the one made from mixed unpurifϊed hematoporphyrin diacetate- monoacetate mixture, hi the course of time, esteric groups in these mixtures turn into ..more stable junctures. These processes proceed more quickly in light, warmth and solutions (D. Kessel, MX. Cheng. Biological and biophysical properties of the tumor localizing component of hematopoφhyrin derivative. Cancer Res. 1985; 45: 3053-3057).
The most exhaustive comparative masspectrometrical oligomeric porphyrin derivative - Photofrin research, employing 5 different and soft ionization methodologies(- FAB, UV- and IR-MALDI, ESI, LD/jet-PI), is performed in M.M. Siegel and colleagues' work (1999 m.). Photofrin composition identification was necessary for substance characterization for medicine controlling institutions. All mass spectrometric methods showed that photofrin II is a mixture of porphyrin oligomers which length go as far as 2,7
± 0,1 porphyrin units. (M.M. Siegel, K. Tabei, R. Tsao, MJ. Pastel, R.K. Pandey, S. Berkenkamp, F. Hillenkamp, M.S. de Vries. Comparative mass spectrometric analyses of Photofrin oligomers by fast atom bombardment mass spectrometry, UV and IR matrix- assisted laser desorption/ionization mass spectrometry, electrospray ionization mass spectrometry and laser desorption/jet-cooling photoionization mass spectrometry. J. Mass Spectrom. 1999; 34 (6): 661-669).
Photofrin was optimized as medicine, firstly intended to photodynamic therapy, the medicine which should possess not only selectiveness for cancerous cell, but also own sufficient absorbing features, required to absorb laser light. According to our data, this oligomer composition is not the most appropriate for ionizing radiation activated different tumour treatment.
The more appropriate for gammadynamic treatment is medicine Photogemum, made by Russian company OOO Photogem (currently Photogem Company) in Moscow.
Our proposed absorbed ionizing radiation doses used in gammatherapy are ten times less than applied during conventional radial treatment. However, they are possible to lessen even ten times more with other sources used instead of radioactive cobalt. Our suggested way to avoid harming healthy tissues is to seek for such ionizing radiating source which enabled to activate sensibiliser with radiating even smaller doses.
Brief description of invention
In gammadynamic therapy we suggest to activate HpD using 0,511 MeV gamma quanta irradiated by a radioactive source, hi the precise discovery realization case, for HpD activation in gammatherapy we suggest using radiating 0,511 MeV gamma quanta radioactive gallium or radioactive krypton or radioactive sodium or radioactive aluminum sources. One more possibility of discovery realization is gammadynamic tumour treatment method, comprising photosensitizer's intrusion into a tumour and tumour irradiation, differing in that 0,511 MeV quanta radiated by radioactive source is used, i.e. 0,511 MeV energy quanta radiated by radioactive sodium or radioactive aluminum sources.
Brief description of figures
Fig. 1. Crimson fluorescence of HpD solutions of different concentrations (ImI), while illuminating them with 405 nm blue light.
Fig. 2. Relational rat C6 glioma subcutaneous tumour volume change after gammadynamic treatment.
Fig. 3. Rat survival after brain C6 glioma gammadynamic treatment.
Fig. 4. 60 Co energetic gamma spectrum, measured for 1024 sec.
Fig. 5. 60 Co spectrum measurement scheme with glass between 60 Co and the detector.
Fig. 6. 60 Co energetic gamma spectrum, with 5mm glass between the source and the detector (glass is placed by the detector).
Fig. 7. 60 Co energetic gamma spectrum, with 5mm glass between the source and detector (glass is placed by the source).
Detailed description of invention
Having started researching the gammadynamic treatment (GDT) in the experiment with implanted laboratory animals tumours, and later having received good GDT results in clinics too, we have no doubt that porphyrins can be activated by gamma rays of certain length (certain energies). In order to verify this, we placed 1 ml ready for infusion HpD solution (concentration lmg/ 1,5ml) into a carpule (for ultracaine) and irradiated it with single 2Gy gamma ray dose. Afterwards we performed a spectroscopic solution analysis, hoping, that conventional HpD spectrum will have changed. Unfortunately, any changes were observed. We repeated the experiments while changing HpD concentration - \n all cases having irradiated solutions in a glass carpule with 2Gy dose afterwards spectroscopic solution analysis showed no changes in HpD spectrum.
Then we posed the hypothesis, that possibly soda-lime glass, that the carpule is made of, acts as a filter, preventing from HpD activating ionizing radiation rays; moreover, that this glass deters ultraviolet rays too, which especially actively activates porphyrines.
Though this hypothesis in physical approach seemed hardly possible, we constructed experiments both with HpD solutions, and in vivo that would make it possible to verify.
At first hematoporphyrin derivative (HpD) solutions researches were carried out, by activating them with electromagnetical waves of different spectrum: visible light and ionizing radiation. HpD solutions of different concentration were analyzed. Changes of HpD solutions were observed, differences and similarities of the changes were compared (in some experiments, spectroscopic analysis of the samples was used; in others - intensity of HpD solution fluorescence. HpD solution compound dependence on the used components concentration, solution temperature, pH and storage time span were also examined.
It is most interesting to observe the investigation of 0,02-0,175 mg/ml .HpD solutions. While illuminating these solutions with blue light (λ=405 nm) in the twilight, crimson light fluorescence is seen with the naked eye. But if a drop (0,005 ml) of such solution to radiate with 2Gy single ray dose (source - radioactive cobalt 60 Co) and afterwards illuminate with blue light (λ=405 nm) , crimson fluorescence would disappear.
What is more, if before irradiating HpD solutions of the said concentration with gamma rays, a solution drop was covered with soda-lime silicate glass and only then affected with
2GY single dose, illuminated with 405nm blue light, fluorescence would remain. It is important to note, that any other type of glass (neither Perspex, nor quartz glass, or NaCl crystals) do not affect fluorescence changes.
This phenomenon is noticed just with appropriate HpD solution concentration, temperature, pH and prepared solution storage time span.
As a drop does not spread evenly on the surface of a slide, wishing to evaluate the phenomenon quantitavely, we carried out one more experiment with HpD solutions, which were placed in plastic bottles. During the experiment we estimated the concentrations, with which ImI volume solution while illuminated with 405nm blue light showed crimson light, seen with a naked eye.
We examined HpD solutions with concentration of 0,00001-10 mg/ml . During the experiments all the solutions were diluted with isotonic NaCl solution (0,9%). We prepared 49 solutions of different HpD concentrations. They were prepared as follows: 10mg/ml HpD solution was diluted a respective number of times from 1,5 till 1 000 000
times (1,5; 2; 10; 20; 30; 40; ... ; 1 000 000 times). Crimson fluorescence was observed in concentrations from 0,0009 mg/ml to 5mg/ml - in 46 solutions of different concentrations
(Fig.l)
Each ImI of these 46 solutions were placed into 46 plastic bottles and they were irradiated with gamma rays 2Gy single dose (source - radioactive cobalt 60 Co ). After 2Gy, solutions which had concentration less than 0,008 mg/ml, illuminated with 405nm blue light, stopped radiating.
Having received such interesting results with HpD solutions, it was decided to verify the hypothesis carrying out the experiments with laboratory animals in vivo. During GDT researches in the experiment, and later on, during GDT application in clinic we noticed that not all but just of some histological types malignant tumours - non-epithelium tumours (melanoma, sarcoma), part of adenocarcinomas - are sensitive to gammadynamic treatment. Especially good were results of the brain malignant tumour GDT treatment: medulloblastoma, malignant ependymomas, and gliomas. Therefore rat glioma was chosen for the experimental work.
Experiments methodology
In experiments in vivo, Vistar clone female or male rats were used (of 300-40Og weight). Experimental animals were kept under Lithuanian bioethics committee confirmed laboratory animal care regulations and requirements. Lithuanian Laboratory Animals Application Ethics Commission under State Veterinary Office permit was received for accomplishing the works. Into every rat's brains or right thigh rat C6 glioma cells were vaccinated subcutaneosly. Rat C6 glioma model is world widely used in new therapeutical effects researches. C6 gliomas (vaccinated into rat brains) are popular ant appropriate animal model, coinciding with human CNS tumours. There are publications by different authors proving that C6 glioma tumour can grow not only in rat brain, but in intraabdomenal and subcutaneous regions too, which facilitates tumour volume growth observation during the period after the therapeutical impact. For rats vaccination a due amount of cells was multiplied by cell culture method. C6 glioma cells are cultivated in DMEM (Dulbecco's Modified Eagle's Medium) with 10% foetal serum, 2 mM glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin, incubating them at 37°C, in 5% CO 2 incubator. After trypsinisation, cell suspensions of appropriate concentration were prepared in 0.9% NaCl solution.
In all experiments rats were anesthetized intraperitoneally by anesthetics and muscle relaxants (50 mg/kg Bioketan+1 mg/kg Xylazin 2%). Stereotaxically the rat brains were vaccinated with 50 μl 5xlO 6 C6 cell suspensions. For subcutaneous vaccination 500 μl of physiological liquid was used, which contained IxIO 7 C6 glioma cells, and it was vaccinated subcutaneously into Wistar line rats right thigh.
In all the experiments a radiosensitizer - hematoporphyrin derivative (HpD) solution (therapeutical dose 5mg/kg) was used, which was injected into rats tail vein. After 24, 48, and72h after HpD injection, rat tumours were three times radiated with single γ ray dose - 2 Gy. Sum dose was 6 Gy. Because of possible due to porphyrin rat sensibility to light, they were kept in a darkened room. In experiments with subcutaneous tumours every two days after the sensitizer injection day, the volume of the tumours was measured 'with special sliding calipers. Tumour volume was estimated according to the formula:
V=l/2 x (4π/3) x (1/2) x (w/2) x h, where:
V - tumour volume; 1 - tumour length; w - tumour width; h - tumour height.
For tumour growth evaluation relational tumour growth was chosen, which was estimated according to the formula S = (S n -So)/S Oj where S n - is a tumour's volume on a respective day, S 0 - initial tumour volume, S - relational tumour growth.
On the 11 th day after C6 vaccination into brains and the 10 th day after subcutaneous vaccination (after developing 100-150 mm 3 size tumours), the rats were divided into 2 explorative and three control groups.
The first explorative group (A) consisted of rats that received gammadynamic treatment: radiosensitizer HpD (5 mg/kg, i/v) was injected, and after 24, 48, 72 h after HpD injection, the tumours were irradiated three times with single γ ray dose - 2Gy. Sum dose was 6Gy.
The second explorative group (B) consisted of rats that received analogical gamma dynamic treatment, but while radiating with gamma rays, all three times the tumours were covered with 0,5cm soda-lime silicate glass.
Alongside the explorative groups, there were three animal control groups:
1. Pure control (C) - the group consisted of rats that, after vaccinating the tumour, no treatment were applied.
2. Sensibiliser control group (D) comprised the rats that after the tumour vaccination received only radiosensitizer and no further treatment was applied.
3. Radiotherapy control group (E) comprised the rats that after vaccinating tumours were not injected with radiosensitizer, and the tumours were analogically to explosive groups three times irradiated with single γ ray dose - 2 Gy. Sum dose was 6 Gy.
During all the experiments the main treatment effectiveness criteria was tumour growth inhibition (if the tumour was vaccinated subcutaneously) and rat survival (if the tumour was vaccinated stereotaxically).
Result statistical analysis was carried out. In experiments, when rats received glioma C6 into their brains, SAS (Statistical Analysis Systems) program was used for the statistical analysis. In experiments when glioma was injected subcutaneously, result statistical analysis was carried out employing computer program SigmaStat (Version 3.0), using disperse analysis method (P<0,05).
In vivo experiments results
While experimenting with rats that received glioma injections stereotaxically into their brains, in control (C) and (D) groups the next day after the injection showed rats behavior changes: reduced agility and activity, they became more irritable, their fur became rumpled, later their coordination and even pace disorganized, diarrhea appeared. This neurologic symptomatic proved the growing intracranial pressure as the effect of growing glioma. During the whole experiment in ( C) and (D) control groups the progress of neurologic symptomatic was observed, which persisted up till the rats death. (A) group rats that were treated by gammadynamic treatment, the next day after HpD injection also showed the first signs of neurologic symptomatic: reduced agility and activity, their fur became rumpled, but these indications disappeared after three days treatment (of CJDT). Some (A) group rats again showed the signs of neurologic symptomatic on 20-22 day after HpD injection.
In radiotherapy control group (E) neurologic symptoms appeared at the same time as in (C ) and (D) control groups and made progress slowly (more slowly than in (C) or (D) during the whole experiment. Parallelly the same situation was observed in investigative (B) group.
After comparing the survival (Fig.2) of the rats under research and in control groups, we observed statistically reliable (A) group rats (that received gammadynamic treatment), survival prolongation in comparison to control (C) and (D) rat groups survival.
Exploratory (B) group rats that received gammadynamic treatment, but during which while irradiating them the tumours were covered with 0,5 cm soda-lime silicate glass, the survival was very similar to radiotherapy control group rats (E) survival, and much shorter than exploratory (A) group rats survival, but the difference was not statistically reliable. The effectiveness criteria of the experiment with C6 glioma, when the tumours were inoculated subcutaneously, was tumour growth inhibition. Tumor growth inhibition was observed in the exploratory (A) group which consisted that rats that received gammadynamic treatment during the first days just beginning the GDT after the tumours were irradiated 2Gy doses. In the other entire groups (except group (E), the first five days since GDT start showed tumour growth (Fig.3). In radiotherapy control group (E) tumour growth was observed the first three days after GDT beginning. After some time, Tumor growth inhibition was observed in other groups too, but it was less statistically reliable than in group (A). The differences between relative tumour growth in exploratory group A and other control groups and exploratory group B was statistically reliable P<0, 05). In 16 days after tumour injection a slow tumour self-regression started. Such phenomenon is described by other authors too (P. Guevara, J. Sotelo). C6 rat glioma grown into the peritoneal cavity, a large source of tumoral cells for subcutaneous transplant of glioma. J Neuro-Oncol 1999; 44: 91-92.). It is important to emphasize that in the exploratory (A) group consisting of rats that received gammadynamic treatment, the first full tumour regression was noticed on the 10* day, and in all the other groups — only the 15 l day.
In the exploratory (B) group (rats that received gammadynamic treatment, but during the gamma rays irradiation, the tumours were covered with 0,05 cm soda-lime silicate glass), tumour regression was very similar to radiotherapy control group rats regression.
Referring to our results we can treat soda-lime silicate glass as a filter that suppresses rays of suitable wave length or , in other words, suitable ionizing radiation spectrum rays, that are able to activate HpD and arise gammadynamic effect. Determining the length of the waves would enable to find other sources of ionizing radiation that are able to activate porphyrines more effectively than the radioactive 60 Co. As the results of the above mentioned experiments enable to draw the conclusion that HpD is activated by those ionizing radiation rays that are radiated by 60 Co, and that do not permeate through soda-lime silicate glass, we researched how the glass changes 60 Co gamma spectrum.
Soda-lime silicate glass, otherwise known as glass in building, Lithuanian standard mark LST EN 572-1, original mark is EN 572-1, is the most widely used type of glass. This glass is made by mixing silicon dioxide, sodium carbonate, calcium carbonate (or calcium oxide) and melting them. The glass possesses such structure, that sodium ions (Na + ) and calcium ions (Ca 2+ ) are interspersed among silicate ion structure in such a way, that silicon and oxygen atoms stretch into a tetrahedron spatial structure.
While researching how glass changes 60 Co gamma spectrum, a series of experiment was accomplished. The experiments with clean germanium gamma detector, covered with lmm aluminum tin were done. It was measured in lead protection. Minimal registered ionizing radiation dose was 0,01 Sv/h. :
At first a simple 60 Co spectrum was recorded (Fig. 4). During this and other experiments a bitmap 60 Co source of 13 kBq activity was used (its activity in comparison with VU Oncology Institute 60 Co source 10 000 Ci = 3,7xlO 14 Bq, is approximately 3 10 10 times less).
60 Co source radiates energy of two types: 1,175 and 1,333 MeV gamma quanta. Gamma quanta absorption is proportional to material thickness and depends on elemental absorption material composition. Flow reduction is described in exhibitor with the sum of absorption coefficient for each element. After recording a simple 60 Co spectrum, three gamma flow reduction experiments were accomplished with soda-lime silicate glass. The experiment was done with a clean germanium gamma detector, covered with lmm aluminum tin, which absorbed electrons sallying from the glass. (Fig.5)
During all the three experiments the source and the detector were 20cm from each other. During the first experiment air was among the source and the detector. During the second experiment 5mm width glass was put by the detector (Fig. 6). During the third experiment 5mm width glass was put by the source. (Fig. 7). The results of the accomplished experiments are presented in table 1.. The changes of the 60 Co spectrum can be evaluated counting three effects: photo effect, Comptons scattering, and electrone- positrone pair forming with gamma quantum appearing in nucleus area.
1 Table. Gamma quanta number per second, registered in 60 Co radiated gamma quanta peaks.
While conducting the experiments and registering the energetic gamma specters we noticed that with 5mm soda-lime glass among 60 Co source and the detector, theife are changes in peaks, corresponding positron annihilation gamma quanta line in 0,511 MeV zone. The glass, especially placed by the detector, significantly reduced the peak. We repeated the experiments with soda-lime glass between 60 Co source and detector while changing duration of the gamma spectrum recording. When gamma spectrum record duration exceeded 800s, an interesting phenomenon was observed: the peak, corresponding positron annihilation gamma quanta line in 0,511 MeV zone, would start growing. If during the experiment we replaced the glass with a new one after the 800s, the peak would not grow. The comparative works were performed with quartz glass, NaCl crystal and wolfram. During those experiments similar phenomena were not observed.
Conclusion: while activating HpD derivatives, 0,511 MeV energy quanta are important. As this peak composes a very poor amount of 60 Co energetical spectrum, it is likely that while applying those energy quanta, for HpD activation just an amount of single 0,02 Gy and sum 0,06 Gy doses were enough. While 0,511 MeV energy quanta are radiated by radioactive sodium and radioactive aluminum, and radioactive gallium, and close energy quanta are radiated by radioactive krypton, it is likely that the sources of these elements are more effective while applying gammadynamic treatment.
Thus, in the invention suggested light source for HpD activation in gammadynamic therapy is different in that the said source emits 0,511 MeV energy quanta, hi single
invention realization cases, light source is radioactive sodium or radioactive aluminum or radioactive gallium. In the other single case it is radioactive krypton.
Therefore, it is possible to create such gammadynamic treatment methodologies during which a single dose of absorbed ionizing radiation would be just ~0,02-0,2 Gy , with sum dose of -0,06-0,6 Gy. This would enable to effectively apply gammadynamic treatment in the future even to those patients that have undergone maximal gamma ray doses and therefore any possibilities of radiotherapy are exhausted. Moreover, gammadynamic treatment would be possible to be repeated multiple (hundreds) times, with malignant tumours recurrent of any location. GDT could be applied as a second prophylaxis, with suspecting possible, but not verified tumour relapse.