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Darbepoetin alfa (Aranesp ® ) molecular characteristics and basic research.

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Presentation on theme: "Darbepoetin alfa (Aranesp ® ) molecular characteristics and basic research."— Presentation transcript:

1 Darbepoetin alfa (Aranesp ® ) molecular characteristics and basic research

2 Presentation overview The evolution of protein therapeutics Structure and function of recombinant human erythropoietin Importance of sialic acid content Discovery and development of darbepoetin alfa –molecular characteristics –implications for clinical use

3 The evolution of protein therapeutics Recognition that proteins can be useful ‘hormone- like’ therapeutics eg, insulin (1920s) First purified from animal and human tissues eg, insulin, growth hormone, factor VIII Recombinant protein therapeutics – Amgen scientists were among the leaders cloning erythropoietin in 1983

4 The evolution of protein therapeutics First purified from animal and human tissues eg, insulin, growth hormone, factor VIII Recombinant protein therapeutics – Amgen scientists were among the leaders cloning erythropoietin in 1983 Now a new era where protein therapeutics are modified to enhance their properties as therapeutics eg, darbepoetin alfa (Aranesp ® ) Recognition that proteins can be useful ‘hormone-like’ therapeutics eg, insulin (1920s)

5 Darbepoetin alfa Darbepoetin alfa is a biochemically distinct recombinant erythropoietic protein that stimulates the production of red blood cells The discovery of darbepoetin alfa resulted from basic research into the structure and function of rHuEPO and its attached carbohydrate The longer serum residence time and greater biological activity of darbepoetin alfa results from the addition of two extra sialic acid-containing carbohydrate side chains

6 Structure of EPO bound to an EPO receptor rHuEPO has four carbohydrate side chains rHuEPO has a theoretical maximum of 14 sialic acids Receptor 1 EPO Receptor 2 Carbohydrate side chains

7 Typical tetra-antennary carbohydrate Sialic acidN-linked carbohydrate The number of branches in carbohydrates and therefore the number of sialic acids is variable

8 In vivo activity in mice increases with greater sialic acid content Adapted from Egrie JC, et al. Br J Cancer. 2001;84(suppl 1):3-10. Isoform Increase in Hct at day 30 (points) Longer serum half-life Higher receptor binding Greater in vivo activity

9 Darbepoetin alfa development strategy Introduce N-linked glycosylation consensus sequences (Asn-Xxx-Thr/Ser) into rHuEPO Identify individual variants that have the desired properties Test optimal combinations of variants

10 Amino acid sequence of rHuEPO N-glycosylation sites Disulphide linkages O-glycosylation site

11 rHuEPO has two receptor binding sites Site 2 Site 1 Site 2 The effect of mutations on in vitro activity is indicated: red <2% active, orange <20% active, yellow <70% active

12 The following needed to be addressed in order to make darbepoetin alfa Would the glycan addition be efficient? Would the molecule be properly folded/stable? Would the ability to stimulate erythropoiesis be retained? Would in vivo activity be increased?

13 Discovery of new glycosylation sites in rHuEPO N-linked carbohydrate consensus sequences were introduced into rHuEPO at positions indicated by vertical lines Each molecule was tested to see if it had the desired properties Two positions, Ala30 and Trp88, were selected for further development work Good bioactivity Poor bioactivity Glycosylated Partial glycosylation Unglycosylated a1 a2 N24T26N38T40N83S85 B1 COOH NH2 S126 a3 B2 a4

14 Optimisation of glycosylation sites A two-fold increase in 9G8A immunoreactivity is suggestive of an altered conformation. Val87 substitutions allow carbohydrate addition at position 88 and normalisation of the conformation Ala = alanine; Asn = asparagine; His = histidine; Leu = leucine; Pro = proline; Ser = serine; Thr = threonine; Trp = tryptophan; Val = valine

15 Comparison of rHuEPO and darbepoetin alfa Three N-linked carbohydrate chains Maximum 14 sialic acids MW ~ 30,400 daltons 40% carbohydrate Five N-linked carbohydrate chains Maximum 22 sialic acids MW ~ 37,100 daltons 51% carbohydrate New carbohydrate side chains Receptor 1 Darbepoetin alfa Receptor 2 Receptor 1 rHuEPO Receptor 2 Carbohydrate side chains

16 1 Heatherington A, et al. Proc Am Soc Clin Oncol. 2002;21:256b. Abstract and poster; 2 Macdougall I, et al. J Am Soc Nephrol. 1999;10: Darbepoetin alfa has a longer half-life than rHuEPO: single-dose PK of IV administration Time post-IV injection (hours) Mean (SD) baseline-corrected serum concentration (ng/mL) Darbepoetin alfa (oncology; 0.5 µg/kg, n = 20)* 1 Darbepoetin alfa (dialysis; 0.5 µg/kg, n = 11) 2 rHuEPO (dialysis; 100 IU/kg, n = 10) 2 t 1/2 = 25.3 hours t 1/2 = 8.5 hours t 1/2 = 38.8 hours *Oncology patients received 2.25 µg/kg Data shown are normalised for 0.5 µg/kg SD = standard deviation; IV = intravenous

17 ,00010,000100,0001,000,000 ng/mL sample rHuEPO (three chains) NM279 (four chains) Darbepoetin alfa (five chains) In vivo activity in mice increases with increasing number of glycans 59 Fe incorporated (% of maximum)

18 The anti-EPO monoclonal Ab F12 does not neutralise EPO bioactivity Amount of Ab added (µg/mL) Effect of antibodies on EPO in vitro bioactivity Non-neutralising anti-EPO monoclonal Ab (F12) Neutralising anti-EPO monoclonal Ab (D11) Neutralising anti-EPO polyclonal Ab (862) In vitro activity (%)* In vitro activity assay measures formation of erythroid colonies from human bone marrow in soft agar * EPO = erythropoietin; Ab - antibody

19 Development of darbepoetin alfa A new erythropoietic protein, biochemically distinct from rHuEPO Increased sialic acid content, resulting in –a longer circulating half-life (2–3-fold greater than rHuEPO) –less frequent dosing requirements –increased biological activity Pharmacokinetics offer potential for higher response rates and faster onset of action

20 Darbepoetin alfa: conclusions Darbepoetin alfa has a similar conformation to rHuEPO Darbepoetin alfa binds to and activates the same receptor as rHuEPO Darbepoetin alfa has increased sialic acid-containing carbohydrate resulting in increased in vivo activity and a prolonged half-life 1 Provides the opportunity to dose less frequently: QW, Q2W, Q3W or Q4W 2–4 Clinical benefits have been demonstrated (high and rapid haematological responses at convenient dosing schedules) 5 QW = once every week; Q2W = once every 2 weeks; Q3W = once every 3 weeks; Q4W = once every 4 weeks 1 Egrie JC, et al. Br J Cancer. 2001;84(suppl 1): Glaspy JA, et al. Br J Cancer. 2002;87: Kotasek D, et al. Eur J Cancer In press. 4 Kotasek D, et al. Proc Am Soc Clin Oncol. 2002;21:356a. Abstract Glaspy JA, et al. Cancer. 2003;97:


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