RU2440158C2 - Method of photodynamic therapy of subjects with malignant tumours - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, photodynamic therapy of tumours. Photosensitiser (PS), in particular radachlorine is added to nanoparticles of emulsion of perfluorocarbons (PFC), consisting of perfluorodecalin and perfluoromethylcyclohexylpiperidine, stabilised with proxanol 268 solution. Emulsion of PFC with PS is added to stem cells of bone marrow (SCBM), in particular, autologic. Their joined cultivation is carried out, after which SCBM is introduced to subject with malignant tumour, in particular breast tumour. Zone of tumour growth is subjected to impact by light irradiation in dose sufficient for complete or partial tumour destruction. Subject can be represented by a mammal, in particular, human being.

EFFECT: method ensures targeted impact, complete or partial tumour regress, excludes systemic phototoxic injuries.

6 cl, 3 ex, 3 dwg

Description

The invention relates to medicine, and is intended for photodynamic therapy of subjects suffering from malignant tumors.

In recent years, researchers have shown increased interest in the experimental and clinical study of photodynamic therapy (PDT), as a promising direction in the treatment of malignant neoplasms, as well as a number of non-cancer diseases, the development of which are based on the processes of active neoangiogenesis and proliferation (Kaplan MA, 1992 -2008, Mironov A.F., 1993-1996; Stranadko E.F., 1993-2008).

According to modern concepts, the mechanisms of PDT action are based on the selective accumulation of photosensitizing drugs introduced into the body in cells with increased mitotic activity (in tumor cells, endothelium of newly formed vessels, etc.). Subsequent irradiation of the pathological focus with light with a wavelength corresponding to the maximum absorption band of the introduced photosensitizer (PS) induces photochemical reactions in sensitized cells and tissues with the release of singlet oxygen and free radicals - highly active biological oxidizing agents, which leads to phototoxic damage to pathologically altered cells (Jori G . et al., 1983; Kessel D., 1997). In addition, there is also destruction of the blood vessel endothelium in the laser irradiation zone, which results in vascular thrombosis and malnutrition in the tumor.

The above selectivity of action determines the undoubted advantages of PDT for use in medicine. First of all, it is the opportunity to achieve the necessary therapeutic effect (obliteration of the neovascular network or radical destruction of the neoplasm) with minimal damage to surrounding structures, which are important for maintaining the necessary functions.

To date, medicine has accumulated sufficient clinical experience in the effective use of PDT in the treatment of malignant neoplasms (skin cancer, lower lip, metastatic skin lesions, breast cancer, in the combined treatment of tracheobronchial cancer, cancer of the esophagus, bladder, etc.), as well as non-tumor diseases.

A significant contribution to the development of this direction was made by Russian scientists: M.A. Kaplan (1993-2008), A.F. Mironov (1996-1998), E.F. Stranadko (1993-2008), M.L. Gelfond (2000 -2008).

The emergence of a new generation of PSs, in particular the photoditazine, Radachlorin (Russia), Photolon (Belarus) chlorine preparations, which have high photodynamic activity with low skin phototoxicity and rapid elimination from the body, as well as the improvement of laser technology, open up prospects for wide introducing the PDT method into practice.

However, the PDT method has a number of significant drawbacks associated with the nature of the active principles used, in particular, there is prolonged skin phototoxicity, requiring strict observance by patients of the limitations of the light regime, or high general toxicity of the drugs themselves (Budzinskaya M.V., Likhvantseva V.G. , 2005; Kliman G., 1994; Puliafito CA et al., 2002).

An illustration of the above is, for example, the method of conducting photodynamic tolerance in patients with malignant tumors, which we selected as a prototype, in which photodynamic therapy is carried out by administering photosensitivity PS at a dose of 0.3-0.8 mg / kg body weight, followed by exposure to the tumor area growth by laser irradiation with a radiation energy density of 300-600 J / cm ² . A feature of the proposed method of treatment is that photodynamic therapy is carried out for 5-10 sessions, with an interval between sessions of 24 hours and fractionation of the therapeutic dose of laser radiation, while the radiation energy density is 30-60 J / cm ² for each treatment session , FS is administered to the patient intravenously once for 1-1.5 hours before the first session (RF patent No. 2161053, 12/27/2000).

However, as can be seen from the analysis of this method, even complex tricks in the form of fractionation of doses do not allow to overcome the restrictions imposed on the use of photosensitizers.

The foregoing served as the basis for research on the search for methods aimed at reducing the side effects inherent in this therapy, while maintaining its effectiveness.

To date, methods based on the use of stem cells have become very widespread.

Stem cells have a number of characteristic features: they are capable of self-renewal and multilinear differentiation. Between a stem cell and its terminal progeny there are usually several intermediate cells with increasing ability to differentiate. Stem cells are undifferentiated and are not capable of performing special functions in most tissues.

Initial studies focused on hematopoietic stem cells of the bone marrow, and today they are fairly well understood. It is now known that stem cells exist in most organs. In organs, they make up a small population, usually 1-2% of the total number of cells. Until recently, it was believed that organ-specific stem cells in differentiation are limited by their origin. Recently, it has been proven that stem cells can have much wider differentiation capabilities. Bone marrow stromal stem cells can differentiate into muscle, bone tissue, liver cells, kidneys, cardiomyocytes, etc. This served as an impetus for the treatment of a number of diseases using stem cells, in particular bone marrow cells (see, for example, Smolyaninov A.B. et al. Cellular technologies for coronary heart disease. AG-info, 2006, No. 2, p.6 -fifteen).

Also, there is the use of another property of stem cells - as process taps associated with active proliferation. A marker “clings” to a stem cell and after that the fate of this cell is monitored.

PFC-based emulsions are traditionally regarded as plasma substitutes with gas transport function (Geyer RP Perfluorochemicals as oxygen transport vehicles // Biomater. Artif. Cells Artif. Organs., 1988, Vol.16, N1-3. P.455-457). In addition, perfluorocarbon nanoparticles (NPs) can be used to visualize body tissues (magnetic resonance imaging, X-ray contrast) (Winter PM, Cai K., Caruthers SD, Wickline SA, Lanza GM // Expert Rev Med Devices. 2007 Mar; 4 ( 2): 137-145). Recently, studies have appeared in which PFC nanoparticles of emulsions are used as stem cell taps (Kathryn S. et al., ¹⁹ F magnetic resonance imaging for stem / progenitor cell tracking with multiple unique perfluorocarbon nanobeacons, The FASEB Journal, 2007 June, vol. 21, p. 1647-1654). Labeling of stem cells with perfluorocarbon nanoparticles, which are easily detected using magnetic resonance imaging, X-ray and gas chromatography, as suggested by the authors of the above article, will allow tracking the fate of transplanted cells in the body, used to activate vascular growth in the extremities of diabetic patients, and restore vessels damaged during myocardial infarction or during shunt surgery, monitoring of tumors This will also allow you to monitor the dynamics of treatment and evaluate its effectiveness.

In addition, it is known that PFC nanoparticles emulsions stabilized with non-ionic surface-active substances (surfactants) (proxanols, pluronics, etc.) have the ability to sorb and firmly hold various biologically active substances (Norman ME, Williams P., Illum L / J. of Biomedical Materials Research, 1993, vol. 27, p. 861-866). It can be assumed that drug substances sorbed on particles with particles of PFC emulsions will penetrate inside the stem cells and be transported with them to organs and tissues.

Based on the foregoing, we proposed using sorbent PFC emulsions nanoparticles to sorb a drug - PS on bone marrow stem cells and with their help deliver this drug to a malignant tumor followed by PDT.

Thus, addressing of the effect is achieved and the presence of systemic phototoxic lesions is excluded. In addition, we unexpectedly found that cells “loaded” with FS retain it for a long time.

In view of the foregoing, we have proposed a method of photodynamic therapy in subjects suffering from malignant tumors, including photodynamic therapy (PDT), in which the following steps are carried out:

a) add FS to an emulsion of perfluorocarbons (PFCs), including at least perfluorodecalin and perfluoromethylcyclohexyl piperedine stabilized with a solution of proxanol 268;

b) add an emulsion of PFCs with FS to bone marrow stem cells (BCCM) and carry out their joint cultivation;

c) administering CCM after cultivation to a subject suffering from malignant tumors;

d) the tumor growth zone is exposed to light radiation in a dose sufficient to completely or partially destroy the tumor

Moreover, a mammal, in particular a human, acts as a subject.

The technical results of our proposed method are addressing effects and the exclusion of systemic phototoxic lesions. In addition, we unexpectedly found that cells “loaded” with FS retain it for a long time. Also, as a result of the implementation of the proposed method, full or partial tumor regression is achieved.

Below this method is confirmed by the following examples of private execution.

Example 1. The first phase of the experiments.

Previously, as a result of our studies, it was shown that cells “loaded” with FS incorporate tropic tissue and the drug will retain its activity.

PFC emulsion. We used nanoparticles of PFC emulsions, consisting of a mixture of perfluorocarbons: perfluorodecalin and perfluoromethylcyclohexyl piperidine, in a ratio of 9: 1 vol.%, Stabilized with a 4% solution of proxanol 268.

Sorption of PS on particles of PFC emulsion. As the FS, Radachlorin was used, which is a composition of three cyclic tetrapyrroles of chlorin nature (with a hydrogenated ring D), the main of which is chlorin E ₆ (70.0-90.0%), which has an intense absorption band in the long-wavelength part spectrum, with an absorption peak of 662 nm, is produced by RADA-PHARMA LLC (Moscow, Russia).

7 mg of Radachlorin was added to 1 ml of PFC emulsion, shaken for 5 minutes. Then, the emulsion particles were precipitated by centrifugation for 30 min at 4000 rpm, the supernatant was drained, and the precipitate was resuspended in Hanks solution. The procedure of deposition and washing of nanoparticles with a Hanks solution was repeated three times.

Obtaining stem and progenitor cells. C 57 black10 GFP mice were used as stem and progenitor cell donors, the cytoplasm of which contains a protein that fluoresces (green) under the influence of UV radiation. Bone marrow cells were isolated from the femur and cultured in DMEM medium containing 10% fetal calf serum, 5 μg / L insulin, 10 nM dexamethasone, for 5 days at 37 ° C.

The introduction of nanoparticles with radachlorin into stem cells. After 5 days of cultivation, nanoparticles containing radachlorin were added to the bone marrow cells and incubated for 30 minutes at 37 ° C. After 5-fold washing with Hanks' solution, the cells were removed with trypsin and transplanted to the recipient ^IV and ^IV at a dose of 2 × 10 ⁴ to 2 × 10 ⁷ cells. The recipients were outbred mice, homogeneous in the group, not containing GFP and contained in vivarium conditions.

3-7 days after transplantation, the animals were sacrificed, bone marrow was removed and a cell suspension was prepared. Under a microscope, in the UV range, cells containing green protein were searched, and after detection, the cells were irradiated with a laser (662 nm) with a power of 1 J / cm ² for 1 minute. Then, photographing the irradiated cell for 3 minutes with an interval of 15 seconds was carried out in visible light.

When conducting research, a LAMI semiconductor laser apparatus with a wavelength of laser radiation of 662 nm was used as a light source, which corresponds to the maximum spectral absorption of the photosensitizers used with a radiation power of 1.3 W at the end of the fiber, reg. number 29/10020203 / 5212-03 (05/20/2003) OKP code 944420, class II A.

In the course of the work, it was shown that PFC nanoparticles containing PS penetrate the bone marrow stem cells (Fig. 1).

After laser irradiation, reactive oxygen species are formed that cause destruction of the cell membrane (FIG. 2).

It was also shown that the absorbed particles remain in the cytoplasm of the cells, while maintaining the ability to form reactive oxygen species throughout the entire cultivation time (up to 20 days of observation).

The presented results show that nanoparticles of PFC emulsion, when incubated with stem cells, are absorbed by them. Donor stem cells containing nanoparticles with PS adsorbed onto them and injected intravenously and intraperitoneally to mice with GFP protein are found in the recipient's bone marrow. This suggests that the cells retained their physiological activity. As shown by experiments, the nanoparticle-bound radachlorin penetrates into stem cells and also retains its activity for a long time (up to 20 days of observation). When processing cells containing nanoparticles with radachlorin, a laser with a power of 1 J / cm ² active forms of oxygen are released, which leads to cell death (figure 2). In vitro experiments also showed that stem cells loaded with free nanoparticles and nanoparticles with sorbed radachlorine did not differ from the control in the rate of division, spreading on a plastic substrate, and the formation of a monolayer. When introduced into the body of the recipient, GFP-protein-labeled cells were found in the bone marrow, liver and spleen, which indicates their free migration. As for the emulsion adsorbed on particles and included in the cell, radachlorin, under cultivation conditions (37 ° C) it retains its activity (the ability to emit reactive oxygen species under laser irradiation) for up to 20 days of cell cultivation. In the body, we also observe the activity of the drug included in the cell for a sufficiently long time (up to 5-7 days of observation). After laser treatment, a cell containing nanoparticles with radachlorine dies, while nearby control cells do not respond to radiation.

Based on the data obtained, the possibility of using stem cells for the transport of drugs in the body becomes apparent.

Example 2. The second phase of the experiments.

The purpose of this phase was to elucidate the question of whether there is an accumulation of stem cells with FS in the tumor tissue.

The preparation of PFC emulsions, the sorption of PS on the particles of PFC emulsion, and the production of stem cells was carried out by the method described in example 1.

The introduction of nanoparticles with radachlorin into stem cells. After 5 days of cultivation, nanoparticles containing radachlorin were added to the bone marrow cells and incubated for 30 minutes at 37 ° C. After 5-fold washing with Hanks's solution, the cells were removed with trypsin and transplanted to the recipient ^iv in a dose of 2 × 10 ⁴ to 2 × 10 ⁷ cells. The recipients were C 57 black mice that did not contain GFP and were kept under vivarium conditions. These animals developed spontaneous breast cancer.

5 days after transplantation, the animals were killed, the tumor, liver, and spleen were removed. The isolated tissues were subjected to gas chromatography in combination with mass spectrometry to quantify the level of fluorocarbons.

As a result of the study, it was shown (Fig. 3) that tumor tissue (105 ± 25 mg) accumulates about 30% of cells containing fluorocarbon particles (32 ± 5%) with PS. In this case, the liver accumulates 8 ± 3%, the spleen 5 ± 2%. The mass of the animals themselves was 28.5 ± 4.2 g.

The introduction of stem cells with FS in healthy outbred mice showed the following cell distribution: liver 7 ± 2%, spleen 6 ± 2%, bone marrow 10 ± 2%. The mass of the animals themselves was 25.4 ± 3.9 g.

It can be seen from the study that the accumulation of stem cells with FS is carried out primarily in actively growing tissue - in the tumor. These data are consistent with the known work on the distribution of stem cells in the body.

Example 3. The third phase of the experiments.

The purpose of this phase was to elucidate the question of whether there is a tumor damage due to the action of the FS when exposed to it by laser radiation.

The preparation of PFC emulsions, the sorption of PS on the particles of PFC emulsion, and the production of stem cells was carried out by the method described in example 1.

The introduction of nanoparticles with radachlorin into stem cells. After 5 days of cultivation, nanoparticles containing radachlorin were added to the bone marrow cells and incubated for 30 minutes at 37 ° C. After 5-fold washing with Hanks's solution, the cells were removed with trypsin and transplanted to the recipient ^iv in a dose of 2 × 10 ⁴ to 2 × 10 ⁷ cells. The recipients were C 57 black mice that did not contain GFP and were kept under vivarium conditions. These animals developed spontaneous breast cancer. In total, 63 mice were taken for the experiment.

5 days after transplantation, the animals were irradiated by contact using a microlens on the surface of the tissue in the tumor area. As the light radiation used a semiconductor laser apparatus "LAMI" with a wavelength of laser radiation of 662 nm.

PDT was carried out with the following parameters: laser energy density of 200-300 J / cm ² , power density of 0.141-0.39 W / cm ² . The duration of irradiation (T, min) was determined by the formula: T = E / P _s , where P _s is the radiation power density (W / cm ² ), E is the specified value of the energy density (laser radiation dose).

P _s = P _in / S, where P _in is the laser radiation power at the output of the fiber (W),

S is the area of the light spot (cm ² ). In order to facilitate the calculations, we used the table of power density (P _s ) depending on the output power at the end of the fiber (P _in ) and the size of the light spot. In order to measure the power of laser radiation, we used a power meter DI-6A. The immediate, immediate and long-term results of PDT were evaluated as:

- complete regression - the complete disappearance of the tumor;

- partial regression - reduction of the tumor by more than 50%;

- no effect - a decrease in the tumor by less than 50%, a state without changes or an increase in the size of the tumor.

The studies showed complete regression - 22% of individuals, partial regression - 46% of individuals, lack of effect - 32% of individuals.

Then the animals were killed and the histological picture of the tumors was examined. The histological picture was consistent with the treatment results.

Thus, the proposed method can be widely used in the treatment of both breast cancer and other types of malignant neoplasms for which the use of PDT is applicable.

Claims (6)

1. The method of photodynamic therapy of subjects suffering from malignant tumors, including photodynamic therapy (PDT), characterized in that the method is as follows:
a) add a photosensitizer (PS) to the nanoparticles of an emulsion of perfluorocarbons (PFCs), consisting of perfluorodecalin and perfluoromethylcyclohexylpiperidine stabilized with a solution of proxanol 268;
b) add an emulsion of PFCs with FS to bone marrow stem cells (BCCM) and carry out their joint cultivation;
c) administering CCM after cultivation to a subject suffering from malignant tumors;
d) the area of tumor growth is affected by light irradiation in a dose sufficient to completely or partially destroy the tumor. 2. The method according to claim 1, characterized in that the subject is a mammal. 3. The method according to claim 2, characterized in that the human being acts as a mammal. 4. The method according to claim 1, characterized in that the quality of the file is radachlorin. 5. The method according to claim 1, characterized in that the cancer of the breast is a malignant tumor. 6. The method according to claim 1, characterized in that autologous CCMs act as SCMS.

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