U.S. patent application number 10/518229 was filed with the patent office on 2005-11-03 for low molecular weight oversulfated polysaccharide.
Invention is credited to Oreste, Pasqua Anna, Zoppetti, Giorgio.
Application Number | 20050245736 10/518229 |
Document ID | / |
Family ID | 29740492 |
Filed Date | 2005-11-03 |
United States Patent
Application |
20050245736 |
Kind Code |
A1 |
Oreste, Pasqua Anna ; et
al. |
November 3, 2005 |
Low molecular weight oversulfated polysaccharide
Abstract
LMW-K5-N,O-oversulfates are described, having a sulfation degree
of from 3.2 to 4 and a mean molecular weight of from about 3,000 to
about 6,000, obtainable by depolymerization of corresponding
K5-N,O-oversulfates or starting from LMW-K5-N-sulfates by
O-oversulfation of a tertiary amine or quaternary ammonium salt
thereof and subsequent N-resulfation of the K5-amine-O-oversulfate
thus obtained. Furthermore, pharmaceutical compositions containing
these LMW-K5-N,Ooversulfates having antiangiogenetic and antiviral,
in particular anti-HIV-1 activity. Intermediate LMW-K5-N-sulfates
are also described.
Inventors: |
Oreste, Pasqua Anna;
(Milano, IT) ; Zoppetti, Giorgio; (Milan,
IT) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Family ID: |
29740492 |
Appl. No.: |
10/518229 |
Filed: |
June 6, 2005 |
PCT Filed: |
June 17, 2003 |
PCT NO: |
PCT/IB03/02347 |
Current U.S.
Class: |
536/54 |
Current CPC
Class: |
A61P 17/16 20180101;
A61P 3/06 20180101; A61P 31/12 20180101; A61K 31/727 20130101; A61P
17/06 20180101; A61P 19/02 20180101; A61P 35/00 20180101; A61P
27/06 20180101; A61P 43/00 20180101; C07H 1/00 20130101; C08B
37/0078 20130101; A61P 7/02 20180101; A61K 31/737 20130101; A61K
31/726 20130101; C08B 37/006 20130101; A61P 27/02 20180101; A61P
9/00 20180101; C08B 37/0075 20130101; A61P 39/06 20180101; A61P
35/04 20180101; A61P 3/10 20180101 |
Class at
Publication: |
536/054 |
International
Class: |
A61K 031/737; C08B
037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 18, 2002 |
IT |
MI2002A001345 |
Jun 18, 2002 |
IT |
MI2002A001346 |
Aug 27, 2002 |
IT |
MI2002A001854 |
Claims
1. A LMW-K5-N,O-oversulfate having a mean molecular weight of from
about 3,000 to about 6,000 and a degree of sulfation of from 3.2 to
4.
2.-55. (canceled)
56. The LMW-K5-N,O-oversulfate of claim 1 having a mean molecular
weight of 3,750-4,250
57. The LMW-K5-N,O-oversulfate of claim 1 having a mean molecular
weight of 4,750-5,250.
58. The LMW-K5-N,O-oversulfate of claim 1 having a mean molecular
weight of 5,750-6,250.
59. The LMW-K5-N,O-oversulfate of claim 1 consisting of a mixture
of chains in which the preponderant species is a compound of
formula I.degree. 18in which R, R' and R" represent hydrogen or
SO.sub.3.sup.-, X' represents a formyl or hydroxymethyl group, for
a degree of sulfation of from 3.2 to 4, m represents 4, 5 or 6 and
the corresponding cation is a chemically or pharmaceutically
acceptable one.
60. The LMW-K5-N,O-oversulfate according to claim 59, having a mean
molecular weight of 3,750-4,250.
61. The LMW-K5-N,O-oversulfate according to claim 59, having a mean
molecular weight of 4,750-5,250.
62. The LMW-K5-N,O-oversulfate according to claim 59, having a mean
molecular weight of 5,750-6,250
63. A LMW-K5-N,O-oversulfate according to claim 1, having a degree
of sulfation of from 3.5 to 4.
64. A LMW-K5-N,O-oversulfate according to claim 1, having a degree
of sulfation of from 3.5 to 3.9.
65. A process for the preparation of a LMW-K5-N,O-oversulfate
having a degree of sulfation of from 3.2 to 4, which comprises (a)
treating a LMW-K5-N-sulfate obtained by nitrous depolymerization of
a K5-N-sulfate and subsequent reduction, in its acidic form, with a
tertiary amine or quaternary ammonium hydroxide, letting the
reaction mixture stand for a period of time of 30-60 minutes by
maintaining the pH of the solution at 7 and isolating its salt with
said organic base; (b) treating said tertiary amine or quaternary
ammonium salt of said polysaccharide with an O-sulfating agent
under O-oversulfation conditions; and (c) treating the product thus
obtained with a N-sulfating agent and isolating the
LMW-K5-N,O-oversulfate thus obtained.
66. A process according to claim 65, which comprises (i) submitting
a K5-N-sulfate to a nitrous depolymerisation followed by a
reduction, for example by sodium borohydride; (ii) treating a
LMW-K5-N-sulfate, in its acidic form, with a tertiary amine or
quaternary ammonium hydroxide, letting the reaction mixture stand
for a period of time of 30-60 minutes, whereby the pH of the
solution is maintained at 7, and isolating the corresponding
tertiary amine or quaternary ammonium salt; (iii) treating said
tertiary amine or quaternary ammonium salt of said LMW-K5-N-sulfate
with an O-sulfation reactant under O-oversulfation conditions; and
(iv) treating the product thus obtained with a N-sulfating agent
and isolating the obtained LMW-K5-N,O-oversulfate.
67. A process according to claim 65, wherein said reduction is
carried out with sodium borohydride.
68. A process according to claim 65, wherein a LMW-K5-N-sulfate
consisting of a mixture of chains in which at least 90% of said
chains has the formula II 19wherein n is an integer from 2 to 20,
containing a 2,5 anhydrommanitol unit of structure (a') 20at the
reducing end of the majority of chains in said mixture of chains,
and the corresponding cation is a chemically and pharmaceutically
acceptable one, is used as starting material.
69. A process according to claim 65, wherein said K5-N-sulfate
starting material is free of lipophilic substances.
70. A process according to claim 65, wherein the LMW-K5-N-sulfate
starting material is used in the form of its sodium salt.
71. A process according to claim 65, wherein said K5-N-sulfate
starting material consists of a mixture of chains in which the
preponderant species is a compound of formula II' 21in which q is
4, 5, 6, 7, or 8, containing a 2,5 anhydrommanitol unit of
structure (a') 22at the reducing end of the majority of the chains
in said mixture of chains, and the corresponding cation is a
chemical or pharmaceutically acceptable one.
72. A process according to claim 65, wherein said K5-N-sulfate
starting material consists of a mixture of chains in which the
preponderant species is a compound of formula II" 23in which m
represents 4, 5 or 6 and the corresponding cation is a chemically
or pharmaceutically acceptable one.
73. A process according to claim 75, wherein the
LMW-K5-N,O-oversulfate is obtained in its sodium salt form and
optionally transformed into another chemically or pharmaceutically
acceptable salt.
74. A LMW-K5-amine-O-oversulfate having a degree of sulfation of
from 2.2 to 3 or one of its chemically or pharmaceutically
acceptable salts.
75. A LMW-K5-amine-O-oversulfate according to claim 74, having a
mean molecular weight of from about 3,500 to about 11,000 or a
chemically or pharmaceutically acceptable salt thereof.
76. A LMW-K5-amine-O-oversulfate according to claim 74 having a
degree of sulfation of from 2.2 to 3 and a molecular weight of from
3,500 to 5,200.
77. A LMW-K5-amine-O-oversulfate according to claim 74,
substantially free of N-acetyl groups.
78. A LMW-K5-amine-O-oversulfate according to claim 74 consisting
of a mixture of chains in which at least 90% of said chains has the
formula III 24in which R, R' and R" represent hydrogen or a
SO.sub.3.sup.- group, n is an integer from 2 to 20, containing a
sulfated 2,5-anhydrommanitol unit of structure (a") 25at the
reducing end of the majority of the chains in said mixture of
chains, for a degree of sulfation of from 2.2 to 3 and the
corresponding cation is a chemically or pharmaceutically acceptable
one.
79. The LMW-K5-amine-O-oversulfate of claim 78, consisting of a
mixture in which the preponderant species is a compound of formula
III' 26in which q is 4, 5, 6, 7, or 8 and the corresponding cation
is a chemically or pharmaceutically acceptable one.
80. The LMW-O-oversulfated-K5 amine of claim 79, consisting of a
mixture in which the preponderant species is a compound of formula
III" 27in which R, R' and R" are hydrogen or SO.sub.3.sup.-, X" is
OH or OSO.sub.3.sup.-, for a degree of sulfation of from 2.2 to 3,
m is 4, 5 or 6 and the corresponding cation is a chemically or
pharmaceutically acceptable one.
81. A LMW-K5-N,O-oversulfate consisting of a mixture of chains in
which at least 90% of said chains has the structure I' 28in which n
is an integer from 2 to 20, R, R' and R" represent hydrogen or a
SO.sub.3.sup.- group, and in which the reducing end of the majority
of said chains has the structure (a") 29in which R" is hydrogen or
a SO.sub.3.sup.- group, for a degree of sulfation of from 3.2 to 4
and the corresponding cation is a chemically or pharmaceutically
acceptable one.
82. The LMW-K5-N,O-oversulfate of the claim 81, consisting of a
mixture of chains in which the preponderant species is a compound
of formula I 30wherein q is 4, 5, 6, 7, or 8 and the corresponding
cation is a chemically or pharmaceutically acceptable one.
83. The LMW-K5-N,O-oversulfate of claim 82, consisting of a mixture
in which the preponderant species is a compound of formula I" 31in
which m is 4, 5, 6, R, R' and R" are hydrogen or SO.sub.3.sup.-,
for a degree of sulfation of from 3.2 to 4 and the corresponding
cation is a chemically or pharmaceutically acceptable one.
84. A LMW-K5-N,O-oversulfate according to claim 81 wherein said
cation is the ion of an alkaline metal, an alkaline-earth metal,
ammonium, tetra(C.sub.1-C.sub.4)alkylammonium, aluminum or
zinc.
85. The LMW-K5-N,O-oversulfate of claim 84, wherein said cation is
the sodium, calcium or tetrabutylammonium ion.
86. A LMW-K5-N,O-oversulfate according to claim 81, having a degree
of sulfation of from 3.5 to 4.
87. A LMW-K5-N,O-oversulfate according to claim 81, having a degree
of sulfation of from 3.5 to 3.9.
88. A process for the preparation of LMW-K5-N-sulfates and of their
chemically or pharmaceutically acceptable salts, which comprises
submitting a K5-N-sulfate to a controlled nitrous depolymerization
optionally followed by a reduction and isolating the product thus
obtained.
89. A process according to claim 88, wherein said K5-N-sulfates are
isolated as their sodium salt which is optionally converted into
another chemically or pharmaceutically acceptable salt.
90. A process according to claim 89, wherein said other salt is
that of an alkaline metal, an alkaline-earth metal, ammonium,
tetra(C.sub.1-C.sub.4)alkylammonium, aluminum or zinc.
91. A process according to claim 90, wherein said other salt is
that of sodium, calcium or tetrabutylammonium.
92. A LMW-K5-N-sulfate substantially free of N-acetyl groups, or a
chemically or pharmaceutically acceptable salt thereof.
93. A LMW-K5-N-sulfate according to claim 92, consisting of a
mixture of chains in which at least 90% of said chains has a mean
molecular weight of from about 1,500 to about 7,500.
94. A LMW-K5-N-sulfate according to claim 93, having a molecular
weight distribution from about 1,000 to about 10,000.
95. A LMW-K5-N-sulfate according to claim 92, having a mean
molecular weight of from about 2,000 to about 4,000.
96. A LMW-K5-N-sulfate according to claim 92, having a mean
molecular weight of from about 4,000 to about 7,500.
97. A LMW-K5-N-sulfate according to claim 92, consisting of a
mixture of chains in which at least 90% of said chains has the
formula II 32wherein n is an integer from 2 to 20, containing an
unit of structure 33in which X represents formyl or hydroxymethyl,
in the majority of said chain and the corresponding cation is a
chemically or pharmaceutically acceptable one.
98. A LMW-K5-N-sulfate according to claim 97, consisting of a
mixture of chains in which the preponderant species is a compound
of formula II' 34wherein q is 4, 5, 6, 7 or 8, containing an unit
of structure 35in which X represents formyl or hydroxymethyl, in
the majority of said chain and the corresponding cation is a
chemically or pharmaceutically acceptable one.
99. A LMW-K5-N-sulfate according to claim 97, consisting of a
mixture of chains in which the preponderant species is a compound
of formula II" 36wherein X represents formyl or hydroxymethyl, m
represents 4, 5, or 6 and the corresponding cation is a chemically
or pharmaceutically acceptable one.
100. A LMW-K5-N-sulfate according to claim 97 wherein, in the
structure (a), X is hydroxymethyl.
101. A LMW-K5-N-sulfate according to claim 92, wherein said salt is
that of an alkaline metal, alkaline-earth metal, ammonium,
tetra(C.sub.1-C.sub.4)alkylammonium, aluminum or zinc.
102. A LMW-K5-N-sulfate according to claim 92, wherein said salt or
cation is that of sodium, calcium or tetrabutylammonium.
103. A pharmaceutical composition comprising, as an active
ingredient, a LMW-K5-N,O-oversulfate according to claim 1, in
admixture with a pharmaceutical carrier.
104. A pharmaceutical composition comprising, as an active
ingredient, a LMW-K5-N,O-oversulfate according to claim 81, in
admixture with a pharmaceutical carrier.
Description
SUMMARY OF THE INVENTION
[0001] The present invention refers to new N,O-oversulfated low
molecular weight polysaccharides derived from K5 polysaccharide, to
a process for their preparation, to new key intermediates in said
process and to pharmaceutical compositions containing said
oversulfated low molecular weight polysaccharides. More
particularly, the present invention refers to a N-deacetylated and
N-sulfated K5 having a degree of sulfation between 3.2 and 4 and a
mean molecular weight from about 3,000 to about 6,000.
BACKGROUND OF THE INVENTION
[0002] Glycosamioglycans such as heparin, heparan sulfate, dermatan
sulfate, chondroitin sulfate and hyaluronic acid are biopolymers
industrially extracted from different animal organs.
[0003] In particular, heparin, mainly obtained by extraction from
pig intestinal mucosa or from beef lung, is a polydisperse
copolymer with a molecular weight distribution from about 3,000 to
about 30,000 D formed by a mixture of chains essentially consisting
of an uronic acid (glucuronic acid or iduronic acid) and of an
aminosugar (glucosamine) linked by .alpha.-1.fwdarw.4 or
.beta.-1.fwdarw.4 bonds. In heparin the uronic unit can be
O-sulfated in position 2 and the glucosamine unit is N-acetylated
or N-sulfated, 6-O sulfated and 3-O sulfated in about 0.5% of the
glucosaminic units present.
[0004] The properties and the natural biosynthesis of heparin in
mammals were described by Lindahl et al., 1986 in Lane, D. and
Lindahl, U. (Editors) "Heparin. Chemical and Biological Properties;
Clinical Applications", Edward Arnold, London, Pages 159-190, by
Lindhal, U, Feingold D. S. e Rodn L, 1986 TIBS, 11, 221-225 and by
Conrad H. E. "Heparin Binding Proteins", Chapter 2: Structure of
Heparinoids. Academic Press, 1998.
[0005] Besides the main anticoagulant and antithrombotic
activities, heparin also exerts antilipemic, antiproliferative,
antiviral, antitumoral and antimetastatic activity, but its use as
a drug for such indications is hampered by the side effects due to
its anticoagulant activity that can cause bleeding.
DESCRIPTION OF THE PRIOR ART
[0006] It is known that the capsular polysaccharide isolated from
strains of Escherichia Coli (herein below also simply referred to
as "K5") described by W. F. Vann et al. (1981) in Eur. J. Biochem
116, 359-364 consists of a mixture of chains formed by a repetitive
disaccharide unit formed by D-glucuronic acid and
N-acetylglucosamine linked .beta.1.fwdarw.4, while the disaccharide
units D-glucuronyl-N-acetylgluco- samine are linked
.alpha.1.fwdarw.4, thus showing the same repeated sequence (A)
1
[0007] as the N-acetylheparosan biosynthetic precursor of heparin
and heparan sulfate. The only difference, which is irrelevant for
the biological activities of the K5 and its derivatives, between
the heparin precursor N-acetylheparosan and K5 polysaccharide, is
the presence of a double bond in position 4(5) at the non reducing
end of some chains of the polymer, as for instance described in EP
489647 and EP 544592 mentioned herein below.
[0008] After these first publications, other papers and patent
applications described the preparation of the E.coli K5
polysaccharide having molecular weight ranges from few thousands to
many hundred thousand Daltons. For example EP 333243, IT 1230785,
EP 489647, EP 544592, WO 92/17507, WO 01/02597, and the paper by M.
Manzoni et al. (1996), Biotechnology Letters, 18(4) 383-386 are
cited.
[0009] Documents EP 489647 and EP 544592 disclose low molecular
weight and high molecular weight N,O sulfate heparosans having
anticoagulant and antithrombotic activity, IT 1230785, WO 92/17507,
WO 96/14425, WO 97/43317, WO 98/42754, WO 01/72848 and US
2002/0062019 describe derivatives of N-deacetylated-K5-N-sulfate,
having a certain number of glucuronic units epimerized in position
C5 to iduronic units, with antithrombotic activity and WO 98/09636
describes N-deacetylated-K5-N-sul- fate with antimetastatic
activity.
[0010] The document US 2002/0062019 describes a process for the
preparation of epiN,O-sulfated-K5 derivatives, active on the
coagulation control, having a degree of sulfation of from 2.3 to
2.9 and a molecular weight of from 2,000 to 30,000, or from 4,000
to 8,000 or from 18,000 to 30,000. Said process comprises the steps
(s-a) a N-deacetylation of K5 polysaccharide and a N-sulfation of
the resulting K5-amine, (s-b) an epimerization of the N-sulfate-K5,
(s-c) an O-oversulfation of the epiK5-N-sulfate, (s-d) a partial
O-desulfation, (s-e) a selective 6-O-sulfation, (s-f) a N-sulfation
of the so obtained product, whatever product obtained at the end of
steps (s-b)-(s-f) being optionally submitted to a depolymerization.
Said document describes a N-sulfated-epiK5 having a molecular
weight of 7,400 and a degree of sulfation of from 2.3 to 2.9,
obtained by the above-mentioned steps (s-a)-(s-f) followed by a
nitrous depolymerization at the end of the (s-f) step.
[0011] The same document describes a K5 fraction with a molecular
weight of about 5,000 that can also be submitted to the (s-a)-(s-f)
steps.
[0012] The pending Italian patent application n. MI2001A/00397 (WO
02/068477), incorporated into the present application as a
reference, describes K5-N,O-oversulfate-derivatives having a degree
of sulfation higher than 3.2, obtained starting from a K5 free from
lipophilic substances or from one of its fractions with a molecular
weight of about 5,000 by (a) N-deacetylation/N-sulfation, (b)
O-oversulfation, and (c) N-resulfation. This document cites
LMW-K5-N,O-oversulfate having a mean molecular weight of from 2,000
to 5,000 obtained by depolymerization of the K5-N,O-oversulfate or
a LMW-K5-N,O-oversulfate of mean molecular weight of about 6,500
directly obtained from the above mentioned fraction of K5 by the
steps (a)-(c).
[0013] None of the above cited documents describe LMW-K5-N-sulfate,
optionally 40%-60% epimerized, in which NH2 or N-acetyl groups are
practically absent.
[0014] Moreover, D. Leali et al., in a paper titled "Fibroblast
Growth Factor 2 Antagonist Activity and Angiostatic Capacity of
Sulfated Escherichia coli K5 Polysaccharide Derivatives" published
in J. Biol. Chem 2001 (October 12), 276(41), 37900-37908 (Leali
2001), described a K5-N,O-oversulfate having a mean molecular
weight of 15,000 and a degree of sulfation of 3.84, which shows a
good antiangiogenic activity expressed as 70% of inhibition of the
formation of new vessels within the 12th day of incubation.
[0015] To make the terminology uniform and the text more
understandable, in the present description meanings and
conventional expressions, in singular or plural form, will be used.
In particular:
[0016] "K5" or "polysaccharide K5" means the capsular
polysaccharide from Escherichia coli obtained by fermentation,
namely a mixture of chains consisting of repetitive disaccharide
units A, optionally containing a double bond at the non reducing
end as illustrated above, whenever prepared and purified according
to the methods described in the literature, in particular according
to Vann 1981, Manzoni M. et al., Journal of Bioactive and
Compatible Polymers, 1996, 11, 301-311 ("Manzoni 1996"), according
to the method described in WO 01/72848 or in example 12 of US
2002/0062019 A1; it is obvious for a skilled in the art that the
matter illustrated herein is applicable to any
N-acetylheparosan;
[0017] "K5 amine" means the K5, N-deacetylated for at least 95%,
but generally in which acetyl groups are undetectable by a current
NMR apparatus;
[0018] "K5-N-sulfate" means the K5, N-deacetylated and N-sulfated
for at least 95% as herein below described, but generally in which
acetyl groups are undetectable by a current NMR apparatus;
[0019] "K5-amine-O-oversulfate" means an O-sulfated-K5-amine with a
degree of sulfation of at least 2.2;
[0020] "K5-N,O-oversulfate" means a N,O-sulfated-K5-amine with a
degree of sulfation of at least 3.2.
[0021] Moreover:
[0022] the terms and conventional expressions herein above defined
refer to K5 polysaccharides as isolated after fermentation,
generally with a distribution of molecular weight from about 1,500
to about 50,000 with a mean molecular weight of 12,000-25,000,
advantageously of 15,000-25,000;
[0023] apart from the specific attribution of the molecular weight,
the terms and conventional expressions herein above defined, when
preceded by the acronym "LMW" (low molecular weight), indicate low
molecular weight products having a mean molecular weight up to
12,000;
[0024] "about" referred to the molecular weight means the molecular
weight measured by viscosimetry.+-.the theoretical weight of a
disaccharide unit, including the weight of sodium, calculated to be
461 in the case of a K5-N-sulfate and 765 in the case of a
K5-N,O-oversulfate with a degree of sulfation of 3.87;
[0025] the terms and conventional expressions as herein above
defined, when are followed by "-derivative" globally designate both
the derivatives from native K5 and those of low molecular weight,
independently of the fact that these are obtained by fractionation
of K5 or of its derivatives or by depolymerization;
[0026] unless otherwise specifically indicated, "degree of
sulfation" means the SO3-/COO-- ratio, that can be expressed also
as the number of sulfate groups per disaccharide unit, as measured
by the conductimetric method described by Casu B. et al. in
Carbohydrate Research, 1975, 39, 168-176 (Casu 1975);
[0027] unless otherwise specifically indicated, the molecular
weight is intended to be measured by viscosimetry according to
Johnson et al. Carb. Res. n.51 (1976) p. 119-127, using samples
whose molecular weight was calculated by HPLC as standards;
[0028] "preponderant species" means the compound that, in the
mixture constituting the LMW-K5-N-sulfate, the
K5-amine-O-oversulfate or the LMW-K5-N,O-oversulfate, is the most
represented species, determined by the peak of the molecular weight
curve measured by HPLC;
[0029] "O-oversulfation conditions" means an O-sulfation performed
for example according to Method C described by B. Casu et al. in
Carbohydrate Research, 1994, 263, 271-284 (Casu 1994);
[0030] the term "alkyl" means a linear or branched alkyl, while
"tetrabutylammonium" means the tetra(n-butyl)ammonium group.
SUMMARY OF THE INVENTION
[0031] It has now been found that it is possible to depolymerize a
K5-N-sulfate to obtain new LMW-K5-N-sulfate derivatives which
constitute useful starting materials for the preparation of new
LMW-K5-N,O-oversulfates. Advantageously, it is also possible to
obtain new LMW-K5-N-sulfates with very low mean molecular weight,
in particular from about 2,000 to about 4,000, more particularly
specific LMW-K5-N-sulfates formed by mixtures in which the
preponderant compound is a decasaccharide or a dodecasaccharide or
a tetradecasaccharide. These new LMW-N-sulfates are useful
intermediates for the preparation of LMW-K5-N,O-oversulfates with
antiviral and/or antiangiogenic activity and devoid of
anticoagulant activity.
[0032] More particularly, it has been found that a
LMW-K5-N,O-oversulfate having a mean molecular weight of from about
3,000 to about 6,000, in particular a LMW-K5-N,O-oversulfate
consisting of a mixture in which the preponderant species is a
deca-, dodeca- or tetradecasaccharide, with a degree of sulfation
of from 3.2 to 4, has an antiangiogenic activity higher than that
of the K5-N,O-oversulfate described by Leali 2001.
[0033] It has also been found that said new LMW-K5-N,O-oversulfates
having a mean molecular weight from about 3,000 to about 6,000, in
particular LMW-K5-N,O-oversulfates consisting of a mixture in which
the preponderant species is a deca-, dodeca- or
tetradecasaccharide, avoid of anticoagulant activity, have a good
activity on HIV-1 virus.
[0034] Moreover it has been found that, starting from said
LMW-K5-N-sulfates, it is possible to obtain new
LMW-O-sulfated-K5-amine with a high degree of sulfation by
preparing a tertiary amine or quaternary ammonium salt of said
LMW-K5-N-sulfate, taking care to keep the reaction mixture for a
period of time of 30-60 minutes by maintaining the pH at about 7,
and then by treating the obtained salt with an O-sulfating reactant
in the conditions of O-oversulfation. By N-sulfating the above
mentioned LMW-K5-amine-O-oversulfate, new LMW-K5-N,O-oversulfate
are obtained.
DETAILED DESCRIPTION OF THE INVENTION
[0035] Thus, it is an object of the present invention to provide
new LMW-K5-N,O-oversulfate having a mean molecular weight of from
about 3,000 to about 6,000 and a degree of sulfation of from 3.2 to
4, advantageously from 3.5 to 4, preferably from 3.7 to 3.9.
[0036] Among these new LMW-K5-N,O-oversulfate of the present
invention, particularly interesting are those having a mean
molecular weight of 3,750-4,250, of 4,750-5,250 or of
5,750-6,250.
[0037] Preferential compounds are LMW-K5-N,O-oversulfates formed by
a mixture of chains in which the preponderant species has the
formula I 2
[0038] in which q is 4, 5, 6, 7 or 8 and R, R' and R" represent
hydrogen or a SO3- group, for a degree of sulfation of from 3.2 to
4, advantageously from 3.5 to 4, preferably from 3.5 to 3.9 and the
corresponding cation is a chemically or pharmaceutically acceptable
one.
[0039] In this context, the term "chemically acceptable" refers to
a cation useful in the chemical syntheses, such as sodium,
ammonium, tetra(C.sub.1-C.sub.4)alkylammonium, or for the
purification of the product, while "pharmaceutically acceptable" is
self explanatory.
[0040] Advantageous cations are those derived from alkaline metals,
earth-alkaline metals, ammonium,
tetra(C.sub.1-C.sub.4)alkylammonium, aluminum and zinc.
[0041] The LMW-K5-N,O-oversulfate of the present invention can be
prepared by depolymerization of K5-N,O-oversulfates of the type
described in Leali 2001 and prepared with the method described in
it.
[0042] Said depolymerization is performed according to the known
methods for the depolymerization of heparin, for instance according
to the method described in EP 37319, in WO 82/03627 or the method
for the depolymerization of a N,O-sulfated-K5 described in EP
544592. Preferably, the depolymerization, performed with sodium
nitrite and hydrochloric acid ("nitrous depolymerization"), is
followed by an in situ reduction with sodium borohydride.
[0043] By suitably controlling the depolymerization reaction, in
particular by using different amounts of sodium nitrite,
LMW-K5-N,O-oversulfates are obtained having the desired molecular
weight.
[0044] According to a preferred embodiment, starting for instance
from 1 g of K5-N,O-oversulfate obtained as described in PREPARATION
IV herein below, the starting product is dissolved in 100-200 ml of
deionized water and thermostated at 4.degree. C. Then an amount of
sodium nitrite to obtain the desired mean molecular weight from
about 3,000 to about 6,000 is added. Consequently, starting from a
K5-N,O-oversulfate having a molecular weight of 20,000 measured by
HPLC equipped with a BioSil 250 Biorad column and using standards
of heparin with known molecular weight, from 160 to 230 mg of
sodium nitrite dissolved in a water solution at 0.2% shall be
added. The solution containing the K5-N,O-oversulfate and sodium
nitrite, kept at 4.degree. C., is brought to pH 2 by adding 0.1N
HCl cooled at 4.degree. C. The reaction is left to react for 20-40
minutes, then neutralized with 0.1 N NaOH. The obtained product is
brought to room temperature and treated with a reducing agent such
as sodium borohydride (250-500 mg dissolved in 50-100 ml of water)
and left to react for 4-8 hours. The excess of sodium borohydride
is eliminated by bringing the pH to 5-5.5 with 0.1N HCl and left to
react for further 2-4 hours. At the end, the solution is
neutralized with 0.1N NaOH and the product is recovered by
precipitation with acetone or ethanol after concentration of the
product by evaporation under reduced pressure.
[0045] The origin of the LMW-K5-N,O-oversulfates from the nitrous
depolymerization of a K5-N,O-oversulfate and subsequent possible
reduction with for instance sodium borohydride implies, at the
reducing end of the majority of the chains in said mixture of
chains, the presence of a sulfated 2,5-anhydromanno unit having the
structure (a.degree.) 3
[0046] in which X' represents formyl or hydroxymethyl and R"
represents hydrogen or SO.sup.-.
[0047] In particular, preferred products are the
LMW-K5-N,O-oversulfate, obtainable by nitrous depolymerization of a
K5-N,O-oversulfate and optional subsequent reduction with, for
instance, sodium borohydride, consisting of mixtures of chains in
which the preponderant species is a compound of formula I.degree.
4
[0048] wherein m is 4, 5 or 6, R,R' and R" are hydrogen or
SO.sup.-, X' is formyl or hydroxymethyl, for a degree of sulfation
of from 3.2 to 4, advantageously from 3.5 to 4, preferably from 3.5
to 3.9 and the corresponding cation is a chemically or
pharmaceutically acceptable one. The mean molecular weight of each
mixture advantageously is 3,750-4,250, 4,750-5,250,
5,750-6,250.
[0049] These new LMW-K5-N,O-oversulfates have high antiangiogenic
activity with a favourable ratio to the global anticoagulant
activity and can be used in the preparation of pharmaceutical
compositions for the treatment of angiogenesis-dependent
pathologies in doses in which the risk of hemorragic side effects
is extremely reduced.
[0050] Angiogenesis dependent pathologies that can be treated with
the LMW-K5-N,O-oversulfates are for example, among those found in
the human beings, diabetic retinopathy, neovascularization of the
transplanted cornea, neovascular glaucoma, trachoma, retrolental
fibrodisplasia, psoriasis, pyogenic glaucoma, development of the
ateroschlerotic plaque, hemangioma and angiofibroma, artero-venous
malformations, arthritis, and in the combinatorial therapy of solid
tumors.
[0051] More particularly, the new LMW-K5-N,O-oversulfates of the
present invention were active in the in vivo test of the inhibition
of the angiogenesis on chicken embryo choroallantioic membrane
(CAM) according to Ribatti D. et al., J. Vasc. Res. 1997, 34,
455-463 (Ribatti 1997). According to this test, sponge implants of
Gelfoam (Upjohn) are applied on the CAM of chicken embryos at the
8th day of development and, immediately after the application, 3
.mu.l of a solution of physiologic saline containing 50 .mu.g of
LMW-K5-N,O-oversulfate are applied or, as reference compound, of
K5-N,O-oversulfate with 15,000 molecular weight and degree of
sulfation of 3.84 described in Leali 2001. The sponges are examined
every day till the 12th day of incubation. The score of
angiogenesis is obtained by counting the number of macroscopic
vessels observable around the sponge at the different days of
development and at the number of embryos (eggs) on which the
compound is active.
[0052] It was observed that the number of vessels that grow around
the sponge embedded of LMW-K5-N,O-oversulfate is equal to those
formed around the sponge embedded of K5-N,O-oversulfate, but the
number of embryos on which the LMW-K5-N,O-oversulfate is active is
higher.
[0053] LMW-K5-N,O-oversulfates having a mean molecular weight of
from about 3,000 to about 6,000 can be also prepared according to a
process that is not described in literature and that is generally
applicable to the preparation of new LMW-K5-N,O-oversulfates having
a mean molecular weight from about 3,000 to about 12,000. Said new
process and said new LMW-K5-N,O-oversulfates represent further
aspects of the present invention.
[0054] Thus the present invention also provides a process for the
preparation of new LMW-K5-N,O-oversulfates having a degree of
sulfation of from 3.2 to 4, advantageously from 3.5 to 4,
preferably from 3.5 to 3.9, which comprises
[0055] (a) treating a LMW-K5-N-sulfate, in its acidic form, with a
tertiary amine or quaternary ammonium hydroxide, letting the
reaction mixture to stand for a period of time of 30-60 minutes,
whereby the pH of the solution is maintained at 7, and isolating
the salt with said organic base;
[0056] (b) treating the tertiary amine or quaternary ammonium salt
of said LMW-K5-N-sulfate thus obtained with an O-sulfation reactant
under O-oversulfation conditions;
[0057] (c) treating the product thus obtained with a N-sulfating
agent and isolating the obtained LMW-K5-N,O-oversulfate. Usually
the final product is isolated as sodium salt that is optionally
transformed in another chemically or pharmaceutically acceptable
salt.
[0058] A LMW-K5-N-sulfate obtained from a K5 by a practically
complete N-deacetylation, subsequent N-sulfation, and final nitrous
depolymerization followed by reduction of the LMW-K5-N-sulfate, as
above illustrated, is used as starting material for the process of
the present invention. Said reduction is necessary since the
LMW-K5-N-sulfate subsequently undertakes reactions wherein the
influence on the formyl group of the 2,5 anhydromannose radical is
unknown. Also in this case, by controlling the depolymerization
reaction as above illustrated, LMW-K5-N-sulfate can be obtained,
having a mean molecular weight in the whole range from about 1,500
to about 10,000, preferably from about 1,500 to about 7,500, as
calculated from the 13C-NMR spectrum by the integration of the
signal attributable to the C-2 of 2,5 anhydromannitol with that of
the anomeric carbon of the glucosamine inside the polysaccharide
chain.
[0059] LMW-K5-N-sulfates having a mean molecular weight from about
1,500 to about 7,500 and their chemically or pharmaceutically
acceptable salts are new products useful as intermediates and also
pharmaceutically active. The molecular weight distribution of said
LMW-K5-N-sulfates can be from about 1,000 to about 10,000.
[0060] According to a general way to proceed, starting for instance
from 1 g of K5-N-sulfate, the starting product is dissolved in
100-200 ml of deionized water and thermostated at 4.degree. C. Then
an amount of sodium nitrite to obtain the desired mean molecular
weight of from about 2,000 to about 4,000 is added. Consequently,
starting from a K5-N-sulfate having a molecular weight of 20,000
measured by HPLC equipped with a BioSil 250 Biorad column and using
standards of heparin with known molecular weight, from 330 to 480
mg of sodium nitrite dissolved in a 0.2% water solution shall be
added. The solution containing the K5-N-sulfate and sodium nitrite,
kept at 4.degree. C. is brought to pH 2 by adding 0.1N HCl cooled
at 4.degree. C. The reaction is left to react for 20-40 minutes,
then neutralized with 0.1 N NaOH. The obtained product is warmed up
to room temperature and treated with a reducing agent such as
sodium borohydride (250-500 mg dissolved in 50-100 ml of water) and
left to react for 4-8 hours. The excess of sodium borohydride is
eliminated by bringing the pH to 5-5.5 with 0.1N HCl and left to
react for further 2-4 hours. At the end the solution is neutralized
with 0.1N NaOH and the product is recovered by precipitation with
acetone or ethanol after concentration of the product by reduced
pressure evaporation.
[0061] Analogously, the amount of sodium nitrite that, starting
from 1 g of K5-N-sulfate, allows to obtain a LMW-K5-N-sulfate with
a molecular weight of from about 4,000 to about 7,500, in
particular of at least 6,000 (6,000-7,500), can be established.
[0062] The K5-N-sulfate is very well known in the literature and it
is described in the documents herein above cited to illustrate the
state of the art. The above cited starting material is invariably
obtained by N-deacetylation and subsequent N-sulfation of the thus
obtained K5-amine. However, it was noted that the preparation of a
K5-N-sulfate practically devoid of acetyl groups or NH2 is
facilitated if the K5 from which it is prepared is particularly
pure, in particular if it does not contain lipophilic substances.
Moreover, it was found that a K5-N-sulfate prepared from a K5 free
from lipophilic substances is easier oversulfated, as described in
the pending patent application IT MI2001A00397 (WO 02/068477). It
is then preferable that the depolymerization is performed starting
from a K5-N-sulfate prepared from a K5 purified as described in
PREPARATION I herein below. Said K5-N-sulfate, whose .sup.13C-NMR
spectrum does not show traces of acetyl groups or NH2, is described
in PREPARATION II herein below.
[0063] Advantageous starting materials of the process of the
present invention are new LMW-K5-N-sulfates obtained by nitrous
depolymerization of a K5 and subsequent reduction, for instance
with sodium borohydride, consisting of mixtures of chains in which
at least 90% of said chains have the formula II 5
[0064] wherein n represents a number from 2 to 20 and the
corresponding cation is a chemically or pharmaceutically acceptable
one.
[0065] More advantageously, the starting materials are new
LMW-K5-N-sulfates consisting of a mixture of chains in which the
preponderant species has the formula II' 6
[0066] wherein q is 4, 5, 6, 7, or 8 and the corresponding cation
is a chemically or pharmaceutically acceptable one. These
LMW-K5-N-sulfates, that constitute an advantageous aspect of the
invention, are obtained from a K5-N-sulfate by nitrous
depolymerization and subsequent reduction for instance with sodium
borohydride as illustrated above. Their mean molecular weight is
from about 2,000 to about 4,000.
[0067] The origin of the LMW-K5-N-sulfates from a nitrous
depolymerization step and subsequent reduction with for instance
sodium borohydride, implies the presence of a 2,5 anhydrommanitol
unit of structure (a') 7
[0068] at the reducing end of the majority of the chains in said
mixture of chains. Consequently, the reducing terminal of the
majority of the chains is actually represented by the structure (b)
8
[0069] The presence of the structure (a') has no influence on the
properties of the LMW-K5-N-sulfates and of their derivatives
because possible sulfations would only involve a possible
introduction of one or more sulfate groups that would not
significantly influence the-degree of sulfation of the O-sulfated
derivatives. The preferred LMW-K5-N-sulfates are practically devoid
of acetyl groups.
[0070] Particularly advantageous LMW-K5-N-sulfates according to the
present invention are formed by mixtures of chains in which the
preponderant species is a compound of formula II' 9
[0071] in which m represents 4, 5 or 6 and the corresponding cation
is a chemically or pharmaceutically acceptable one.
[0072] According to another of its aspects, the invention relates
to a process for the preparation of new LMW-K5-N-sulfates and of
their chemically or pharmaceutically acceptable salts, which
comprises submitting a K5-N-sulfate to a controlled nitrous
depolymerization optionally followed by a reduction and isolating
the product so obtained. Said products usually are in form of their
sodium salt, that can be transformed into another chemically or
pharmaceutically acceptable salt. At the reducing end of the
majority of the chains by which they are composed, said
LMW-K5-N-sulfates have a 2,5-anhydromanno unit having the structure
(a) 10
[0073] in which X represents a formyl or hydroxymethyl group and,
preferably, they are formed by mixtures of chains in which at least
90% of said chains has the formula II or by mixtures of chains in
which the preponderant species has the formula II' or II". These
new LMW-K5-N-sulfates can be used as active ingredients of
pharmaceutical compositions.
[0074] If, in the structure (a) above, X represents hydroxymethyl,
the new LMW-K5-N-sulfates are the starting material of the process
for the preparation of the LMW-K5-N,O-oversulfates of the present
invention.
[0075] Advantageously, the LMW-K5-N-sulfate has a molecular weight
distribution from about 1,000 to about 10,000.
[0076] The LMW-K5-N-sulfates starting materials of the process of
the present invention are preferably used as sodium salt, unless a
tertiary amine or quaternary ammonium salt prepared according to
step (a) above illustrated, preferably the tetrabutylammonium salt,
is already available.
[0077] According to another advantageous embodiment of the process
of the present invention, step (a) is performed by passing a
solution of the sodium salt of the starting LMW-K5-N-sulfate
through an acid ionic exchange resin, for example of the type
IR-120 H+, by collecting the eluate comprising also the waste of
the resin and by neutralizing the eluate with a tertiary amine or
quaternary ammonium hydroxide, preferably with an aqueous solution
of tetrabutylammonium hydroxide. The solution is let to stand for
one hour, by concurrently maintaining the pH at 7 by adding the
same base and the so obtained salt is isolated by freeze
drying.
[0078] In step (b), the O-oversulfation is performed using an
excess of O-sulfating agent at a temperature from 20 to 70.degree.
C. for a period of time up to 24 hours in a polar aprotic solvent.
Advantageously, the tertiary amine or quaternary ammonium salt of
the LMW-K5-N-sulfate so obtained in step (a), is dissolved in
dimethylformamide and treated with 2-10 moles of an O-sulfating
agent for each free hydroxy group at a temperature of 40-60.degree.
C. for 10-20 hours. As O-sulfating agent, the adduct
pyridine.SO.sub.3 is advantageously used, in amounts of 2.5-5
moles, preferably 2.5-4 moles per free hydroxy group and the
reaction is advantageously performed at 50-60.degree. C.,
preferably at 55.degree. C. overnight. The product obtained at the
end of the reaction is isolated by addition of 0.1-1 volume of
water and neutralization, preferably with sodium hydroxide, by
precipitation with a saturated solution of sodium chloride in
acetone and filtration optionally followed by an
ultrafiltration.
[0079] The so obtained product is preferably the sodium salt of a
LMW-K5-amine-O-oversulfate having a degree of sulfation of from 2.3
to 3. The sodium salt so obtained can be converted into another
salt. The mean molecular weight of such a product can be from about
3,500 to about 11,000.
[0080] In step (c), the LMW-K5-amine-O-oversulfates are submitted
to a N-sulfation, performed by treating their aqueous solution with
sodium carbonate and a N-sulfating agent, for example a
(C.sub.1-C.sub.4)trialky- lamine.SO.sub.3 or pyridine.SO.sub.3, by
maintaining the mixture at 30-50.degree. C. for 8-24 hours and by
isolating the LMW-K5-N,O-oversulfate for example by
diafiltration.
[0081] The LMW-K5-amine-O-oversulfates and their chemically or
pharmaceutically acceptable salts obtainable according to the above
said process, limited to steps (a) and (b), are new products which
constitute a further aspect of the present invention. Surprisingly,
it was found that LMW-K5-amine-O-oversulfates, besides being useful
intermediates, also have pharmacobiological activities.
[0082] Thus, the present invention also provides new
LMW-K5-amine-O-oversulfates having a degree of sulfation of from
2.3 to 3 and their chemically or pharmaceutically acceptable salts.
Advantageously, their mean molecular weight is from about 3,500 to
about 10,000, more advantageously from about 3,500 to about
5,200.
[0083] Preferably they are substantially free of N-acetyl
groups.
[0084] If a LMW-K5-N-sulfate consisting of a mixture of chains in
which at least 90% of said chains has the above mentioned formula
II is used as starting material, a new LMW-K5-amine-O-oversulfate
consisting of a mixture of chains in which at least 90% of said
chains have the formula III 11
[0085] in which n is an integer from 2 to 20, R, R' and R"
represent a hydrogen or a SO3- group, for a degree of sulfation of
from 2.2 to 3, and the corresponding cation is a chemically or
pharmaceutically acceptable one, is obtained at the end of step
(b).
[0086] If an advantageous LMW-K5-N-sulfate consisting of a mixture
of chains in which the preponderant species has the formula II' is
used as starting material, a new LMW-K5-amine-O-oversulfate
consisting of a mixture of chains in which the preponderant species
has the formula III' 12
[0087] in which q is 4, 5, 6, 7, or 8, R, R' and R" are as defined
above, the degree of sulfation is from 2.3 to 3, and the
corresponding cation is a chemically or pharmaceutically acceptable
one, is obtained at the end of step (b).
[0088] These LMW-K5-amine-O-oversulfates are new products useful as
intermediates in the preparation of their N-sulfated derivatives,
but they have per se interesting pharmacological properties, in
particular against free radicals.
[0089] The origin of the new LMW-K5-amine-O-oversulfates from
LMW-K5-N-sulfates obtained by nitrous depolymerization and
subsequent reduction with, for instance, sodium borohydride,
involves the presence of a 2,5 anhydromannitol sulfated unit of
structure (a") 13
[0090] in which R" represents hydrogen or SO.sup.-, at the reducing
end of the majority of the chains in said mixture of chains.
[0091] Thus, the reducing end of the majority of the chains in said
mixture of chains is represented by the structure (b') 14
[0092] Among the new above mentioned LMW-K5-amine-O-oversulfates,
those consisting of mixtures in which the preponderant species is a
compound of formula (III") 15
[0093] wherein R, R', R" are hydrogen or SO.sup.-, X" is OH or
OSO.sup.-, for a degree of sulfation of from 2.2 to 3, m is 4, 5,
or 6 and the corresponding cation is a chemically or
pharmaceutically acceptable one, are preferred.
[0094] According to the present invention, starting from a
K5-N-sulfate it is possible to prepare LMW-K5-N,O-sulfates and
their chemically or pharmaceutically acceptable salts by a process
which comprises
[0095] (i) submitting a K5-N-sulfate to a nitrous depolymerisation
followed by a reduction, for example by sodium borohydride;
[0096] (ii) treating a LMW-K5-N-sulfate, in its acidic form, with a
tertiary amine or quaternary ammonium hydroxide, letting the
reaction mixture to stand for a period of time of 30-60 minutes,
whereby the pH of the solution is maintained at 7, and isolating
the corresponding tertiary amine or quaternary ammonium salt;
[0097] (iii) treating said tertiary amine or quaternary ammonium
salt of said LMW-K5-N-sulfate with an O-sulfation reactant under
O-oversulfation conditions;
[0098] (iv) treating the product thus obtained with a N-sulfating
agent and isolating the obtained LMW-K5-N,O-oversulfate.
[0099] The new LMW-K5-N,O-oversulfates obtained at the end of the
process of the present invention are generally present as sodium
salt. Said sodium salt can be converted into another chemically or
pharmaceutically acceptable salt. For example, an exchange with the
calcium ion can be performed using ultrafiltration membranes.
Particularly advantageous salts are those of alkaline metals,
earth-alkaline metals, ammonium,
tetra(C.sub.1-C.sub.4)alkylammonium, aluminum, and zinc. The
sodium, calcium and tetrabutylammonium are preferred salts.
[0100] The LMW-K5-N,O-oversulfates obtained according to the
process of the present invention consist of mixtures of chains in
which at least 90% of said chains has the structure I' 16
[0101] in which n is an integer from 2 to 20, R, R' and R"
represent hydrogen or a SO.sup.-- group, in which the reducing end
of the majority of said chains has the structure (a"), for a degree
of sulfation of from 3.2 to 4, advantageously from 3.5 to 4,
preferably from 3.5 to 3.9, and the corresponding cation is a
chemically or pharmaceutically acceptable one.
[0102] Among these new LMW-K5-N,O-oversulfates obtained according
to the process of the present invention those consisting of
mixtures of chains in which the preponderant species has the
formula I and in which the reducing end of the majority of said
chains has the structure (a") are particularly advantageous.
[0103] Preferred LMW-K5-N,O-oversulfates among those obtained
according to the process of the present invention consist of
mixtures of chains in which the preponderant species is a compound
of formula I" 17
[0104] wherein R, R' and R" are hydrogen or SO3-, X" is OH or
OSO3-, for a degree of sulfation of from 3.2 to 4, advantageously
from 3.5 to 4, preferably from 3.5 to 3.9, m is 4, 5 or 6 and the
corresponding cation is a chemically or pharmaceutically acceptable
one.
[0105] The new LMW-K5-N,O-oversulfates of the present invention are
interesting as active principles useful in therapy since they
possess antiviral, especially anti HIV-1, activity and an excellent
antiangiogenic activity. Particularly interesting are the
LMW-K5-N,O-oversulfates with a molecular weight of 3,750-4,250
(m=4), of 4,750-5,250 (m=5) or of 5,750-6,250 (m=6) and a degree of
sulfation of from 3.5 to 3.9.
[0106] All the LMW-K5-N,O-oversulfates above illustrated, due to
their antiviral and antiangiogenic activity, constitute interesting
drugs for the treatment of the above mentioned pathologies. For the
intended therapeutic uses, the active principles of the present
invention and their salts will be formulated according to
conventional techniques in suitable forms of administration such as
for example sterile solutions, topic dosage forms and, in general,
in all of those forms proposed until today for
glycosarninoglycan-type derivatives. Also the therapeutic dosages
will be chosen in analogy with those already studied for the known
natural compounds.
[0107] The administration of the active principle can be performed
by oral, transdermic or preferably parenteral, in particular
subcutaneous, intramuscular or intravenous, or topic route.
[0108] In man, the intended daily dosage for the parenteral
administration is of 0.5-500 mg/Kg/die, advantageously of 5-250
mg/kg/die, preferably of 10-150 mg/kg/die, while the dosage
intended for the topical route is of 1-1,000 mg/Kg/die,
advantageously 10-500 mg/Kg/die, preferably 20-100 mg/Kg/die.
[0109] Thus, according to another of its aspects, the present
invention provides a pharmaceutical composition comprising, as an
active ingredient, a pharmacologically effective amount of a
LMW-K5-N,O-oversulfate advantageously consisting of mixture of
chains in which the preponderant species is a compound of formula
I, I.degree., I" or by mixtures of chains in which at least 90% of
said chains has the formula I', for a degree of sulfation of from
3.2 to 4, advantageoulsy from 3.5 to 4, preferably from 3.5 to 3.9,
or of its pharmaceutically acceptable salts, in admixture with a
pharmaceutical vehicle. Said LMW-K5-N,O-oversulfates and their
salts are largely illustrated herein above. Advantageous
pharmaceutically acceptable salts are sodium, potassium, calcium,
magnesium, aluminum and zinc salts.
[0110] In the pharmaceutical compositions of the present invention
for oral, subcutaneous, intravenous, transdermic or topic
administration, the active ingredients are preferably administered
as dosage units, in admixture with the classic excipients or
pharmaceutical vehicles. The dose can amply change in function of
age, weight, and health conditions of the patient, as much as of
severity of the infection and of route of administration. This dose
comprises the administration of a dosage unit of from 1 to 1,000
mg, advantageously from 10 to 750 mg, preferably from 250 to 500
mg, once to three times per day, by intravenous, intramuscular,
subcutaneous, oral, transdermic, transmucosal or topical route.
[0111] The pharmaceutical compositions of the present invention are
formulated with the pharmaceutical carriers suitable for the
various administration routes. Formulations in form of cream,
pomade, ointment, gel, intravaginal ovules, suppositories, solution
or suspension adapted to local administration.
[0112] In particular, the present invention provides a
pharmaceutical composition for the treatment of angiogenesis
dependent pathologies or for the treatment of HIV infection which
comprises, as an active ingredient, a pharmacologically effective
amount of a LMW-K5-N,O-oversulfate advantageously consisting of
mixtures of chains in which the preponderant species is a compound
of formula I, I.degree., I" or by mixtures of chains in which at
least 90% of said chains has the formula I', for a degree of
sulfation of from 3.2 to 4, advantageously from 3.5 to 4,
preferably from 3.5 to 3.9, or of one of its pharmaceutically
acceptable salts, in admixture with a pharmaceutical vehicle or
carrier.
[0113] Finally, the present invention relates to a pharmaceutical
composition comprising, as one of its active ingredients, a new
LMW-K5-amine-O-oversulfate obtainable according to steps (a) and
(b) of the process above described, especially a
LMW-K5-amine-O-oversulfate having a degree of sulfation of from 2.2
to 3, advantageously having a mean molecular weight from about
3,500 to about 11,000, more advantageously from about 3,500 to
about 5,200, preferably consisting of a mixture of chains in which
at least 90% of said chains has the formula III or in which the
preponderant species is a compound of formula III' or III", or one
of its pharmaceutically acceptable salts, in admixture with a
pharmaceutical carrier. Preferably said LMW-O-oversulfated-K5 amine
with a degree of sulfation of from 2.2 to 3 is substantially free
of N-acetyl groups.
[0114] The following examples illustrate the invention without,
however, limiting it.
Preparation I
[0115] One gram of K5 obtained as described in paragraphs [0251]
and [0252] of example 12 of US 2002/0062019, giving a .sup.1H-NMR
spectrum (FIG. 3) in which signals due to lipophilic substances are
present in the region below 1.5 ppm, is dissolved in 100 ml of a
saturated aqueous solution of sodium chloride, thermostated at
4.degree. C. To the so obtained solution, 3 volumes of cold
isopropanol are added. The salt concentration of the solution is
brought to 3 M by adding a calculated amount of a saturated sodium
chloride solution and the cooled solution is kept at cold
temperature (about 4.degree. C.) overnight. The precipitate formed
is separated by centrifugation at 10,000 rpm for 20 minutes and the
purity of the product is controlled by overnight dialysis and
subsequent .sup.1H-NMR analysis in order to ascertain that signals
in the region below 1.5 ppm are absent. If necessary, the procedure
of dissolution in water containing 4M NaCl and precipitation with
isopropanol is repeated. The precitate is dissolved in water and
ultrafiltrated on a Miniplate membrane Millipore with a 10,000 D
cut off till disappearance of the salts. A K5 is obtained having a
purity of at least 99% and giving a .sup.1H-NMR spectrum in which
traces of lipophilic impurities in the region below 1.5 ppm are
undetectable.
Preparation II
Preparation of a K5-N-sulfate
[0116] (i) N-deacetylation
[0117] One gram of pure K5 polysaccharide prepared as described in
PREPARATION I are dissolved with 100 ml of 2 N sodium hydroxide and
the solution thus prepared is kept at 60.degree. C. for 24 hours.
The solution is brought to room temperature and then to neutral pH
with 6N hydrochloric acid.
[0118] (ii) N-sulfation
[0119] To the solution containing the deacetylated K5, kept at
40.degree. C., 1.6 g of sodium carbonate and, subsequently, 1.6 g
of pyridine.sulfur trioxide are added in 4 hours. At the end of the
reaction, after 24 hours, the solution is brought to room
temperature and then to pH 6.5-7 with a 5% solution of hydrochloric
acid. The K5-N-sulfate thus obtained is purified from salts by
diafiltration using a spiral membrane of 1,000 D (Prepscale
Cartridge-Millipore). The process is ended when the conductivity of
the permeate is below 1,000 .mu.S, preferably below 100 .mu.S. The
intradialysis is reduced till a polysaccharide concentration of 10%
using the same in concentration dialysis system. The concentrated
solution is freeze dried. The .sup.13C-NMR spectrum of the
K5-N-sulfate does not show any signal of residual N-acetyl or
NH.sub.2 groups.
Preparation III
LMW-K5-N-sulfate
[0120] The product obtained in PREPARATION II is depolymerized by
the degradation method with nitrous acid and subsequent reduction
of the formed aldehyde. One gram of K5-N-sulfate is dissolved in
200 ml of distilled water and 480 mg of sodium nitrite dissolved in
240 ml of distilled water are added thereinto. The solution is then
cooled to 4.degree. C. and the pH is brought to 2 with 0.1N HCl and
maintained for 30 minutes. At the end of the reaction the solution
is brought to pH 7 with 0.1N NaOH and then to room temperature. The
solution is then added with 450 mg of NaBH.sub.4 and left to react
for 4 hours. The excess NaBH4 is eliminated with HCl by bringing
the pH to 5-6. The product, neutralized with 0.1 M NaOH, is
recovered by precipitation with 3 volumes of acetone at 4.degree.
C., filtration with a glass funnel and dried at 40.degree. C. in
vacuum oven. Thus, 900 mg of a LMW-K5-N-sulfate having a mean
molecular weight of about 2,000 are obtained.
Preparation IV
K5-N,O-oversulfate
[0121] One gram of K5-N-sulfate obtained as described in
PREPARATION II is dissolved in 100 ml of deionized water and the
solution is cooled to 10.degree. C. with a cooling bath and then
passed through a cationic exchange resin IR 120 H.sup.+ or
equivalent (50-200 ml). Both the column and the reservoir of the
eluate are maintained at 10.degree. C. After the passage of the
solution containing the sample, the resin is washed with deionized
water till the pH of the permeate is higher than 6 (about 3 volumes
of deionized water). The acidic solution is brought to neutrality
with tetrabutylammonium hydroxide (15% aqueous solution), then
reduced to the minimum volume and freeze dried. The
tetrabutylammonium salt is dissolved in 40 ml of DMF and added with
3.5 g of adduct pyridine.SO.sub.3 in solid form. The solution is
kept at 50.degree. C. for 24 hours. At the end of the reaction the
solution is cooled to room temperature and added with 3 volumes of
a saturated sodium chloride solution in acetone till complete
precipitation. The precipitate is separated from the solvent by
filtration, solubilized with the minimum amount of deionized water
(for example 100 ml) and added with sodium chloride to obtain a
0.2M solution. The solution is brought to pH 7.5-8 with 2N sodium
hydroxide and added with 2 volumes of acetone till complete
precipitation. The precipitate is separated from the solvent by
filtration. The solid obtained is solubilized with 100 ml of
deionized water and purified from residual salts by ultrafiltration
using a spiral membrane of 1,000 D cut-off (prepscale
cartridge-Millipore).
[0122] The solution containing the O-sulfated product is treated
for the N-sulfation as previously described in step (ii) of
PREPARATION II. The product has a sulfate to carboxyl ratio of 3.87
measured by conductimetry according to Casu et al. and a mean
molecular weight of 20,000 measured by molecular exclusion
HPLC.
EXAMPLE 1
[0123] One gram of K5-N,O-oversulfate obtained as described in
PREPARATION IV is dissolved in 200 ml of deionized water and
thermostated to 4.degree. C. Then 230 mg of sodium nitrite
dissolved in an aqueous solution at the concentration of 0.2% are
added. The solution containing the K5-N,O-oversulfate and sodium
nitrite, kept at 4.degree. C., is brought to pH 2 by addition of
0.1N HCl cooled to 4.degree. C. The solution is left to react under
slow stirring for 30 minutes, then neutralized with 0.1N NaOH. The
solution containing the LMW-K5-N,O-oversulfate so obtained,
consisting of a mixture of chains in which the preponderant species
is a decasaccharide of formula I.degree. in which m is 4 and X' is
formyl, is brought to room temperature and treated with 250 mg of
sodium borohydride dissolved in 50 ml of water and left to react
for 4 hours. The excess sodium borohydride is eliminated by
bringing the pH to about 5 with 0.1N HCl and left for further 2
hours. At the end the solution is neutralized with 0.1N NaOH and
the product is recovered by precipitation with acetone after
concentration of the product by evaporation under reduced pressure.
The LMW-K5-N,O-oversulfate so obtained shows characteristics of
sulfation similar to those of the K5-N,O-oversulfate starting
material, a mean molecular weight of about 4,250 measured by
viscosimetry and consists of a mixture of chains in which the
preponderant species is a decasaccharide of formula I' in which m
is 4 and X' is CH2OH.
EXAMPLE 2
[0124] One gram of K5-N,O-oversulfate obtained as described in
PREPARATION IV is treated as described in EXAMPLE 1 using 200 mg of
sodium nitrite. The product obtained shows characteristics of
sulfation similar to those of the K5-N,O-oversulfate starting
material, a mean molecular weight of about 5,000 measured by
viscosimetry and consists of a mixture of chains in which the
preponderant species is a dodecasaccharide of formula I' in which m
is 5 and X' is CH.sub.2OH.
EXAMPLE 3
[0125] One gram of K5-N,O-oversulfate obtained as described in
PREPARATION IV is treated as described in Example 1 using 160 mg of
sodium nitrite. The LMW-K5-N,O-oversulfate thus obtained shows
characteristics of sulfation similar to those of the
K5-N,O-oversulfate starting material, a mean molecular weight of
about 6,000 measured by viscosimetry and consists of a mixture of
chains in which the preponderant species is a tetradecasaccharide
of formula I' in which m is 6 and X' is CH.sub.2OH.
EXAMPLE 4
[0126] (a) Tetrabutylammonium Salt of LMW-K5-N-sulfate
[0127] A solution of 500 mg of LMW-K5-N-sulfate obtained as
described in PREPARATION III in 50 ml of water is thermostated to
4.degree. C., then passed through a IR 120 H.sup.+ ionic exchange
resin preconditioned with water at 4.degree. C. The eluate
obtained, consisting of 125 ml of a solution at a pH of about 2, is
neutralized with a 15% solution of tetrabutylammonium hydroxide and
left at room temperature for one hour, by maintaining the pH at 7
by addition of a 15% tetrabutylammonium hydroxide solution and
finally is freeze dried. One gram of the tetrabutylammonium salt of
LMW-K5-N-sulfate is obtained.
[0128] (b) LMW-K5-amine-O-oversulfate
[0129] A solution containing one gram of the so obtained salt in 20
ml of dimethylformamide is kept at 55.degree. C. and treated with
20 ml of dimethylformamide containing 1.7 g of pyridine.SO.sub.3
adduct. The reaction at 55.degree. C. is performed overnight, then
40 ml of water are added to the mixture. After neutralization with
1N NaOH, the product is precipitated with 3 volumes of saturated
NaCl solution in acetone and kept at 4.degree. C. overnight. The
precipitate is recovered by filtration on a G4 glass funnel and
then ultrafiltrated with a TFF Millipore system with 1,000 D cut
off and dried at reduced pressure. Thus, there are obtained 683.2
mg of LMW-K5-amine-O-oversulfate consisting of a mixture of chains
in which the preponderant species is a decasaccharide of formula
II" wherein m is 4, for a degree of sulfation of about 2.9.
[0130] (c)LMW-K5-N,O-oversulfate
[0131] To a solution of 500 mg of the LMW-K5-amine-O-oversulfate
obtained in step (b) in 30 ml of water, 800 mg of sodium carbonate
are added, then 800 mg of pyridine.SO.sub.3 adduct in solid form
are added stepwise in 4 hours to the so obtained solution. The
reaction mixture is kept at 55.degree. C. overnight, then the
reaction is stopped by bringing its pH to 7 by addition of 0.1 N
HCl. After ultrafiltration on a 1,000 D membrane, 3 volumes of a
saturated solution of NaCl in acetone are added and the precipitate
is recovered by centrifugation at 5,000 rpm for 5 minutes. Thus,
there are obtained 502 mg of a LMW-K5-N,O-oversulfate having a mean
molecular weight of about 4,100 measured by viscosimetry,
consisting of a mixture of chains in which the preponderant species
is a decasaccharide of formula I" wherein m is 4, with a degree of
sulfation of about 3.9.
EXAMPLE 5
[0132] One gram of LMW-K5-N-sulfate obtained as described in
PREPARATION III is submitted to steps (a) and (b) as described in
Example 4. The LMW-K5-amine-O-oversulfate is recovered by
precipitation with 3 volumes of a saturated solution of NaCl in
acetone, dissolution of the precipitate obtained in water,
ultrafiltration on a 1,000 D cut-off membrane and freeze drying.
The so obtained product is a LMW-K5-amine-O-oversulfate having a
mean molecular weight of about 3,600 measured by viscosimetry and
consists of a mixture of chains in which the preponderant species
is a decasaccharide of formula III wherein m is 4.
* * * * *