- Email: [email protected]
Optimizing the management of renal anemia: challenges and new opportunities
review
http://www.kidney-international.org & 2008 International Society of Nephrology
Optimizing the management of renal anemia: challenges and new opportunities Francesco Locatelli1 and Lucia Del Vecchio1 1
Department of Nephrology, Dialysis and Renal Transplant, Ospedale A. Manzoni, Lecco, Italy
Erythropoiesis stimulating agents (ESAs) are the main tool to achieve anemia correction in CKD patients. At present six different ESAs are available: epoetin alpha, epoetin beta, epoetin omega, epoetin delta, darbepoetin alpha, and very recently CERA. From one side the patent of older ESAs have expired, and biosimilars (for the moment only of epoetin alpha) have been approved for use in Europe by the European Medicines Agency. However, a number of issues about bioequivalence and how to test it are still to be solved completely. In the mean time pharmaceutical research has kept on working, developing new ESAs and alternative strategies for stimulating erythropoiesis. In this review we present and discuss these points.
Anemia, resulting primarily from insufficient production of erythropoietin (EPO) to support erythropoiesis, is a common complication of chronic kidney disease (CKD). Approximately 50% of patients with CKD stages 3–5 have anemia.1 An even greater proportion of patients starting dialysis are anemic.2 Anemia may significantly impair quality of life, increase cardiovascular risk, and reduce long-term survival, when left untreated. Earlier, treatment options were essentially limited to blood transfusions; however, since the late 1980s the availability of recombinant human EPO (rHuEPO) has revolutionized the management of anemia in patients with CKD. Today, erythropoiesis-stimulating agents (ESAs) are the main tools to achieve anemia correction in CKD patients.
Kidney International (2008) 74 (Suppl 111), S33–S37; doi:10.1038/ki.2008.525 KEYWORDS: anemia; chronic kidney disease; erythropoietin; erythropoiesisstimulating agents; hematide; continuous erythropoiesis receptor activator
Correspondence: Francesco Locatelli, Department of Nephrology, Dialysis and Renal Transplant, Ospedale A. Manzoni, Via dell’Eremo 9, 23900 Lecco, Italy. E-mail: [email protected] Kidney International (2008) 74 (Suppl 111), S33–S37
TYPES OF HUMAN RECOMBINANT EPO
The gene encoding for HuEPO was cloned in 1985.3 This opened the way to the synthesis of rHuEPO.4,5 This molecule has been proven effective and relatively safe in correcting anemia and maintaining stable hemoglobin (Hb) levels in CKD patients. Currently, four different types of rHuEPO are available in the market: epoetin alpha, epoetin beta, epoetin omega, and epoetin delta. Both epoetin alpha and beta are synthesized in Chinese hamster ovary cells and share the same amino acid sequence as endogenous EPO, but differences in the manufacturing process between the two glycoproteins reflect into differences in their carbohydrate moieties.6 Epoetin omega is synthesized in baby hamster kidney cells.7 Differing from epoetin alpha and beta, only 60% of epoetin omega is O-glycosylated at the serine residue, and a higher proportion of carbohydrate residues are tetrasialylated when compared with endogenous EPO, as has been reported for the other epoetins.8 Epoetin delta shares the same amino acid sequence as endogenous EPO, but is synthesized in human cells.9,10 This process may circumvents problems arising from species-dependent differences in protein-folding or post-translational modification. Despite these differences in the glycosylation pattern, all rHuEPO have similar half-life (6–8 h intravenously and 19–24 h subcutaneously), pharmacodynamic and pharmacokinetic characteristics. BIOSIMILARS
In December 2004, the patent of epoetin alpha expired in Europe, followed by epoetin beta in 2005 in many European S33
countries. This has opened the way to biosimilars. Currently, two biosimilars of epoetin alpha, HX575 (Rentschler Biotechnologie, Laupheim, Germany) and Epoetin zeta (Silapo by STADA Arzneimittel AG, Bad Vilbel, Germany and Retacrit by Hospira Enterprises B.V., Hoofddorp, the Netherland), received full marketing authorization valid throughout the European Union. HX575, which is already available in some European countries, is marketed by three different companies (‘Binocrit’ from Sandoz GmbH, Holzkirchen, Germany, ‘Epoetin alfa Hexal’ from Hexal Biotech Forschungs GmbH, Holzkirchen, Germany and ‘Abseamed’ from Medice Arzneimittel Pu¨tter GmbH & Co., Iserlohn, Germany). Both drugs are not inferior to epoetin alpha in maintaining target Hb concentrations; they can be administered only intravenously in CKD patients. Unfortunately, the fulfillment of the European Medicines Agency requirements for approval does not solve completely the issue of what bioequivalence really means and how it has to be tested, as quality assurance assays for biopharmaceuticals are generally less sensitive and precise than those for small molecules, and many of them still require development and standardization.11 THE NEW GENERATION OF ESAs
Even though it is very effective in correcting anemia, rHuEPO cannot be considered as the ideal ESA. It requires a relatively frequent administration and this has been claimed as a possible reason of high Hb variability. Moreover, as its oral bioavailability is insufficient, it can be administered only by the subcutaneous or intravenous route, the latter requiring higher doses than the subcutaneous one. In the setting of hemodialysis, where the intravenous route is more convenient, physicians have to balance increased costs because of extra dose needs with patient discomfort and risk of bloodrelated infections. Another important limitation of rHuEPO is that it is unstable at room temperature. A strict cold chain is thus necessary, starting from the manufacturer till the moment of administration; an inadequate handling of the drug may increase the risk of immunization in patients selfadministering the drug at home. Finally, the synthesis of rHuEPO, as that of other biomolecules, is complex and thus expensive. Altogether, these points have urged pharmacological research in developing new ESAs with improved characteristics. LONG-ACTING EPOs
Darbepoetin alpha is an ESA with prolonged half-life. As in epoetin alpha and beta, it is produced in Chinese hamster ovary cells but it differs from EPO in the amino acid sequence at five positions allowing the adding of two extra N-linked carbohydrate chains.12 These molecular changes result in longer circulating half-life (B25 h when given intravenously and B48 h by the subcutaneous route) and in a 4.3-fold lower relative affinity for the EPO receptor. Given its longer half-life, darbepoetin alpha can be administered less often than rHuEPO.13–16 Differing from rHuEPO, dose requirements are S34
F Locatelli and L Del Vecchio: Optimizing renal anemia management
independent of the administration route.14,17 According to the label, the drug can remain at room temperature (up to 251C) for a maximum single period of 7 days. Continuous erythropoiesis receptor activator (CERA) is another new ESA recently become available that is obtained by adding a large water-soluble polyethylene glycol moiety to the EPO beta molecule. This agent has a much higher molecular weight than that of EPO (B60,000 Da vs 34,000 Da) and a longer half-life (B130 h when administered either intravenously or subcutaneously). Moreover, it has a reduced binding affinity for the EPO receptor, which is B45-fold lower than that of epoetin beta, mainly because of a much slower association rate.18 Given these binding properties, the molecule activates the EPO receptor but is not internalized into the cell, remaining are available for binding to other EPO receptors. Results of clinical trials indicate that CERA is effective in maintaining Hb levels after switching from rHuEPO therapy (one to three times a week) or darbepoetin alpha (once a week or once every other week) when administered once every 4 weeks;19,20 and in correcting anemia in EPO-naive patients when administered once in every 2 weeks.21 Since the publication in 2006 of two trials aimed at testing the effect of complete anemia correction on cardiovascular status and survival in CKD patients not on dialysis,22,23 a lively debate is going on in the nephrological community about the fear of complications consequent to anemia overcorrection. In this perspective, it is of interest that the drug achieves the Hb target more smoothly than other ESAs, reducing the risk of overshooting (Figure 1).21 Moreover, despite a longer half-life than that of other ESAs, when the drug is halted because Hb levels exceed the target, the rate of decrease in Hb levels is similar to that observed with short-acting ESAs. This suggests that the half-life of erythrocytes is more important than that of the drug (Figure 2). As for darbepoetin alpha, the dose does not need to be modified according to the adminisCERA 1x/2wk Mean (s.d.) Hb (g per 100 ml)
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Darbepoetin alfa 1x/wk
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Figure 1 | Hb concentrations during correction phase with CERA, rHuEPO, and darbepoetin alpha in EPO-naive CKD patients not on dialysis. The observed raise in Hb concentration is smothering with CERA than with other ESAs. Data are obtained from reference Macdougall et al.21 and from reference Locatelli et al.13 CERA, continuous erythropoiesis receptor activator; CKD, chronic kidney disease; Hb, hemoglobin; rHuEPO, recombinant human erythropoietin. Kidney International (2008) 74 (Suppl 111), S33–S37
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F Locatelli and L Del Vecchio: Optimizing renal anemia management
CERA QM Darbepoetin Q2W rHuEPO TIW
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Figure 2 | Hb decrease rate after drug withdrawal in patients exceeding Hb levels of 14 g per 100 ml with different ESAs. The rate of decrease is influenced by erythrocyte- and not by drug half-life. Data are obtained from reference Locatelli et al.13 and reference Heifets et al.49 CERA, continuous erythropoiesis receptor activator; ESAs, Erythropoiesis-stimulating agents; Hb, hemoglobin.
tration route. The drug can remain at room temperature for a maximum single period of nearly one month. FUSION PROTEINS AND SYNTHETIC EPO
Beyond increasing the carbohydrate content of EPO, various strategies have been pursued to increase the yield of EPO produced by gene transfer and to enhance its intrinsic activity. Erythropoietin binds to the EPO receptor through two binding domains, one with high affinity for the receptor and the other with low affinity; the latter is required for the activation of the receptor. On the basis of the hypothesis that the presence of two molecules of EPO might bring together two high-affinity binding sites and facilitate binding to the EPO receptor, one approach is the production of a molecule consisting of two EPOs linked by small flexible peptides.24,25 Experimental data indicate that EPO dimers induce a much higher hematocrit increase than the EPO monomer without differences in pharmacokinetics.25 It has been shown that fusing the carboxyl-terminal peptide of human chorionic gonadotropin beta-subunit (human chorionic gonadotropin beta) to a number of biologically active proteins increased in vivo potency and circulatory half-life of the proteins containing this peptide without affecting assembly, secretion, receptor binding affinity, or in vitro bioactivity. Recently, Fares et al.26 obtained a fusion protein by adding the carboxyl-terminal peptide of human chorionic gonadotropin beta to HuEPO-coding sequence. This chimeric protein had both enhanced in vivo potency and half-life compared with wild-type EPO in mice. Another approach for enhancing serum half-life includes increasing the molecular weight of EPO by fusion to the Fc part of an antibody.27,28 Interestingly, this approach has been used to create an Fc fusion protein that can be administered by aerosol inhalation.29 Attempts have also been made to synthesize artificially a glycoprotein similar to EPO, called synthetic erythropoiesis Kidney International (2008) 74 (Suppl 111), S33–S37
protein. This is a 51-kDa protein-polymer consisting of 166 amino acids with two other polymer moieties attached covalently.30 In cell and animal assays for erythropoiesis, synthetic erythropoiesis protein displayed potent biological activity and had significantly prolonged duration of action in vivo.31 Even though it is effective in correcting anemia, all these EPO-modified proteins raise some concerns about immunogenicity.32 NON-EPO-DERIVED EPO RECEPTOR AGONISTS: HEMATIDE
Hematide is a pegylated synthetic dimeric peptide with no sequence homology to EPO. In CKD patients not on dialysis, it has an intermediate mean half-life amid darbepoetin alpha and CERA (B59 and B70 h by the intravenous and subcutaneous route, respectively) and longer half-life than that measured in healthy individuals; this is consistent with the hypothesis that hematide is cleared, at least partially, through the kidney.33 Phase II trials showed that the drug given either intravenously or subcutaneously was effective in both the correction and maintenance phase of anemia treatment at dose ranges between 0.025 and 0.05 mg/kg; phase III trials are ongoing. Even though antibodies against hematide have been found in patients receiving the drug, they do not cross react with EPO.34 For this reason, hematide has been proposed as a possible treatment of anemia in pure red cell aplasia.34,35 Potential advantage of this drug is that its manufacturing process is simpler and cheaper than that of current ESAs. Recently, in the attempt to create other non-EPO-derived EPO receptor agonists, two sequences of EPO mimetic peptide-1, which is a 20-amino acid peptide with weak EPOlike bioactivity, were inserted on a human IgG4 Fc framework. The obtained molecule, called CNTO 530, was shown to bind selectively the EPO receptor, to mediate EPO receptor signaling, and to support proliferation of EPO-dependent cells.36 In vivo it stimulated erythropoiesis, providing a longlived increase in Hb in mice. OTHER STRATEGIES TO INCREASE ERYTHROPOIESIS
The inhibition or activation of transcription factors modulating EPO gene expression may be an alternative for stimulating erythropoiesis. The hypoxia-inducible transcription factors (HIFs) are central components in the cellular responses to hypoxia; under normoxic conditions, EPO gene expression is suppressed as a result of HIF inactivation because of O(2)dependent enzymatic hydroxylation and subsequent degradation of their alpha-subunit. 2-oxoglutarate analogs, which prevent HIF alpha hydroxylation, have emerged as promising tools for the stimulation of erythropoiesis and angiogenesis (the so called HIF stabilizers). Experimental data indicate that these agents stimulate erythropoiesis in vivo.37 These agents can be administered by the oral route. Unfortunately, the development of the first candidate molecule of this class, FG-2216, was halted after the occurrence of a case of fatal S35
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hepatic necrosis. Another concern related to HIF stabilizers is related to the fact that the HIF system promotes the transcription of many other genes; careful evaluation of other effects than erythropoiesis stimulation seems mandatory. GATA genes are an evolutionarily conserved family, which encode a group of important transcription factors involved in the regulation of diverse processes including the development of the heart, hematopoietic system, and sex gonads. Some of these factors also regulate EPO gene transcription.38,39 Oral administration of a GATA-specific inhibitor, K-7174, was found effective in reversing anemia induced by IL-1 beta or TNF-a in mice.40 Another possible strategy to increase erythropoiesis is the inhibition of the hematopoietic cell phosphatase. This protein, also known as src homology domain 2-containing tyrosine phosphatase 1, binds and activates the negative regulatory domain of the EPO receptor, inhibiting transduction inside the cell.41 Interestingly, a defective expression of hematopoietic cell phosphatase was found in patients with polycythemia vera.42 In contrast, hematopoietic cell phosphatase expression is increased in CD34 þ cells from patients with EPO resistance; the adding of a src homology domain 2-containing tyrosine phosphatase 1 antisense oligonucleotide suppressed src homology domain 2-containing tyrosine phosphatase 1 protein expression and partial recovered BFU-E colonies.43 Hematopoietic cell phosphatase inhibitors may be thus a novel target molecule to treat renal anemia and/or sensitize patients to EPO action. Finally, EPO gene therapy has been proposed for the management of anemic patients with CKD. A number of different techniques have been tried, such as the delivering of naked plasmid DNA into skeletal muscles,44,45 the subcutaneous implantation of autologous bone marrow stromal cells genetically engineered to secrete EPO,46 viral transfection,47 and the use of artificial human chromosome.48 EPO gene therapy has the theoretical advantage of releasing small but continuous amount of EPO into the circulation, but has still a number of problems, such as immunogenicity and the difficulty to exactly tune the exact amount of EPO needed to correct anemia and maintaining a level of expression that sufficiently promotes erythropoiesis in the long term.
F Locatelli and L Del Vecchio: Optimizing renal anemia management
DISCLOSURE
Francesco Locatelli has received consulting fees from Affimax, Roche, Amgen, and Shire. Francesco Locatelli has also received lecture fees from Roche, Amgen, and Shire. Lucia Del Vecchio has declared no financial interests. REFERENCES 1.
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CONCLUSIONS
Currently, the treatment of anemia with rHuEPO is quite expensive, given that its synthesis, as that of other biomolecules, is complex. New ESAs are, or will be, more expensive than rHuEPO, because of the high costs of pharmacological research, not yet covered by years of selling, but offer improved pharmacological characteristics that may help in the management of anemia in CKD patients. The synthesis of simpler molecules that is not related to ESA structure or the development of alternative strategies of erythropoiesis stimulation may lead to the creation of cheaper agents that would revolutionize the market. S36
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http://www.kidney-international.org & 2008 International Society of Nephrology
Optimizing the management of renal anemia: challenges and new opportunities Francesco Locatelli1 and Lucia Del Vecchio1 1
Department of Nephrology, Dialysis and Renal Transplant, Ospedale A. Manzoni, Lecco, Italy
Erythropoiesis stimulating agents (ESAs) are the main tool to achieve anemia correction in CKD patients. At present six different ESAs are available: epoetin alpha, epoetin beta, epoetin omega, epoetin delta, darbepoetin alpha, and very recently CERA. From one side the patent of older ESAs have expired, and biosimilars (for the moment only of epoetin alpha) have been approved for use in Europe by the European Medicines Agency. However, a number of issues about bioequivalence and how to test it are still to be solved completely. In the mean time pharmaceutical research has kept on working, developing new ESAs and alternative strategies for stimulating erythropoiesis. In this review we present and discuss these points.
Anemia, resulting primarily from insufficient production of erythropoietin (EPO) to support erythropoiesis, is a common complication of chronic kidney disease (CKD). Approximately 50% of patients with CKD stages 3–5 have anemia.1 An even greater proportion of patients starting dialysis are anemic.2 Anemia may significantly impair quality of life, increase cardiovascular risk, and reduce long-term survival, when left untreated. Earlier, treatment options were essentially limited to blood transfusions; however, since the late 1980s the availability of recombinant human EPO (rHuEPO) has revolutionized the management of anemia in patients with CKD. Today, erythropoiesis-stimulating agents (ESAs) are the main tools to achieve anemia correction in CKD patients.
Kidney International (2008) 74 (Suppl 111), S33–S37; doi:10.1038/ki.2008.525 KEYWORDS: anemia; chronic kidney disease; erythropoietin; erythropoiesisstimulating agents; hematide; continuous erythropoiesis receptor activator
Correspondence: Francesco Locatelli, Department of Nephrology, Dialysis and Renal Transplant, Ospedale A. Manzoni, Via dell’Eremo 9, 23900 Lecco, Italy. E-mail: [email protected] Kidney International (2008) 74 (Suppl 111), S33–S37
TYPES OF HUMAN RECOMBINANT EPO
The gene encoding for HuEPO was cloned in 1985.3 This opened the way to the synthesis of rHuEPO.4,5 This molecule has been proven effective and relatively safe in correcting anemia and maintaining stable hemoglobin (Hb) levels in CKD patients. Currently, four different types of rHuEPO are available in the market: epoetin alpha, epoetin beta, epoetin omega, and epoetin delta. Both epoetin alpha and beta are synthesized in Chinese hamster ovary cells and share the same amino acid sequence as endogenous EPO, but differences in the manufacturing process between the two glycoproteins reflect into differences in their carbohydrate moieties.6 Epoetin omega is synthesized in baby hamster kidney cells.7 Differing from epoetin alpha and beta, only 60% of epoetin omega is O-glycosylated at the serine residue, and a higher proportion of carbohydrate residues are tetrasialylated when compared with endogenous EPO, as has been reported for the other epoetins.8 Epoetin delta shares the same amino acid sequence as endogenous EPO, but is synthesized in human cells.9,10 This process may circumvents problems arising from species-dependent differences in protein-folding or post-translational modification. Despite these differences in the glycosylation pattern, all rHuEPO have similar half-life (6–8 h intravenously and 19–24 h subcutaneously), pharmacodynamic and pharmacokinetic characteristics. BIOSIMILARS
In December 2004, the patent of epoetin alpha expired in Europe, followed by epoetin beta in 2005 in many European S33
countries. This has opened the way to biosimilars. Currently, two biosimilars of epoetin alpha, HX575 (Rentschler Biotechnologie, Laupheim, Germany) and Epoetin zeta (Silapo by STADA Arzneimittel AG, Bad Vilbel, Germany and Retacrit by Hospira Enterprises B.V., Hoofddorp, the Netherland), received full marketing authorization valid throughout the European Union. HX575, which is already available in some European countries, is marketed by three different companies (‘Binocrit’ from Sandoz GmbH, Holzkirchen, Germany, ‘Epoetin alfa Hexal’ from Hexal Biotech Forschungs GmbH, Holzkirchen, Germany and ‘Abseamed’ from Medice Arzneimittel Pu¨tter GmbH & Co., Iserlohn, Germany). Both drugs are not inferior to epoetin alpha in maintaining target Hb concentrations; they can be administered only intravenously in CKD patients. Unfortunately, the fulfillment of the European Medicines Agency requirements for approval does not solve completely the issue of what bioequivalence really means and how it has to be tested, as quality assurance assays for biopharmaceuticals are generally less sensitive and precise than those for small molecules, and many of them still require development and standardization.11 THE NEW GENERATION OF ESAs
Even though it is very effective in correcting anemia, rHuEPO cannot be considered as the ideal ESA. It requires a relatively frequent administration and this has been claimed as a possible reason of high Hb variability. Moreover, as its oral bioavailability is insufficient, it can be administered only by the subcutaneous or intravenous route, the latter requiring higher doses than the subcutaneous one. In the setting of hemodialysis, where the intravenous route is more convenient, physicians have to balance increased costs because of extra dose needs with patient discomfort and risk of bloodrelated infections. Another important limitation of rHuEPO is that it is unstable at room temperature. A strict cold chain is thus necessary, starting from the manufacturer till the moment of administration; an inadequate handling of the drug may increase the risk of immunization in patients selfadministering the drug at home. Finally, the synthesis of rHuEPO, as that of other biomolecules, is complex and thus expensive. Altogether, these points have urged pharmacological research in developing new ESAs with improved characteristics. LONG-ACTING EPOs
Darbepoetin alpha is an ESA with prolonged half-life. As in epoetin alpha and beta, it is produced in Chinese hamster ovary cells but it differs from EPO in the amino acid sequence at five positions allowing the adding of two extra N-linked carbohydrate chains.12 These molecular changes result in longer circulating half-life (B25 h when given intravenously and B48 h by the subcutaneous route) and in a 4.3-fold lower relative affinity for the EPO receptor. Given its longer half-life, darbepoetin alpha can be administered less often than rHuEPO.13–16 Differing from rHuEPO, dose requirements are S34
F Locatelli and L Del Vecchio: Optimizing renal anemia management
independent of the administration route.14,17 According to the label, the drug can remain at room temperature (up to 251C) for a maximum single period of 7 days. Continuous erythropoiesis receptor activator (CERA) is another new ESA recently become available that is obtained by adding a large water-soluble polyethylene glycol moiety to the EPO beta molecule. This agent has a much higher molecular weight than that of EPO (B60,000 Da vs 34,000 Da) and a longer half-life (B130 h when administered either intravenously or subcutaneously). Moreover, it has a reduced binding affinity for the EPO receptor, which is B45-fold lower than that of epoetin beta, mainly because of a much slower association rate.18 Given these binding properties, the molecule activates the EPO receptor but is not internalized into the cell, remaining are available for binding to other EPO receptors. Results of clinical trials indicate that CERA is effective in maintaining Hb levels after switching from rHuEPO therapy (one to three times a week) or darbepoetin alpha (once a week or once every other week) when administered once every 4 weeks;19,20 and in correcting anemia in EPO-naive patients when administered once in every 2 weeks.21 Since the publication in 2006 of two trials aimed at testing the effect of complete anemia correction on cardiovascular status and survival in CKD patients not on dialysis,22,23 a lively debate is going on in the nephrological community about the fear of complications consequent to anemia overcorrection. In this perspective, it is of interest that the drug achieves the Hb target more smoothly than other ESAs, reducing the risk of overshooting (Figure 1).21 Moreover, despite a longer half-life than that of other ESAs, when the drug is halted because Hb levels exceed the target, the rate of decrease in Hb levels is similar to that observed with short-acting ESAs. This suggests that the half-life of erythrocytes is more important than that of the drug (Figure 2). As for darbepoetin alpha, the dose does not need to be modified according to the adminisCERA 1x/2wk Mean (s.d.) Hb (g per 100 ml)
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Darbepoetin alfa 1x/wk
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14 13 12 11
10 9 8 7 Months Weeks
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Figure 1 | Hb concentrations during correction phase with CERA, rHuEPO, and darbepoetin alpha in EPO-naive CKD patients not on dialysis. The observed raise in Hb concentration is smothering with CERA than with other ESAs. Data are obtained from reference Macdougall et al.21 and from reference Locatelli et al.13 CERA, continuous erythropoiesis receptor activator; CKD, chronic kidney disease; Hb, hemoglobin; rHuEPO, recombinant human erythropoietin. Kidney International (2008) 74 (Suppl 111), S33–S37
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F Locatelli and L Del Vecchio: Optimizing renal anemia management
CERA QM Darbepoetin Q2W rHuEPO TIW
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Figure 2 | Hb decrease rate after drug withdrawal in patients exceeding Hb levels of 14 g per 100 ml with different ESAs. The rate of decrease is influenced by erythrocyte- and not by drug half-life. Data are obtained from reference Locatelli et al.13 and reference Heifets et al.49 CERA, continuous erythropoiesis receptor activator; ESAs, Erythropoiesis-stimulating agents; Hb, hemoglobin.
tration route. The drug can remain at room temperature for a maximum single period of nearly one month. FUSION PROTEINS AND SYNTHETIC EPO
Beyond increasing the carbohydrate content of EPO, various strategies have been pursued to increase the yield of EPO produced by gene transfer and to enhance its intrinsic activity. Erythropoietin binds to the EPO receptor through two binding domains, one with high affinity for the receptor and the other with low affinity; the latter is required for the activation of the receptor. On the basis of the hypothesis that the presence of two molecules of EPO might bring together two high-affinity binding sites and facilitate binding to the EPO receptor, one approach is the production of a molecule consisting of two EPOs linked by small flexible peptides.24,25 Experimental data indicate that EPO dimers induce a much higher hematocrit increase than the EPO monomer without differences in pharmacokinetics.25 It has been shown that fusing the carboxyl-terminal peptide of human chorionic gonadotropin beta-subunit (human chorionic gonadotropin beta) to a number of biologically active proteins increased in vivo potency and circulatory half-life of the proteins containing this peptide without affecting assembly, secretion, receptor binding affinity, or in vitro bioactivity. Recently, Fares et al.26 obtained a fusion protein by adding the carboxyl-terminal peptide of human chorionic gonadotropin beta to HuEPO-coding sequence. This chimeric protein had both enhanced in vivo potency and half-life compared with wild-type EPO in mice. Another approach for enhancing serum half-life includes increasing the molecular weight of EPO by fusion to the Fc part of an antibody.27,28 Interestingly, this approach has been used to create an Fc fusion protein that can be administered by aerosol inhalation.29 Attempts have also been made to synthesize artificially a glycoprotein similar to EPO, called synthetic erythropoiesis Kidney International (2008) 74 (Suppl 111), S33–S37
protein. This is a 51-kDa protein-polymer consisting of 166 amino acids with two other polymer moieties attached covalently.30 In cell and animal assays for erythropoiesis, synthetic erythropoiesis protein displayed potent biological activity and had significantly prolonged duration of action in vivo.31 Even though it is effective in correcting anemia, all these EPO-modified proteins raise some concerns about immunogenicity.32 NON-EPO-DERIVED EPO RECEPTOR AGONISTS: HEMATIDE
Hematide is a pegylated synthetic dimeric peptide with no sequence homology to EPO. In CKD patients not on dialysis, it has an intermediate mean half-life amid darbepoetin alpha and CERA (B59 and B70 h by the intravenous and subcutaneous route, respectively) and longer half-life than that measured in healthy individuals; this is consistent with the hypothesis that hematide is cleared, at least partially, through the kidney.33 Phase II trials showed that the drug given either intravenously or subcutaneously was effective in both the correction and maintenance phase of anemia treatment at dose ranges between 0.025 and 0.05 mg/kg; phase III trials are ongoing. Even though antibodies against hematide have been found in patients receiving the drug, they do not cross react with EPO.34 For this reason, hematide has been proposed as a possible treatment of anemia in pure red cell aplasia.34,35 Potential advantage of this drug is that its manufacturing process is simpler and cheaper than that of current ESAs. Recently, in the attempt to create other non-EPO-derived EPO receptor agonists, two sequences of EPO mimetic peptide-1, which is a 20-amino acid peptide with weak EPOlike bioactivity, were inserted on a human IgG4 Fc framework. The obtained molecule, called CNTO 530, was shown to bind selectively the EPO receptor, to mediate EPO receptor signaling, and to support proliferation of EPO-dependent cells.36 In vivo it stimulated erythropoiesis, providing a longlived increase in Hb in mice. OTHER STRATEGIES TO INCREASE ERYTHROPOIESIS
The inhibition or activation of transcription factors modulating EPO gene expression may be an alternative for stimulating erythropoiesis. The hypoxia-inducible transcription factors (HIFs) are central components in the cellular responses to hypoxia; under normoxic conditions, EPO gene expression is suppressed as a result of HIF inactivation because of O(2)dependent enzymatic hydroxylation and subsequent degradation of their alpha-subunit. 2-oxoglutarate analogs, which prevent HIF alpha hydroxylation, have emerged as promising tools for the stimulation of erythropoiesis and angiogenesis (the so called HIF stabilizers). Experimental data indicate that these agents stimulate erythropoiesis in vivo.37 These agents can be administered by the oral route. Unfortunately, the development of the first candidate molecule of this class, FG-2216, was halted after the occurrence of a case of fatal S35
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hepatic necrosis. Another concern related to HIF stabilizers is related to the fact that the HIF system promotes the transcription of many other genes; careful evaluation of other effects than erythropoiesis stimulation seems mandatory. GATA genes are an evolutionarily conserved family, which encode a group of important transcription factors involved in the regulation of diverse processes including the development of the heart, hematopoietic system, and sex gonads. Some of these factors also regulate EPO gene transcription.38,39 Oral administration of a GATA-specific inhibitor, K-7174, was found effective in reversing anemia induced by IL-1 beta or TNF-a in mice.40 Another possible strategy to increase erythropoiesis is the inhibition of the hematopoietic cell phosphatase. This protein, also known as src homology domain 2-containing tyrosine phosphatase 1, binds and activates the negative regulatory domain of the EPO receptor, inhibiting transduction inside the cell.41 Interestingly, a defective expression of hematopoietic cell phosphatase was found in patients with polycythemia vera.42 In contrast, hematopoietic cell phosphatase expression is increased in CD34 þ cells from patients with EPO resistance; the adding of a src homology domain 2-containing tyrosine phosphatase 1 antisense oligonucleotide suppressed src homology domain 2-containing tyrosine phosphatase 1 protein expression and partial recovered BFU-E colonies.43 Hematopoietic cell phosphatase inhibitors may be thus a novel target molecule to treat renal anemia and/or sensitize patients to EPO action. Finally, EPO gene therapy has been proposed for the management of anemic patients with CKD. A number of different techniques have been tried, such as the delivering of naked plasmid DNA into skeletal muscles,44,45 the subcutaneous implantation of autologous bone marrow stromal cells genetically engineered to secrete EPO,46 viral transfection,47 and the use of artificial human chromosome.48 EPO gene therapy has the theoretical advantage of releasing small but continuous amount of EPO into the circulation, but has still a number of problems, such as immunogenicity and the difficulty to exactly tune the exact amount of EPO needed to correct anemia and maintaining a level of expression that sufficiently promotes erythropoiesis in the long term.
F Locatelli and L Del Vecchio: Optimizing renal anemia management
DISCLOSURE
Francesco Locatelli has received consulting fees from Affimax, Roche, Amgen, and Shire. Francesco Locatelli has also received lecture fees from Roche, Amgen, and Shire. Lucia Del Vecchio has declared no financial interests. REFERENCES 1.
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Currently, the treatment of anemia with rHuEPO is quite expensive, given that its synthesis, as that of other biomolecules, is complex. New ESAs are, or will be, more expensive than rHuEPO, because of the high costs of pharmacological research, not yet covered by years of selling, but offer improved pharmacological characteristics that may help in the management of anemia in CKD patients. The synthesis of simpler molecules that is not related to ESA structure or the development of alternative strategies of erythropoiesis stimulation may lead to the creation of cheaper agents that would revolutionize the market. S36
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