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1. WO2002066046 - CANCER THERAPEUTIC AGENT COMPRISING THIOPEPTIDE WITH MULTIPLE THIAZOLE RINGS

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CANCER THERAPEUTIC AGENT COMPRISING THIOPEPTIDE
WITH MULTIPLE THIAZOLE RINGS

FIELD OF THE INVENTION
The present invention relates to cancer therapeutic agent comprising thiopeptide with multiple thiazole rings, its derivative or its pharmaceutically acceptable salt as an effective ingredient.

BACKGROUND OF THE INVENTION

Due to the increase of senile population and pollution of the environment, it has been estimated that the world's cancer incidence rate is increasing more than 5 to 7% every year. People dying of cancer estimate 6 million to 7 million by the statistics in 1997, which makes up 12 to 14% of the world's death rate.
Surgical operation, chemotherapy, biological therapy and radiotherapy are typically used for the treatment of cancer in modern medical science. Each therapy can be applied independently or can be combined with each other to treat cancer. However, there is still no perfect method for a complete recovery from cancer. Accordingly, cancer has become one of the main themes of highest priority which should be overcome to lengthen human life span in the 21st century.

Cancer therapeutic agents refer to any compound exerting a cytotoxic or cytostatic effect against cancer cells by activating various metabolic pathways. Cancer therapeutic agents developed until now can be classified as metabolic antagonists, plant alkaloids, topoisomerase inhibitors, alkylatmg agents, cancer therapeutic antibiotics, hormonal agents and other drugs according to their action mechanism and chemical structure. The above-mentioned cancer therapeutic agents have various target sites in the cells depending on the action mechanism. Hereupon, the specific action mechanism of cancer therapeutic agents prevents DNA synthesis, transcription or translation of the cells, or inhibits the function of proteins having central roles in cell survival. By the above-mentioned action mechanisms, cancer cells die of necrosis or apoptosis.

In general, the metabolic pathways wherein cancer therapeutic agents act on against cancer cells do not differ from those of normal cells. Therefore, the damage (i.e. toxicity) of normal cells by cancer therapeutic agents cannot be avoidable. However, since there is a significant difference between the metabolisms of cancer cells and normal cells, which makes cancer therapeutic agents display more toxicity to "cancer cells than normal cells do, such selective toxicity of the cancer therapeutic agent makes cancer therapeutic chemotherapy clinically effective. Therefore, the bigger the specific therapeutic index, the safer the cancer therapeutic agents, wherein the specific therapeutic index means the selective killing of cancer cells without any toxicity against normal cells .

Meanwhile, since cancer cells show higher protein synthesis rate than that of normal cells, the protein synthesis inhibitor can show cancer therapeutic effects with selective toxicity against cancer cells.
As for procaryotes, kanamycm, tetracyclme, chloramphenicol, erythromycin, streptomycin, lincomycm, clindamycin, thiostrepton and micrococcm are used as antibiotics inhibiting protein synthesis. Among them, thiostrepton, a thiopeptide antibiotic separated from microorganisms such as Streptomyces la uren tn (ATCC No.

31,255), Streptomyces actuosus (ATCC No. 25,421) and Streptomyces azureus (ATCC No. 40,485), shows antibiotic effects mainly against gram positive bacteria (J. Chem .

Sue , Chem . Commun . , 1993, 1612-1614) .
Thiostrepton targets the GTPase center which s well reserved among prokaryotic and eucaryotic cells as its target site. Ribosome, a protein synthesis machinery, is consisted of two subunits, and the GTPase center is located at the rRNA located in the bigger unit of the two subunits . The GTPase center is located at the double hairpin structure of domain II in the 23 S-like rRNA, and the pentameric protein complex of Lll and L10(L12)4 protein combines to the double hairpin structure, performing its biological function.
As for protozoa, thiostrepton serves as an antibiotic by inhibiting protein synthesis by preventing the GTPase activity at the ribosome in the presence of EF-G by being combined at the said GTPase center. Furthermore, it has been reported that thiostrepton inhibits the growth of protozoa by inhibiting protein synthesis at the plastid-like organelle of the malarial parasite { Proc . Na tl . Acad . Sci . USA, 1999, 96, 9586-9590) . However, there has been no report that thiopeptide such as thiostrepton and its derivative show inhibition against eukaryotic cells.

So, the present inventors have designed the present invention to show that the side effects or problems in the clinical use of known chemotherapy can be overcome by confirming that cancer therapeutic agent having thiopeptide with multiple thiazole rings and its derivative which inhibits the function of ribosome in protein synthesis shows a cancer therapeutic effect completely different from that of the known antibiotic effects .

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a cancer therapeutic agent comprising thiopeptide with multiple thiazole rings, its derivative or its pharmaceutically acceptable salt.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The terminology and technology referred in the present detailed description are used as general meaning of the technical field which includes the present invention. In addition, references mentioned in the present detailed description are all included in the present detailed description for describing the present invention .

The present invention provides a cancer therapeutic agent comprising thiopeptide with multiple thiazole rings, its derivatives or its pharmaceutically acceptable salt.
Thiopeptide and its derivative of the present invention is preferably selected from a group consisting of compounds represented by formula 1 to formula 15, thiostrepton, micrococcin P, nosiheptide, siomycin, sporangiomycin, althiomycin, Promoinducin, Berninamycin, glycothiohexide alpha, Sch40832, GE37468A, GE37468B, GE37468C, GE2270, GE2270 factors C2a, GE2270 factors D2, GE2270 factors E, thiocillins, thiopeptin, amythiamicin A, sulfomycin, Thioxamycin and MJ3471F4A. More preferably, it can be selected from a group consisting of the compounds represented by formula 1 to formula 15, but is not limited to this compound, and can be selected from any known thiopeptide compound and its derivative.

<Formula 1>


<Formula 2>

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<Formula 3>


<Formula 4>



<Formula 5>


<Formula 6>

<Formula 7>


<Formula 8>

<Formula 10>



<Formula 11>


<Formula 12>



<Formula 13>


<Formula 14>


<Formula 15>


Thiopeptide and its derivative of the present invention may be used in the form of pharmaceutically acceptable salts. As for said salts, the acid salt formed by a pharmaceutically acceptable free acid is useful. Thiopeptide and its derivative may form pharmaceutically acceptable acid salts using methods well known in the art. Both organic and inorganic acids may be used as free acids. Hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid can be used as inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, proprionic acid, oxalic acid, trifluoroacetic acid, benzonic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluene sulfonic acid, galacturonic acid, glutamic acid or aspartic acid can be used as organic acid .

Thiopeptide and its derivative of the present invention can be used as a cancer therapeutic agent and can be extracted from microorganisms. For example, thiopeptide and its derivative can be obtained by a process comprising gathering mycelium after separating supernatant from the culture medium of a kind of actinomyces, Streptomyces lauren tii (ATCC NO. 31,255) by centrifugation, obtaining the mycelium extract by treating the gathered mycelium with an organic solvent, dissolving the said extract in the organic solvent again after concentrating it, eliminating impurities, and gathering crystallized thiopeptide by adding a small dosage of alcohol. Thiopeptide and its derivative of the present invention can be obtained from other microorganisms such as Streptomyces a ctuosus (ATCC No. 25,421) and Streptomyces azureus (ATCC NO. 40,485) as well .

Thiopeptide and its derivative of the present invention show useful effects as a cancer therapeutic agent by inducing apoptosis of cancer cells. Cancer therapeutic effect is confirmed by showing selective toxicity against cancer cells, when cancer cells and normal cells were treated with thiopeptide and its derivative of the present invention dissolved in a pharmaceutically acceptable solvent, such as dimethyl sulfoxide .

Cancers, wherein thiopeptide and its derivative of the present invention can be applied, can be selected from a group consisting of gastric cancer, liver cancer, colon cancer, lung cancer, cervical cancer, breast cancer, ovarian cancer and leukemia. Particularly, thiopeptide and its derivative of the present invention can be effectively applied, but is not limited, to gastric cancer and liver cancer. In addition, it can be effectively applied to any known cancer.

The IC50 value of thiopeptide and its derivative of the present invention against cancer cells range from 0.2 μmol to 12 μmol (see table 1) .

According to the present invention, a pharmaceutical composition containing thiopeptide with multiple thiazole rings and its derivative as an effective ingredient can be prepared by mixing thiopeptide with multiple thiazole rings and its derivative with a pharmaceutically acceptable carrier.

Cancer therapeutic agents comprising thiopeptide with multiple thiazole rings and its derivative may further include generally-used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactant, or excipients, and may be formulated with tablets, coated tablets, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, dusting powders and sprays by conventional methods .

Specifically, solid formulations for oral administration of the cancer therapeutic agent of the present invention are tablets, pill, dusting powders and capsules, liquid formulations for oral administration are suspensions, solutions, emulsions and syrups, and the abovementioned formulations can include various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to the generally-used simple diluents such as water and liquid paraffin.
Tablets, coated tablets, capsules, pills and granules can contain the active compound or compounds in addition to the customary excipients, such as (a) fillers and extenders, for example starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, for example carboxymethylcellulose, alginates, gelatine and polyvinylpyrrolidone, (c) humectants, for example glycerol, (d) disintegrating agents, for example agar-agar, calcium carbonate and sodium carbonate, (e) solution retarders, for example paraffin, and (f) absorption accelerators, for example quaternary ammonium compounds, (g) wetting agents, for example cetyl alcohol and glycerol monostearate, (h) adsorbents, for example kaolin and bentonite, and (i) lubricants, for example talc, calcium stearate, magnesium stearate, and solid polyethylene glycols, or mixtures of the substances listed under (a) to (i) .
The tablets, coated tablets, capsules, pills and granules can be provided with the customary coatings and shells, optionally containing opacifying agents, and can also be of a composition such that they release the active compound or compounds only or preferentially in a certain part of the intestinal tract, if appropriate in a delayed manner. Examples of embedding compositions which can be used being polymeric substances and waxes.

Further, cancer therapeutic agent comprising thiopeptide with multiple thiazole rings and its derivative can be parenterally administered. Parenteral administration can be performed by hypodermic, intravenous, intramuscular or subcutaneous injection.

The cancer therapeutic agent of the present invention can be prepared for parenterally administration by being mixed with generally-used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactant, or excipients.

In general, it has been proven to be advantageous to administer the active compound or compounds according to the present invention in a total amount of about 1.5 to 8.5 mg, preferably 10 to 100 mg/kg of body weight, 1-6 times every 24 hours, if appropriate, in several individual doses, to achieve the desired results. However, the dosage may be determined in light of various relevant factors including the absorption rate of the active ingredient, the inactivity rate, the excretion rate, the age, sex and body weight of the object to be treated, the nature and severity of the disease, the nature of the formulation and of the administration of the medicament and the period or interval wherein the administration takes place.

Hereinafter, the present invention is described in detail.

EXAMPLES

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Separation of thiopeptide with multiple thiazole rings
After culturing Streptomyces la urentii (ATCC No. 31,255) for 65 hours by a well-known standard culturing method (J. Chem . Soc . Chem . Commun . , 1993, 1612-1614), the cultured mycelium was gathered by separating supernatant from the culture medium by centrifugation .

After that, the mycelium extract was obtained using chloroform, and the chloroform extract was concentrated in vacuum at 40°C. The impurity was eliminated using methanol, and the extract was dissolved again in the chloroform. Finally, the extract was crystallized by adding methanol .

As a result, thiopeptide derivatives represented by formula 1 to formula 15 were separated.

Example 2: Measurement of cancer therapeutic effect of thiopeptide
The present inventors estimated the effect of thiopeptide and its derivative on the growth of cancer cells by measuring the growth rate of cancer cells . To do this, the present inventors treated gastric cancer, liver cancer, colon cancer, lung cancer, cervical cancer, breast cancer, ovarian cancer, leukemia cell lines and the monocyte (MNC) separated from the blood of a healthy person as a control with thiopeptide and its derivative of the present invention dissolved in DMSO (dimethyl sulfoxide) at various concentrations (from 10 nmol to 10 μmol) for 72 hours.
Then, various concentrations of thiopeptide and its derivative were treated to the culture media and the cell proliferation rate was examined by MTT assay after culturing them at 37 °C , 5% C02 for 72 hours.

As a result, it has been found out that thiopeptide and its derivative of the present invention showed specific toxicity against various types of cancer cells in proportion to the concentration of thiopeptide and its derivative within the range of the concentration being administered. As for MNC used as the control group, MNC did not show toxicity from 10 nmol to 10 μmol (Table 1 and Table 2) .

<Table 1>
Cancer therapeutic effect of thiopeptide and its

<Table 2>
Cancer therapeutic effect of thiostrepton against various cancer cell lines (ICso=μM)


Example 3: Acute toxicity in rats tested via oral

administration

The following experiment was performed to see if thiopeptide and its derivative of the present invention have acute toxicity m rats. Six-week old specific- pathogen free (SPF) SD line rats were used in the tests for acute toxicity. Thiopeptide and its derivative of the present invention were suspended in a methylcellulose solution of 0.5% and orally administered once to six rats per group at the dosage of 5 g/kg/15 ml. Death, clinical symptoms, and weight change in the tested animals were observed. In addition, hematological and biochemical tests of blood were performed, and any abnormal signs in the gastrointestinal organs of chest and abdomen were visually (with the naked eye) checked during autopsy.
The results showed that the tested compounds did not cause any specific clinical symptoms, weight changes, or death in rats. No change was observed in hematological tests, biochemical tests of blood, and autopsy. Therefore, the compounds used in this experiment are evaluated to be safe substances, since they do not cause any toxic change in rats up to the level of 2 g/kg and their estimated LD50 value is much higher than 2 g/kg in rats.

INDUSTRIAL APPLICABILITY

As described above, thiopeptide and its derivative of the present invention can be usefully used as a cancer therapeutic agent inducing the selective death of cancer cells, since they show selective toxicity against various types of cancer cells without showing any toxicity against normal cells.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .