Technical Field of the Invention

The present invention relates to the use of indium (III) phthalocyanine compound in photodynamic therapy for the treatment of cervical cancer.

State of the Art

Cancer is abnormal cell growth that is caused by multiple changes in gene expression, which is defined as a phenomenon involving nearly a hundred complex diseases exhibiting different behaviors depending on the cell type they originate from. It disrupts the balance between cell proliferation and cell death. It may spread to various regions of the body due to its ability of invasion and metastasis, and cause tissue and organs to lose their function in the region where they spread. Cancers vary by their age of onset, growth rate, spread, stage, and response to treatment. However, all cancer types have common characteristics at the molecular level which unite all cancer types in a single unit family. Cancer, if not treated, generally results in the death of people, and today, the second leading cause of death worldwide is cancer-related deaths. Death caused by cancer can occur as a result of the side effects of existing conventional treatments as well as the damage caused by the disease. Patients cannot be administered sufficient doses in order to avoid the fatal results which may be induced by the side effects of the conventional drugs and as a result, complete and effective treatment cannot be provided. The disease may relapse due to the fact that all cells in the tumor population cannot be killed even in cases where the disease is thought to be cured.

Today, three main treatment methods are implemented in cancer treatment, which include surgery, chemotherapy, and radiotherapy. Less frequently, hormonal therapies, biological therapies, and targeted therapies are also employed. Said treatment methods may be implemented individually or in combination. In the surgical method, cancerous tissue is removed from the body. The surgical method is the first method applied in many types of cancer. Radiotherapy is a cancer treatment method in which high-energy X-rays or other types of radiation are used in order to kill cancerous cells or prevent their growth. This treatment method is divided into two types as external radiation and internal radiation. In the external radiation method, X-rays are directed from a machine directly to the cancerous cell and surrounding tissues. In internal radiation, capsules that contain radioactive material are placed into the body cavity of a patient, on and around the tumor. Some patients experience side effects such as fatigue, skin rash, and nausea after radiotherapy. Chemotherapy is a cancer treatment method that is applied in order to prevent tumor cell growth by either killing cancerous cells or stopping their division. Chemotherapy, as another cancer treatment method, is performed in order to destroy cancer cells or to control the growth of said cells by means of using anticancer drugs. Chemotherapy can be administered to the patient in different ways. The drugs enter the bloodstream and may reach cancerous cells in the body (systemic chemotherapy) when chemotherapy is administered orally or injected into a vein or muscle. When chemotherapeutic drugs are placed directly in the cerebrospinal fluid, an organ, or a body cavity such as the abdomen, they mainly affect cancer cells in said areas (regional chemotherapy). The method of administration of chemotherapy depends on the type and stage of cancer that is being treated. Chemotherapeutic drugs stop cell division by inhibiting enzymes involved in DNA replication or metabolism. However, chemotherapeutic drugs do not specifically target tumor cells, but also damage normally dividing cells of rapidly regenerating tissues such as bone marrow, intestinal mucosa, and hair follicles. Therefore, chemotherapy brings many problems along. Cancer cells and rapidly dividing normal cells are difficult to distinguish from each other due to their similarity, and cells may also develop resistance to drugs. Every drug cannot be applied to every patient since every person and tumor is genetically different. For this reason, some cells treated with chemotherapy can survive and turn into drug-resistant cancer cells, and even treatment-related diseases may develop afterwards. In addition, many of the drugs that kill tumors can also cause mutations that turn normal cells into cancerous cells. Consequently, all of the aforementioned reasons necessitate developing new drugs that target cancer cells but do not affect normal cells.

Cervical cancer is one of the most common cancer types among women, in which 98% of said cancer is caused by HPV (Human Papillomavirus). It occurs as a result of the proliferation of cancer cells in the cervix that connects the uterus and the genital tract (vagina). Cervical cancer is a type of cancer that usually develops slowly. Cervical cells go through changes called dysplasia before cancer appears in the cervix. In cervical cancer, abnormal cells begin to appear in the cervical tissue and may become cancerous cells over time, and said abnormal cells begin growing and spreading more extensively in the cervix and surrounding areas. In some cases, the fact that being unable to provide a complete treatment for cervical cancer with the treatments available in the state of the art may result in a relapse of the disease just like in other types of cancer.

There are various treatment options for patients with have cervical cancer. As in other types of cancer, standard treatment options include surgery, radiotherapy, and chemotherapy. An early diagnosis is the most effective way of the treatment process. In the surgical treatment method, some part or all of the cancerous tissue in the cervix can be removed by means of administering general anesthesia to the patient. Chemotherapy or radiotherapy methods are applied to patients after surgical intervention if considered necessary. In chemotherapy, it is required to search for alternative short-term and effective treatment methods since drugs that are not related to cancer but used against cancer cause deaths due to side effects (heart attack, etc.) thereof, and cause reduced quality of life.

When compared to other treatment methods employed nowadays, the photodynamic therapy (PDT) method is a less harmful and effective cancer treatment method, which is performed in the existence of photosensitizer (photosensitive substance), light and oxygen molecules. Said photosensitizers are defined as chemical compounds that absorb the energy of light with a wavelength that suits their structure, that transform from the ground state to the excited state, and that transfer the energy they absorb into biomolecules. The first example of photodynamic therapy was recorded in 1900 when Raab has observed a toxic effect as a result of reacting the acridine orange with light. In addition, Raab’s study has shown that it is possible to kill paramecium, which is a unicellular animal species, by means of using acridine dye and sunlight. When the same experiment was repeated in an environment where there was no light, it was revealed that the dye has interacted with the light since no change was observed (Rasmussen-Taxdal, 1955). In 1905, photodynamic therapy was applied to malignant tumors in facial basal cells on 6 patients by T appeiner and Jesionek. In this study, 1 % eosin solution was used, and sunlight and arc lamps were utilized for a long period of time, and as a result of the studies conducted, and improvement was observed in tumors of four patients (Tappeiner, 1909). The first steps of modern photodynamic therapy were taken by Lipson et al. through their studies regarding hematoporphyrin derivative (HPD) in 1961 , and an important step was taken in photodynamic therapy when Parker et al. have achieved regression in experimental iris neovascularization using the hematoporphyrin derivative in 1984. In 1987, Dougherty et al. conducted research on 1 13 skin and subcutaneous tumors and as a result of these studies, partial or complete recovery was observed in 11 1 tumors. However, none of these studies were put into clinical practice. In PDT applications, a photosensitive substance called a photosensitizer, which does not have a toxic effect individually, is exposed to light. The resulting free radicals and singlet oxygen interact with many biological molecules and cause the death of cancerous cells through apoptosis (programmed cell death) or necrosis. In other words, PDT is based on a principle in which the drug accumulates in the tumor tissue and destroys the tumor by being stimulated with a light source of a certain wavelength after the photosensitive drug is administered to the patient through intravenous route. Said photosensitive drugs have a much higher tendency to accumulate and be retained in tumor tissues compared to normal tissues. Light may stay longer in tumor tissue compared to normal tissue. Singlet oxygen, which is released as a result of occurring reactions, is extremely toxic when the light is absorbed by the photosensitive drug. Singlet oxygen causes necrosis in the desired region only and although its life in the tissue is noticeably short, it is local. Thus, the cells in the tumor region are destroyed without damaging the healthy tissues.

In photodynamic therapy, light energy is converted into chemical energy by means of a non-toxic photosensitive substance, the photosensitizer, and said chemical energy is transferred to the target tissue. In photodynamic therapy, the administration of the photosensitizer into the body consists of two basic steps, including the production of a photochemical effect in the targeted area and the activation of said substance. Basically, there are two selectivity features. The first feature is that the photosensitizer, which is a light-sensitive substance administered to the body, is accumulated especially in the target region, and the other is that the light activating said substance is applied only to the desired region. The mechanism of action of photodynamic therapy occurs such that the photosensitive substance, which is in the ground state, becomes a triplet with high energy after being excited by a light source of a wavelength that it can absorb, and activated photosensitizers follow two different paths, which are Type I and Type II reactions, so as to interact with biomolecules. One of the three essential elements in photodynamic therapy is oxygen, and the importance of oxygen is stemming from its transition to singlet oxygen form. There are two unpaired electrons in the outer antibonding orbitals when oxygen is in the ground state. The singlet oxygen, which is formed as a result of changing the direction of one of said outer electrons, is highly reactive. Singlet oxygen interacts with other molecules and transfers its energy due to its high reactivity. The creation of singlet oxygen constitutes the basis of the PDT method.

Type I reactions create electron transfer reactions which lead to the formation of ROS (Reactive Oxygen Species). Type II reactions are energy transfer reactions that cause ( ¹ O2) formation. In Type I reactions, radicals with highly reactive properties appear when the hydrogen atom of the excited photosensitizer is transferred to a molecule in the cell and said radicals react with molecular oxygen. As a result, oxygenated products are formed. Several photosensitive substances used in photosensitive substances show their effects through Type II reaction mechanisms over ¹ O2, which is ROS most of the time. In Type II reactions, the excited photosensitizer gives its energy directly to molecular oxygen (O2) and induces the formation of singlet oxygen ( ¹ O2). Singlet oxygen ( ¹ O2), which is the electronically excited state of the molecular oxygen, oxidizes the biomolecules and causes damage in the cell. Type III reactions are immune complex related reactions. These reactions are also known as hypersensitivity reactions. Type III reaction occurs when the antibody against a soluble antigen binds with the antigen and activates the complex system. An immune complex is formed as a result of antigenantibody binding. The intensity of the reaction is associated with the size and distribution of the immune complex. Immune complexes are observed quite frequently in the blood vessel wall, the synovial membrane of the joints, and the choroidal plexus of the brain. Type III reactions may be local or systemic. Type IV reactions are known as cell-mediated or delayed-type hypersensitivity. The conventional example of these reactions is the tuberculin (Mantoux) reaction, which peaks 48 hours after antigen injection. Type IV reactions are associated with many autoimmune and infectious diseases, foreign antigens, and infection-induced granuloma pathogenesis.

The wavelength of the light used in photodynamic therapy is particularly important in terms of the depth that the light may reach in the tissue. However, the wavelength and the energy of the light used are inversely proportional. Therefore, the use of a light source with high wavelength should be preferred in order to minimize the side effects of the therapy that is being administered. It has been determined that the optimal wavelength range is between 650 and 850 nm for PDT applications, and said range is defined as 'Phototherapeutic window'. Arc lamps are utilized to stimulate photosensitizers since they are easy to use and inexpensive. However, UV and IR filters must be used in order to prevent heating since such lamps are light sources with a broad spectrum. Additionally, LEDs with a wide wavelength range may also be used as a light source in PDT. Another important light source is laser light that may be emitted in a single color and in synchronized light waves with little deviation, which facilitates focusing the light rays. Lasers have characteristic features such as the operation of optical fibers in photodynamic therapy, the treatment of endoscopic internal tumors, and the placement of a light source in tumor tissue with respect to their use in photodynamic therapy. Moreover, one of the reasons to use the laser light for the activation of the photosensitizer in PTD is that the laser light has high power.

The photosensitizer used in the region of the body, which is threatened and damaged by cancer cells may vary based on the application of the light and photosensitizer agent that activates it. The type of photosensitizer agent also varies according to the light source and the type of treatment applied. After the photosensitizer agent used in the treatment is received by cancer cells, the light with a wavelength, which can be absorbed by the agent, is applied only to the part with cancerous cells. Photodynamic therapy is an important treatment method for inducing damage to the blood vessels feeding cancer cells and enabling the immune system to attack cancer cells. The time period between the administration of the photosensitizer agent and the application of light to the patient varies depending on the photosensitizer agent used. Photosensitizer to be used is determined based on characteristics such as; accumulation in tumor tissues, accumulation in the desired region in a short period of time, not containing any impurities, not getting accumulating in healthy tissues, not having any toxic effects in case there is no stimulating light, and tending to form singlet oxygen with high efficiency.

Phthalocyanines structures of which do not deteriorate when exposed to heat, light, acids, and bases, were discovered by Braun and Tchemiac in 1907 in a study conducted for obtaining o-cyanobenzamide from phthalimide and acetic acid, however, it was the study conducted by Linstead and Robertson which shed light upon the structures thereof between the years of 1933-1940. Phthalocyanines are a class of compounds consisting of a 16-membered planar macro ring having a highly conjugated 18 electron system. Phthalocyanines are not found in nature although they are structurally similar to porphyrins such as hemoglobin, chlorophyll a, and B12. Today, phthalocyanines with the characteristics of blue and green colors are used in applications such as lasers, sensors, nanotubes, self-emitting compounds (OLED), photodynamic therapy (PDT), cancer treatment, and catalysts, in addition to their use as pigments in printing inks, coatings, paints, and plastics.

The reasons such as insufficient treatment methods used in cervical cancer treatment in the state of the art, side effects caused by chemotherapeutic drugs, deaths stemming from side effects (heart attack, etc.) necessitated developing a drug for use in the treatment of cervical cancer.

Brief Description and Objects of the Invention

The present invention discloses the use of indium (III) phthalocyanine compound in photodynamic therapy for the treatment of cervical cancer. In the present invention, 335ln(lll)Pc compound was applied to HeLa cells originating from human cervical carcinoma by means of employing the PDT method, and it was demonstrated that the cell viability decreased depending on the dose as a result of the proliferation rate analysis and that 100 pM of product concentration cause the survival of 47.45 % of the cells, in other words, the death of 52.55 % of the cells. An evaluation of cell index parameters demonstrated that different concentrations both show antiproliferative effects on cells and lead to cell death through various mechanisms. It has been demonstrated that the cells undergo mitosis division decreased significantly depending on time by means of the mitosis index parameter. As such, it has been found that it leads to the death of cancer cells by using various mechanisms in different concentrations.

The most important object of the present invention is to ensure that cervical cancer is treated effectively. In the present invention, more than half of HeLa cancer cells are killed by means of the indium (III) phthalocyanine compound, which is the active ingredient that is being synthesized.

Another object of the present invention is to produce a drug with an anticarcinogenic effect for use in cervical cancer treatment by means of the photodynamic therapy method. A drug that contains indium (III) phthalocyanine compound ensures that the cancerous cells causing cervical cancer are killed and cancer is treated. The IC50 concentration of indium (III) phthalocyanine compound applied to HeLa cells, which is a cervical cancer strain, was determined as 100 pM.

Another object of the present invention is to obtain an anticancer drug by means of an eco-friendly synthesis method. In the present invention, no solvent is used in the synthesis of indium (III) phthalocyanine compound, which is the active ingredient of the drug synthesized for use in cervical cancer treatment. Thus, it is eco-friendly within the scope of green synthesis.

Yet another object of the present invention is to obtain an active ingredient that has anticancer effects and high singlet oxygen production for being used in photodynamic therapy for the treatment of cervical cancer. The use of indium (III) phthalocyanine compound in photodynamic therapy produces exceedingly high amounts of singlet oxygen.

Description of the Figures

FIGURE 1 illustrates the chemical structures of 3-(3,5-di-tert-butylphenoxy) phthalonitrile (Compound 1 ) and 11 ,8(1 1 ), 15(18),22(25)-tetra-(3,5-di- tert-butylphenoxy) ln(lll)phthalocyanine (Compound 2) compounds.

FIGURE 2 illustrates the FT-IR spectrum of Compound 1 . FIGURE 3 illustrates the FT-IR spectrum of Compound 2.

FIGURE 4 illustrates the NMR spectrum of Compound 2.

FIGURE 5 illustrates the mass spectrum of Compound 2.

FIGURE 6 illustrates the fluorescence spectrum of Compound 2. FIGURE 7 illustrates the absorbance chart of Compound 2 under the light.

FIGURE 8 illustrates the photodegradation spectrum of Compound 2 under the light.

Detailed Description of the Invention

The present invention relates to a 1 ,8(11 ), 15(18),22(25)-tetra-(3,5-di-tert- butylphenoxy) indium (III) phthalocyanine (Compound 2, ln(lll)Pc or 335ln(lll)Pc) compound in order to be applied with photodynamic therapy for use in the treatment of cervical cancer. The present invention further relates to an anticarcinogenic drug in which the 1 ,8(1 1 ),15(18),22(25)-tetra-(3,5-di-tert-butylphenoxy) indium (III) phthalocyanine compound is used as an active ingredient. The chemical structure of Compound 2 is as shown in Formula 1 :

Formula 1 Synthesis method of said indium (III) phthalocyanine compound comprises the process steps of;

Powdering 0,508 g of 3-(3,5-di-tert-butylphenoxy) phthalonitrile compound (Compound I), and 0,101 g of indium (III) acetate inside a mortar,

Filling the powdered mixture into the reaction tube,

Adding ten drops of 1 ,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) to the reaction tube and passing through Argon gas for 5 minutes,

Heating it in a fume cupboard at 360°C after the reaction vessel is tightly closed,

Continuing the heating for 8 more minutes after the color turns green,

Checking its purity by means of the thin-layer chromatography (TLC) after cooling it to room temperature, and purifying the green product obtained as a result by means of column chromatography with chloroform,

Drying the purified 1 ,8(11 ),15(18),22(25)-tetra-(3,5-di-tert-butylphenoxy) indium (III) phthalocyanine (Compound 2) by using phosphorus pentoxide (P2O5) in vacuum for one week.

The compound obtained by means of the aforementioned method is in the form of a dark green crystal, and the yield thereof is 82 mg (16,14%). The closed formula of Compound 2, which has a melting point >300°C is CsgHgglnNsOe, and its molecular weight is 1491 ,61 g/mol. It has been determined that C, 71.66; H, 6.69; 7.70; N, 7.51 ; O, 6.44 when the theoretical elemental analysis of the compound was performed. FTIR analysis was conducted in order to shed light upon the structure of said Compound 2 and it is illustrated in Figure 3. FTIR results have indicated that the Compound 2 structure was synthesized correctly. Said compound is capable of getting dissolved in common solvents such as chloroform (CHCI3), acetone (C3H6O), tetrahydrofuran (THF), ethanol (C2H5OH), methanol (CH3OH), dimethylformamide (DMF), or dimethyl sulfoxide (DMSO) (Table 1 ). Table 1. The solubility data of the In(lll) Pc compound.

Equation-1 was used in order to calculate the fluorescence quantum yield (<t>F) of Compound 2. Hence, approximately 10-6 molar solution of Compound 2 complex and standard Zinc (II) phthalocyanine (ZnPc) in dimethyl sulfoxide (DMSO) was prepared, 639 nm was chosen as the most suitable excitation wavelength in order to form the fluorescence spectrum. Thus, emission and excitation spectra were obtained. Equation-1 is as follows: wherein, <t>F; the fluorescence quantum yield of the sample,

<t> F(std) ; the fluorescence quantum yield of the standard compound,

F; the area under the fluorescence emission curve of the sample,

Fstd; the area under the fluorescence emission curve of the standard compound,

A; the absorbance of sample, Astd; the absorbance of the standard compound, q; the refractive index of the solvent in which the sample is dissolved, r|std; the refractive index of the solvent in which the standard compound is dissolved.

Singlet oxygen quantum yields of the inventive Compound 2 were calculated by means of Zinc (II) phthalocyanine (ZnPc) standard and 1 ,3-diphenylisobenzofuran (DPBF) singlet oxygen quencher at room conditions via using Equation-2 below: wherein,

<t> ^std A; the singlet oxygen yield (ZnPc <t>A = 0,53 in THF)

<T>A; for example, singlet oxygen yield,

RDPBF; the degradation rate of DPBF in the presence of the sample,

RDPBFstd ; the degradation rate of DPBF in the presence of the standard, labs; the light absorption rate of sample and standard, labstd ; the light absorption rate of the standard.

DPBF with a concentration of 3 x10 ⁵ mol.L ¹ was used in order to prevent chain reactions caused by the presence of singlet oxygen. Solutions of a sensitizer containing DPBF (absorbance at irradiation wavelength = 2.0) were prepared in a dark environment and irradiated in the Q band area by means of using the setup described above. This yield was found as 49%, according to Figure 7. The maximum absorbance of the Q band in the solutions of all complexes was set to 1 .0 in the solvent used in order to determine the photodegradation quantum yield (<t>d), and the decrease in the intensity of the Q band under irradiation with time is observed by means of using the following Equation-3: wherein,

Co (mol. dm ^-3 ); concentration of Compound 2 before irradiation,

Ct (mol. dm ^-3 ); concentration of Compound 2 before irradiation,

V; reaction volume (3.0 cm ³ ),

S; irradiated cell area, t; irradiation time,

NA; Avogadro's number, labs; the overlapping integration of the radiation source intensity. Accordingly, the photodegradation quantum yield was found as (<t>d) = 2,0017x10 ⁰⁵ , and this result is an acceptable value for phthalocyanine compounds and indicates that the In(lll) Pc complex is stable under the light. It has been observed that Compound 2 is active in destroying cancer cells, and as such may have a potential for the treatment of cancer with PDT when considering the PDT characteristic measurement results described above.

Different concentrations of In (III) Pc complex were applied to cultured human cervical cancer originated HeLa cells for 24 hours in order to determine the changes in mitochondrial dehydrogenase enzyme activity of cancer cells by means of the ln(lll)Pc complex obtained. For proliferation rate analysis, routinely cultured HeLa cells were cultivated in culture plates with 96 wells such that the growth medium is 3x10 ⁴ cell/ml per well. ln(lll)Pc complex prepared at 50 pM, 100 pM, and 150 pM concentrations were applied to the cells after waiting for the cells to adhere and spread. The growth medium in the wells was withdrawn and 40 pl of 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5mg/ml) was added at the end of the experiment periods. 160 pl of dimethyl sulfoxide (DMSO) was added to the wells with MTT and incubated for 1 hour in a shaker incubator after waiting for approximately 4 hours. The absorbance values of the formed experimental groups were carried out by means of the spectrophotometer measurements at 570 nm with reference to the 690 nm wavelength after the formazan crystals formed are dissolved for the 1 hour incubation period. The absorbance values obtained as a result of this application were measured as 590x10 ^-3 for the control group; 460 x10 ^{_ 3} for 50 pM; 280 x10 ^-3 for 100 pM, and 170 x10 ^-3 for 150 pM. According to the control group, which is considered 100% for HeLa cells, it was observed that 50 pM of Compound 2 concentration reduced the viability of cells to 78% that 100 pM of Compound 2 concentration increases the viability of cells to 47.45%, and 150 pM of Compound 2 concentration reduced the viability of cells to 28.81 %.

In the present invention, the cell index parameter was evaluated by means of using a real-time cell analysis system in order to both support the results obtained and to determine the types of cell death, and an E-Plate with 16 wells was used in order to evaluate this parameter. Primarily, 100 pl of growth medium was added to each well of the E-Plate, and background measurements were taken in the cell analyzer device. Then, cultivation is performed in each well such that the 100 pl of growth medium has 6000 cells for HeLa cells. The E-Plates were incubated for 20 minutes in a sterile working cabinet at room temperature, and then placed in the cell analyzer device and incubation was continued at 37°C under ambient conditions of 5% carbon dioxide (CO2) after the cultivation process. The cell analyzer was issued a command to take the measurement every 15 minutes after the cell cultivation to the E-Plates was performed. After approximately 24 hours of cell cultivation, the growth medium in the E-Plates was changed with a growth medium containing different concentrations of ln(lll)Pc complex at the 1 /3rd slice of the proliferation phase of the cells, and measurements were continued at 15-minute intervals for 72 hours. Subsequently, graphics of concentration and time-dependent cell index values were transferred to the computer screen. The antiproliferative effects of the ln(lll)Pc complex on HeLa cells are observed when the cell index values obtained from the real-time cell analysis system are examined. The 50 pM of ln(lll)Pc complex applied does not show a significant effect when the cell index values obtained are compared with the standard curves, however, 150 pM of ln(lll)Pc complex concentration produces a cytoskeletal effect, while 100 pM of ln(lll)Pc complex concentration, which is an optimum concentration, is causing DNA damage on cells. As a result of applying the optimum concentration of the product(100 pM) on HeLa cells for 0-72 hours, the preparations prepared according to the experimental groups were primarily hydrolyzed with 1 N hydrochloric acid (HCI) at room temperature for 1 minute and then with 1 N HCI at 60°C for 10 minutes in order to determine the effects on the mitotic index values of the cells. After hydrolysis, the Feulgen method was applied to the preparations for 1 hour. Then, these preparations were washed 3 times for 2 minutes with a washing solution, which is specific to the Feulgen method, and air-dried. After the preparations were dried, they were prepared for counting by means of staining with Giemsa dye for 2 minutes. Late prophase, metaphase, anaphase, and telophase stages were counted in order to determine the mitosis index (Ml) values of said preparations. The early prophase stage was evaluated together with this stage, as it was morphologically similar to the cells in the interphase group.

Three preparations were evaluated for each experimental group and mitosis index values were determined by means of counting 3000 cells on average from each preparation. The mitosis index values have been decreased from 6% to 3,7% at the 24th hour; from 6,68% to 2,21 % at the 48th hour, and from 7,56% to 1 ,19% at the 72nd hour.

The values of cell kinetic parameters determined according to concentration and time applied in all experimental groups were evaluated according to control and each other, and to that end, a one-way ANOVA test was applied to the values determined from all experimental groups. The significances of the groups according to the control were evaluated with the DUNNETT'S test, and the significances of the groups with each other were evaluated with the t-test. A significance level of p<0.01 was selected as a basis in statistical evaluations. The anticancer effects of Compound 2 in HeLa cell cultures originated from human cervical cancer were investigated by means of evaluating various cell kinetic parameters. The data obtained has indicated that Compound 2 caused a significant decrease in cell proliferation depending on the concentration and time, and at the same time, cell death has occurred with different death mechanisms at different concentrations. 100 pM of Compound 2 concentration had a DNA damaging effect on cells while 150 pM of Compound 2 concentration had a cytoskeletal effect. In addition, the mitosis index parameter using 100 pM of Compound 2 concentration, which is the optimum concentration, indicates that mitosis cell division decreases significantly over time. Consequently, the significant death of human cervical cancer cells via different mechanisms indicates that the inventive compound may be a drug that can be used in the treatment of this cancer.

In the second stage, after administering the 335ln(ll) Pc substance to the body, only the cancerous region is exposed to red light, and therefore, the treatment of cervical cancer is provided effectively.

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