NZ625096B2 - Low anticoagulant heparins - Google Patents
Low anticoagulant heparins Download PDFInfo
- Publication number
- NZ625096B2 NZ625096B2 NZ625096A NZ62509612A NZ625096B2 NZ 625096 B2 NZ625096 B2 NZ 625096B2 NZ 625096 A NZ625096 A NZ 625096A NZ 62509612 A NZ62509612 A NZ 62509612A NZ 625096 B2 NZ625096 B2 NZ 625096B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- heparin
- chemically modified
- kda
- ppm
- solution
- Prior art date
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- 239000003085 diluting agent Substances 0.000 description 1
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- VGGSQFUCUMXWEO-UHFFFAOYSA-N ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
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- ICIWUVCWSCSTAQ-UHFFFAOYSA-M iodate Chemical compound [O-]I(=O)=O ICIWUVCWSCSTAQ-UHFFFAOYSA-M 0.000 description 1
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- AAEVYOVXGOFMJO-UHFFFAOYSA-N prometryn Chemical compound CSC1=NC(NC(C)C)=NC(NC(C)C)=N1 AAEVYOVXGOFMJO-UHFFFAOYSA-N 0.000 description 1
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- RONWGALEIBILOG-VMJVVOMYSA-N quinine sulfate Chemical compound [H+].[H+].[O-]S([O-])(=O)=O.C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21.C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21 RONWGALEIBILOG-VMJVVOMYSA-N 0.000 description 1
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- AEMOLEFTQBMNLQ-VCSGLWQLSA-N α-L-iduronic acid Chemical compound O[C@@H]1O[C@@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-VCSGLWQLSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/727—Heparin; Heparan
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
- A61P33/06—Antimalarials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
- C08B37/0078—Degradation products
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
Disclosed herein is chemically modified heparin, with an antifactor II activity of less than 10 IU/mg, an antifactor Xa activity of less than 10 IU/mg and an average molecular weight (Mw) between about 6.5 and 9.5 kDa. Also disclosed is a method of preparing the heparin and its medical use, including treatment of malaria. g treatment of malaria.
Description
LOW ANTICOAGULANT HEPARINS
Technical field
The present invention relates to chemically d heparins with low anticoagulant ty
and methods of its production. The chemically modified heparins are useful for treating
disorders where n has been regarded as effective, but considered too prone to side
effects, such as malaria.
Background of the invention
Heparin is a naturally ing GAG (glucosaminoglycan) that is synthesized by and stored
intracellulary in so-called mast cells in humans and animals. Prepared industrially from
porcine intestinal mucosa, heparin is a potent anticoagulant and has been used clinically for
more than 60 years as the drug of preference for prophylaxis and treatment of
thromboembolic disorders. The major potential adverse effects of heparin treatment are
bleeding complications caused by its anticoagulant properties. Heparin is highly polydisperse
and composed of a heterogeneous population of polysaccharides with molecular weights
ranging from 5 to 40 kDa, with the average being approximately 15 to 18 kDa.
Low molecular weight/mass ns (LMWH) according to European pharmacopeia 6.0 are
defined as “salts of sulfated GAGs having a mass-average molecular mass less than 8 and for
which at least 60 per cent of the total mass has a molecular mass less than 8 kDa.” Low
molecular mass heparins display different chemical structures at the reducing or the non-
reducing end of the polysaccharide chains.” “The potency is not less than 70 IU of anti-factor
Xa activity per milligram calculated with reference to the dried substance. The ratio of anti-
factor Xa activity to anti-factor IIa activity is not less than 1.5.” Clinically used LMWHs
have molecular weights ranging from 3 to 15 kDa with an average of approximately 4 to 7
kDa. ed by lled depolymerization/fractionation of heparin, LMWHs exhibits
more favorable pharmacological and pharmacokinetic properties, including a lower tendency
to induce hemorrhage, increased bioavailability and a prolonged half-life following
aneous injection.
Heparin exerts its anticoagulant activity primarily through ffinity g to and
activation of the serine proteinase inhibitor, antithrombin (AT). g is mediated by a
specific pentasaccharide sequence. AT, an important physiological inhibitor of blood
coagulation, neutralizes activated coagulation factors by forming a stable complex with these
factors. Binding of heparin causes a conformational change in AT that dramatically enhances
the rate of inhibition of coagulation factors, thereby ating blood coagulation and the
formation of blood clots.
Infection caused by Plasmodium falcz'parum frequently gives rise to severe a in
humans. tized ocytes (pE) have the ability to bind (in vivo: sequestrate) in the
deep microvasculature as well as to uninfected erythrocytes, so called rosetting. The
sequestration and rosetting of pE augments the generation of severe disease when binding is
1O excessive, blocking the blood- flow, ng oxygen delivery and causing tissue damage.
Heparin has been ted as a useful agent in the treatment of the pathology occurring
during severe malaria. Heparin was previously used in the treatment of severe malaria
because of the suggested presence of disseminated intravascular coagulation (DIC) in malaria
patients but it was discontinued due to the occurrence of severe side effects such as
intracranial bleedings. Moreover, it was found that pE ation is not primarily due to
blood coagulation, but to noncovalent interactions n a parasite-induced protein on pE
surfaces and heparan sulfate (a heparin-related GAG) on erythrocytes and vascular endothelial
cells. The effect of heparin is ed to its ability to compete out this interaction (Vogt et al.,
PloS Pathog. 2006, 2, e100). Hence, there is a medical need for a heparin derivative with a
markedly reduced anticoagulant activity and bleeding inducing potential designed with
respect to its distribution of suitable sized and charged chains. US Patent No. 5,472,953 (Ekre
et al) ses the use of heparins with reduced anticoagulant ty for the treatment of
malaria.
AM Leitgeib et al. in Am. J. Trop. Med. Hyg. 2011, vol. 84(3), pp. 380-396 report promising
studies with low anticoagulant heparins which are found to disrupt rosettes of fresh al
isolates from patients with malaria.
In summary, a heparin derivative that carries the polyanionic es of heparin in essential
respects, but lacks an agulant effect would be an excellent candidate for treating
maladies in which the anticoagulant effect of heparin would be considered as a serious side
effect.
ption of the invention
The present ion relates to ally modified heparins that is selectively prepared to
retain therapeutic effects from the polysaccharide chains, while having a low anticoagulant
effect.
In the t of the t invention, anti-coagulant ty of heparin relates to the clinical
function of potentiating inhibition of coagulation factors Xa and 11a (thrombin) by AT. Other
terms will be defined in relevant contexts in the following description.
In one , the invention relates to a method of preparing chemically modified heparin with
an antifactor II activity of less than 10 IU/mg, an antifactor Xa activity of less than 10 IU/mg
and an average molecular weight (weight average, Mw) from about 6.5 to about 9.5 kDa. The
method generally comprises a step of selectively oxidizing heparin present in an aqueous
solution by subjecting it to an oxidizing agent e of oxidizing non-sulfated saccharide
residues and followed by reducing the resulting oxidized saccharide residues. The method
also lly comprises merizing the oxidized and reduced heparin chains by
hydrolysis at an acid pH from about 3 to about 4. The method can be performed in the general
sequence, consecutively by oxidizing, reducing and depolymerizing with hydrolysis in the
manners just described, while other complementary process steps may be added in any
suitable order.
The depolymerization is performed at a temperature of at least about 20 °C in order to obtain
suitably fractioned chains with desirable molecular s. In order to support selection of
desirable chains, the method generally can also e a step of enriching polysaccharide
chains having a molecular weight of about from 5.5 to about 10.5 kDa. The enrichment step
generally includes conventional chromatographic, filtering or sieving procedures well known
to those skilled in biopolymer manufacturing.
The methods according to the ion can further comprise at least one step of eliminating
remaining ing agent.
In addition, the methods according to the invention may comprise at least one elimination step
which includes removing reduced forms of the oxidation agent. In this context reduced forms
means oxidation agent transformed to reduced forms from contributing to oxidation of
targeted saccharide residues in heparin. Also in this context, the reduction step can comprise
on of a reducing agent which apart from reducing the oxidized heparin, contribute to
consumption (reducing) of remaining oxidizing agent.
In one aspect, the method ing to the invention comprises a step of eliminating any
remaining oxidizing agent and ng reduced forms of ing agent between the
described reducing step and the described depolymerization step. The merization can
be performed with hydrolysis at pH from to 3.0 to 3.5.
Accordingly, in one aspect, the invention is directed to a method comprising the consecutive
steps of selectively ing an unfractionated n by subjecting it to an oxidizing agent
capable of ing non-sulfated saccharides, reducing the resulting oxidized saccharides,
1O eliminating remaining oxidizing agent and reduced forms of oxidizing agent, and
depolymerizing the heparin chains by hydrolysis at an acidic pH from about 3 to about 3.5.
The elimination step may se adding an l in an amount sufficient for the
chemically modified heparin to precipitate. The alcohol can be methanol, ethanol or similar
alcohols and admits the chemically modified heparin to precipitate, while the oxidizing agent
and its reduced forms are removed with the alcohol.
The elimination step can also include addition of a quenching agent capable of chemically
inactivating the oxidizing agent to further exert oxidizing effects on the heparin. It is generally
considered by the inventors that the so described elimination step or elimination steps would
contribute to counteract or minimize non-specific merization of heparin, i.e.
depolymerization effects not attributable to the predictable results of the acidic hydrolysis.
Non-specific depolymerization may result in unpredictable loss in lar weight,
discolored products (with unstable absorbance values), other problems with stability and the
appearance of unidentified residues not predicted to arrive in heparin or low molecular weight
The introduction of an elimination step after the oxidation step enables an improved control
over any non-specific depolymerization. Another way of controlling non-specific
depolymerization, applicable with any earlier described method, is to reduce the temperature
significantly below ambient (room) temperature during the previous precipitation step or steps
when adding an alcohol. For example, the temperature can be reduced to about 5 °C in order
to prevent unwanted ons ing in non-specific depolymerization.
In accordance with the present invention, heparin is selectively oxidized, thereby inhibiting
the agulant effect mediated by the interaction between AT and the specific
pentasaccharide. The oxidation selectively splits glycols with 2 adjacent free hydroxyls and
the resulting product is referred to as a “glycol split” product. For this purpose the
composition of unfractionated heparin is treated with a periodate compound, such as sodium
metaperiodate in a suitable reaction medium, for example following the disclosures in US
Patent 4,990,502. Other oxidation agents would be useful if they have the same chemical
impact on the non-sulfated residues, without damaging critical levels of sulfates as ed in
the final product. When a periodate compound is used as an oxidizing agent, it is reduced to
iodate and subsequently, in the reducing step, to other inert forms of iodine, tively
referred to as “iodine compounds”. The elimination step of the inventive processes serves to
1O eliminate or ze the oxidative effect of any iodine compounds and to remove the iodine
compounds from the process in a way that counteracts of zes ecif1c
depolymerization. For this reason, the elimination step can comprise one or two precipitation
steps with l. It can also include addition of a quenching agent with two vicinal hydroxyl
groups, such as ethylene , glycerol and similar agents, in order to chemically and
selectively ate oxidizing agents.
The oxidized heparin, for e after isolation through alcohol precipitation, subsequently
is treated with a reducing agent, ly sodium borohydride, for example according to the
protocols of US Patent 4,990,502. Other ng agents may be used if they are capable of
performing similar ion of oxidized onic/iduronic acid residues as sodium
borohydride without unnecessarily modifying or destroying the sulfate groups of other
saccharide residues. The so reduced chains can be isolated, for example by alcohol
precipitation and transferred to the depolymerisation step.
The employment of unfractionated heparin in the so bed methods is regarded as
generally advantageous for the invention, since it will contribute towards reducing waste of
material and increasing cost efficacy and support the provision of a composition product with
desirable ccharide chain length and with retained sulfate groups
The depolymerization step can be performed in an s solution at a concentration from
about 15 to about 25% w/v of the d heparin. A strong acidif1er is then admixed to the
solution to a pH of from about 3 to about 4. A suitable pH range is from about 3.0 to about
3.5. A pH value of about 3.0 is suitable according to the inventive method, while pH 3.5 also
has been found suitable and admits production of a chemically modified heparin within the
outlined molecular weight range. It has been found that the inventive process admits
flexibility in this pH range that can be controlled by the process time of the hydrolysis step
when operating within a time frame of 4 to 10 hours. Hydrochloric acid is a suitable acid with
the inventive process, however other strong acids can be found useful if they do not
substantially destroy sulfate groups. By applying the above specified conditions, a t
with suitable chain lengths and storage stability is ved for subsequent work up to a
pharmaceutically useful composition.
The methods yield an overall enrichment in sulfate groups within the polysaccharide chain
length as non-sulfated iduronic/glucuronic acid is chemically modified and appears mainly as
reducing end, remnant als. The methods accordingly involve ions that retain
sulfate groups and thus to retain the sulfated domains of native heparin. The methods also yield
chains with an advantageous size distribution which supports a desirable therapeutic efficacy
and is considered to improve the therapeutic index ed to other described low
anticoagulant ns. The invention does in general terms extend to chemically modified
heparins prepared with the recited methods.
The invention is directed to chemically modified heparins with an antifactor II activity of less
than 10 IU/mg, an antifactor Xa activity of less than 10 IU/mg and an average molecular
weight (Mw) between about 6.5 and about 9.5 kDa which can be manufactured with the
described methods. Chemically modified heparin according to the invention has
polysaccharide chains which retain at least 90 % of the sulfate groups. Chemically modified
heparin according to the invention have a loss of sulfate groups of about one sulfate group per
haride unit of 100 disaccharide units, corresponding to a loss of sulfate groups of less
than 1 % of the total sulfate content, when assuming that heparin contains in e 2.4
sulfate groups per disaccharide unit and that there is one sulfate group per iduronic acid, 12S
and 2 sulfate groups for the predominant glucosamine variant, GlcNS.
According to a first aspect of the present invention there is provided a chemically modified
heparin with an antifactor IIa ty of up to 10 IU/mg, an antifactor Xa activity of up to
IU/mg and a weight average molecular weight from about 6.5 to about 9.5 kDa, wherein
the polysaccharide :
(i) retain at least 90 %, of the sulfate groups of the corresponding native heparin;
(ii) have a reduction in chemically intact accharide ces providing an
antithrombin mediated anticoagulant , when compared to the polysaccharide chains
of native heparin; and
(iii) have a ion in unsulfated iduronic and/or glucuronic acid units when compared to
native heparin;
wherein the predominant disaccharide of the polysaccharide is a disaccharide having the
chemical ure:
wherein n is an integer of from 2 to 25, such that it ses from 2 to 25 disaccharide
units corresponding to lar weights from 1.2 to 15 kDa; and
wherein the chemically modified heparin has, in a 1H-NMR spectrum, no unidentified
signals in the ranges 0.10-2.00 ppm, 2.10-3.10 ppm and 5.70-8.00 ppm larger than 4 per
cent when compared to the height of the signal present in native heparin at 5.42 ppm.
According to a second aspect of the present invention there is provided a method of preparing
a chemically modified heparin with an antifactor IIa activity of up to 10 IU/mg, an
antifactor Xa activity of up to 10 IU/mg and an average molecular weight (weight average,
Mw) from about 6.5 to about 9.5 kDa, sing the consecutive steps of:
(a) selectively oxidizing unfractionated heparin by subjecting it to an
oxidizing agent e of oxidizing non-sulfated saccharide residues;
(b) reducing the resulting ed saccharide residue;
(c) eliminating any remaining oxidizing agent and removing reduced forms of
oxidizing agent; and
(d) merizing the polysaccharide chains by hydrolysis at an acidic pH of
from about 3 to about 4.
According to a third aspect of the present invention there is provided a chemically modified
heparin obtainable by a method according to the second aspect .
According to a fourth aspect of the present invention there is ed the use of a chemically
modified heparin according to the first or third aspect in the manufacture of a medicament
for therapy.
According to a fifth aspect of the present invention there is provided the use of a ally
ed heparin according to the first or third aspect in the manufacture of a medicament
for the ent of malaria.
According to a sixth aspect of the present ion there is provided a pharmaceutical
composition comprising a therapeutically ive amount of a chemically modified
heparin according to the first aspect or the third , together with a pharmaceutically
and/or pharmacologically acceptable carrier.
According to an seventh aspect of the present invention there is provided a combination
comprising a chemically modified heparin according to the first aspect or the third aspect,
and another medicament for the ent of malaria.
An aspect of the invention is a chemically modified heparin with an antifactor II activity of less
than 10 IU/mg, an antifactor Xa activity of up to 10 IU/mg and an average molecular weight
from about 6.5 to about 9.5 kDa, wherein the polysaccharide chains:
(i) retain at least 90 %, of the sulfate groups of the corresponding native heparin;
(ii) comprise from 2 to 25 disaccharide units corresponding to molecular weights from 1.2 to
kDa;
(iii) have a reduction in chemically intact saccharide sequences providing an antithrombin
mediated anticoagulant effect, when compared to the polysaccharide chains of native heparin;
WO 95276
(iv) have a reduction in unsulfated iduronic and/or glucuronic acid units when compared to
native heparin.
A ally modified heparin has from 2 to 25 disaccharide units corresponding to
molecular weights from about 1.2 to aboutl5 kDa. A chemically modified heparin has
polysaccharide chains with a reduction in chemically intact accharide ces
responsible for the anti-thrombin (AT) mediated anticoagulant effect, when compared to the
chains of native heparin and have polysaccharide chains with a reduction in unsulfated
iduronic and onic acid residues when compared to native heparin.
An aspect of the invention is that ally modified n having predominantly
occurring polysaccharide chains with between 6 and 16 disaccharide units with molecular
weights between 3.6 and 9.6 kDa. The term “predominantly” does in this context have the
g of “the frequently most present” polysaccharide chains.
An aspect of the invention is a chemically modified heparin having at least 30 % of the
polysaccharide chains with a molecular weight of at least 8 kDa.
An aspect of the invention is a chemically modified heparins comprising chains terminated by
a threonate residue or by a derivative of ate, such as esters or amides thereof The
threonate residue is depicted below as a terminal group.
In an aspect of the invention, from 3 to 15 % of the polysaccharide chains of the chemically
modified heparin have a molecular mass of at least 15kDa.
In an aspect of the invention, from 25 to 47 % of the polysaccharide chains of the chemically
modified heparin have a molecular mass of at least 9 kDa.
In an aspect of the invention, from 40 to 60 % of the polysaccharide chains of the chemically
modified heparin have a molecular mass of at least 7 kDa.
In an aspect of the invention, from 60 to 80 % of the polysaccharide chains of the chemically
modified heparin have a molecular mass of at least 5 kDa.
In an aspect of the invention, 85 % of the polysaccharide chains of the chemically modified
heparin have a molecular mass of at least 3 kDa.
In an aspect of the invention, 95 % of the polysaccharide chains of the chemically modified
heparin have a lar mass of at least 2 kDa.
In yet an aspect, chemically modified heparin of the invention have a distribution of
polysaccharides and their corresponding molecular mass expressed as cumulative % of weight
according the table:
Molecular mass, Cumulative weight,
In yet an aspect, chemically modified n of the invention have a distribution of
ccharides and their corresponding molecular mass expressed as cumulative % of weight
according the table:
Molecular mass, Cumulative weight,
Chemically modified heparin according to the invention has polysaccharide chains with the
disaccharide depicted below as the predominant structure with a terminal threonate residue.
The predominant disaccharide has a molecular weight of about 600 Da.
(n is an integer of 2-25).
According to yet an aspect of the invention, chemically modified heparin according to the
invention comprises glycol-split residues with the chemical structure:
Glycol-split residues appear in polysaccharide chains of chemically ed heparins, as a
result of the oxidation and reduction processes, as earlier discussed in the context with the
method and the specific hydrolysis step. They can also be regarded as indicative of the
efficacy of the earlier described depolymerization lysis) step. It is further referred to
US Patent 4,990,502 for a chemical nce of the appearance of glycol-split residues.
The depicted glycol-spilt residue arrives from oxidation and reduction of unsulfated
iduronic acid and onic acid.
Chemically modified n according to the invention has a 1H -NMR spectrum in the
range of from 5.0 to 6.5 ppm that es with a 1H -NMR spectrum from native heparin
by the absence of any proton signals with a magnitude above 0.1 (mol) %.
In one aspect of the invention, chemically modified heparin as herein described es
with tly accepted heparin standards by having an 1H-NMR spectrum meeting the
heparin
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acceptance criterion set out by EDQM, Council of Europe, 2012, for e by not having
any unidentified signals larger than 4 per cent compared to the height of the heparin signal at
.42 ppm in the ranges 0.10-2.00 ppm, 2.10-3.10 ppm and 5.70-8.00 ppm.
In one aspect, chemically modified heparin according to the invention has a relative average
molecular mass range of approximately 7,500 daltons with about 90% ranging n 2,000
and 15,000 daltons, the degree of sulfation is 2 to 2.5 perdisaccharidic unit.
In one aspect of the invention, a chemically modified heparin as herein described, may be
useful for therapies previously disclosed as associated with other regions of heparin than the
binding site to AT. Examples include, but are not limited, to such areas as ent of
inflammation, treatment of neurodegenerative diseases, tissue repair, stroke, prevention and
treatment of shock, especially septic shock and prevention of the development of metastases.
An aspect of the invention, is a chemically modified n for use in the treatment of
a. Chemically modified heparins as herein disclosed, may be useful in the preventionor
treatment of occlusive effects from malaria, caused by abnormal adhesive effects in the blood.
An aspect of the invention is a ation of chemically modified heparin as herein
disclosed, with another malaria medicament. In one aspect of the invention, such
combinations comprise chemically modified heparin and atovaquone/proguanil or artesunate
(parenteral). Examples of a medicaments in combination aspects of the inventions
medicaments are, for use alone, or combinations with each other, are artemether,
lumefantrine, amodiaquine, mefioquine, oxine, pyrimethamine, tetracycline,
doxycycline, dapsone, clindamycin, quinine, tetracycline, atovaquone, proguanil, quine,
primaquine, sulfadoxin, amodiaquine, dihydroartemisini, piperaquine, dihydroartemisinin,
and piperaquine
In still an aspect of the ion, a ally modified heparin as herein disclosed may be
administered simultaneously or sequentially with a malaria medicament, i.e. in an t
therapy with a malaria medicament.
The term “malaria medicament” includes agents conventionally used for ng malaria,
such as agents already established for treating the parasite infection. Yet an aspect of the
invention, is a method for the treatment of malaria, comprising the administration to patient in
need of such treatment, a therapeutically effective amount of a ally modified heparin as
herein described.
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Yet an aspect of the invention is a pharmaceutical composition sing a chemically
modified heparin as herein described, together with a pharmaceutically and pharrnacologically
acceptable carrier. In yet an aspect of the invention, a pharmaceutical composition as herein
described, may be administered systemically by parenteral administration, such as by
subcutaneous or intravenous injection. In yet an , a pharmaceutical ition as
herein described, may be administered orally. For parenteral stration, the active
compounds can be incorporated into a on or suspension, which also n one or more
adjuvants such as sterile diluents such as water for injection, saline, fixed oils, hylene
glycol, glycerol, propylene glycol or other synthetic solvents, cterial agents,
antioxidants, ing agents, buffers and agents for ing the osmolality. The parenteral
preparation can be delivered in ampoules, vials, prefilled or disposable syringes also for self
administration, or as infusion arrangements, such as for intravenous or subcutaneous infusion.
Chemically modified heparins according to the invention may be administered
subcutaneously and with suitable self-administration tools, such as injectors.
Pharmaceutical compositions comprising a chemically modified heparin as herein described,
can comprise combinations of one or several conventional pharmaceutically acceptable
carriers. The rs or excipients can be a solid, semisolid or liquid material that can serve as
a vehicle for the active substance. The itions can be stered in a single dose
every 24 h for a period of 1-30, preferably 1-10 days. The dose may be between 0.5-6 mg/kg
bodyweight given, either enously every 6 or 8 hours, or 1-4 times daily given
subcutaneously. An estimated single dose is 25-100 mg/d of a chemically modified heparin,
but may be up to l g or more. The dose is related to the form of administration. The described
pharmaceutical compositions can r se additional agents suitable for treating
malaria with supplementary or complementary therapies as outlined in the previous section.
A chemically modified heparin of the invention would need to retain a sufficient amount of
the sulfate groups included in the native form, in order to exert a therapeutic activity unrelated
to anticoagulant effects, for example by targeting P. falciparum erythrocyte membrane protein
1 (PfEMPl), and, at the same time have the anticoagulant activity inherent in the
pentasaccharide abolished or largely reduced. Also, the inventors understand that selectin
inhibition, as well as other heparin-dependent biological effects, correlate to polysaccharide
chain length, so the chemical modification cannot result in extensive fragmentation of the
native molecules. The bioavailability of hain heparins after subcutaneous dosing is low
and the possibility of heparin induced thrombocytopenia (HIT) is positively correlated to
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chain , the chemically d heparin derivatives according to the invention should
not be of full length. The present chemically modified heparin is the result of a number of
important considerations 1. Initially, in order to satisfy the process economy criteria, the target
heparin had to be able to be produced from tionated heparin. 2. The process can not
yield too abundant short chains as the eutic effect is positively correlated with
sufficiently long saccharide chain lengths. 3. The process should not yield too abundant long
chains as the desirable subcutaneous dosing regime is not possible with longer chains. 4.
Similarly, long chain length is correlated with undesirable side-effects such as HIT. 5. The
process should eliminate the anticoagulant effect inherent in the AT—binding pentasaccharide.
6. The s shall avoid desulfatation of the polymer, but should rather increase the
proportion of the sulfated residues, as therapeutic effects are positively correlated with the
degree of sulfatation, that provides negative charge density. The invention as described above
and to be described in the following detailed experimental section demonstrates that it is
possible to overcome the s that are outlined above and thus to e a successful drug
candidate, for treating malaria.
Detailed and exemplifying description of the invention
One aspect of the invention is chemically modified heparins having the International
proprietary name (INN) sevuparin sodium also given the code DF02. These terms are used
hangeably and shall have same meaning.
ption of the drawings
Fig. 1 shows a representative example of heparin sequence
Fig. 2 shows the structure of the pentasaccharide unit in heparin required for its binding to AT.
Fig. 3 shows a scheme of the synthesis of the chemically d heparin DF02.
Fig. 4 shows the predominant structure of DF02.
Fig. 5 shows how rosettes of the parasite FCR3Sl.2 were disrupted by DF02 and heparin in a
dose dependent manner.
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Fig. 6 shows how rosettes of fresh isolates of children with severe, complicated or mild
malaria are sensitive to the treatment with DF02 (100 (dark bars) and 1000 (grey bars) ug/ml).
Fig. 7 shows cytoadherence disruption: binding of the pE of parasite FCR3Sl.2 to endothelial
cell can be inhibited or reversed by DF02 or n in a dose dependent manner.
Fig. 8 demonstrates merozoite invasion of parasite FCR3Sl.2 into fresh red blood cells can be
inhibited by DF02 or heparin in a dose dependent .
Fig. 9 demonstrates that sequestration of P.falciparum-infected erythrocytes in the lungs of
rats can be inhibited by the treatment with chemically modified heparin.
Example 1
Both heparin and LMWH are composed of repeating disaccharide units containing one uronic
acid residue curonic or L-iduronic acid, UA) and one D-glucosamine moiety (GlcN)
that is either N—sulfated or N—acetylated. These carbohydrate residues may be further 0-
sulfated, at the C-6 and C-3 positions in the case of amine and the C-2 on of the
UA. The structure of n is variable regarding distribution ofUA and sulfate residues; a
representative partial sequence is shown in Fig. l (which also illustrates the mode of
numbering of carbon atoms in a monosaccharide residue). Fig. 2 shows the unique,
pentasaccharide sequence distributed within heparin polymers which is ial for the
binding to AT. Several structural characteristics of this sequence have been shown to be
crucial for the interaction of heparin with AT. Notably, one of the two UA residues present in
this pentasaccharide sequence is consistently sulfated at the C-2 position, whereas the
hydroxyl groups at both C-2 and C-3 of the other uronic moiety are unsubstituted
ed ption of the manufacturingprocess ofchemically modified heparins according
to the invention
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Fig 3 schematically shows the manufacturing of a chemically modified heparin according to
the present invention, hereinafter designated DF02, while the following sections outline the
manufacturing steps.
The substance is prepared from Heparin Sodium. The preparation involves selective oxidation
of non-sulfated ironic acid residues in heparin by period ate, including the glucuronic acid
moiety in the pentasaccharide sequence that binds AT. Disruption of the structure of this
residue lates the high-affinity interaction with AT and, consequently, the anticoagulant
effect (measured as a-FXa or a-FIIa, see Table 4 and 5). Subsequent reduction and treatment
by acid results in cleavage of the polymer at the sites that has been oxidized by periodate.
1O Together, these manipulations lead to a loss of anticoagulant activity along with adequate de-
polymerization of the heparin chain.
Subsequently, additives, impurities and side-products are removed by repeated precipitations
with l, tion and centrifugations. Thereafter the substance is obtained in powder
form by drying with vacuum and heat. The drug substance DF02 is dissolved in a sterile
aqueous buffer to yield the drug product, which is intended for intravenous or aneous
administration.
Oxidation of glucuronic and iduronic acid gresidues), deletion of anticoagulant activity
A quantity of about 3000 grams of Heparin is dissolved in d water to obtain a 10-20 %
w/v solution. The pH of this solution is adjusted to 4.5-5.5. The sodium metaperiodate
) is uently added to the process solution,, quantity of periodate 15-25% of the
weight of heparin. The pH is again adjusted to 4.5-5.5. The reactor is d in order to
protect the reaction from light. The process solution is reacted during 22 — 26 hours with
nt stirring and maintenance of the temperature at 13 — 17 °C. The pH at the end of the
on period is measured and recorded.
Termination of the oxidation reaction and removal of iodine-containing compounds
Ethanol (95-99.5%) is added to the reaction mixture over a period of 0.5 — 1 hour, with
careful ng and at a temperature of 20 — 25 °C. The volume of ethanol to be added is in the
range 1-2 volumes of ethanol per volume of process solution. The oxidized heparin is then
W0 2013/095276
allowed to precipitate and sediment for 15 — 20 hours, after which the mother liquor is
decanted and discarded.
Next, the sediment is dissolved in purified water to obtain a 15-30% w/v process solution.
Then NaCl is added to obtain a tration of .30 mol/liter in the process solution.
Stirring continues for another 0.5 — 1 hour while maintaining the temperature of 20 — 25 °C.
Subsequently 1.0-2.0 volumes of ethanol (95-99.5%) per volume of process solution
is added to this solution with careful stirring, during a period of 0.5 — 1 hour. This precipitates
the product from the solution. This precipitation continues for >1 hour.
Reduction of oxidized glucuronic/iduronic acids
After the mother liquor has been decanted and discarded, the sediment is dissolved by
addition of purified water until a concentration of the process solution of 15-30% w/v is
obtained. While maintaining the temperature at 13-17 °C, the pH of the solution is adjusted to
.5-6.5. A quantity of 130-150 grams of sodium borohydride is then added to the solution and
dissolved, the pH will ately se to a pH of 10-1 1, and the reaction is continued
for 14-20 hours. The pH of the solution, both prior to and after this reaction period, is
recorded. After this reaction time, a dilute acid is added slowly in order to adjust the pH to a
value of 4, this degrades remaining sodium borohydride. After maintaining a pH of 4 for 45 —
60 minutes, the pH of the solution is ed to 7 with a dilute NaOH solution.
Acid hydrolysis to e depolymerization of the polysaccharide chains
A dilute acid is added to the on until a pH of 3.5 (acceptable range 3.2-3.8) is obtained.
The temperature is kept at 50-5 5°C while ng the solution for 3 hours +/- 10 minutes. A
dilute NaOH solution is then added until a pH of 7.0 is obtained and the reaction solution is
cooled down to a ature of 13-17 °C. Sodium chloride (NaCl) is then added until a
concentration of 0.2-0.3 mol/liter is obtained.
Purification of the t
Removal of process additives and impurities, addition of counter-ions and filtration
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One volume of process solution is then added to 1.5-2.5 volumes of l (95-99.5%)
followed by centrifugation at >2000 G, and at <20°C for 20 — 30 minutes, after which the
supernatant is decanted and discarded.
The product paste obtained by centrifugation is then dissolved in purified water to obtain a
product concentration 10-20% w/v. Then NaCl is added to obtain a concentration of 0.20-0.35
mol/liter. Further, 1.5-2.5 s of ethanol (95-99.5%) is added per volume of process
solution which precipitates the product from the solution. Centrifugation s at >2000 G,
and at <20°C for 20 — 30 minutes after which the supernatant is decanted and ded.
1O Next the remaining paste is added purified water to dissolve. The product concentration
would now be in the range of 10-20% w/v. The pH of the product solution is now adjusted to
6.5-7.5. The solution is then filtered to remove any particulates. Then, to one volume of
process solution is added 1.5-2.5 volumes of ethanol (95-99.5%). Centrifugation follows at
>2000 G, and at <20°C for 20 — 30 minutes after which the atant is decanted and
discarded.
Reduction of the size and water content of the precipitate paste
A glass reactor is then filled with ethanol, volume 2 liter. While stirring the ethanol, the
precipitate paste is added. The mechanical stirring solidifies the paste and replaces the water
present by the l giving a homogenous particle suspension. The stirring is discontinued
after 1-2 hours after which the particles are allowed to sediment, then the mother liquor is
decanted. This procedure is repeated twice. The precipitate is isolated on a polypropylene
(PP) filter. This procedure was repeated two more times. After removal of excessive liquid,
the particles are passed through a sieve to obtain smaller and uniform sized particles.
Vacuum drying
The product is buted evenly onto two pre-weighed trays, and placed in a vacuum cabinet.
The pressure is reduced with a vacuum pump, the pressure actually obtained being noted, and
the trays are heated to 35 — 40°C, with nt recording of the temperature. A stream of
nitrogen is passed through the drier at this time while maintaining the low pressure in the
dryer. When a constant weight is ed, i.e. no r evaporation is noticed, the drying is
W0 2013/095276
considered complete. The dry product is dispensed, packed and protected from moisture.
Storage is performed in a dry area at a temperature of 20-25°C.
The so manufactured product can prepared as drug product by a conventional aseptic s,
such as on comprising 150 mg/mL of chemically modified heparin active agent and Na
phosphate till 15 mM, pH 6-8. The so obtained drug product is intended for intravenous or
subcutaneous administration. The resulting ally modified n, DF02, is a
merized form of heparin with a ted average molecular weight of 6.5-9.5 kDa and
with essentially no anticoagulant activity.
DF02 has a size distribution of polysaccharide polymers, with a range for n of 2-25
corresponding to molecular weights of 1.2-15 kDa. The predominant size is 6-16 disaccharide
units corresponding to molecular weights of 3.6-9.6 kDa.
By practical tests it can be found that reaction of the oxidized heparin preparation in alkaline
solution gives rise to chains that are too short, or lack the proper degree of sulfatation, for the
optimal pharmaceutical function of the resulting heparin. Further by practical tests, it can be
shown that treatment of the heparin preparation in a solution of less than pH 1, leads to
desulfatation of the product, and thus giving rise to a chemically modified heparin with less
than optimal pharmaceutical effect.
Table 1. Distribution of ccharides and their corresponding molecular mass in DF02
(several batches) as cumulative % of weight
Molecular mass, tive weight,
kDa %
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The corresponding value for weight average molecular weight, Mw falls in the range 6.5-9.5
Table 2 Distribution of polysaccharides and their corresponding molecular mass in DF02 as
cumulative % of weight for an individual batch
Molecular mass, Cumulative weight,
kDa %
The corresponding value for lar weight average weight, Mw is 7.4 kDa
Example 2
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Example 2 represents a modified version of the manufacturing process according to Example
1. n s parameters have been modified, such as process temperatures, with the
purpose of preventing any non-specific depolymerization in the initial part of the process
Oxidation of glucuronic and iduronic acid gresidues), deletion of anticoagulant activity
A quantity of about 3000 grams of Heparin is dissolved in purified water to obtain a 10-20 %
w/v solution. The pH of this solution is adjusted to 4.5-5.5. The sodium metaperiodate
(NaIO4) is subsequently added to the process solution; quantity of periodate 15-25% of the
weight of n. The pH is again ed to 4.5-5.5. The reactor is covered in order to
protect the reaction from light. The process solution is reacted during the 22 — 26 hours with
constant stirring and maintenance of the temperature at 13 — 17 °C, while the temperature is
lowered to about 5 °C during the last two hours. The pH at the end of the reaction period is
measured and recorded.
Termination of the oxidation reaction and removal of iodine-containing compounds
Ethanol .5%) is added to the reaction mixture over a period of 0.5 — 1 hour, with
careful stirring and at a ature of about 5 °C. The volume of ethanol to be added is in the
range 1-2 volumes of ethanol per volume of process solution. The oxidized heparin is then
allowed to itate and sediment for 15 — 20 hours, after which the mother liquor is
decanted and discarded.
Next, the sediment is dissolved in d water to obtain a 15-30% w/v process solution.
Then NaCl is added to obtain a concentration of 0.15-0.30 mol/liter in the process solution.
Stirring continues for another 0.5 — 1 hour while ining a temperature of about 5 °C.
Subsequently 1.0-2.0 volumes of ethanol (95-99.5%) per volume of process solution
is added to this solution with careful stirring, during a period of 0.5 — 1 hour. This precipitates
the product from the on. This itation continues for >1 hour.
Reduction of oxidized glucuronic/iduronic acids
This step is made in accordance with Example 1.
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Acid hydrolysis to e depolymerization of the polysaccharide chains
This step is med in accordance with Example 1 with the difference that the process time
may be extended about two hours before pH is raised to 7.0 with NaOH.
The further process steps towards a drug product comprising for example 150 mg/ml
chemically modified heparin active agent is cal to the steps outline in Example 1.
By performing the process steps according to Example 2, a chemically modified heparin with
1O a polysaccharide molecular weight distribution demonstrated in Table l of Example 1 is
obtained.
Example 3
Example 3 represents another method to cture chemically modified heparins ing
to the invention modified by directly subjecting the process solution arriving from the
oxidation step to a strong reducing agent, before any precipitation step is introduced.
Oxidation of glucuronic and ic acid gresidues), on of anticoagulant activity
A quantity of about 3000 grams of Heparin is dissolved in purified water to obtain a 10-20 %
w/v solution. The pH of this solution is adjusted to 4.5-5.5. The sodium metaperiodate
(NaIO4) is subsequently added to the process solution; quantity of periodate 15-25% of the
weight of heparin. The pH is again adjusted to 4.5-5.5. The reactor is covered in order to
protect the reaction from light. The process solution is reacted during the 22 — 26 hours with
nt stirring and maintenance of the temperature at 13 — 17 °C. The pH at the end of the
reaction period is measured and recorded.
Reduction of oxidized glucoronic/iduronic acids and elimination of oxidizing iodine
containing compounds
While maintaining the temperature at 13-17 °C, the pH of the on is adjusted to 5.5-6.5.
A quantity of 130-200 grams of sodium borohydride is then added to the solution and
dissolved, the pH will immediately increase to a pH of 10-1 1, and the reaction is continued
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for 14-20 hours. The pH of the solution, both prior to and after this reaction period, is
recorded. After this on time, a dilute acid is added slowly in order to adjust the pH to a
value of 4, this degrades remaining sodium borohydride. After maintaining a pH of 4 for 45 —
60 minutes, the pH of the solution is adjusted to 7 with a dilute NaOH solution.
Removal of iodine-containing compounds
l (95-99.5%) is added to the reaction mixture over a period of 0.5 — 1 hour, with
careful stirring and at a temperature of 20 — 25 °C. The volume of ethanol to be added is in the
1O range 1-2 s of l per volume of process solution. The oxidized and subsequently
reduced heparin is then allowed to precipitate and sediment for 15 — 20 hours, after which the
mother liquor is decanted and discarded.
Next, the sediment is dissolved in purified water to obtain a 15-30% w/v process solution.
Then NaCl is added to obtain a concentration of 0.15-0.30 mol/liter in the process solution.
Stirring continues for another 0.5 — 1 hour while ining the temperature of 15 — 25 °C.
Subsequently 1.0-2.0 volumes of ethanol (95-99.5%) per volume of process solution
is added to this solution with careful stirring, during a period of 0.5 — 1 hour. This precipitates
the product from the solution. This precipitation continues for >1 hour.
Acid ysis to achieve depolymerization of the polysaccharide chains
After the mother liquor has been decanted and discarded, the sediment is dissolved by
addition of purified water until a concentration of the process solution of 15-30% w/v is
obtained.
A dilute acid is added to the on until a pH of 3.0 is obtained. The temperature is kept at
50-55°C while stirring the solution for 5 to 10 hours. The progress of depolymerization may
be followed by in-process analyses of the molecular weight, by GPC-HPLC as to determine
the actual time of reaction ed. A dilute NaOH solution is then added until a pH of 7.0 is
ed and the reaction solution is cooled down to a temperature of 13-17 °C. Sodium
de NaCl is then added until a concentration of 0.2-0.3 mol/liter is obtained.
Alternatively, in order to rly control the e molecular weight, the dilute acid can
be added to obtain a pH of 3.5, but to accomplish a comparable level of hydrolysis the process
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time is extended from 5 to 6 hour to 8 to 9 hours. According to both alternatives, the average
molecular weight is kept well within the specification range of 6.5 and 9.5 kDa.
The ing process steps towards a drug product comprising for example 150 mg/ml
chemically modified heparin active agent is identical to the steps outline in Example 1.
By performing the process steps according to Example 3, a chemically d heparin with
a polysaccharide molecular weight distribution demonstrated in Table l of Example 1 is
obtained.
Table 3. Intensity of signals present in 1H-NMR spectra compared to heparin in the range of
5 to 6.5 ppm
produced Intensity of signals %
6 14 ppm 6.00 ppm 5. 94 ppm
according to:
——---
——---
——---
——---
Table 3 is a result of comparing studies of 1H-NMR spectra in the range of 5.0 to 6.5 ppm, of
chemically modified heparins produced according to es 1 to 3.
Table 3 confirms that a chemically d heparin as manufactured with the process
according to Example 3 results in a 1H-NMR spectrum with absence of cted signals in
the range 5.90 ppm to 6.14 ppm equivalent to that of heparin. These signals show a
correlation to partially unsaturated, double bond structures containing e amines, which
may undergo r chemical modifications and contribute to discoloration of the product.
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In other terms, the s according to Example 3, does not result in unidentified residues or
ures that are unexpected in the proton spectra from conventional heparins or low-
molecular weight n.
In order to confirm that methods according to the invention contribute to retain a desired level
of sulfated polysaccharide , tests was performed with a sulfate measuring electrode on
samples of process liquid from the step of acidic hydrolysis, i.e. samples from process liquid
not subjected to the directly subsequent steps of work-up and purification to a chemically
modified heparin product. The results demonstrate levels of ed (lost) sulfate from
1O polysaccharides generally below 1500 ppm. In other terms the tests confirm that the inventive
methods induce a loss of sulfate groups not exceeding one sulfate group per disaccharide unit
of 100 disaccharide units. chemically modified heparins according to the ion n
one sulfate group per iduronic acid, 128 and 2 e groups for the predominant glucosamine
variant, GlcNS. Accordingly, the chemically modified heparins according to the invention
retain at least 90 % sulfate groups corresponding to heparin.
Chemically modified heparin produced in accordance with processes of Example 3 and
worked up to a product exhibit a very low absorbance at 400 nm (10% solution). ance
values vary between 0.02 AU and 0.04 AU for a product when subjected to the process
including the hydrolysis at pH 3.5 or 3.0 respectively. The low absorbance values confirm that
effects from any non-specific depolymerization associated with discoloration from side
reactions of Maillard type (measured as absorbance at 400 nm) are minimized and that
suitable stability of the chemically modified heparin ts ing to the ion are
expected.
Antihaemostatic and anticoagulation effects
Studies on effects on coagulation parameters and on bleeding of DF02 were performed in
male, adult and juvenile, Sprague-Dawley rats. Heparin and a LMWH preparation (Fragmin)
were also studied for comparison. Basic test procedures were as follows:
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Fifteen minutes after i.v. dosing of test article the rats had a longitudinal on made at the
dorsal mid-section of the tail. The on was 9 mm long and 1 mm deep and was
standardized using a te device. Blood was blotted from the incision until bleeding
stopped. The time during which visible bleeding could be observed was measured, for up to
minutes. The longer the ng time, the more pronounced the anti-coagulant effects of
the administered agent.
Adult rat
Forty minutes after dosing, the rats were sacrificed by full bleeding. Citrate stabilized
plasma was prepared from the blood. Plasma was stored in aliquots of l or 0,5 mL at -
70 °C until analysis of APTT and PT.
The following compounds and doses were tested (each in groups of 8 rats) in adult rats:
0 Saline: (Negative Control)
0 m: 0.7, 1.5, 3.5, and 7.0 mg/kg
0 Mgm_in: 1.5, 3.5, 7.0 and 35 mg/kg
. m: 3.5, 7.0, 35, 70, 105,210, 350 and 700 mg/kg
Juvenile rat
The following compounds and doses were tested (each in groups of 8 rats) in le
rats of age 14 :1 days:
2. Saline: (Negative l)
3. DF02: 7.0, 35, 70 and 105 mg/kg
Bleeding time and coagulation parameters as measured in adult animals revealed that DF02
has a low anti-coagulant effect in rats. The potency of DF02 was less than that of the
anticoagulants Heparin and Fragmin though, both of which had a profound effect on all
parameters, the effect being directly correlated with the dose in question. The effect on PT
was too weak to allow for comparative estimates.
Established bleeding time and coagulation parameters in juvenile animals, indicate that DF02
has a low anti-coagulant effect also in juvenile rats. The change in bleeding time and
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coagulation parameters in the juvenile rats are in the same range as in adult rats. As in the
adult rats also in the juvenile rats the effect on PT was weak.
To further understand the ence in agulant potency of the chemically modified
heparin compounds, an estimation of equipotent relative doses was calculated (Table 4). The
relation between the estimated equipotent relative doses was calculated with respect to effects
on bleeding time and APTT as measured in the rat bleeding model. The normalization or
comparator was set to unfractionated n see Table 4, below.
Table 4
Bleeding time 30-50 1 5
(min)
Table 5 below show the specific anti-coagulant activities of DF02 by anti-factor Xa and anti-
factor IIa assays.
Table 5
Drug substance Batch Results
Property Batch 1 Batch 2 Batch 3
Anti- PhEur. 4.6 5.0 3.8 IU/mg
coagulant (chromoge IU/mg IU/mg
FIIa nic assay)
activity
Anticoagul Ph. Eur. 3.9 4.9 5.5 IU/mg
ant activity IU/mg IU/mg
anti-factor
For ison, the corresponding value for Unfractionated Heparin (UFH) is at least 180
IU/mg.
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Example 5
igation of rosetting and cytoadherence in malaria infected blood
DF02 has been investigated for s in vitro malaria models, e.g. disruption of rosettes of
infected and uninfected erythrocytes, and prevention or disruption of cytoadherence of
infected erythrocytes to the endothelium. In both models DF02 has shown efficacy in a dose-
dependent manner. DF02 demonstrated significant potency in field studies, where rosetting in
fresh parasitized erythrocytes (pE) from patients with mild or complicated malaria were tested
1O in vitro. DF02 has also been tested for blocking effects on merozoite invasion of erythrocytes
in vitro. DF02 demonstrated equal potency per mg to heparin in this model.
Results
A highly rosetting and multi-adhesive parasite clone (FCR3SI.2) as well as te isolates
from ly ill patients have been tested for their sensitivity to DF02 in rosetting and
cytoadherence assays. DF02 disrupts rosettes of many tested parasite cultures in a dose
dependent manner and total or close to total disruption of rosettes was reached at 1000 ug/ml
with some parasites (Fig. 5). The rosettes of clinical es were also sensitive to DF02.
DF02 has r been investigated in the field. Forty-seven parasites from children with
malaria showing the rosetting phenotype were treated with DF02. 91% of the rosetting blood
samples collected from children with severe/complicated malaria showed 50% rosette
disruption at the highest concentration tested (1000 pg /ml) (Fig. 6 ). The effect of DF02 on
the binding of pE to endothelial cells dherence) has similarly been evaluated by
c tion in order to mimic in vivo blood flow conditions. The direct effect on
primary binding to endothelial cells was examined by adding pE together with DF02
simultaneously to the endothelium dherence blocking). Up to 80% of the binding of pE
could be blocked by DF02 as compared with untreated samples. In order to test the efficiency
of the DF02 to ge already bound pE from endothelium, pE were allowed to adhere to
the endothelium, before incubation with the DF02 at different final concentrations
(cytoadherence disruption). herence tion with DF02 resulted in up to 80%
reduction of binding (Fig. 7). Some parasite cultures were more sensitive than others.
Example 6
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Effects of DF02 on Merozoite Invasion of Erfihrocfles In Vitro
The intra-erythrocyte lifecycle of P. falciparum is short and the pE burst every 48 h and
released parasites have to reinvade fresh erythrocytes. Heparin has previously been
trated to inhibit continuous cultivation of P. falciparum in vitro by blocking merozoite
invasion of erythrocytes. DF02 was therefore tested for their blocking effects on merozoite
on of erythrocytes using an in vitro assay (Fig. 8). DF02 blocked merozoite invasion in
a dose dependent manner and the inhibition was more than 80%. The inhibitory effects of the
DF02 were found to be equal to those of standard heparin.
Method
1O The merozoite invasion inhibition assay was performed with chemically d heparin and
tionated heparin Mature pE (trophozoite) synchronized P. falciparum es with a
parasitemia of 0.4% and a hematocrit of 2% were grown in micro-cultures (100 ul) in the
presence of increasing concentrations of chemically modified heparin or unfractionated
n at 37° C for 24—30 h. In order to quantify the parasitemia, the samples were stained
for 10 s with acridine orange and then analyzed using a FACS instrument from Becton
son. A minimum of 50,000 cells per sample were collected.
Results
DF02 and standard heparin, were tested for their blocking effects on merozoite invasion of
erythrocytes using an in vitro assay. DF02 blocked merozoite invasion in a dose-dependent
manner and reached up to 80% inhibition. The inhibitory effects of the DF02 were found to be
equal to those of standard heparin.
Example 7
In vivo release of seguestered infected erythrocytes
The cy of DF02 to release bound infected erythrocytes from lung micro-vessels into the
blood ation has been studied in vivo in the rat. DF02 demonstrated a release into the
ation of pE. In the rat model, an injection of the substance together with the pE blocked
up to 80% of pE from binding in the lung of the rat. Similarly, when the pE were first
injected, and allowed to bind in the microvasculature of the animals for 60 minutes, followed
W0 2013/095276
by an enous injection of DF02 up to 60% of the previously sequestered pE were found
to be released by the treatment (Fig. 9).
Method
Human pE were cultivated in vitro and enriched to a parasitemia above 70%. Prior to
injection into the animals, human infected erythrocytes were ctively labeled with 99mTc.
The rats were anaesthetized and the labeled pE erythrocytes were injected intravenously into
the tail vein. The treated rats were either co-injected with labeled pE together with different
concentrations of the chemically modified heparin, or first injected with pE and, after 3 min,
injected with different concentration of chemically modified heparin, unfractionated heparin,
or dextran e, whereas control animals were injected with labeled pE without DF02,
heparin, or dextran sulfate. The distribution of the labeled cells was monitored using a gamma
camera for 30 min. The relative amount of d cells sequestered in the lungs was
calculated by comparing the activity of excised lungs to that of the whole animal.
Effect of chemically modified heparin s on Sequestration of QB in Rats In Vivo
Studies of pE sequestration, including both rosetting and cytoadherence were performed in the
rat. In this in vivo system pE of different strains and clones robustly adhere in the rat lungs in
a - dependent manner. The system shows a sequestration-blocking effect of the
ally modified heparin DF02 on pE with a maximal 80% (approximately) average
reduction of sequestration. Co-injection of uninfected labeled human ocytes with
chemically d heparin was compared with injection of d uninfected erythrocytes
without chemically d heparin. No difference was seen, and the overall amount
retained was very low. Rats were also treated with chemically modified n s after the
labeled pE had sequestered in order to study the capacity of the chemically d heparin
to release pE into circulation. Sequestration was reduced by approximately 50%.
Example 8
Clinical investigation of sevuparin sodium in malaria patients
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A Phase I/II, Randomized, Open Label, Active Control, Parallel Assignment, Safety/Efficacy
Study of Sevuparin/DF02 as an Adjunctive Therapy in Subjects Affected with Uncomplicated
Falciparum Malaria.
P. falciparum infected erythrocytes (pEs) have the ability to sequestrate in the deep
microvasculature in many of the vital . The sequestration property is involved in the
generation of e severity and pathology, through hampered blood flow, reduced oxygen
delivery and consecutive tissue damage, and is based on the ability of trophozoite pEs to
adhere to the ar endothelium and to uninfected erythrocytes. The combined effect of
endothelial and ocyte adhesion of pEs, is the pivotal ism leading to the
obstruction of the asculature, and thereby the al symptoms of severe malaria.
Sevuparin sodium is administered as an iv. infusion in combination with
atovaquone/proguanil il®) as anti-malarial treatment to female and male subjects
(18 to 65 years of age) affected with uncomplicated malaria. A dose escalation part (part 1) is
followed by an open labelled, randomized comparison of treatment with sevuparin sodium
and Malanil® versus Malanil® alone (part 2). Sevuparin sodium is administered to each
patient 4 times a day and atovaquone/proguanil (Malanil® ) is administered to each patient
according to its ed indication. The study arms are sevuparin sodium in combination with
atovaquone/proguanil (Malanil® ) and atovaquone/proguanil il® ) alone as control.
Method
Parasite clearance curves and sequential peripheral blood parasite staging of DF02 treated
patients are compared with the control group. Cytoadherence and thus sequestration of pEs
containing the more mature forms of the parasite is affected by DF02, a temporary rise in
parasitemia and ance of more mature stages of the parasite in the peripheral blood.
The clearance curves in relation to the eral blood staging are modeled using stage
distribution, proportion of stage specific sequestration and stage specific parasite clearance
through quinine as parameters. A similar approach has been trialed in the evaluation of
sole as anti-adhesive adjuvant therapy in falciparum malaria (Dondorp et al. J Infect
Dis 2007, 196:460—6). Differences in sequestration between DF02 d patients and the
control group are evaluated by comparing the integrated numbers (in parasites per microliter)
and parasitemia (in percentages) of trophozoite- and nt-stage parasites seen in the
W0 95276
peripheral blood over time up to 72 h, determined as the area under the time-parasitemia
curve. The efined morphological stages of the parasite consist of the ing: tiny
rings, small rings, large rings, early trophozoites, midtrophozoites, late trophozoites, and
schizonts ut K, et al. Am J Pathol 1999, 155:395—410]. The parasite asexual-stage ages
(from merozoite invasion) bordering the morphological stages, as assessed by in vitro culture,
are, respectively, 12, 17, 22, 28, 37, and 42 h. A cohort of large-ring forms on admission
evolves to the early trophozoite stage 6 h later. Other matching cohorts include tiny rings on
admission and small and large rings combined after 12 h, small rings on admission and large
rings after 6 h, early trophozoites after 12 h and midtrophozoites after 18 h, and large rings on
admission and either midtrophozoites after 12 h or late trophozoites after 18 h. Assessment of
peripheral blood slides is performed by 2 independent microscopists, who are blinded to the
study drug allocation.
Claims (2)
1. Chemically modified heparin with an antifactor IIa activity of up to 10 IU/mg, an antifactor Xa activity of up to 10 IU/mg and a weight average lar weight from about 6.5 to about 9.5 kDa, wherein the polysaccharide chains: (i) retain at least 90 %, of the sulfate groups of the corresponding native heparin; (ii) have a reduction in ally intact pentasaccharide sequences providing an antithrombin mediated anticoagulant effect, when compared to the polysaccharide chains of native heparin; and (iii) have a reduction in unsulfated ic and/or glucuronic acid units when compared to native heparin; wherein the predominant disaccharide of the polysaccharide is a disaccharide having the chemical structure: wherein n is an integer of from 2 to 25, such that it comprises from 2 to 25 disaccharide units corresponding to molecular weights from 1.2 to 15 kDa; and wherein the chemically modified n has, in a 1H-NMR spectrum, no unidentified signals in the ranges 0.10-2.00 ppm, 2.10-3.10 ppm and 5.70-8.00 ppm larger than 4 per cent when compared to the height of the signal present in native n at 5.42 ppm.
2. Chemically modified n according to claim 1, wherein the predominantly ing polysaccharide chains have from 6 to 16 disaccharide units with molecular weights from about 3.6 to about 9.6 kDa. H:\mm\Interwoven\NRPortbl\DCC\MM\9410213_1.docx-
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SEPCT/SE2011/051538 | 2011-12-19 | ||
PCT/SE2011/051538 WO2013095215A1 (en) | 2011-12-19 | 2011-12-19 | Low anticoagulant heparins |
PCT/SE2012/051428 WO2013095276A1 (en) | 2011-12-19 | 2012-12-19 | Low anticoagulant heparins |
Publications (2)
Publication Number | Publication Date |
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NZ625096A NZ625096A (en) | 2016-07-29 |
NZ625096B2 true NZ625096B2 (en) | 2016-11-01 |
Family
ID=
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