WO2013157967A1

WO2013157967A1 - The use of dextran derivatives in preventing or treating anemia.

Info

Publication number: WO2013157967A1
Application number: PCT/PL2013/000049
Authority: WO
Inventors: Maria Nowakowska; Krzysztof SZCZUBIATKA; Kamil KAMIŃSKI; Włodzimierz BUCZKO; Andrzej MOGIELNICKI; Bartłomiej KATASKA
Original assignee: Uniwersytet Jagielloński; Medical University Of Białystok
Priority date: 2012-04-16
Filing date: 2013-04-11
Publication date: 2013-10-24
Also published as: PL398814A1

Abstract

The present invention relates to the use of modified dextran to prevent or treat anemia in mammals by increasing the number of red blood cells and/or hemoglobin and/or the hematocrit levels in blood. Dextran is modified by substitution of the cationic compound containing amino and/or ammonium groups.

Description

The use of dextran derivatives in preventing

or treating anemia.

The present invention relates to the use of modified dextran derivatives to prevent or treat anemia of diverse origin.

Anemia is a decrease in the concentration of hemoglobin (Hb) , hematocrit (Ht) and the number of erythrocytes in the blood by more than two standard deviations from the norm. Anemia may have essentially two origins: it can either occur from the loss of erythrocytes as a result of bleeding or hemolysis, or from the decrease or disorder of erythropoiesis in the bone marrow (Szczeklik A. [ed] Choroby wewnetrzne, Krakow 2006, p 1441) .

In particular, the main causes of anemia are: a relative erythropoietin deficiency, functional or absolute iron deficiency, visible or hidden blood loss, the inhibition of bone marrow function by uremic toxins (such as PTH, spermine) , shortened life span of erythrocytes or a deficiency of folic acid or vitamin B12 . Anemia should be recognized and treated at an early stage, because it disturbs systemic homeostasis, particularly metabolic and gas homeostasis, carrying, in particular, the risk of left ventricular hypertrophy and deteriorating the quality of life.

The treatment aims at maintaining the Hb level in the range of 11-12 g/dl . Medications used in the treatment of anemia are preparations containing iron and/or erythropoiesis -stimulating agents (ibid., pp. 1268-1269).

For more than 20 years recombinant human erythropoietin has been mainly used as treatment for anemia, and only the last decade has seen the launch of its molecularly engineered analogues: darbepoetin and human recombinant erythropoietin 6.

Table 1 shows erythropoiesis-stimulating agents available on the market today (Macdougall, IC, Am J Kidney Dis., 2012, 59 (3) , p 444) . Table 1 Erythropoiesis-stimulating agents available on the market today.

Drug Ac ive Production Year license

compound process obtained

Erythropoietin 1989 (Epogen, in

alfa/beta Recombinant USA) ; 1990

Recombinant

(Epogen, Eprex, DNA (Eprex/Erypo/NeoR

human EPO

Erypo, technology ecormon, in

NeoRecormon) Europie)

2006 (outside of

Recombinant

Erythropoietin Recombinant USA) ; product

DNA

delta (Dynepo) human EPO technology recalled by Shire

w 2009 r.

"Biocompatible"

erythropoietin

Recombinant

(Binocrit , Recombinant 2009 project

DNA

Hexal, Retacrit, human EPO under development

technology

Silapo,

Eporatio)

Available in many

Not allowed or countries outside only locally Recombinant of USA and

Recombinant

authorized DNA Europa , i.e.

human EPO

"copies" of technology India, China, erythropoietin Thailand,

Argentina, Brazil

Darbepoetin Hyper- Recombinant 2001 (outside of alfa (Aranesp) glycosylated DNA USA and Europe)

recombinant technology

DNA

human EPO

analogue

Pegylated

2009 (available

C.E.R.A. recombinant

( ircera) only outside of

human EPO

USA)

analogue

In the prior art it is known that polysaccharides are used for various medical applications. For example, dextran, which is a glucose polymer of high molecular weight, is used as the liquid blood substitute.

Chitin, a polysaccharide of animal origin, thanks to its biodegradability, non- toxicity, lack of physiological activity, anti-bacterial properties, ability to form gels and affinity to protein is used for the immobilization of enzymes. Another use of chitin is the treatment of wounds. Chitosan, which is obtained from chitin, is also used for this purpose.

Another example of a polysaccharide with a very large number of medical applications is hyaluronic acid. These include, for example, viscosupplementation of joints, smoothing of wrinkles, and moisturizing skin.

This polysaccharide is commonly used in ophthalmology, i.e. in cataract surgery, to accelerate the healing of the cornea and to increase the stability of atropine.

Gels made from alginate, a polysaccharide obtained from seaweed, are used for encapsulation enzymes and cell cultures.

Many other polysaccharides, such as pectins, glucomannans , galactomannans , xanthans also have medical applications. Recently it was found, that the removal of heparin from aqueous solutions can take can be conducted with the use of cross- linked genipin (Kamil Kaminski, Karolina Zazakowny, Krzysztof Szczubiatka, Maria Nowakowska Biomacromolecules 2008, 9 (11), 3127-3132. ) .

This polymer is used in the form of microspheres or in the form of film, and in these forms it can be potentially applied in devices used for extracorporeal removal of heparin.

The prior art also knows the use of dextran for complexing iron, which is the active ingredient in preparations for the treatment of anemia resulting from its deficiency (CN 101215338, RU2338516, CN1353194, US6291440, KR20010058414 ) and the use of dextran in preparations containing erythropoietin as an active component for the treatment of anemia (US6291440, JP 3063299, JP61097229) . However, none of the above patent applications has demonstrated a direct activity of dextran or derivatives thereof to increase the number of red blood cells, hemoglobin or hematocrit levels, and thus to treat anemia.

Unfortunately, the administration of erythropoietin in treating anemia can cause side effects in the form of hypertension in 20-30% of patients, hypercoagulability with vascular access thrombosis in 5-10% of patients, seizures in ~ 3% of patients, which usually accompany hypertensive encephalopathy, or selective red blood cell aplasia caused by the presence of anti -erythropoietin antibodies. In addition, there are a number of objections against the treatment of anemia with erythropoietin (Szczeklik A. [ed] , Choroby wewn^trzne , Krakow 2006, p 1269) . Given the occurrence of side effects and objections against treatment of anemia with erythropoietin, new methods are being continually sought to prevent and treat this disease.

The types of drugs that are being sought are characterized by a smaller number of side effects and the possibility for use in anemia of unknown origin, or in cases where the use of known methods of treatment is impossible or difficult (e.g., pregnant women) .

Successively, the methods of using dextran known from the prior art are limited to perform auxiliary functions in relation to known methods of treatment - by the administration of iron erythropoietin, and do not relate to the application of modified dextran directly for the prevention or treatment of anemia. The developed invention can be safely used for all applications in patients with anemia. The beneficial impact on the morphological parameters of blood would constitute a great advantage over other drugs and the reason for preference, as opposed to using several drugs, as it is currently practiced.

The aim of the invention is to provide a method for the prevention or treatment of anemia in mammals by increasing the number of red blood cells as well as hemoglobin and hematocrit levels in the blood.

The essence of the invention is the use of dextran derivatives for the prevention or treatment of anemia by increasing the number of red blood cells as well as hemoglobin and hematocrit levels in the blood of mammals .

Preferably, polysaccharides modified with compounds containing ammonium cationic group or modified through grafting polymer containing amino and/or ammonium groups are used, but these two types of modification can be used together or separately.

Preferably, ammonium halogens, and especially ammonium chlorides are used as compounds containing cat ionic ammonium groups and monomers used for obtaining grafter polymers .

Preferably, glycidyl trimethylammonium chloride (GTMAC) or chloride (3 -acrylamidopropyl) ammonium is used as ammonium chloride .

Preferably, dextran modified with glycidyl trimethylammonium chloride is used.

Preferably, a modified polysaccharide is used in the form of an intravenous solution.

Dextran is preferably used as polysaccharide, but it is also possible to use other polysaccharides. Then, for modification, any ammonium compound can be used as longs as it contains a functional group capable of reacting with the functional groups of the given polysaccharide. For example, in the case of GTMAC the glycidyl group can react with the OH or NH₂ groups of dextran, causing their substitution with GTMAC. In the general case, the compounds, which can be used in cationic polysaccharide modification may prove to be of very diverse nature, thus the modifying reactions of polysaccharide can be carried out in very different experimental conditions generally known in literature. Thanks to the cationic modification, dextran acquired the ability to increase the number of red blood cells.

It is possible to enter low molecular weight cationic groups into polysaccharide, or, alternatively, graft polysaccharide with polymer containing cationic moieties, thanks to which entire polymer side chains containing multiple cationic groups are entered.

It is highly preferable to combine those two modification methods because the ammonium compounds used for low molecular weight modification react easily with amino groups present in the polymer used for grafting polysaccharide.

For example, grafting of polyvinylamine onto hydroxypropyl cellulose allowed for easy cationic modification with the use of glycidyl trimethylammonium chloride (GTMAC) of the obtained polymer, because GTMAC reacts easily with amino groups present in polyvinylamine .

In turn, for low molecular weight modification any ammonium compound can be used as long as it contains a functional group capable of reacting with the functional groups of the given polysaccharide. For example, in the case of GTMAC the glycidyl group can react with NH₂ groups of chitosan, or OH groups of dextran causing their substitution with GTMAC. In the general case, the compounds, which can be used in cationic polysaccharide modification may prove to be of very diverse nature, thus the modifying reactions of polysaccharide can be carried out in very different experimental conditions generally known in chemical literature. Successively, for grafting the polysaccharide any ammonium compound can be used as long as it is capable of polymerization, e.g., radical polymerization, controlled radical polymerization, ring-opening polymerization, polycondensation or polyaddition . This could be, e.g., ammonium compound containing a vinyl group or a hydroxy1 acid containing an ammonium group. In the case of radical polymerization grafting consists of formation of radicals along the polysaccharide chain (e.g., by adding a BPO or KMn0₄) , which initiate growth of side chains when an appropriate vinyl monomer is present.

The invention is further described in the following example.

Preparation of modified dextran

2 g of dextran with the molecular weight of 40 000 Da, was dissolved in 100 ml of distilled water, then 400 mg of NaOH and 12 ml GTMAC was added.

The reaction mixture was heated to 60°C. Reactions were carried out for 4 hours. The solution containing the product was dialyzed against distilled water until the conductivity dropped to the value of 2 . The resulting polymer was isolated by freeze-drying under reduced pressure.

The resulting polymer was determined as Dex-GTMAC.

For the obtained polymer a degree of modification was determined based on elemental analysis. The results the determination of elemental composition for the both polymers obtained are shown in Table 2.

Table 2 - Elemental composition of the modified dextran

The degree of substitution of dextran by GTMAC, as calculated on the basis of the obtained results of elemental analysis (defined as the number of GT AC particles attached to the glucose unit) is 0.48.

Pharmacological studies

According to the present invention, the effect that modified dextran has on the increase of the number of red blood cells and the increase of hemoglobin was observed during the experiments whose objective was to examine the impact of the reversal of heparin activity in experimental models of arterial thrombosis induced by electrical stimulation. The experiment was carried out in accordance with the procedure shown in Figure 1, where in 3 minutes after administration of heparin, the rats were given an agent reversing its effects, and then, in the 10^th minute of the experiment the induction of arterial thrombosis was begun, which was triggered for 3-10 minutes, and then after 45 minutes the clot was removed, weighed after 24 hours and blood was collected for analysis.

Heparin was intravenously administered to rats 10 minutes before inducing arterial thrombosis in two doses of 150 U/kg (n=7) and 300 U/kg (n=5) either alone or before the intravenous administration of the modified dextran - respectively - at a dose of 6.25 mg/kg (n=6) and 12.5 5 mg/kg (n=5) , and Protamine - respectively - in the dose of 1.5 mg/kg (n=7) and 3 mg/kg (n=5) . The control group was given 0.9% NaCl (VEH) (n=10) . Modified dextran was also administered alone at a dose of 6.25 mg/kg (n=6) and 12.5 mg/kg (n=5) at a concentration of 5% or 10% in the volume of less than 0.5 ml.

The results of the analysis of morphological parameters of blood are shown in Table 3 , where in the case of heparin administered to rats at a dose of 300 U/kg with modified dextran in a dose of 12.5 mg / kg or the modified dextran in a dose of 12.5 mg/kg an increase was observed in the number of red blood cells (RBC) from the value of 7,09±0,2 10⁶/mm³, to the respective values of 8,24+0,15 10⁶/mm³ and 7,95±0,12 10⁶/mm, an increase in hemoglobin (HGB) was observed from the value of 13.7±0,3 g/dl to the respective values of 15,6+0,3 g/dl and 15,0±0,1 g/dl, as well as an increase in hematocrit (HCT) with from the value of 41.1+0,9% to the respective values of 46,1±0,8% and 44,9±0,4%. However, when protamine with heparin were administered to rats, or they were administered independently there was a decrease in the number of red blood cells to the respective levels of 6,75±0,1 10⁶/mm³ and 6,42+0,1 10⁶/mm³ and, respectively, a decrease in hemoglobin (HGB) was observed to the values of 13,5±0,3 g/dl and 12,9±0,4 g/dl.

Table 3 - Morphological parameters measured in rats with develo ing arterial thrombosis.

The impact of modified dextran, according to the present invention, on the increased number of red blood cells and the increase of hemoglobin was also observed during the experiments on healthy rats without developing arterial thrombosis.

The results are illustrated in Table 4, where it is shown that when heparin is administered at a dose of 300 U/kg, along with the modified dextran in a dose of 12.5 mg/kg or the modified dextran alone in a dose of 12.5 mg/kg there is an increase in the number of red blood cells (RBC) from the value of 6.12+0,11 10^e/mm³ to the respective values of 7,18±0,23 10⁶/mm³ and 6,96±0,26 10⁶/mm, there was an increase in hemoglobin (HGB) from the value of 12,8±0,1 g/dl to the respective values of 14,9±0,5 g/dl and 14,8±0,6 g/dl, as well as an increase in hematocrit (HCT) from the value of 36,5±0,5% to the respective values of 41,8±1,4% and 41,9±2,1%.

Table 4 - Morphological parameters measured in rats without developing arterial thrombosis.

As described above, with the administration of modified dextran, according to the invention, the number of red blood cells, hemoglobin and hematocrit increased relatively to the control sample.

Claims

Patent Claims

1. The use of dextran derivatives for prevention or treatment of anemia in mammals by increasing the number of red blood cells and/or hemoglobin and/or hematocrit levels in the blood.

2. The use according to claim 1, characterized in that modified dextran is applied by means of compounds containing cationic ammonium group and/or modified by grafting with a polymer containing amino and/or ammonium groups .

3. The use according to claim 2, characterized in that ammonium halogens are used as the compounds containing cationic ammonium groups and as monomers used for the preparation of grafted polymers .

4. The use according to claim 3, characterized in that ammonium chlorides are used as the ammonium halogens .

5. The use according to claim 4, characterized in that glycidyl trimethylammonium chloride or (3-acrylamidopropyl) ammonium chloride is used as ammonium chloride.

6. The use according to claim 1, characterized in that the dextran used is modified with glycidyl trimethylammonium chloride .

7. The use according to claim 1, characterized in that the modified dextran is used in the form of an intravenous solution .