DE60317624T2 - New chitosans - Google Patents

Description

BACKGROUND

chitin is a very insoluble N-acetylated polymer of β- (1,4) -D-glucosamine. Chitosan is an acid-soluble, deacetylated form of chitin. Chitin is commonly found in exoskeletons of invertebrate marine organisms and the cuticle of Insects. In the cell walls Most Zygomycetes species are also chitin and chitosan available.

chitosan can be obtained by deacetylation of chitin derived from crab or shrimp shells is made. However, this process creates no chitosan of consistent quality. The composition of Crab or shrimp shells is also dependent heavily dependent on seasonal and environmental factors. It is therefore difficult from crab or shrimp chitosan with consistent physiochemical To get properties. Chitosan can also be made from a filamentous fungus the Mucoraceae family. In this method is no chemical deacetylation required. As a result, the quality of mushroom chitosan more consistent.

The US-A-2 255 085 provides a method of producing chitin or chitosan by cultivating a Rhizopus azygosporus fungus or an Actinomucor taiwanensis fungus and isolating chitosan or chitin from the culture.

The WO 02/34287 provides a method and formulation for inducing cytotoxic T cell responses to a polypeptide antigen of choice. The formulations are characterized by containing chitosan mixed with the polypeptide antigen, preferably in the form of microparticles, which may be cross-linked.

The EP-A-0 531 991 provides a method for producing chitosan according to the present invention, wherein chitosan is prepared by inoculating a filamentous fungus belonging to the family Mucoraceae into a culture medium for preculture, containing the yeast extract, malt extract, peptone, glucose and magnesium to carry out the preculture, and then inoculating the preculture into a culture medium to the main culture having a composition substantially identical to that of the medium for the preculture to perform the main culture.

SUMMARY

The The present invention relates to a novel chitosan, the preparation of chitosan from a mushroom and the use of chitosan to Treatment of cancer.

One Chitosan according to the invention has an average molecular weight (weight average) of 50,000 to 140,000 g / mol and a degree of deacetylation of 85 to 95%.

The weight average molecular weight ( _Mw ) is the first moment of a plot of the weight of the polymer in each molecular weight range versus molecular weight. It is calculated as follows:

The summations are made over all polymer molecules (i = 1 to i = ∞) of different sizes, and N _i is the number of molecules (in moles) whose weight is M _i . The value of M _w can be z. B. by light scattering, Sedimentierungsgleichgewichts measurements or any other method are known, which are known in the art.

Deacetylation involves the removal of acetyl groups from the chitin molecular chain, leaving an amino group (-NH ₂ ). The Deacetylierungsgrad (DD), which stands for the content of polysaccharides on free amino groups, z. Example by Ninhydridtest, linear potentiometric titration, colloidal titration, near-infrared spectroscopy, nuclear magnetic resonance spectroscopy, hydrogen bromide titrimetry, infrared spectroscopy roscopy, UV spectrophotometry, first derivative UV spectrophotometry, enzymatic determination, or any other method known in the art.

Chitosan according to the invention can z. B. made from a fungus. To suitable mushrooms belong Mushrooms of the family Mucoraceae, such as those of the genus Actinomucor. A special example of a usable according to the invention Mushroom is Actinomucor taiwanesis. Chitosan can by breeding a Fungus culture at a temperature of 15 to 24 ° C and isolating chitosan obtained from the culture.

chitosan can be in a pharmaceutical composition with a pharmaceutical acceptable vehicle used for the treatment of cancer. The invention includes according to one Aspect of a method of treating breast cancer. The procedure involves the administration of an effective amount of a chitosan, z. A chitosan of average molecular weight (Weight average) of 50,000 to 140,000 g / mol and a degree of deacetylation from 85 to 95% to a subject / person who needs it. The invention includes according to a Another aspect is a method of treating cervical cancer (Cervical cancer) by a person / subject who / that required, an effective amount of a chitosan according to the invention is administered.

The The present invention provides a novel chitosan with anti-cancer effects. The details of one or more embodiments of the invention are in the subsequent appended Description explained. Other Features, objects and advantages of the invention will become apparent from the Description and also from the claims.

DETAILED DESCRIPTION

The Invention is based on the unexpected finding that made Actinomucor taiwanensis chitosan produced the growth of human Cervical epithelioid carcinoma cells (HeLa) and human breast carcinoma cells (MCF-7) inhibited in vitro.

The The present invention accordingly includes a method of manufacture a chitosan from a fungus of the genus Actinomucor. In which Method, a fungal culture is grown at a temperature of 15 to 24 ° C and a chitosan isolated from the culture.

mushrooms belonging to the genus Actinomucor to the family Mucoraceae. These mushrooms can be from different public accessible Mushroom picking points are obtained. The mushroom (Actinomucor taiwanensis), used in the examples described below is for example available on request from the Bioresource Collection and Research Center (BCRC, Catalog No. BCRC31159), Food Industry and Research Development Institute, No. 331, Shih-Ping Road, Hsinchu 300, Taiwan Republic of China, available.

The fungi can be cultured according to methods well known in the art. For example, YM agar can be inoculated with a fungus and the inoculated agar incubated for 3 to 6 days at 25 to 37 ° C. Spores obtained from the fungus are suspended in liquid to obtain a stock solution of 10 ⁴ to 10 ⁷ colony forming units (cfu) / ml. This stock solution is inoculated directly into a fermentation medium.

The fermentation medium has an initial pH in the range of 3 to 6 (eg, between 3 and 5) and can be 5 to 50 g / L (eg, 20 g / L) of glucose, 5 to 60 g / L (eg 10 g / L) peptone, 0.1 to 5 g / L (eg 1 g / L) yeast extract or other suitable ingredients. The medium may further comprise 0.01 to 30 g / L (NH ₄ ) ₂ SO ₄ , 0 to 3 g K ₂ HPO ₄ , 0 to 3 g NaCl, 0 to 15 g MgSO ₄ .7H ₂ O and 0 to 0, 3 g / L CaCl ₂ included. The fungus is cultured for a further 2 to 4 days in the fermentation medium at 15 to 24 ° C.

Chitosan can be obtained by alkali and acid treatment, as in the US-B1-6,255,085 is isolated from mushroom mycelium and purified. The cell mass is separated from the fermentation broth and washed with distilled water. The cells are then treated with 0.5 to 2N NaOH and the alkaline mixture is incubated at 121 ° C for 15 minutes. The solid material is then pelleted by centrifugation and washed with distilled water and ethanol. The washed material is treated with 2% acetic acid solution and incubated for 12 hours at 95 ° C. The resulting slurry is then isolated by centrifugation, yielding an acid-soluble supernatant. The pH of the supernatant is adjusted to 10 with 2N NaOH, whereby the chitosan precipitates. The purified chitosan is finally washed with distilled water and lyophilized.

The weight-average molecular weight ( _Mw ) and degree of deacetylation (DD) of chitosan may be e.g. Example, by the method GPC / SEC ³ or first derivative UV spectrophotometry or by any other method are known, which is known in the art. As described in the following examples, the M _w and DD of the fungal chitosan prepared by the above method are 50,000 to 140,000 g / mol and 85 to 95%.

The Mushroom chitosan unexpectedly exhibits potent cytotoxic activity the growth of HeLa and MCF-7 cells in a concentration of 10 ppm (about 10 during Crustacean chitosan does not show this effect. The mushroom chitosan also has no cytotoxicity against human embryonic lung cells (MRC-5) or other tumor cell lines on how the human gastric carcinoma cell line (AGS) and human Hepatoma cell line (Hep G2).

The present invention accordingly comprises a pharmaceutical composition containing a pharmaceutically acceptable carrier and an effective amount of a chitosan having a weight average molecular weight of 50,000 to 140,000 g / mole and a degree of deacetylation of 85 to 95%, the Chitosan is made from a fungus of the genus Actinomucor. The pharmaceutical composition can be used for the prevention and treatment of breast or cervical cancer. The pharmaceutically acceptable carrier includes a solvent, a dispersion medium, a coating, an antibacterial and antifungal agent, and an isotonic and absorption delaying agent. An "effective amount" is the amount required to achieve a therapeutic effect. The relationship of cans for animals and humans (based on milligrams per m ² body surface area) is described in Freireich et al., (1966) Cancer Chemother. Rep., 50: 219. The body surface may be determined approximately from the height and weight of the person / subject. See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardley, NY, 1970, page 537. Effective dosages also vary, as known to those skilled in the art, depending on the route of administration, the use of excipients, and the like.

One Chitosan according to the invention can according to conventional methods to dosage forms for different Routes of administration are formulated. It can for example a capsule, a gel seal material or a tablet for oral Administration formulated. Capsules can be any pharmaceutical contain acceptable standard materials, such as gelatin or cellulose. Tablets can according to conventional Process by pressing chitosan with a solid support and formulated with a lubricant. Examples of fixed carrier belong Strength and sugar bentonite. A chitosan according to the invention can also be in the form a hard coat tablet or a capsule to be administered a binder, for example lactose or mannitol, a conventional one filler and a tabletting agent. The pharmaceutical composition can be administered by the parenteral route become. Examples of parenteral Dosage forms include aqueous Solutions, isotonic saline solution or 5% glucose solutions the active ingredient or other well-known, pharmaceutically acceptable Excipient. Cyclodextrins or other solubilizing agents that Professionals familiar with this sector can act as pharmaceutical adjuncts be used to deliver the therapeutic agent.

The Effectiveness of a composition according to the invention can be evaluated both in vitro and in vivo. See, for example, For example, the following examples. The composition can be short on her ability for inhibiting the growth of breast or cervical cancer cells be tested in vitro. The composition may be for in vivo studies be injected into an animal, and then their cancer-inhibiting effects rated. Based on the results, a suitable dosing range and route of administration.

One Method for the prevention and treatment of breast cancer or cervical cancer belongs also to the scope of the invention, wherein a person / subject, which requires an effective amount of a chitosan (eg. the said mushroom chitosan) is administered. A person to be treated / a person to be treated Subject may be a cancer patient or an individual who is through genetic or environmental factors carries the risk of breast or cervical cancer to develop. This procedure may be alone or with other medicines or other therapy become.

The Use of a chitosan that has an average molecular weight Weight average) of 50,000 to 140,000 g / mol and a degree of deacetylation from 85 to 95% has, for the preparation of a drug for treatment breast cancer or cervical cancer, wherein the chitosan is from a fungus the genus Actinomucor is also part of the scope of Invention.

Generally, a chitosan is dissolved in a pharmaceutically acceptable carrier (e.g., physiological Saline) and administered orally or by intravenous infusion, or injected or subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrially, intratracheally or intrapulmonaryly. The dose required depends on the choice of route of administration, the nature of the formulation, the nature of the person / subject's disease, the size, weight, surface area, age and sex of the person / subject, the administration of other medicinal products and the Judgment of the attending physician. Suitable dosages are in the range of 0.01 to 100.0 mg / kg. Considering the variety of available chitosans and the different efficacies of the different routes of administration, large variations in the required dose are to be expected. For example, oral administration is expected to require higher dosages than administration by intravenous injection. Variations of these dosage levels may be adjusted according to standard empirical routines for optimization, as known in the art. Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) can increase the efficiency of delivery, particularly in oral delivery.

The The following specific examples are intended to be illustrative only and not the rest of the disclosure in any way restrictive become. It is believed that a professional based on the Here given description without further explanation, the present invention in its full extent.

Production of mushroom chitosans

(1) Production of mushroom chitosan A

Sporensuspension of Actinomucor taiwanensis from 4-day slant cultures was inoculated directly into 1 L shake flasks containing 400 ml of fresh fermentation medium. The fermentation was carried out for 50 hours with shaking at 200 revolutions / minute at a temperature of 22 ° C. Each liter of medium contained 10 g of peptone, 20 g of glucose, 1 g of yeast extract, 5 g of (NH ₄ ) ₂ SO ₄ , 1 g of K ₂ HPO ₄ , 1 g of NaCl, 5 g of MgSO ₄ .7H ₂ O and 0.1 g CaCl ₂ . The initial pH was adjusted to 4.0.

The Cell mass was recovered from the fermented broth and 15 minutes long with 1 N sodium hydroxide solution at 121 ° C treated. The alkali-insoluble Material was in 2 acetic acid and the mixture was incubated for 12 hours at 95 ° C, to solubilize the chitosan. The solubilized chitosan was precipitated by adjusting the pH of the acid soluble supernatant to 10. Of the Precipitate (fungal chitosan A) was then washed and dried.

(2) Production of mushroom chitosan B

Sporensuspension of Actinomucor taiwanensis from 4-day slant cultures were inoculated directly into 5 L fermentors containing 3 L of fresh fermentation medium. The fermentation was carried out for 50 hours at a temperature of 22 ° C, mixing at 400 rpm and aeration of 3 L / min. Each liter of medium contained 10 g of peptone, 20 g of glucose, 1 g of yeast extract, 5 g of (NH ₄ ) ₂ SO ₄ , 1 g of K ₂ HPO ₄ , 1 g of NaCl, 5 g of MgSO ₄ .7H ₂ O and 0.1 g CaCl ₂ . The initial pH was adjusted to 4.0.

The Cell mass was separated from the fermented broth, and the chitosan (Fungal chitosan B) was prepared as described above in section (1).

Antitumor effects of fungal chitosans

HeLa cells (BCRC60005) and MCF-7 cells (BCRC60436) were obtained from the Bioresource Collection and Research Center, Taiwan, ROC. The cell suspension was trypsinized with 0.05% trypsin / 0.53 mM EDTA · 4Na in Hank's salt solution (Hanks Balanced Salt Solution, HBSS), disaggregated by pipetting and counted with a hemacytometer. The cells were resuspended in MEM medium (with 10% serum) at 8.35 x 10 ³ (for HeLa) or 1.67 x 10 ⁴ (for MCF-7) cells per ml. The cell suspension (180 μl) was inoculated into a 96-well tissue culture plate and incubated at 37 ° C in a 5% CO ₂ atmosphere for 17 to 20 hours.

Into the wells were added various chitosan solutions (in Dulbecco's phosphate buffered saline (D-PBS) containing 5 × 10 ^-3 M acetic acid, 20 μl each), and the cells were placed in a 5% CO ₂ atmosphere for 3 days incubated at 37 ° C. The final concentration of each chitosan was 10 ppm (about 10 ^-7 M).

The viability of the cells was analyzed by the MTT- (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) method. 20 μl of 5 mg / ml MTT solution in D-PBS were added to each well and the cells were incubated for 4 hours in a 5% CO ₂ atmosphere at 37 ° C. Then the medium was removed. 100 μl of DMSO was added to each well and the plate was shaken for 5 minutes. The optical density of each well was then measured at both a test wavelength (540 nm) and a reference wavelength (690 nm).

To The chitosans used in these studies included mushroom chitosan A, mushroom chitosan B, crab shell chitosan I, crab shell chitosan II, crab shell chitosan III and crab shell chitosan IV.

Of the Acetylation level of each chitosan was through the first derivative UV spectrophotometry, as described in Tan et al. (1998) Talanta 45: 713-719 is described.

The molecular characteristics of each chitosan were determined by triple detection GPC / SEC ³ . The chitosan samples were prepared at 2.5 mg / ml in a mobile phase and heated to 90 ° C for 3 hours. The mobile phase was 0.35 N acetic acid, 8.2 g / L sodium acetate, pH 4.1, and the flow rate was 0.7 ml / min. The sample injection volume was 100 μl and the SEC ³ separation was performed on a TSK GPC column GMPW _XL from Tosoh, Japan. The detectors used for the triple detection analysis were a refractive index detector (Waters Co.), a differential viscometer (Viscotek Model T60) and a light scattering detector (Viskotek Model T60). The data were acquired using the three detectors simultaneously and processed with TriSEC software.

The weight average molecular weight (M _w ), the number average molecular weight (M _n ), the polydispersity index (M _w / M _n ), the radius of gyration (Rgw), the intrinsic viscosity (weight average) ([η] w) and the degree of acetylation ( DD) of each chitosan are listed in Table 1 below. Table 1 Molecular characteristics and degree of deacetylation for chitosans used for anti-tumor efficacy assays chitosan M _w × 10 ^-3 (g / mol) M _n × 10 ^-3 (g / mol) M _w / M _n Rgw (nm) [η] _w (ml / g) DD (%) Mushroom chitosan A 116.5 58.0 2.01 18.0 1.70 90.1 Mushroom Chitosan B 94.4 89.4 1.06 11.0 0.44 87.0 Crab shell chitosan I 317.2 286.3 1.12 41.1 6.40 79.7 Crab shell chitosan II 163.5 115.3 1.42 28.9 4.66 83.5 Crab shell chitosan III 140.7 80.5 1.75 24.7 3.56 78.1 Crab shell chitosan IV 88.5 48.6 1.83 17.1 1.90 78.5

• a. jeder Prozentsatz steht für das durchschnittliche prozentuale Zellwachstum (%) von 4 oder 6 Wiederholungen und die Standardabweichung.
• b. prozentuales Zellwachstum = (Zahl der Chitosanbehandelten Zellen/Zahl der Kontrollzellen) × 100%.

It was unexpectedly found that both HeLa cells and MCF-7 cells were sensitive to fungal chitosans (A and B), but not to crab shell chitosans (I to IV). The fungal chitosans (A and B) selectively inhibited tumor growth with inhibition ratios greater than 30% (see Table 2 below). On the other hand, the fungal chitosans (A and B) did not significantly inhibit the growth of human embryonic lung cells (MRC-5) or other tumor cell lines, such as human gastric carcinoma cell line (AGS) and human hepatoma cell line (Hep G2). Table 2 Effects of different chitosans on the growth of HeLa cells and MCF-7 cells Percent cell growth (mean ± standard deviation) ^{a, b} rehearse n = HeLa cells MCF-7 cells Control (no chitosan) 6 100 ± 4 100 ± 5 Mushroom chitosan A 6 65 ± 3 57 ± 6 Mushroom Chitosan B 6 69 ± 2 69 ± 4 Crab shell chitosan I 4 98 ± 11 96 ± 3 Crab shell chitosan II 4 95 ± 7 98 ± 7 Crab shell chitosan III 4 94 ± 9 92 ± 6 Crab shell chitosan IV 4 99 ± 5 98 ± 3

• a. each percentage represents the average percentage cell growth (%) of 4 or 6 repetitions and the standard deviation.
• b. % cell growth = (number of chitosan-treated cells / number of control cells) × 100%.

Claims (10)

Chitosan with an average molecular weight from 50,000 to 140,000 g / mol and a degree of deacetylation of 85 to 95%, the chitosan from a fungus of the genus Actinomucor has been produced. Pharmaceutical composition containing chitosan according to claim 1 and a pharmaceutically acceptable carrier. Use of chitosan, which is an average Molecular weight of 50,000 to 140,000 g / mol and a degree of deacetylation from 85 to 95% has, in the manufacture of a drug for the treatment of Breast cancer, wherein the chitosan from a fungus of the genus Actinomucor has been produced. Use according to claim 3, wherein the chitosan from Actinomucor taiwanesis. Use of chitosan, which is an average Molecular weight of 50,000 to 140,000 g / mol and a degree of deacetylation from 85 to 95%, in the manufacture of a medicament for Treatment of cervical cancer (Cervical cancer), wherein the chitosan from a fungus of the genus Actinomucor has been produced. Use according to claim 5, wherein the chitosan from Actinomucor taiwanesis. Process for the preparation of chitosan from a Mushroom, the method comprising: growing the culture of the fungus of the genus Actinomucor at a temperature of 15 to 24 ° C and isolation of Chitosan from the culture. The method of claim 7 wherein the fungus is Actinomucor taiwanesis is. Chitosan obtained by the process according to claim 7 or claim 8 has been prepared, wherein the chitosan a average molecular weight of 50,000 to 140,000 g / mol and having a deacetylation degree of 85 to 95%. Pharmaceutical composition according to claim 2, Chitosan was made from Actinomucor taiwanesis is.