WO1992008741A1

WO1992008741A1 - Method for preparing compounds for gradually releasing glucosamine oligomers, compounds thereby obtained and uses thereof

Info

Publication number: WO1992008741A1
Application number: PCT/FR1991/000908
Authority: WO
Inventors: Alain Domard; Noël CARTIER
Original assignee: Centre National De La Recherche Scientifique
Priority date: 1990-11-19
Filing date: 1991-11-18
Publication date: 1992-05-29
Also published as: FR2669340B1; FR2669340A1

Abstract

Glucosamine oligomers having relatively low degrees of polymerization are provided as active principles or agents in a treatment. It has been shown that by allowing such compounds to rest once isolated, a mixture can be obtained which includes, firstly, at least one isomolecular aggregate which directly and physically combines several molecules of the basic oligomer, whose total molar mass is a multiple of that of said basic oligomer, and secondly, the isolated form of the same oligomer, i.e. in an unaggregated state. In solution, this mixture leads to a pseudo-equilibrium between the above-mentioned components. Therefore, the method provides a controlled-release form of the glucosamine oligomers.

Description

COMPOUNDS WITH PROGRESSIVE RELEASE OF GLUCOSAMINE OLIGOMERS, PREPARATION METHOD AND APPLICATIONS In general, the present invention relates to glucosamine oligomers, as active agents or principles in various applications, in particular therapeutic, cosmetic, food and dietetics, and treatment of plants and plants.

The term “glucosamine oligomer” is understood to mean a polymer of the latter, having a degree of polymerization less than or equal to 100, distinguishing it from polymers of higher molecular weight, which can be assimilated to practically completely N-deacetylated chitosans.

We sense all the interest and the action of the glucosamine oligomers, when we know that in many applications of chitosan, it is in fact the degraded forms of the latter, corresponding to a reduction in the degree of polymerization, which are actually active. Thus, we will refer to the following documents:

- Document WO-A-8907395 demonstrating that a chitosan has a beneficial effect on plants and plants, in particular their growth, their resistance to frost, etc.

- the publication of MM. Thomas CHANDY and Chandra P. SHARMA, entitled "Chitosan as a biomaterial", BIOMAT. , ART. CEL LS,

ART.ORG., 18 (1), 1-24 (1990), which indicates the favorable role of chitosans, in particular for epithelization, in particular of periodontal tissues, to treat liver disorders, bone diseases.

In the present discussion and description, the term "chitosan" will be reserved, without further qualifying addition, for partially

(but never completely) N-deacetylated chitin. In the description below, when the chitosan is completely deacetiated, this will be said expressly.

Various documents have described, and it is known to obtain glucosamine oligomers, according to the following general process:

- obtaining a completely deacetiated chitosan; see publication of MM. DOMARD and RINAUDO, having for title "Preparation and characterization of fully deacetylated chitosan", In 3. Biol.Macromol., 19S3, Vol 5, February

- then hydrolysis or enzymatic degradation of the completely N-de-sacetylated chitosan, to obtain a mixture of glucosamine oligomers, of different degrees of polymerization; separation by chromatography of this mixture, to obtain different fractions of oligomers; isolation of oligomers from different separate fractions; cf "Glucosamine oligomers: 1. Preparation and characterization", Int. J. Biol, Macromol., 1989, Vol I, October.

In all the applications explained above, using glucosamine oligomers, a central problem, as for chitosan, concerns the lifetime of said oligomers, as active agents or principles. However, this problem is in the present case exacerbated by the relatively low molecular weight of the oligomers considered. It is known in fact that these oligomers can be quickly entrained in solution, degraded, altered under the effect of different physical, chemical agents, etc. And it is therefore essential to find a form of these oligomers allowing progressive, controlled release , or delayed by these agents or active ingredients, "in situ", that is to say at the very place where they act or intervene.

In the case of a chitosan, various solutions with a delay effect have been proposed, such as crosslinking between them macromolecows of different degrees of polymerization by means of covalent bonds, complexation of the same macro molecules with heavy metals, or even association of electrostatic type by polyanion-polycation. Thus, if one refers to the document WO-A-8907395, it is proposed to introduce into the treating chitosan solution, a fixative whose role is to prevent, or at least limit the degradation of the chitosan, once the solution applied treatment. The fixatives selected are chosen from tribasic potassium oxide, etc .; such a fixer interacts with the various macromolecusses of chitosan, by crosslinking or complexing with the metals of the fixer, to lead to the desired delay effect. Such solutions are very often unacceptable for the medium or the treated substrate, especially if it is alive, because of the toxicity of the binding agents used.

The object of the present invention was to find a means or solution of controlled release, often called "delay", remedying the drawbacks discussed above, and specific to the oligomers of glucosamine.

By characterizing a new physicochemical form of the oligomers in question, demonstrated by the experimental protocol set out below, the present invention has provided a particularly simple solution to the problem identified previously. This new form can be obtained, in the solid state, according to the general method described above, but by observing, which had never been done before, a time of rest or latency for the one or more isolated oligomers, before any subsequent use or consumption of the oligomer.

The dissolution of the new form identified previously generates a pseudo-equilibrium between the isomolecular aggregate or aggregates of the oligomer on the one hand, and the isolated form of the same oligomer ^" on the other hand, gradually leading to the dissociation of said aggregates, and the release of the isolated form of the same oligomer.

A particularly effective retard form is thus obtained, without any addition of a binding or crosslinking agent, or of another nature; in the form according to the invention, the oligomers are associated with each other, isomolecularly, that is to say with the same degree of polymerization or the same molecular mass, only by physical forces as it appears today difficult to characterize, but nonetheless existing as shown below. By "direct association" is meant that between the associated oligomers, of the same degree of polymerization, there is no direct or indirect covalent bond, for example a crosslinking agent. As shown below, moreover, the new form according to the invention, in the solid state, shows itself to be very stable over time. The present invention also has the following characteristics, considered in isolation or in combination:

the compound according to the invention, dissolved, comprises a mixture of several isomoiecular aggregates constituted respectively by multiples different from the basic oligomer, for example in decreasing weight proportions for the highest multiples

the compound according to the invention comprises several oligomers of respectively different degrees of polymerization, each oligomer being present in solution in the form of a mixture of at least one isomolecular aggregate as defined above, and of the isolated form of said oligomer - the amino groups of the oligomer are in neutral form, in the -NH_ state, in which case, in solution, the weight proportion of the aggregate associating two molecules of the basic oligomer is at least equal to 10% , or the amino groups of the same oligomer are in cationic form, that is to say in the -NH state, in which case, in solution, the weight proportion of the aggregate associating two molecules of the basic oligomer is at least 2%

- the basic oligomer has a degree of polymerization of between 2 and 100, and preferably between 20 and 30

- The compound according to the invention is at _. solid state, after precipitation followed by possible lyophilization

- In its various applications, the compound according to the invention can be used in the form dissolved in a solvent, or supported in ^" one or on a support in suspension in a dispersion medium.

With regard to the method according to the invention, the following additional characteristics can also be noted:

- the mixture of oligomers is separated by steric exclusion chromatography, in which case the elution phase of the chromatography is an ammonium acetate-acetic acid system

the oligomers of the same degree of polymerization are isolated, in neutral form, that is to say in the -NF state for the amino groups, in particular by precipitation in isopropyl alcohol, then treatment of the precipitate in an ammoniacal solution

- it is also possible to isolate the oligomers of the same degree of polymerization in cationic form, that is to say in the -NH- state, for the amino groups, in particular by precipitation in isopropyl alcohol, then treatment of the precipitate with a strong mineral acid. In a preferred embodiment of the process, a chromatography column is chosen for the separation of the oligomers, having the following characteristics in combination:

- a silica support grafted using hydrophilic organic groups - a number of trays per meter greater than 16,000

- a resolution range between DP 2 and DP 100 for polysaccharides.

The present invention is now described in a general and experimental manner, with reference to the results of the chromatographic and spectral analyzes represented by the attached figures 1 to 7, according to which:

- Figure 1 is the chromatogram obtained on a "Protein-Pac" 200 SW column (Waters), corresponding to the total distribution of a completely deacetiated chitosan, hydrolyzed by 12N HCl at 72 ° C for one hour

- Figure 2 is the chromatogram obtained on a "Protein-Pack" 200 SW column (Waters), corresponding to an oligomer of DP 39.5 with a polymolecularity index of 1.03, prepared by a single fractionation of Phydrolysate, according to Figure 1 - Figure 3 is an X-ray diffraction pattern of an oligomer of DP 39 isolated in the form -NH ~, and kept at rest for several months

- Figure 4 is the chromatogram obtained on a "Protein-Pack" 200 SW column (Waters) of an oligomer of DP 9 (C = 5 g / 1) isolated in -NH ₂ form, and kept at rest for 4 days

- Figure 5 is the chromatogram obtained on a "Protein-Pack" 200 SW column (Waters) of an oligomer of DP 39 (C = 5 g / 1) isolated in the form -NH _2> and kept at rest for several months

- Figure 6 is the chromatogram obtained on column "Protein-Pack" WS 803 F (Shodex) of an oligomer of DP 39 (C = 5 g / 1) isolated in the form

-NH- ,, and kept at rest for several months

- Figure 7 is the chromatogram obtained on a "Protein-Pack" 200 SW column (Waters) of an oligomer of DP 39 (C = 25 g / 1) isolated in -NH ₂ form. First of all, the process for obtaining the compounds according to the present invention is generally in accordance with that described in the publication introduced previously, and having as title: "Glucosamine oligomers: 1. Preparation and characterization"; but as already said, it is the addition of a step of resting the isolated oligomer in the solid state, which has led to the identification of the retard form according to the present invention.

The separation of the oligomer fractions can be obtained more quickly and with better efficiency, on steric exclusion chromatography columns of a particular type, identified below, and this for degrees of polymerization between 15 and 100. We start with chitosans produced in France by the Abertechnologies Company.

Their molecular mass doesn't matter; on the other hand, for reasons of convenience, it is preferable that their residual acetylation rate is less than 15% or, better still, less than 3%. In the first case, it will be possible to obtain their total deacetylation after two successive stages, whereas a single stage will suffice in the second case.

The chromatography columns selected according to the preceding characteristics are, for example, those of the 200 SW or 300 SW "Frotein-Pack" type sold by the company Waters (Millipore), or WS S00 sold by the company Shodex, or any other support. silica grafted using hydrophilic organic substances. For separation on the preparative scale, the corresponding systems existing in the trade are used. The chromatography eluent is an ammonium acetate / acetic acid buffer (0.15 M in ammonium acetate, pH 4.5).

Using an acetic acid-ammonium acetate buffer for eluent, it is possible to obtain a chromatographic separation of the glucosamine oligomers, on grafted silica columns, of the "Protein-Pack" type, as sold by Waters or Shodex. The separation, obtained according to a steric exclusion process, proves to be more efficient, while covering a much wider separation domain than on a Biogel P60 type column; an example of a chromatogram is given in FIG. 1, for a chitosan hydrolyzate of approximately one hour in hydrochloric acid approximately 12 N at 72 ° C. The log (molecular mass) calibration curves as a function of the elution volume show that the separation limit (volume excluded) corresponds for example to a degree of polymerization (DP) close to 100, for a column of the 200 SW type ( Waters), and 500 on a column of type WS 803 F (Shodex). It is also possible, with this type of column, to separate a given distribution into very low polymolecularity fractions. This separation is also much more effective here than with a Biogel P60 type column, since in a single operation, it is possible, as the example in FIG. 2 shows, to collect a fraction of PD centered on 39.5 with a polymolecularity index equal to 1.03; this is usually only obtained after three successive fractionations on a column of the Biogel P60 type.

The compounds contained in the fractions separated by chromatography can be isolated according to two types of structure: the saline or cationic structure corresponding to the ammonium (-NI-) form of the primary amine function of the glucosamine motif, and the neutral structure corresponding to the form was free (-NHL) for the same reason.

The anion associated with the ammonium form can be chosen from any of the acids. If we take the example of a chloride, the fraction is concentrated to the maximum, then precipitated with isopropyl alcohol, washed and dried. The precipitate is redissolved in water, and the solution is lyophilized, after addition of the stoichiometric amount (in very slight excess) of hydrochloric acid. It should however be noted that it is preferable to use strong, non-organic acids, which give much more stable salts in the solid state. For the free amino form, it is sufficient after concentration of the fraction, to add dilute ammonia until reaching a pH of 9,, where the precipitation is complete. The precipitate is then washed with water, then lyophilized after centrifugation and redispersion in water.

In both cases, the solid compounds obtained are stored in stoppered bottles.

If we consider the glucosamine oligomers, as previously obtained, whatever their degree of polymerization (DP -2 to 100 for example), or the form in which they are isolated, we note the appearance of structures crystalline in the samples, with a crystallinity rate which depends on the duration of storage of the sample, reaching a plateau before

3 months. This solid phase crystallization evolves over time, from germs created during the isolation of the compounds, and thanks to the intervention of water molecules present in the samples, at levels always greater than or equal to 10% in weight. The diffraction patterns in FIG. 3 show that the crystalline forms obtained are in fact allomorphs of chitosan, of the "tendon" type. When a glucosamine oligomer is dissolved in water, immediately after its isolation, the chromatographic analysis of the solution obtained gives a chromatogram entirely superimposable on that which had been obtained before its isolation. This result is observed whatever the form (-NH., Or -NHL) of the isolated fraction, independently of the various parameters for preparing the solution (pH, temperature, concentration, etc.).

When one operates in the same way with an oligomer having been stored, in solid form, after several days or even months, one obtains a chromatogram like those represented on figure 4. As for the preceding case, one finds a peak corresponding exactly to the distribution from which the oligomer originates. On the other hand, this peak is accompanied by a succession of secondary peaks, appearing at increasing elution volumes of molecular weights, the proportion of which by weight decreases with it. The mass calibration curve of the chromatographic column used makes it possible to show that these peaks are centered on elution volumes corresponding to molar masses exactly multiple of those of the main peak. There is thus a solution containing a distribution of isomolecular aggregates of the same oligomer, which has the molecular mass that of the isolated oligomer contained in the main peak. The use of a chromatographic column having a wider separation domain (FIG. 4) makes it possible to demonstrate that one can thus observe the elution of aggregates having up to more than fifteen times the molecular mass corresponding to the peak. main. The infrared and NMR spectra make it possible to show that no modification of the chemical structure has occurred, and that the phenomenon is only due to reversible associations of oligomers, mainly depending on the structure of the product. starting (-NH or NH _? ), and as a result of the nature and energy of the cohesion of the crystals contained in ^" the solid sample of the compound having served to prepare the solutions. This phenomenon takes place in a limited way an oligomer isolated in acid form, whereas for the same oligomer isolated in -NH form, depending on the conditions, it can represent up to more than 60% of the behavior of the sample in solution (FIG. 7). therefore to a delayed dissolution process, and cannot be compared to the behavior of conventional associations of high molecular weight chitosans, since it is not created in the solution and evolves contrary to what is observed for a high molecular weight chitosan This phenomenon decreases for example over time, contrary to the process observed with a high molecular weight chitosan.

The behavior described above exists for all the oligomers of DP between 2 and 100, in particular when they are isolated in the form -NH _? , however with an optimum lying in the PD range between 20 and 30. Beyond a PD of 100, this phenomenon decreases progressively as the mass increases.

The phenomenon also decreases when the solutions are heated, when the concentration is reduced, or when the solutions are allowed to age. However, in any case, it never disappears completely and there always exists ^" a pseudo-balance between the aggregated oligomers and the isolated oligomers. Thus, if the proportion of isolated oligomers increases with time, to the detriment of the aggregated oligomers, this increase does not exceed 25%, even after several weeks of storage of the solution. This effect is all the less important as the initial concentration of oligomer is high. The phenomenon observed is a pseudo equilibrium corresponding to a delayed dissolution mechanism. Indeed, if, by chromatography, the fraction comprising the isolated oligomers is separated from the fraction containing all the aggregated oligomers, it is shown that in the second fraction the equilibrium is shifted towards the formation of isolated oligomers, after dissolution of this fraction without isolating it, and that on the other hand the first fraction corresponding to the isolated oligomers undergoes no modification in solution. In addition, if you prepare a solution at a given concentration, the same solution, after having been concentrated ten times, gives exactly the same chromatogram as that of the starting solution.

. Finally, if the products contained in an aged solution are isolated so that the proportion of isolated oligomers has greatly increased, the crystallization process in the solid state develops again, and the chromatogram of a solution (at the same concentration as above) prepared for example one week after isolation again shows an increase in the share of aggregated oligomers.

Finally, the mixture characterizing the present invention can be obtained in a controlled manner, whether in solution, in the solid state, or during processes alternating successively solution and solid state.

The invention described above finds a very wide field of applications including the fields of cosmetology, pharmacy, medicine, agriculture and food (dietetics). In general, the forms according to the invention of the glucosamine oligomers, and their particular properties, can be used: in solution, in emulsion, in encapsulation (liposomes, gels, etc.), or in solid state. The invention relates to the use of oligomers as active principles or agents, either directly or with a delayed effect, alone or combined with other active principles or agents.

In pharmacy, in addition to what has been stated in the general case, it is possible to use jointly chitosan, already known to promote the delay effect mechanism of various active ingredients.

In medicine, it applies more particularly to wound healing and to any cell development process.

In agriculture, the compounds according to the invention will be used as biostimulants or bioprotectors, either in solid form, in particular in the case of ornamental plants, or in foliar spraying where the mechanism of alternating solid solution will occur in situ . In the experimental process described below, by DP is meant a degree of polymerization of a given molecule, in all cases where it will be less than or equal to 15, while above this value it will correspond to the geometric mean the number and weight average degree of polymerization (DP = y. \ ln.Mp). By "nmère" we call any aggregation of n molecules of the same oiomer, between them. EXAMPLE 1

Preparation of trace mothers

A chitosan sample from an Abertechnologies batch is totally deacetiated according to the method described by A. DOMARD et al., Int. J. Biol. Macromol. (1983) _5, 49. The totally deacetiated chitosan thus prepared is hydrolyzed according to the method proposed by A. DOMARD et al, Int. 3. Biol. Macromol. (1989) _U, 297, in 12 N hydrochloric acid at 72 ° C. An example of distribution obtained for a hydrolysis time of approximately 1 hour is given in FIG. 1.

The chromatographic study is carried out with 5g / l solutions, of which 20 μl are injected into a "Protein Pack" column 200 SW Waters (Millipore), with a flow rate of 0.5 mi / minute and a recording of 1 cm /minute. The oligomers of DP ^ 15 are prepared according to the method described above. For the DP- ^ 15 fractions, an example of separation is carried out by injecting 20 μl of 50 g / l solution of a given hydrolyzate. A fraction, always the same, is collected several times in succession, corresponding to an interval of 250 μl of eluted volume. The solution is then concentrated and reinjected for analysis. An example of the fraction obtained is shown in FIG. 2. The calculation of the average masses using the calibration curve M = f (Log Ve) gives for this example DPp = 40.2 and DPn = 38.9, or a polymolecularite index Ip = 1.033, much less than 1.1. The operation is advantageously carried out on preparatory equipment. Whatever they are, the oligomers can be isolated in acid form, after concentration of the solutions containing them up to the limit of solubility of ammonium acetate, using isopropyl alcohol as precipitant. The suspension is then washed and centrifuged three times in succession with this alcohol, then lyophilized after redissolution in water. After a new dissolution, the quantity of acid chosen (barely greater than the stoichiometry) is added so as to prepare a given salt form. The product is then isolated by lyophilization. It will be taken into account that certain salt forms are unstable over time and release the acid by regenerating the free amino form. The hydrochloride can, for example, be considered to be stable. For the form -NH_, in the case of oligomers of DP - <. 15, the saline acetate form, initially obtained after lyophilization of the precipitate in isopropanol, is dissolved in water and brought to pH = 9.5 to 1 using a dilute ammonia solution, then lyophilized. For DP ^" ^ 15, the oligomers can be precipitated after concentration of the initial solution, by adding dilute ammonia until reaching a pH of 9.5. The precipitate obtained is then centrifuged and then washed several times with water at pH = 9.5, and lyophilized after redispersion in water.

EXAMPLE 2

Solid state crystallization of oligomers of glucosam ine

Any oligomer of the glucosamine of DP between 2 and 100, whatever its chemical structure (-NH, or -NH_), gives, after several months of storage, an X-ray diffraction pattern (in the form of a powder) highlighting the presence of crystal structures. FIG. 3 gives an example of a photograph produced with a fraction of DP 39, having been stored for several months after having been isolated in the free amine form. The same study carried out in the form (-NH, ⁺ ) also gives an intense line at 4.35 Λ.

EXAMPLE 3

Phenomena of late dissolution

i - An oligomer of DP 9 isolated in -NH_ form gives, after 4 days of storage, the chromatogramτιe represented in FIG. 4 where we note, in addition to the main peak, those corresponding to the elution volumes of "n-mothers "of DP 18, 27 and 36, ie" dimers "," trimers "and" tetramers "of DP 9.

ii - A fraction of oligomers centered on DP 39, isolated in -NH_ form, in the solid state, gives after 6 months of storage the chromatogram represented in FIG. 5, in which we note in addition to the main peak corresponding to isolated molecules, the presence of peaks centered on the elution volumes of molecules of DP SI, 1 19, etc., corresponding to the "dimer", "trimer", etc. of the DP 39 oligomer.

iii - The use of a wider separation domain column

(FIG. 6) makes it possible to highlight the decreasing proportion, with their mass, of the higher "n-mothers". iiii - The proportion of the main peak corresponding to the isolated molecules is always greater than or equal to 90%, when the oligomer has been isolated in acid form. When isolated in free amino form, a solution of a fraction centered on DP 39, prepared at a concentration of 25 g / 1, shows that the proportion of the peaks of "n-mothers" can exceed 65% of - the sample (Figure 7).

EXAMPLE 4

Stability of the phenomenon

i - Role of the concentration

The role of concentration on the distribution of molecules between the peak corresponding to the isolated molecules and those of "n-mothers" is given in the example shown in the table below for a fraction of DP 39.

Conversely, if a solution is prepared at a given concentration, for example 5 g / l, and if it is then concentrated 10 times (50g / l) with a rotary evaporator, the chromatograms obtained from the two solutions are all completely stackable.

ii - The effect of the aging of the solutions over time is illustrated in the table below for a fraction of DP 54 prepared at 5 g / 1.

13 iii - If we inject a solution of a fraction of DP 28, and if we collect the volumes corresponding to "n-mothers", we can observe the regeneration of the main peak over time as shown in the table next.

iiϋ - We can finally show the role of temperature on a solution of DP 39 (1 g / 1)

In addition, a solution of the fraction of DP 39 prepared at 2 ° C shows an increase of 12% in the proportion of the peaks of the higher masses, compared to the proportion which is obtained at room temperature.

Claims

1 / Compound comprising at least one oligomer of glucosamine of relatively low degree of polymerization, characterized in that, when dissolved, it comprises, in mixture, on the one hand at least one isomolecular aggregate associating directly and solely by physical means several molecules of the basic oligomer, the total molecular mass of which is a multiple of that of said basic oligomer, and on the other hand the isolated form of said basic oligomer, that is to say in the non-aggregated state.

2 / A compound according to claim 1, characterized in that, dissolved, it comprises in mixture several isomolecular aggregates, constituted respectively by different multiples of the basic oligomer.

3 / A compound according to claim 1, characterized in that it comprises several oligomers of respectively different degrees of polymerization, each oligomer being present in solution in the form of a mixture of at least one isomolecular aggregate and of the isolated form of said oligomer.

4 / A compound according to claim 1, characterized in that the amino groups of the oligomer are in neutral form, in the -NH „state.

5 / A compound according to claim 4, characterized in that, in solution, the weight proportion of the aggregate associating two molecules of the basic oligomer is at least equal to 10%.

6 / A compound according to claim 1, characterized in that the amino groups of the oligomer are in cationic form, in the -NH state ,.

7 / A compound according to claim 6, characterized in that, in solution, the weight proportion of the aggregate associating two molecules of the basic oligomer is at least equal to 2%.

8 / A compound according to claim 1, characterized in that the basic oligomer has a degree of polymerization between 2 and 100, and preferably between 20 and 30.

9 / A compound according to claim 1, characterized in that it is in solid form.

10 / Compound according to claim 1, characterized in that it is in dissolved form in a solvent, or supported in or on a support in suspension in a dispersion medium.

11 / Treatment product comprising as active agent at least one oligomer of glucosamine of relatively low degree of polymerization, characterized in that the active agent is a compound according to any one of claims 1 to 10, allowing a gradual release of said oligomer. 12 / Product according to claim 11, as a medicament. 13 / Product according to claim 1 1., as an agrochemical or phytosanitary product.

14 / Product according to claim I I, as a food or dietetic product.

15 / Product according to claim 1 1, as a cosmetic product. 16 / Process for the preparation of a compound according to any one of claims 1 h 10, characterized in that:

- In a manner known per se, one obtains or one starts from a completely deacetylated chitosan, one hydrolyzes the latter to obtain a mixture of glucosamine oligomers, of different degrees of polymerization respectively, this mixture is separated to obtain different fractions of oligomers, the oligomers of the different separate fractions are isolated

- In a new way, an oligomer thus isolated is left to stand, in the solid state, to obtain a mixture, in particular crystallized, of at least one aggregate and of the isolated form of said oligomer.

17 / A method according to claim 16, characterized in that the mixture of oligomers is separated by chromatography, steric exclusion.

18 / A method according to claim 7, characterized in that a chromatography column is chosen having in combination the following characteristics:

- It is obtained from a silica support grafted using hydrophilic organic groups

- it has a number of plates per meter at least equal to 16000. - its resolution range is between DP 2 and DP 100 for polysaccharides.

19 / A method according to claim 17, characterized in that the elution phase of the chromatography is an onium ametate-acetic acid acetate system. 20 / A method according to claim 16, characterized in that the oligomers of the same degree of polymerization are isolated, in neutral form, that is to say in the -NH_ state for the amino groups, in particular by precipitation in the isopropyl alcohol, then treatment of the precipitate in an ammoniacal solution. 21 / A method according to claim 16, characterized in that the oligomers of the same degree of polymerization are isolated in cationic form, that is to say in the -NH state, for the amino groups, in particular by precipitation in isopropyl alcohol, then treatment of the precipitate with a strong mineral acid.

22 / A method of using a compound according to any one of claims 1 to 10, characterized in that said compound is put in solution or in suspension, and optionally mixed with other products, of the excipient type, adjuvant, and packaged in a form of administration or treatment.