RU2713941C2 - Method for determining maximum concentration time of photosensitizer chlorin e6 lysine dimeglumine salt in tumor - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to oncology, and can be used to determine time of reaching maximum concentration of photosensitizer (PS) of chlorine series – chlorine e6 lysine dimeglumine salt in body tissues after its introduction. PS chlorin e6 lysine dimeglumine salt is introduced into the body. 30 minutes after the PS administration, a tumor / standard fluorescent contrast value is calculated. If the tumor / standard fluorescent contrast is up to 3.0, the maximum PS accumulation in the tumor is considered to be 3 hours from the moment of PS administration, with the tumor / standard fluorescent contrast more than 3.0–4 hours.EFFECT: method provides higher clinical effectiveness ensured by possibility of individual determination of time of maximum accumulation of PS chlorine e6 dimeglumine salt in tumors.1 cl, 3 ex

Description

The invention relates to medicine, namely to oncology, and can be used to determine the time to reach the maximum concentration of the chlorine-type photosensitizer (PS) - chlorin e6 lysine dimeglumine salt in the body tissues after its introduction, which in turn is necessary to determine the start time of the photodynamic therapy (PDT) with its use.

The PDT method is based on the use of drugs - PS, which, when introduced into the body, accumulate mainly in the tumor. After the introduction of PS into the body and achieving its maximum accumulation in tissues, the pathological area is irradiated, mainly by laser radiation. In this case, PS molecules catalyze the formation of cytotoxic agents, in particular, singlet oxygen, which destroy tumor cells. To obtain the most pronounced positive effect, it is necessary to carry out PDT at the time of maximum accumulation of PS in the tumor tissue.

The effectiveness of PDT in many respects depends on the correct implementation of the treatment technique, one of the important components of which is the correct choice of the optimal time interval between the introduction of PS and the conduct of a laser irradiation session. This interval is characterized by an indicator of the maximum accumulation of PS in the tumor. The time of maximum accumulation of PS in the tumor can vary depending on the characteristics of the body’s metabolism and the biodistribution of the photosensitizer, which are individual for a particular patient. In this connection, it is not uncommon in practice that the selected interval during the 1st and 2nd phases of clinical trials of FS in a certain group of patients, subsequently, may not be optimal in other patients. To solve this problem, methods have been developed for determining the hourly kinetics of the time of maximum accumulation of the photosensitizer in the tumor in each patient. However, such methods are a high-cost procedure, both financially and in terms of labor.

There is a method for determining the accumulation of FS in tumor tissue (Morozova NB "Experimental study of a new photosensitizer" Phthalosens "for photodynamic therapy of malignant neoplasms." Abstract of dissertation for the title of senior scientific candidate of biological sciences, M., 2007 ) We studied the kinetics of the distribution of the photosensitizer by measuring fluorescence in tumor and normal tissues for various periods after administration of the drug after the killing of animals (mice). Thus, each animal was measured only once for a certain period after administration of the photosensitizer. Thus, the methods for studying the photosensitizer presented in this work cannot be used in clinical oncology.

The closest is a method for determining the optimal modes of fluorescence diagnostics and photodynamic therapy (RU 2376044, Filonenko E.V., Chissov V.I., Sokolov V.V., Yakubovskaya R.I.). In the work, the authors determined the kinetics of tissue distribution of various PS by the method of local fluorescence spectroscopy.

A significant drawback of this method is that, firstly, the study of kinetics implies hourly changes in the level of PS accumulation in the tumor tissues over a period of 7-8 hours, which is not applicable in everyday clinical practice. Secondly, because fluorescence contrast indices were not taken into account in this technique, i.e. it is impossible to determine the exact time of maximum accumulation of FS in the tumor. In this regard, the data obtained describe only the time range in which the maximum values of PS accumulation in the tumor can be achieved, and not a specific time.

Currently, a promising area of medicine is personalized treatment, taking into account the characteristics of a particular patient in order to increase efficiency and achieve the best results.

Thus, the technical problem we were solving was the individual determination of the optimal time interval between the introduction of the photosensitizer and the laser irradiation session, i.e. the time of maximum accumulation of FS chlorin e6 lysine dimeglumine salt in the tumor.

As FS, we studied preparations of the chlorin series, in particular, chlorin e6 lysine dimeglumine salt (CML). This drug has a high rate of excretion from normal tissues, provides a deep therapeutic effect on tumor tissue, is non-toxic and promising for use in oncology.

The technical result is achieved by the fact that a chlorin e6 photosensitizer is introduced into the body, lysine dimeglumine salt, and 30 minutes after the administration of PS, the tumor / normal fluorescence contrast value is calculated:

- up to 3.0 inclusive - 3 hours,

- from 3.0 and more - 4 hours.

The method is as follows.

FS chlorin e6 lysine dimeglumine salt is introduced into the body. 30 minutes after the introduction of the FS, the optimal time for the laser irradiation session is determined, then the magnitude of the tumor / normal fluorescence contrast is calculated (for example, V. V. Lukin. Laparoscopic fluorescence diagnosis of peritoneal dissemination of malignant neoplasms. Abstract of dissertation for the degree of candidate of medical sciences ., Moscow, 2010), measured 30 min after the introduction of FS.

With the intravenous administration of FS, it circulates in the systemic circulation. Moreover, it takes time to achieve a certain concentration of PS in the tumor, which will characterize the rate and kinetics of PS accumulation in the future. This time is 30 minutes from the time of the introduction of the FS, because during this time, blood circulation through the tumor in a sufficient volume occurs.

With fluorescence contrast up to 3.0 inclusive, the optimal interval between the administration of PS and the laser irradiation session (maximum level of PS accumulation in the tumor) is 3 hours. With fluorescence contrast more than 3.0 - 4 hours.

The proposed method was tested in in vivo studies on transplantable tumors in animals. As the FS of the chlorin series, CML was administered at a dose of 12.0 mg / kg.

Fluorescence contrast was measured by the following procedure.

CML fluorescence was recorded in the tumor and healthy surrounding tissues of mice at various time intervals using a laser spectral analyzer for fluorescence diagnosis of tumors and monitoring PDT LESA-06 (CJSC Biospek, Russia). For this purpose, fluorescence was excited by the radiation of a He-Ne laser (lasing wavelength 632.8 nm, spectral range of measurements 640-900 nm). Mathematical processing of the fluorescence spectra was performed using the LESA-06 program. Upon excitation of fluorescence in the red region of the spectrum, the integrated fluorescence intensity in the range 645-680 nm was normalized to the integrated intensity of the back diffuse scattering signal in the tissue of the exciting laser radiation (λ = 632.8 nm).

Fluorescence contrast was calculated as the ratio of normalized fluorescence in the tumor to normalized fluorescence in the surrounding tissue (skin).

The optimal time for a laser irradiation session was determined by the magnitude of the fluorescence contrast of the tumor / norm, measured 30 minutes after administration of CML (hereinafter referred to as fluorescence contrast).

With fluorescence contrast up to 3.0 inclusive, the optimal interval between the introduction of PS and the laser irradiation session (maximum level of CML accumulation in the tumor) is 3 hours; and more than 3.0-4 hours

Example 1

Spectrophotometric examination was performed in an F1 mouse (C57Bl / 6j × CBA), a female, inoculated with S37 sarcoma. CML injected a mouse at a dose of 12.0 mg / kg.

The fluorescence contrast, measured 30 minutes after the administration of the FS solution, was 2.2, which allows us to determine the maximum level of PS accumulation in the tumor - 3 hours after the introduction of the FS.

To confirm this fact, then every hour the intensity of normalized CML fluorescence in the tumor was recorded, which amounted to:

1 hour after administration of the photosensitizer 2.1 conv. units

after 2 hours - 2.9 conv. units

after 3 hours - 4.1 conv. units

after 4 hours - 3.6 conv. units

after 5 hours - 3.2 srvc. units

after 6 hours - 2.7 srvc. units

Thus, the maximum CML fluorescence intensity, corresponding to the maximum accumulation of PS in the tumor, was achieved 3 hours after the introduction of PS, which corresponded to a predetermined value of fluorescence contrast.

At 2 and 4 hours after the administration of PS, the fluorescence intensity in the tumor was 29% and 12% less than at the time of the maximum accumulation of the photoactive PS form in the tumor, respectively. When the fluorescence contrast value was 30 minutes after the administration of CML 2.2 (less than 3.0), the time to reach the maximum intensity of CML fluorescence in the tumor was 3 hours. A PDT session was performed 3 hours after the administration of CML, resulting in complete regression of the tumor in the mouse (tumor growth inhibition (TRO) values were 100% during 21 observation days after PDT).

Example 2

Spectrophotometric examination was performed in an F1 mouse (C57Bl / 6j × CBA), a female, inoculated with S37 sarcoma. CML injected a mouse at a dose of 12.0 mg / kg.

The fluorescence contrast, measured 30 minutes after the administration of the FS solution, was 4.2, which allows us to determine the maximum level of PS accumulation in the tumor - 4 hours after the introduction of the FS.

To confirm this fact, each hour, the intensity of normalized CML fluorescence in the tumor was recorded, which amounted to:

1 hour after the introduction of the photosensitizer 2.4 srvc. units

after 2 hours - 3.3 srvc. units

after 3 hours - 3.7 conv. units

after 4 hours - 4.3 srvc. units

after 5 hours - 3.2 srvc. units

after 6 hours - 2.6 conv. units

Thus, the maximum CML fluorescence intensity, corresponding to the maximum accumulation of PS in the tumor, was achieved 4 hours after the introduction of PS, which corresponded to a predetermined value of fluorescence contrast.

At 3 and 5 hours after the administration of PS, the fluorescence intensity in the tumor was 14% and 26% less than at the time of the maximum accumulation of the photoactive PS form in the tumor, respectively. When the fluorescence contrast value was 30 minutes after the introduction of CML 4.2 (more than 3.0), the time to reach the maximum intensity of CML fluorescence in the tumor was 4 hours. A PDT session was performed 4 hours after the administration of CML, resulting in complete regression of the tumor in the mouse (TPO values were 100% for 21 days of observation after PDT).

Example 3

Spectrophotometric examination was performed in an F1 mouse (C57Bl / 6j × CBA), a female, inoculated with S37 sarcoma. CML injected a mouse at a dose of 12.0 mg / kg.

The fluorescence contrast measured 30 minutes after the administration of the FS solution was 4.3, which allows us to determine the maximum level of PS accumulation in the tumor - 4 hours after the introduction of the FS.

Then, every hour, the intensity of normalized CML fluorescence in the tumor was recorded, which amounted to:

1 hour after the introduction of the photosensitizer 2.2 srvc. units

after 2 hours - 2.6 srvc. units

after 3 hours - 3.5 srvc. units

after 4 hours - 4.4 conv. units

after 5 hours - 3.3 srvc. units

after 6 hours - 3.0 conv. units

Thus, the maximum CML fluorescence intensity, corresponding to the maximum accumulation of PS in the tumor, was achieved 4 hours after the introduction of PS, which corresponded to a predetermined value of fluorescence contrast.

At 3 and 5 hours after the administration of PS, the fluorescence intensity in the tumor was 21% and 25% less than at the time of the maximum accumulation of the photoactive PS form in the tumor, respectively.

With the fluorescence contrast value 30 minutes after the introduction of CML 4.3 (more than 3.0), the time to reach the maximum intensity of CML fluorescence in the tumor was 4 hours.

However, the PDT session was conducted at a different time than previously identified, namely 3 hours after the introduction of CML, as a result of which the mouse achieved only partial tumor regression (TPO values were 83-91%) during 21 days of observation after PDT).

Thus, the proposed method has been successfully tested on a model of tumor-bearing mice and can be extrapolated to humans, taking into account the use of CML doses in animals for animal studies of the equivalent therapeutic dose by recalculation according to the method of Freireich at al. This invention is scientifically based and appropriate for practical use.

Claims (1)

The method for determining the time of the maximum concentration of the chlorin e6 photosensitizer lysine dimeglumine salt in the tumor, including the administration of FS chlorin e6 lysine dimeglumine salt, 30 minutes after the administration of FS, the tumor / normal fluorescence contrast value is calculated, with tumor / normal fluorescence contrast up to 3.0 inclusive The maximum level of PS accumulation in a tumor is considered to be 3 hours from the moment of PS administration, with a fluorescence contrast ratio tumor / norm of more than 3.0 - 4 hours.