Patent Application: US-87544097-A

Abstract:
the invention is to a method for stimulating the production of fetal hemoglobin in a mammal having a hemoglobinopathy , comprising administering heme or a derivative thereof .

Description:
it was discovered that a heme preparation ( heme arginate ) was able to decrease incidence of mutagenesis ( mouse micronucleus test ) in the mouse in vivo ( table 2 ). in the mouse the antimutagenic effect was demonstrated at rather high dose level of 36 mg / kg . in humans the active dose level can , however , be much lower ( even 10 - times lower ) because the elimination rate is slower in humans ( auc 998 μg / ml / h ) than in rodents ( auc in the rat 64 μg / ml / h ). the both auc values were observed after the intravenous dose of 3 mg / kg ( 21 and kangasaho , unpublished information ). thus heme preparations can be used for decreasing incidence of mutagenesis in patients treated with known mutagenic therapies like cytostatics and / or radiotherapy . in addition heme preparations can be used in prevention of mutagenesis or progress of mutagenic development in persons exposed to mutagenic chemicals or radiation in accidents or via environmental or occupational circumstances . it was also discovered that a heme preparation ( heme arginate ) was able to decrease proportion of blasts in bone marrow of some patients with myelodysplastic syndromes ( mds ). it is known that heme arginate is clinically effective in part of mds patients by increasing the blood cell counts . however , there has been fear that the use of heme therapy ( 13 , 14 , 15 ) like the use of other hematopoietic growth factors in mds patients may increase the risk of disease progression into leukemias via enhancing the proportion of blasts . surprisingly it was noticed in a clinical study that heme arginate was able to decrease proportion of blasts ( blasts and myeloblasts ) in bone marrow of some mds patients ( table 3 ). this finding is clinically significant because the proportion of blasts in the bone marrow is strongly correlated into the expected survival time of the patient ( table 1 ) and the natural course of mds varies from a relatively static to aggressive progression i . e . spontaneus remission can not be expected ( 14 ). in the published studies mds progression was seen in 4 / 26 patients ( 13 ) and in 4 / 14 patients ( 15 ) which is considered to be normal in these patients . in the latter study ( 15 ) decrease of blasts in 1 / 14 patient was reported but considered to be an occasional event . in the data presented here a clear decrease was reported in 5 / 21 patients ( table 3 ) with a smaller decrease in 12 / 21 , no effect in 1 / 21 and slight increase in 3 / 21 patients ( table 4 ). thus some decrease was seen totally in 17 / 21 patients . however , the clinically significant bone marrow blast decreasing activity of heme seems to be limited to a patient subpopulation . thus treatment with heme preparations by decreasing the proportion of blasts in bone marrow in a patient subpopulation can prevent transformation of &# 34 ; milder &# 34 ; subtypes of the mds into the more malignant subtypes ( raeb , raeb - t ) and further into leukemia . because increase in the proportion of blasts in bone marrow is the most critical factor in development of leukemia , heme preparations can be active also in other preleukemic conditions than the mds . in one of the patients the proportion of blasts ( blasts + myeloblasts ) in bone marrow was 30 % before start of the treatment with heme arginate and it decreased into 10 % after the treatment . because 30 % proportion of blasts in bone marrow is the limit value between mds ( raeb - t ) and acute leukemia ( table 1 ) it can be assumed that treatment with heme preparations can be effective also in leukemias ( slowing down or prevention of disease progression or even transformation of leukemia back into preleukemic or non - leukemic state ) and not only in preleukemic diseases like the mds . it was also discovered that heme arginate can affect the globin chain expression in erythroid precursor cells originating from patients with hemoglobinopathies ( β - thalassemias , sickle cell anemia ). heme arginate increases the amount of total hemoglobin ( fig1 ) produced by these cells and increases the proportion of fetal hemoglobin ( fig2 ). it is especially surprising how strongly the effect of heme arginate on the proportion of fetal hemoglobin was potentiated by the presence of hydroxyurea ( fig3 ). the ability of heme arginate alone or in combination with hydroxyurea ( or other agents ) to induce fetal hemoglobin production is of considerable clinical importance . an increase in fetal hemoglobin in patients with hemoglobinopathies ( like β - thalassemia and sickle cell disease ) may ameliorate the clinical symptoms of the disease . in addition to heme preparations also other tretments can be used concomitantly to enhance the therapeutic potential of heme . such concomitant treatments can be e . g . inhibitors of heme catabolism ( like tin or zink - protoporphyrins , tin or zink - mesoporphyrins etc .) or cytostatics ( especially at low dose ) etc ., retinoids or other differentiation inducing agents , cytokines , biological growth factors etc . and in the case of hemoglobinopathies hydroxyurea or other pharmacological agents known to induce production of fetal hemoglobin . in mitotic cells in which chromosomal damage has been caused acentric fragments of the chromosomes do not separate at the anaphase stage of cell division . after telophase these fragments may not be included in the nuclei of the daughter cells and hence will form single or multiple micronuclei ( howell - jolly bodies ) in the cytoplasma of these cells . micronuclei are seen in a variety of cells , but erythrocytes are chosen for examination since micronuclei are easily detected in this cell type . a few hours after the last mitosis is completed , erythroblasts expel their nucleus . young erythrocytes stain blue with giemsa due to the presence of ribonucleic acid , which gradually disappears so that more mature erythrocytes stain pink with giemsa . the young blue - staining cells are known as polychromatic erythrocytes and micronuclei are readily detected in this cell type . the test animals were specific pathogen free cd - 1 outbred mice of swiss origin weighing between 22 and 24 grams and approximately 40 days old during the study . the mice were acclimatized for four days and after that dosed by intravenous injection with a volume of 10 ml / kg body weight either with saline solution ( vehicle control ) or with heme arginate solution at the dose 36 mg hemin / kg body weight . five male and five female mice were sacrificed by cervical dislocation 24 , 48 , and 72 hours after dosing . a direct bone marrow smear was made from femur onto a slide containing a drop of calf serum . the smears were fixed in methanol , air dried , and stained for 10 minutes in 10 % giemsa solution . the stained smears were examined ( under code ) by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal . in a clinical study patients with myelodysplastic syndromes were treated with intravenous infusions of heme arginate at the dose level 3 mg hemin / kg . the infusions were given first on four consecutive days and after that once a week for eleven weeks . thus totally 15 infusions were given to each patient . bone marrow sample was collected from each patient before start of the heme arginate infusions and one week after the last infusion . proportion of blasts and myeloblasts were determined in the bone marrow samples by using standard hematological laboratory procedures . the definition of &# 34 ; a blast &# 34 ; as used in the classification of myelodysplastic syndromes includes both blasts and myeloblasts ( 9 ). peripheral blood mononuclear cells obtained from normal volunteers and either sickle cell anemia or β - thalassemia patients were isolated by centrifugation on a gradient of ficoll - hypaque and seeded at a density of 5 × 10 6 cells / ml in alpha - minimal essential medium supplemented with 10 % fetal calf serum ( fcs ), 1 μg / ml cyclosporin a , and 10 % conditioned medium collected from cultures of the 5637 bladder - carcinoma cell line . the cultures were incubated at + 37 ° c . in an atmosphere of 5 % co 2 in air with extra humidity . following 7 - day incubation in this phase i culture , the non - adherent cells were harvested , washed , and recultured in fresh medium composed of alpha - medium , 30 % fcs , 1 % deionized bovine serum albumin , 1 × 10 - 5 m β - mercaptoethanol , 1 . 5 mm glutamine , 1 × 10 - 6 m dexamethasone , and 1 u / ml human recombinant erythropoietin . hemoglobin containing cells were determined by using the benzidine - hcl procedure . the effect of heme arginate on fetal hemoglobin ( hb - f ) production was determined by growing human erythroid precursors in the second phase of the above described liquide cell culture in the presence of 1 u / ml erythropoietin and different concentrations of heme arginate . the combination effect of heme arginate and hydroxyurea was investigated with 100 μm heme arginate added on day 0 and various concentrations of hydroxyurea added on day 5 . cells were harvested on day 13 and lysed , and their total hemoglobin and hb - f content was analyzed by cation - exchange hplc . the results are presented in the tables 2 , 3 and 4 and in fig1 to 4 enclosed . table 2______________________________________activity of heme arginate in the mouse micronucleus test in vivo . time of killing themice after dosing compound dosage incidence *. sup . ) ______________________________________24 hours vehicle control -- 1 . 2 ‰ heme arginate 36 mg / kg i . v . 1 . 1 ‰ 48 hours vehicle control -- 1 . 0 ‰ heme arginate 36 mg / kg i . v . 0 . 5 ‰ 72 hours vehicle control -- 1 . 0 ‰ heme arginate 36 mg / kg i . v . 0 . 4 ‰ ______________________________________ *. sup .) incidence : number of micronucleated polychromatic erythrocytes observed in bone marrow smears per 1000 polychromatic erythrocytes examined . table 3______________________________________effect of heme arginate treatment in blasts the proportion of blasts inbonemarrow of some patients with myelodysplastic syndromes ( raeb orraeb - t ). for classification of the myelodysplastic syndromes see table myeloblastsblasts in in sum of blastsbone marrow bone marrow and myeloblasts myelodysplasticpa - be - be - in bone marrow syndrome subtypetientfore after fore after before after before after______________________________________1 4 % 0 % 6 % 4 % 10 % 4 % raeb rars2 3 % 0 % 8 % 4 % 11 % 4 % raeb rars3 6 % 1 % 4 % 3 % 10 % 4 % raeb rars4 15 % 4 % 3 % 4 % 18 % 8 % raeb raeb5 10 % 6 % 20 % 4 % 30 % 10 % raeb - t raeb______________________________________ table 4______________________________________effect of heme arginate treatement in blasts . the proportion of blastsinbone marrow of myelodysplastic syndrome ( rars or raeb ) patientswho were not considered as good responders . for classification of themyelodysplastic syndrome see table 1 . myeloblastsblasts in in sum of blastsbone marrow bone marrow and myeloblasts myelodysplasticpa - be - be - in bone marrow syndrome subtypetientfore after fore after before after before after______________________________________6 5 5 4 3 9 8 raeb raeb7 5 5 4 3 9 8 raeb raeb8 8 8 4 3 12 11 raeb raeb9 2 0 2 2 4 2 rars rars10 3 2 2 1 5 3 rars rars11 3 0 4 7 7 7 raeb raeb12 1 2 8 6 9 8 raeb raeb13 5 3 9 10 14 13 raeb raeb14 4 1 3 3 7 4 raeb rars15 3 3 10 9 13 12 raeb raeb16 0 1 5 3 5 4 raeb rars17 0 3 3 3 3 6 rars raeb18 1 1 4 5 5 6 raeb raeb19 2 1 1 0 3 1 rars rars20 0 2 1 2 1 4 rars rars21 5 4 5 5 10 9 raeb raeb______________________________________ for the purpose of the invention heme or its derivative can be administered by various routes . the suitable administration forms includes parenteral injections and implants including intravenous , intramuscular , intradermal and subcutanous injections ; oral formulations and suppositories . the required dose of heme or its derivative will vary with the particular condition being treated , the severity of the condition , the duration of the treatment , the administration route , and the specific heme compound , the specific pharmaceutical composition , or the specific combination of concomitant treatments being employed . possible use of heme catalysis inhibitors ( as part of the pharmaceutical composition or concomitant use ) decreases the preferred dose of heme or derivatives thereof . for intravenous administration the preferrable daily doses of heme or derivatives thereof range from 0 . 1 to 15 mg / kg calculated as hemin equivalents . for other administration routes ( p . o ., s . c ., i . p ., i . m .) the preferrable daily dose may vary depending on each specific pharmaceutical composition of heme or a derivative thereof . however , the preferrable daily dose will be one resulting in systemic daily release (&# 34 ; bioavailable dose &# 34 ;) of 0 . 1 - 15 mg / kg calculated as hemin equivalents . it will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments , only a few of which are disclosed herein . it will be apparent for the person skilled in the art that other embodiments exist and do not depart from the spirit of the invention . thus , the described embodiments are illustrative and should not be construed as restrictive . 1 . sievers g , hakli h , luhtala j , tenhunen r . optical and epr spectroscopy studies on haem arginate , a new compound used for treatment of porphyria . chem biol interactions 1987 ; 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