Hematopoietic Growth Factors - EPO & G-CSF

Question 1

Hematopoiesis

Answer 1

EPO & G-CSF play major roles in hematopoiesis

CSFs affect the differentiation of stem-derived cells into a range of mature blood cell types

Question 2

EPO function

Answer 2

stimulates cell proliferation and differentiation within the erythroblast cell lineage

does so by
increasing number of committed cells capable of differentiating into erythrocytes
accelerating rate of differentiation of such precursors
increasing rate of Hgb synthesis in developing cells

Question 3

EPO therapeutic uses

Answer 3

anemia associated with CKD
anemia from chemo, AIDS virus, prematurity
myelodysplasia - bone marrow dysfunction
perioperatively to reduce the need for transfusion
improve autologous blood donation

Question 4

EPO synthesis

Answer 4

produced and excreted in the fetal liver and the adult kidney

synthesis is increased by kidney when oxygen content of blood is low

• when o2 abundant HIF-1a is hydroxylated and recognized by ubiquitin
• when O2 is scarce HIF-1a remains unmodified and translocates to nucleus where it increases EPO transcription

Question 5

EPO receptor activation

Answer 5

recall hGH receptor activation
JAK phosphorylated STAT
phosphorylized STATs spontaneously dissociate from the receptor and dimerize
STAT dimer moves into the nucleus where it activates transcription of target genes

Question 6

What expression host to use for EPO?

Answer 6

Bacterial cells don’t have N glycosylation needed
yeast do but it is the high mannose type
CHO & HEK cells useful

Question 7

Mammalian expression plasmid

Answer 7

CMV promoter for gene expression in mammalian cells
FLAG sequence that serves as a tag
polyA tract for efficient mRNA stability

Question 8

rhEPO types

Answer 8

same amino acid sequence by glycosylation differs and determines fate of EPO
Epoetin-a
Darbepoetin-a produced by CHO cells

Question 9

what factors contribute to immunogenicity

Answer 9

• sequence variation
• glycosylation
• contaminants
• formulation
• route
• dose
• length of treatment
• assay tech
• pt characteristics

Question 10

G-CSF

Answer 10

stimulates production of neutrophils that are important in combating infections via phagocytosis, antibact protein, and ROS secretion

acts through JAK-STAT signaling

used to treat neutropenia

Question 11

rG-CSF production

Answer 11

used in pts receiving chemotherapy. these agents reduce the duration of severe neutropenia, decrease the incidence of febrile neutropenia, and facilitate on-time delivery of scheduled doses of chemo

Question 12

rG-CSF agents

Answer 12

Filgrastim - produced by rDNA tech in E. coli

lenograstim - produced by rDNA tech in CHO cells

Pegfilgrastim - filgrastim with PEG covalently bonded

• greater activity
• lower peak plasma conc
• reduced rates of enzymatic degradation
• lower immunogenicity and antigenicity
• less toxicity
• greater solubility
• less frequent admin