Title: A Systems Pharmacology Model of Erythropoiesis in Mice Induced by the Small Molecule Inhibitors of
Prolyl Hydroxylase Enzymes
Indrajeet Singh* (1), Pratap Singh (2), Wojciech Krzyzanski (1)
(1) Department of Pharmaceutical Sciences, State University of New York at Buffalo, Buffalo, NY, USA;
(2) Pharmacokinetics, Dynamics and Metabolism (PDM), Pfizer Inc, Cambridge, MA, USA.
Objective: Hypoxia-Inducible-Factor (HIF-1) is an important target for drug development due to its crucial role in
angiogenesis, erythropoiesis, glucose metabolism, ischemia, and cell proliferation/survival . The intra-cellular levels
of HIF1α, one of the subunits of hetero-dimeric HIF-1 transcription factor, are tightly controlled by the Prolyl-
Hydroxylases (PHDs) through a highly regulated oxygen sensing mechanism [2, 3]. The small molecule inhibitors of
PHDs result in the accumulation of HIF1α levels, and lead to the initiation of erythropoiesis. Thus small molecule
inhibitors of PHDs (PHI) have the potential to serve as viable alternatives to the currently approved erythropoietins
suffering from safety liabilities.
The main objective of this study was to develop a mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model
describing the effect of a novel small molecule inhibitor (PHI-1) on erythropoietic response in mice. This model employs
the systems biology based approach to vertically integrate the intracellular HIF1α and EPO mRNA responses with in
vivo markers of erythropoiesis such as EPO plasma levels and reticulocyte counts.
Methods: A novel inhibitor of PHDs (PHI-1) was given orally at three single doses of 10, 30 and 100mg/kg to male
BALB/C mice (N=3-6, per time point). The blood plasma and kidney tissue samples were collected upto 96hr. A series
of phamarcodynamic responses were measured to quantify the system components varying from cellular level mRNA
and proteins to plasma proteins and blood cell populations. The measured PD markers were: kidney HIF1α, kidney
erythropoietin (EPO) mRNA, plasma EPO, reticulocyte, hemoglobin and red blood cells. A two compartment model
with first order absorption was selected to describe the PK in plasma. For PD effects, our model includes direct effect,
indirect response, enzyme kinetics and cell life span models. ADAPT5 was used for all model fittings.
Results: At higher PHI doses, ~30-40 fold increase in HIF1α was observed which caused ~1000 fold increase in EPO
mRNA and plasma EPO within 6-8 hr whereas the reticulocytes increased by 3-4 fold within 3-5 days. Model parameters
such as PHI elimination rate (kel, 0.43hr-1), the drug potency (IC50, 0.68μM ) for the degradation of PHDs; and the mean
life span of reticulocytes (Tr~90hr) were estimated with high precision. The PK/PD model was reduced to the point
where all system compenent parameters were estimated by fitting the experimental data. Simulations were performed to
validate the model by predicting RBC and hemoglobin time profiles.
Conclusion: This study marks the first scientific report on a novel mechanism-based PK/PD model to explain the
stimulation of erythropoietic response induced by the small molecule inhibitors of PHDs enzymes. The quantitative
framework of this model can be applied towards efficient design of pre-clinical studies for the development of novel
therapeautics for critically ill anemia patients.
1. Semenza, G.L., Targeting HIF-1 for cancer therapy. Nat Rev Cancer, 2003. 3(10): p. 721-732.
2. Hsieh, M.M., et al., HIF prolyl hydroxylase inhibition results in endogenous erythropoietin induction,
erythrocytosis, and modest fetal hemoglobin expression in rhesus macaques. Blood, 2007. 110(6): p. 2140-7.
3. Schofield, C.J. and P.J. Ratcliffe, Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol, 2004. 5(5): p.