Title of Invention

"PROCESS FOR PREPARING A POLYMERIC GUANIDIUM DERIVATIVE"

Abstract

The invention relates to a medicament containing, as an active ingredient, a polymer guanidine derivative or the pharmaceutical ly acceptable salts of the same, based on a diamine containing oxyalkylene chains between two amino groups. Said guanidine derivative is a product of the polycondensation of a guanidine acid addition salt with a diamine containing polyalkylene chains between two amino groups

Full Text

The invention relates to a drug composition, in particular a cytostatic drug. In the Western civilization, a third of the population suffers from cancer with a mortality rate of no less than 75%. Malignant tumors are nowadays treated cytostatically. The main problem with the chemotherapy of those diseases is that the cancer cells respond to the applied cytostatic drug only to a certain percentage. Moreover, a complete remission often cannot even be expected if the tumor does respond to the treatment. A current trend toward an efficiency increase of the chemotherapy consists in the polychemotherapy, i.e. the use of several cytostatic drugs. More and more frequently, various cytostatic drugs with different application points are combined in order to improve the cancer therapy. Thereby, better efficacy is achieved on the one hand and on the other hand the problem of a progressively developing resistance is countered. A further possibility consists in selectively protecting the healthy cells from the cytostatic drug by a simultaneous administration of cytoprotectors, whereby a higher dose can be administered, at the same time involving fewer side effects (f.i. taxanes). Despite those measures, the side-effect rate of chemotherapy is still very high. Precisely for this reason it is of utmost importance to develop drug substances which exhibit good efficacy as well as good tolerance, i.e. which possess a therapeutic window that is as broad as possible. The invention aims at providing a cytostatic drug which, in comparison with conventional cytostatic drugs such as 5-fluorouracil, cisplatin, epirubicin and mitomycin C, has a broader therapeutic window. The drug composition according to the invention contains as a drug substance a polymeric guanidine derivative based on a diamine containing oxyalkylene chains between two amino groups, with the guanidine derivative representing a product of polycondensation between a guanidine acid addition salt and a diamine containing polyalkylene chains between two amino groups, as well as the pharmaceutically acceptable salts thereof. A preferred embodiment of the drug composition according to the invention is characterized in that, among the representatives of the family of polyoxyalkylene guanidine salts, there are such using triethylene glycol diamine (relative molecular mass: 148), polyoxypropylene diamine (relative molecular mass: 230) as well as polyoxyethylene diamine (relative molecular mass: 600). Most preferably poly-[2-(2-ethoxyethoxyethyl)guanidinium hydrochloride] comprising at least 3 guanidinium groups is contained as the drug substance, with the average molecular mass in particular ranging from 500 to 3.000 D. Furthermore, the invention relates to the use of a polymeric guanidine derivative based on a diamine containing oxyalkylene chains between two amino groups, with the guanidine derivative representing a product of polycondensation between a guanidine acid addition salt and a diamine containing polyalkylene chains between two amino groups, as well as the pharmaceutically acceptable salts thereof, for the preparation of a cytostatically active drug composition. Furthermore, the invention relates to the use of polyoxyalkylene guanidine salts produced by using triethylene glycol diamine (relative molecular mass: 148), polyoxypropylene diamine (relative molecular mass: 230) as well as polyoxyethylene diamine (relative molecular mass: 600). The polymeric guanidine derivatives used in accordance with the invention are known from PCT/ATO1/00134. By way of reference, the content of said literature is incorporated in the present specification. The preparation of a preferred representative of the compounds used in accordance with the invention as well as the detection of the cytostatic activity are described in the following. Substitutionally for the class of compounds used in accordance with the invention, the cytostatic activity of poly-[2-(2-ethoxyethoxyethyl)guanidinium hydrochloride] with an average molecular mass of 1000 D is described hereafter (CAS No. 374572-91-5). In order to prepare said compound, 4.43 moles of guanidinium hydrochloride were dissolved in 4.03 moles of triethylene glycol diamine at 50°C. Subsequently, this was heated to 120°C and stirred for 2 hours at said temperature. Thereafter, said temperature was maintained for 2 hours, then a vacuum (0.1 bar) was applied and stirring under vacuum was continued for 2 more hours at 170°C. Subsequently, this was aerated at normal pressure, cooled to 120°C and diluted with demineralized water to appx. 50%. It was neutralized to a pH of appx. 6 with phosphoric acid, allowed to cool and diluted to the desired concentration. The molecular weight was determined to be 1000 D. Statement of the Invention An embodiment of the present invention relates to A process for preparing a polymeric guanidine derivative comprising the following steps: (a) dissolving guanidinium hydrochloride in polyalkylene glycol diamine at 50°C to obtain a mixture; (b) heating the mixture of step (a) at 120° C, under stirring and maintaining under vacuum for set time; and (c) aereating the product of step (b) at normal pressure, diluting the mixture and neutralizing to a set pH to obtain the derivative. In another embodiment, the the polyalkylene glycol diamine is triethylene glycol diamine. In yet another embodiment, the stirring is carried for 2 hours. In yet another embodiment, the amount of guanidine hydrochloride is 4.43 moles and the amount of triethylene glycol is 4.03 moles. In yet another embodiment, the set time is 2 hours and the vacuum is 0.1 bar. In yet another embodiment, the neutralization is carried using phosphoric acid and the pH is 6.0. The present invention does not provide a composition, which is a mere admixture, nor it's a new use of known substance. The present invention provides a first new medical use of a substance has never been used as drug before. The present invention combines and provides a composition wherein two compounds i.e. first compound, which is a product arriving from product of polymeric guanidine derivative based on diamine oxyalkylene chains between two amino groups, with the guanidine derivative representing a product of polycondenation between a guanidine acid addition salt and second compound which is a diamine containing polyalkylene chains between two amino groups. Here the first compound is not a product of chemical reaction. Had this been a product of chemical reaction the application of the product would have been identified along with the chemical reaction itself. In other words, had this been obvious such application of the product or method by which is produced would have been known before. But there are no prior teachings. The composition of the present invention actually describes the various attributes of the first and the second compounds, which have been combined. There are no prior arts, which envisage such application of disclosed new composition in the present invention. There are no prior art teachings, wherein such composition has been envisioned. Considering this the present invention is non-obvious and novel. Establishing the cytostatic activity Cell lines of a colon carcinoma and a pancreas carcinoma, such as Capan-1, DLD-1, HT 29, HCT-8, MIA-PA-CA2, PANC1, BXPC-3, ASPC-1, and HT-29, were examined. The tested cancer cell lines were stored in liquid nitrogen. After defrosting, the cancer cells were cultivated in culture flasks with RPMI-1640 + glutamine medium (Gibco No. 5240025) at 37°C/5 % CO2 atmosphere for up to 14 days so that a monolayer of cells was able to form. Thereupon, the cells were harvested with trypsin + EDTA (Gibco No. 15400-054) and washed twice with an RPMI-medium. In addition, lymphocytes of healthy test persons were examined. In doing so, a total amount of 100 ml blood was drawn into EDTA test tubes. The lymphocytes were isolated from the full blood by means of a Mono-Poly Resolving Medium/Ficol-Hypaque gradient, were washed three times with an HBSS buffer and, prepared in this manner, they were added to the test stock. Apart from poly-[2-(2-ethoxyethoxyethyl)guanidinium hydrochloride], the chemotherapeutic agents cisplatin, epirubicin, mitomycin C and 5-fluorouracil were tested by way of comparison. Therefor, the compounds were at first dissolved according to the respective instructions of the manufacturer and were then stored as a stock solution in aliquots of 1 ml each (concentration of drug substance: 1000 ug/ml) in liquid nitrogen at -180°C. The dissolved substances were used on the very same day. In order to determine the cytotoxic activity of the substances, the cancer cells were transferred from the culture flasks with an RPMI-washed suspension at a concentration of 20.000 cells in 200 ul onto microtiter plates. In the presence of varying concentrations of the test substances, the cancer cells were subsequently incubated for three days at a temperature of 37°C and in an atmosphere of 5 % CO2. For the poly-[2-(2-ethoxyethoxyethyl) guanidinium hydrochloride], concentrations of 0.12 to 500 ug/ml (factor 2 dilutions) were used in all test arrangements. After three days of incubation, the evaluation was carried out by means of the nonradioactive cell proliferation and cytotoxicity test EZ4U (Biomedica No. BI-5000 10 x 96 determinations). After three hours of incubation with EZ4U, the evaluation was carried out photometrically in percent at a wavelength of 49/630 nm in a slide-photometer (extinction of the test sample divided by extinction of the control-sample blank value). The vitality of the lymphocytes was determined in a suspension of 1.2 x 107/ml at varying concentrations of the test substances. In the presence of the test substances, the cell suspensions were incubated for 24 hours at 37°C and in an atmosphere of 5% CC>2 and were then dyed with trypan blue. After dyeing, the lymphocytes were applied onto the counting chamber and their vitality was evaluated in percent. The drug substance poly-[2-(2-ethoxyethoxyethyl)guanidinium hydrochloride] exhibits favourable pharmacodynamic properties, along with low toxicity and good tolerance from a pharmacological point of view, and can therefore be used as a medicine in oncological therapy. The substance shows in particular an excellent cytostatic activity, as can be demonstrated by tests using several cancer cell lines, f.i. of a colon carcinoma (HT-29, HCT-8, DLD-1) or a pancreas carcinoma (ASPC-1, BXPC-3, CAPAN-1, PANC-1). In addition, the drug substance used in accordance with the invention possesses a broad therapeutic window since in healthy endogenous cells such as lymphocytes the cytostatic activity has been observed only at concentrations starting from 100 ug/ml (table 4) while in cancer cells said activity occurs already at concentrations starting from 2 to 16 ug/ml (tables 1 and 2). By way of comparison, table 3 shows the cytostatic activity of 5-fluorouracil, cisplatin, epirubicin and mitomycin C. After an (intravenous or intraperitoneal) systemic administration of poly-[2-(2-ethoxyethoxyethyl)guanidinium hydrochloride] in amounts of up to 15 mg/kg body weight, serum concentrations of up to 100 ug/ml are measured in the rat's blood after two hours, whereby, at the same time, the tolerance is good. Therefore, poly-[2-(2-ethoxyethoxyethyl) guanidinium hydrochloride] can be used as a cytostatic drug. The drug substances used in accordance with the invention can be processed to pharmaceutical preparations in a manner known per se, either alone or together with inorganic or organic pharmacologically indifferent adjuvants. Table 1. Cytostatic activity in comparison with different cell lines of a colon carcinoma (vitality in %) (Table Removed) * Poly-[2-(2-ethoxyethoxyethyl)guanidinium hydrochloride] Table 2. Cytostatic activity in comparison with different cell lines of a pancreas carcinoma (vitality in %) (Table Removed)Poly-[2-(2-ethoxyethoxyethyl)guanidiniumhydrochloride] Table 3. Cytostatic activity of standard chemotherapentic agents in comparison with different cell lines of pancreas carcinoma (vitality in %) (Table Removed)Table 4. Vitality of lymphocytes in % (Table Removed)* Poly-[2-(2-ethoxyethoxyethyl)guanidinium hydrochloride] We claim: 1. A process for preparing a polymeric guanidinium hydrochloride derivative comprising the following steps: a. dissolving guanidinium hydrochloride in triethylene glycol diamine at 50°C to obtain a mixture, b. heating the mixture of step (a) at 120°C, under stirring for 2 hours at the said temperature and maintaining the set temperature for 2 hours, c. stirring the mixture of step (b.) under vacuum (0.1 bar ) for 2 more hours at 170°C, d. aerating the mixture of step (c.) at normal pressure, cooling the mixture upto 120°C and diluting the same with demineralised water to approximately 50% and e. neutralizing the mixture of step (d.) with phosphoric acid to pH of 6.0 and allowing the mixture to cool and diluted to obtain the final product 2. A process for preparing a polymeric guanidinium derivative substantially as herein described with reference to the foregoing description.

Documents:

3718-DELNP-2005-Abstract-(30-04-2009).pdf

3718-delnp-2005-abstract.pdf

3718-DELNP-2005-Claims (26-05-2009).pdf

3718-delnp-2005-claims.pdf

3718-DELNP-2005-Correspondence-Others-(26-05-2009).pdf

3718-DELNP-2005-Correspondence-Others-(30-04-2009).pdf

3718-delnp-2005-correspondence-others.pdf

3718-DELNP-2005-Description (Complete)-(30-04-2009).pdf

3718-delnp-2005-description (complete).pdf

3718-DELNP-2005-Form-1-(30-04-2009).pdf

3718-delnp-2005-form-1.pdf

3718-delnp-2005-form-13-(30-04-2009).pdf

3718-delnp-2005-form-13.pdf

3718-delnp-2005-form-18.pdf

3718-DELNP-2005-form-2-(30-04-2009).pdf

3718-delnp-2005-form-2.pdf

3718-delnp-2005-form-26.pdf

3718-DELNP-2005-Form-3-(30-04-2009).pdf

3718-delnp-2005-form-3.pdf

3718-delnp-2005-form-5.pdf

3718-delnp-2005-pct-210.pdf

3718-delnp-2005-pct-304.pdf

3718-delnp-2005-pct-308.pdf

3718-delnp-2005-pct-311.pdf

3718-delnp-2005-pct-332.pdf

3718-DELNP-2005-Petition-137-(30-04-2009).pdf