U.S. patent application number 14/115184 was filed with the patent office on 2014-10-23 for biotechnological sulphated chondroitin sulphate at position 4 or 6 on the same polysaccharide chain, and process for the preparation thereof.
This patent application is currently assigned to GNOSIS S.P.A.. The applicant listed for this patent is Paola Bazza, Davide Bianchi, Niccolo Miraglia, Marco Valetti, Ermanno Valoti. Invention is credited to Paola Bazza, Davide Bianchi, Niccolo Miraglia, Marco Valetti, Ermanno Valoti.
Application Number | 20140315854 14/115184 |
Document ID | / |
Family ID | 46208441 |
Filed Date | 2014-10-23 |
United States Patent
Application |
20140315854 |
Kind Code |
A1 |
Bianchi; Davide ; et
al. |
October 23, 2014 |
BIOTECHNOLOGICAL SULPHATED CHONDROITIN SULPHATE AT POSITION 4 OR 6
ON THE SAME POLYSACCHARIDE CHAIN, AND PROCESS FOR THE PREPARATION
THEREOF
Abstract
The present invention discloses a process for the production of
chondroitin sulphate with an average molecular weight (Mw) of 10-30
kDa by chemical sulphation starting from an unsulphated chondroitin
backbone, obtained in turn by acid hydrolysis of capsular
polysaccharide K4 made directly from E. coli strain O5:K4:H4, or
directly produced from a genetically modified strain of E. coli.
Sulphation of the N-acetyl-D-galactosamine residue at position 4 or
6 takes place simultaneously in the same polysaccharide chain,
simulating the sulphation pattern observed in natural chondroitin
sulphate, unlike the sulphation obtained with the synthesis methods
described to date.
Inventors: |
Bianchi; Davide; (Milano,
IT) ; Valetti; Marco; (Desio (MI), IT) ;
Bazza; Paola; (Desio (MI), IT) ; Miraglia;
Niccolo; (Desio (MI), IT) ; Valoti; Ermanno;
(Dalmine (BG), IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bianchi; Davide
Valetti; Marco
Bazza; Paola
Miraglia; Niccolo
Valoti; Ermanno |
Milano
Desio (MI)
Desio (MI)
Desio (MI)
Dalmine (BG) |
|
IT
IT
IT
IT
IT |
|
|
Assignee: |
GNOSIS S.P.A.
Milano
IT
|
Family ID: |
46208441 |
Appl. No.: |
14/115184 |
Filed: |
May 10, 2012 |
PCT Filed: |
May 10, 2012 |
PCT NO: |
PCT/EP2012/058654 |
371 Date: |
December 4, 2013 |
Current U.S.
Class: |
514/54 ;
536/53 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61P 31/04 20180101; A61P 19/02 20180101; A61P 35/00 20180101; A61K
31/737 20130101; A61P 25/28 20180101; A61P 25/00 20180101; C08L
5/08 20130101; A61P 29/00 20180101; Y02A 50/473 20180101; A61P 9/10
20180101; A61P 21/00 20180101; A61P 19/10 20180101; C08B 37/0069
20130101 |
Class at
Publication: |
514/54 ;
536/53 |
International
Class: |
C08B 37/00 20060101
C08B037/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 12, 2011 |
IT |
MI2011A000829 |
Feb 2, 2012 |
IT |
MI2012A000136 |
Claims
1. Process for the preparation of chondroitin sulphate sodium salt
in which all the N-acetyl-D-galactosamine units in the same
polysaccharide chain are monosulphated either randomly or at the 4-
or 6- position, said process comprising the following steps: a.
transforming chondroitin sodium salt into its free acid or a salt
thereof with a quaternary ammonium cation selected from
tetramethylammonium, tetraethylammonium or tetrabutyl-ammonium, or
into the pyridinium salt or the methyl ester; b. reacting the
compound obtained in step a) with an orthoester of formula
RC(OR.sub.1).sub.3, in which R is selected from hydrogen, methyl,
ethyl or phenyl and R.sub.1 is selected from methyl or ethyl, in
the presence of acid catalysis, to give a compound in which the
repeating disaccharide unit present in chondroitin has the formula
I ##STR00006## in which R and R1 are as defined above; c.
protecting the hydroxy groups at the 2'- and 3'-positions of the
glucuronic acid units of the compound obtained in the previous step
by reaction with an anhydride of formula (R.sub.2CO).sub.2O in
which R.sub.2 is selected from methyl, ethyl or propyl, in the
presence of pyridine or an organic tertiary base selected from
triethylamine or triisopropylamine and of 4-dimethylaminopyridine
(DMAP), to give a compound in which the repeating disaccharide unit
present in chondroitin has the formula II ##STR00007## in which R,
R.sub.1 and R.sub.2 are as defined above; d. rearranging the
orthoester functionality present in the product obtained in step c)
with an organic water-soluble acid to give an ester derivative in
which the repeating GalNAc units in the polysaccharide consist of
triacyl derivatives having formula IIIIa or IIIb ##STR00008## in
which R and R.sub.2 are as defined above; e. mono-sulphating the
compound obtained in step d) followed by removal of the O-acyl
groups present in compounds IIIa and IIIb obtained in the previous
step.
2. The process of claim 1 wherein the chondroitin sodium salt of
step a) is obtained starting either from the capsular
polysaccharide K4 produced by a culture broth of E. Coli strain
O5:K4:H4, or from the polysaccharide produced by a culture broth of
E. Coli strain DSM23644.
3. The process of claim 1 wherein step b) is carried out with an
orthoester selected from trimethyl orthoacetate, triethyl
orthoacetate, trimethyl orthoformate, triethyl orthoformate,
trimethyl orthopropionate, triethyl orthopropionate or trimethyl
orthobenzoate.
4. The process of claim 1 wherein the acid catalysis of step b) is
carried out with an acid selected from camphorsulphonic acid,
para-toluenesulphonic acid, methanesulphonic acid or with a
sulphone resin.
5. The process of claim 1 wherein step c) is effected with acetic
anhydride.
6. The process of claim 1 wherein step d) is effected at 20 to
40.degree. C., preferably at room temperature.
7. The process of claim 1 wherein step d) is effected at 40 to
70.degree. C., preferably at 60.degree. C.
8. The process of claim 1 wherein step d) is effected in a
water/organic water-soluble acid mixture or in water alone.
9. The process of claim 8, wherein the organic acid is selected
from acetic, formic, propionic, tartaric, citric acid or a
propionic resin.
10. The process of claim 1 wherein the obtained chondroitin
sulphate sodium salt has an average molecular weight (Mw) of 10-30
kDa.
11. The process of claim 10 wherein chondroitin sulphate sodium
salt has a distribution of monosulphate groups whose ratio ranges
from 90/10 4S/6S to 10/90 4S/6S.
12. The process of claim 1 wherein the ratio between the sulphated
N-acetyl-D-galactosamine units at the 4-position and the 6-position
in the obtained chondroitin sulphate sodium salt is lower than
1.
13. The process of claim 1 wherein the ratio between the sulphated
N-acetyl-D-galactosamine units at the 4-position and the 6-position
in the obtained chondroitin sulphate sodium salt is higher than
1.
14. Chondroitin sulphate sodium salt obtained according to the
process of claim 1.
15. Chondroitin sulphate having an average molecular weight
comprised between 4000 and 9000 daltons of biotechnologic origin in
which all the N-acetyl-D-galactosamine units in the same
polysaccharide chain are monosulphated either randomly or at the 4-
or 6- position.
16. Chondroitin sulphate according to claim 15 having a
distribution of monosulphate groups whose ratio ranges from 90/10
4S/6S to 10/90 4S/6S.
17. Method of preventing or treating osteoarthritis and/or
maintaining the well-being of the musculoskeletal system in
patients in need thereof, said method comprising: administering an
effective amount of the chondroint sulphate of claim 14 to said
patients; and preventing or treating said osteoarthristis and/or
mantaining said well-being of the musculoskeletal system in said
patients.
18. Composition comprising the chondroitin sulphate as claimed in
claim 14 and one or more pharmaceutically or nutraceutically
acceptable excipients.
19. (canceled)
20. The method of claim 17, further comprising administering
chondroint sulphate together with one or more pharmaceutically or
nutraceutically acceptable excipients.
Description
TECHNICAL FIELD OF INVENTION
[0001] The present invention relates to a method for the production
of chondroitin sulphate by chemical sulphation starting from an
unsulphated chondroitin backbone. The process according to the
invention allows simultaneous sulphation, within the same
polysaccharide chain, of position 4 or position 6 of the
N-acetyl-D-galactosamine residue. The chondroitin sulphate thus
obtained presents the same sulphation pattern as observed in
natural chondroitin sulphate, unlike that obtained with the
synthesis methods described so far.
[0002] The invention also relates to a chondroitin sulphate which
has an average molecular weight determined by SEC (Mw) of 4-9 kDa,
and a distribution of mono-sulphated groups ranging from 90%
4-sulphate and 10% 6-sulphate to 10% 4-sulphate and 90%
6-sulphate.
TECHNICAL BACKGROUND
[0003] Chondroitin sulphate (CS) is a complex natural
polysaccharide belonging to the glycosaminoglycan (GAG) class,
consisting of disaccharide sequences formed by residues of
glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc)
sulphated in different positions and bonded by beta 1-3 bonds.
[0004] CS is present in animal tissues, with structural and
physiological functions. Depending on its origin, CS mainly
consists of variable percentages of two types of disaccharide unit
monosulphated at position 4 or position 6 of GalNAc (disaccharides
A and C respectively). However, disaccharides in which the sulphate
groups are present in different numbers and different positions may
be present in various percentages in the polysaccharide chains. The
CS backbone also contains unsulphated disaccharide, generally in
small quantities. Disulphated disaccharides having two sulphate
groups bonded through the oxygen atom in various positions, such as
position 2 of GlcA and 6 of GalNAc (disaccharide D), position 2 of
GlcA and 4 of GalNac, or positions 4 and 6 of GalNAc (disaccharide
E), can be present in the CS backbone in variable percentages,
depending on the specific animal sources (Volpi N. J Pharm
Pharmacol 61, 1271, 2009. Volpi N. J Pharm Sci 96, 3168, 2007.
Volpi N. Curr Pharm Des 12, 639, 2006).
[0005] The repeating disaccharide unit found in CS has the
following chemical formula:
##STR00001##
[0006] wherein R.sub.2, R.sub.4 and R.sub.6 are independently H or
SO.sub.3.sup.-.
[0007] The negative charges of the carboxylate and sulphate groups
in the repeating disaccharide unit are neutralised by sodium
ions.
[0008] The meanings of the acronyms most commonly used to identify
the variously sulphated disaccharides are set out below:
[0009] Di-0S (R2=H; R4=H; R6=H)
[0010] Di-6S (C) (R2=H; R4=H; R6=SO3-)
[0011] Di-4S (A) (R2=H; R4=SO3-; R6=H)
[0012] Di-4,6diS (E) (R2=H; R4=SO3-; R6=SO3-)
[0013] Di-2,6diS (D) (R2=SO3-; R4=H; R6=SO3-)
[0014] Di-2,4diS (B) (R2=SO3-; R4=SO3-; R6=H)
[0015] Di-2,4,6triS (R2=SO3-; R4=SO3-; R6=SO3-)
[0016] Samples of CS originating from different animal sources are
also characterised by different molecular weights and charge
densities, this latter parameter being directly correlated with the
specific sulphated groups.
[0017] Table 1 shows the main disaccharides found in natural CS
extracted from cartilage and other tissues of various animal
species:
TABLE-US-00001 TABLE 1 Bovine Chicken CS Porcine CS CS Shark CS
Skate CS Squid CS Mn (kDa) 12-17 9-14 8-13 25-40 27-34 60-80 Mw
(kDa) 20-26 14-20 16-21 50-70 50-70 80-120 Polydispersity 1.8-2.2
1.4-1.8 1.6-2.0 1.0-2.0 1.2-2.5 0.8-1.3 index Di-0S 6 6 8 3 3 13
Di-6S 33 14 20 44 39 15 Di-4S 61 80 72 32 43 50 Di-2,6diS ND ND ND
18 13 0 Di-4,6diS ND ND ND 2 1 22 Di-2,4diS ND ND ND 1 1 0 Charge
density 0.90-0.96 0.92-0.96 0.90-0.94 1.15-1.25 1.08-1.20 1.00-1.20
Ratio 4S/6S 1.50-2.00 4.50-7.00 3.00-4.00 0.45-0.90 1.00-1.40
2.50-4.00 Mn = number average molecular weight; Mw = weight average
molecular weight; polydispersity index = Mw/Mn; the charge density
is the number of sulphate groups per disaccharide unit; ND = not
identified
[0018] As shown in Table 1, CS derived from land animals has
similar molecular mass parameters (Mn and Mw), whereas it is
different from that originating from fish species, which have
higher molecular mass values. The terrestrial CS samples are also
characterised by charge density (CD) values below 1.0, whereas the
marine CS samples always have CD values exceeding 1.0. This
characteristic is due to the different distribution of the
sulphated disaccharides. Generally, disulphated disaccharides are
found in trace amounts in terrestrial CS, and no polysulphated
disaccharides (tri- and tetra-sulphates) are observed in natural
CS.
[0019] The absence of tri- and tetra-sulphated disaccharides can
easily be evidenced by analysis following digestion of the
polysaccharide with chondroitinase ABC, a lytic enzyme specific for
monosulphated disaccharides (Di-4S and Di-6S) and for unsulphated
disaccharides (Di-0S), which are able to digest disulphated
disaccharides but unable to hydrolyse the polysaccharide chain in
correspondence with the polysulphated disaccharides. FACE
(Fluorophore-Assisted Carbohydrate Electrophoresis) analysis of
natural CS digested with chondroitinase ABC does not detect the
electrophoresis bands characteristic of the partly undigested
oligosaccharides which are found in synthetic or semisynthetic CS
deriving from the prior art.
[0020] It is also well known that, due to biosynthesis processes,
all natural CSs always show the simultaneous presence of
monosulphated disaccharides at position 4 or 6 of GalNAc on the
same polysaccharide chains (D'Arcy S M et al., Carbohydr Res. 1994
Mar. 4; 255:41-59. Hardingham T E et al., Carbohydr Res. 1994 Mar.
4; 255:241-54. Cheng F, et al., Glycobiology. 1992 December;
2(6):553-61. Chai W et al., Anal Biochem. 1996 May 15;
237(1):88-102. Zaia J et al., Anal Chem. 2001 Dec. 15;
73(24):6030-9. Desaire H et al., Anal Chem. 2001 Aug. 1;
73(15):3513-20).
[0021] Different activities have been reported for CS in relation
to its molecular structure (Kimata K et al., Mol Cell Biochem 1,
211, 1963. Volpi N. Biomaterials 23, 3015, 2002. Volpi N, Tarugi P.
Biochimie 81, 955, 1999. Volpi N. Biomaterials 20, 1359, 1999.
Suzuki S et al., J Biol Chem 243, 7, 1968).
[0022] CS has anti-inflammatory activity, and is currently
recommended in the treatment of osteoarthritis (OA) as a
Symptomatic Slow-Acting Drug for OsteoArthritis (SYSADOA) in
Europe, in particular for the treatment of osteoarthritis of the
knee (Jordan K M et al., Ann Rheum Dis 62, 1145, 2003), hip (Jordan
K M et al. Ann Rheum Dis 62, 1145, 2003) and hand (Zhang W et al.,
Ann Rheum Dis 66, 377, 2007) on the basis of clinical evidence and
corresponding meta-analyses of numerous clinical trials. CS is also
widely used as a nutraceutical in Europe and the USA, either alone
or in combination with other ingredients (McAlindon T E et al.,
JAMA 283, 1469, 2000. Volpi N et al., Food Anal Meth 1, 195, 2008.
Volpi N et al., Separation Sc 1, 22, 2009).
[0023] Commercial CS is obtained by extraction from animal tissue,
such as bovine and porcine tissue (Fuentes E P et al., Acta Farm
Bonaerense 17, 135, 1998), bird tissue (Luo X M et al., Poult Sci
81, 1086-1089, 2002) and fish cartilage (Sugahara K et al., Eur J
Biochem 239, 871, 1996. Lignot B et al., J Biotechnol 103, 281,
2003).
[0024] The animal origin of commercial CS involves safety problems
associated with transmissible infectious agents that cause diseases
such as bovine spongiform encephalopathy (BSE), and restricts the
possible sources available to meet the growing worldwide demand.
These factors have stimulated research into alternative methods of
producing CS.
[0025] Intensive efforts have been made to find a biotechnological
method of producing CS, using a micro-organism as source of a
precursor polysaccharide which has a structure partly similar to
that of CS and conducting chemical sulphation to produce a CS
similar to the natural one.
[0026] One example of this strategy is the production of
biotechnological CS from capsular polysaccharide K4 of E. coli
O5:K4:H4, as described in EP 1304338 B1. Said patent discloses a
process wherein polysaccharide K4 produced in liquid cultures is
extracted and purified, and then redissolved and subjected to acid
hydrolysis to eliminate the fructose residues bonded to the GlcA
residues of the polymer. The defructosylated polymer, identical to
the unsulphated backbone of CS (CH), is then sulphated at position
4 or position 6 of the GalNAc residue according to two different
chemical synthesis methods. Said patent also discloses a third
method whereby a disulphated CS in both positions 4 and 6 is
obtained. The CS described therein has a content of at least 70% of
sulphated polysaccharides consisting of mono- and/or disulphated at
position 4 and 6 of the GalNAc residue, position 2' of the GlcA
residue being unsulphated, and has a molecular weight (Mw) of 6-25
kDa and a charge density (CD) of 0.7-2.0.
[0027] In EP 1304338 B1 the authors disclose and claim, depending
on the synthesis strategy used, the possibility of:
[0028] a) synthesising CS 4S by selectively protecting position 6
of all the N-acetylgalactosamine (GalNAc) residues present, thus
obtaining a polymer selectively sulphated only at position 4 of all
the N-acetylgalactosamine (GalNAc) residues
[0029] b) obtaining a polymer in which, similarly, the hydroxyl
groups at position 6 of all the GalNAc residues are sulphated,
suitably protecting the hydroxyl residues present at position
4.
[0030] In the process described in EP 1304338 B1, simultaneous
sulphation therefore never takes place at positions 4 or 6 in the
same chain, unlike the situation with natural CS.
[0031] A recent publication (Bedini E et al., Angew Chem Int Ed
Engl. 2011 May 18) describes a process wherein the polysaccharide
K4 produced is sulphated at position 4 and/or position 6 of the
GalNAc residue in the same chain. However, the biotechnological CS
described by Bedini et al. has a molecular weight similar to that
of natural CS, namely around 17 kDa, leading to the low
bioavailability typical of natural extracted products. Bedini et
al. do not report any pharmacological characterisation of the
product they obtained.
LIST OF FIGURES
[0032] FIG. 1 relates to natural chondroitin sulphate of bovine
origin treated with chondroitinase C. Various oligosaccharides of
different length demonstrating the presence of sulphate groups at
position 4 or 6 of the GalNAc residue on the same polysaccharide
chain are formed.
[0033] The chromatogram was obtained by gradient separation on a
strong anion-exchange column (SAX-HPLC) and UV detection at 232 nm.
The gradient was obtained by 50 mM NaCl up to 1.2 M NaCl from 0 to
60 minutes.
[0034] FIG. 2 relates to natural chondroitin sulphate of porcine
origin treated with chondroitinase C. Various oligosaccharides of
different length demonstrating the presence of sulphate groups at
position 4 or 6 of the GalNAc residue on the same polysaccharide
chain are formed.
[0035] The chromatogram was obtained by gradient separation on a
strong anion-exchange column (SAX-HPLC) and UV detection at 232
nm.
[0036] FIG. 3 relates to biotechnological chondroitin sulphate
according to the present invention treated with chondroitinase C.
Also for this polysaccharide various oligosaccharides of different
length demonstrating the presence of sulphate groups at position 4
or 6 of the GalNAc residue on the same polysaccharide chain are
formed.
[0037] The chromatogram was obtained by gradient separation on a
strong anion-exchange column (SAX-HPLC) and UV detection at 232
nm.
DESCRIPTION OF THE INVENTION
[0038] The present invention describes a method for the production
of CS following chemical sulphation starting from an unsulphated
chondroitin backbone (CH), this CH being obtained by acid
hydrolysis of a natural microbial polysaccharide i.l. (K4), or
produced directly from a genetically modified E. coli, such as E.
coli strain DSM23644, described in patent applications
MI2010A001300 and MI2010A001264. The bacterial strain described
therein carries a mutation that causes the inactivation of the KfoE
gene for fructosylation of K4.
[0039] The CS obtained by the process according to the invention
presents the characteristics of a natural CS with a titre exceeding
95% on the basis of the analytic methods described in the European
Pharmacopoeia.
[0040] The CS obtained with the process according to the invention
has an average molecular weight (Mw), measured by SEC, of 10-30
kDa, preferably 20-30 kDa, and presents a distribution of
mono-sulphated groups ranging from 90% of 4-sulphate and 10% of
6-sulphate to 10% of 4-sulphate and 90% of 6-sulphate (Table
2).
TABLE-US-00002 TABLE 2 Characteristics of the CS described in this
invention Mw (kDa) 10-30 Digestibility with chondroitinase ABC
>95% Di-0S <10% Di-6S 10-90% Di-4S 90-10% Di-2,6diS <5%
Di-4,6diS <5% Di-2,4diS <5% Di-triS ND Di-tetraS ND Titre
(w/w) >95% (o.d.b.)* Charge density 0.8-1.0 Ratio 4S/6S 0.1-9.0
*(o.d.b.): on dry basis
[0041] The CS obtained with the process according to the invention
contains a small amount (<10%) of unsulphated disaccharide and
very low percentages (<5%) of disulphated disaccharides;
trisulphated disaccharides cannot be identified.
[0042] The CS obtained with the process according to the invention
is characterised by charge density values of 0.8-1.0.
[0043] In some forms of implementation of the present invention,
the CS obtained shows a ratio between the sulphated disaccharide at
position 4 (Di-4S) and the sulphated disaccharide at position 6
(Di-6S) of less than 1, whereas in other forms it shows a ratio
between (4S) disaccharide and (6S) disaccharide greater than 1.
[0044] The process according to the present invention allows
site-specific sulphation to be modulated to produce a CS with a
specific 4S/6S ratio within the range specified above.
[0045] The present invention also relates to the production of
chondroitin sulphate (CS) with low molecular weight (LMW-CS BIOTEC,
4,000-9,000 daltons) by chemical sulphation from a non-sulphated
chondroitin backbone, which in turn is obtained by acid hydrolysis
of the capsular polysaccharide K4 produced by E. coli strain
O5:K4:H4, or directly produced from a genetically modified E. coli.
The chondroitin sulphate with low molecular weight that is object
of the invention is characterised by a molecular weight interval of
4,000-9,000 daltons, which is much less than that of chondroitin
sulphates of natural origin, whether terrestrial, in particular of
bovine, porcine or avian origin (14,000-26,000 daltons) or of
marine origin, for example obtained from sharks, squid, rays or
bony fish (generally >40,000 daltons). In view of these
characteristics, the chondroitin sulphate according to the
invention presents higher absorption after oral administration and
therefore better bioavailability in humans than highly pure natural
chondroitin sulphate or chondroitin sulphate produced by
biotechnological/chemical processes. The chondroitin sulphate
according to the invention possesses anti-inflammatory and
antiarthritic activity comparable with those of highly pure natural
chondroitin sulphate. The chondroitin sulphate according to the
invention is suitable for use in the treatment of inflammatory and
osteoarthritic/arthritic processes.
[0046] The LMW-CS BIOTEC according to the invention has an average
molecular weight, measured by SEC (Mw), of 4-9 kDa, and a
distribution of mono-sulphated groups ranging from 90% 4-sulphate
and 10% 6-sulphate to 10% 4-sulphate and 90% 6-sulphate. The
characteristics of the low molecular weight CS according to the
invention are substantially identical to those of the higher
molecular weight derivatives reported in Table 2 above.
[0047] The LMW-CS BIOTEC according to the invention has a small
quantity (<10%) of non-sulphated disaccharide and very low
percentages (<5%) of disulphated disaccharides, while no
trisulphated disaccharides are identifiable.
[0048] LMW-CS BIOTEC is characterised by charge density values of
0.8-1.0, which are comparable with those of natural CS of
terrestrial origin (see Table 1).
[0049] The process according to the invention also allows
site-specific sulphation to be modulated in order to supply a CS
with a specific 4S/6S ratio within the limits specified above,
which are similar to those present in CS of natural origin.
[0050] The LMW-CS BIOTEC according to the invention is recognised
and digested by chondroitinase ABC, a lytic enzyme which has the
task of catabolising the natural CS in specific organisms, thus
demonstrating that the polysaccharide chains of biotechnological
LMW-CS have not undergone structural modifications liable to
prejudice the specific, highly sensitive recognition of natural
enzymes.
[0051] Finally, the LMW-CS BIOTEC digested with chondroitinase C,
an endolyase that hydrolyses the polysaccharide in residues
sulphated in position 6, but not in position 4, produces
oligosaccharide sequences typical of the presence of Di-4S units
alternating with Di-6S units on the same polysaccharide chain, as
occurs in natural CS (FIGS. 1, 2 and 3). FIG. 1 in particular
describes natural chondroitin sulphate of bovine origin treated
with chondroitinase C. Oligosaccharides of different lengths can be
seen which indicate the presence of sulphate groups in position 4
or 6 of the GalNAc residue on the same polysaccharide chain. The
chromatogram was obtained by gradient separation on strong
anion-exchange column (SAX-HPLC) and UV detection at 232 nm. The
gradient was obtained with 50 mM NaCl to 1.2 M NaCl from 0 to 60
minutes;
[0052] FIG. 2 describes natural chondroitin sulphate of porcine
origin treated with chondroitinase C. Oligosaccharides of different
lengths can be seen which indicate the presence of sulphate groups
in position 4 or 6 of the GalNAc residue on the same polysaccharide
chain. The chromatogram was obtained by gradient separation on
strong anion-exchange column (SAX-HPLC) and UV detection at 232
nm;
[0053] FIG. 3 describes the LMW-CS BIOTEC of the present invention,
treated with chondroitinase C. Once again, oligosaccharides of
different lengths are visible which indicate the presence of
sulphate groups in position 4 or 6 of the GalNAc residue on the
same polysaccharide chain.
[0054] The chromatogram was obtained by gradient separation on
strong anion-exchange column (SAX-HPLC) and UV detection at 232
nm.
[0055] The LMW-CS BIOTEC according to the invention has been
evaluated for oral absorption and bioavailability in humans by
comparison with highly pure natural CS of bovine origin, the first
standard of the European Pharmacopoeia. This is particularly
important because the presence of a bacterium able to biosynthesise
a lytic enzyme specific for the breakdown of CS (and derivatives
with low molecular weight) has been described in human but not
animal bacterial flora (Ahn M Y, et al., Can J Microbiol 1998; 44:
423-9).
[0056] The oral absorption and bioavailability of LMW-CS BIOTEC
have been evaluated in humans by known techniques.
[0057] The LMW-CS BIOTEC according to the invention was evaluated
for possible anti-inflammatory activity using specific tests such
as:
[0058] the ability to inhibit a proteolytic enzyme produced during
inflammatory processes by the leucocytes, namely human leucocyte
elastase (Kostoulas G. et al., Biol Chem 378, 1481, 1997; Volpi N.
Chem Biol Interact 105, 157, 1997; Ying Q L et al., Am J Physiol.
272, L533, 1997); the ability to inhibit antichemotactic,
phagocytic activity, lysozyme release and damage to the biological
membrane by free radicals in human neutrophils (Matzner Y. et al.,
Thromb Haemost 52, 134, 1984; Ronca F, Palmieri L et al.,
Osteoarthritis Cartilage 6 Suppl A, 14, 1998).
[0059] These tests were conducted on the LMW-CS BIOTEC according to
the invention by comparison with a reference compound, a highly
pure natural CS of bovine origin which is the first standard of the
European Pharmacopoeia.
[0060] The LMW-CS BIOTEC according to the invention was also
evaluated for antiarthritic properties in an animal model, the
"Adjuvant Arthritis (AA) model", which is widely recognised by the
scientific community and has been published in numerous scientific
papers. Once again, the results were compared with those previously
obtained with the reference molecule: the European Pharmacopoeia
standard, a highly pure natural CS of bovine origin (Volpi N. J
Pharm Sci 96, 3168, 2007). In fact, animal models of OA and
rheumatoid arthritis (AR) are useful tools for the study of these
pathogenic processes. "Adjuvant Arthritis" (AA) is one of the most
commonly used models. AA in the rat is an experimental model of
polyarthritis which has been widely used to test numerous
antiarthritic agents and medicaments before and after thorough
clinical trials (Bendele A et al., Toxicol Pathol 27, 134, 1999;
Rovensky J et al., Rheumatol Int. 31, 507, 2011; Bauerova K et al.,
Interdisc Toxicol 4, 101, 2011). Numerous studies have also been
conducted wherein the data on animals obtained with the AA test
were compared with the results in humans (Kannan K et al.,
Pathophysiology 12, 167, 2005).
[0061] Simultaneous monosulphation in position 4 or 6 of the
polymer chain, purity and low molecular weight give the LMW-CS
BIOTEC according to the invention greater oral absorption and
better bioavailability.
[0062] One aspect of the present invention relates to the
composition of the CS according to the invention and a carrier
acceptable in the pharmaceutical or nutraceutical field. Said
composition can be formulated in various solid forms, such as
tablets, rigid capsules, soft gelatin capsules or powdered mixtures
for drinks, or in liquid forms (solutions), preferably in the form
of pharmaceutical or nutraceutical preparations for parenteral or
oral administration. The composition can contain other active or
inactive ingredients.
[0063] The composition can also, preferably, contain at least one
of the following substances: glucosamine hydrochloride, glucosamine
sulphate, N-acetyl glucosamine, hyaluronic acid, heparin, keratin,
dermatin, methyl sulphonyl methane, folates and reduced folates,
Group B vitamins, S-adenosylmethionine (SAMe), ascorbic acid or
manganese ascorbate. The composition can be administered to
patients in effective quantities based on their needs.
[0064] For example, but without limiting its use, the CS or the
composition described in the present invention can be administered
in a quantity of between 100 and 3000 mg a daily, preferably
between 1000 and 2000 mg a daily, and more preferably between 1250
and 1750 mg a daily, divided into two doses of approx. 600 mg or
three doses of 400 mg a daily.
[0065] The present invention also relates to the use of the CS
described, or a composition thereof, for the treatment or
prevention of osteoarthritis or for the maintenance of
musculoskeletal well-being as an ingredient of a medicament or
nutritional supplement.
[0066] For example, the CS described or a composition thereof can
be used to make a pharmaceutical preparation, dietary additive or
nutritional supplement for the prevention and/or treatment of
osteoarthritis of the hip, hand or knee and the main symptoms
thereof (pain, joint swelling, inflammation), Alzheimer's disease,
microbial infections, arteriosclerosis and osteoporosis, and as
adjuvant in antitumoral treatment and tissue regeneration,
including nerve tissue.
[0067] An advantageous characteristic of the process according to
the invention is that the sulphation at position 4 or 6 of the
GalNAc residue takes place simultaneously in the same
polysaccharide chain, simulating the sulphation pattern observed in
natural CS, unlike that obtained with the synthesis methods
described to date. This aspect is confirmed by the data obtained
with the use of two different enzymatic systems, namely
chondroitinase ABC, which is able to digest units sulphated at
position 6 and position 4 and unsulphated units, and chondroitinase
C, an endolyase which is able to hydrolyse in correspondence with
the residues sulphated at position 6 and unsulphated residues, but
unable to perform similar lytic cleavage in correspondence with the
residues sulphated at position 4. The products of digestion,
obtained with chondroitinase ABC and with chondroitinase C alone,
are analysed with HPLC chromatography techniques, as described by
Joon-Soo Sim et al. (J. Chromatography B, 2005 vol. 818, 133-139),
qualitatively and quantitatively indicating the presence of
disaccharides Di-0S, Di-4S and Di-6S and any oligosaccharides not
digested by the enzymes.
[0068] Analysis of the products of digestion with chondroitinase
ABC demonstrates almost total digestion of the product with
formation of the unsulphated disaccharide Di-0S, monosulphated
disaccharides Di-4S and Di-6S, and traces of disulphated
disaccharide Di-4,6S.
[0069] However, the same analysis conducted on the products of
digestion with chondroitinase C clearly shows the presence of
disaccharide sequences, and above all of oligosaccharide sequences,
indicating the inability of the enzyme to break down the
polysaccharide completely due to the presence on the same chains of
GalNAc sulphated in 4. This is because when a sulphated residue is
present in 4, the enzyme is unable to act, and consequently leaves
oligosaccharide residues. Said residues are also clearly detected
by chromatography and electrophoresis techniques, such as gel
chromatography and capillary electrophoresis (CE), as shown, for
example, in the chromatographic tracings in FIGS. 1, 2 and 3
relating to digestion with chondroitinase C of natural CS (bovine
and porcine) and biotechnological CS obtained according to the
present invention. They contain various oligosaccharides of
different lengths wherein sulphate groups are present at position 4
or 6 of the GalNAc residue on the same polysaccharide chain.
[0070] All these properties give the CS obtained with the process
according to the present invention the structure of a natural CS
having the following characteristics:
[0071] a) all or nearly all the GalNAc residues are monosulphated
at position 6 or 4;
[0072] b) depending on the synthesis conditions used, the ratio
between residues 4S and 6S (4S/6S) is completely analogous to that
found in CS of both terrestrial and fish origin.
[0073] Typically, the CS according to the present invention can be
obtained using as starting substrate the capsular polysaccharide K4
naturally produced by E. coli strain O5:K4:H4 (EP 1304338 B1) or
another polysaccharide having the structure of unsulphated
chondroitin (CH).
[0074] In the first case, polysaccharide K4, obtained from a
culture broth of E. coli strain O5:K4:H4, is defructosylated at the
end of fermentation by thermoacid hydrolysis, and the chondroitin
is purified in accordance with an adaptation of the methods
described by Rodriguez and Jann (Eur. J. Biochem. 117, 117-124,
FEBS 1988).
[0075] Alternatively, the starting polysaccharide is obtained, for
example, from the culture of E. coli strain DSM23644 described in
MI2010A001300 which, due to a mutation induced in the KfoE gene
responsible for the fructosylation of K4, produces a polysaccharide
identical to natural unsulphated CH. Defructosylation is not
necessary in this case; however, the thermoacid hydrolysis step is
maintained to eliminate some impurities, including the bacterial
endotoxins that precipitate as a result of the treatment. The
chondroitin (CH) is then purified by centrifugation, dialysis and
spray drying.
[0076] Hydrolysis is conducted on the culture supernatant,
separated from the biomass by continuous centrifugation. Partial
hydrolysis and defructosylation of K4 is performed by incubation at
90-95.degree. C. for 30-50 min at pH 2.8-3.0.
[0077] After the incubation period, the resulting suspension is
cooled at a temperature below 40.degree. C., preferably
20-30.degree. C., to quench the hydrolysis reaction, and the pH is
simultaneously adjusted to 4-4.5. The resulting suspension
undergoes, in sequence, clarification by continuous centrifugation,
ultrafiltration and finally, dialysis with water through a 30 kDa
membrane. The dialysed retentate (approx. 1/10th of the volume of
the initial culture broth) is filtered and finally dried with a
spray dryer to obtain a polysaccharide having the structure of CH,
to be subjected to the sulphation process. The CH obtained has a
titre of 80-90% on a dry basis (w/w), as determined by capillary
electrophoresis (CE) or HPLC.
[0078] The CH thus obtained takes the form of the sodium salt, and
in order to be sulphated needs to be converted to free acid or a
salt thereof.
[0079] The sulphation process according to the present invention,
which allows positions 4 or 6 of the GalNAc residue of the same
polysaccharide chain to be monosulphated randomly, comprises the
formation of an orthoester which simultaneously involves GalNAc
positions 4 and 6 and its subsequent rearrangement to an ester
which, surprisingly, can be modulated to release mainly the
hydroxyl in 4 or in 6, thus allowing selective sulphation of those
hydroxyls.
[0080] The process according to the invention comprises the
following steps:
[0081] a) Conversion of the chondroitin sodium salt to free acid
or, alternatively, to a salt thereof with a quaternary ammonium
ion, such as tetramethyl-, tetraethyl- or tetrabutyl-ammonium, or
with pyridine. Tetrabutylammonium (TBA) salt is preferably
used.
[0082] Alternatively, chondroitin (CH) in acid form is converted to
its methyl ester after reaction in methanol and acetyl
chloride.
[0083] b) Reaction of the chondroitin salt, or chondroitin methyl
ester, with an orthoester of formula RC(OR.sub.1).sub.3, wherein R
is selected from hydrogen, methyl, ethyl or phenyl, and R.sub.1 is
selected from methyl or ethyl, in the presence of acid catalysis,
thus obtaining a cyclic orthoester formed by the movement of two
alkoxyls of the starting orthoester by alcohol functions 4 and 6 of
the GalNAc residue. In the compound obtained in this step, all or
nearly all the disaccharide units present possess a cyclic
orthoester structure represented by formula I,
##STR00002##
[0084] wherein R, R.sub.1 are as defined above.
[0085] Examples of orthoesters which can be used are trimethyl
orthoacetate, triethyl orthoacetate, trimethyl orthoformate,
triethyl orthoformate, trimethyl orthopropionate, triethyl
orthopropionate or trimethyl orthobenzoate. Trimethyl orthoacetate
or triethyl orthoacetate is preferably used. The use of trimethyl
orthoacetate is particularly preferred.
[0086] An acid selected from camphorsulphonic acid,
paratoluenesulphonic acid, methanesulphonic acid or a sulphone
resin, preferably camphorsulphonic acid or a sulphonic resin, more
preferably camphorsulphonic acid, is used as acid catalyst.
[0087] c) Protection of the alcohol groups at positions 2' and 3'
of the GlcA residue by acylation with an anhydride of a carboxylic
acid of formula (R.sub.2CO).sub.2O, wherein R.sub.2 is preferably
selected from methyl, ethyl or propyl in the presence of pyridine
or a tertiary organic base, such as triethylamine or
triisopropylethylamine, and of catalytic quantities of
4-dimethylaminopyridine (DMAP), to give a product wherein the
repeating disaccharide unit found in the chondroitin has a cyclic
orthoester structure acylated in 2' and 3' which is represented by
formula II
##STR00003##
[0088] wherein R, R.sub.1 and R.sub.2 are as defined above.
[0089] Acetic anhydride is preferably used.
[0090] d) Rearrangement from cyclic orthoester to ester, a reaction
which is performed in a mixture of a water-soluble organic acid and
water, or in water only. This rearrangement, which takes place
randomly on the various GalNAc units of the polysaccharide
sequence, can be modulated to promote the release of one or other
hydroxyl (in 4 or 6 respectively), with simultaneous formation of
the ester with the soluble organic acid used in the remaining
position (6 or 4 respectively). The result is the formation, in the
same polysaccharide chain, of two different disaccharide units,
namely: [0091] those with a structure wherein the hydroxyls at
positions 6, 2' and 3' are acylated and the hydroxyl in 4 is free,
said units being represented by formula IIIa;
##STR00004##
[0092] wherein R and R.sub.2 are as defined above; or [0093] those
with a structure wherein the hydroxyls at positions 4, 2' and 3'
are acylated and the hydroxyl in 6 is free, said units being
represented by formula IIIb
##STR00005##
[0094] wherein R and R.sub.2 are as defined above.
[0095] By conducting the reaction at a temperature of between 20
and 40.degree. C., preferably at room temperature for a time of
between 1 and 48 hours, preferably between 3 and 38 hours, and more
preferably for 38 hours, a larger amount of compound having the
free hydroxyl in 6 is surprisingly observed, whereas when the
reaction is conducted at a temperature of between 40 and 70.degree.
C., preferably 60.degree. C., for a time of between 1 and 48 hours,
preferably between 3 and 38 hours, and more preferably for 18
hours, the product with the free hydroxyl at position 4 prevails.
The water-soluble organic acid is selected from acetic, formic,
propionic, tartaric citric acid or a cationic resin such as for
example Sepra SCX 50 .mu.m 65A, preferably acetic acid or propionic
acid, and more preferably acetic acid.
[0096] d) This is followed by sulphation with pyridine sulphur
trioxide in DMF according to the method already described in EP
1304338 B1, or with the DMF-sulphur trioxide complex, to obtain a
CS which, according to the rearrangement conditions used and
consequently the percentage of structures IIIa and IIIb present
therein, will be simultaneously and variously sulphated at position
4 of disaccharide IIIa or position 6 of disaccharide IIIb. The
sulphation reaction is followed by removal, by basic treatment, of
the acyl groups present at positions 2' and 3' of the GlcA residue
and positions 4 or 6 of the GalNAc residue, according to the
procedures described in EP 1304338 B1, giving CS sodium salt which
is partly sulphated in 4 and 6.
[0097] Some techniques used during the process lead to
depolymerisation of the polysaccharide chain so as to produce a
sulphated CS in position 4 or 6 of the GalNAc residue characterised
by a low molecular weight (LMW).
[0098] Chondroitin can also be depolymerised at the orthoester
rearrangement stage, using the acid as solvent or co-solvent of the
reaction. The high concentration of acid at this stage leads to
rupture of the polysaccharide chain, with consequent production of
low molecular weight chains, in the 4-9 kD range.
[0099] The LMW-CS BIOTEC, 4,000-9,000 daltons, obtained by the
process described, was evaluated for efficacy in an experimental
animal arthritis model (Adjuvant Arthritis AA) in the rat, and the
results were compared with those for pharmaceutical grade natural
CS of extracted origin used in the same experimental model
(Bauerova K. et al., Osteoarthritis Cartilage 2011, Epub ahead of
print) after daily oral treatment with 900 mg/kg.
[0100] AA was induced by a single intradermal injection of
Mycobacterium butyricum in incomplete Freund's adjuvant. The
experiments comprised healthy animals, untreated arthritic animals
and treated arthritic animals. Among the treated animals, one group
of animals was subjected to pre-treatment consisting of
administration of 900 mg/kg of LMW-CS BIOTEC a day for 14 days
before arthritis was induced, continuing for 28 days after the
induction of AA. Another group of animals was treated with 900
mg/kg of LMW-CS BIOTEC a day solely during the 28 days after the
induction of AA.
[0101] The oedema that developed in the hind paw was significantly
reduced in the pretreated animals. Pre-treatment with the LMW-CS
BIOTEC according to the invention (900 mg/kg/day) significantly
reduced oedema throughout the experiment compared with the
untreated controls. Pre-treatment with LMW-CS BIOTEC also restores
the body weight by approx. 8-15% compared with the untreated
arthritic control.
[0102] The severity of the arthritis was quantified on the basis of
increasing levels of swelling and periarticular erythema. 900
mg/kg/day of LMW-CS BIOTEC, administered as both pre-treatment and
treatment, is significantly effective in reducing the arthritis
score. Moreover, pre-treatment is effective throughout the subacute
stage (from day 14 to day 28 after induction of AA), whereas the
treatment is only effective in the medium-long term, in days 21-28
after induction of AA, not at the acute stage (the first 14 days
after induction of AA).
[0103] Oxidative stress, a consequence of the chronic inflammatory
processes that take place in arthritic/osteoarthritic processes,
significantly increases in the animal model at both the acute and
the subchronic stage. Increased oxidative stress induces high
consumption of endogenous antioxidants in the plasma, and
consequently causes a reduction in the plasma antioxidant capacity,
measured as the total antioxidant status. Pre-treatment with LMW-CS
BIOTEC is effective in correcting the total antioxidant status in
the animal model, significantly reducing the consumption of
endogenous antioxidants. The activity of .gamma.-glutamyl
transferase, which increases in correspondence with oxidative
stress and is therefore considered to be a good marker for
oxidative stress, measured in joint tissue homogenates, proved
considerably greater in animals with experimentally induced
polyarthritis, and considerably lower in the animals treated with
LMW-CS BIOTEC, by comparison with the untreated animals.
[0104] Interleukin-1.beta. (IL-.beta. and interleukin-6 (IL-6),
pro-inflammatory cytokines, significantly increased in the animal
model of experimentally induced arthritis, with a dramatic increase
in IL-6 at the acute stage, presenting a level 10 times higher than
the healthy controls. The therapeutic effect of LMW-CS BIOTEC was
already evident from day 14, at the acute stage, reducing the IL-6
concentration by approx. 30-40% compared with the animals suffering
from AA.
[0105] The basic marker for inflammatory proteins, namely
C-reactive protein (CRP), has a very similar time profile to IL-6.
The increase at the acute stage was approx. 7.5 times greater in
the experimental arthritis model than the healthy controls. The
effect of LMW-CS BIOTEC on CRP, like its effect on the IL-6 level,
is observed at the acute stage, with a significant reduction in the
plasma CRP concentration.
[0106] As regards the phagocytic activity and intracellular
oxidative increase of the neutrophils, the differences observed
between the healthy control and the control suffering from induced
experimental AA were significant in the case of increased
phagocytic activity. The administration of LMW-CS BIOTEC on a
pre-treatment basis induced a significant reduction in phagocytosis
and the oxidative burst.
[0107] The LMW-CS BIOTEC according to the invention significantly
reduces the severity of the arthritic processes and the oxidative
stress generated as a result of chronic inflammatory processes.
Pre-treatment with LMW-CS BIOTEC is effective throughout the
subacute stage, whereas treatment from day 1 of onset of AA is only
effective during the chronic period. The effects are confirmed by
an improvement in the total antioxidant status and activity of
.gamma.-glutamyl transferase. LMW-CS BIOTEC, administered as a
pre-treatment, also reduces the production of pro-inflammatory
cytokines, C-reactive protein in the plasma, phagocytic activity
and the intracellular oxidative burst of the neutrophils. Finally,
LMW-CS BIOTEC has proved effective in slowing the development of
experimental arthritis/osteoarthritis at both the acute and the
subchronic stage, and in reducing the markers of the disease, thus
supporting its beneficial activity, on a par with that of the
reference compound.
[0108] The invention will now be further illustrated by the
following examples.
Example 1
Preparation of a Tetra-Alkyl Ammonium or Pyridinium Salt of
Chondroitin
[0109] The CH sodium salt obtained after hydrolysis, purification
and drying by the methods described above, starting from
polysaccharide K4 or the polysaccharide obtained from fermentation
of E. Coli strain DSM23644, is dissolved in an aqueous medium.
After complete dissolution, the solution is introduced into a
column packed with a cation-exchange resin, such as Amberjet 1200
H, Rohm and Haas, or equivalent.
[0110] The fractions eluted at pH 1.5-4.0, or preferably at pH
1.5-2.0, are collected, and an aqueous solution of an ion selected
from tetramethyl-, tetraethyl- and tetrabutyl-ammonium or
pyridinium is added until a pH of 6.0-8.0, or preferably 6.5-7.0,
is obtained. The solution is then evaporated to complete dryness by
freeze-drying or spray drying to obtain the corresponding salt.
Example 2
Protection of the Hydroxylated Functions (4 and 6) of the GalNAc
Portion with Formation of the Corresponding Cyclic Methyl
Orthoester CH (CH-cMOE)
[0111] The salt obtained from chondroitin, such as tetrabutyl
ammonium (TBA) salt, is mixed with dimethylformamide (DMF) in a
flask in the quantities of 5.2 g and 130 ml respectively. 8.49 g of
trimethyl orthoacetate is dripped into the flask, followed by the
addition of 300 mg of camphorsulphonic acid, and the reaction
mixture is maintained at 70.degree. C. for 72 h. The reaction is
then evaporated under vacuum to dryness, and further stove-dried at
40.degree. C. for 20 h to obtain 6.1 g of chondroitin-MOE TBA in
the form of a solid.
[0112] The analyses on the product of reaction were conducted to
confirm that protection had taken place. The disappearance of the
starting product and the appearance of a new product with a higher
molecular weight (48 KDa) was established with SEC-HPLC. The
analyses performed by digestion with chondroitinase ABC, an enzyme
able to hydrolyse free but not protected CH, demonstrated that the
unprotected percentage of starting CH molecules was under 15%.
Example 3
2',3' Acetylation of chondroitin cyclic orthoester (2',3'diacetyl
CH-cMOE)
[0113] The chondroitin originating from the preceding step,
protected as cyclic methyl orthoester (CH-cMOE) (4.79 g), is
introduced into a reaction flask with 23.95 ml of acetonitrile,
15.69 ml of triethylamine (TEA), 6.21 ml of acetic anhydride and
78.96 mg of 4-dimethylaminopyridine (DMAP). After 2 hours' stirring
at 25-26.degree. C., 94 ml of di-isopropyl ether is added to obtain
a viscous solid, which is then filtered through filter paper and
stove-dried under vacuum at 45.degree. C. for 24 h. The
intermediate cyclic orthoester thus obtained has the appearance of
a pink solid.
Example 4
Rearrangement from Cyclic Methyl Orthoester to Ester with Prevalent
Formation of Acetate at Position 4, and with the Free Hydroxyl at
Position 6 (see Figure IIIB)
[0114] The intermediate obtained from the preceding step (2.42 g)
is introduced into a reaction flask, to which 18.8 ml of 96% acetic
acid and 2.35 ml of demineralised water are added. The mixture is
stirred for 38 h at room temperature, after which 100 ml of an 0.6
M solution of NaCl are added and the mixture is ultrafiltered
through a 5 kDa membrane and dialysed, to recover a retentate with
a pH of 3.32.
[0115] The solution is evaporated under vacuum at 45-50.degree. C.;
after further stove-drying overnight, 1.38 g of a product with the
appearance of a vitreous solid are obtained.
Example 5
Rearrangement from Cyclic Methyl Orthoester to Ester with Prevalent
Formation of the Acetate at Position 6, and with the Free Hydroxyl
at Position 4 (See Figure IIIA)
[0116] 2.42 g of intermediate cyclic orthoester obtained from the
preceding step are introduced into a reaction flask with 14.52 ml
of 96% acetic acid and 9.8 ml of demineralised water and heated to
60.degree. C. for 17.5 h, 100 ml of 0.6 M NaCl are then added and
the solution (pH 2.27) is ultrafiltered and dialysed to recover a
retentate with a pH of 3.56.
[0117] The solution is evaporated under vacuum at 45-50.degree. C.,
and after further stove-drying overnight, 1.12 g of a product with
the appearance of a vitreous solid are obtained.
Example 6
Preparation of Chondroitin Sulphate with Sulphur Trioxide
Pyridinium Complex
[0118] The intermediate obtained as described in example 4 (0.76 g)
is introduced into a flask with 46.0 ml of DMF starring the mixture
at 30.degree. C. for 10 min. 0.72 g of sulphur trioxide pyridinium
are added and when the starting material has dissolved (approx. 10
min), the solution is left under stirring at 30.degree. C. for 1 h.
A further 0.72 g of sulphur trioxide pyridinium are then added,
followed by a further 0.72 g of sulphur trioxide pyridinium. The
solution is stirred for a further hour at 30.degree. C.
[0119] The reaction is quenched by pouring the mixture into 50 ml
of 10% NaHCO.sub.3 in water at room temperature (pH 7.81). After
filtration the solution is evaporated under vacuum (10 mBar) to
dryness, the residue redissolved with 150 ml of 0.6 M NaCl and,
finally, the solution is ultrafiltered.
[0120] After 6 changes of volume the retentate has a pH of 9.22;
the pH is adjusted to 6.7 with 1N HCl and ultrafiltration
continues, replacing the 0.6N NaCl solution with demineralised
water.
[0121] The resulting solution is ultrafiltered again for 2 volumes,
and then dialysed to a volume of 20 ml. The dialysed solution is
concentrated to dryness under vacuum (10 mBar, 45.degree. C.).
[0122] The product thus obtained (0.88 g) is dissolved with 34.0 ml
of 0.2N soda (NaOH) and heated to 40.degree. C. under stirring for
2 h. Finally, the solution is diluted with an 0.6M aqueous solution
of sodium chloride, ultrafiltered through a 5 kDa membrane, and
dialysed with demineralised water. The retentate is concentrated to
dryness under vacuum (45.degree. C., 10 mBar), to obtain 0.67 g of
chondroitin sulphate. The end product, which has a molecular weight
of 29 kDa, determined by HPLC-SEC, shows: [0123] digestibility with
chondroitinase ABC exceeding 95%; [0124] a 4S/6S ratio of 18/82;
[0125] a total charge density value of approx. 0.9; [0126] only
partial digestibility with chondroitinase C, demonstrated by the
presence of oligosaccharides due to the presence on the same
polysaccharide chain of both 4-sulphated and 6-sulphated units,
characteristic of the present invention.
Example 7
Preparation of Chondroitin Sulphate with Sulphur Trioxide
Pyridinium Complex
[0127] The intermediate obtained as described in example 5 (1.12 g)
is introduced into a flask with 67.2 ml of DMF, stirring the
mixture at 50.degree. C. for 10 min. 1.05 g of sulphur trioxide
pyridinium are added, and when the starting material has dissolved
(approx. 10 min), the solution is left under stirring at 50.degree.
C. for 1 h. A further 1.05 g of sulphur trioxide pyridinium are
then added. The solution is stirred for a further hour at
50.degree. C.
[0128] The reaction is quenched by pouring the mixture into 60 ml
of 10% NaHCO.sub.3 in water at room temperature (RT) (pH 7.81).
After filtration the solution is evaporated under vacuum (10 mBar)
to dryness, and the residue is redissolved with 30 ml of 0.6 M
NaCl. Finally, the solution is ultrafiltered.
[0129] After 6 changes of volume the retentate has a pH of 9.22;
the pH is adjusted to neutrality (7.5) with 1 N HCl and
microfiltration continues, replacing the 0.6 N NaCl solution with
demineralised water.
[0130] The resulting solution is ultrafiltered again for 2 volumes,
and then dialysed to a volume of 20 ml. The dialysed solution is
concentrated to dryness under vacuum (10 mBar, 45.degree. C.), to
obtain 1.53 g of product.
[0131] This residue is dissolved in 59.6 ml of 0.2 N soda (NaOH)
and heated at 60.degree. C. for 2 h. Finally, the solution is
diluted with an 0.6M aqueous solution of sodium chloride,
ultrafiltered through a 3 kDa membrane, and dialysed with
demineralised water. The retentate is concentrated to dryness under
vacuum (45.degree. C., 10 mBar), to obtain 0.76 g of chondroitin
sulphate.
[0132] The product thus obtained has a molecular weight of 15.4
kDa, determined by HPLC-SEC; digestibility with chondroitinase ABC
exceeding 95%; a 4S/6S ratio of 82/18; and a total charge density
value of approx. 1.09. The almost complete digestion obtained with
chondroitinase ABC (over 95% of the product is broken down),
together with reduced digestibility with chondroitinase C, which
are characteristic of the present invention, demonstrate the
existence of both 4-sulphated and 6-sulphated units on the same
polysaccharide chain.
[0133] Over 95% digestibility with chondroitinase ABC also
demonstrates the absence of polysulphated (tri- and
tetra-sulphated) disaccharides in the CS polysaccharide chain to
which the present invention relates.
Example 8
Preparation of Chondroitin (CH) Methyl Ester
[0134] 10.0 g of CH in acid form are added to a solution of 1.3 L
of methanol and 14.43 g of acetyl chloride placed under stirring at
room temperature for 2 hours in a 3 litre flask, and the suspension
obtained is left under stirring for 20 hours.
[0135] When that time has elapsed, the suspension is filtered and
the solid is washed with 100 ml of methanol (2.times.50 ml) and
dried at 50.degree. C. under vacuum to recover 9.4 g of dry
solid.
[0136] The reaction is repeated a second time with the same
procedure, and when the second period has elapsed, the suspension
is cooled at between 0 and 5.degree. C. for 60 minutes before
filtration. The solid obtained is washed with cold methanol
(0-5.degree. C.) and stove-dried under vacuum for 3 hours at
50.degree. C. to recover 6.3 g of solid.
Example 9
Protection of the Hydroxylated Functions (4 and 6) of the GalNAc
Portion of CH Methyl Ester by Orthoester Formation
[0137] 150 ml of dimethylformamide (DMF) and 6.0 g of the product
obtained in the preceding step are introduced into a 500 ml flask
with a calcium chloride valve and nitrogen flow. 20.06 g of
trimethyl orthoacetate and 0.71 g of camphorsulphonic acid are then
added. The solution obtained is heated at 50.degree. C. (internal
temperature) for 18 hours.
[0138] At the end of that period it is left to cool at RT and
concentrated under vacuum to obtain 8.5 g of product.
Example 10
Acetylation of the 2',3' Hydroxyls of the Product Deriving from
Example 9
[0139] 8.0 g of the product obtained in the preceding step, 40 ml
of DMF, 28.6 g of triethylamine, 17.15 g of acetic anhydride and 96
mg of dimethylaminopyridine are introduced into a 250 ml flask with
a calcium chloride valve and nitrogen flow at room temperature.
[0140] The solution obtained is left under stirring for 3 hours;
when that time has elapsed, 150 ml of isopropyl ether are added to
the flask and an amorphous solid precipitates. The waters are
eliminated by decanting and 100 ml of isopropyl ether are added to
the solid and left under stirring for 1 hour. The solid is then
filtered and washed with 50 ml of isopropyl ether and dried under
vacuum at 40.degree. C. to recover 8.52 g of product.
Example 11
Rearrangement of Orthoester Deriving from Example 10
[0141] 7.0 g of the product obtained in the preceding step, 72.8 g
of glacial acetic acid and 8.7 ml of water are introduced into a
250 ml flask to obtain a solution which is left under stirring at
RT for 3 hours. The solution is then diluted to 150 ml with 0.6 M
sodium chloride and the resulting solution is purified by
ultrafiltration through a 5 KD membrane. After dialysis, the
solution obtained is concentrated under vacuum and 6.7 g of solid
product are recovered.
Example 12
Sulphation of Triacetyl Methyl Ester
[0142] 670 mg of the product obtained in the preceding step are
introduced into a 250 ml flask with nitrogen flow and calcium
chloride valve with 40 ml of DMF.
[0143] 630.44 g of sulphur trioxide pyridinium complex are added to
the solution obtained and the resulting solution is heated at
50.degree. C. (internal temperature) for 1 hour. 630.44 g of
sulphur trioxide pyridinium complex are then added to the flask at
the same temperature and again left under stirring for 1 hour.
[0144] When that time has elapsed, the solution is cooled to RT and
40 ml of 3% NaHCO.sub.3 are added to the flask at the same
temperature to produce a solution which is concentrated under
vacuum to obtain 2.3 g of solid mixed with inorganic salts. The
product obtained is diluted to 150 ml of 0.6 M sodium chloride and
ultrafiltered through a 5 KDa membrane.
[0145] After dialysis, the solution obtained is concentrated under
vacuum and 1.32 g of solid product are recovered.
Example 13
To Obtain Chondroitin Sulphate
[0146] The product obtained in the preceding step is introduced
into a 100 ml flask with 33 ml of 0.2 M soda. The solution is
heated at 40.degree. C. (internal temperature) for 2 hours, after
which it is cooled to RT and neutralised with 1M HCl.
[0147] The solution is diluted to 150 ml of 0.6 M sodium chloride
and ultrafiltered through a 5 KDa membrane. After dialysis and
concentration of the solution under vacuum, 350 mg of solid are
obtained.
[0148] The product obtained in this example has a molecular weight
of 11 KDa, a 4S/6S ratio of 47/53, and a charge density value of
0.9.
Example 14
Formation of Cyclic Orthoester on the Hydroxyl Functions in 4 and 6
of the GalNAc Portion, with Simultaneous Depolymerisation of the
Polysaccharide Chain
[0149] A suspension of chondroitin tetrabutylammonium salt,
obtained as described above (4.07 g; 6.535 mmols), in
dimethylformamide (101 ml), was maintained under stirring and under
nitrogen flow at ambient temperature (20-25.degree. C.). Trimethyl
orthoacetate (9.03 ml, 71.89 mmols) and camphorsulphonic acid (1.82
g; 7.84 mmols) were added. The suspension was heated to 70.degree.
C. (internal temperature), and complete dissolution was observed
after only a few minutes. The reaction was maintained under
stirring at the same temperature for 18-20 h. The next day, the
reaction was concentrated by removing the solvent by evaporation
under vacuum, providing 13.67 g of the product in the form of a
bright yellow rubbery residue.
[0150] The residual content of unprotected chondroitin after
digestion is 4.6%. The presence of the orthoester is demonstrated
by the corresponding signal in FTIR.
[0151] The product thus obtained was used in the subsequent steps
as described above, until a LMW-CS BIOTEC sulphated in position 4
or 6 on the GalNAc residue was obtained.
Example 15
Opening of the Cyclic Orthoester of Chondroitin to Ester with
Prevalent Formation of Acetate in Position 4 or 6 of the GalNAc
Portion, and Simultaneous Depolymerisation of the Polysaccharide
Chain
[0152] Chondroitin orthoester (3.00 g), water (3.14 ml) and acetic
acid (26.25 g; 437 mmols) were introduced into a 250 ml
three-necked flask. The suspension obtained was heated for 36 h at
ambient temperature (20-25.degree. C.). Water was then added to
make up the solution to a total volume of 100 ml. The solution thus
obtained was ultrafiltered (5 KD membrane). The retentate collected
was dialysed to a small volume (20 ml), and then concentrated until
dry by evaporation under vacuum, providing 1.55 g of solid residue
corresponding to the desired product (triacetyl chondroitin).
[0153] The product thus obtained was used in the subsequent steps
as described above, until a LMW-CS BIOTEC sulphated in position 4
or 6 on the GalNAc residue was obtained.
Example 16
Induction of Arthritis (Adjuvant Arthritis, AA) in Rats, and
Treatment with LMW-CS BIOTEC
[0154] 40 male Lewis rats weighing between 150 and 190 g were
randomised to four groups of 10 animals each, housed in
polypropylene cages in a environment maintained at the temperature
of 22.+-.2.degree. C., and fed on a standard laboratory diet with
unlimited access to water.
[0155] The experimental groups were as follows:
[0156] 1) An untreated healthy control group.
[0157] 2) An untreated control group with adjuvant-induced
arthritis (AA).
[0158] 3) A group of arthritic rats treated orally with LMW-CS
BIOTEC at the dose of 900 mg/day per kg of body weight for 28 days
after induction of AA (days 0-28 of the experiment).
[0159] 4) A group pretreated orally with LMW-CS BIOTEC at the dose
of 900 mg/day per kg of body weight for 14 days preceding the
induction of Articles of Association, and for the 28 days after
induction of AA (days -14 to +28 of the experiment).
[0160] Arthritis was experimentally induced in the rats on day 0 by
a single intradermal injection of 1 ml of a mixture consisting of
Mycobacterium butyricum inactivated by heat in incomplete Freund's
adjuvant.
[0161] The LMW-CS BIOTEC was dissolved in distilled water at the
concentration of 20 mg/ml and administered orally as a single daily
dose by gavage.
[0162] At the end of 28 days' treatment the rats were sacrificed
under anaesthesia and the blood and tissues concerned were
collected and analysed to evaluate the parameters observed in the
study.
Example 17
Effects of LMW-CS BIOTEC on the Assessment of AA in Rats by
Recording the Oedema Developed, Body Weight and the Arthritis
Score
[0163] The oedema that developed as a consequence of arthritis was
measured by observing the increase in volume of the hind paw with a
caliper suitable for the measurement. The measurements were
performed before the induction of AA and on day 28 of the
study.
[0164] The body weight of the rats was measured before induction of
AA and at the end of the treatment (day 28). The effect of the
treatment on this parameter was evaluated by comparing the various
weight increases of the different groups during the treatment
period.
[0165] The arthritis score was evaluated by attributing a score to
the paw joint swelling and the extent of the periarticular
erythema. The arthritis score or arthrogram was measured as the sum
total of oedema (in ml, max. 8 points), plus the diameter of the
forepaw (in mm, max 5 points), plus the diameter of the scab at the
site of application of Mycobacterium butyricum measured parallel to
the spinal column (in mm, max 5 points), for each animal.
Example 18
Effect of LMW-CS BIOTEC on the Activity of .gamma.-Glutamyl
Transferase as a Marker for Oxidative Stress Induced by AA
[0166] Oxidative stress was evaluated by measuring the activity of
.gamma.-glutamyl transferase in homogenates of joint tissue taken
from the rats at the end of the treatments with LMW-CS BIOTEC.
.gamma.-glutamyl transferase is considered to be a marker for
oxidative stress.
[0167] The activity of the cell .gamma.-glutamyl transferase was
determined in homogenates of tissue taken from the hind paw, and
evaluated by the Orlowski and Meister method (Orlowski M, Meister
A. The gamma-glutamyl cycle: a possible transport system for amino
acids. Proc Natl Acad Sci USA 1970; 67: 1248-1255) as modified by
Ondrejickova et al. (Cardioscience 1993; 4: 225-230). The samples
were homogenised in a buffer (2.6 mM NaH.sub.2PO.sub.4, 50 mM
Na.sub.2HPO.sub.4, 15 mM EDTA, 68 mM NaCl, pH 8.1) in a 1:9 (w/v)
solution with UltraTurax TP 18/10 (Janke & Kunkel, Germany) for
1 min at 0.degree. C. The substrates, 8.7 mM of .gamma.-glutamyl
p-nitroanilide and 44 mM of methionine, were added to 65% of
isopropyl alcohol at final concentrations of 2.5 mM and 12.6 mM
respectively. After incubation for 60 min at 37.degree. C., the
reaction was stopped by adding 2.3 ml of cold methanol, and the
test tubes were centrifuged for 20 min at 5000 rpm. The absorbance
of the supernatant was measured with a Specord 40 spectrophotometer
(Jena, Germany) in 0.5 cm cuvettes at 406 nm. Reaction mixtures in
the absence of substrate or acceptor were used as reference
samples.
Example 19
Effect of LMW-CS BIOTEC on the Inflammatory State Induced by AA by
Evaluating the Levels of Pro-Inflammatory Cytokines (I-1, IL-6) and
C-Reactive Protein (CRP) in the Plasma
[0168] Blood samples were drawn from the rats at the end of the
experiment and placed in test tubes containing heparin as
anticoagulant; the plasma was separated from the corpuscular part
consisting of blood cells by centrifugation, and the inflammatory
cytokines (IL-1, IL-6) were assayed with the ELISA technique using
specific commercial kits.
[0169] C-reactive protein was assayed in the rat plasma with an
ELISA kit (Immunology Consultant Laboratories, Inc., ICL). The
reaction of the biotin-conjugated secondary antibody with anti-rat
C-reactive protein antibodies was evaluated by means of the
activity of streptavidin-horseradish peroxidase (HRP). The reaction
of methyl-benzidine with HRP bonded to immune complexes was then
measured at 450 nm using a Labsystems Multiskan RC microplate
reader. The results were calculated using the standard calibration
curve in accordance with the ELISA kit instructions.
Example 20
Effect of LMW-CS BIOTEC on Phagocytic Activity and on the
Neutrophil Oxidative Burst Induced by AA
[0170] The neutrophil population was extracted from the blood of
the rats at the end of the evaluation of their phagocytic activity
and oxidative burst. The measurement of phagocytosis, namely
ingestion of bacteria, was performed under controlled conditions
using opsonised Staphylococcus aureus labelled with fluorescein
(SPA-FITC) (Invitrogen Molecular Probes, USA). Aliquots of
peripheral blood in lithium-heparin were then incubated with
hydroethidine (Invitrogen molecular probes, USA) (15.75 mg in 5 ml
of dimethylformamide, Merck, Germany) for 15 minutes at 37.degree.
C. After treatment with SPA-FITC for 15 minutes at 37.degree. C.,
the reaction was interrupted by placing the test tubes in ice. The
subsequent lysis of the erythrocytes was performed for 15 min with
a lysis solution consisting of cold ammonium chloride/potassium
chloride (200 ml deionised water, 1.658 g NH.sub.4Cl, 0.2 g
KHCO.sub.3 and 7.4 mg Na.sub.2EDTA, pH 7.2-7.4). The average
percentage of phagocyte cells represents the percentage of
granulocytes which ingested at least one particle of SPA-FITC, and
the average percentage of the respiratory burst represents the
percentage of granulocytes labelled with ethidium.
* * * * *