WO1998011903A1

WO1998011903A1 - Chitosan derivatives for treating malignant tumours

Info

Publication number: WO1998011903A1
Application number: PCT/EP1997/004577
Authority: WO
Inventors: Claus Timmermans
Original assignee: Chicura Cooperatie Ua
Current assignee: Chicura Cooperatie Ua
Priority date: 1996-09-18
Filing date: 1997-08-15
Publication date: 1998-03-26
Anticipated expiration: 1999-03-18
Also published as: AU4381197A

Abstract

The invention relates to the use of a chitosan derivative comprising at least approximately 20 % of structural elements having general formula (I) wherein: R is a hydrogen atom, or a (C1-C4) alkylcarbonyl group wherein the alkyl group may be substituted with carboxy, hydroxy or (C1-C4)alkoxy; or wherein OR is a sulfated or phosphated hydroxy group; p and q are each independently 0 or 1; R1 is a straight or branched, saturated or unsaturated (C1-C16)hydrocarbylene group, optionally substituted with 1-3 hydroxy groups; and R2 represents 1-8 moieties, selected from amino acids and peptideous compounds; as well as functional derivatives thereof; for preparing a pharmaceutical composition for the treatment of malignant tumours in a warm-blooded living being. The invention further relates to a pharmaceutical composition, to new chitosan derivatives, and to their method of preparation.

Description

CHITOSAN DERIVATIVES FOR TREATING MALIGNANT TUMOURS

The invention relates to the use of chitosan derivatives for treating malignant tumours, to a pharmaceutical composition to be used therefor, as well as to new chitosan derivatives and their method of preparation.

The results of cancer control are relatively poor, in spite of the world-wide efforts in combating malignant tumours. Various types of cancer with a high incidence, such as lung and intestinal cancers, cannot be controlled effectively as yet, so that the mortality percentages are still very high. In the control of some other types of cancer indeed significant results have been obtained, as well as in the prevention of metastasation of tumours in general. A main problem in cancer control, however, is the presence of small (minor) malignant tumours, remaining behind after an apparently successful removal of the major tumours, e.g. by an operation. Therefore, it is a challenge to many research people active in this field to find substances with an increased tumoricidal or tumour-growth inhibiting activity. The tumoricidally active pharmaceuticals, generally called cytostatica, at present available and in use in hospitals and clinics for the chemotherapeutic treatment of cancer and related diseases, are generally not sufficiently effective in the dosages which are acceptable with regard to their often serious side-effects.

Tumoricidal properties are ascribed to chitosan and its oligomers. In this connection, Suzuki and coworkers (A.

Tokoro et al . ; Chem . Pharm. Bull., 1988; 26. * 784-790) have disclosed that hexa-N-acetylchitohexaose, i.e. the chitosan hexarner, presents a growth- inhibiting effect against certain solid tumours. This effect is attributed by the authors to the acceleration of the production and response to IL-1 (interleukin-1) and IL-2 for maturation of splenic T-lymphocytes to killer T-cells. It will be evident from the above, that the development of tumour-growth inhibiting substances with an increased activity and decreased side-effects is of utmost importance in cancer control. It is the objective of the present invention to provide a method of efficaciously combating tumours by using substances with improved properties for this purpose.

It has now been found, that this objective can be achieved by treating a warm-blooded living being wherein the presence of tumours has been observed or is expected, with a pharmaceutical composition comprising in a therapeuti- cally effective amount a chitosan derivative. This chitosan derivative comprises at least approx . 20% of structural elements having the general formula I

wherein :

R is a hydrogen atom, or a (C -C, ) alkylcarbonyl group wherein the alkyl group may be substituted with ca^'rboxy, hydroxy or (Ci-C,,) alkoxy; or wherein OR is a sulfated or phosphated hydroxy group; p and q are each independently 0 or 1; R, is a straight or branched, saturated or unsaturated aliphatic (C, -C_l6) hydrocarbylene group, optionally substituted with 1-3 hydroxy groups; and R represents 1-8 moieties, selected from amino acids and peptideous compounds; as well as functional derivatives thereof. It has appeared, that the above chitosan derivatives have strong tumour-growth inhibiting properties, as opposed to chitosan or its oligomers. Therefore, even at low dosages these chitosan derivatives can be used to reach an effective therapy. As will be clear from the Examples, chitosan derivatives to be used according to the invention, such as N-carboxybutyl chitosan and the lacta e of chitosan - levulinic acid - condensate (containing 2 - methylpyrrolidon-1-yl-deoxyglycose residues) , can inhibit tumour-growth with over 50% when used in acceptable dosages .

The above-defined chitosan derivatives are to be understood to comprise, in addition to derivatives of chitosan itself, derivatives of chitosan oligomers from six derivatized glucosamine residues up.

The above-defined chitosan derivative preferably consists of at least 40% of the above structural elements of formula I, most preferably of at least 60% thereof; the remainder may consist of unconverted or acetylated glucosamine residues without adversely influencing its properties.

Functional derivatives of the above chitosan derivatives comprise esters with lower alkanols, cyclic condensation products such as lactones and lactames, amides with lower alkylamides, and salts, in particular salts with basic substances such as alkali metal hydroxides or ammonia.

The term peptideous compound or peptide-like compound is to be understood to mean a compound having at least one peptide bond in its molecule, such as glutathione and its thiol-derivatives . Examples of such derivatives are S- alkylated glutathione, e.g. the S-methyl and S-ethyl ethers of glutathione, S-acylated glutathione, e.g. S- acetylglutathione, and S-sulfated and S-phosphated glutathione . Preferred chitosan derivatives to be used according to the method of the invention are derived from a chitosan oligomer having 8-30 glucosamine residues, which derivative comprises the structural elements of formula I above, wherein : R is a hydrogen atom or an acetyl group; p and q are each independently 0 or 1; R. is a straight or branched ( C_l - C ) alkylene group, and R represents 1-4 moieties, derived from glutathione, alanme, lysme or leucme; as well as salts or cyclic condensation products thereof.

Various chitosan derivatives to be used according to the present invention are known per se Muzzarelli has investigated the wound-healing properties of N-carboxybutyl chitosan (Carbohydrate Polymers 1993, 20. 7-16). In the European patent application no. 0563013 various N-subst - tuted chitosan derivatives are described, e.g as wetting (moistening) agents for the skin and mucous membrane.

Part of the above-defined chitosan derivatives, however, is new Therefore, the present invention also relates to a pharmaceutical composition to be used m the above-defined method of combating tumours, comprising m addition to a pharmaceutically acceptable carrier and optional auxilia- ries, as the active ingredient a new chitosan derivative comprising at least approx. 20% of structural elements having the general formula II

- C 0- V_jL-. O ^A ₁ t^J

wherein the symbols have the meanings given above, or its salts or cyclic condensation products. The invention further relates to the above new active ingredient per se .

The above new chitosan derivatives of the invention can be prepared in a manner known per se for related compounds, e.g. in that an N-carboxyalkylchitosan or an N- carboxyalkylcarbonylchitosan is reacted with an amino acid, a peptide or a peptideous compound, after which the deriva- tive obtained is functionalised, if desired, by a conversion into its salt or in its cyclic condensation product. The formation of the starting N- carboxyalkyl (carbonyl) chitosan can be performed, e.g. in a corresponding manner as described in U.S. patent 4,835,265, from chitosan and a keto-acid or a dicarboxylic acid, respectively, such as levulinic acid or sebacic acid, if necessary under reducing conditions. In a convenient manner said formation may be carried out simultaneously with the formation of the final chitosan derivative. Chitosan can be used as such or, if desired, partly depolymerized. The term "chitosan", as used in this patent application, comprises partly depolymerized chitosan.

The present invention is now described in more detail with reference to the following specific Examples.

Example I

Preparation of chitosan butylcarbonylglutathione.

Chitosan flakes (10 g, MW approx. 300,000, deacetylation 80%) is suspended in a solution of 5 g levulinic acid and 1 g glutathione in 1 1 water. The suspension is stirred for 6 hours to give a viscous solution. Under vigorous stirring a solution of 500 mg of NaN0₂ in 50 ml water is added to achieve partial depolymerization of the chitosan; the viscosity of the solution decreases gradually. After stirring for 30 min. the chitosan has a MW of beneath 30,000. By adding a solution of 1 g NaBH, in 50 ml water further depolymerization is stopped. The desired reactions take place under the reducing conditions of the added borohydrate. The stirring is continued until the develop- ment of gas has stopped. The sodium salt of the title product is precipitated by the addition of 10% aqueous NaOH (to pH 8.0). The product is obtained as a microcrystalline material by filtration, e.g. by using a glass filter, and is washed with water (lx) and 70% aquous ethanol (lx) . If desired, an aqueous solution of the free acid can be obtained by acidification with 10% aqueous HC1 to pH 5-6, producing a clear, slightly viscous solution. In a corresponding manner chitosan sebacoylalanine is prepared from chitosan, sebacic acid and alanine .

Example II

In vitro experiments.

The proliferation of malignant epithelial cells, isolated from mice (C26), is investigated in vitro. Two cell- densities are used: 6,000 cells/well and 12,000 cells/well. The following chitosan derivatives are investigated: chitosan sebacoylalanine, obtained according to Example I, and carboxybutylchitosan, known per se, the former substance in a 1% aqueous solution, the latter in a 2% aqueous solution. The test concentration is 4 mg of solution per ml. A photometric test method is used to determine the inhibition of the proliferation of the cells, applying MTT as the vital colouring agent. Addition of chitosan sebacoylalanine results in cell-growth inhibitions of 23 and 30%, respectively, for the above two cell- densities and compared to the blank, addition of carboxybutylchitosan in cell -growth inhibitions of 49 and 36%, respectively.

Claims

1 . Use of a chitosan derivat ive comprising at least approx . 20 % of structural elements having the general formula I

wherein :

R is a hydrogen atom, or a (C -C ) alkylcarbonyl group wherein the alkyl group may be substituted with carboxy, hydroxy or (C -C,)alkoxy; or wherein OR is a sulfated or phosphated hydroxy group; p and q are each independently 0 or 1; R- is a straight or branched, saturated or unsaturated aliphatic (C_; -C-. ) hydro- carbylene group, optionally substituted with 1-3 hydroxy groups; and R represents 1-8 moieties, selected from amino acids and peptideous compounds; as well as functional derivatives thereof;

for preparing a pharmaceutical composition for the treatment of malignant tumours in a warm-blooded living being.

2. Use according to claim 1, wherein the chitosan derivative is derived from a chitosan oligomer having 8-30 glucosamine residues, which derivative comprises the structural elements of formula I, shown in claim 1, wherein: R is a hydrogen atom or an acetyl group; p and q are each independently 0 or 1; R-_t is a straight or branched (Ci-Cjn) alkylene group; and R represents 1-4 moieties, derived from glutathione, alanine, lysine or leucine,- as well as salts or cyclic condensation products thereof.

A method of treating tumours m warm-blooded living beings, by administering to said beings a pharmaceutical composition comprising in a therapeutically effective amount a chitosan derivative as defined in claim 1 or 2.

4. A pharmaceutical composition to be used m the method of claim 3, comprising in addition to a pharmaceutically acceptable carrier and optional auxiliaries, as the active ingredient a chitosan derivative comprising at least 20% of structural elements having the general formula II

wherein the symbols have the meanings given in claim 1 or 2, or its salts or cyclic condensation products.

A chitosan derivative comprising at least 20% of structural elements having the general formula II, shown in claim 4, wherein the symbols have the meanings given in claim 1 or 2; as well as its salts or its cyclic condensation products. A method of preparing a chitosan derivative according to claim 5, characterized in that an N-carboxyalkyl- chitosan or an N-carboxyalkylcarbonylchitosan is reacted with an amino acid, a peptide or a peptideous compound, if necessary under reducing conditions, after which the derivative obtained is functionalised, if desired, by a conversion into its salt or in its cyclic condensation product .