HK1088915B - Polymer guanidine derivatives as a cytostatic medicament - Google Patents

Description

Polymeric guanidine derivatives as cytostatic drugs

The invention relates to a medicament, in particular to a cell inhibition medicament.

1/3 people in western societies have cancer with a fatality rate of not less than 75%. Currently malignant tumors are treated with cytostatic approaches.

The main problem with chemotherapy for these diseases is that only a certain percentage of cancer cells respond to the administered cytostatic drugs. Furthermore, even if the tumor responds to treatment, complete remission of symptoms is often not desired.

In order to increase the efficacy of chemotherapy, the current trend is towards a variety of chemotherapies, i.e., the use of a variety of cytostatic drugs. More and more often, a number of cytostatic drugs with different points of action are combined to improve cancer treatment. This results in better efficacy on the one hand and the problem of progressive resistance on the other hand.

An additional possibility is to selectively protect healthy cells from cytostatic drugs by simultaneous administration of cytoprotective agents, whereby higher doses can be administered with lower side effects (e.g. taxanes).

Despite these measures, the side-effect rate of chemotherapy is still high. For this reason, it is of utmost importance to study active ingredients which have both good efficacy and good tolerability, i.e. a therapeutic window which is as wide as possible.

The object of the present invention is to provide a cytostatic drug having a wider therapeutic window than conventional cytostatic drugs such as 5-fluorouracil, platinium, epirubicin and mitomycin C.

The pharmaceutical composition according to the invention contains, as active ingredient, a polymeric guanidine derivative based on a diamine containing an oxyalkylene chain between two amino groups, wherein the guanidine derivative represents a polycondensation product of a guanidine acid addition salt with a diamine containing a polyalkylene chain between two amino groups, and pharmaceutically acceptable salts thereof.

A preferred embodiment of the pharmaceutical composition according to the present invention is characterized by triethylene glycol diamine (relative molecular weight: 148), polyoxypropylene diamine (relative molecular weight: 230) and polyoxyethylene diamine (relative molecular weight: 600) as representative substances of polyoxyalkylene guanidine salts.

More preferably, the active ingredient contains poly [2- (2-ethoxyethoxyethyl) guanidine hydrochloride ] having at least 3 guanidinium groups, the average molecular weight of which is in the range of 500-3000D.

Furthermore, the invention relates to the use of polymeric guanidine derivatives based on diamines containing an oxyalkylene chain between two amino groups, wherein the guanidine derivatives represent the polycondensation product of a guanidine acid addition salt and a diamine containing a polyalkylene chain between two amino groups, and pharmaceutically acceptable salts thereof, for the preparation of cytostatic-active medicaments.

Further, the present invention relates to the use of polyoxyalkylene guanidine salts prepared using triethylene glycol diamine (relative molecular weight: 148), polyoxypropylene diamine (relative molecular weight: 230) and polyoxyethylene diamine (relative molecular weight: 600).

Polyguanidine derivatives used in the present invention are known from PCT/AT 01/00134. The content of said document is incorporated into the present description by reference.

The preparation methods and determination of the cytostatic activity of preferred representatives of the compounds employed according to the invention are described below.

As representative of the class of compounds employed in the present invention, the cytostatic activity of poly [2- (2-ethoxyethoxyethoxyethyl) guanidine hydrochloride ] (CAS number 374572-91-5) having an average molecular weight of 1000D is described below.

To prepare the compound, 4.43mol of guanidine hydrochloride were dissolved in 4.03mol of triethylene glycol diamine at 50 ℃. It was then heated to 120 ℃ and stirred at said temperature for 2 hours. The temperature was then maintained for 2 hours, followed by application of vacuum (0.1bar) and continued stirring in vacuo at 170 ℃ for a further 2 hours. Then aerated to atmospheric pressure, cooled to 120 ℃ and diluted to about 50% with deionized water. Neutralized to a pH of about 6 with phosphoric acid, allowed to cool and diluted to the desired concentration. The molecular weight was found to be 1000D.

Determination of cytostatic Activity

Experiments were performed on cell lines of colon and pancreatic cancer such as Capan-1, DLD-1, HT 29, HCT-8, MIA-PA-CA2, PANC1, BXPC-3, ASPC-1 and HT-29. The cancer cell lines tested were stored in liquid nitrogen. After the thawing, the mixture is thawed,cancer cell lines were cultured in RPMI-1640+ Glutamine Medium (Gibco No. 5240025) at 37 deg.C/5% CO₂The atmosphere was cultured in a culture flask for no more than 14 days so that a cell monolayer could be formed. Subsequently, cells were harvested with Trypsin + EDTA (Gibco No. 15400) -054) and washed twice with RPMI medium.

In addition, experiments were performed on lymphocytes from healthy subjects. For this purpose, a total of 100ml of blood was drawn into EDTA tubes. Lymphocytes were isolated from whole blood samples using a Mono-Poly-ResolvingMedium/Ficol-Hypaque gradient and washed three times with HBSS buffer, prepared in this manner and added to the test material.

Except for poly [2- (2-ethoxyethoxyethoxyethyl) guanidine hydrochloride ], the chemotherapeutic agents, schlatinum, epirubicin, mitomycin C and 5-fluorouracil, were tested as comparative examples. For this purpose, the compounds were first dissolved according to the instructions of the respective manufacturer and stored as stock solutions in 1ml portions (active substance concentration 1000. mu.g/ml) in liquid nitrogen at-180 ℃. The dissolved material was used within the day.

To determine the cytostatic activity of the substances, cancer cells were transferred from culture flasks with RPMI-washed suspensions to microtiter plates at a concentration of 20000 cells in 200. mu.l. The cancer cells were then incubated at 37 ℃ and 5% CO in the presence of varying concentrations of the test substance₂Cultured in the atmosphere for three days. For poly [2- (2-ethoxyethoxyethyl) guanidine hydrochloride]Concentrations of 0.12-500. mu.g/ml (2-fold dilution) were used in all experiments. After a three-day culture period, evaluation was performed by the nonradioactive cell proliferation and cytotoxicity assay EZ4U (Biomedica No. BI-500010X 96 assay). After incubation for three hours with EZ4U, the evaluation was carried out photometrically at a wavelength of 49/630nm in a Dias-Photometer (extinction of the test sample divided by the blank extinction of the control sample).

The activity of the lymphocyte is 1.2 multiplied by 10⁷The measurement was carried out in different concentrations of the test substance in the suspension per ml. Cell suspensions at 37 ℃ and 5% CO in the presence of test substances₂Culturing in the atmosphere for 24 hoursAnd then stained with trypan blue. After staining, lymphocytes were applied to a cell counting chamber and viability was measured as a percentage.

In addition to having low toxicity and good resistance from a pharmacological point of view, the active ingredient poly [2- (2-ethoxyethoxyethoxyethyl) guanidine hydrochloride ] has sufficient pharmacodynamic properties and can therefore be used as a medicament for the treatment of tumors. The substance particularly shows excellent cytostatic activity, which can be confirmed in experiments using multiple groups of cancer cell lines such as colon cancer (HT-29, HCT-8, DLD-1) or pancreatic cancer (ASPC-1, BXPC-3, CAPAN-1, PANC-1).

Furthermore, the active ingredients employed in the present invention possess a wide therapeutic window, since the cytostatic activity in cancer cells already occurs at concentrations of 2-16. mu.g/ml (tables 1 and 2), whereas this activity is observed only at concentrations above 100. mu.g/ml in healthy endogenous cells such as lymphocytes (Table 4). By way of comparison, Table 3 shows the cytostatic activity of 5-fluorouracil, platinium, epirubicin and mitomycin C.

After systemic administration (intravenous or intraperitoneal) of poly [2- (2-ethoxyethoxyethyl) guanidine hydrochloride ] in an amount of not more than 15mg/kg body weight, serum concentrations of up to 100. mu.g/ml were measured in the rat blood after two hours, while the drug resistance was good. Therefore, poly [2- (2-ethoxyethoxyethoxyethyl) guanidine hydrochloride ] can be used as a cytostatic drug.

The medicaments used in the present invention can be processed into pharmaceutical preparations in a manner known per se, i.e. alone or together with inorganic or organic pharmacologically neutral adjuvants.

TABLE 1 cytostatic Activity against different colon cancer cell lines (% viability)

Concentration of test Compound (. mu.g/ml)	HAT-29	HCT-8	DLD-1	HCT-15	Colon320DM	Colon205
							64321684210.50.250.125	11132147767788988576	111534911149085797088	1517264986103----	161825698181----	212224263779----	151616275970----

Poly- [2- (2-ethoxyethoxyethoxyethyl) guanidine hydrochloride ]

TABLE 2 cytostatic activity (% viability) against different pancreatic cancer cell lines

Concentration of test Compound (. mu.g/ml)	AsPc-1	BxPc-3	Capan-1	Panc-1	MIA PaCa-2
						643216842	131215507092	91118447463	467911495111115	111630559088	1112165897106

Poly- [2- (2-ethoxyethoxyethoxyethyl) guanidine hydrochloride ]

TABLE 3 cytostatic Activity of standard chemotherapy against different pancreatic cancer cell lines (% viability)

Substance concentration in μ g/ml		AsPc-1	BxPc-3	Capan-1	Panc-1
						5-Fluorouracil	6060.6	718892	677986	233846	627780
Platinum ingot	2.50.250.025	538985	936587	365494	---
						Epirubicin	11.91.190.12	5559105	625576	543550	6143111
Mitomycin C	1.50.150.015	658998	4791100	223059	55103104

TABLE 4 lymphocyte viability%

Concentration of test Compound (. mu.g/ml)
		1000500250100502512.5	11630989610098

Poly- [2- (2-ethoxyethoxyethoxyethyl) guanidine hydrochloride ]

Claims (6)

1. A pharmaceutical composition comprising, as an active ingredient, a polyguanidine derivative based on a diamine containing an oxyalkylene chain between two amino groups, wherein the guanidine derivative represents a product of polycondensation of a guanidine-acid addition salt and a diamine containing a polyalkylene chain between two amino groups, and pharmaceutically acceptable salts thereof.

2. Pharmaceutical composition according to claim 1, characterized in that as polyoxyalkylene guanidine salts are used those which employ triethylene glycol diamine having a relative molecular weight of 148, polyoxypropylene diamine having a relative molecular weight of 230 and polyoxyethylene diamine having a relative molecular weight of 600.

3. Pharmaceutical composition according to claim 1 or 2, characterized in that it comprises as active ingredient poly [2- (2-ethoxyethoxyethyl) guanidine hydrochloride ] having at least 3 guanidinium groups.

4. Pharmaceutical composition according to claim 3, characterized in that the average molecular weight of the active ingredient is in the range of 500-3000.

5. Use of a polyguanidine derivative based on a diamine containing an oxyalkylene chain between two amino groups, wherein the guanidine derivative represents a product of the polycondensation of a guanidine-acid addition salt and a diamine containing a polyalkylene chain between two amino groups, and of a pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical composition having cytostatic activity.

6. Use according to claim 5, characterized in that as polyoxyalkylene guanidine salts are used those which employ triethylene glycol diamine having a relative molecular weight of 148, polyoxypropylene diamine having a relative molecular weight of 230 and polyoxyethylene diamine having a relative molecular weight of 600.