Patent Application
20080051350
The following examples are merely illustrative and do not represent a limitation on the scope of this invention.
1-A: Preparation of Inulin Polysulphate Sodium Salt Using Chlorosulphonic Acid/Pyridine
Under constant stirring, at a temperature <6° C., 88 mL (1.32 mol; 1.8 eq/OH) of chlorosulphonic acid were added drop by drop to 580 mL (7.20 mol) of dry pyridine. The resulting mixture was heated to 75° C. and, subsequently, 40 g (0.25 mol) of inulin (Fibruline Standar, marketed by Trades S.A.) were added. The stirring and the heating to 100° C. were continued for 5 h. Once this time elapsed, the reaction mixture was allowed to cool down to approximately 50° C. and 50 mL of de-ionised water were added in order to destroy the chlorosulphonic acid excess. During this step, the ambient temperature was slightly increased and two phases appeared.
The crude reaction product was allowed to rest until it reached ambient temperature. The upper phase (pyridine) was separated by suction, while the oily lower phase was poured over a 10% solution of sodium acetate in methanol. The precipitate that was formed was allowed to sediment. The supernatant was separated by decanting and discarded.
Treatment with the sodium acetate methanolic solution was repeated twice and, in the last step, the solid was separated by vacuum filtering by means of a type k-100 depth filter (Pall Corporation. Seitz-k-100).
The filter produced (brown solid) was dissolved in distilled water and the resulting solution was vacuum filtered by means of a k-100 depth filter in order to eliminate the insoluble matter remainders resulting from the reaction.
The filtrate resulting from the preceding step was a dark amber colour solution which was treated with quaternary ammonium (Quartamin). As a consequence, a very abundant solid was formed which was isolated by vacuum filtering using a Hyflo earth pre-layer (100 g). The filter was abundantly washed with distilled water
The inulin polysulphate-Quartamin complex was carefully separated from the filtering earths and treated with a 20% NaCl aqueous solution, at 80° C., for two hours.
Once the two hours of reaction elapsed, the heating was stopped, and when the ambient temperature reached 60° C., isopropanol was added. Stirring was maintained for 30 min. and, subsequently, the mixture was poured into a decanting funnel in order to separate the aqueous phase from the organic one.
The organic phase (isopropanol+quaternary ammonium) was discarded. The aqueous phase was vacuum filtered in order to eliminate the Hyflo earth remainders.
A mixture of methanol/acetone was poured over the aqueous phase. The brown precipitate that was formed was allowed to sediment. The supernatant was separated by decanting and discarded. The sediment was washed with methanol and subsequently dissolved in de-ionised water.
The pH of the resulting solution was adjusted between 10.5-11 with 10% NaOH. The solution was heated to 50° C. and treated with H2O2 for 15 min. Finally, the heating was stopped and the pH of the medium was adjusted to 5.5 with a 2% acetic acid solution.
When the discoloured solution reached ambient temperature, a 10% methanol/sodium acetate solution was poured thereon. A white or slightly yellow precipitate immediately formed which was allowed to sediment.
The supernatant was separated by decanting and the sediment was abundantly anhydrated with methanol.
The inulin sulphate was separated by vacuum filtering with a No. 3 porous plate and dried in a vacuum heater at 30° C. until the methanol concentration was equal to or lower than 0.3%.
The product was produced in the form of a fine, amorphous powder, white or slightly yellow in colour.
Exactly 150 mg of the product were weighed, they were dissolved in water, and the resulting solution was taken to 250 mL with the same solvent. 5 mL of this solution were pipetted, they were transferred to a 50 mL beaker, and 25 mL of water were added. It was photometrically assessed at 420 nm, with a 0.1% (0.00279 M) N-cetylpyridinium chloride (CPC) solution.
The sulphate content was calculated using the following equation:
1-B: Preparation of Inulin Polysulphate Sodium Salt Using Chlorosulphonic Acid/Dimethylformamide (DMF)
In a three-neck reactor (2 L), having a thermometer and two addition funnels, 1,000 mL of dry DMF were added under a nitrogen current. The solvent was cooled to a temperature of <6° C., and 112 mL (1.68 mol; 1.8 eq/OH) of chlorosulphonic acid were added drop by drop under constant stirring.
The reactor temperature was allowed to reach 15° C., and, subsequently, a solution of 52.6 g of inulin in 400 mL of dry DMF was added. The reaction was maintained at ambient temperature for less than 3 h.
Once this time elapsed, the reaction was poured over cold water. The pH of the resulting solution was increased to 7-8 with 30% NaOH and, subsequently, the solution was poured over a 10% solution of sodium acetate in methanol. The solid that precipitated was allowed to sediment and was separated from the supernatant.
The solid was separated by vacuum filtering through a Büchner funnel. It was dissolved in 1,500 mL of process water and the solution was treated with 350 mL of quaternary ammonium (Quartamin). The white solid that was formed was isolated through vacuum filtering using a Hyflo earth pre-layer (100 g).
The solid retained in the filter, jointly with the Hyflo earth pre-layer, were transferred to a reactor endowed with coolant and mechanical stirring, to which 3,000 mL of a 20% NaCl solution were added. The mixture was stirred at 80° C. for 6 h and it was subsequently vacuum- and heat-filtered, using filter paper. The filtrate (A) was saved, and the solid retained in the Büchner funnel was transferred once again to the reactor in order to repeat the complex breakdown step under the same exact conditions. The filtrate (B) which was produced from this repetition was mixed with (A), and the residual solid was discarded.
3.3 volumes of a methanol/acetone mixture were poured over the solution produced from the union of filtrates (A) and (B). The solid which precipitated was allowed to sediment and was isolated by suction of the supernatant. The sediment was washed with methanol, vacuum-drained through a Büchner funnel, and dissolved in process water. The NaCl content in this solution was determined and adjusted to 10% (w/V).
The precipitation step using methanol/acetone was repeated, and the solid deposited on the bottom of the contained was dissolved in de-ionised water.
The sodium chloride concentration in this solution was adjusted to 2% (w/V) and, subsequently, 2 volumes of methanol were added. The mixture was allowed to rest. The product was deposited on the bottom of the container. The supernatant was separated by suction and the product was dissolved in process water in order to obtain a solution with an approximate concentration of 5% (w/V). This solution was diafiltered through a regenerated cellulose membrane MWCO=5,000-1,000 Da. The permeate generated during this step was periodically assayed with AgNO3. The diafiltering was considered to be finished when the chloride assay gave a negative result.
The diafiltered solution was lyophilised. The lyophilate that was produced was ground and sieved. The inulin polysulphate sodium salt was produced in the form of an amorphous powder, white or slightly yellow in colour.
The cartilage's resistance and repair capacity are determined by the proteoglycans of the extracellular matrix, particularly the aggrecans. Synthesis of these aggrecans by the articular chondrocytes, and their quality, diminishes with age, which is one of the main factors involved in the development of arthrosis.
This procedure may be applied to the evaluation of any sulphated polysaccharide of this invention.
1A: Aggrecan Levels Determined by the Incorporation of 35S
Materials and Methods
Human articular chondrocytes were isolated following the methods described by W. T. Green Jr. (Clin. Orthop., 75, 248-260 (1971)) and K. E. Kuettner et al. (J. Cell. Biol., 93, 743-750 (1982)).
These chondrocytes were cultured in an agarose gel following the method described by P. D. Benya et al. (Cell, 30, 215-224, (1982)), and modified by G. Verbruggen et al. (Clin. Exp. Rheumatol., 8, 371-378 (1990)) and by M. Cornelissen et al. (J. Tiss. Cult. Meth., 15, 139-146 (1993)).
The synthesis of aggrecans was determined by the incorporation of 35S, using labelled sodium sulphate Na235SO4 as radioactive precursor. After two weeks of culture, 10 μCi/mL of radio-labelled precursor were introduced in the culture medium for 48 h, as was the compound to be assayed (inulin polysulphate in Chemical Example 1) at different concentrations (0.0, 0.1, 0.5, 1.0, 5.0 μg/mL).
The synthesised 35S aggrecans once again partially accumulated in the intercellular agarose matrix or else were released in the incubation medium.
Once the incubation period ended, the agarose gel was mechanically broken down and subsequently digested by means of 3 mL of a 50 U/mL agarose solution in a 0.067 M phosphate buffer, at pH 6.0, in the presence of proteinase inhibitors.
The suspension thus produced was centrifuged; the supernatant containing the inter-territorial matrix's 35S aggrecans and the incubation medium containing the 35S aggrecan metabolites released in the extracellular matrix were subsequently united by means of chromatography.
The residue, which contained the chondrocytes and the associated 35S aggrecans, was treated for 48 hours with 1 mL of a 4.0 M guanidium chloride solution in a 0.05M acetate buffer at pH 5.8 containing the proteinase inhibitors.
The purpose of this operation is to extract the 35S aggrecans associated with the cells. The solution produced was centrifuged in order to separate the cells from the supernatant, which was subsequently separated by means of chromatography. The chromatography operations for the different fractions produced were performed on Sephadex G25 gel in a phosphate buffer pH 6.8 containing 0.01 M of Na2SO4, in order to separate the 35S aggrecans from the free Na235SO4.
The radioactivity of each of the macromolecular eluants produced was measured and related to the number of chondrocytes contained in the initial culture, expressed in pg of 35SO4 incorporated into the aggrecans, per million chondrocytes per hour.
Results
They are shown in Tables 1, 2, and 3.
The tables show the remarkable effectiveness of the inulin polysulphate in Chemical Example 1, in the total production of re-synthesised aggrecans (Table 3). The inulin polysulphate in Chemical Example 1 is also capable of increasing production of aggrecans in the inter-territorial matrix (Table 1) as well as of the aggrecans associated with the cells (Table 2).
The optimum activity is found at a concentration of about 0.5 μg/mL.
1B: Aggrecan Levels Determined by Means of Immunocytochemistry
Materials and Methods
The human articular chondrocytes were isolated and cultured following the previously described methods.
The accumulation of aggrecans associated with the chondrocytes was measured, after a week of culture, by means of the immunocytochemistry technique described by L. Wang et al. (Osteoarthritis Cart., 9, 248-260 (2001)). The chondrocytes were treated with interleukin-1 (IL-1) in order to simulate a situation of inflammation and catabolism. The inulin polysulphate in Chemical Example 1 was added jointly with IL-1.
Once the incubation period ended, the chondrocytes were released from the agarose gel following the described method. The chondrocytes and the associated aggrecans were studied by means of flux cytometry using monoclonal antibodies specifically directed against the aggrecans' proteic part.
Results
They are shown in Table 4.
This Table confirms the effectiveness of the inulin polysulphate in Chemical Example 1 on the production of aggrecans associated with the cells.
Moreover, it can be seen that there is a dose-effect relationship, since, with greater doses of the compound, there is a greater increase in the production of aggrecans associated with the cells.
This procedure may be applied to the evaluation of any sulphated polysaccharide of this invention.
Materials and Methods
The assays were performed on chondrocyte cultures from a rat chondrosarcoma cell line called LTC.
The LTC cells were kept in a monolayer culture in a Gibco DMEM medium (Dulbecco's Modification of Eagle's Medium) (containing 4.5 g/L of glucose) supplemented with sodium bicarbonate (3.7 g/L), glutamine (2 mM), ascorbic acid (50 mg/L), gentamicin (50 mg/L), and bovine fetal serum Hyalone (10%), at pH 7.4.
The confluent LTC cell cultures were trypsinised and 40,000 cells per 0.5 mL of culture were seeded in 48-well plates. They were maintained for 5 days, during which time they deposited 15-30 μg of glycosaminoglycans (GAG) in each well.
The medium was separated, the cell membranes were washed by adding 5×1 mL of a catabolic medium {DMEM+4.5 g/L glucose supplemented with sodium bicarbonate (3.7 g/L), glutamine (2 mM), and 10 ng/mL of IL-1α (interleukin-1α) at pH 7.4). Subsequently, the cultures were kept in 200 μL of this medium, supplemented or not with inulin polysulphate sodium salt in Chemical Example 1, for 4 days, without changing the medium, at 37° C.
In order to verify inulin polysulphate sodium salt's effect on aggrecan degradation, a Western Blot was performed.
For the Western Blot, 20 μL of 50 mM tris-acetate at pH 7.3 were added to each well. The cultures (medium+cell membrane) were deglycosylated with 50 mU of chondroitinase ABC at 37° C. for 4 hours. Subsequently, the samples in each well were recovered and centrifuged at 1,460 g for 10 minutes. 25 μL-fractions of the supernatant were separated by electrophoresis (gel gradient 8-12% SDS PAGE) and analysed by means of a Western Blot using anti-aggrecan antibody G1. G1 is the product of the aggrecanase activity, also verified by immunoreactivity with the anti-NITGE neo-epitope-specific antibody. The antiserum was used at a 1:5,000 dilution and the peroxidase-conjugated goat anti-(rabbit IgG) was detected by means of the Amersham ECL Kit.
Results
They are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| P 200400464 | Feb 2004 | ES | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP05/01390 | 2/11/2005 | WO | 00 | 8/23/2006 |
