Pharmacologic Modulation of Fetal Hemoglobin

Introduction Fetal hemoglobin (HbF; α 2 γ 2 ) inhibits the polymerization of sickle hemoglobin (HbS; α 2 β S2 ) and is a physiologic substitute for normal adult hemoglobin (HbA; α 2 β 2 ) in erythrocytes of patients with β thalassemia and sickle cell anemia. Evidence for the potential therapeutic utility of HbF began to accumulate more than 50 years ago when it was recognized that infants with sickle cell anemia had few symptoms during early life and that the red cells of infants with sickle cell trait failed to sickle in vitro like cells of their mothers who carried the trait. Watson et al (169) hypothesized that these observations were a result of high HbF levels in infant blood. Also, persons compound heterozygous for sickle cell trait and gene-deletion hereditary persistence of HbF (HPFH) who had more than 20% HbF in all of their erythrocytes were clinically normal despite having nearly 80% HbS in their blood (43,144). It was appreciated that enough HbF, if evenly distributed among sickle erythrocytes, might “cure” sickle cell disease. HbF and its γ-globin chains interfere with HbS polymerization. HbS polymer concentrations sufficient to injure the red cell are a prerequisite for causing sickle cell disease. Besides reducing HbS concentration, the sole effect of HbA or HbC when present with HbS, HbF also inhibits HbS polymerization (120). HbS polymer is in equilibrium with a solution of HbS monomers whose concentration defines the solubility of HbS. Fully oxygenated HbS cannot enter the polymer phase, while partially or fully deoxygenated HbS can. If HbF is mixed with HbS, neither the HbF tetramer nor the α 2 β S γ hybrid tetramer enters the polymer phase. In contrast, the hybrid tetramer α 2 β S β A has a 0.5 probability of entering the polymer. Inhibition of polymerization has been localized to the acidic residues γ80 (Asp instead of β80 Asn) and γ87 (Gln instead of β87 Thr); however, other residues may also play some role (97). Some homozygotes with β 0 thalassemia who also inherit the ability to synthesize large amounts of HbF have a milder than expected disease (17,121,136). The hallmark of β thalassemia is imbalanced α- toalpha;-to non-α-globin chain synthesis. Increasing the ability to synthesize more γ-globin chains and HbF reduces this imbalance and decreases the concentration of free α-globin chains that cause most of the pathophysiology of β thalassemia. Therefore, sufficiently high HbF concentrations should also “cure” severe β thalassemia (170). A search was launched for pharmacologic agents that could reverse the neonatal switch from γ- togamma;-to β-globin chain synthesis (11). Based on studies in prokaryotes and in cell cultures, it was hypothesized that cytosine hypomethylation might induce γ-globin gene expression. The first “hemoglobin switching” agent, a nucleoside analog, 5-azacytidine, was postulated to work by hypomethylation of the γ-globin genes. In sickle cell anemia and β thalassemia, this drug increased HbF concentration, but its toxicity and carcinogenicity made it difficult to use. Other cytotoxic drugs incapable of directly causing gene hypomethylation were tried that might promote HbF production indirectly by perturbing the maturation of erythroid precursors. In sickle cell anemia, these drugs, of which hydroxyurea is the prototype, increased HbF level, and controlled clinical trials confirmed that hydroxyurea improved some symptoms of disease. Mechanisms of Action of HbF-Activating Agents ( Figure 1)Fig. 1: Mechanisms of action of fetal hemoglobin (HbF) activating agents. Three general mechanisms explain the reactivation of HbF synthesis. In the left column, methylated cytidine residues are generally associated with failure of gene transcription (Pre-Drug). Following the incorporation of 5-azacytidine, gene hypomethylation occurs and transcription takes place (Post-Drug). The center column illustrates the effects of a cytotoxic drug like hydroxyurea on erythroid precursors cells. Pre-drug, there exist fewer precursors like BFU-e that have the capacity to make large amounts of HbF. Post-drug, more cells that can synthesize HbF are present. By inhibiting histone deacetylases (right column), short-chain fatty acids like sodium butyrate promote histone acetylation and gene expression.Methylation and gene expression Reactivating HbF synthesis in cells where it is dormant is a problem in the regulation of gene expression in a multigene cluster under tight developmental control. Cytosine methylation and demethylation at CpG dinucleotides may repress transcription (14,34,47,68). Hypomethylation is a general feature of the “housekeeping” genes that are usually expressed in all cells while tissue-specific genes, for example, β-like globin genes, are variably methylated (142). In fetal tissues, human γ-globin genes are hypomethylated; promoter methylation silences transcription of embryonic globin genes (29,92,140,161). However, hypomethylation does not accompany all expressed genes, and whether gene hypomethylation is a primary cause or secondary effect of gene expression is still unclear (47,53). Hypomethylation can by cytidine like has a in the of the and at not cause hypomethylation than incorporation in more than that to the of to induce γ-globin gene expression in and in patients with sickle cell anemia and β thalassemia of erythroid and erythroid γ-globin gene expression to than of the some HbF, to erythrocytes is present and of adult erythrocytes are of HbF. with sickle cell anemia have more than and a switch from γ-globin gene expression to β S gene expression In erythroid from adult erythroid high levels of than present in in some a erythroid cell and Figure or of the erythroid production observations that the of erythroid whether a cell or early erythroid the capacity for γ-globin gene expression but this is and adult globin gene expression erythroid cells fetal erythroid that γ-globin gene expression while other erythroid that β-globin gene expression the of in production of Figure agents erythroid cells erythroid and is of a cytotoxic that high HbF levels for the erythroid of action of HbF at for agents incapable of causing hypomethylation fatty acids a fatty analog, can induce gene expression in cells gene expression is of histone histone and in Figure Other histone like A can also induce gene expression is by and butyrate the embryonic gene in adult cells with 5-azacytidine, that gene by butyrate may directly to globin gene expression by to the butyrate in the of the γ-globin gene histone In genes that are not residues and the histone are not In that is and are and is does not cytotoxic and is to HbF by cell like hydroxyurea and other agents. It can induce by of and is have been for the effects of of butyrate and the of this by A of butyrate of has a more studies have that 2 general of short-chain fatty acids may have effects on HbF production by mechanisms of inhibits cell by inhibiting histone and and Other short-chain fatty like and the of butyrate in that can induce HbF but also cell of the genes, and and not histone If therapeutic this of have the of cell and of HbF Agents was to HbF concentration increased to and of hemoglobin in HbF and to in a result of γ-globin gene to clinical trials of in patients with sickle cell anemia and β thalassemia. In patients with sickle cell anemia or β thalassemia, in γ-globin in methylation in the γ-globin gene increased and increased hemoglobin levels and HbF concentration of 5-azacytidine, may have fewer effects than the In patients who failed to to hydroxyurea with in HbF synthesis were for for 2 patients with a of HbF of nearly by and was the sole effect these drugs are to use. fatty acids and that the switch from γ-globin chain to β-globin chain synthesis failed to in of infants of present in increased concentrations in of infants of was fetal in during the normal globin gene The globin gene switch was in of with and was in in the was the globin switch was under butyrate increased HbF in and γ-globin gene expression in erythroid cells of patients with sickle cell anemia and β thalassemia but the butyrate trials in sickle cell anemia and β thalassemia that of drug were can at the of the cell that its might induce a of to butyrate was in sickle cell anemia. butyrate was or of patients with sickle cell anemia with a in HbF from to and in some this was for years In with sickle cell sodium at to to in some patients is to butyrate and is to this compound had with levels of in to is to of the by but not patients with these have levels than agent, and can HbF production in sickle cell anemia and β thalassemia with and to of that fatty acids had HbF levels of anemia or during has been associated with in HbF, and can in and HbF in cell of that levels of short-chain fatty acids and their have been associated with increased HbF In where is by levels of and more than a in HbF to was when and high concentrations were present A with thalassemia and when 2 years had HbF the of butyrate or its in sickle cell anemia and β thalassemia and cannot clinical in sickle cell disease is a and a cytotoxic that synthesis. It is is to a free in is the cells by It the free at the of the a of that to for its nearly it a drug to studies in that hydroxyurea increased HbF levels In studies in sickle cell anemia, hydroxyurea increased and HbF trials it increased HbF in patients and toxicity and A of hydroxyurea in with sickle cell anemia that hydroxyurea by nearly the of and the of and blood was the to the first or the to the first and the first with this of in the had in HbF from a of to 2 years of HbF increased to a of in the of HbF and to in the but in the 2 of HbF may not of all patients with sickle cell anemia with this for this were with a of years who had severe disease and were to the of with of and not all patients the in the the was to the effects of to have a HbF than their more and is to may also more to with cell and increased while and In patients who there is usually a in with in hemoglobin concentration, a in and a in also have a in Some patients had improved capacity with increased and for were for patients and for of and blood were the were for and for of hydroxyurea in and adult studies in the of clinical by controlled trials patients were or in but patients years were in a with drug or trials had HbF increased from to to of in and toxicity was A of hydroxyurea in years has been with to in patients the and of 20% of patients from the of of not drug HbF was at the increased to Other in and hemoglobin concentration and a in were of and were at the of hydroxyurea in the in HbF was the adult patients with the HbF concentration had the HbF levels with and the in not the HbF In the and of hydroxyurea have been in HbF levels were increased or but all disease were not It is to that the effects of hydroxyurea in with sickle cell anemia at in the result in clinical to in the and are not and The on the of hydroxyurea and of the effect of this drug on from sickle cell anemia and from the of patients in the and have been in the effects of patients in the were the of the for in and of 2 and patients had a by the of patients the was by β-globin gene α-globin production HbF level, and but this may a of the of and the of the in patients on hydroxyurea and were in patients on a effects have not been of the in nearly years of disease was the most cause of In other hydroxyurea not reverse in patients with HbF levels were hydroxyurea reduces the of and by nearly in adult sickle cell anemia, and are the most of a was whether the associated with hydroxyurea was by this at years of to the concentration of HbF was The for this was the that HbF levels had to HbF at when HbF was at its level, and hydroxyurea when the was years and HbF concentrations were by this agent, were to its effects on In with HbF at had a at years of with in patients whose HbF was In patients who had HbF during to hydroxyurea or a patients with HbF had a at years with in patients whose HbF was of the effects of hydroxyurea on HbF, patients who had HbF at had HbF at of years who this drug in or of had a with patients not hydroxyurea during this The of was to a in of years that hydroxyurea in with sickle cell anemia with severe to severe disease. However, the is not a and the of the with the that patients were free to of the of whether the of hydroxyurea should to patients with sickle cell disease and fewer severe not whether hydroxyurea or reverse of in with of years not In studies of in some to or was in 2 with sickle cell anemia who had HbF levels of hydroxyurea In sickle disease despite a to hydroxyurea does not to the of sickle cell anemia of who to hydroxyurea with in HbF is a that is not in The HbF were in with the and and the in these with the in and also had the in In HbF HbF levels and blood from the patients and and failed to the HbF but the with patients to hydroxyurea with increased of but there was a patients who were and who not their patients with in HbF level, and to early during and the of of some patients may to of hydroxyurea may prerequisite for of HbF with the and also had the decreases in these during the in HbF. In the and in with were also of the HbF levels with hydroxyurea of all patients not to or had a in HbF. However, the and of among studies patients who to have a of and drug in that may or to the β-like globin gene cluster may for in HbF and the HbF to with the HbF were to have a HbS gene on a was a of HbF but not the HbF to In with sickle cell anemia a in HbF and in their and hydroxyurea was and a effect in hemoglobin concentration, and HbF was present of the or had that not the HbF to In of where the were had β-globin gene cluster that of the HbF to hydroxyurea may to the β-globin gene these observations that a search for to the β-globin gene may of the HbF to hydroxyurea and potential for In the ability to to hydroxyurea in may the capacity of the to of with precursors that synthesize HbF to may by the and HbF during hydroxyurea can when is sufficient to with and the with of HbF synthesis. Mechanisms of clinical may work by a clinical its effects on HbF concentration is by the hydroxyurea was to by HbF In most in clinical symptoms or the in HbF studies that the in and may also from patients having a had increased to cells and expressed levels of that may promote By reducing blood hydroxyurea might the of may also these cells may A in of normal and in sickle cell anemia and disease patients with and in sickle cell anemia, this was with the in place in the sickle the in HbF is a primary effect of it difficult to if the other effects are primary or secondary to increased HbF. HbF there is a and of HbS polymerization. cell decreases a in HbF a effect on red cell or cell In hydroxyurea may cells their and and a for sickle cell cell α β or the and also early during hydroxyurea in HbF and are to secondary to increased HbF and may play a role in the of and the levels are increased in sickle cell anemia with and a was during with hydroxyurea not levels or on the by its cytotoxic a of that can synthesize increased amounts of fetal hemoglobin It has effects on gene expression. may also concentrations of HbF the of sickle hemoglobin polymer and the of and with a high HbF and to a in and are erythrocytes are to to and the reducing the of vitro studies of the of sickle erythrocytes to and a in when patients with sickle cell anemia with hydroxyurea were with who were and during with hydroxyurea were was associated with a in mechanisms of action of or is a of hydroxyurea can In hydroxyurea to hemoglobin could early and to of to patients with sickle cell anemia In increased the 2 of HbS, reducing its polymerization but these have not been In vitro studies have that HbS is also to sickle and that the of this from the of hydroxyurea is in the while in tissues, is to the by the 2 of HbS or by reduces polymerization and the for studies have that hydroxyurea can induce but early to sickle cell anemia by was Other work has the that hydroxyurea production to levels It was hypothesized that this the of erythroid precursors with the capacity to synthesize HbF in other sickle In thalassemia, hydroxyurea to work in sickle cell anemia In a in patients with 0 thalassemia and thalassemia, HbF were than in with sickle cell anemia, to from the of patients were in these but were to patients with thalassemia have HbA and a disease not hydroxyurea studies of disease that was associated with a in and red and in that hydroxyurea may in a in and a in the hemoglobin of all by in the of the hemoglobin of all erythrocytes to hemoglobin a in HbF not was in a few with disease whose was years and were with a of of hydroxyurea for concentration increased nearly increased from to to and HbF in increased from to not ability in with but it was that was present at the of hydroxyurea the cell is However, on of the pathophysiology of of cells and a polymerization of HbS should this disease. sickle cell anemia with hydroxyurea for hydroxyurea are to of its and hydroxyurea should for and who can with the whose are sufficiently severe to the of the of controlled trials in with and a to of the of this and should in that at a and and other severe like are the for A controlled hydroxyurea with the 2 other of sickle cell anemia, blood and has not been 1: of hydroxyurea in sickle cell hydroxyurea to to or when are not Three of hydroxyurea to had a early during HbF was Other have been of in patients with sickle cell anemia with hydroxyurea who had HbF levels that most on the of HbF among erythrocytes in these or in most of hydroxyurea of sickle cell anemia, or the concentration of is but in with a high of If these cells all HbF concentrations sufficient to the effects of this of HbF should to a where 80% of cells are incapable of and 20% of cells were HbS cells. high HbF levels in patients with sickle cell anemia not from is HbF levels of 20% not from other disease is a more of the of a that does not the of the of sickle cell disease. the of may from that of other and might not to the effects of HbF Some patients have at there is to at a can with in a or can on the of and hydroxyurea it is to more the of adult patients cannot a if blood are the may patients who to hydroxyurea blood at and have been but the sole controlled drug and the of toxicity has that to levels is not for a therapeutic effect in most The therapeutic should a of hydroxyurea and of HbF. when hydroxyurea is and usually the in HbF, this is a for HbF that can the HbF and is not and HbF should at and at during the first of and is for hydroxyurea to HbF. It has not been but hydroxyurea in patients on or is not that some may have a HbF when also hydroxyurea a is blood should when the is should at to from in or some of which may to Some adult patients not to with in HbF or when their failure to the to for the of may and 20% of A may on the of the and drug should to that not among may to the of with blood and the and effects of are If a has not blood or had that a should sufficient to if hydroxyurea to have been effects effects of hydroxyurea are still not but the expected and effects of have been effects in the of the of example, and the of in and is patients The of is 50 and patients The the of patients have of for all patients and of for patients to If the of and in with sickle cell anemia is to that in the general the has to in the of or that may in or in the genes, have not been in sickle cell anemia patients with A was of gene and the β and γ gene of the in with sickle cell anemia to hydroxyurea for with sickle cell anemia for or and with for who were to hydroxyurea for had more with with of drug and to drug other in were these and or to the that the potential of the drug is was for more than years to in with disease of of hydroxyurea in patients with that in this the drug can of patients with with hydroxyurea In of patients hydroxyurea the sole to of patients with to with hydroxyurea a of and of had a the with 2 had and if these observations in to a disease but with to of at patients with sickle cell disease with hydroxyurea who or a years of and 2 of these have been in had patients with sickle cell anemia are hydroxyurea it is difficult to this If hydroxyurea a or this is most a and of to with a the of this in patients may is a in and and should not if is has been in at most for but in with sickle cell anemia in this not should by and hydroxyurea and the of If a who is hydroxyurea is on which to a for or effects of hydroxyurea on and have been studies in the patients have not been of the and localized in have been and may despite and have been in patients with with hydroxyurea were when these patients hydroxyurea for of when the drug was but when it was In the hydroxyurea had effect on or of with the observations in the was in β thalassemia A therapeutic role for hydroxyurea in the severe β is that patients with β thalassemia to this drug with in hemoglobin In β thalassemia the of is to globin chain synthesis imbalance by non-α-globin chain synthesis. HbF have been there is that β-globin gene expression can patients with β thalassemia with hydroxyurea have been In these the of thalassemia was and patients had β 0 thalassemia, β thalassemia, and hemoglobin of the were compound for and β 0 thalassemia patients were concentrations from to some patients were it difficult to their hemoglobin was with and patients were for than but had nearly years of were Some patients had in and had of clinical in the most of β thalassemia that hydroxyurea is not a generally for these In some of β thalassemia there may a effect on hemoglobin concentration and HbF. may and may The for hydroxyurea it is a sole of severe β thalassemia does not trials of hydroxyurea in thalassemia in with other agents and for more may of and and Other was with hydroxyurea in and in HbF levels In a of 2 who were made by but were hydroxyurea or a in with agents increased with sickle cell disease with and hydroxyurea and β thalassemia patients not of with hydroxyurea had in HbF work that might with agents that cause and erythroid to a hydroxyurea and were to patients with sickle cell anemia, in HbF concentration that with hydroxyurea of in a it more to the effects of with hydroxyurea and and in with hydroxyurea and were also was to patients with β thalassemia to their hemoglobin concentration In of patients patients who were increased their hemoglobin levels from to in a for HbF levels not but in some patients blood are to who to this with the result of Other and may HbF in and in cell cultures, from patients with sickle cell disease but neither nor to work with a potential to the may the of and in sickle cell anemia and in other severe β-globin gene where may the of disease In vitro studies a of BFU-e and γ-globin chains of erythroid to and and a in and γ-globin was associated with of that was by the of sodium butyrate The effect of on was by increased expression of the and genes, genes that may play a role in γ-globin gene expression. β increased the of during the first of in vitro not on of and the of in was than in Three patients with sickle cell anemia with hydroxyurea had butyrate to their drug increased their HbF concentrations when butyrate was who was to with butyrate In 2 of patients to of hydroxyurea to in HbF The of HbF in 2 for with hydroxyurea and butyrate and a of in a compound for and β thalassemia hydroxyurea that the may the to for have levels of HbF than for β thalassemia to a few patients with have been with and whether this is a general is not Other than these observations in of the there is to that patients with β thalassemia are to to butyrate with in hemoglobin or HbF HbF genes early in HbF levels to a concentration and that sickle has 20% HbF, should “cure” sickle cell anemia. If β a concentration of α-globin this also of HbF production hydroxyurea is a for pharmacologic of sickle cell disease. in with sickle cell anemia its effects are not and the of HbF with hydroxyurea may have been In its is and its early in life is does not in the most severe β to HbF concentration has a but studies of its clinical have not been In this cytotoxic drugs are to a of precursors that the capacity to make HbF. with short-chain fatty acids or to expression of the γ-globin genes is agents that or reverse sickle cell might to the concentration of HbS and polymerization. Fetal hemoglobin (HbF) inhibits the polymerization of sickle hemoglobin and can substitute for normal adult hemoglobin in erythrocytes of patients with β thalassemia and sickle cell anemia. these pharmacologic agents that can reverse the switch from γ- togamma;-to β-globin chain synthesis chains HbF and sickle β-globin chains are present in or adult erythroid precursors that the ability to HbF are The first “hemoglobin switching” agent, a nucleoside analog, 5-azacytidine, was postulated to HbF by causing gene Other drugs, of which hydroxyurea is the prototype, promote HbF production indirectly by perturbing the maturation of erythroid precursors. fatty acids may directly HbF gene expression. all agents that HbF gene hydroxyurea has been clinically in controlled clinical trials in patients with sickle cell anemia. of HbF production hydroxyurea is a for pharmacologic of in with sickle cell anemia its effects are not and the of HbF with hydroxyurea may have been In its is and its and when early in life is still