WO2008082200A1

WO2008082200A1 - Antitumoric compositions for oral administration comprising chlorin compounds

Info

Publication number: WO2008082200A1
Application number: PCT/KR2007/006964
Authority: WO
Inventors: Yong-Chul Kim; Zee-Yong Park; Hyo-Jun Kim
Original assignee: Gwangju Institute Of Science And Technology
Priority date: 2006-12-28
Filing date: 2007-12-28
Publication date: 2008-07-10
Also published as: KR100808630B1

Abstract

The present invention relates to an antitumoric composition for oral administration comprising a chlorin compound, or its pharmaceutically acceptable salt as an active ingredient, wherein the chlorin compound or its pharmaceutically acceptable salt exhibits the antitumoric activity without light irradiation. This invention suggests a novel use and application of the chlorin compound, which is conventionally used in a photodynamic therapy for cancer as photosensitive substances. Thus, according to this invention, the chlorin compound is orally administrated, and may have antitumoric effects without light irradiation. The composition of this invention is orally administrated, well absorbed in human through gastro-intestinal tract, and showed an excellent antitumoric effect. The composition of this invention does not have a cytotoxicity and an immunetoxicity, so that the composition has a higher safety to human.

Description

ANTITUMORIC COMPOSITIONS FOR ORAL ADMINISTRATION COMPRISING

CHLORIN COMPOUNDS

FIELD OF THE INVENTION The present invention relates to antitumoric compositions comprising chlorin compounds as an active ingredient.

DESCRIPTION OF THE RELATED ART

The risk groups of cancer diseases could be generally classified into two representative groups. One group of risk groups is constantly and continuously exposed to external and internal environment factors, and the other group of risk groups includes individuals suffering from cancer diseases who are in the stage of remission.

In the former group of risk groups, the external environment factors include ecological factors, for example, the atmosphere and water from chemical industry, vehicles exhausts, radiation background and foodstuffs containing synthetic preservatives.

The internal environment factors include chronic metabolic disorders that cause the generation of endogen products hostile to the main functional systems {e.g., cell structures) of the human body.

The cancer development in the first risk group is greatly affected by impairments of redox processes in the human body, activating excess generation of free radicals which degenerates normal cells and induces tumorigenesis.

Meanwhile, the second risk group is very susceptible to cancer recurrence.

Throughout this application, several patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications is incorporated into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THIS INVENTION

For developing antitumoric compositions with safety to human, particularly compositions showing excellent applicability to healthy food, the present inventors have made intensive studies for various substances with safety and antitumoric activities. As a result, the inventors have found that chlorin compounds (in particular, chlorin e6) conventionally used in cancer therapy as photosensitive substances, showed excellent antitumoric activities and safety to human with no help of irradiation. Accordingly, it is an object of this invention to provide an antitumoric composition for oral administration comprising a chlorin compound as an active ingredient.

It is another object of this invention to provide a method for treating tumors. It is still another object of this invention to provide a use of a composition comprising a chlorin compound or its pharmaceutically acceptable salt for manufacturing a composition for tumor treatment.

Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.

In one aspect of the present invention, there is provided an antitumoric composition for oral administration comprising a chlorin compound, or its pharmaceutically acceptable salt as an active ingredient, wherein the chlorin compound or its pharmaceutically acceptable salt exhibits the antitumoric activity without light irradiation.

In another aspect of the present invention, there is provided a method for treating tumors, which comprises orally administering a patient an antitumoric composition comprising (a) a therapeutically effective dose of a chlorin compound, or its pharmaceutically acceptable salt; and (b) a pharmaceutically acceptable carrier, wherein the composition exhibits the antitumoric activity without light irradiation.

In still another aspect of the present invention, there is provided as a use of a composition comprising a chlorin compound or its pharmaceutically acceptable salt for manufacturing a composition for tumor treatment, wherein the chlorin compound or its pharmaceutically acceptable salt exhibits the antitumoric activity without light irradiation.

For developing antitumoric compositions with safety to human, particularly compositions showing excellent applicability to healthy food, the present inventors have made intensive studies for various substances with safety and antitumoric activities. As a result, the inventors have found that chlorin compounds (in particular, chlorin e6) conventionally used in cancer therapy as photosensitive substances, showed excellent antitumoric activities and safety to human with no help of irradiation. An ingredient used as an active ingredient in the composition of this invention is chlorin compounds. The term "chlorin" or "chlorin compound" as used herein, refers to a heterocyclic aromatic ring consisting of 3 pyrroles at the core, and one reduced pyrrole coupled through 4 methine linkages. In general, the chlorin compound has a structure represented by the following formula 1. In addition, the chlorin compound formed being modified chemically the following formula 1 is comprised in the scope of this invention.

(1) Since chlorin exhibits a photosensitivity generally, it is used in a photodynamic therapy of cancer. By conventional use of a photosensitivity of chlorin, chlorin was administrated (generally, injection) in body, and then light was irradiated, thereby treating cancer.

The most striking feature or discovery of this invention is based on the finding in which chlorin compounds administrated via oral route rather than injections are absorbed in a living body through gastro-intestinal tract and the absorbed chlorin exhibits an antitumoric effect without light irradiation.

Comparing to the use and application of the conventional chlorin photodynamic therapy, the feature or discovery of this invention is very interesting.

The chlorin compounds used in this invention may include various chlorin compounds known to one of skill in the art. Preferably, the chlorin compound used in this invention is chlorin e6, sulfonated meso-tetraphenyl chlorin or β, β'-dihydroxy meso-substituted chlorin.

Chlorin e6 is represented by the following formula 2:

Chlorin e6 used in the present invention includes chlorin e6 substituted on a functional group (i.e. -COOH, etc) of the formula 1 as well as the structure of the formula 2. Preferably, the compound of the formula 2 which is not substituted with substituents is chlorin e6 used in this invention. Sulfonated meso-tetraphenyl chlorin was known as a photosensitive chlorin compound to one skilled in the art, details are disclosed in EP 1 420 824, which is incorporated herein by reference.

Sulfonated meso-tetraphenyl chlorin capable of being used in this invention, was represented by the following formula 3:

wherein X represents -SO₃H; and n, p, q and r independently is an integer of o or 1.

β, β'-Dihydroxy meso-substituted chlorin was known as a photosensitive chlorin compound to one skilled in the art, details are disclosed in US 5,648,485, which is incorporated herein by reference. β, β'-Dihydroxy meso-substituted chlorin capable of being used in this invention, was represented by the following formula 4:

wherein A represents one of the following 3 pyrroles

and D represents one of the following 3 pyrroles

According to the most preferred embodiment, the chlorin compound comprised in the antitumoric composition is chlorin e6.

Chlorin e6 used in this invention may be synthesized by a variety of chemical methods well-known to one skilled in the art, preferably prepared by chemically modifying chlorophyll-a obtained from chlorella.

The present inventors have already developed methods for preparing chlorin e6 in much higher efficiency, which have been filed for patent applications. The method for preparing chlorin e6 from chlorophyll-a of chlorella developed by the present inventors comprise the steps of: (a) treating chlorella with 30-68% ethanol to remove other ingredients than chlorophyll-a from the chlorella; (b) treating the chlorella with 80-100% ethanol to extract chlorophyll-a, thereby obtaining a chlorophyll-a extract; (c) adding an acid to the chlorophyll-a extract to eliminate Mg²⁺ from chlorophyll-a, thereby obtaining pheophytin a; and (d) adding a base to pheophytin a to obtain chlorin e6.

According to the method of the present invention, the yield of chlorin e6 from chlorella is 8.2-8.4%, which is higher than other conventional methods.

Chlorin used in this invention may be in a type of a pharmaceutically acceptable salt. The pharmaceutically acceptable salt in this invention may be prepared by conventional methods. For example, the pharmaceutically acceptable salt may be prepared by use of inorganic acids or organic acids. The inorganic acids may include hydrochloric acid, hydrobromic acid, sulfuric acid, sodium hydrogen sulfate, phosphoric acid and carbonic acid. As the organic acid, formic acid, acetic acid, oxalic acid, benzoic acid, citric acid, tartaric acid, gluconic acid, gestisic acid, fumaric acid, lactobionic acid, salicylic acid, or acetylsalicylic acid (aspirin) may be used. In addition, metal salt may be formed by reacting with alkali metal ions such as sodium and potassium. Another type of the pharmaceutically acceptable salt may be prepared by reacting with ammonium ion.

Most preferably, the pharmaceutically acceptable salt of chlorin used in this invention is a sodium salt. Where chlorin e6 is used as chlorin compounds, chlorin e6 tri sodium salt is the most preferable. According to a preferred embodiment, the composition of this invention further comprises at least one ingredient selected from a group consisting of β-sitosterol, glycyrrhizic acid, lentinan, reishidin, letiporin, acacetin, zinc, selenium and amino acid.

Among of the additional ingredients, preferably, β-sitosterol, glycyrrhizic acid, lentinan, reishidin, letiporin, acacetin and amino acid are obtained from nettle, licorice, lentinus edodes, ganoderma lucidum, Latiporus sulphereus , agastache rugosa and yeast, respectively.

According to a preferred embodiment, the composition of this invention exhibits little or no immunotoxicity and also improves an activity of macrophage. The composition of the present invention may be prepared as a food and a pharmaceutical composition.

The antitumoric composition of this invention may be prepared as a food, in particular a functional food composition. The functional food composition of this invention may comprise conventional additives for preparing food compositions, e.g., protein, carbohydrates, lipids, nutritive substances and flavors. For example, where the food composition of this invention is provided as a drink, it may further comprise flavors and natural carbohydrates as well as chlorin compound as an active ingredient. Non-limiting examples of natural carbohydrates include, but not limited to, monosaccharide {e.g., glucose and fructose); disaccharide (e.g., maltose and sucrose); oligosaccharide; polysaccharide {e.g., dextrin and cyclodextrin); and sugar alcohol (e.g., xylitol, sorbitol and erithritol). Non-limiting examples of Flavors include, but not limited to, natural flavors {e.g., thaumatin and extract of stevia) and synthetic flavors {e.g., saccharin and aspartame). Considering availability to food, the food composition of this invention is very useful in preventing and treating cancer.

The antitumoric composition of this invention may be prepared as a pharmaceutical composition, and the pharmaceutically acceptable carrier as well as the active ingredient contained in the pharmaceutical composition. The pharmaceutically acceptable carrier, which is commonly used in pharmaceutical formulations, but is not limited to, includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils. The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995).

A pharmaceutical composition of this invention is orally administered and exerts antitumoric efficacies in human body.

A suitable dosage amount of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, pathogenic state, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition, and physicians of ordinary skill in the art can determine an effective amount of the pharmaceutical composition for desired treatment. According to a preferred embodiment of this invention, suitable dosage unit is to administer once a day with 0.001-200 mg/kg (body weight).

According to the conventional techniques known to those skilled in the art, the pharmaceutical composition of the present invention may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms a unit dose form and a multi-dose form. Non-limiting examples of the formulations include, but not limited to, a solution, a suspension or an emulsion in oil or aqueous medium, an extract, an elixir, a powder, a granule, a tablet and a capsule, and may further comprise a dispersion agent or a stabilizer.

The cancers or tumors prevented and treated by the present pharmaceutical composition include stomach cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchogenic cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer and ureter cancer, but not limited to.

The summary of features and advantages of this invention is as follows:

(i) This invention suggests a novel use and application of a chlorin compound, which is conventionally used in a photodynamic therapy for cancer as photosensitive substances.

(ii) Thus, according to this invention, the chlorin compound is orally administrated, and may have antitumoric effects without light irradiation.

(iii) The composition of this invention is orally administrated, well absorbed in human through gastro-intestinal tract, and showed an excellent antitumoric effect.

(iv) The composition of this invention have no cytotoxicity and immunetoxicity, so that the composition has a higher safety to human.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. Ia shows the result analyzing ingredients contained in seawater chlorella using HPLC (high pressure liquid chromatography).

Fig. Ib represents the HPLC analysis result removing other ingredients except chlorophyll-a by washing chlorella with 60% ethanol. The HPLC analysis is results concerning supernatants formed by washing. Fig. Ic shows the HPLC analysis result extracting chlorophyll-a with 100% ethanol.

Fig. 2 is the result analyzing the molecular weight of chlorin e6 prepared finally according to the method of this invention.

Figs. 3 and 4 are a graph showing the analysis result of chlorin e6 pharmadynamics in animals. Chlorin e6 was orally administrated to rats, and measured the concentration of chlorin e6 in blood serum at time interval.

Fig. 5 is a graph showing antitumoric effects of the present composition in animals level. The composition of this invention was administrated to rats transplanted tumor cells, and size of transplanted tumor tissues was estimated at time interval.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples. EXAMPLES Example 1: Preparation of chlorophyll-a from chlorella

Example 1-1: Elimination of other ingredients except chlorophyll-a from chlorella First, to remove salt in seawater Chlorella purchased from Chlorland co, Ltd, the

Chlorella was washed with distilled water and then precipitated centrifuging at 5000 rpm. The precipitate was resuspended in distilled water and recentrifuged. After washing five times with distilled water and then taken a small amount, the contents of chlorophyll-a and other ingredients were analyzed using HPLC (high pressure liquid chromatography). HPLC was performed using HPLC system (Dong-il Shimadzu Corp.) having SPD-MlOAVP column, and setting up 20 μl input volume, 1 ml/min flow velocity. An eluant is a mixture containing methanol (67.5), dichloromethane (22.5), acetonitrile (9.5) and water (0.5). Chlorophyll-a commercially available from Fluka was used as a reference. HPLC results were shown in Fig. Ia. As shown in Fig. Ia, the pick of chlorophyll-a was observed at about a retention time of 9.7 min and picks of other ingredients at retention times of 2.5-7.5 min.

100 g chlorella removed of seawater (intact chlorella cells not lysated) were washed with 300 ml of 60% ethanol and then precipitated by centrifugation at 5000 rpm, thereby eliminating other ingredients at retention times of 2.5-7.5 min. The washing procedures using ethanol were conducted six times. Fig. Ib is the HPLC results for the supernatant produced by centrifuging a product of the final six-run ethanol washing.

As shown in Fig. Ib, it was found that other ingredients shown at 2.5-7.5 min retention time were abundantly removed by washing procedures using 60% ethanol. Where the washing procedure was performed using ethanol of more than 70% concentrations, chlorophyll-a was extracted and other ingredients could not be eliminated. Where the washing procedure was carried out using ethanol solutions of 20%, 40% and 50% concentrations, the removal of other ingredients became poor compared with 60% ethanol.

These results demonstrated that 60% ethanol is the most preferable washing solution. Namely, 60% ethanol as washing solutions effectively extracts other ingredients from chlorella cells except chlorophyll-a, leading to relatively higher content of chlorophyll-a in chlorella cells washed.

Example 1-2: Extraction I of chlorophyll-a from chlorella removed of other ingredients Following the addition of 1 L of 100% ethanol to the 100 g chlorella eliminated of other ingredients, chlorophyll-a was extracted by stirring for 3 hr. Chlorella cells used for extraction were not lysated by sonication and their undisrupted form itself was used. Although chlorphyll-a is generally extracted by treating cell lysates with organic solvents, the present invention uses chlorella cells per se rather than their lysates. The extraction results were shown in Fig. Ic.

As represented in Fig. Ic, the pick of chlorophyll-a was observed at about a retention time of 8.4 min and picks of other ingredients abundantly removed at a retention times of 2.5-7.5 min. Furthermore, it was found that contents of chlorophyll- a are 76.92% in chromatogram of HPLC.

Example 2: Preparation chlorin e6 from chlorophyll-a The mixtures were adjusted to pH 2-3 by adding 2-5 ml of 1 N HCI per 1 L of the ethanol extract comprising chlorophyll-a obtained in Example 1, and then Mg²⁺ was eliminated from chlorophyll-a by stirring for 3 hr, thereby obtaining pheophytin a. Then, 1 L of the pheophytin a solution with black color was neutralized by adding 2-5 ml of 1 N NaOH, followed by filtration. Next, the filtered solution comprising the pheophytin a solution was adjusted to pH 11-12 by adding 2-5 ml of 1 N NaOH and then stirring for 12 hr for producing chlorin e6 from pheophytin a. The resultants were neutralized with 1 N HCI and then filtered, followed by eliminating completely ethanol, finally giving 8.16 g of chlorin e6 from 100 g of chlorella (yield: 8.2 %).

To analyze the finally obtained chlorin e6, the molecular weight (MW) of chlorin e6 was measured using LCQ DecaXP Plus, Thermo Finnigan. As shown in Fig. 2, the measured MW is 597.4 which is consistent with a theoretical MW, 596.97 (in the measurement of MW, the analyzer recognizes +1 MW, so that the measured MW is accurately consistent with a theoretical MW). Accordingly, it was found that chlorin e6 was prepared in high yield by the method of the present invention.

In addition, to prepare the salt form of chlorin e6, 3 equivalent of NaHCO₃ were dissolved in ice water, added to chlorin e6, and then water was filtered out to yield the salt form of chlorin e6 by freeze-drying.

Example 3: Preparation of compositions for preventing and treating cancer comprising chlorin e6 as a main ingredient

Compositions for preventing and treating cancer comprising chlorin e6 as a main ingredient were prepared as formulation described in the following Tables 1 and

2. The following Table 1 is a syrup composition in the total volume of 100 ml, and

Table 2 is a capsule composition in the total weight of 400 mg. Particularly, the compositions of Tables 1 and 2 are suitable as a food composition.

Table 1 Syrup composition

Example 4: Experiment of acute toxicity

The analysis of acute toxicity for the composition of Table 1 was performed. Wistar rats (195 ± 5 g) and mice were used, and total 12 animals (6 male and 6 female) were used for each experimental group. The sample was administrated to rats and mice as a single oral dose at the following volume: 6.0 ml for rats and 0.8 ml for mice. As a result, doses of administrated sample were 30 ml/kg for rats and 40 ml/kg for mice. To the control animals, distilled water was administrated in the same volumes. Observations over experimental animals were continued for 14 days after the sample administration. The experiment results were summarized in the Table 1.

Table 3

Study of acute toxicity

As represented in Table 3, the present composition has low toxicity and death of animals was not observed in any group. In addition, the constant survey over the animals during the experiment did not detect any changes in the behavior compared to the control. After completion of administration, internal organs of animals were examined, but it was not to find out any visible changes of organs compared to the control.

Example 5: Experiment of subchronic toxicity

The experiment of subchronic toxicity was performed using wistar rats (195 ± 5 g), 5 animals. The sample was administrated to rats daily for 60 days in dose of 26.3 ml/kg. To the control animals, a sugar syrup or distilled water was administrated. The systematic survey for the experimental animals was carried out during the whole period of the experiment. Parameters examination for the experimental animals was conducted on the 30-th, 60-th days and recovery period (30 days after completion of experiment), respectively after administration.

Body weight changes of experimental animals, weight changes of specific organs, a number of biochemical and hematological parameters were considered as integral indices showing general condition of experimental animals. Urea, creatinine, glucose, whole protein and activity of enzymes were estimated using Cormay kits (Poland). The concentration of hemoglobin in blood was measured by hemiglobincyanide method. The number of erythrocytes and leukocytes, and erythrocyte sedimentation rate were estimated according to standard procedures. The experiment results were summarized in the Tables 4-13.

Table 4 After oral administration, body weight changes of rats (g)

Agent, dose ienderNumber Terms of survey (ml/kg) of

Original data 30 days animals

175.0± 191.5±

Control, Male 5.56 5.50

Distilled water

173.5± 189.3±

(26.3) Female 7.60 6.64

171.2± 210.2-t

Control, Male 8.85 5.02^*

Sugar syrup

169.8± 211.8±

(26.3) ^:emale 5.14 6.54"

170.9± 210.5±

Composition of Male 6.34 5.23^** this invention

172.6± 208.4*

(26.3) Female 5.47 3.75"

Note: " significant difference with the control at p<0.05 *significant difference with the control at p<0.01

Table 5

Weighting coefficient of rats organs (%) at oral administration (30 days of administration)

Organs

Liver Kidneys

4.22± 0.7± CK 12 0.03

3.80± 0.72± JλO7_ 0.02

3.80± 0.75± 0.2 0.04

3.60± 0.67± 0.1 0.04

3.76± 0.67± 0.07 0.03

3.44± 0.65± 0.10 0.02

Table 6 Weighting coefficient of rats organs (%) at oral administration (60 days of administration)

Table 7

Weighting coefficient of rats organs (%) at oral administration (Recovery period)

Table 8a

Study of hematological indices in rats at oral administration (30days of administration)

Table 8b

Agent, dose Leukocytic blood formula

Gender (ml/kg) stab.% segm.% eos.%

Control, Male 2.0±0.57 25.8±0.95 2.440.57

Distilled water

(26.3) Female 0.8±0.19 26.6±3.45 4.6i 1 72

Control, Male 2.4±0.57 30.0±2.87 7.Oi 1.34

Sugar syrup

(26.3) Female 2.2±0.38 25.6±4.02 2.0:1 0 38

Composition of Male 1.8±0.38 1.6±5.93 3.4±1.15 this invention

(26.3) Female 0.6±0.0 25.6±4.8 3.4±1.15

Table 9a

Study of hematological indices in rats at oral administration (60days administration)

Table 10a

Study of hematological indices in rats at oral administration (recovery period)

7.60±0.69 0.58±0.02 14.0±0.96

7.84±0.34 0.58±0.01 10.8±0.96

Table 10b

Agent, dose /tic blood formula lender (ml/kg) stab.% segm.% eos.% lymph. % mon.%

Control, Male 0.4±0.24 24.6±4.6 4.0±0.96 70.6±6.7 0.4±0.24

Distilled water

(26.3) Female 1.0±0.77 25.0±7.85 8.2±2.3 65.4±6.5 0.4±0.24

Control, Male 0.6±0.24 23.0±7.0 3.0±0.57 73.0±6.5 -

Sugar syrup

(26.3) Female 1.7±0.75 23.6±5.22 4.4±0.97 69.8±5.2 0.6±0.6

Composition of Male 1.6±0.33 22.6±5.4 4.2±0.96 71.4±5.3 2.0±1.0 this invention

(26.3) Female 1.75±0.^ 21.4±6.14 4.4±1.36 68.0±6.7 4.0±1.6

Table 11a

Blood biochemical indices in rats at oral administration (30 days of administration)

Agent, dose Urea Creatinine Bilirubin Glucose

Gender

(ml/kg) mM/l mcM/l mcM/l mM/l

Control, Male 4.60±0.12 38.78±2.0 4.04±0.34 4.01±0.42

Distilled water

Female

(26.3) 4.13±0.33 39.22±2.6 4.06±0.3 3.96±0.30

Control, Male 4.43±0.27 38.06±2.0 4.75±0.07 4.32±0.31

Sugar syrup

(26.3) Female 3.19±0.14 38.48±2.32 4.71±0.34 4.44±0.23

Composition of Male 3.15±0.12 39.72±2.6 4.04±0.26 4.21±0.51 this invention

(26.3) Female 3.94±0.27 35.72±2.66 3.96±0.21 4.15±0.21

Table lib

Table 12a

Blood biochemical indices in rats at oral administration (50 days of administration)

Agent, dose Urea Creatinine

Gender (ml/kg) mM/l mcM/l

Control, Male 4.20±0.42 39.27±1.03

Distilled water

Female

(26.3) 3.89±0.15 38.16±0.55

Control, Male 4.21±0.55 36.64±4.6

Sugar syrup

Female 5.16±0.3 36.5±4.23

(26.3)

Composition of Male 3.51±0.25 26.77±1. this invention

(26.3) Female 5.43±0.18 32.49±4.43

Table 12b

Alkaline

Agent, dose AsAT AIAT Whole protein

Gender phosphatase (ml/kg) mM/l mM/l g/i mM/l

Control Male 3.73±0.34 1.53±0.4 2.04±0.38 56.7±2.73

Distilled water

(26.3) Female 3.90±0.35 1.43±0.3 1.93±0.30 52.72±4.7 Control, Male 3.48±0.3 1.42±0.15

Sugar syrup

(26.3) Female 3.94±0.3 1.18±0.2

Composition of Male 3.49±0.13 1.31±0.13 this invention

(26.3) Female 4.20±0.24 1.70±0.2

Table 13a

Blood biochemical indices in rats at oral administration (recovery period)

Agent, dose Urea Creatinnine

Gender (ml/kg) mM/l mcM/l

Control Male 4.80±0.33 24.15±2.24

Distilled water

(26.3) Female 4.96±0.16 27.07±1.50

Control, Male 5.12±0.16 22.97±3.46

Sugar syrup

(26.3) Female 4.89±0.29 23.62±3.36

Composition of Male 4.39±0.36 29.97±3.06 this invention

Female 22.75±2.62

(26.3) 4.28±0.33

Table 13b

Alkaline

Agent, dose AsAT AIAT Whole protein lender phosphatase (ml/kg) mM/l mM/l g/i mM/l

Control Male 4.05±0.13 1 85±0.06 2.01±0.11 66.40±4.15

Distilled water

(26.3) Female 4.91±0.13 2 33±0.20 2.06±0.15 67.67±1.93

Control, Male 4.24±0 15 1.94±0.14 1 85±0.08 64.72±5.66

Sugar syrup

(26.3) Female 4.27±0.24 2.05±0.02 1 95±0.08 66.52±4 57

Composition of Male 4.03±2.06 2 45±0.19 1.93±0.56 62.26±2.53 this invention

(26.3) Female 4.14±0.20 2 09±0.11 I 2 25±0.17 56.52±2.55 These experiment results demonstrate that the behavior of the experimental animals has no difference from the control. Rats were active, ate up food and had smooth wooly cover. As shown in Table 4, the body weight of the experimental animals introduced with the composition of this invention was a little bit higher than the control. This change is caused by sugar (high-calorific stuff) contained in the composition of this invention.

Furthermore, the experimental animals had no difference from the control in the survey for internal organs (Tables 5-7). In addition, the experimental animals introduced with the composition of this invention had not any significant differences from the control (Tables 8-10).

In contrast, in the study of biochemical indices for blood serum of experimental rats, there was detected a slight increase of glucose concentration on 30-th day of the sample administration, and a significant increase of glucose concentrations at introduction of the sample for 60 days (Tables 11-13). However, considering the fact that administrated doses of the sample to experimental animals exceed a maximal recommended daily dose for human by over 30 times, it was not considered that this results caused toxicity.

The experiment results of sub-chronic toxicity urge us to reason that the composition comprising chlorin e6 of this invention is a safe material with no adverse effects to human body.

Example 6: Cytotoxicity experiment of chlorin e6

Final cone. 10, 25, 50, 75 or 100 μg/ml of chlorin e6 was treated to a monolayer cultured HeLa tumor cell lines (100,000 cells in 2.0 ml of medium) on plates. The plates were wrapped in light-protective cover, and incubated in incubator for 1 hr at 37.5⁰C. Then, the monolayer of tumor cell lines was dispersed by 0.02% Versene solution, and the cell number was calculated in Gorieav's chamber. The survival rate of cells in the experimental group was estimated through the following formula: (N _eχ_Peπm_en_t/N con_tro_l) x 100 (%), where N is number of cells. After estimation of cells survival rate, EC₅₀ (effective concentration causing death of 50% of cells) was estimated using regression analysis of data. The experiment results were summarized in Table 14.

Table 14

After incubation with the agents for 24 hours, number of HeLa cells (percent to the control, %)

As represented in Table 14, it could be appreciated that chlorin e6 exhibit the cytotoxicity for tumor cell lines. In addition, EC₅₀ was found 69.1 ± 1.8, 63.1 ± 4.6 or 66.4 ± 6.6 μg/ml.

Example 7: Pharmacokinetics of chlorin e6 in cells

Final cone. 10 μg/ml of chlorin e6 was treated to monolayer cultured HeLa tumor cell lines (100,000 cells in 2.0 ml of medium) on plates, and incubated for 1, 2, 3, 4, 5 or 24 hr at 37⁰C. Then, chlorin e6 was washed up by Hank's solution four times, and 2 ml of 0.5 % Triton X-100 solution was added to plates. The plates were incubated for 2 hr at room temperature and the fluorescence was measured at 540 nm. The experiment results were summarized in Table 15.

Table 15 After incubation with the agents for 24 hours and further photoexposure (Ph#) at dose of 3,3 joule/ciif, the number of HeLa cells (percent to the control, %) 2 Chlorin sample 3

mcg/ml

0.1 83.1 81.1 90 .9

0.5 83.6 76.3 86 .8

1.0 44.7 51.6 1. 0

5.0 1.9 0.9 0. 3

10.0 0.5 0.3 0. 5

EC₅₀ (PhE) mcg/ml 0.78±0.5 O.δ±l.l l.l±l.l

EQo/ECso (PhE) 78.9 60 .4

As shown in Table 15, chlorin accumulation was initiated within 1 hr post- administration and reached maximum values at 4-5 hr post-administration. Afterwards, the intracellular level of chlorin was reduced. Such decrease is due to intracellular metabolism of chlorin.

Example 8: Pharmacokinetics of chlorin e6 in animals

Chlorin e6 was injected intragastrically to 190-210 g weighted, 12 wistar rats in the dose of 300 mg/kg using probe. After 0.5, 1, 2, 3 and 5 hr, blood was collected, and concentrations of chlorin e6 in blood were measured. The concentrations of

K) chlorin e6 were estimated using spectrophotometer at 664 nm, or fluorometer at 675 nm. The concentrations of chlorin e6 were calculated by applying a measured absorbance to the following formula: X (chlorin e6 concentration in sample, %) = [(D₆₆₄ x 662 x V_sampi_e)/(34230 x L x 1000 x V_totai)] x 100. D₅₆₄: absorbance of chlorin e6 solution at 664 nm; 662: molar mass of chlorin e6 trisodium salt, g/mol; 34230: molar

15 extinction coefficient of chlorin e6 solution at 664 nm; l/mol x cm^'1; L: flask coating thickness, cm; 1000: conversion factor L to ml; V_totai : total volume of spectrophotometry sample, ml; V_sampι_e: volume of serum aliquot added to the sample, ml.

The experiment results were summarized in Tables 16-17 and Figs. 3-4.

20 Table 16

Dynamics of chlorin concentration in rats blood serum

Index Time of blood collection, hours 0 hour 0 hour 1 hour 2 hours 3 hours 5 hours chlorin 0 0 0. 0002 0.0011 0 .0012 0.0017 concentration, % 0 0 0 0.0009 0 .0026 0.0009

Average value 0 0 0. 0001 0.001 0 .0019 0.0013

Table 17

Dynamics of fluorescence intensity of rats blood serum

As shown in Tables 16-17 and Figs. 3-4, it was found that chlorin e6 oral administrated to rats was absorbed in gastro-intestinal tract. Chlorin e6 was detected by spectrophotometer at 1 hr after administration, and the concentration of chlorin e6 in blood reached maximum values by the 3-rd hr. Maximal chlorin e6 concentration in rats blood serum was 0.0019% (19 μg/ml).

Example 9: Anti-tumor effects of the present composition in animals level.

The composition of this invention of Table 1 was injected to 190-210 g weighted, 12 wistar rats with transplanted Waker 256 carcinosarcoma cells in the dose 500 mg/kg using probe. The composition of this invention was continuously administrated to rats one month before tumor transplantation and during the experiment. Tumor size was measured, and tumor volume was calculated according to Schrek formula:

V (tumor volume, cm²) = (a x b x c) i /6. a, b, c: linear tumor size, cm.

Percentage of tumor growth inhibition was calculated from the tumor volume:

[(Vav(Control)-V_av(Experιmental))/Vav(Control)] ^x 100. The experiment results were summarized in Table 18-21 and Fig. 5.

Table 18

Changes of W -256 growth in the control

Table 19

Changes of W -256 growth in the experimental group, received the drug in the dose of 500 mg/kg of animal weight

Table 20

After the drug administration, lifetime duration of rats with W-256

## Control 500 mg/kg administration

L. 14 21

Table 21

After the drug administration, percentage of W-256 growth inhibition in rats (by volume vs. the control)

Tables 18-19 and Fig. 6 represented that tumor was significantly decreased by the composition of this invention. Table 20 showed that the lifetime of rats transplanted tumor was considerably prolonged by the composition of this invention. Furthermore, it was observed to two recovered rats (complete elimination of tumor in 120 days after tumor transplantation). In contrast, the percentage of dead animals was reached by 50% in the control during the experiment.

Table 21 presented that percentage of tumor growth inhibition was reached about 60-70% by the composition of this invention. According to these results, in spite of oral administration of the composition of this invention, it was found that the present composition exhibited very excellent anti-tumor effects.

Example 10: Immunotoxicity analysis of the composition of this invention

It was examined whether chlorin e6 and the composition of this invention showed the immunotoxicity. Each mouse with 19-20 g weight was used, and each group consisted of 7 animals. The samples were administrated for 10 days, both chlorin e6 and the composition were administrated once a day in the dose of 170 mg/kg. On the 3-rd day of the experiment, the mice were immunized by ram erythrocytes (1 x 10⁷ erythrocytes in 0.1 ml of isotonic solution of NaCI). On the 8-th day of the experiment, delayed-type hypersensitivity (DTH) was induced. On the 11-th day, the animals were taken out from the experiment.

Table 22

Change of the amount and formula of leukocytes in peripheral blood at administration (average value ± standard deviation)

Groups of mice

## Indices Composition of

Control Chlorin this invention

Number of leukocytes,

6.7±2.8 6.7±2.4 4.8±1.7 x 10⁹/l

Stab neutrophils, % 7.9±2.5 5.1±1.2 4.6±2.4

Segmental 44.7±9.2 34.6±9.8^Λ 35.1±12.4^Λ neutrophils, % eosinophil, % 0.57±0.98 2.6±5.6 1.7±2.2 monocytes, % 10.7±4.8 8.0±4.0 10.6±3.4 lymphocytes, % 36.1±8.4 49.7±7,90^* 46.6±8.3^*

10 difference is significant as compared to the control group, Student criteria, p < 0.05 Λ difference is significant as compared to the control group, Fisher criteria, p < 0.05

Table 23

Effect of the drugs on macrophage (average value ± standard deviation)

Groups of mice

I T) difference is significant as compared to the control group, p < 0.05

Table 24 Effect of the drugs on the complement system (average value ± standard deviation)

As shown in these Tables, number of leukocytes in peripheral blood, neutrophils, eosinophils, monocytes and lymphocytes in administration groups of the compositions of this invention and chlorin e6 has no significant difference from the control. However, there was recorded an increase in number of leukocytes and eosinophils compared to the control, neutrophils number showed a tendency to decrease. In addition, little or no differences from the control were found in the complement system.

However, in the macrophage, it was examined to have very high activity in the present composition and chlorin e6. These results suggested that the composition of this invention and chlorin e6 could increase in the immunity (resistance) against bacteria.

In contrast, Table 25-27 were summarized results analyzing effects to antigen- specific immune system, which was induced an immune response by introducing ram erythrocytes.

Table 25

Drug effect to the body weight, spleen weight and splenic index in the course of immunization by ram erythrocytes (average value ± standard deviation)

Table 26 Drug effect to hummoral immune response in the course of immunization by ram erythrocytes (average value ± standard deviation)

APC number

## Groups of animals Hemagglutinin tter (log.) (per 10⁵ splenocytes)

Control 237.1±92.4 4. 7±0 76

Composition of this

267.0±166.7 4 .3±1 .4 invention

Chlorin 241.0±111.7 4 .li_¬ .7

Table 27

Drug effect to cellular immune response (DTH index, %) in the course of immunization by ram erythrocytes (average value ± standard deviation)

Number of mice, not responded by DTH (abs. - % for group)

0

2 - 28.6%

As results described in these Tables, the administration group had no significant difference from the control in antigen-specific immune systems. However, a spleen index and a DTH index showed a tendency to decrease.

The experiment results of the immunotoxicity suggest that the composition of this invention and chlorin e6 exhibit no immunotoxicity and instead enhance cellular immune system with no considerable influence on humoral immune system.

As described hereinabove, the present invention provides an antitumoric composition for oral administration comprising chlorin compound as an active ingredients. This invention suggests novel use and use method of chlorin compound, which is conventionally used in a photodynamic therapy for cancer as photosensitive substances. Thus, according to this invention, chlorin compound is orally administrated, and may have antitumoric effects without irradiation of light. The composition of this invention is orally administrated, well absorbed in a living body through gastro- intestinal tract, and showed an excellent antitumoric effect. The composition of this invention does not have cytotoxicity and an immunetoxicity, so that the composition has a higher safety to human.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. An antitumoric composition for oral administration comprising a chlorin compound, or its pharmaceutically acceptable salt as an active ingredient, wherein the chlorin compound or its pharmaceutically acceptable salt exhibits the antitumoric activity

5 without light irradiation.

2. The antitumoric composition for oral administration according to claim 1, wherein the chlorin compound is chlorin e6, sulfonated meso-tetraphenyl chlorin or β, β'- dihydroxy meso-substituted chlorin.

10

3. The antitumoric composition for oral administration according to claim 2, wherein the chlorin compound is chlorin e6.

4. The antitumoric composition for oral administration according to claim 1, wherein ] 5 the antitumoric composition is a food composition.

5. The antitumoric composition for oral administration according to claim 1, wherein the antitumoric composition is a pharmaceutical composition.

20 6. The antitumoric composition for oral administration according to claim 3, wherein the chlorin e6 is prepared by chemically modifying chlorophyll-a obtained from chlorella.

7. The antitumoric composition for oral administration according to claim 1, wherein 5 the composition further comprises at least one ingredient selected from a group consisting of β-sitosterol, glycyrrhizic acid, lentinan, reishidin, letiporin, acacetin, zinc, selenium and amino acid.

8. The antitumoric composition for oral administration according to claim 7, wherein β-sitosterol, glycyrrhizic acid, lentinan, reishidin, letiporin, acacetin and amino acid are obtained from nettle, licorice, lentinus edodes, ganoderma lucidum, Latiporus sulphereus , agastache rugosa and yeast, respectively.

5

9. The antitumoric composition for oral administration according to claim 1, wherein the composition improves an activity of macrophage.

10. A method for treating tumors, which comprises orally administering a patient an0 antitumoric composition comprising (a) a therapeutically effective dose of a chlorin compound, or its pharmaceutically acceptable salt; and (b) a pharmaceutically acceptable carrier, wherein the composition exhibits the antitumoric activity without light radiation. 5

11. The method for treating tumors according to claim 10, wherein the chlorin compound is chlorin e6, sulfonated meso-tetraphenyl chlorin or β, β'-dihydroxy meso- substituted chlorin.

12. The method for treating tumors according to claim 11, wherein the chlorinQ compound is chlorin e6.

13. The method for treating tumors according to claim 10, wherein the antitumoric composition is a food composition. 5

14. The method for treating tumors according to claim 10, wherein the antitumoric composition is a pharmaceutical composition.

15. The method for treating tumors according to claim 12, wherein the chlorin e6 is prepared by chemically modifying chlorophyll-a obtained from chlorella.

16. The method for treating tumors according to claim 10, wherein the composition further comprises at least one ingredient selected from a group consisting of β-

5 sitosterol, glycyrrhizic acid, lentinan, reishidin, letiporin, acacetin, zinc, selenium and amino acid.

17. The method for treating tumors according to claim 16, wherein β-sitosterol, glycyrrhizic acid, lentinan, reishidin, letiporin, acacetin and amino acid are obtained

10 from nettle, licorice, lentinus edodes, ganoderma lucidum, Latiporus sulphereus , agastache rugosa and yeast, respectively.

18. The method for treating tumors according to claim 10, wherein the composition improves an activity of macrophage.

I o

19. A use of a composition comprising a chlorin compound or its pharmaceutically acceptable salt for manufacturing a composition for tumor treament, wherein the chlorin compound or its pharmaceutically acceptable salt exhibits the antitumoric activity without light irradiation.

20

20. The use according to claim 19, wherein the chlorin compound is chlorin e6, sulfonated meso-tetraphenyl chlorin or β, β'-dihydroxy meso-substituted chlorin.

21. The use according to claim 20, wherein the chlorin compound is chlorin e6.

22. The use according to claim 19, wherein the composition is a food composition.

23. The use according to claim 19, wherein the composition is a pharmaceutical composition.

24. The use according to claim 21, wherein the chlorin e6 is prepared by chemically modifying chlorophyll-a obtained from chlorella.

25. The use according to claim 19, wherein the composition further comprises at least one ingredient selected from a group consisting of β-sitosterol, glycyrrhizic acid, lentinan, reishidin, letiporin, acacetin, zinc, selenium and amino acid.

26. The use according to claim 25, wherein β-sitosterol, glycyrrhizic acid, lentinan, reishidin, letiporin, acacetin and amino acid are obtained from nettle, licorice, lentinus edodes, ganoderma lucidum, Latiporus sulphereus , agastache rugosa and yeast, respectively.

27. The use according to claim 19, wherein the composition improves an activity of macrophage.