WO2003053449A1

WO2003053449A1 - Use of 7-monohydroxyethylrutoside for the treatment of haematological disorders, particurlarly beta-thalassemia

Info

Publication number: WO2003053449A1
Application number: PCT/EP2002/013872
Authority: WO
Inventors: Willem Jan Frederik Van Der Vijgh; Aalt Bast; Carlo Brugnara; Lucia De Franceschi
Original assignee: Vereniging Voor Christelijk Wetenschappelijk Onderwijs
Priority date: 2001-12-10
Filing date: 2002-12-04
Publication date: 2003-07-03
Also published as: AU2002358102A1

Abstract

Described are novel uses of 7-monohydroxyethylrutoside for the manufacture of a medicament for treatment of haematological disorders, associated with oxidative damage of the red blood cell membrane and/or with iron storage, in particular β thalassemia. Further, 7-monohydroxyethylrutoside can be used for decreasing the bilirubin level and/or the LDH level, the plasma iron level in the peripheral blood, for decreasing the degradation of red blood cells and/or for increasing the erythrocyte K content in the peripheral blood of a subject.

Description

E OF 7-MONOHYDROXYETHYLRUTOSIDE FOR THE TREATMENT OF HAEMATOLOGICAL DISORDERS, PARTICULARLY BETA-THALASSEMIA

SUMMARY OF THE INVENTION

The invention relates to novel uses of 7- monohydroxyethylrutoside (monoHER) for the manufacture of a medicament for treatment of haematological disorders associated with oxidative damage of the red blood cell membrane and/or with iron storage, in particular for treatment of β thalassemia.

The compound monoHER, the anti-oxidant function and iron chelating properties thereof is described in S.A.B.E. van Acker et al., Biochem. Pharmacol. 56, 935-943 (1998), herein incorporated by reference.

The formula of monoHER is as follows

wherein Rl is O-rutinose, R2 is -OCH₂CH₂OH, R3 and R7 are both H and R4, R5 and R6 are OH.

Further, the invention relates to the use of monoHER for the manufacture of a medicament for the above identified treatment, in particular for treatment of β thalssemia, the treatment being for decreasing the bilirubin level, the LDH level and/or the plasma iron level in the peripheral blood, for increasing the erythrocyte K content in the peripheral' blood, and/or for decreasing the degradation of red blood cells, of a subject in need thereof.

A "subject" can be any vertebrate; preferably the subject is a mammal, most preferably human. However, the medicament according to the present invention can also be used for veterinary purposes. Oxidation plays a crucial role in the membrane damage and reduced life span of β thalassemic erythrocytes. We have characterised erythrocyte membrane transport function and oxidative damage in a mouse model that closely resembles human β thalassemia (β thai) intermedia. As in the human counterpart, dehydrated erythrocytes, reduced cell K content and upregulated K-Cl cotransport were present in β thai mouse erythrocytes. The effects on in vivo administration of a novel flavonoid antioxidant, 7- monohydroxyethylrutoside (monoHER) were studied. MonoHER was administered to wild-type mice (n=6) at the dosage of 500 mg/Kg i.p. once a day and to β thai mice at the dosage of either 500 mg/Kg i.p. once a day (n=6) or 250 mg/Kg i.p. twice a day (n=6) . MonoHER treatment of β thai mice significantly decreased total bilirubin, LDH and plasma iron and increased erythrocyte K content and cell hydration. Phosphatidylserine exposure on the outer side of the membrane, measured by Annexin V binding, was significantly elevated in β thai erythrocytes compared to wild-type (11.7 ± 5.2 %, n=8 vs 0.49 ± 0.19%, n=S , p<0.05) and was markedly decreased by MonoHER treatment (500mg: 1.13 ± 0.5%, n=6; 250mg: 0.3 ± 0.03 %, n=6) . β thai mice showed reduced plasma levels and membrane content of vitamin E compared to wild-type, consistent with oxidative membrane damage. These values returned to values close to normal after MonoHER therapy. These data indicate that monoHER is an effective in vivo anti-oxidant agent and that monoHER may be used in the manufacture of a medicament for treatment of haematological disorders as identified above, such as oxidative damage of the red cell membrane.

Ineffective erythropoiesis and reduced survival of circulating erythrocytes are responsible for the anemia of β thalassemia (β thai) . [Orkin, 1998 #44] The membrane damage induced by the excess of free alpha chains plays a crucial role in the shortening of erythrocyte life span, but the relative contribution of the multiple membrane alterations to the pathogenesis of this disease remains still undetermined. [Shinar, 1990 #871; Rachmilewitz, 1976 #46;Rouyer- Fessard, 1990 #1304;Advani, 1992 #49;Schrier, 1992 #1900]

Red cells from β-thalassemia intermedia patients show aggregated clusters of haemichrome and band 3, presumably as a result of oxidative injury. [Kuypers, 1998 #1693;Yuan, 1992 #868; Mannu, 1995 #862] Immunoglobulin and complement components localise at the membrane surface over these clusters, mediating the removal of the damaged β thalassemic erythrocytes by macrophages . [Kuypers, 1998 #1693;Yuan, 1992 #868] Membrane lipid peroxidation, loss of phospholipid asymmetry, and externalisation phosphatidylserine (PS) have been demonstrated in β thalassemic red cells . [Kuypers, 1998 #1693;Yuan, 1992 #868;Mannu, 1995 #862;Zwaal, 1997 #653] PS externalisation in thai erythrocytes is believed to play a significant role in their premature removal and reduced survival, as it has been demonstrated for dense sickle erythrocytes . [de Jong, 2001 #1684;de Jong, 2001 #1683] PS externalisation may promote macrophage recognition with removal of erythrocytes, cell apoptosis and activation of coagulation. [Frank, 1985 #657; Schroit, 1991 #1923]

In human β thalassemia, the portion of PS positive erythrocytes varies from patient to patient and appears to be either distributed over the entire cell membrane or localised in areas with abundant deposits of α-globin chains . [Kuypers, 1998 #1693] Different factors may modulate the extent of membrane oxidative damage and loss of phospholipid asymmetry, such as the co-inheritance of other mutations affecting the production of β or α chains or the presence/absence of the spleen. [Kuypers, 1998 #1693]

It has been shown that oxidative damage plays a significant role in the upregulation of K-Cl cotransport and associated cell dehydration of β thai erythrocytes . [Olivieri, 1993 #67;01ivieri, 1994 #269; De Franceschi, 1996 #192] In vitro treatment of β thai erythrocytes with dithiothreitol (DTT) reduced K-Cl cotransport activity to almost normal values, indicating that the functional changes induced by oxidation are irreversible. [Olivieri, 1994 #269] Studies have been published on the use of antioxidants such as vitamin E, flavonoid(s) and polyphenols in β thalassemia,

[Rachmilewitz, 1979 #1297;Grinberg, 1994 #1732;Grinberg, 1997 #1924;van Acker, 2000 #1932] but limited data are available for in vivo use in either animal models or patients. It has been shown that iron chelation reduces oxdative damage in a mouse model of β thalassemia and in patients . [Browne, 1997 #233;De Franceschi, 1999 #676] .

It has recently been shown that monohydroxyethylrutoside confers protection against chronic doxorubicin cardiotoxicity, which is caused by the formation of oxygen free radicals. In addition, MonoHER exhibited minimal toxicity and did not affect the anti- proliferative activity of doxorubicin [van Acker, 1997 #1734] [van Acker, 2000 #1931] . The invention will now be further illustrated by the following non-limiting examples as figures.

FIGURES

Fig. 1: Gating strategy for analysis of the Annexin-V positive mouse red cells. Erythrocyte population was identified based on forward and side scatter (gate Rl) and staining for TER-119 (gate R2) (31) . Expression of Annexin-V was examined on cells double gated for Rl and R2. Fig. 2: Effect of monoHER treatment on phosphatidylserine (PS) exposing red cells in wild-type and in β thalassemic mouse strains. The average of PS positive erythrocytes were at baseline: wild-type mice: 0.49+0.2 % (n=8 ) , β thalassemic mice: 11.7+0.4 % (n=8); after 21 days of treatment: wild-type plus monoHER 500 mg/Kg once a day: 0.63+0.19 (n=8) (MH500) , β thalassemic mice plus 250 mg/Kg twice a day: 1.13+0.5% (n=8) (MH250) and β thalassemic mice plus 500 mg/Kg once a day: 0.26+0.3% Fig.3: Functional characterisation of K-Cl cotransport in β thai mouse erythrocytes. K-Cl cotransport was measured as okadaic acid-sensitive (100 nM) efflux in isotonic medium, as chloride- dependent K efflux in hypotonic conditions, and as K efflux in isotonic conditions stimulated by the presence of 2.5 μM staurosporine. The sensitivity of chloride-dependent K efflux to DTT treatment (10 itiM preincubation, 1 mM in flux media) was assessed in hypotonic conditions. Fig. 4: Phthalate density profiles in β thai mice treated with

MonoHER at either 250 mg/Kg twice a day (4A) or 500 mg/Kg once a day (4B) . Data are from one single mouse, representative of the six animals studied in each group. Fig. 5: Changes in PS exposure following treatment with monoHer in β thai mice. PS exposing red cells were double-labelled with Annexin-V FITC and TER-119 antibody. Shown are the results with one typical animal, representative of the six studied in each group. Panel A: Annexin-V "positive control" normal red cells; Panel B: wild-type erythrocytes; Panel C, LEFT: β thai untreated; MIDDLE: β thai, treatment with monoHER, 250 mg/Kg twice a day; RIGHT: β thai, treatment with monoHER 500 mg/Kg twice a day. Fig.6: Effects of monoHER treatment on plasma and membrane vitamin E levels in wild-type and β thalassemic mouse strains. A: Plasma vitamin E levels at baseline in wild-type: 3931 μg/L (n=6) and after 21 days of treatment with monoHER 500 mg/Kg once a day: 3879 μg/L (n=6) . Plasma vitamin E levels at baseline in β thalassemic mice: 1557 μg/L (n=6) and after 21 days of treatment with monoHER 500 mg/Kg once a day: 3923 μg/L (n=6) and with monoHER 250 mg/Kg twice a day: 4442 μg/L (n=6) . Data are presented as mean of n experiments; p<0.05, β thai untreated mice vs. β thai treated mice. B: Membrane vitamin E levels at baseline in wild-type: 22,607 μg/L, (n=6) and after

21 days of treatment with monoHER 500 mg/Kg once a day: 21,154 μg/L (n=6) . Membrane vitamin E levels at baseline in β thai mice: 14,254 μg/L (n=6) and after 21 days of treatment with monoHER 500 mg/Kg once a day: 18,017 μg/L (n=6) and with monoHER 250 mg/Kg twice a day: 19,777 μg/L (n=6) . Data are presented as mean of n experiments; p<0.05, β thai untreated mice vs. β thai treated mice.

EXAMPLES

In the following examples the ion transport and cell volume characteristics are characterised in a mouse model with deletion of both bl and b2 mouse β globin genes; this mouse model exhibits clinical and biological features similar to those observed in the human β thalassemia intermedia [Yang, 1995 #1836] . Also the effects of in vivo administration of MonoHER to control and β thai mice, examining several membrane parameters which are indicative of oxidative damage, are examined.

MATERIALS AND METHODS

Drugs and Chemicals:

NaCl, KC1, okadaic acid (OA) , staurosporine, N-ethylmaleimide (NEM^, dithiothreitol (DTT) , ouabain, bumetanide and nystatin were purchased from Sigma Chemical Co (St. Louis, Mo). MgCl₂, Mg(N0₃)₂, dimethylsulphoxide (DMSO) , and n-butyl phthalate were purchased from Fisher Scientific Co. Sulphamic acid (SFa) , A21387 ionophore, Tris (hydroxymethyl) aminomethane (Tris) and 3 (N-morpholino) propanesulphonic acid (MOPS) were purchased from Sigma Chemical Co. Choline chloride was purchased from Calbiochem-Boehring (San Diego, CA) . Bovine serum albumin fraction V was purchased from Boehringer Mannheim (Mannheim, Germany) . Monohydroxyethylrutoside (MonoHER, MH was kindly provided by Prof. Bast, Maastricht, The Netherlands. Annexin-V antibody was kindly provided by Frans A. Kuypers, Children's Hospital Oakland Research Institute, (Oakland, CA) . Phycoerythrin-conjugated anti-Terll9 was purchased from Pharmingen (San Diego, CA) . All solutions were prepared using double-distilled water.

Animals: Mice heterozygous for deletion of both bl and b2 were obtained from Jackson Laboratories (Bar Harbor, ME, USA) . [Yang, 1995 #1836] Animals between 4 and 6 months of age, the females weighing 25 to 28 grams and the males weighing 28 to 30 grams were selected for the study. C57B6/2J mice were used as controls. Haematological parameters and red cell cation content:

Blood was collected from ether-anaesthetised mice by retro- orbital venipuncture into heparinised microhaematocrit tubes. Haemoglobin (Hb) concentration was determined by the spectroscopic measurement of the cyanmet derivative. Haematocrit (Hct) was determined by centrifugation in a micro-Hct centrifuge. Distribution of cell volume, haemoglobin concentration and reticulocytes were performed on an ADVIA 120 haematology analyser (Bayer Diagnostics, Tarrytown, NY, USA) . Density distribution curves and median erythrocyte density (D₅₀) were obtained using phthalate esters in microhaematocrit tubes, after washing the cells three times with PBS (330 mOsm) at 25°C. [Danon, 1964 #190] Osmotic fragility was determined by addition of 40 μl of whole blood to 1 ml of media with different NaCl concentrations, followed by incubation for 20 minutes at room temperature, and centrifugation at 1,200 x g at 4°C. The absorbance of the supernatant was measured at 546 nm, and the percentage haemolysis was calculated in relation to the absorbance of cells lysed in distilled water. The T₅₀%_L is the tonicity (in mOsm) yielding 50% cell lysis.

Characterization of ion content and transport:

Erythrocyte Na and K contents were determined after 5 washes in 172 mM choline chloride, 1 M MgCl₂, and 10 mM Tris (hydroxymethyl) aminomethane-3 (N-morpholino) propanesulphonic acid (Tris-MOPS) pH 7.4 at 4°C (choline washing solution) . Rates of Na-K pump and Na-K-2C1 cotransport activity were measured in cells containing equal amounts of Na and K by the nystatin technique, loading the cells with "nystatin solution" (77 mM NaCl, 77 mM KC1, and 55 mM sucrose) . The Na-K pump activity was estimated as the ouabain-sensitive (1 mM) fraction of Na efflux into a medium containing 165 mM choline chloride and 10 mM KC1. Na-K-2C1 cotransport activity was estimated as bumetanide-sensitive (10 μM) . Na efflux into medium containing 174 mM choline chloride and 1 mM ouabain. K-Cl cotransport activity was measured in fresh erythrocytes as chloride- and volume-dependent K efflux. Net K efflux from fresh cells was measured in hypotonic (260 mOsm) Na solution (340 mOsm is isotonic for mouse erythrocytes) . Chloride dependent- and volume-dependent K effluxes were calculated as the difference between K efflux in chloride or sulphamate hypotonic media and in isotonic or hypotonic chloride media, respectively. All media contained (in mM) : 1 MgCl₂, 10 glucose, 1 ouabain, 0.01 bumetanide, and 10 Tris-MOPS (pH 7.40 at 37° C) . Efflux was calculated from the K concentrations in the supernatant at 5 and 25 min.

Experimental design: Monohydroxyethylrutosisde (MonoHER) was dissolved in sterile water containing 36 mM NaOH to a final concentration 33 mg/ml, pH 7.8-8, and administered intra-peritoneally (i.p.). [van Acker, 1997 #1734;van Acker, 2000 #1931] The β thalassemic mice were divided into 2 groups of six mice each and treated with two different MonoHER dosing protocols: either 250 mg/Kg twice a day, i.p. or 500 mg/Kg once a day, i.p.. Control mice (C57B6/2J) were treated with MonoHER 500 mg/Kg a day, i.p. (n =6) . Total bilirubin, serum LDH and serum iron, haematological parameters, red cell cation content, red cell density profile, phosphatidylserine (PS) exposing mouse red cells, plasma and membrane vitamin E levels were determined at baseline and after 3 weeks of treatment with MonoHER. Measurement of phosphatyldyl serine (PS) exposure in mouse red cells with Annexin-V Labeling:

Erythroid PS exposure was assessed as previously described with minor modifications. [Kuypers, 1998 #1693] To generate positive control erythrocytes that expose PS on their outer surface, wild-type mouse red cells were incubated with NEM, which inhibits the aminophospholipid translocase by reacting with a sulphhydryl group necessary for its activity. Red cells from normal wild-type mice (Hct: 30%) were incubated in buffer (10 mmM Hepes, 170 mM NaCl, pH7.4, HBS buffer) containing NEM 10 mmol/L for 30 min at 37°C and subsequently washed in buffer without NEM. Cells exposing PS can also be generated by treatment A23187 in the presence of Ca. Control mouse erythrocytes were equilibrated at 16% haematocrit in HBS buffer with 1 mM CaCl₂ for 3 min at 37 °C: A23187 was then added to the red cell suspension to a final concentration of 4 μmol/L and the suspension was incubated for 15 min at 37°C. Cells were washed with HSM buffer containing 1 % bovine serum albumin (BSA) , 5 mM EDTA, and resuspended in HSM buffer. [Kuypers, 1998 #1693]

PS positive control red cells and freshly drawn whole blood from control and β thalassemic mouse groups (1.5 μml, containing < 1 x 10⁷ cells) were incubated for 30 min in ice in the dark with 1 μL of Phycoerythrin-conjugated anti -Terll9. Terll9 is a cell-surface erythroid-specific antigen expressed in terminally differentiating erythroblasts and associated closely with glycophorin A. [Kina, 2000 #1940;Auffray, 2001 #1941] Cells were then incubated in HBS-binding buffer (lOmM Hepes, 170 mM NaCl, 2.5 mM CaCl₂, pH 7.4) and 2 μl of (FITC) -labelled annexin V (Alexa 488) for 30 min in the dark at room temperature. [de Jong, 2001 #1684] Two-colour immunofluorescence analysis was used to quantify the fraction of erythrocytes binding Annexin-V. The erythrocyte population was defined based on the Forward, vs. Side Scatter characteristic (Gate Rl) . Events that correlated with intact red blood cells were further analysed for the expression of TER-119 antigen (Gate R2) . Only events which fell into gate Rl and were positive for the TER-119 (Gate R2) were analysed for the Annexin-V binding. 10,000 events were acquired for each sample analysed. The threshold line was based on the maximum fluorescence of the cells incubated without Annexin-V or in some cases, based on the maximum fluorescence of the cells negative for the phosphatidylserine (PS) incubated with Annexin-V. The threshold line for defining Annexin-V positive and negative cells was set such that less than 0.5% of negative control cells was present to the right of the threshold line. Stained cells that were brighter than negative control (present to the right of the negative control histogram) were defined as positive for Annexin-V.

The data were acquired using MoFlo flow cytometer (Cytomation, Fort Collins, CO) and analysed using Summit (Cytomation, Fort Collins, CO) software. The cytometer was aligned using AlignFlow Beads (Molecular Probes, Eugene, OR) .

Measurements of plasma and red cells membrane vitamin E levels in wild-type and β thalassemic mice:

Vitamin E was quantified in plasma and red cell membranes with HPLC following ethyl ether extraction.

Histology, electron microscopy and tissue iron Content studies:

Mouse tissues (spleen and liver) were fixed in phosphate- buffered formaldehyde (3.7%) before dehydration and paraffin embedding using standard technology. Sections were cut at 4 μm and stained with haematoxylin and eosin or Perl's iron stain.

Iron content was determined on fresh frozen liver samples as described by Levy et al.[Levy, 1999 #1956]

For electron microscopy studies of erythrocytes, washed red cells were suspended in 2.5% glutaraldehyde (in cacodylate buffer, pH 7.4), treated with 1% osmium tetraoxide solution, and embedded in Araldite (Ciba-Geigy, Switzerland) . Statistical analysis: All values are presented as mean ± standard deviation (SD) .

Data on vitamin E are presented as median (ι.=6) . For each group of mice, after 21 days of treatment comparison of more than two groups was performed by one-way analysis of variance (ANOVA) with Tukey' s test for post hoc comparison of the means.

Example 1: Haematological features and biochemistry of wild-type and β thai mice:

Table 1 presents the baseline haematological profile and erythrocyte cation content in wild-type and in β thai mice, β thai mice showed a significant reduction in haematocrit, haemoglobin, mean corpuscular volume (MCV) and an increase in red blood cell distribution width (RDW) , haemoglobin concentration distribution width (HDW) , and total reticulocyte count. As in human β thalassemias, erythrocyte K content was significantly decreased in β thai mice compared to wild-type, while the red cell Na content was similar, β thai mice showed increased serum total bilirubin and LDH, indicating the presence of haemolysis and/or ineffective erythropoiesis . (Table 2) Decreased plasma and/or membrane levels of vitamin E have been observed in disorders characterised by oxidative damage. Compared with control mice, β thai mice exhibit a marked reduction in vitamin E levels both in plasma (1.56 mg/L vs 3.93 mg/L) and in the erythrocyte membrane (14.25 mg/L vs 22.61 mg/L), suggesting significant oxidative damage.

Example 2: Phosphatidylserine (PS) exposure in normal and β thai mouse erythrocytes:

Fig. 1 shows the gating strategy for analysis of the annexin-V labelling of mouse erythrocytes. The erythrocyte population was identified based both on forward and side scatter (gate Rl) and staining for Terll9 antigen (gate R2) . Expression of Annexin-V was examined on cells double gated for Rl and R2. As shown in Fig. 2, the percentage of red cells exposing PS (Annexin-V positive) was significantly higher in β thalassemic mice than in wild-type mice (0.49+0.2% vs. 11.7+5.2%; n=8).

Example 3: Characterisation of K-Cl cotransport in β thai mouse red cells:

K-Cl cotransport activation and cell dehydration are characteristic features of human β thai erythrocytes. [Olivieri, 1994 #269] K-Cl cotransport activation can be reproduced in vitro in normal human erythrocytes with agents that mimic the oxidative damage seen in α and β thalassemias . [Olivieri, 1993 #67;01ivieri, 1994 #269] As shown in Fig. 3, β thai mouse erythrocytes exhibit a marked upregulation of K-Cl cotransport. K efflux mediated by this transporter is greatly increased in both isotonic and hypotonic conditions and is inhibited by the protein phosphatase inhibitor okadaic acid and stimulated by the protein kinase inhibitor staurosporine. Interestingly, only limited inhibition of K-Cl cotransport was observed when β thai erythrocytes were treated in vitro with 10 mM DTT, suggesting that a significant portion of the upregulation of K-Cl cotransport in β thai mouse erythrocytes is not due to reversible sulphhydryl oxidation (Fig. 3) .

Example 4: Effects of in vivo MonoHER treatment on wild-type and β thai mice:

MonoHER was administered daily for 21 days: wild-type mice were treated with 500 mg/Kg i.p. once a day, while β-thal mice were divided into two groups, one treated with 250 mg/Kg i.p., twice a day and the other one with 500 mg/Kg i.p., once a day. Table 1 presents the effects of MonoHER treatment on the haematological parameters of wild-type and β-thal mice. No statistically significant changes were observed in either Hct or Hb levels with MonoHER treatment. The reticulocyte count was significantly reduced in the β-thal mice treated with MonoHER at 250 mg/Kg twice a day, while it showed a non-statistically significant trend for reduction in the β-thal mice treated with 500 mg/Kg once a day. HDW and RDW showed a significant decrease with MonoHER treatment, most likely as a consequence of the reduced reticulocyte count and decreases dehydration (see below). Erythrocyte Na content was unchanged while erythrocyte K content was significantly increased after 21 days of treatment with MonoHER (Table 1) . The improved hydration state of the β thai erythrocytes was associated with a measurable and significant shift in the density distribution of the erythrocytes. As shown in Fig. 4, MonoHER treatment at 500 mg/Kg once a day determined a leftward shift in the density curves, which interested the fraction of red cell with a density ranged between 1.096 and 1.112. In the β-thal mice treated with monoHER at the dosage of 250 mg/Kg twice a day, the changes in red cell density profiles were similar but less pronounced (Fig.4).

MonoHER therapy resulted in a statistically significant decrease in total bilirubin and in plasma LDH in both groups of β thai mice, suggesting an improvement of the haemolysis and/or reduction in ineffective erythopoiesis (Table 2) .

No significant changes in either haematological or biochemical parameters were noted in the wild-type mice treated with MonoHER at 500 mg/Kg once a day (Table 1 and 2) . Example 5: Effects of MonoHER treatment on membrane and plasma indicators of oxidative damage:

As shown in Fig.2, the percentage of PS-exposing red cells in β thai mice was markedly higher than that observed in wild-type controls, indicating a significant loss of phospholipid asymmetry, similarly to what observed in human β thalassemia and sickle cell disease. [Kuypers, 1998 #1693] As shown in Fig. 5, the pattern of PS positivity in this β thai mouse model exhibited a shift of the entire population of red cells toward increased Annexin V binding, rather than the appearance of a distinct fraction of PS positive cells on a background of PS negative cells. This was not due to autofluorescence, since β thai mouse erythrocytes without Annexin V staining showed minimal fluorescence, that could account for a maximum of 0.2% of their Annexin V positivity (data not shown).

In β thalassemic mice, MonoHER treatment determined a marked reduction in the percentage of PS-exposing erythrocytes, to values that were essentially the same as those on wild-type control mice (Fig. 2 and 5) . Control experiments showed that addition of MonoHER to Annexin V "positive control" cells did not affect Annexin V binding, ruling out a direct effect of this compound on the binding of Annexin V to β thai erythrocytes (data not shown) . No changes in the percentage of PS exposing red cells were detectable in wild-type treated group (Fig. 2) .

Plasma and the red cell membrane vitamin E levels were markedly reduced at baseline in β thai mice, an indication of severe membrane oxidative damage. MonoHER treatment resulted in a marked increase in plasma (Fig. 6A) and erythrocyte membrane (Fig. 5B) vitamin E levels to values which were close to those observed in wild-type mice. No changes in plasma and in membrane vitamin E levels were detectable in wild-type mice treated with MonoHER (Fig. 5A-B) .

Example 6: Effects of MonoHER treatment on liver and spleen histopathology and erythrocyte morphology: Histopathological examination of liver sections of control and treated mice revealed no discernible difference in the extent of hepatic extramedullary haematopoiesis or significant difference in either the degree or the distribution of iron staining (Kupfer cell vs. hepatocyte) . There was no light microscopic evidence or toxic hepatocellular injury in the treated groups. Treated and untreated animals also had similar histological features in the spleen, including comparable amounts of micrographs or peripheral blood demonstrated no difference in red cell membrane ultrastructure or the frequency or quality of intracellular globin precipitates. The frequency of RBC's containing residual organelles and/or abundant ribosomes appeared decreased in the treated β thalassemic groups, compatible with a decrease in reticulocyte count.

MonoHER treatment did not produce significant changes in tissue iron content: iron contents of liver and spleen (meant ± SD) were 249 ± 109 μg/g and 1,644 ± 354 μg/g, respectively in untreated β thai mice. In β thai mice treated with 500 mg monoHER/kg/day, liver and spleen iron contents were 231 + 104 μg/g and 1,743 ± 392 μg/g, respectively. In β thai mice treated with 250 mg monoHER/kg/BID, liver and spleen iron contents were 199 + 67 μg/g and 1,659 ± 394 μg/g^ respectively.

Summarising, the red cell features of a mouse model for β thai which closely resembles human β thai intermedia are characterised. [Yang, 1995 #1836] β thai mice showed presence of dehydrated erythrocytes, decreased erythrocyte K content and increased activity of K-Cl cotransport (Table 1, Fig. 3) . Presence of significant oxidative damage in this mouse model was demonstrated by the reduced plasma and erythrocyte membrane levels of vitamin E (Fig. 6) and the marked positivity of erythrocytes for Annexin V binding, suggesting loss of phospholipid asymmetry and PS externalisation (Fig. 2 and 4). In this β thai mouse model, activation of K-Cl cotransport was clearly present at baseline in isotonic conditions and became more evident with hypotonic challenge or pretreatment with the protein kinase inhibitor staurosporine (Fig. 3) . The functional up-regulation of K-Cl cotransport seen in β thai mouse erythrocytes differs from that seen in vitro with human thalassemic erythrocytes, since there was little effect of in vitro DTT treatment on K-Cl cotransport activity, while a significant effect has been described in the human counterpart . [Olivieri, 1994 #269] K-Cl cotransport activation by oxidative damage has been demonstrated in vitro with a variety of agents known to induce oxidative damage of the red cell membrane. [Olivieri, 1993 #67;Haas, 1989 #790;Lauf, 1990 #66;Bize, 1998 #316;Lauf, 2000 #1621] The main regulatory phosphatase for K-Cl cotransport, PP-1, is stimulated by in vitro exposure to H₂0₂.[Bize, 1998 #316] Iron chelation therapy diminishes free iron-mediated membrane damage and K-Cl cotransport activity both in murine and human β thalassemia. [Browne, 1997 #233;De Franceschi, 1999 #676] K- Cl cotransport activation due to oxidative membrane damage was also demonstrated in the anemia associated with ribavarin therapy. [De Franceschi, 2000 #785]

Presence of significant structural alterations of the erythrocyte membrane induced by oxidative damage was demonstrated by the marked Annexin V positivity of β thai mouse erythrocytes. (Fig. 2 and 4) The Annexin V positivity of erythrocytes in this mouse model differs from that of the human counterpart: in human β thalassemia (and in sickle cell disease) , Annexin V positivity seems to be limited to a defined subpopulation of erythrocytes. [Kuypers, 1998 #1693;de Jong, 2001 #1684] Annexin V positivity in this β thai mouse model was more uniformly distributed and appeared as a shift of the entire red cell population (Fig. 4) . It is possible that this difference may be related to the more homogeneous genetic background of this mouse model and to the fact that, contrary to most of the human patients, these mice were not splenectomised. It may also be expression of a more generalised and significant oxidative damage, as demonstrated by the marked decrease in plasma and erythrocyte membrane vitamin E levels observed in this mouse model (Fig. 6) .

The presence of marked oxidative damage in β thalassemia provides the rationale for testing anti-oxidant therapies . [Shinar, 1990 #871;Scott, 1993 #1308;Scott, 1995 #1305] A pilot trial with large doses of oral vitamin E, prompted by the abnormally low levels of this vitamin in plasma of patients with β thai intermedia, showed a decrease in the levels of malonylaldehyde, but not in transfusion requirements. [Rachmilewitz, 1979 #1297] Rutin, a quercitin rutoside member of the flavonoid family prevented primaquine-induced methaemoglobin formation in vitro, but could not restore normal haemoglobin from preformed methaemoglobin. [Grinberg, 1994 #1732] The polyphenol curcumin caused a significant inhibition of lipid peroxidation in β thalassemic red cell ghost [Grinberg, 1996 #1942]

Tea polyphenols have shown antioxidant properties in vitro. [Grinberg, 1997 #1924] N-allylsecoboldine seemed to protect β thalassemic erythrocytes from peroxyl, hydroxyl radicals and H ₂0₂ -induced damage and decreased haemolysis and lipid peroxidation in vitro. [Teng, 1996 #1318]

Semisynthetic flavonoids, such as 7-monohydroxyethylrutoside (MonoHER) and related compounds have been reported to be powerful in vitro antioxidants and to prevent doxorubicin cardiotoxicity in vivo in mice. [van Acker, 2001 #1927;Heijnen, 2001 #1928;van Acker, 2000 #1930;van Acker, 2000 #1931;van Acker, 1997 #1734] MonoHER and related compounds possess both iron chelating and radical scavenging properties, although iron chelation does not seem to play a role in the antioxidant activity, at least in vitro, [van Acker, 1998 #1933] MonoHER treatment of β thai mice resulted in marked changes of several parameters indicative of membrane oxidative damage. Both plasma and erythrocyte membrane vitamin E levels rose to values close to those of normal control mice with monoHER therapy (Fig. 6A-B) and the percent of Annexin V positive cells decreased markedly to levels close to normal controls (Fig. 2 and 5) . Although we did not measure K-Cl cotransport activity in monoHer treated β thai mice, the improvement of cell hydration status (Fig. 4) and the increased erythrocyte K content (Table 1) indicate that monoHER therapy affected ion transport and volume control of β thai erythrocytes. In an in vitro system to study red cell K loss induced by lipid peroxidation, it has been elegantly shown that the ability of flavonoids to reduce free radical-stimulated K loss is strictly associated with their ability to prevent membrane lipid peroxidation. [Maridonneau-Parini, 1986 #1967] Although there were laboratory signs of decreased haemolysis (reduced serum bilirubin, LDH, and absolute reticulocyte counts) , no significant changes in Hct or Hb were seen in β thai mice treated with monoHER (Table 1 and 2) . It may be possible that more than three weeks of treatment are necessary to demonstrate change in Hb/Hct. Normal erythrocyte survival in mice is approximately 40 days, and after 3 weeks, 50% of the original cells could still be present in a normal mouse. Although the survival of β thai mouse erythrocytes is greatly reduced, it is possible for a significant fraction of the original cells to still be present after 21 days of monoHER therapy, indicating a possible effect of monoHER on circulating erythrocytes as well as on newly produced erythrocytes. Future studies will need to include longer treatment times and red cell survival studies to address these important issues. However, this is the first demonstration that a specific treatment can restore normal lipid asymmetry in erythrocytes exposed to oxidative damage in vivo. Recently, studies with a different class of antioxidants which possess catalytic superoxide dismutase and catalase activities, have shown partial correction of anemia and haemolysis in a mouse model with absent mitochondrial superoxide dismutase and oxidative stress- induced haemolytic anemia. [Friedman, 2001 #1674]

The results presented here are relevant not only for thalassemias but also for other anemias and in particular for sickle cell disease. The role of oxidation in the pathophysiology of sickle cell disease [Hebbel, 1990 #613] and the evidence of reduced antioxidant defenses in patients with sickle cell disease [Natta, 1992 #1738;Chiu, 1990 #1251;Adelekan, 1989 #1739] suggest that antioxidant therapy should be considered. Transgenic sickle mice show typical patterns of "reperfusion injury" with excessive reactive oxygen species (ROS) generation following hypoxia. [Osarogiagbon, 2000 #1099] Sickle erythrocytes have a decreased number of titrable thiol residues carried by membrane proteins such as spectrin, ankyrin, protein 3 and protein 4.1. [Rank, 1985 #621] Thiol oxidation is believed to play an important role in the membrane transport abnormalities of sickle erythrocytes, contributing to the activation of the K-Cl cotransport and Gardos channel . The thiol compound N- acetyl cysteine exhibits in vitro antioxidant properties, in sickle and normal erythrocytes under oxidative stress . [Udupi, 1992 #1321] Gardos channel-mediated dehydration of sickle cells induced by in vitro oxidation is reduced by n-acetylcysteine . [Shartava, 1999 #1066;Shartava, 2000 #1357]

By the above date it is shown that MonoHER is an attractive compound for the in vivo prevention of oxidative damage in human β thalassemias and in other haematological disorders characterised by oxidative damage to the erythrocyte (red blood cell) .

Claims

C L A I S

1. Use of 7-monohydroxyethylrutoside for the manufacture of a medicament for treatment of haematological disorders, associated with oxidative damage of the red blood cell membrane and/or with iron storage, in a subject in need thereof.

2. Use of 7-monohydroxyethylrutoside for the manufacture of a medicament according to claim 1, for treatment of β thalassemia in a subject in need thereof.

3. Use of 7-monohydroxyethylrutoside for the manufacture of a medicament according to claim 1, for decreasing the bilirubin level in the peripheral blood of a subject in need thereof.

4. Use of 7-monohydroxyethylrutoside for the manufacture of a medicament according to claim 1, for decreasing the LDH level in the peripheral blood of a subject in need thereof.

5. Use of 7-monohydroxyethylrutoside for the manufacture of a medicament according to claim 1, for decreasing the plasma iron level in the peripheral blood of a subject in need thereof.

6. Use of 7-monohydroxyethylrutoside for the manufacture of a medicament according to claim 1 for increasing the erythrocyte K level in the peripheral blood of a subject in need thereof.

7. Use of 7-monohydroxyethylrutoside for the manufacture of a medicament according to claim 1 for decreasing the degradation of red blood cells of a subject in need thereof.