Thermosetting neutralized chitosan composition forming a hydrogel, lyophilizate, and processes for producing the same
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- 1Patent claims Zastrzeżenia patentowe 1. An aqueous thermosetting neutralized chitosan composition forming a phosphate-free transparent hydrogel at a temperature higher than 5aboutC, wherein the composition contains from 0.1 to 5.0% w / w relative to the entire composition of reacetylated chitosan with a molecular weight not less than 100 kDa and a degree of deacetylation from 40 to 70%, neutralized with a hydroxylated base, and from 1 to 30% w / w, based on the total composition, of a complexing agent selected from polyoses and polyols derived from polyoses. 1. Wodna termoutwardzalna zobojętniona kompozycja chitozanu tworząca wolny od fosforanów przezroczysty hydrożel w temperaturze wyższej niż 5oC, przy czym kompozycja ta zawiera od 0,1 do 5,0% w/w w stosunku do całej kompozycji reacetylowanego chitozanu o masie cząsteczkowej nie mniejszej niż 100 kDa i o stopniu deacetylowania od 40 do 70%, zobojętnionego za pomocą hydroksylowanej zasady, oraz od 1 do 30% w/w, w stosunku do całej kompozycji, środka kompleksotwórczego wybranego spośród polioz i polioli pochodzących od polioz. 2. The aqueous thermosetting neutralized chitosan composition according to claim 1, which contains reacetylated chitosan in an amount of 0.5 to 3.0% w / w relative to the total composition. 2. Wodna termoutwardzalna zobojętniona kompozycja chitozanu według zastrzeżenia 1, która zawiera reacetylowany chitozan w ilości od 0,5 do 3,0% w/w w stosunku do całej kompozycji. 3. The aqueous thermosetting neutralized chitosan composition according to claim 1 or 2, wherein the deacetylation degree of the reacetylated chitosan is from 45 to 65%. 3. Wodna termoutwardzalna zobojętniona kompozycja chitozanu według zastrzeżenia 1 albo 2, w której stopień deacetylowania reacetylowanego chitozanu wynosi od 45 do 65%. 4. The aqueous thermosetting neutralized chitosan composition according to any one of claims 1 to 3, wherein the molecular weight of the reacetylated chitosan is not less than 200 kDa. 4. Wodna termoutwardzalna zobojętniona kompozycja chitozanu według któregokolwiek z zastrzeżeń od 1 do 3, w której masa cząsteczkowa reacetylowanego chitozanu jest nie mniejsza niż 200 kDa. 5. The aqueous thermosetting neutralized chitosan composition according to any one of claims 1 to 4, which contains a complexing agent in an amount of 5 to 15% w / w relative to the total composition. 5. Wodna termoutwardzalna zobojętniona kompozycja chitozanu według któregokolwiek z zastrzeżeń od 1 do 4, która zawiera środek kompleksotwórczy w ilości od 5 do 15% w/w w stosunku do całej kompozycji. 6. The aqueous thermosetting neutralized chitosan composition according to any one of claims 1 to 5, wherein the complexing agent is polyose. 6. Wodna termoutwardzalna zobojętniona kompozycja chitozanu według któregokolwiek z zastrzeżeń od 1 do 5, w której środkiem kompleksotwórczym jest polioza. 7. The aqueous thermosetting neutralized chitosan composition according to claim 6, wherein the polyose is selected from monosaccharides and disaccharides. 7. Wodna termoutwardzalna zobojętniona kompozycja chitozanu według zastrzeżenia 6, w której polioza jest wybrana spośród monosacharydów i disacharydów. 8. The aqueous thermosetting neutralized chitosan composition according to claim 7, wherein the polyose is a monosaccharide selected from Dglucose, fructose and tagatose. 8. Wodna termoutwardzalna zobojętniona kompozycja chitozanu według zastrzeżenia 7, w której polioza jest monosacharydem wybranym spośród Dglukozy, fruktozy i tagatozy. 9. The aqueous thermosetting neutralized chitosan composition according to claim 7, wherein the polyose is a disaccharide selected from trehalose, sucrose, maltose and lactose. 9. Wodna termoutwardzalna zobojętniona kompozycja chitozanu według zastrzeżenia 7, w której polioza jest disacharydem wybranym spośród trehalozy, sacharozy, maltozy i laktozy. 10. The aqueous thermosetting neutralized chitosan composition according to claim 9, wherein the disaccharide is trehalose. 10. Wodna termoutwardzalna zobojętniona kompozycja chitozanu według zastrzeżenia 9, w której disacharydem jest trehaloza. 11. The aqueous thermosetting neutralized chitosan composition according to any one of claims 1 to 5, wherein the complexing agent is a polyol derived from polyose, selected from glycerin, mannitol, sorbitol, xylitol, erythritol, lactitol and maltitol. 11. Wodna termoutwardzalna zobojętniona kompozycja chitozanu według któregokolwiek z zastrzeżeń od 1 do 5, w której środkiem kompleksotwórczym jest poliol pochodzący od poliozy, wybrany spośród gliceryny, mannitolu, sorbitolu, ksylitolu, erytrytolu, laktitolu i maltitolu. 12. The lyophilisate obtained by drying by freezing the aqueous thermosetting neutralized chitosan composition of any one of claims 1 to 11, except for the composition containing glycerin as a complexing agent. 12. Liofilizat otrzymany drogą suszenia przez wymrażanie wodnej termoutwardzalnej zobojętnionej kompozycji chitozanu z któregokolwiek z zastrzeżeń od 1 do 11, z wyjątkiem kompozycji zawierającej glicerynę jako środek kompleksotwórczy. 13. A method for producing an aqueous thermosetting neutralized chitosan composition according to any one of claims 1 to 11, which comprises the following steps:13. Sposób wytwarzania wodnej termoutwardzalnej zobojętnionej kompozycji chitozanu według któregokolwiek z zastrzeżeń od 1 do 11, który obejmuje następujące etapy: a) dissolving the right amount of reacetylated chitosan with a molecular weight of not less than 100 kDa and a degree of deacetylation of 40-70% in aqueous HCl and cooling this acidic chitosan solution to a temperature lower than 5aboutC;a) rozpuszczanie odpowiedniej ilości reacetylowanego chitozanu o masie cząsteczkowej nie mniejszej niż 100 kDa i stopniu deacetylowania 40-70% w środowisku wodnego HCl i chłodzenie tego kwasowego roztworu chitozanu do temperatury niższej niż 5oC;b) neutralizing the cooled chitosan solution obtained in step a) by adding an aqueous hydroxylated base previously cooled to a temperature lower than 5aboutC to the chitosan cooled solution until the chitosan cooled solution has a pH of 6.7-7.1;b) zobojętnianie ochłodzonego roztworu chitozanu otrzymanego w etapie a) przez dodawanie wodnej hydroksylowanej zasady uprzednio ochłodzonej do temperatury niższej niż 5oC do ochłodzonego roztworu chitozanu aż do chwili, gdy ochłodzony roztwór chitozanu wykaże wartość pH równą 6,7-7,1;c) adding a suitable amount of a complexing agent selected from polyoses and polyols derived from polyoses during or after the dissolution step a) or before, during or after the neutralization step b), and c) dodawanie odpowiedniej ilości środka kompleksotwórczego wybranego spośród polioz i polioli pochodzących od polioz podczas albo po etapie rozpuszczania a), albo przed, podczas lub po etapie zobojętniania b), i d) ewentualnie zamrażanie otrzymanej wodnej termoutwardzalnej zobojętnionej kompozycji chitozanu. d) optionally freezing the resulting aqueous thermosetting neutralized chitosan composition. 14. The method of claim 13, further comprising the step of sterilizing the reacetylated chitosan prior to the dissolution step a). 14. Sposób według zastrzeżenia 13, obejmujący ponadto etap sterylizowania reacetylowanego chitozanu przed etapem rozpuszczania a). 15. The method of claim 13 or 14, wherein in step b) the hydroxylated base is NaOH. 15. Sposób według zastrzeżenia 13 albo 14, w którym w etapie b) hydroksylowaną zasadą jest NaOH. 16. A method for producing a lyophilisate according to claim 12, which comprises freeze-drying an aqueous thermoset neutralized chitosan composition obtained by a method according to any one of claims 13 to 15, wherein the composition does not contain glycerin as a complexing agent. 16. Sposób wytwarzania liofilizatu według zastrzeżenia 12, który obejmuje suszenie przez wymrażanie wodnej termoutwardzalnej zobojętnionej kompozycji chitozanu otrzymywanej sposobem według któregokolwiek z zastrzeżeń od 13 do 15, przy czym kompozycja nie zawiera gliceryny jako środka kompleksotwórczego. 17. Use of an aqueous thermosetting neutralized chitosan composition according to any one of claims 1 to 11 for the preparation of a drug delivery system. 17. Zastosowanie wodnej termoutwardzalnej zobojętnionej kompozycji chitozanu według któregokolwiek z zastrzeżeń od 1 do 11 do wytwarzania układu dostarczającego leki. 18. Use of an aqueous thermosetting neutralized chitosan composition according to any one of claims 1 to 11 in the preparation of an injectable preparation. 18. Zastosowanie wodnej termoutwardzalnej zobojętnionej kompozycji chitozanu według któregokolwiek z zastrzeżeń od 1 do 11 do wytwarzania preparatu nadającego się do iniekcji. 19. Use of the lyophilisate according to claim 12 for the preparation of a drug delivery system. 19. Zastosowanie liofilizatu według zastrzeżenia 12 do wytwarzania układu dostarczającego leki. 20. Zastosowanie liofilizatu według zastrzeżenia 12 do wytwarzania preparatu nadającego się do iniekcji. twenty. Use of the lyophilisate according to claim 12 for the preparation of an injectable preparation. Laboratoire Medidom S.A.;Szwajcaria Pełnomocnik: Laboratoire Medidom SA;Switzerland Representative: Z-6506 Z-6506 EP 1978925 B1 EP 1978925 B1 FIG. 1 FIG. 1 Z-6506 Z-6506 EP 1978925 B1 EP 1978925 B1 Temperatura r40 «ί Temperature r40 «ί Ο. Ο. + in + w ο ο ό with trehalose 8% (w / w) z trehalozą 8 % (w/w) SA ++ '+4 ++++++ ι ° £ ^ *++++++++^ ο ^ °°Γ10 , ...... r ........ ί ι SA ++' +4++++++ ι°£^*++++++++^ ο^°°Γ10 , ...... r........ί ι 60 90 120 150 180 60 90 120 150 180 Czas [min.] Time [min.] FIG. 2 o FIG. 2 sts FIG. 3 FIG. 3 Z-6506 Z-6506 EP 1978925 B1 EP 1978925 B1 4o 4o g about ó FIG. 4 FIG. 4 Temperaturar 40 with trehalose 8% (w / w) Temperaturar 40 z trehalozą 8 % (w/w) ') bez trehalozy ') without trehalose 1 +++ - ++++ 1 ι i nń / tt litu ++ n + i + ++++ oooooooooooooooooooooooooooooooo 1+++-++++1 ι i nń/tt litu ++n + i + ++++ oooooooooooooooooooooooooooooooo 0' 0' 30 60 90 120 150 180 30 60 90 120 150 180 Czas [min.] Time [min.] FIG. 5 nj FIG. 5 nj CL + CL + b θ 'b b θ' b by 30 60 90 120 150 180 o 30 60 90 120 150 180 Czas [min.] Time [min.] T [° CJ T[°CJ Z-6506 Z-6506 EP 1978925 B1 EP 1978925 B1 FIG. 6 flj FIG. 6 flj ABOUT. O. + + ABOUT O 3) 3) O o Oh o (B o (B ABOUT. O. b o because Czas [min.] [3„] 1 Time [min] [3 "] 1 Z-6506 Z-6506 EP 1978925 B1 EP 1978925 B1 +++++++ £ # * 'oaboutoo °° +++++++£#*’ oooo°° 30-, 30-, FIG. 7 FIG. 7 Temperatura Γ4θ rf>' ,cP' o°° o° ++++++++++ ++ τ-1-1-r Temperature Γ4θ rf> ', cP' at ° happiness at ° ++++++++++ ++ τ-1-1-r 60 90 120 150 180 60 90 120 150 180 Czas [min.] Time [min.] FIG. 8 (Fr. FIG. 8 (O ABOUT. O. + b + b b b Temperatura r40 Temperature r40 90 120 150 180 90 120 150 180 Czas [min.] Time [min.] T [° C] T PC] T [°C] T PC]
150 paragraphs in 1 section, as filed
[0001] The present invention relates to an aqueous thermosetting neutralized chitosan composition forming a phosphate-free transparent hydrogel at a temperature higher than 5.<sup>about</sup>C and the method of producing this composition.
[0002] Furthermore, the present invention relates to a lyophilisate obtained by drying by freezing the thermosetting neutralized chitosan composition of the present invention and a process for preparing this lyophilisate.
Background Art [0003] Hydrogels are attractive for biomedical applications.
[0004] Furthermore, it is believed that hydrogels having a specific ability to increase viscosity with temperature, also known as "thermosensitive / thermoregulating / pseudo-thermosetting / thermogelling" hydrogels, have improved application ability combined with an increased duration of application and with this they can advantageously be used for drug delivery or tissue enlargement.
[0005] As known from O. Felt et al. in The Encyclopedia of Controlled Drug Delivery, 1999, these thermosensitive hydrogels can preferably be based on polymers of natural origin, e.g., chitosan, which is a commercially available inexpensive polymer derived from chitin, which is the second most abundant polysaccharide after cellulose.
[0006] Chitosan is known as a chitin derivative obtained by partial to substantial N-deacetylation of chitin also called poly- (N-D-glucosamine), which is a naturally occurring biopolymer.
[0007] Chitosan contains free amino groups (-NH2) and can be characterized by the ratio of N-acetyl-D-glucosamine units and Dglucosamine units and expressed as the degree of deacetylation (DD) of a fully acetylated chitin polymer.
[0008] Chitosan parameters that affect important properties such as solubility and viscosity are the degree of deacetylation (DD), which should be understood as representing the percentage of deacetylated monomers, and molecular weight (Mw).
[0009] It is known that chitosan is biodegradable, biocompatible, bioadhesive, bacteriostatic, and further enhances wound healing, drug absorption and tissue reconstruction.
[0010] Due to the abovementioned internal properties, it is known that chitosan has many cosmetic and pharmaceutical effects and that it is also widely used for various applications as a gel.
[0011] Thus, when considering the beneficial properties of chitosan, there is a continuing need to improve the properties of known thermosensitive chitosan hydrogels that are still considered as very promising for a wide range of biomedical applications.
[0012] WO-A-99/07416 (BIOSYNTHEC) describes a pH-dependent temperature-controlled chitosan hydrogel that exhibits thermosensitive properties at neutral pH in such a way that it exhibits low viscosity at low temperature but gels at body temperature.
[0013] This thermosensitive chitosan hydrogel is obtained by neutralizing commercial chitosan having a deacetylation degree of about 80% with monophosphate dibasic salts of polyols or sugars especially represented by β-glycerophosphate (β-GP).
[0014] However, the presence of β-GP in the hydrogel leads to the following disadvantages.
[0015] β-GP is a negatively charged unit that can react with a positively charged bioactive component, leading to its precipitation or to interference with its release from the hydrogel.
[0016] Thus, the presence of β-GP makes chitosan / e-GP hydrogels unsuitable for use with numerous drugs.
[0017] Furthermore, the properties of this hydrogel, such as gelation time and viscosity, depend on the concentration of β-GP and are therefore limited by the solubility of β-GP.
[0018] In particular, a high concentration of β-GP is desirable for a low gelation time preventing rapid elimination of the hydrogel after its administration.
[0019] However, high concentration of β-GP also reduces the mechanical properties of the hydrogel.
[0020] Thus, the gel time must balance with the consistency of the hydrogel and it is not possible to obtain gels that have both low gel time and high viscosity, which would be a desirable combination of characteristics.
[0021] Too high a β-GP concentration may also cause precipitation of the hydrogel at the site of administration.
[0022] In addition, these thermosensitive chitosan / e-GP hydrogels have been found to be cloudy, which makes their use unsuitable for some applications, such as ocular or topical administration.
[0023] In addition, phosphate-containing substances may be inappropriate in terms of biocompatibility (G. Molinaro et al., Biomaterials, 23: 2717-2722 (2002)).
[0024] To overcome the disadvantages of chitosan / e-GP hydrogels, WO-A2005 / 097871 (UNIVERSITE DE GENEVE) proposes a thermosetting neutralized chitosan composition forming a phosphate-free transparent hydrogel at a temperature higher than 5<sup>about</sup>C, wherein this composition contains homogeneously reacetylated chitosan with a molecular weight not less than 200 kDa and a degree of deacetylation of 30-60%, neutralized with a hydroxylated base.
[0025] WO-A-2005/097871 also discloses that this composition may further comprise 1,3-propanediol to modulate the viscoelastic properties of the hydrogel.
[0026] However, 1,3-propanediol is not mentioned either as "generally known as safe" (GRAS) or known as an additive mentioned in the American Pharmacopoeia, European Pharmacopoeia or Japanese Pharmacopoeia, so that its use is restricted for biomedical applications.
[0027] In addition, thermosetting neutralized chitosan compositions containing 1,3-propanediol weaken their thermosensitive properties after freeze drying.
[0028] Due to the constant concern for improving thermosensitive chitosan hydrogels for biomedical applications, the inventors have now conducted further research to overcome the disadvantages of known thermosensitive hydrogels.
[0029] It is an object of the present invention to provide an aqueous thermoset neutralized chitosan composition forming a phosphate-free transparent hydrogel exhibiting improved properties and being acceptable for biomedical applications.
[0030] Another object of the present invention is to provide an aqueous thermosetting neutralized chitosan composition that is easily stored and that retains its thermogelling properties after storage.
[0031] Still another object of the present invention is to provide an aqueous thermosetting neutralized chitosan composition exhibiting facilitated use, for example by injection using needles or by minimally invasive techniques.
[0032] These objects are achieved by the present invention.
Summary of the Invention [0033] According to a first aspect, the present invention includes an aqueous thermosetting neutralized chitosan composition (hereinafter also referred to as "the composition of the present invention") as defined in independent claim 1 and dependent claims 2-11.
[0034] According to a second aspect, the present invention comprises a lyophilisate of the aqueous thermosetting composition of the first aspect as defined in independent claim 12.
[0035] According to a third aspect, the present invention includes a method of producing an aqueous thermosetting neutralized chitosan composition according to the first aspect as defined in independent claim 13 and dependent claims 14-15.
[0036] According to a fourth aspect, the present invention includes a method of producing a lyophilisate according to the second aspect as defined in independent claim 16.
[0037] According to a fifth aspect, the present invention includes the use of the composition of the first aspect or the lyophilisate of the second aspect as defined in independent claims 17-20.
[0038] According to the present invention, the addition of a complexing agent selected from polyoses and polyols derived from polyoses to said chitosan compositions advantageously results in a phosphate-free transparent hydrogel having improved properties that is acceptable for biomedical applications and that can be easily stored.
[0039] Other advantages of the present invention will be described in the following description.
[0040] The present invention will now be described in more detail.
Brief description of the figures [0041] Fig. 1 shows a device for measuring the injectability of a composition according to the invention.
Fig. 2 shows the evolution of the elastic modulus G '(storage module) and viscosity modulus G' (loss module) of the transparent trehalose containing hydrogel obtained in Example 1, compared to the same hydrogel without trehalose as a function of time when the temperature rises from 4 to 37<sup>about</sup>C.
Fig. 3 shows the evolution of the modulus of elasticity G '(storage module) and viscosity module G' (loss module) of the transparent trehalose-containing hydrogel obtained in Example 1, after production (A) and after thawing (B) as a function of time as the temperature rises from 4 to 37<sup>about</sup>C.
Fig. 4 shows the evolution of the elastic modulus G '(storage modulus) and viscosity modulus G' (defect modulus) of the transparent trehalose hydrogel obtained in Example 1, after freeze drying and restoring to its original form, compared to the same hydrogel without trehalose, as a function of time when the temperature rises from 4 to 37<sup>about</sup>C.
Fig. 5 shows the evolution of the modulus of elasticity G '(storage module) and viscosity module G' (loss module) of the transparent trehalose containing hydrogel obtained in Example 4, compared to the same hydrogel without trehalose as a function of time when the temperature rises from 4 to 37<sup>about</sup>C.
Fig. 6 shows the evolution of the modulus of elasticity G '(storage module) and viscosity module G' (loss module) of the transparent 1,3-propanediol hydrogel obtained in Example 5 (comparative), after preparation (A) and after freeze drying and restoration to its original form (B) as a function of time when the temperature rises from 4 to 37<sup>about</sup>C.
Fig. 7 shows the evolution of the modulus of elasticity G '(storage module) and viscosity module G "(loss module) of the transparent hydrogel containing mannitol obtained in Example 6, after freeze drying and restoring to its original form as a function of time when the temperature rises from 4 up to 37<sup>about</sup>C.
Fig. 8 shows the evolution of the modulus of elasticity G '(storage module) and viscosity module G' (loss module) of the transparent glycerin containing glycerin obtained in Example 7 as a function of time when the temperature rises from 4 to 37<sup>about</sup>C.
Detailed description of the invention [0042] It should be noted that in the present description and claims the expression "thermosetting" in combination with the composition according to the invention means that the temperature does not cause gelation of the composition, but rather acts as a catalyst that dramatically reduces the gelation time as it increases.
[0043] It should also be noted that in the present description the term "hydrogel" or "hydrogel according to the invention" is used instead of "the composition according to the invention" as appropriate.
[0044] It is further noted that in the present description and claims the term "neutralized" means pH = 6.7-7.1.
[0045] According to the present invention, an aqueous thermosetting neutralized chitosan composition forming a phosphate-free transparent hydrogel at a temperature higher than 5<sup>about</sup>C contains reacetylated chitosan neutralized with a hydroxylated base and a complexing agent selected from polyoses and polyols derived from polyoses.
[0046] The average molecular weight (Mw) of the reacetylated chitosan contained in the composition of the present invention is usually not less than 100 kDa.
[0047] The molecular weight of chitosan can be determined by flow fractionation in an asymmetric current field (AF-FF) coupled with polygonal light scattering (MALS), as exemplified by
B. Wittgren and K.-G. Wahlund in Journal of Chromatography A 760: 205-215 (1997).
[0048] Reacetylated chitosan having an Mw usually not lower than 100 kDa is particularly suitable for use in the present invention because it allows the formation of a thermosetting composition forming a compact hydrogel.
[0049] Preferably, the reacetylated chitosan used in the present invention has a Mw of not less than 200 kDa.
[0050] The upper limit of Mw of the reacetylated chitosan used in the present invention depends on the amount of reacetylated chitosan contained in the composition of the invention and is determined by the ease of administration which depends on the chosen application.
[0051] The reacetylated chitosan used in the present invention must have a deacetylation degree of 40-70%, which means that the chitosan contains 40 to 70% D-glucosamine units and 60 to 30% neutral N-acetyl-D-glucosamine units respectively.
[0052] The degree of deacetylation of chitosan can be determined by nuclear magnetic resonance, as described in the literature by Lavertu et al., Journal of Pharmaceutical and Biomedical Analysis 32: 1149-1158 (2003).
[0053] If the deacetylation degree of reacetylated chitosan is less than 40%, then the reacetylated chitosan becomes a chitin-like polymer that is insoluble under acidic conditions and is therefore not suitable for use in the present invention.
[0054] If the degree of deacetylation of reacetylated chitosan is higher than 70%, then the reacetylated chitosan does not allow the formation of a composition forming a phosphate-free transparent hydrogel.
[0055] Preferably, the deacetylation degree of the reacetylated chitosan contained in the composition of the invention is from 45 to 65%.
[0056] Reacetylated chitosan with a molecular weight usually not less than 100 kDa and a degree of deacetylation of 40-70% for use in the present invention can be produced, for example, by the method described in WO-A-2005/097871 or can be obtained from Novamatrix (Oslo, Norway).
[0057] The amount of reacetylated chitosan contained in the composition of the present invention must be from 0.1 to 5.0% w / w relative to the total composition.
[0058] An amount of reacetylated chitosan lower than 0.1% w / w does not allow the formation of a hydrogel, and an amount of reacetylated chitosan higher than 5.0% w / w results in a composition that is too difficult to inject.
[0059] The amount of reacetylated chitosan contained in the composition of the invention is selected depending on the Mw of chitosan and the intended use.
[0060] Preferably the amount of reacetylated chitosan contained in the composition of the present invention is from 0.5 to 3.0% w / w relative to the total composition.
[0061] The amount of complexing agent contained in the composition of the present invention must be from 1 to 30% w / w relative to the total composition and depends on the concentration and molecular weight of the reacetylated chitosan as well as the desired gelation time and hydrogel consistency.
[0062] The amount of complexing agent contained in the composition of the present invention is preferably from 5 to 15% w / w relative to the total composition.
[0063] This complexing agent selected from polyoses and polyols derived from polyoses contained in the composition of the present invention allows modulating hydrogel properties such as gelation time and hydrogel viscosity.
[0064] In one preferred embodiment of the present invention, the complexing agent that can be used in the present invention is polyose, particularly preferably a polyose selected from monosaccharides and disaccharides.
[0065] As preferred examples of monosaccharides that can be used in the present invention, mention may be made of D-glucose (also called dextrose), fructose and tagatose, which are known as vehicles for pharmaceutical compositions according to European, American or Japanese pharmacopoeia.
[0066] As preferred examples of disaccharides that can be used in the present invention, mention may be made of trehalose, sucrose, maltose and lactose, which are known as vehicles for pharmaceutical compositions according to European, American or Japanese pharmacopoeia, with trehalose being particularly preferred.
[0067] As other examples of polyoses that can be used in the present invention, mention may be made of polysaccharides selected from polydextrose and amylose which are known as vehicles for pharmaceutical compositions.
[0068] In another preferred embodiment of the present invention, the complexing agent that can be used in the present invention is a polyol derived from polyose (also called sugar alcohol) selected from glycerin, mannitol, xylitol, erythritol, lactitol and maltitol, which are known as substrates for pharmaceutical compositions according to European, American or Japanese pharmacopoeia, with glycerin being particularly preferred.
[0069] The compositions of the present invention can be frozen for storage while maintaining their thermogelling properties and should be thawed at 4<sup>about</sup>C before using them.
[0070] Furthermore, the compositions of the present invention, with the exception of those containing glycerin as a complexing agent, can be advantageously freeze dried to obtain a lyophilisate for easier storage and distribution, and they are returned to their original form by adding cooled water to the lyophilisate with stirring at 4<sup>about</sup>C before use, while their thermogelling properties are preserved.
[0071] The composition of the present invention may be prepared according to a method forming part of the present invention.
[0072] In step a) of this method, reacetylated chitosan with a molecular weight usually not less than 100 kDa, preferably not less than 200 kDa, with a deacetylation degree of 40-70%, preferably 45-65%, is dissolved in aqueous HCl and after total dissolution of chitosan, the temperature of the chitosan solution is lowered to a temperature lower than 5<sup>about</sup>C, for example in an ice bath.
[0073] Then, in step b) of this method, the pH value of this cooled chitosan solution is neutralized until a pH of 6.7-7.1, preferably pH 6.8, by dropping while stirring at a temperature lower than 5<sup>about</sup>C of the desired amount of an aqueous solution containing a hydroxylated base previously cooled to less than 5<sup>about</sup>C.
[0074] A higher pH value is not suitable as it may cause precipitation of chitosan.
[0075] According to this method, the hydroxylated base used for neutralization is preferably NaOH.
[0076] Inadequate mixing or too fast addition of an aqueous hydroxylated base causes precipitation of chitosan.
[0077] In step c) of this method, the complexing agent from polyoses and polyols derived from polyoses is added during or after the dissolution step a), or before, during or after the neutralization step b).
[0078] It should be noted that the description of chitosan, complexing agent and their amounts in connection with the composition of the present invention also relates to the method of the present invention.
[0079] The method of preparing the composition of the present invention may further comprise, if desired, a step of sterilizing reacetylated chitosan prior to step a) regarding dissolution. In order to obtain a sterile hydrogel, the production process is carried out under aseptic conditions (e.g. with laminar flow) and each solution added is previously filtered through a 0.22 μm filter or steam sterilized.
[0080] For example, sterilization may be carried out by radiation or, preferably, by steam sterilization of reacetylated water-suspended chitosan as described by Yen (Yen SF et al., 1998, US-A-5773608).
[0081] The method of making the composition of the present invention may further comprise, if desired, a step of freezing the composition to facilitate storage.
[0082] In this case, the frozen composition should be thawed at 4<sup>about</sup>C before use.
[0083] In a particularly preferred embodiment, the method of the present invention can be supplemented by further drying by freezing the compositions of the present invention, with the exception of the compositions of the present invention containing glycerin as a complexing agent, to obtain a lyophilisate forming part of the present invention.
[0084] This lyophilisate can be advantageously stored and spread for medical use and can be returned to its original form by adding cold water while stirring at 4<sup>about</sup>C.
[0085] When the temperature of the thermoset neutralized chitosan composition of the present invention increases, for example after application, thermogelling occurs leading to the formation of a phosphate-free transparent compact hydrogel. The higher the temperature, the shorter the gelation time.
[0086] According to the present invention, the composition of the invention can be advantageously used as a drug delivery system and, due to its specific properties, can be advantageously used in the preparation of injectables.
[0087] In addition, since the lyophilisate of the present invention retains its thermogelling properties after restoring its original form, it can be advantageously used for the preparation of a drug delivery system and for the preparation of injectables.
[0088] To demonstrate the improved elastic properties of the phosphate-free transparent chitosan hydrogels of the present invention, rheological measurements of various hydrogels of the present invention and comparative hydrogels are carried out according to the following method, unless otherwise indicated.
[0089] Viscoelastic properties of hydrogels are determined immediately after the production of the hydrogels using the Rheostress 1 method (Haake, Karlsruhe, Germany) using a cone / plate device (diameter 60 mm, angle 4<sup>about</sup>). The temperature is controlled by means of a Haake DC30 thermostatic bath and a Haake K10 cooling device (Haake, Karlsruhe, Germany) coupled with a rheometer. Hydrogels are placed between the cone and the plate (cooled to 4<sup>about</sup>C) and is measured after 10 minutes. All measurements are carried out in the linear viscoelastic range and G '(storage module) and G' (loss module) are determined at constant strain (y = 0.05) at 1.00 Hz for 180 minutes. The temperature is raised from 4 to 37<sup>about</sup>C at 6.6<sup>about</sup>C / min during the first 5 minutes and stays at 37<sup>about</sup>C in the next 175 minutes. Water evaporation leading to drying of the hydrogels is minimized by using a covering surrounding the cone / plate.
[0090] The following hydrogels are tested:
(1) the hydrogel of the present invention obtained in Example 1, containing 2% w / w chitosan (DD = 47%) obtained from Novamatrix and 8% w / w trehalose, and a comparative hydrogel without trehalose (see Fig. 2);
(2) the hydrogel of the present invention obtained in example 1, containing 2% w / w chitosan (DD = 47%) obtained from Novamatrix and 8% w / w trehalose, after receiving and after the freezing-thawing process according to example 2 (see Figures 3A and 3B);
(3) the hydrogel of the present invention obtained in example 1, containing 2% w / w chitosan (DD = 47%) obtained from Novamatrix and 8% w / w trehalose, after freeze drying and restoring to the original form according to example 3, and comparative hydrogel without trehalose (see Fig. 4);
(4) the hydrogel of the present invention obtained in Example 4, containing 0.9% w / w chitosan (DD = 61%) obtained according to Production Example 1 and 5% w / w trehalose, and a comparative hydrogel without trehalose (see Fig. 5 );
(5) comparative hydrogel obtained in Example 5 (comparative), containing 1% w / w chitosan (DD 47%) obtained according to the production example 2 and 10% w / w 1,3-propanediol, after obtaining and freezing drying and restoring to its original form (see Figures 6A and 6B);
(6) the hydrogel of the present invention obtained in Example 6, containing 0.9% w / w chitosan (DD = 61%) obtained according to Production Example 1 and 5% w / w mannitol; after freezing drying and restoring to its original form (see Fig. 7);
(7) the hydrogel of the present invention obtained in Example 7, containing 2% w / w chitosan (DD = 47%) obtained from Novamatrix and 10% glycerin, and a comparative hydrogel without glycerin (see Fig. 8).
[0091] Figs. 2-8 show the evolution of the modulus of elasticity G '(storage module) and viscosity module (loss module) of the tested hydrogels as a function of time when the temperature rises from 4 to 37<sup>about</sup>C. The onset of gel network formation that determines the gel time is given by the intersection of G 'and G'.
[0092] As shown in Figure 2, the addition of a polyose, such as trehalose, in accordance with the present invention increases the G 'and G "values of the hydrogel compared to the same hydrogel without trehalose.
[0093] Fig. 3A shows the viscoelastic properties of the hydrogel as soon as it is obtained (as shown in Fig. 2), while Fig. 3B shows the viscoelastic properties of the same hydrogel after freezing and thawing.
[0094] As shown in Fig. 3B, the thermogelling properties of the compositions of the present invention are maintained after freezing and thawing.
[0095] Fig. 4 shows the viscoelastic properties of the hydrogel shown in Fig. 2 after freeze drying and restoring it to its original form.
[0096] As shown in Fig. 4, the thermogelling properties of the compositions of the present invention are maintained after freeze drying and return to their original form.
[0097] As shown in Figure 5, the composition of the present invention containing trehalose has a gel point of 30 minutes, while the same formulation without trehalose forms a gel after 150 minutes.
[0098] As shown in Figs. 6A and 6B, a comparative composition containing 1,3-propanediol forms a hydrogel when the viscoelastic properties are measured after it is obtained (Fig. 6A), but does not retain thermogelling properties after lyophilization and restoration to its original form, as indicated by the absence of a gel point in Fig. 6B.
[0099] As shown in Figure 7, the composition of the present invention containing mannitol retains its thermogelling properties after lyophilization and restoration to its original form, as indicated by the presence of a gelation point.
[0100] As shown in Figure 8, the composition of the present invention containing glycerin forms a hydrogel, as indicated by the presence of a gel point, while the same formulation without glycerin does not show a gel point after 180 minutes.
[0101] The following examples explain the present invention in more detail.
Examples [0102] In the following examples, the degree of deacetylation of chitosan is determined by nuclear magnetic resonance (NMR) as described in the literature by Lavertu et al., Journal of Pharmaceutical and Biomedical Analysis 32: 1149-1158 (2003).
[0103] The molecular weight of chitosan is determined by flow fractionation in an asymmetric current field (AF-FF) coupled with polygonal light scattering (MALS) as follows:
Fractionation of the chitosan solution (2 mg / ml in acetate buffer pH 4.5) is carried out in a trapezoidal channel, 26.5 cm long and 350 μm high, connected to the ecliptic system F (Wyatt Technology Europe, Dernbach, Germany). The bottom of the canal is lined with regenerated cellulose membrane with a 10 kDa cut-off (Microdyn-Nadir GmbH, Wiesbaden, Germany). The elution medium consists of pH 4.5 acetate buffer. The channel flow is set at 1 ml / min and the injection flow at 0.2 ml / min. The separation starts at a focal flow of 1 ml / min within 3 minutes and occurs at a cross flow of 0.2 ml / min within 15 minutes. The Dawn EOS polygonal light scattering detector (Wyatt Technology, Santa Barbara, USA) and refractive index (RI) detector (Waters differential refractometer, Milford, MA, USA) couples directly to the flow fractionation channel. The light scattering detector is equipped with a GaAs laser (wavelength: 690 nm) and eighteen detectors. The scattered light is focused at angles between 14 and 163 degrees. The RI detector is calibrated with sodium chloride. Data is collected and analyzed using the Astra program version 4.90.08, using a refractive index increase (dn / dc) of 0.153 ml / g.
[0104] In the following examples, the injectability of some compositions is determined by means of a device consisting of a vertical support for a luer lock syringe filled with a hydrogel at 3<sup>about</sup>C and the disc resting on the syringe plunger as shown in Figure 1. 27G needle<sup>1/2</sup> x 0.5 inches is attached to the syringe, which is placed in the support. A load (500 g or 1 kg) is placed on this disk and the time required for expelling the composition from the syringe is measured.
[0105] In the following examples 1-3 and 7, the reacetylated chitosan used to make the hydrogel is a reacetylated chitosan obtained from Novamatrix (series FU-507-03) with DD 47% (measured by NMR) and average molecular weight (Mw) 3600 kDa (measured with AFFF-MALS).
[0106] In the following examples 4 and 6, the reacetylated chitosan used to make the hydrogel is reacetylated chitosan produced according to Production Example 1.
[0107] In the following example 5, the reacetylated chitosan used to make the hydrogel is reacetylated chitosan produced according to Production Example 2.
Production example 1
Preparation of "Fagal Lot 21" reacetylated chitosan with DD 61% according to the method disclosed in WO-A-2005/097871 [0108] 25.5 g of chitosan flakes (Sigma-aldrich, Saint Louis, Missouri, USA, Product No. 41,941-9 , series 14418LB) dissolves in 1 liter of 10% acetic acid and methanol (50/50) in one hour with stirring. 550 ml methanol are added. After 2 hours of stirring, the mixture is filtered through a 100 Pm filter to remove insoluble particles. The viscous solution is then dialyzed (Spectra / Por<sup>®</sup> 1 dialysis membrane 6,000-8,000 MWCO, n<sup>about</sup>132665, Spectrum Laboratories, Rancho Dominguez, USA) against deionized water for 72 hours, with daily water change. The solution is then filtered through a 5 μm filter.
[0109] With stirring, 400 ml of a 0.2M NH4OH / methanol (50/50) mixture is added to induce precipitation. After stirring for 1 hour, the suspension is filtered through a 100 μm filter. The precipitate is washed with methanol until neutral. The obtained purified chitosan is dried in the presence of silica gel in vacuo at room temperature and protected from light.
[0110] 10 g of this purified chitosan are dissolved in 500 ml of a 10% acetic acid / methanol (50/50) mixture. The mixture is stirred for 1 hour and left overnight. 400 ml methanol are added. The solution is stirred for several hours and left overnight. 150 ml methanol are added and the chitosan solution is cooled to less than 5<sup>about</sup>C using an ice bath. The solution with 2.4 ml acetic anhydride and 200 ml methanol is cooled to less than 5<sup>about</sup>C and added dropwise to the chitosan solution, using vigorous mechanical stirring. This solution containing homogeneously reacetylated chitosan is kept under stirring at a temperature lower than 5<sup>about</sup>C within one hour to ensure completion of the reaction and left overnight at room temperature. To eliminate salts formed during reacetylation and to further eliminate insoluble particles, this viscous solution is dialyzed against deionized water for 12 days (using the same dialysis membranes as above) with daily water exchange. The viscous chitosan solution is then filtered through a 5 μm filter. 200 ml NH mixture is added with stirring<sub>4</sub>OH 0.2M / methanol (50/50) to induce precipitation of chitosan. After 4 hours of stirring, the chitosan is passed through a 100 Pm filter and washed with methanol. Finally, homogeneously reacetylated chitosan is dried in the presence of silica gel under vacuum at room temperature and protected from light.
[0111] The resulting reacetylated chitosan exhibits DD 61% (measured by NMR) and average molecular weight (Mw) of 7900 kDa (measured by AFFF-MALS).
Production example 2
Preparation of the reacetylated "Fagal Lot 25" chitosan with DD 47% according to the method disclosed in WO-A-2005/097871 [0112] Chitosan flakes (Sigma-aldrich, Saint Louis, Missouri, USA, Product No. 41,941-9, series 14418LB) Purify as in Preparation Example 1. Then the chitosan is reacetylated with a solution made of 2.0 mL acetic anhydride and 200 mL methanol, following the same procedure as in Production Example 1.
[0113] The resulting reacetylated chitosan shows DD 47% (measured by NMR).
Example 1
Preparation of a composition according to the present invention containing 2% w / w reacetylated chitosan (DD = 47% <sub>RMN</sub>) obtained from Novamatrix and
8% w / w trehalose [0114] 700 mg of reacetylated chitosan obtained from Novamatrix (series FU-507-03) with a DD value of 47% (measured by RMN) is suspended in an autoclave in 4% water (in /in). 145 μΐ HCl is added and the suspension is kept under stirring for 18 hours at room temperature to allow complete dissolution of the chitosan. 3.09 g trehalose is dissolved in 6.5 ml 0.15M NaOH. This solution is cooled in an ice bath and added dropwise to the cooled chitosan solution while stirring. The pH of the gel is then adjusted to 6.8 by the addition of cooled diluted NaOH. Finally, cold water is added to obtain a total weight of 35 g. The resulting transparent gel shows an increase in viscoelastic properties over time at 37<sup>about</sup>C as shown in Fig. 2 and Fig. 3A and shows gelation in 111 minutes. Injection capacity measurement shows 30 seconds for a kilogram of weight.
Example 2
Freezing-thawing the hydrogel obtained in Example 1 [0115] 10 g of the preparation of Example 1 is frozen in liquid nitrogen and kept at -20<sup>about</sup>C. It is then thawed at 4<sup>about</sup>C, after which its rheological properties are determined. As shown in Fig. 3B, its thermogelling properties were maintained.
Example 3
Freeze drying the hydrogel obtained in Example 1 [0116] 10 g of the preparation of Example 1 is frozen in liquid nitrogen and kept at -20<sup>about</sup>C, after which it is freeze-dried for 24 hours using an Edwards Modulyo freeze-drying device (-50 plate temperature<sup>about</sup>C, vacuum 10<sup>-1</sup> mbar). The lyophilisate obtained is restored to its original form by the addition of cold water while stirring at 4<sup>about</sup>C. As shown in Figure 4, the thermogelling properties have been maintained.
Example 4
Preparation of a composition of the present invention containing 0.9% w / w of "Fagal lot 21" reacetylated chitosan (DD = 61%<sub>RMN</sub>_) obtained according to Production Example 1 and 5% w / w trehalose [0117] 270 mg of reacetylated chitosan obtained according to Production Example 1 and showing DD 61% (measured by RMN) are dissolved in 15 ml HCl 0.1N, under stirring for 18 hours at room temperature. 1.66 g of trehalose dihydrate are dissolved in 8 ml of 0.15M NaOH. This solution is cooled in an ice bath and added dropwise to the cooled chitosan solution while stirring. Then the pH of the gel is adjusted to 6.8 by the dropwise addition of cooled diluted NaOH. Finally, cold water is added to give a total weight of 30 g. The resulting transparent hydrogel shows an increase in viscoelastic properties over time at 37<sup>about</sup>C as shown in fig. 5. The trehalose formulation has a gel point after 30 minutes, while the trehalose formulation forms a gel after 150 minutes. Measurement of injectability shows 30 seconds for 500 g of weight.
Example 5 (comparative)
Preparation of a comparative composition containing 1% w / w of "Fagal lot 25" reacetylated chitosan (DD = 47%<sub>RMN</sub>) obtained according to production example 2 and 10% w / w 1,3-propanediol [0118] 200 mg of reacetylated chitosan obtained according to production example 2 and showing DD 47% (measured with RMN) is dissolved in 10 ml HCl 0.1N, stirring for 18 hours at room temperature. 2 g of cooled 1,3-propanediol are added to the dissolved chitosan. Then the pH of the gel is adjusted to 6.8 by the dropwise addition of cooled diluted NaOH. Finally, cold water is added to obtain a total weight of 20 g. The resulting transparent hydrogel shows an increase in viscoelastic properties over time at 37<sup>about</sup>C as shown in Fig. 6A.
[0119] After freezing (at -80 temperature<sup>about</sup>C), freeze-drying (within 24 hours) and restoring it to its original form with cold water, stirring at 4<sup>about</sup>C, the resulting formulation was no longer injectable (27G<sup>1/2</sup>, kilogram weight) according to the test for measuring the ability to inject. In addition, it no longer had thermogelling properties as shown in Fig. 6B.
Example 6
Preparation of a composition of the present invention containing 0.9% w / w of "Fagal lot 21" reacetylated chitosan (DD = 61%<sub>RM</sub>n) Mannitol obtained according to Production Example 1 and 5% w / w [0120] 630 mg of reacetylated chitosan obtained according to Production Example 1 are dissolved in 35 ml HCl 0.1N with stirring for 18 hours at room temperature. The chitosan solution is cooled to about 5<sup>about</sup>C followed by 3.5 g of mannitol. Then the pH of the gel is adjusted to 6.8 by the dropwise addition of cooled diluted NaOH. Finally cold water is added to obtain a total weight of 70 g. 10 g of this preparation are frozen in liquid nitrogen and kept at a temperature of
-20<sup>about</sup>C, after which it is freeze-dried within 2 hours using an Edwards Modulyo freeze-drying device (-50 plate temperature<sup>about</sup>C, vacuum 10<sup>-1</sup> mbar). The lyophilisate obtained is restored to its original form by the addition of cold water while stirring at 4<sup>about</sup>C. The obtained transparent hydrogel shows an increase in viscoelastic properties with time at 37<sup>about</sup>C, with a gel time of 1 hour, as shown in Figure 7.
Example 7
Preparation of a composition according to the present invention containing 2% w / w reacetylated chitosan (DD = 47%<sub>RMN</sub>) obtained from Novamatrix and
10% w / w glycerin [0121] 700 mg of reacetylated chitosan obtained from Novamatrix is suspended in an autoclave in 4% water (w / w). After cooling the suspension to room temperature, 145 Pl HCl is added and the mixture is stirred for 18 h to complete dissolution of the chitosan. The chitosan solution is cooled to about 5<sup>about</sup>C using an ice bath and 3.5 g of cooled glycerin are added. The pH of the gel is adjusted to 6.8 by the addition of cooled diluted NaOH. Finally, cold water is added to obtain a total weight of 35 g. The transparent glycerin-containing hydrogel shows an increase in viscoelastic properties over time at 37<sup>about</sup>C and a gel time of 135 minutes, as shown in Figure 8, while the glycerin-free formulation did not show a gel point after 180 minutes.
Laboratoire Medidom SA; Switzerland Representative:
Z-6506 EP 1978925 B1
18 members in 12 offices
Priority claims4
| Document | Office | Kind | Date |
|---|---|---|---|
| 05824526 | European Patent Office (EPO) | A | |
| 2005013980 | European Patent Office (EPO) | W | |
| EP20050824526 | – | – | – |
| WO2005EP13980 | – | – | – |
Members18
| Document | Office | Kind | |
|---|---|---|---|
| CA2634479A1 | Canada | A1 | |
| WO2007073749A1 | World Intellectual Property Organization (WIPO) | A1 | |
| EP1978925A1 | European Patent Office (EPO) | A1 | |
| US2009004230A1 | United States of America | A1 | |
| CN101360479A | China | A | |
| JP2009520705A | Japan | A | |
| EP1978925B1 | European Patent Office (EPO) | B1 | |
| AT439122T | Austria | T | |
| ATE439122T1 | Austria | T1 | |
| DE602005016030D1 | Germany | D1 | |
| PT1978925E | Portugal | E | |
| ES2331620T3 | Spain | T3 | |
| BRPI0520742A2 | Brazil | A2 | |
| PL1978925T3This record | Poland | T3 | |
| CN101360479B | China | B | |
| CA2634479C | Canada | C | |
| JP5474353B2 | Japan | B2 | |
| US8945609B2 | United States of America | B2 |
Numbers
- Publication, DOCDB
- 1978925
- Publication, EPODOC
- PL1978925T
- Application
- 824526
- Application, DOCDB
- 05824526
- Application, EPODOC
- PL20050824526T
Titles2
- English
- THERMOSETTING NEUTRALIZED CHITOSAN COMPOSITION FORMING A HYDROGEL, LYOPHILIZATE, AND PROCESSES FOR PRODUCING THE SAME
- Polish
- Termoutwardzalna zobojetniona kompozycja chitozanu tworzaca hydrozel, liofilizat i sposoby ich wytwarzania