text:
FUNK, Felix (Römerstrasse 177b, Winterthur, CH-8404, CH)
KURTZHALS, Jørgen (Strandvejen 59, 1.tv, Copenhagen ø, DK-2100, DK)
SØRENSEN, Lasse Maretty (Rådmansgade 40C, 145, Copenhagen N, DK-2200, DK)
BURCKHARDT, Susanna (Ackersteinstrasse 207, Zürich, CH-8049, CH)
FUNK, Felix (Römerstrasse 177b, Winterthur, CH-8404, CH)
KURTZHALS, Jørgen (Strandvejen 59, 1.tv, Copenhagen ø, DK-2100, DK)
SØRENSEN, Lasse Maretty (Rådmansgade 40C, 145, Copenhagen N, DK-2200, DK)
1 . Iron carbohydrate complex compounds, wherein the carbohydrate is selected from the group which consists of natural carbohydrates or synthetic carbohydrate derivatives, such as starch, hydrolyzed starches, dextrins (in particular maltodextrln, maltose syrup, glucose syrup, cyc!odextnns), dextrin derivatives (in particular oxidized dextrins, especially oxidized maitodextrins or hydrogenated dextrins), and pullulan and derivatives thereof, for the use in the intravenous therapy of malaria patients.
2. Iron carbohydrate complex compounds according to claim 1 for the use in the treatment of iron deficiency with or without anaemia in malaria patients,
3. Iron carbohydrate complex compounds according to claim 1 or 2 for the use in the intravenous therapy of patients with acute malaria, in particular, severe and complicated maiaria, and, in particular, of severe malarial anaemia ,
4. iron carbohydrate complex compounds according to any of the
preceding claims for the use in the intravenous therapy of patients with chronic Plasmodium infection and symptoms thereof .
5. Iron carbohydrate complex compounds according to any of the
preceding claims for the use In the intravenous iron supplementation of patients suffering from malaria .
6. Iron carbohydrate complex compounds according to any of the
preceding claims for the use in the intravenous iron supplementation of asymptomatic or symptomatic patients suffering from chronic Plasmodium infection. , Iron carbohydrate complex compounds according to any of the preceding claims for the use in the treatment of organ failure, in particular, suppression of erythropoiesis in malaria patients, , Iron carbohydrate complex compounds according to any of the
preceding claims for the use in reducing outbreak of malaria attacks, , Iron carbohydrate complex compounds according to any of the
preceding claims for the use in reducing frequency and/or intensity of recurrence of malaria attacks and/or re-infection with Plasmodium . 0, Iron carbohydrate complex compounds according to any of the
preceding claims for the use in the prophylactic and/or routine iron supplementation in malaria patients as well as in patients without malaria infection in areas where malaria is endemic . 1 , Iron-carbohydrate complex compounds according to any of the
preceding claims, characterized In that the carbohydrate is chosen from oxidised or hydrogenated dextrins, 2. Iron-carbohydrate complex compounds according to any of the
preceding claims, which are selected from iron(lll)-compIex
compounds of poiymaltose or oxidized poiymaltose. 3, Iron-carbohydrate complex compounds for the use according to any of the preceding claims having a molecular weight in the range from 20,000 to 500,000 Daltons 4. Iron-carbohydrate complex compounds for the use according to any of the preceding claims, which are selected from iron(lll)-complex compounds of poiymaltose or oxidized poiymaltose preferably having a molecular weight in range of 80 kDa to 400 kDa, preferably 1 00 kDa to 350 kDa . 5 , lron-carbohydra†e complex compounds for the use according to any of the preceding claims, which are selected from iron-carbohydrate complex compounds with an oxidation product of one or more maltodextr s, which are ootainable from an aqueous iron(lll) salt solution and an aqueous solution of the product of the oxidation of one or more maltodextrins with an aqueous hypochlorite solution at a pH value in the alkaline range, where when one maltodextrin Is used Its dextrose equivalent is from 5 to 37, a nd when a mixture of a plurality of maltodextrins is used, the average dextrose equivalent of the mixture is from 5 to 37 and the dextrose equivalent of the
Individual maltodextrins contained in the mixture is from 2 to 40 , 6. Iron-carbohydrate complex compounds for the use according to any of the preceding claims which is a polynuclear iron(I I I)-oxyhydroxy complex compound comprising carboxy polymaltose as !igands and having a weight average molecular weight of about 1 00 to 200 kDa . 7 , Iron-carbohydrate complex com pounds for the use according to any of the preceding claims for the therapy of infants, children and pregnant women , 8 , Combination preparation comprising an iron-carbohydrate complex according to any of the preceding claims and at least one further pharmaceutically active compound, In particular a compound useful for the treatment or prevention of malaria .
The present invention relates to iron carbohydrate complex compounds, wherein the carbohydrate is selected from the group which consists of natural carbohydrates or synthetic carbohydrate derivatives, such as starch,
hydroiyzed starches, aextrins (in particular maitodextrin, maltose syrup, glucose syrup, cyclodextrins), dextrin derivatives (in particular oxidized dextrins, especially oxidized maltodextrins or hydrogenated dextrins), and puliulan and derivatives thereof, for the use in the intravenous therapy of malaria patients ,
Today malaria remains a leading cause of death in endemic countries, being responsible for In the order of 500 million clinical cases and 1 million deaths annually (www.who.org). Human malaria is caused by four different species of the protozoan parasite genus, Plasmodium (P. Falciparum, P, Vivax, P. Ovale and P , alariae) that infects and multiplies in red blood cells (RBCs) .
P, falciparum is the most virulent species and is associated with the highest mortality rates due to life threatening complications such as cerebral malaria, lactic acidosis, and severe malarial anaemia (S A) that appear especially among infants, children and pregnant women , SMA is the most common life- threatening malaria complication in infants and young children and develops when the bone marrow (BM) is unable to compensate for an accelerated loss of erythrocytes from circulation; the last being caused mainly by an increase in erythrophagocytosis of uninfected RBCs (Jakeman et al . , 1 99; Price et a!,, 2001 ). The bone marrow constitutes a unique environment possible of
supporting proliferation and differentiation of haematopoietic stem ceils to mature blood cells. This process involves the coordinated action of several signalling molecules assisted by supporting stroma cells, all adding to the complexity of critical events as self-renewal, proliferation, differentiation and apoptosis. Erythropolesis designates the differentiation pathway from
pluripotent haematopoietic stem cell to mature erythrocytes. Erythropolesis is mainly regulated by the hormone erythropoietin (Epo), which is being produced in the kidney in response to changes in oxygen tension In the blood . In the mouse model, during certain stress conditions such as hypoxia due to severe blood loss, Epo cooperates with stem cell factor (SO " ), an early acting cytokine, and glucocorticoids to induce expansion of erythroid progenitors in the spleen, so-called stress erythropolesis (Bauer et al . , 1 99; Broudy et al , , 1 996) , Epo exerts its effect mainly by inhibiting apoptosls and stimulating proliferation of erythroid progenitors and an increase i n erythropolesis due to elevated Epo levels is normally reflected In the circulation as an elevated number of reticulocytes .
In addition to Epo and SCF , later erythropoietic stages require large amounts of iron for haemoglobin synthesis. The majority of this iron is supplied by recirculation from the reticuloendothelial system (RES), which is responsible for the disposal of senescent RBCs , Here iron is released from the heme moiety and exported to the blood, where it, bound to transferrin, is shuttled back to the erythron for haemoglobin synthesis , High Epo levels directly translate erythropoietic iron needs Into RES Iron export and Intestinal absorption, by lowering the recently identified hormone, hepcidin (Pinto et al , , 2008) .
Inflammatory cytokines, however, Increases hepcidin levels and hence lowers circulating iron levels to reduce iron availability to invading pathogens (Lee et al . , 2006) , Possibly prior to the inflammatory hepcidin response, a direct stimulation of the inflammatory transcription factor NF-KB In macrophages may exaggerate iron seq uestration In these by an increased iron uptake from plasma (Tacchlnl et al , , 2008) . Despite severe anaemia and concurrently elevated Epo levels during malaria, the number of reticulocytes in circulation remains low, Indicating a defective erythropoietic response, as Epo secretion Is apparently adequate to the degree of anaemia (Burchard et al , , 1 995;
Kurtzhals et al , , 1 997 ) , Despite improvement of reticulocyte counts rapidly after initiation of anti- malarial treatment, peak reticulocytosis is not achieved until approximately seven days post treatment [Casals- Pascual et al , , 2006;
Kurtzhals et al , , 1 997) . Histological and kinetic studies in humans and mouse models point towards a combination of dyserythropolesis (irregularly shaped nuclei, karyormexis etc ), suboptimal proliferation of precursors and a left-shift in overall precursor kinetics is responsible for erythropoietic suppression (Chang e† αΙ , , 2004a; Wickramasinghe et al. , 1 987) , The underlying mechanisms remain unclear, however a number of responsible erythroid suppressors have been proposed including Tumor Necrosis Factor-alpha (TNF-a), Interferon- gamma (IFN-γ), Macrophage migration Inhibitory Factor (MIF), malaria parasite Rhoptry Protein-2 (RSP- 2) and hemozoin (Lamikanra et al . , 2007) ,
In semi-immune children, malaria parasites can cause chronic infections that are usually asymptomatic . Such i n fections are associated with moderate immune stimulation and with a moderate but significant reduction in
haemoglobin levels, which are associated with erythropoietic suppression ( urtzhals et al . Trans R Soc Trop Med Hyg 1 999; 93: 623-627 )
Experimental evidence suggest that anaemia and hence erythropoietic suppression constitute a double-edged sword during malaria infection, being host-protective by limiting the availability of RBCs for parasite invasion but concurrently posing a direct threat†o host survival due to SMA (Chang et al .. 2004b). Iron may play a central role within this interface. Indeed, this paradox may well explain the highly contradicting outcomes of clinical studies of iron supplementation and iron deficiency (ID) in relation to malaria susceptibility. Specifically, some investigators have reported a decrease in severe anaemia incidence In iron-supplemented individuals without any change in malaria susceptibility (Menendez et al . , 1 997; Menendez et al . , 1 994), while others observed increased malaria incidence with improvement of iron status f Kabyemela et al , , 2008; Oppenheimer et al . , 1 986; 80 81 8-822). For example Oppenheimer et al (Trans R Soc Trop Med Hyg; 1 86 80 (4), 603-61 2) carried out a placebo-controlled trial of intramuscular Iron dextran prophylaxis for two- month-old infants on the north coast of Papua New Guinea where there is high transmission of malaria . The results gave evidence for a protective role of iron deficiency against malaria and would argue against the injudicious use of Iron replacement In areas where malaria is endemic . The intramuscular Injection of iron given to children suffering from anaemia and its negative effects in the form of exacerbated malaria, are also mentioned by Scrimshaw ( 1 991 ) .
Further, a clinical trial with 1 2 weeks or oral iron supplementation to children with severe malaria-associated anaemia was carried out in Tanzania (van den Hombergh et al . , 1 996) . The results indicated that oral iron supplementation did not have any effect on the rate of parasitaemia and on parasite density during the 1 2 weeks, However, the iron-supplemented group had a significantly increased morbidity from other causes than malaria and it appeared that supplemented iron had no effect on the recovery of haemoglobin level In the children with malaria-associated anaemia , Accordingly, the study provided no evidence supporting routine iron supplementation to these children ,
Experimental studies of parasite proliferation kinetics have confirmed that ID confers protection against malaria by increasing RBC turnover and possibly by producing erythropoietic suppression ( oka et al . , 2007 ) , Furthermore, studies of genetic polymorphisms at loci directly or indirectly involved in iron handling, further point towards a strong selection pressure for variants increasing protection against malaria at the expense of anaemia (Atkinson et al . , 2006; Atkinson et al , , 2008; Cox et al., 2007) , The high incidence and mortality of SMA clearly underlines the limitations of this protective strategy and hence therapeutic measures capable of reversing this condition are highly warranted ,
The specific role of iron in the pathogenesis of malarial erythropoietic
suppression remains poorly defined; however two recent studies by de Mast et al . did observe increased hepcidin levels and suppressed plasma iron levels in malaria anaemic individuals combined with a decrease in reticulocyte haemoglobin content, an indicator of iron-restricted erythropoiesis (de Mast et al . , 2008; de Mast et al . , 2009) . These studies were conducted in non-severe anaemia cases and it can be speculated that a further amplification of these mechanisms contribute to the often detrimental outcome of SMA, A further investigation of de Mast et al . (201 0) relates to increased serum hepcidin and altered blood iron parameters in asymptomatic P. falciparum and P. vivax malaria ,
Further studies of erythroid iron supply in SMA are thus needed as this may facilitate the identification of a therapeutic window for iron treatment, This would provide an important alternative to blood transfusions, which remain largely unavailable due to logistical and financial constraints in malaria endemic areas, and hence enhance adjuvant treatment for an otherwise often fatal malaria complication, Besides the investigations in the treatment of SMA with iron supplementation, several studies have been carried out, relating to routine oral iron
supplementation, either as a prophylaxis In malaria endemic regions or as a routine treatment of iron deficiency anaemia besides malaria or of malaria- associated anaemia in earlier stages (see e .g . IDS Health and Development Information, 2009 : Stoltzfus et a! . , 2001 ) .
In this context it has to be pointed out, that OJukwu et a l . ( 2009) analysed 68 different randomized and controlled trials evaluating oral iron supplementation for preventing or treating anaemia among children in malaria-endemic areas, As result the authors concluded that orally given iron supplementation does not adversely affect children living in malaria-endemic areas, especially no increase in the risk of clinical malaria was found . Nevertheless, the analysis revealed that an inc reased risk of malaria with iron was observed in trials that did not provide malaria surveillance and treatment, The conclusion of Ojukwu et al . is in contrast to the recently changed WHO guidelines which are based on a large trial in Zanzibar (see above ; van den Hombergh et al . ), according to which the WHO now recommend to withheld iron supplements from children under two years, who are not iron deficient, in areas where they are at high risk of contracting malaria (ScienceDaiiy, 2009; WHO, 2006 Food Nutr Bull . 2007 Dec ; 28 (4 SuppI) :S489-631 ) ,
These findings indicate that iron supplementation in any case seems
disadvantageous in patients suffering from malaria but not being supplied with an adequate malaria therapy. Accordingly, until now it can not be excluded that iron supplementation In patients suffering from e.g . unrecognized or simply untreated malaria infection will be adversely affected f rom iron supplementation and thus, in any case, routine or prophylactic iron
supplementation in malaria endemic areas remains risky.
Cercamondi et al . (201 0) further investigated the absorption and utilization of iron in asymptomatic female malaria patients (no severe anaemia) by oral and intravenous application of extremely low doses isotope-labelled iron as iron citrate ( 1 00 μg iron citrate labelled as 58Fe) , The treatment was accompanied by malaria treatment with Malarone tablets , The used iron amounts are too low to act as iron supplementation but solely function as markers for the evaluation of iron uptake from regular nutrition in patients suffering from malaria . A decrease of dietary iron absorption of approximately 40 % was found, which is assumed to mainly result from low grade inflammation and circulating hepcidin. It was further found that oral iron supplementation in malaria patients is of low effectiveness as no sufficient iron utilisation can be reached , The use of iron carbohydrate complex compounds for intravenous iron supplementation is not described and can neither be concluded from the presented results. Byles et al . ( 1 979) described the use of iron dextran (Smferon ®) for the intraveneous (total dose Infusion) application to anaemic pregnant women in regions where malaria due to Plasmodium falciparum is hoioendemic . In this context the authors investigated the possibility to reduce the negative reactions due to the intraveneous iron dextran application by of the
application of either the antihistamine promethazine or the anti-malaria agent chloroauine. In this context the authors explicitly refer to the disadvantage of the incidence of generalized reaction, especially In pregnant patients, due to the iron dextran infusion and pointed out that in tropical areas where malaria is endemic the additional load of iron dextran on the lymphoreticular system may disturb the altered cell-mediated immune mechanism of pregnancy and thus may precipitate clinical malaria , They further explained that total dose infusion of iron dextran increases the incidence of circulating malarial parasites and induces inflammatory response reaction both at the site of the infusion and in the generalized reaction. Further, Byles et al . ( 1 975) investigated the possibilities of managing severe anaemia in pregnancy in developing countries considering the three main causes of anaemia in the tropics, iron deficiency, folic acid deficiency and haemolysis due to malaria , In this context the iron deficiency was treated by parenteral application of iron dextran (!mferon®) as total dose infusion and herein, too, the incidence of the negative reactions had to be managed by a combined treatment with promethazine. The presented results revealed that only a poor response to parenteral iron therapy alone could be achieved. In addition, the authors reported that one patient of the group, which received Imferon® only, died from congestive cardiac failure due to anaemia although she received Imferon® 5 days previously. The authors finally concluded that the use of iron dextran (Imferon®) by total-dose infusion ensures a readily available supply of iron for the requirements of pregna ncy and correction of the anaemia , Nevertheless, the authors pointed out that other methods of administration of parenteral iron are impractica l ,
Fleming ( 1 989), too, mentioned the intravenous use of iron dextran as total dose infusion (TDI), but explicitly recommend that such treatment Is indicated only when severe iron deficiency anaemia has been proven and further point out that such treatment must be accompanied by antimalarial therapy and folic acid supplements to reduce severe side effects as well as by prophylaxis to prevent rec urrence during the rest of pregnancy. They pointed out that TD! in iron-sufficient patients is not beneficial , and in patients with malaria is even likely to be harmful, as parenteral iron has been shown to be followed by increased frequency and intensity of malaria .
The use of total dose intravenous infusion of iron-dextran (Imferon®) in severe anemia is further generally disc ussed by Bhatt et al . ( 1 966), without mentioning the treatment of malaria patients or in regions where malaria is endemic .
Therein, the authors discussed the total dose infusion of iron-dextran compared to the intravenous use of saccharated oxide of iron or to the intramuscular use of iron-dextran complex and clearly stated that the intravenous use of saccharated oxide of iron was diminished due to severe reactions and even deaths and that the intramuscular use of iron-dextran complex is
accompanied by sarcomatous effects at the site of injection , For the
intravenous use of iron-dextran the incidence of severe reactions and as a result the recommendation, not to use it routinely, is mentioned, too.
Taking these findings into consideration, it becomes obvious that in addition to improved therapeutic methods in the treatment of S A, Improved therapeutic methods for the prophylactic and routine treatmen - of iron deficiency or iron deficiency anaemia in mild to moderate and thus early stages or therapeutic and/or prophylactic methods to generally improve the iron status in areas where malaria is endemic without causing the described side effects or detrimental development in malaria infections are necessary. Nevertheless, although some of the aforementioned authors already referred to the intravenous use of iron-dextran even In the treatment of malaria patients and in regions where malaria is endemic, all of them referred to severe side effects and harmful reactions in context with the iron-dextran infusion, too. Byles et al, (1975) even more explicitly stated that other methods of
administering parenteral iron are impractical and from Bhatf et al. (1966) it becomes obvious that a further iron-carbohydrate compound, namely saccharated oxide of iron, is clearly disadvantageous In the treatment of iron deficiency at all. Accordingly, starting from the disclosure of these documents a skilled person would clearly not be motivated to use an alternative Iron compound of the group of Iron carbohydrate compounds, namely any of the iron carbohydrate complex compounds according to the present invention or as mentioned In US 2010/0099647 Al , in the treatment of malaria patients or In regions where malaria Is endemic, From the patent literature the treatment of malaria with iron chelating or iron complexing agents is known, The use of such iron chelating agents is suitable to remove iron from the body and thus to inhibit the proliferation of the malaria parasites. Such iron complexing agents for the treatment of malaria are e.g. subject of WO99/39706 Al , WOl 0/011684 A2, WOO 0/16763 A2,
WO05/051411 Al , WO98/09626 Al , WO09/055863 Al , VVO93/C0327A1 ,
WO93/00082 Al , EP 0214101 A2, W098/11066 Al , WO04/041151 A2 or
WO02/26225 Al describes methods for reducing free radical levels or free radical overproduction associated with e.g. malaria by administration of physiologically active dlthiocarbamate compounds which may comprise dlthiocarbamate- ferrous iron complexes which bind non covalently to free radicals, forming stable, water-soluble dlthiocarbamate- iron-free radical complexes. The use of such dithiocarbamate-ferrous iron complexes for the treatment of iron deficiency or anaemia in malaria patients is not mentioned. W096/35698 Al relates to organometallic iron complexes with advantageous properties as antimalarial agents, and in particular to the insertion of
ferrocenyl groups into the structure of molecules possessing antimalarial properties. By combining molecules having a structure close to that of antimalarials with one or more iron atoms, for example by means of the insertion of a ferrocenyl group inside analogs of substances such as quinine, chloroquinine, mepacrine and primaquine, the therapeutic activity of these substances was substantially increased. The use of such organometallic iron complexes for the treatment of Iron deficiency or anaemia in malaria patients is not mentioned ,
Further, W098/1 1 066 A1 relates to the treatment of iron overload by
administering a dithiocarbamate-macromolecule-containing composition wherein the macromolecule may be selected from polysaccharides such as e.g . dextran, cellulose, starch, glycogen, cycloaextran etc , The definition of iron overload comprises inter alia blood transfusions, anaemia, sickle cell anaemia and dietary iron uptake, As sickle cell anaemia is a disease which is accompanied by Iron overload it can be concluded that the treatment of iron deficiency anaemia is not comprised hereunder,
WO09/01 2953 AT relates to nutritional blends for fortifying food comprising iron, e.g in form of sodium iron(lll) EDTA, ferrous sulphate or ferric pyrophosphate, pnytase, vitamin C and optionally further nutrients and further relates to the use of such blends for supplying humans In malaria endemic areas with the nutritional need of iron without the risk of a bolus effect. The use of iron carbohydrate complex compounds for intravenous iron supplementation is not described.
EP 1 61 441 4 A2 relates to compositions comprising nanoparticles of a fixed copper compound core or a fixed copper-iron compound core, which can inter alia be used in the treatment of malaria , Such copper compound can further be administered intravenously in combination with an iron compound such as e.g . iron dextran . Nevertheless, the active principle is the fixed copper compound and the suitability of such copper compound In the malaria treatment is the object of the invention . No indication for any beneficial effects of the iron dextran compound in the malaria treatment is mentioned therein . Further, no hint can be taken from this document to use any other iron carbohydrate complex compound in the treatment of malaria , Objectives
The present Inventors thus attempted to overcome the disadvantages of known iron supplementation in malaria patients and thus to provide a beneficial therapy alternative to blood transfusions and routine oral or intramuscular iron supplementation that would be useful in the treatment of malaria patients, in particular the treatment of SMA and suppression of erythropoiesis and for improving bone marrow function in malaria patients or in areas where malaria is endemic, respectively.
The present inventors surprisingly found that intravenous iron supplementation with iron carbohydrate complex compounds, wherein the carbohydrate is selected from the group which consists of natural carbohydrates or synthetic carbohydrate derivatives, such as starch, hyo!rolyzed starches, dextrins (in particular maltodextrin, maltose syrup, glucose syrup, cycicdextrins), dextrin derivatives (in particular oxidized dextrins, especially oxidized maltodextrlns or hydrogenated dextrins], and pullulan and derivatives thereof, in malaria patients can overcome, in particular, the disadvantages of conventional known oral and intramuscular iron supplementation therapies, as well as intravenous iron-dextran therapy, in malaria patients, Without being bound to theory, the present inventors believe that a decreased erythropoietic Iron availability contributes to experimental malarial anaemia and that this effect can be alleviated with the presently suggested intravenous iron therapy, In contrast oral iron supplementation is likely to be of low value in the acute malaria setting as intestinal iron absorption is severely suppressed (Doherty et al , , 2008). It is believed that iron availability is indeed a rate-limiting factor in erythropoiesis during malarial anaemia and that intravenous iron therapy can alleviate this effect, and that the increased plasma iron levels during
intravenous iron therapy will not directly affect parasite proliferation kinetics,
So far, neither the scientific literature nor the patent literature provides improved therapeutic methods in the treatment of SMA as well as improved therapeutic methods for the prophylactic and routine treatment of iron deficiency or iron deficiency anaemia in malaria patients or in areas where malaria is endemic which are based on the intravenous iron supplementation with iron carbohydrate complex compounds of satisfying physiological and biological compatibility and safety,
Ds t a ij . ecl .... i e s c notion of the invention Thus the present invention provides an improved method of treating malaria patients or patients in regions where malaria is endemic, by providing iron carbohydrate complex compounds, wherein the carbohydrate is selected from the group which consists of natural carbohydrates or synthetic carbohydrate derivatives, such as starch, hydrolyzed starches, dextrims (in particular maitodextrin, maltose syrup, glucose syrup, cyc!odextrins), dextrin derivatives (in particular oxidized dextrins, especially oxidized maltodextrins or
hvdrogenated dextrins), and pullulan and derivatives thereof, for the
intravenous therapy of malaria or of patients suffering from malaria,
respectively, and symptoms thereof, in particular, for the treatment of Iron deficiency with or without anaemia (iron deficiency anaemia) in malaria patients. Furthermore, the iron carbohydrate complex compounds
administered according to the invention are used in particular for the intravenous therapy of patients with acute malaria, in particular, severe and complicated malaria and, in particular, of severe malarial anaemia (SMA) , (As is known by the skilled person In the art the definition of severe and
complicated malaria Is based on clinical presentation, where the patient presents with any of the following clinical features:
* A change In behaviour, confusion or drowsiness;
* Impaired consciousness or unarousable coma;
* Multiple/recurrent convulsion;
* Deep breathing or respiratory distress;
* Difficulty In breathing or demonstrable pulmonary oedema as may be seen radiologically;
* Circulatory collapse or shock;
* Jaundice;
* Haemoglobinuria;
* Bleeding tendency; * Prostration i ,e generalized weakness so the patient cannot walk, or sit up without assistance;
* Severe anaemia with or without congestive cardiac failure,
See e,g . 'Severe and complicated malaria, third edition . Trans Soc Trop Med Hyg 2000; 94 supplement 1 ; Page 1 - Page 90),
Aditionally, the iron carbohydrate complex compounds administered
according to the invention are used for the intravenous therapy of patients with chronic Plasmodium infection and symptoms thereof ,
A further aspect of the present Invention relates to the selected iron
carbohydrate complex compounds for the intravenous iron supplementation of patients suffering from acute, in particular, severe and complicated malaria as well as of symptomatic or asymptomatic patients suffering from chronic Plasmodium infection , Furthermore the iron carbohydrate complex compounds intravenously administered according to the invention are used in particular for the treatment of organ failure, in particular, suppression of erytnropoiesis in malaria patients. Thus the iron carbohydrate complex compounds are
intravenously administered also to improve bone marrow function and also weight gain in malaria patients,
In a further aspect of the invention the iron carbohydrate complex compounds administered according to the invention are used for the prophylactic and/or routine iron supplementation, in particular for the treatment of iron deficiency or iron deficiency anaemia, in patients suffering from malaria and symptoms thereof, as well as in patients without (clinical) malaria infection in areas where malaria is endemic ,
A further aspect of the present invention is directed to the use of the selected iron carbohydrate complex compounds in reducing prevalence and/or outbreak of malaria attacks, Further, the selected iron carbohydrate complex compounds of the present invention turned out to be suitable for the use in reducing frequency and/or intensity of recurrence of malaria attacks or reinfection, respectively, preferably in patients which already suffered from malaria and/or already passed through at least one malaria attack and have been treated according to the present invention previous, during or subsequent to such at least one malaria attack.
Accordingly, the present invention comprises a post-treatment subsequent to at least one malaria attack in order to prevent or at least reduce the
recurrence and/or the degree of subsequent malaria attacks and/or of reinfection as well as of further symptoms following a maiaria attacks,
Iron-carbohydrate complex compounds used in accordance with the present invention are complex compounds of in particular I ron (III) (where (III) indicated the formal oxidation number 3 (three)) having at least one carbohydrate ligand coordinatively bond to iron atoms, usually via oxygen atoms that are part of the carbohydrates, In particular, of hydroxy groups but also oxo and/or carboxylate groups, Such iron-carbohydrate complex compounds may be mono- or polynuclear, I e. may have one or more iron atoms in average per molecule, They are particularly prepared by reaction iron(lll) salts with carbohydrates In the presence of a base, upon which, in general, polynuclear iron oxy hydroxy-carbohyarate complex compounds of higher molecular (i.e, w of more than 20000 Dalton weights) are formed, which are particularly preferred,
In accordance with the present invention the iron-carbohydrate complex compounds used according to the Invention are characterized in that the carbohydrate is selected from the group which consists of: natural
carbohydrates or synthetic carbohydrate derivatives, such as starch,
hydrolyzed starches, dextrins (in particular maltodextrin, maltose syrup, glucose syrup, cyclodextrins), dextrin derivatives (In particular oxididized dextrins, especially oxidized maltodextrins or hydrogenated dextrins), and pullulan and derivatives thereof.
In principle, the carbohydrate may be a dextran or a derivative thereof, too. Nevertheless, dextrans and derivatives thereof are for the aforementioned reasons not preferred. Such iron-carbohydrate complex compounds are known and are described, in particular, in the applicants * patent applications WO 2004/037865 related†o iron(l l l) complex compounds of oxidized maltodextrins (carboxypolymaltose); WO 2006/084782 related to the use of !ron(lll) complex compounds with carbohydrates or derivatives thereof in the preparation of a medicament for improving immune defence and/or brain performance; WO 2008/087 1 35 related to lron{lll]/(i ! )-carbohydrate complex compounds; WO 2007/0231 54 related to the use of iron(l ll) complex compounds with carbohydrates or derivatives thereof for the preparation of a medicament for treatment of iron deficiency states in patients with chronic inflammatory bowel disease;
WO 2007/060038 related to preparations comprising one or more iron(III) carbohydrate complex compounds; WO 2008/1 45586 related to iron- carbohydrate derivative complex compounds; and of other applicants like WO 2004/082693 or WO 2007/055804A2 related to a method of treating
Restless Leg Syndrome - the contents of all documents are included herewith completely by making reference to them . Particularly preferred iron- carbohydrate complex compounds are chosen from iron complex compounds of oxidised or hydrogenated dextrlns (the latter being low-molecular-weight fractions produced by the enzymatic or acid hydrolysis of starch, including in particular maltodextrins - as is known by a skilled person in the art (see for example Wikipedia entry on dextrin] , Particularly preferred iron-carbohydrate complex compounds are further preferably selected from iron(lIl)-complex compounds, optionally comprising to some extent iron(l l ) ions (generally less than 2 weight percent calculated on the basis of total iron), among which iron(ll l)-complex compounds of polymaltose and In particular oxidized
polymaltose (i .e, carboxy polymaltose) are again more preferable (in particula r those described in WO 2004/037865) , Still more preferred are iron(ll l)-compiex compounds of polymaltose or oxidized polymaltose having an weight average molecular weight in the range from 20 to 500 kilo Daltons (<Da) more
preferably in the range of 80 kDa to 400 kDa , and still more preferably in the range of 1 00 kDa to 350 kDa, and most preferably in the range of 1 00 to 200 kDa (wherein the molecular weight is measured by gel permeation
chromatography, e .g . as described by Geisser et al, in Arzneim , Forsch/Drug Res . 42(11 ), 1 2, 1 439- 1 452 ( 1 92), paragraph 2.2.5) . Most preferred are iron(l l l) complex compounds with an oxidation product of one or more ma!todextrins as a ligand (i ,e, carboxy-functional maltodextrins) as described In particular in WO 2004/037865, Accordingly such Iron-carbohydrate complex compounds are obtainable from an aqueous iron(lll) salt solution and an aqueous solution of the product of the oxidation of one or more maltodextrins with an aqueous hypochlorite solution at a pH value in the alkaline range, where when one maltodextrin is used its dextrose equivalent is from 5 to 37, and when a mixture of a plurality of maltodextrins is used, the average dextrose equivalent of the mixture is from 5 to 37 and the dextrose equivalent of the individual
maltodextrins contained in the mixture Is from 2 to 40 , The reaction products obtained are polynuclear i ron [III) oxy hydroxy complex compounds with oxidized maltodextrins ligands where part or all of the aldehyde groups in the maltodextrins are oxidized to carboxyiic acid groups (thus being a carboxy- functional polymaltose) and which have molecular weights in the ranges indicated before preferably In the range of 1 00 kDa to 300 kDa, and even more preferred in the range of 1 00 to 200 kDa (determined as indicated above), As described In WO 2004/037865 these polynuclear iron(lll) oxy hydroxy complex compounds having oxidized maltodextrins as ligands are obtained in particular by a process wherein one or more maltrodextrins are oxidized in an aqueous solution at an alkaline pH-value using an aqueous hypochlorite solution and the obtained solution is reacted with an aqueous solution of an Iron (III) salt. Preferably when one maltodextrin is applied, its dextrose
equivalent lies between 5 and 20, and when a mixture of several maltodextrins is applied, the average dextrose equivalent of the mixture lies between 5 and 20 and the dextrose equivalent of each individual maltodextrins contained in the mixture lies between 2 and 40, Preferably the average DE value of the maltodextrins subjected to oxidation lies between 7 and 1 5, Iron(lll) oxy hydroxy complex compounds prepared with starting maltoxetrins outside these ranges of the DE values are less suitable for intravenous administration , The term dextrose equivalent of a polysaccaride designates the percentage of reducing functional sugar groups (terminal aldehyde groups) In a dry substance and corresponds to the amount of glucose which has the same reduction
capability per 1 00 g dry substance, The DE-value is thus a measure of the degree of degradation of a starch product. Products with a low DE-value have a higher molecular weight (lower relative content of terminal reducing aldehyde groups) and products with a high DE-vaiue have a lower molecular weight (higher relative content of terminal reducing aldehyde groups), For example the DE value of starch is zero (almost no reducing capability), and the DE value of glucose in accordance with the above definition is 1 00.
Depending on the particular degree of hydrolysis maltodextrins usually hove an average DE value of 3 to 20, Products with an average DE-value above 20 (glucose sirups) are less favourable. In the present invention the dextrose equivalents are measured gravimetrically. In order to do so, the maltodextrins are reacted in a boiling aqueous solution with Fehiing's solution. The reaction is carried out quantitatively, i .e. until the Fehiing's solution is no longer
discoloured. The precipitated copper (I) oxide is dried at 1 05°C until a constant weight is achieved and measured gravimetrically, The glucose content (dextrose equivalent) is calculated from the obtained results as % weight/weight of the maltodextrln dry substance, it is, for example, possible to use the following solutions; 25 ml Fehiing's solution I, mixed with 25 ml Fehiing's solution II; 1 0 ml aqueous maltodextrin solution ( 1 0 % mol/vol) (Fehiing's solution I : 34 , 6 g copper (II) sulfate dissolved in 500 ml water; Fehiing's solution II ; 1 73 g potassium sodium tartrate and 50 g sodium hydroxide dissolved in 400 ml water) ,
In a preferred process of preparing the preferred iron(lll) carboxy polymaltose complex compounds the oxidized maltrodextrin and the iron (III) salt is mixed to form an aqueous solution having a pH-value so low that no hydrolysis of the iron (III) salt occurs, whereafter the pH is raised to 5 to 1 2 by the addition of a base. The reaction is preferably carried out at a temperature of 1 5 °C up to boiling point (of water) for 1 5 minutes up to several hours. The maltodextrins having suitable DE values are commercially available, They are oxidized in an aqueous solution with a hypochlorite solution . Suitable examples are solutions of alkali hypochlorites such as a solution of sodium hypochlorite. Commercially available solutions can be used . The concentration of the hypochlorite solution Is, e.g . at least 1 3 % by weight, preferably in the order of 1 3 to 1 6 % by weight, calculated as active chlorine. Preferably the solutions are used in such an amount that about 80 to 1 00 %, preferably about 90 % of one aldehyde group per molecule of maltodextrin is oxidized (to a carboxyl group). The oxidation is carried out in an alkaline solution, e.g. at a pH of 8 to 12, for example 9 to 11 , As an example, oxidation can be carried out at temperatures in the order of 15 to 40 °C, preferably of 25 to 35 °C. The reaction times are, e.g. In the order of 10 minutes to 4 hours, e.g. 1 to 1.5 hours By this procedure the degree of depoiymerisation of the starting maltodextrins is kept at a minimum. It Is assumed that the oxidation occurs mainly at the terminal aldehyde group (aceta! or semiacetal group respectively) of the maltodextrin molecules. It is also possible to catalyse the oxidation reaction of the
maltodextrins. The addition of bromide ions Is suitable, e.g. in the form of alkali bromides, for example sodium bromide. The added amount of bromide is not critical. The amount is kept as low as possible in order to achieve an end product (Fe-complex) which can easily be purified. Catalytic amounts are sufficient, The process to oxidize maltodextrins cataiytically with alkali bromides or with the ternary TEMPO system is described e.g. by Thaburet et al In
Carbohydrate Research 330 (2001 ) 21 - 29, which method can be used for the present Invention. Water soluble salts of inorganic or organic acids, or mixtures thereof, such as halides, e.g. chloride and bromide or sulfates can be used as iron (III) salts. It is preferred to use physiologically acceptable salts. If is especially preferred to use an aqueous solution of iron (III) chloride. For instance, the aqueous solution of the oxidized maltodextrin can be mixed with an aqueous solution of the iron (III) salt in order to carry out the reaction. Here, if is preferred to proceed in a manner so that during and Immediately after mixing of the oxidized maltodextrin and the iron (III) salt, the pH is strongly acid or so low that no hydrolysis of the iron (III) salt occurs, e.g. 2 or less, in order to avoid an undeslred precipitation of iron hydroxides. In general, it is not necessary to add an acid, if iron (III) chloride is used, since aqueous solutions of Iron (III) chloride can be sufficiently acid, Only after mixing, the pH is raised to values of e.g. in the order of at least 5, for example up to 11 , 12, 13 or 14. The pH is preferably raised slowly or gradually which, for example, can be achieved by first adding a weak base, for example, up to a pH of about 3, and then neutralizing further using a stronger base. Examples of weak bases are alkali - or alkaline earth - carbonates, bicarbonates, such as sodium and potassium carbonate or bicarbonate, or ammonia. Examples of strong bases are alkali - or alkaline earth - hydroxides such as sodium, potassium, calcium or magnesium hydroxide. The reaction can be improved by heating . For example, temperatures in the order of 1 5 °C up to boili ng point can be used , It is preferred to raise the temperature gradually. Thus, for example. It Is possible to heat to about 1 5 to 70 °C and then raise the temperature gradually up to boiling point. The reaction times are, for example. In the order of 1 5 minutes up to several hours, e .g . 20 minutes to 4 hours, such as 25 to 70 minutes, e.g . 30 to 60 minutes, The reaction can be carried out in a weakly acid range, for example, at a pH in the order of 5 to 6. However, it has been found, that it is useful , but not necessary, to raise the pH during the formation of the
complexes to higher values of up to 1 1 , 1 2, 1 3 or 1 4. In order to complete the reaction, the pH can be lowered then by addition of an acid, for example, to the order of 5 to 6. It is possible to use inorganic or organic acids or mixture thereof , especially hydrogen halide acids such as hydrogen chloride or aqueous hydrochloric acid respectively. As stated above, the formation of the complexes is usually improved by heating . Thus, at the preferred embodiment of the invention, wherein the pH Is raised during the reaction to ranges of at least 5 and above up to 1 1 or 1 4, if is, for instance, possible to work at first at lower temperatures in the order of 1 5 to 70°C, such as 40 to 60°C, e .g . about 50 °C, where after the pH is reduced to values in the order of at least 5 and the temperature Is gradually raised over 50 °C up to boiling point. The reaction times are in the order of 1 5 minutes up to several hours and they can vary depending on the reaction temperature. If the process is carried out with an intermediate pH of more than 5, it is, for example, possible to work 1 5 to 70 minutes, e .g . 30 to 60 minutes, at the enhanced pH, for exa mple at
temperatures of up to 70°C, where after the pH is lowered to a range in the order of at least 5 and the reaction is carried out for a further 1 5 to 70 minutes, e.g . 30 to 60 minutes, at temperatures e .g . up to 70 I "C, and
optionally a further 1 5 to 70 minutes, e .g . 30 to 60 minutes, at higher
temperatures up to boiling point. After the reaction the obtained solution can be cooled to e .g . room temperature and can optionally be diluted and optionally be filtered . After cooling, the pH can be adjusted to the neutral point or a little below, for example, to values of 5 to 7 , by the addition of an acid or base. It is possible to use e .g . the acids and bases which have been mentioned for carrying out the reaction , The solutions obtained are purified and can directly be used for the production of medica ments . However, it is also possible to isolate the Iron (I I I) complexes from the solution e. g , by precipitation with an alcohol such as an alkanol, for exa mple, ethanol .
Isolation can also be effected by spray-drying . Purification can take place in the usual way, especially in order to remove salts. This can, for example, be carried out by reverse osmosis . It is, for example, possible to carry out the reverse osmosis before spray-drying or before a direct application in
medicaments. The iron content of the obtained iron (I I I ) carbohydrate complexes is, for example, 1 0 to 40 % weight/weight, especially, 20 to 35 % weight/weight. They can easily be dissolved in water. It is possible to prepare neutral aqueous solutions which, e.g . have an Iron content of 1 % weight/voi . to 20 % weight/vol , e .g . at room temperature (20°C) , Suc h solutions can be sterilised thermically. It Is possible for example, to fill solutions having an iron content of 1 to 20 % by weight, e .g . 5 % by weight, into vessels such as ampoules or phials of e .g . 2 to 1 00 ml, e.g . , up to 50 ml . The preparation of the intravenous applicable solutions can be carried out as known in the art, optionally using additives which are normally used for parenteral solutions . The solutions can be formulated in such a way that they can be administered by injection or in the form of an Infusion, e .g . , In brine solution . A particularly preferred I r n (111) carbohydrate complex compound, obtained according to description of WO 2004/037865, is marketed for example by the applicant under the tradename Ferinject(®) . According to the Product Monograph of Ferinject(®) (ferric carboxy maltose), Ferinjec†(®) is a Iron( l l l ) complex compound having an Iron oxy hydroxy-core, wherein carboxypolymaltose ligands are coordinatively bond to iron . Ferlnject(®) has a weight average molecular weight of about 1 50 kDa and is readily soluble In water and insoluble in most of organic solvents . Similar processes apply for the manufacture of other iron carbohydrate complex compounds according to the present invention. I .e . , in particular, subjecting iron(lllj-salts to the reaction with carbohydrates in the presence of a base. Also similar dosage forms for intravenous administration as described before are applicable for other iron carbohydrate complex compounds . For example, iron complex compounds of hydrogenoted dextrins are described for example in US 2003 1 91 090 (Al ) which is incorporated by reference herein . Accordingly iron complex compounds of hydrogenoted dextrins may be used, wherein the hydrogenoted dextrins preferably have weight average molecular weights (Mwj equal to or less than 3,000 Doltons, and such iron complex compounds of hydrogenoted dextrins may be prepared for example by a process, comprising the steps of :
(a ) hydrolysing starch or dextrin ,
(b) hydrogenating the resulting hydrolysed dextrin to convert functional aldehyde groups into alcohol groups,
(c) fractloning of the hydrogenoted hydrolysed mixture,
(d) reacting the resulting fractionated hydrogenoted dextrin as an aqueous solution with at least one water soluble ferric salt in the presence of a base .
Average single dosages of the iron-carbohydrate complex compounds used in accordance with the invention may comprise 1 to 30 mg iron per kg body weight, Frequency of administration is determined on the basis of the need of the individual patient i .e . the iron status of the patient. In a preferred aspect of the invention the iron carbohydrate complex compounds can be administered, for example, with a maximal single dose of up to 1 5 mg Iron/kg body weight if administered by intravenous injection or up to 20 mg iron/kg body weight If administered by intravenous drip infusion, for example administering these doses once a week until the cumulative dose for repletion of iron is reached . However, the dosage, frequency and treatment period can be increased or reduced , depending on the age, weight, condition of the patient or severity of the disease ,
The iron-carbohydrate complex compounds used in accordance with the invention can be administered in combination with at least one further active ingredient . Preferably, such at least one further active ingredient is selected from usual antimalarial drugs to prevent or cure malaria, for example quinine and related agents, chloroquine, amodiaquine, pyrimethamine, proguanil, sulfona mides, like sulfadoxine and sulfa methoxypyridazine, preferably in co- administration with the antifoiate pyrimethamine (Fansidar), mefloquine, atovaquone in combination with proguanil (Malarone), primaquine, artemether and artemisinin and derivatives thereof, both preferably in co-administration with longer acting antimalarials such as e.g . !umefantrine, halofantrine, doxycycline, clindamycin, iumefantrine ( iamet / Coartem), azithromycin, pyronaridine, piperaquine, fosmidomycin etc . , and combinations thereof .
Further, such at least one further active ingredient may be selected from antihistamines such as for example azelastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetlrizlne, cyclizlne,
chlorpheniramine, chlorodiphenhyarami e, clemastine, cyproheptadine, desloratadine, dexbrompheniramine, deschlorpheniramine,
dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamlne, ePastine, embramine, fexofenadine, levocetirizine, loratadine, meclozine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine (Phenergan), pyrilamine, quetlapine, rupatadine, tripelennamine, triprolidine and combinations thereof. The at least one further active ingredient may further be selected from the group of usual dietary supplements, comprising minerals, trace elements, vitamins and glucose ,
Co-administration of the iron-carbohydrate complex compounds of the present invention with a combination of one or more selected ingredients of one or more of the above mentioned groups of ingredients is possible, too,
Accordingly, a further object of the invention are combination preparations comprising the Iron-carbohydrate complex compounds used In accordance with the invention and at least one further pharmaceutically active compound, in particular a compound useful for the treatment or prevention of malaria .
Nevertheless, due to the improved physiological and biological compatibility of the selected iron carbohydrate complex compounds it is now possible to administer the iron compounds alone, preferably without co- administration of antihistamines such as e .g . promethazine or anti-malaria agents such as e .g chloroquine and/or artemislnln . Accordingly, one further aspect of the invention relates to the use of the selected iron carbohydrate complex compounds alone in the intravenous therapy of malaria patients or preferably of Individuals at risk of malaria either due to asymptomatic carriage of malaria parasites or due to risk of transmission, Preferably, the I nvention relates to the use of the selected iron carbohydrate complex compounds, wherein the coadministration of antihistamines, such as preferably promethazine, and/or of anti-malaria agents, such as preferably chloroquine and/or artemlsinin, is excluded ,
The Iron-carbohydrate complex compounds used in accordance with the invention are administered intravenously in the form of an aqueous solution or emulsion , They are particularly useful for the therapy as well as for the prophylactic and/or routine iron supplementation of infants, children and pregnant women suffering from malaria and symptoms thereof or living In areas where malaria is endemic ,
The present invention is illustrated in detail by the following examples,
Example 1 Description of the study:
Iron Carboxymaltose (Ferinjec†®) was used for the Treatment In Experimental
Malarial Anemia in mice, 1 . Experiment Design Overview
Table 1 ; Experiment design overview Abbreviations: VCC = trials, Baib/C, C57BI/6, A/J = inbred mouse strains, PBA
= P berghei ANKA, PCA = P chabaudi AS, INF = infected, AM = anti-malarial treatment (sulfasalazine), IT = Ferinject treatment,
*Five mice from group 5-1 and 5-2 were euthanized on day 9 for cross- sectional analyses, Values in brackets denote the number of remaining individuals after day 9,
2, Methods
2.1 Animals
All mice were kept in individually ventilated cages at standard diet and conditions and subjected to frequent Inspections during experiments, Detailed logs of weight, health and intravenous administrations were kept at all times,
2.2 Parasites
Parasites were stored in liquid nitrogen and passed according to standard protocols. Representative batches were tested for non-malarial pathogens according to the Federation of European Laboratory Animal Science
Association standards, Identical (strain-specific) parasite batches were used for ail experimental infections,
2.3 Treatment
All mice enrolled for iron treatment received a total dose of 1800 μg of iron as iron-carboxymaltose (Ferinject) by tall-vein injection administered in three-day regimens. Blood sampling was always performed before intravenous
administrations to avoid assay interference, For anti-malarial chemotherapy, Sulfasalazine (30 mg/l) was delivered in the drinking water ad libitum. All treatments were provided non-blinded and without parallel placebo
administration to controls, 2.4 Haemoglobin measurements
Haemoglobin concentration in whole blood was determined by a modified, plate-based version of Alkaline-Haema†ln-Detergen†-575 (AMD)
spectrophotometry validated in our lab (Zander et al, 1984; Wolf et al. 1984). In brief, 2 jul's of tail-vein blood was obtained in doublets and transferred to 96-weli, round-bottom plates containing 248 μΙ of AHD reagent (2,5 % Triton X- 100 In 0,1 M NaOH) in each well. A chlorohaemin dilution series (0-15 mM) was added to each plate in doublets and used as standard. Absorbance was measured using an ELISA (Enzyme Linked Immunosorbent Assay) reader equipped with a 595 nm filter. Haemoglobin levels were expressed as m monomeric haemoglobin.
2.5 Reticulocyte and parasite counts
Parasite and reticulocyte fractions were counted by flow cytometry as previously described (Hein-Kristensen et al. 2009). A representative subset of data points were selected for reticulocyte count validation by microscopy (see Fig.1 ), In brief, 45 thin blood smears (blinded, 600 counts each) were counted by microscopy and subjected to linear regression against the corresponding flow cytometric estimates. There was an overall good correlation between the two methods (R 2 = 0,92). However, in agreement with the literature, higher reticulocyte counts (i.e. > 40 %) were associated with increased variance and FCM: icroscopy count ratio (overall slope = 0,92); flow cytometry is generally accepted to perform better in this context. For optimal interpretation, fractions were converted to cell concentrations by deriving a Red Biood Cell
concentration from haemoglobin concentration (assuming constant RBC volume, 20 pg hgb monomerlc per RBC & M hgb,monomerlc - 16400 g/mol) and multiplying by fraction (Riley et al. 2001 ). Reticulocyte and parasite
concentrations were expressed as million cells/ml.
2.6 Blood smears
Blood smears were fixed in methanol, stained with 8 % Giemsa under standard conditions and stored for quality control purposes. 2,7 Flow cytometric evaluation of erythroid precursor kinetics
A flow cytometric assay for enumeration of erythroid precursors and their differentiation kinetics was set up as described by (Chen et al. 2009) 2 , 8 Graphics and statistics
Statistical analyses were conducted using SAS software (SAS V, 9 , 2, SAS
Institute, Gary, NC, USA) by application of a mixed effect model (SAS Proc
Mixed) with mouselD and treatment group included as random and fixed effects, respectively, In brief, weight data was normalised to baseline, while the other variables were analysed with no standardisation , To maximise
Information yield, a dynamic control group approach, where all mice are treated as controls until their first treatment, was chosen , Treatment effects were assessed both generally (treated vs , non-treated) and in individual treatment groups compared with controls, using three different time groupings ; First, the data was compared independently of the time variable , Secondly, the infection period was separated Into three phases (aO-9 : "parasitemic", d l 1 - 1 7 ; "recovery" and d l 9 : "re-patency" ) based on data from control mice and analysed , Finally, the specific interactions between treatment and individual days were investigated . Residuals for all variables analysed were normally distributed ,
3 , Results
3. 1 VCC 1 & VCC2 : Ferinject trial In the Balb/C ■ P , berghei ANKA (PBA) malaria model
Two different regimes of Ferinject In combination with anti-malarial treatment were tested . The data revealed no negative effects of Ferinject treatment on parasite proliferation and overall survival , No significant effects of Ferinject treatment on haemoglobin and reticulocyte concentrations were observed (data not shown) . Control animals only developed moderate anaemia (data not shown) ,
3.2 VCC3 ; Test of P . chabaudi AS (PCA) malaria model in Balb/C, C57 BI/6 and A/J mice
The obtained batch of PCA was tested in three mouse strains at different inoculi to identify the optimal model for erythropoietic suppression and test the in vivo characteristics of the parasite batch in relation to the existing literature (Yap et al , 1 992) , The A/J low Inoculum model was chosen due to its ability to induce severe erythropoietic suppression and due to its comprehensive literature base (Chang et al . 2004)(data not shown) , At the low inoculum, A/J mice cleared the parasite spontaneously without any need for antl-maiarial therapy,
3.3 VCC4 : Ferinject trial in the A/J ■ P , chabaudi AS (PCA) malaria model To search for therapeutic effects of Ferinject treatment, three Interventions groups were subjected to three days of Iron treatment at different time points between peak parasitaemia and the haemoglobin nadir, Interestingly, the data showed Increased haemoglobin (d l 0 - d l 4) and reticulocyte (d l 0-d l 2) concentrations in the group receiving Ferinject on day 5- 7 (data not shown) , These elevations occurred simultaneously with a steep i ncrease in weight, Importantly, no differences in parasite concentrations were observed between groups (data not shown) , ANOVA analyses confirmed that haemoglobin and reticulocyte concentrations in the early treatment were significantly higher than the other groups (p < 0 , 05) , A similar tendency was also observed in the group receiving treatment on day 7-9, however this finding was non-significant,
3.4 VCC5 : Main Ferinject trial in the A/J - P. chabaudi AS (PCA) malaria model Based on our findings from VCC4, in the main experiment three infected groups (n = 1 0- 1 5 mice) were subjected to three days of iron treatment at Inoculation ( ίΤ0- 2), at peak parasitaemia (ITS- 7), and at the haemoglobin nadir (IT9- 1 1 ), respectively, along with an untreated, infected control group , Five mice from control and ITO-2 were euthanized on day 9 for cross-sectional analyses; the same procedure was intended for 5- 7 but was cancelled due to logistical problems ,
3 , 4 , 1 Iron treatment generally ■ proves weight, haemoglobin and reticulocyte counts
All mice enrolled survived the infection without any adverse effects, except one mouse that had to be euthanized at the beginning of the experiment due to internal bleedings caused by inoculation ,
All mice exhibited a weight nadir on day 1 1 coinciding with severe anaemia . Iron treated mice generally exhibited significantly less weight loss (p< 0,05), faster weight and hemoglobin recovery (p < 0,01 ) and a strong tendency towards less severe anaemia (p = 0,054). These improvements coincided with increased reticulocyte concentrations from day 11 in ITS 7 and IT9-11 (p < 0,05); this was delayed In group ITC-2 until day 13 (p < 0,02), Furthermore, while controls and ITQ-2 both exhibited significant drops in hemoglobin levels from day 9 to 11 (p <0,05), IT5-7 and 1T9-11 stabilised around day 9 levels. The control, IT5-7 and ΙΪ9-11 groups showed uniform parasite concentrations, !TO-2 exhibited lower parasite concentration on day 7 (p < 0,01). Parasite concentration increased In all groups from day 15 until the end of study; all treatment groups exhibited lower parasite concentrations at day 1 compared to controls, however this did not reach statistical significance (see Table 2),
Treatment of S A still relies on blood transfusion, which remains largely unavailable due to logistical and financial constraints and poses a high risk of blood borne infections including HIV.
Here we show that intravenous iron carboxymaltose (Ferinject®) accelerates both general and erythropoietic recovery in P. chabaudi AS infected A/J mice. Significant treatment effects were observed for three independent outcome measurements (weight, haemoglobin and reticulocyte counts) without any adverse events. Animal studies using the PCA · A/J model have underlined the potentially lethal consequences of erythropoietic suppression caused by malaria (Chang et ctl. 2004). The fact that Iron treatment resulted in a prompt burst in reticulocyte counts compared to controls in our study, lends support to the existence of an important window for intravenous iron treatment in malarial anaemia,
3.4,2 Iron pre-treatment is associated with decreased parasite concentration and accelerated recovery
To look into the effects of early iron administration, a group receiving iron treatment from day 0-2 was included. Overall weight recovery was significantly faster in the pre-treated mice (p < 0,01 ) and hemoglobin levels significantly higher on day 9, 13 and 15 (p < 0,05) as compared with controls (fig, 3A & 3B), Surprisingly, this finding was not accompanied by an onset of retlculocytosis until day 13 (p < 0,01 ), but by a lower parasite concentration compared to controls on day 7 (p < 0,01 , fig, 3C & 3D), Thus, early iron treatment resulted in reduced initial parasitemia which was further underlined by the finding that rebound parasitemia was modulated by Iron treatment in all treatment groups,
3.4.3 Iron treatment In the parasltemic phase is associated with less severe anaemia and accelerated recovery
Iron treatment during the clinical cause of the disease was associated with a significantly higher global weight and hemoglobin minimum (p < 0,05) as well as accelerated recovery when estimated by both weight and haemoglobin indices (p < 0,01 » fig. 2A & 2B); this coincided with a robust reticulocyte response (p < 0,01» fig. 2C), Parasite concentration In this group was not significantly different from controls at any time point during the study (fig. 2D). 3.4,5 Iron treatment during severe anaemia triggers prompt retlculocytosis and accelerated recovery
Iron treatment administered immediately prior to the nadir in hemoglobin concentration (IT9-11), exhibited effects similar to those observed with the IT5-7 treatment regimen. Specifically, weight and hemoglobin recovery was significantly faster compared to controls (p < 0,01 ) and was accompanied by a strong reticulocyte response on day 11 (p< 0,05). No differences in parasite concentrations compared to controls were observed,
Table 2:P-values for treatments effects by day (left section) and phases (right section, phases:"parasitemic": day 0-9, "recovery"; day 11-17 & re patency: day 1 ) on all major outcome parameters, Asterisks Indicate statistical significance at the 5 % level, P-va!ues for analyses, where control outcomes were higher than the corresponding treatment outcomes, are shown in brackets,
4 , Conclusion
Intravenous therapy with iron carbohydrate com plex compounds, in particular, iron carboxymaitose (Ferinjec†®) accelerates recovery from experimental malarial anaemia without any observed negative effects ,
Iron treatment at inoculation was associated with lower parasite concentrations compared to controls .
intravenous therapy with iron carbohydrate complex compounds therefore appears to be an important (adjunct) therapy for malaria patients, in
particular, patients suffering from life-threatening severe malarial anaemia ,
Although the present study was d irected to the treatment of severe malarial anaemia, the findings further give clear indication for the suitability of such intravenous iron supplementation in routine and prophylactic treatment of iron deficiency and anaemia in patients suffering from malaria, even in early stages of Infection as well as in non-infected patients in areas where malaria is endemic .
Example 2 :
Furtj2eMnv ^
Experimental Malarial Anaemia
iLeihal Qdeil
1 , Methods
1 , 1 Experimental Infections and laboratory analyses Experimental Infections were carried out essentially as described In Example 1 , except that initial parasite passage was conducted in A/J mice, which were infected with PCA (Plasmodium chabaudi AS).
All laboratory analyses were carried out as described in Example 1 , except that temperature measurement was conducted using a rectal probe in VCC 7-9 and Infrared scanning in VCC 1 0- 1 1 , The latter was obtained by recording the maximum temperature measured in the infra-sternal region within a 1 x1 x1 cm triangular area ,
1 0 5 was selected as the lethal inoculum for survival studies (unless otherwise indicated below) . A rectal body temperature < 30° C was selected as the main humane endpolnt for these survival studies,
1 , 2 Statistical analyses
Statistical analyses were carried out using R 2, 1 3.1 (R Development Core Team, 201 1 ) with figures created using the R package w ggplo†2" (Wickham , 2009) , Selection of humane endpolnts was conducted using standard logistic regression Implemented in the "glrn" package in R, Survival analyses was conducted by palrwise estimation of the "logrank" test-statistic between treatment groups as implemented in the "survival" package (Therneau &
Lumley, 201 1 ) , The significance of continuous outcome measures was estimated using a linear "mixed-effects" model with treatment groups and mouselD included as fixed and random effects, respectively; models were fitted using the "nlme" package in R (Pinheiro, Bates, DebRoy, Sarkar & R
Development Core Team, 201 1 ) . Significance of individual terms was assessed using the standard conditional t-tesf using a 5% significance level ,
2 , Investigation of ferric -carboxymaltose (Ferlnjec†®) treatment in a lethal model of malarial anaemia 2 , 1 Effects of ferric-carboxymaltose treatment on survival from experimental malaria (VCC8)
To investigate the effects of ferric-carboxymaltose (Ferinject® / FC) treatment on survival, weight, temperature, haemoglobin levels, reticulocyte and parasite concentration in a partially lethal model of PCA,
Based on the previous findings of Ferinject® treatment being mostly effective when administered prior to peak parasitaemia (VCC5, day 5-7), a simple two- group study on mice was conducted, the animals receiving either 600 μg iron as FC or the equivalent volume of saline i.v. every day from day 4-6. Based on power-calculations, a group size of 20 mice was chosen , Each mouse was inoculated with 1 05 parasites and weight, body temperature (rectal), haemoglobin concentration, reticulocyte and parasite levels measured dally from day 3. One mouse from the FC group died before signs of severe malaria had developed, probably due to intestinal perforation, shortly after
measurement of temperature, Results :
Parasite concentrations exhibited profound heterogeneity and a threshold of
5 · 1 08 parasltes/ml on day 6 was used to identify potential outliers. Using this criterion, two mice were excluded from the control group (VCC8_A) and six mice excluded from the FC-treatment group (VCC8 B) . Although this points towards some assoc iation with treatment group, this difference in exclusion frequency was not significant (Fisher's Exact Test, p = 0, 1 3) and as five of the excluded mice likely shared inoc ulation syringe, the eight outliers were excluded from downstream analysis. For completeness, the statistical analyses were also applied to the full dataset with no implications for the overall conclusions , Survival at day 1 5 was approximately 39% and 54% in saline and FC treated groups, respectively, however this difference was not significant (figure 4, p = 0 ,67).
Figure 4 shows summaries of all continuous outcome measures after outlier exclusion , No obvious explanation for the insignificant difference in survival was observed , Body temperature exhibited an alternating pattern with a significant swift decrease on day 8 followed by a significant increase on day 9 In the treatment group relative to controls A similar pattern was observed for reticulocyte concentrations (and fractions) with significantly more pronounced reticulocytosis in the controls on day 1 2 followed by significantly higher counts in treated mice on day 1 4 and 1 5. Logistic regression analysis was applied to the haemoglobin data of terminally ill mice and the minimum value of survivors and no significant association between anaemia and death was detected although this approach is slightly biased by the use of the
temperature end- point.
Conclusion ;
There was an insignificant trend towards a modest survival effect of FC treatment and some significant oscillations in reticulocyte levels and
temperature were observed ,
3 Investigation of ferric-carboxymaltose (Fennject® ) treatment in
combination with anti-malaria! therapy in a lethal model of malarial anaemia
3. 1 Effect of ferric -c a r boxymaltos© and artesunate on survival from malarial anaemia (VCC9J
Objective :
To investigate the effect of FC and F C/artesunate combination therapy on survival, weight, temperature and haemoglobin, reticulocyte and parasite concentrations in a partially lethal model of PCA.
As VCC8 did not provide conclusive evidence of a treatment effect and to search for treatment effects in a more clinically relevant context, both treatments were to be administered at the onset of anaemia defined as a 20% mean drop in haemoglobin concentration , which In the current case occurred on day 7 ,
The standard dose of 600 μg iron as FC was administered i ,v. from day 7 to 9. Artesunate (Sigma-Aldric h, Germany) was suspended in saline and 60 jyg administered by i . p. injection on day 7 to 9. Four groups of ten mice were set to receive saline, sahne/FC, artesunate/saline or both artesunate and FC , Each mouse was inoculated with 1 05 parasites and weight, body temperature, haemoglobin concentration, reticulocyte and parasite levels measured daily from day 3 ,
Results ;
Two outliers were excluded using the threshold from VCC8 , Both artesunate and FC treatment alone or in combination exhibited significant effects on survival compared to saline-only controls (figure 6, p < 0, 06); these effects were insensitive to exclusion of the outliers, Parasite concentrations were
significantly lower on day 6 (i .e . prior to treatment) in the FC and artesunate mono-treatment groups (figure 7A) . Artesunate treatment (VCC9_C and
VCC9_D) caused a decrease In parasite fraction from day 8; a significant decrease in parasite fraction for FC treatment was observed on day 9. in line with the survival data, temperatures were significantly elevated in all treatment groups on day 8 and 9. Finally, FC treatment was associated with significant increase in reticulocyte fraction and concentration on day 1 2.
Investigation of combination treatment effects relative to artesunate mono- treatment showed that the FC group had significantly higher parasite fractions on day 6 and 8, however the first occurred before administration of treatment and the effect hence appear unrelated to treatment (figure 7B) , Interestingly, FC treatment caused a significant increase in reticulocyte fraction and concentration on day 1 3 following a significant increase In haemoglobin concentration as compared to artesunate mono-treatment (figure 7B).
Conclusion :
Both artesunate and FC - alone or in combination - was associated with a significantly increased survival compared to saline-only controls. No
indications of erythropoietic effects mediating the increase In survival due to FC-treatment were observed , indeed, haemoglobin levels remained stable for vehic!e/FC vs. vehicle/vehicle and Ar†/FC vs , Art/vehicle and reticulocyte production remained largely suppressed until day 1 0, The lower parasite fractions in all treatment groups - although modest in the FC group - points towards direct effects on the parasite with unknown secondary effects on survival rather than effects mediated through enhanced erythropoiesis, As this was also observed before administration of treatment, a systematic bias in the treatment groups cannot be ruled out,
Interestingly, some indications of erythropoietic effects of FC were observed during the recovery phase, where FC appeared to provide a modest increase in haemoglobin and reticulocyte concentrations independent of parasite densities around day 1 2 and 1 3 in both the FC mono- (vehicle/FC vs.
vehicle/vehicle) and combination therapy (Art/FC vs Art/vehicle) setups (figure 7 A and figure 7B) . 3 , 2 Effect of ferric-carboxymaltose (Ferinject®) and artesunate on survival from malarial anaemia (VCC 1 0)
Objective ;
To attempt to reproduce the effects of FC mono-therapy observed in VCC9 using an inoculum of 1 0 5 parasites and investigate the effects of all four treatment regimens tested in VCC9 at a lower inoculum (5* 1 0 4 parasites) ,
Methods ;
Four groups of ten mice were inoculated with 5* 1 0 4 parasites and set to receive vehicle, vehicle/FC, artesunate/vehicle, or both artesunate and FC; two groups of ten mice were inoculated with 1 0 5 parasites and set to receive either vehicle or FC . Weight, body temperature (infrared), haemoglobin concentration, reticulocyte and parasite levels were measured daily from day 3. As in VCC9, treatments were administered at the onset of anaemia defined as a 20% mean drop In haemoglobin concentration, which in the current case occurred on day 7 and 8 for high and low inocuia, respectively. The standard dose of 600 μg iron as FC was administered i .v, for three days; artesunate (Slgma -Aidrlch, Germany) was suspended in saline and 60 μg administered by i . p. injection for three days ,
Results :
Again, substantial heterogeneity in parasite concentrations was observed around day 6 and 7 especially in the mice receiving the low inoculum . Using the threshold from earlier experiments on day 7 - instead of day 6 due to the slower progression of infection at the lower inoculum - three mice were excluded . As there was no evident outlier pattern supporting the use of the day 6 threshold used in the earlier experiments in the high inoculum groups, the low inoculum threshold was used for these groups as well leading to the exclusion of a single mouse from group VCC 1 0 E . In the low inoculum groups, survival was significantly higher in the FC mono-therapy and combination treatment groups (figure 8A, p < 0.05) and borderline significant in the artesunate mono-therapy group (p = 0,057) , As inspection of the data identified other putative outliers in the low inoculum g roups, the analysis was repeated using a threshold of T O 9 parasites/ml on day 7. For this set, the combination therapy group remained statistically significant, whereas the two other groups were only borderline significant (p < 0 , 1 ) , In the high inoculum groups, survival in the saline-treated group was larger than the previous experiments and the difference In survival was not significant (figure 8B) , However, the trend was increased survival In the FC mono-therapy group .
For the high inoculum case there was significantly higher parasite fractions and concentrations on day 7 i .e . prior to treatment hence again pointing towards a systematic bias between groups (figure 9) . Later in the infection, FC treatment was associated with significantly higher haemoglobin levels on day 1 1 , 1 3 and 1 4, and a significantly increased reticulocyte concentration on day 1 4.
Reticulocyte fractions were significantly elevated in the saline -treated group on day 1 1 and 1 2 ,
In the mice receiving the low inoculum , there were significantly lower parasite concentrations prior to treatment in treatment groups VCC 1 0 B and VCC T 0_C as compared to controls (figure 1 0) . As expected from the survival data, temperature was significantly higher in all treatment groups . Surprisingly, reticulocyte fractions were significantly higher in the saline-treated group compared to all other treatment groups. This was probably caused by
significantly lower haemoglobin in these mice. The reticulocyte concentrations were only significantly different between saline- and artesunate treated groups. Generally, artesunate treated mice exhibited a prolonged course of severe disease as judged by weight and haemoglobin levels that were significantly lower in these groups compared to saline-treated mice. The analysis was repeated using the higher outlier exclusion threshold with no major impact on the results .
Conclusion :
I n the low inocu lum groups, all treatment regimens were associated with significantly - or borderline significantly - increased survival . No obvious cause of this survival effect was identified , A non-significant survival effect was observed in the high-inoculum sub-study, and haemoglobin and reticulocyte concentrations were significantly higher In the FC treated mice, 3.3 Effect of ferric-carboxymaltose (Ferinjec†®) and a rtesunate on the course of re-infection with PCA (VCC 1 1 )
To investigate the course of PCA re-infection in mice previously infected with PCA and treated with either artesunate or FC-arresunate combination therapy.
Methods ; After termination of VCC 1 0, treatment groups VCC 1 0_C and
VCC 1 0_D received an additional course of artesunate treatment (60 μg x 3), to ensure clearance of parasites from the last infection, and all mice were re- inoculated with 1 0 5 parasites on day 0 (day 26 after the initial inoculation) , Standard parameters were measured from day 3 to 1 2 at which point no parasites were detectable in any mice,
Results ;
No deaths were observed in any of the treatment groups . As seen in figure 1 1 , FC-ar†esuna†e combination treatment was associated with significantly lower parasite fraction and concentration on day 6 and 7 after inoculation , This was further associated with significantly increased weight levels, Significantly increased reticulocyte levels were observed in the artesunate only treated mice likely in response to the lower haemoglobin levels observed prior to this response.
Again, the included mice exhibited heterogeneous parasite levels likely resulting at least partly from varying degrees of immunity as the mice were challenged with the same batch as used for their primary infection , An outlier exclusion threshold of 2* 1 0 8 parasites/ml on day 6 was used due to the lower magnitude of parasite levels in the re- Infection . The results presented above were relatively robust towards higher exclusion thresholds, Conclusion ;
Mice receiving FC-ar†esuna†e combination treatment exhibited significantly lower parasite levels and higher weight compared to artesunate mono-treated controls.
4. Summary and Conclusion
These studies provide substantial evidence for a surviva l effect of intravenous ferric-carboxyma!tose (Ferinject® / FC) in a lethal malaria model , Signs of late erythropoietic effects of FC treatment were observed, however no linkage between erythropoietic effects of FC and survival could be established , Instead, sparse observations suggested a negative effect of FC on parasite growth indicating a candidate mechanism behind the survival effect, however data was inconclusive on this matter .
Interestingly, re-infection of FC treated mice after clearance of the primary infection by artesunate caused a decrease in parasltaemia and an increase in weight as compared to artesunate-only treated mice, Such findings indicate the suitability of FC for reducing prevalence and/or outbreak of malaria attacks as wel l as frequency and/or Intensity of recurrence of malaria attacks.
No observations of negative outcomes of FC treatment were made in any of these studies, Safety of FC has proven excellent in all these trials, in particular re-infections after FC treatment appear to be milder than In control groups . Description of the Figures; Fig, 1 :
Reticulocyte count validation by microscopy. 45 thin blood smears, 600 counts each, were counted and plotted against the corresponding flow cytometric estimates,
Figs, 2;
Overview of central outcome parameters In VCC5 in treatment groups ΊΤ a ' 5-7" (Infected, iron treated day 5-7) and "IT d9-11 " (infected, iron treated day 9-11) vs, controls (infected),
Fig 2A: Mean weight (normalisedto baseline) + /- SEM,
Fig 2B: Mean hemoglobin concentration (m ) +/- SEM,
Fig 2C: Mean reticulocyte concentration (million cells/ml) +/- SEM,
Fig 2D: Mean parasite concentration (million cells/ml) +/- SEM.
Figs, 3:
Overview of central outcome parameters in VCC5 in treatment groups "IT d0-2 " (infected, iron treated day 0-2) vs, controls (infected),
Fig 3A: Mean weight (normalised to baseline) +/- SEM,
Fig 3B: Mean hemoglobin concentration (mM) +/- SEM,
Fig 3C: Mean reticulocyte concentration (million cells/ml) +/- SEM,
Fig 3D: Mean parasite concentration (million ceils/ml) +/- SEM, Fig. 4:
Cumulative survival of A/J mice infected with 10 5 PCA and treated with either FC (600 μg x 3) or an equivalent volume of saline from day 4-6 (VCC8, n = 31 ), Nine mice were excluded, Fig, 5:
Continuous outcome measures for A/J mice Infected with 10 6 PCA and treated with either FC (600 jug x 3) or an equivalent volume of saline from day 4-6 (VCC8, n = 31). Nine mice were excluded, Asterisks indicate statistical significance relative to VCC8_A (5% level).
Fig. 6:
Cumulative survival for A/J mice infected with 10 5 PCA and treated with either saline, FC (600 pg x 3), artesunate (60 μg x 3) or both FC and artesunate from day 7-9 (VCC9, n = 38), Two mice were excluded.
Fig, 7A:
Continuous outcome measures for A/J mice infected with 10 5 PCA and treated with either saline, FC (600 jug x 3), artesunate (60 pg x 3) or both FC and artesunate from day 7-9 (VCC9, n = 38). Two mice were excluded, Asterisks indicate statistical significance relative to VCC9_A (5% level), Fig. 7B;
Excerpt - Data VCC9_C and VCC9_D; asterisks indicate statistical significance relative to VCC9_C (5% level).
Fig. 8A:
Cumulative survival for four groups of A/J mice infected with 5*10 4 (VCC I 0__A - VCC10_D) and treated with saline, FC (600 μ x 3), artesunate (60 μg x 3) or both FC and artesunate from day 8-10;), Two mice were excluded (n = 38),
Fig. 8B;
Cumulative survival for two groups of A/J mice infected with 10 5 PCA (VCC10_E & VCC 10_F) and treated with either saline or FC (600 jjg x 3) from day 7-9. One mouse was excluded (n= 19),
Fig. 9;
Continuous outcome measures for A/J mice infected with TO 5 PCA and treated with either saline or FC (600 μg x 3) from day 7-9 (VCC10 high, n = l 9), One mouse was excluded, Asterisks indicate statistical significance relative to VCC10 E (5% level), Fig, 10;
Continuous outcome measures for A/J mice infected with 5*10 4 PCA and treated with either saline, FC (600 μg x 3), artesunate (60 jug x 3) or both FC and artesunate from day 8-10 (VCC1 QJow, n = 38), Two mice were excluded, Asterisks indicate statistical significance relative to VCC10_A (5% level),
Fig, 11 :
Continuous outcome measures for A/J mice re-infected with TO 5 PCA on day 26 after initial infection with artesunate or FC-artesunote combination treatment, Five mice were excluded, Asterisks indicate statistical significance relative to VCC ! 1 A (5% level),
Atkinson, S.H., ockett, K , Sirugo, G., Bejon, P. A.. Fulford, A,, O'Connell, M.A., Baiiey, R,, Kwiatkowski, D.P., and Prentice, A.M. (2006), Seasonal childhood anaemia in West Africa Is associated with the haptoglobin 2-2 genotype, PLoS Med 3, el 72.
Atkinson, S.H., Rocket! K.A., Morgan, G., Bejon, P. A , Sirugo, G., O'Connell, M.A , Hancnard, N,, Kwiatkowski, D. P. , and Prentice, A.M. (2008), Tumor necrosis factor SNP haplotypes are associated with iron deficiency anemia in West African children, Blood 112, 4276-4283,
Bauer, A,, Tranche, F., Vessely, 0., Kellendonk, C, Reicnardt, H.M., Stelnlein, P., Schurz, G,, and Beug, H, (1 99). The glucocorticoid receptor Is required for stress erythropoiesis. Genes Dev 13, 2996-3002,
Bhcftt R. V., Joshi S, K , Shah M. C, (1966). Total Dose Intravenous infusion of Iron-Dextran (Imferon) in Severe Anemia, Am, J, Obst & Gynec, 94 (8), 1098- 1102,
Byles A, B, and Avita D ' Sa (1970), Reduction of Reaction due to Iron Dextran
Infusion using Chloroquine, British Medical Journal 3, 625-627,
Byles A. B, and Avita D'Sa (1975), A Case for Empirical Therapy in the yanagement of Anaemia in Pregnancy in Developing Countries, East African Medical Journal 5 (10), 556-563.
Broody, V.C., Lin, N.L., Priestley, G.V., Nocka, K., and Wolf, N.S, (1996), Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spieen. Blood 88, 75-81 , Burchard, G . D . , Radloff, P., Philipps, J,, Nkeyi, M., Knobloch, J., and Kremsner,
P.G (1995), Increased erythropoietin production in children with severe malarial anemia, Am J Trop Med Hyg 53, 547-551.
Casals-Pascual, C, Kai, 0,, Cheung, J.0,, Williams, 5,, Lowe, B., Nyanoti, M. , Williams, T.N., Maitland, K., Molyneux, M., Newton, C.R., et al. (2006),
Suppression of erythropoiesis in malarial anemia Is associated with hemozoin in vitro and in vivo, Blood 108, 2569-2577,
Cercamondi, C.I., Egli, I.M., Ahouandjinou, E,, Dossa, R,, Zeder, C, Salami, L., Tjalsma, H,, Wiegerinck, E,, Tan no, T., Hurrell, R., Hounhouigan, J. and
Zimmermann, M.B. (2010), Afebrile Plasmodium falciparum parasitemia
decreases absorption of fortification iron but does not affect systemic iron utilization: a double stable-Isotope study in young Beninese women. Am J Clin Nutr 10,3945/AJCN.2010.30051.
Chang, K.H., Tarn, ., and Stevenson, MM, (2004a), Inappropriately low reticulocytosis in severe malarial anemia correlates with suppression in the development of late ©rythroid precursors. Blood 103, 3727-3735,
Chang, Κ,Η,, Tarn, M., and Stevenson, M.M. (2004b), Modulation of the course and outcome of blood-stage malaria by erythropoietin -induced reticulocytosis. J Infect Dis 189, 735-743.
Chen, K. , Liu, J,, Heck, S., Chasis, J. A., An, X,, and Mohandas, N, (2009), Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis, Proceedings of the National Academy of Sciences 106, 17413-17418. Available at:
http://www.pnas.org/content/! 06/41/17413. abstract,
Cox, S.E., Doherry, C, Atkinson, S.H., Nweneka, C.V,, Fulford, A, J,, Ghattas, H,, Rocken, K.A., Kwiatkowski, D.P., and Prentice, A M, (2007), Haplotype
association between haptoglobin (Hp2) and Hp promoter 8NP (A-61C) may explain previous controversy of haptoglobin and malaria protection, PLoS ONE 2, Θ362, de Mast, Q., Nadjm, B., eyburn, H., Kemna, Ε,Η,, Amos, B,, Laarakkers, CM,, Si!olye, S., Verhoef, H,, Sauerwein, R.W., Swinkels, D.W., et al. (2008),
Assessment of Urinary Concentrations of Hepcidin Provides Novel Insight Into Disturbances in Iron Homeostasis during Malarial Infection, J Infect Dis, de Mast, Q., van Dongen-Lases, E.G., Swinkels, D.W., Nieman, A E.,
oestenberg, M,» Druilhe, P , Arens, T.A., Luty, AU, Hermsen, C.C., Sauerwein, R.W., et al. (2009). Mild Increases In serum hepcidin and ipterleukin-6
concentrations impair iron Incorporation in haemoglobin during an
experimental human malaria Infection. Br J Haematol.
de Mast, Q., Syofruddin D,, eijmel, S., iekerink, T. O., Deky, O., Asih, P. B., Swinkels, D. W, and van der Ven, A. J. (2010). Increased serum hepcidin and alterations in blood iron parameters associated with asymptomatic P.
falciparum and P. vivax malaria. Haematologlca 95 (7), 1068-1074.
Doherty, CP., Cox, S.E , Fulford, A, J., Austin, S,» Hllmers, D C, Abrams, S.A., and Prentice, A.M. (2008). Iron Incorporation and post-malaria anaemia. PLoS ONE 3, Θ2133.
Evans, K.J,, Hansen, D.S., van ooijen, N., Buckingham, L.A., and Schofield, L,
(2006). Severe malarial anemia of low parasite burden in rodent models results from accelerated clearance of uninfected erythrocytes. Blood 107, 1192-1199. Fleming A. F. (1 89), The Aetiology of Severe Anaemia in Pregnancy in Naola, Zambia, Annals of Tropical Medicine and Parasitology 83 (1), 37-49.
IDS Health and Development Information (2009). Iron supplements for
childhood anaemia in malaria endemic areas. Health reporter, 13, August 2009.
Jakeman, G.N., Saul, A., Hogarth, W.L., and Collins, W.E. (1999). Anaemia of acute malaria Infections in non-immune patients primarily results from
destruction of uninfected erythrocytes. Parasitology 119 (Pt 2), 127-133.
Kabyemela, E. ., Fried, M., urtis, J.D., Mutabingwa, T.K., and Duffy, P.E.
(2008). Decreased susceptibility to Plasmodium falciparum infection in pregnant women with iron deficiency, J Infect Dis 198, 163-166.
Koka, S., Poller, M., Lamprecht, G., Bolni, K.M., Lang, C, Huber, S. ., and Lang, F. (2007). Iron deficiency influences the course of malaria in Plasmodium berghei infected mice. Blochem Blophys Res Commun 357, 608-614.
Kurtzhals, J, A., Adabayerl, V., Goka, B.Q., Akanmori, B.D., Oliver-Commey, J.O., Nkrumah, F.K , Behr, C, and Hvlid, L, (1998), Low plasma concentrations of interleukin 10 in severe malarial anaemia compared with cerebral and uncomplicated malaria. Lancet 351 , 1768-1772.
Kurtzhals, J. A., odrigues, 0,, Addae, ML, Commey, J.O., Nkrumah, F.K., and Hvlid, L, (1997), Reversible suppression of bone marrow response to
erythropoietin in Plasmodium falciparum malaria. Br J Haematol 97, 169-174, Lamikanra, A. A,, Brown, D., Potocnik, A,, Casals-Pascual, C, Langhorne, J,, and Roberts, D.J. (2007). Malarial anemia: of mice and men. Blood 110, 18-
Lee, P., Peng, H., Gelbart, T,, Wang, L, and Beutler, E. (2005). Regulation of hepcldin transcription by in†erleukin-l and interleukin-6. Proc Natl Acad Sci USA 102, 1906-1910.
Lee, T.S., and Chau, L.Y. (2002), Heme oxygenase- 1 mediates the antiinflammatory effect of interleukin- 10 in mice. Nat Med 8, 240-246.
Liu, Y., Pop, R., Sadegh, C, Brugnara, C, Haase, (Fl and Socolovsky, M.
(2006). Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo. Blood 108, 123-133.
Menendez, C Kahigwa, E,, Hirt, R,, Vounatsou, P , Aponte, J.J,, Font, F., Acosta, C.J,, Schellenberg, D.M., Galindo, CM., Kimario, J., et a!. (1997), Randomised placebo-controlled trial of iron supplementation and malaria chemoprophylaxis for prevention of severe anaemia and malaria in Tanzanlan infants. Lancet 350, 844-850,
Menendez, C, Toda, J,, Aionso, P.L., Francis, N., Lulat, S,, Ceesay, S., M'Boge, B., and Greenwood, B.M, (1994), The effects of iron supplementation during pregnancy, given by traditional birth attendants, on the prevalence of anaemia and malaria, Trans R Soc Trop Med Hyg 88, 590-593,
Ojukwu, J. U,, Okebe, J. U., Yahav, D,, Paul, M, (2009). Oral Iron
supplementation for preventing or treating anaemia among children in
malaria-endemic areas. Cochrane Database of Systematic Reviews, issue 3. Oppenheimer, S.J., Gibson, F.D., Macfanane, S.B , Moody, J.B,, Harrison, C, Spencer, A. and Bunari, O. (1986). Iron supplementation increases prevalence and effects of malaria; report on clinical studies in Papua New Guinea, Trans R Soc Trop Med Hyg 80 (4), 603-612. Oppenheimer, S.J., Macfarlane, S.B., Moody, J.B,, and Harrison, C. (1 86).
Total dose Iron infusion, malaria and pregnancy in Papua New Guinea. Trans R Soc Trop Med Hyg 80, 818-822.
Othoro, C, Lai, A. A., Nah!en, B., Koech, D., Orago, A.S., and Udhayakumar, V, (1999). A low interleukin-10 tumor necrosis factor-alpha ratio is associated with malaria anemia in children residing in a ho!oendemic malaria region in western Kenya. J Infect Dis 179, 279-282,
Pinhelro, J., Bates, D., DebRoy, S., Sarkar, D,, & R Development Core Team.
(2011). R package nlme; Linear and Nonlinear Mixed Effects Models.
Pinto, J, P., Ribeiro, S., Pontes, H., Thowfeequ, S., Tosh, D,, Carvalho, F., and
Porto, G. (2008). Erythropoietin mediates hepcidin expression in hepatocytes through EPO signaling and regulation of C/EBPalpha. Blood 111 , 5727-5733.
Price, R.N., Simpson, JA, Nosten, F., Luxemburger, C, Hkirjaroen, L, ter Kuile, F.,
Chongsuphajaisiddhi, T,, and White, N.J, (2001), Factors contributing to anemia after uncomplicated falciparum malaria. Am J Trop Med Hyg 65, 614-
R Development Core Team (2009). R: A Language and Environment for
Statistical Computing. Available at: http://www.r- project .org.
R Development Core Team (2011 ), R: A Language and Environment for
Statistical Computing, Vienna Austria; R Foundation for Statistical Computing Sazawal, S. et ai. (2006). Effects of routine prophylactic supplementation with iron and folic acid on admission to hospital and mortality in preschool children in a high malaria transmission setting: community-based, randomised, placebo-controlled trial. Lancet 367, 133-43. Available at:
http://www.ncbi.nlm.nih.gov/pubmed/! 6413877,
ScienceDaily (July 9, 2009). Give Children Iron Supplements: They Don't
Incease larla Risk, Study Suggest.
Scrimshaw, N, S. (1 91), Iron Deficiency. Scientific American 265 (4) 46-52, Stadie, VV.C. (1 20). A method for the determination of methemoglobin in whole blood. J Biol Chem 41 ,
Stoltzfus, R. J., Kvaisvig, J. D., Chwaya, H. M., Montresor, A,, Albonico, M., Tielsch, J. M, , Ravioli, L,, Poliitt, E. (2001 ). Effects of iron supplementation and anthelmintic treatment on motor and language development of preschool children In Zanzibar; double blind, placebo controlled study, B J 323 1-8, Tacchini, L., Gammella, E,, De Ponti, C, Recalcari, S,, and Cairo, G, (2008), Role of HIF-1 and NF-kappaB transcription factors in the modulation of transferrin receptor by inflammatory and anti-inflammatory signals. J Biol Chem 283» 20674-20686.
Therneou, I,, & Lumley. T. (2011 ), package survival; Survival analysis, including penalised likelihood.
Van den Hombergh, J., Dalderop, E, and Smit Y, (1996), Does Iron Therapy Benefit Children with Seere Malaria-associated Anaemia? A Clinical Trial with 12 Weeks Supplementation of Oral Iron In Young Children from the Turiani Division, Tanzania. Journal of Tropical Pediatrics 42 (4), 220 - 227,
WHO (2006), Iron supplementation of young children In regions where malaria transmission Is intense and infectious disease highly prevalent,
Wickham, H, (2009), ggplo†2; elegant graphics for data analysis, Springer New York,
Wickramasinghe, S.N., Phillips, R.E., Looareesuwan, S, , WarrelL D.A., and
Hughes, M, (1987). The bone marrow in human cerebral malaria; parasite sequestration within sinusoids, Br J Haematol 66, 295-306,
Yap, G, S., and Stevenson, M. M, (1992), Plasmodium chabaudi AS:
Erythropoietic responses during infection in resistant and susceptible mice, Experimental Parasitology 75» 340-352,