Axis Shield : Preclinical Characterization of Vadadustat (AKB-6548), an Oral Small Molecule Hypoxia Inducible Factor Prolyl-4-Hydroxylase Inhibitor, for the Potential Treatment of Renal Anemia

Preclinical Characterization of Vadadustat (AKB-6548), an Oral Small Molecule Hypoxia Inducible Factor Prolyl-4-Hydroxylase Inhibitor, for the Potential Treatment of Renal Anemia

A. Zuk, Z. Si, S. Loi, S. Bommegowda,

S. Danthi, G. Molnar, M. Rabinowitz

Research and Development

Akebia Therapeutics

Cambridge, MA

Disclosures

• The authors are employees of Akebia Therapeutics, which funded the studies

Disclaimers

• Vadadustat is an investigational drug. Vadadustat is not approved by the United States Food and Drug Administration or any regulatory authority.

Confidential - Not for Distribution

Objective

• To summarize the preclinical pharmacological characterization of vadadustat

HIF and the prolyl-4-hydroxylase domain enzymes

Normal O2

HIF-α

Abbreviations:

Low O2

O2 = oxygen

PHD = prolyl-4-hydroxylase domain HIF = hypoxia inducible factor EPO = erythropoietin

Hb = hemoglobin RBC = red blood cell

O2 O2 O2

O2 PHD O2

O2O2

HIF-α

OH

OH

HIF-α

OO

Vadadustat2 PHD 2

HIF-α

HIF-α

HIF-α

…combines with other factors…

HIF-α is degraded by proteasome

α

HIF translocates to nucleus

Activates protein production

Protects against low oxygen

EPO

Iron utilization/absorption

Hepcidin

Hb and RBC

production

Vadadustat inhibits recombinant human PHD1, PHD2 and PHD3 at equivalent nanomolar concentrations*

PHD1

PHD2

PHD3

% Normalized Response

100			Expt #1			Response	100			Expt #1			Response	100			Expt #1
			Expt #2							Expt #2							Expt #2
75			Expt #3				75			Expt #3				75			Expt #3
					Normalized%						Normalized%
50						50						50
25							25							25
0							0							0
-10	-9	-8	-7	-6	-5		-10	-9	-8	-7	-6	-5		-10	-9	-8	-7	-6	-5
	Vadadustat Log [M]					Vadadustat Log [M]					Vadadustat Log [M]

*Measured by Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay. Data represent Mean +SD.

Abbreviations:

PHD = prolyl-4-hydroxylase domain

IC50 = half maximal inhibitory concentration pIC50 = negative log of the IC50 value in molar

Mean (95% Confidence Interval)

	IC50 value (nM)	pIC50 value
PHD1	15.36 (11.96, 19.73)	7.81 (7.71, 7.92)
PHD2	11.83	(8.20,	17.07)	7.93 (7.77, 8.09)
PHD3	7.63	(7.21,	8.07)	8.12 (8.09, 8.14)

Vadadustat-O-glucuronide inhibits recombinant human PHD2 at micromolar concentration*

Vadadustat-O-glucuronide

s e						E x p t # 1
n
1 0 0
p o
					E x p t # 2
R e s	7 5					E x p t # 3
liz e d	5 0
o rm a	2 5
N
%	0
	- 1 0	- 9	- 8	- 7	- 6	- 5

Inhibition is approximately 200-fold less potent than the parent compound at the IC50

V a d a d u s ta t -O-G lu c u r o n id e L o g [M ]

*Measured by TR-FRET Assay. Data represent Mean +SD.

	IC50 value (μM)	pIC50 value
Mean (95%	2.31 (1.74, 3.08)	5.64 (5.51, 5.77)
Confidence Internal)

Abbreviations:

PHD2 = prolyl-4-hydroxylase domain 2

IC50 = half maximal inhibitory concentration pIC50 = negative log of the IC50 value in molar

Vadadustat is a competitive inhibitor of 2-oxoglutarate for recombinant human PHD2*

Ratio (665nm/615nm)

				[Vadadustat] Log [M]
6000				-5
				-6
4000				-7
			-8
				-9
2000				-10
				-11
0				-12
-10	-9	-8	-7	-6

2-Oxoglutarate Log [M]

*Measured by TR-FRET Assay. Data represent Mean +SD.

Abbreviations:

PHD2 = prolyl-4-hydroxylase domain 2

Vadadustat inhibition of recombinant human PHD2 is not sensitive to iron concentration in vitro*

% Normalized Response

100	+ 100nM Fe	2+
	+ 1μM Fe	2+
50	+ 10μM Fe	2+

0

-15	-10	-5
	Vadadustat Log [M]

*Measured by TR-FRET Assay. Data represent Mean +SD.

		Vadadustat +	Vadadustat +	Vadadustat +
		100 nM Fe2+	1 µM Fe2+	10 µM Fe2+
Abbreviations:	IC50 (nM)	19.25 ± 5.74	3.91 ± 0.38	3.26 ± 0.24
PHD2 = prolyl-4-hydroxylase domain 2
IC50 = half maximal inhibitory concentration

Vadadustat was shown to stabilize both HIF-1α and HIF-2α in Hep3B and HUVEC cell lines in a dose and time dependent manner*

		HIF-1α Hep3B
g)	10	6 Hours
(EC		= 44 μM)
μ	50
(pg/			50
		24 Hours
		(EC		= 67 μM)
HIF1α	5
	0
		-6		-5	-4	-3
		Log Vadadustat [M]

		HIF-2α Hep3B
g)	1.0	6 Hours
(EC		= 51 μM)
μ	50
(pg/
		50
		24 Hours
		(EC		= 54 μM)
HIF2α	0.5
	0.0
		-6		-5	-4	-3
		Log Vadadustat [M]

Abbreviations:

HIF1α = hypoxia inducible factor-1 alpha HIF2α = hypoxia inducible factor-2 alpha Hep3B = human hepatocarcinoma cell line HUVEC = human umbilical vein endothelial

cell

		HIF-1α HUVEC				HIF-2α HUVEC
g)	60	6 Hours			g)	10
	(EC50 = 25 μM)
μ				μ
(pg/	40	(EC50 = 71 μM)			(pg/
	24 Hours
HIF1α	20				HIF2α	5
					6 Hours	μM)
						24 Hours
								(EC50 = 21
	0					0		(EC50 = 38 μM)
	-6	-5	-4	-3		-6	-5	-4	-3
		Log Vadadustat [M]				Log Vadadustat [M]

*Measured by Mesoscale Discovery (MSD) Electrochemiluminescence Assay. HIF1α and HIF2α were normalized to total cellular protein (pg/μg). Data represent Mean +SD.

Erythropoietin (EPO) secretion is increased in vitro after exposure of Hep3B cells to vadadustat*

EPO (mIU/mL)

30	Expt #1	+	+
	Expt #2
25
20
15		+ +
10		+
5
0

Medium	0.1%	0.1	0.3	1	3	10	0.3%	30μM
DMSO
			Vadadustat (μM)		DMSO	Vada

			EPO EC50	(μM)	EPO release
			(mIU/mL)/EC50
Abbreviations:		Vadadustat	9.97		12.04
DMSO = dimethylsulfoxide vehicle
*Measured by an Enzyme Linked ImmunoSorbent Assay (ELISA) after 24 hrs incubation. Data
EC50 = half maximal effective
represent Mean +SD. + P < 0.05 vs respective DMSO Control, Tukey's Multiple Comparisons Test
concentration

Production of vascular endothelial growth factor (VEGF) was not observed to increase in vitro after exposure of Hep3B cells to vadadustat*

VEGF (pg/mL)

1000	+ +
Expt #1
Expt #2
800

600

400

200

0

0.1%		3 μM 10 μM		0.3% 30 μM	1% O2
DMSO		Vadadustat		DMSO Vadadustat	Hypoxia

Abbreviations:

*Measured by Enzyme Linked ImmunoSorbent Assay (ELISA) after 24 hrs incubation. Data

represent Mean +SD. + P < 0.05 vs 0.1% DMSO, Tukey's Multiple Comparisons Test.

DMSO = dimethylsulfoxide vehicle

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Single-dose administration of vadadustat in rats was shown to increase the circulating levels of EPO in a time and dose dependent manner*

EPO (pg/mL)

30000	50 mg/kg
	150 mg/kg
20000

10000

0

0	1	3	6	24	72
		Hours Post Dose

*Measured by Enzyme Linked ImmunoSorbent Assay (ELISA). Data represent Mean +SD.

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Multi-dose exposure to vadadustat in mouse, rat and dog demonstrated increases in hemoglobin and hematocrit

Hemoglobin

Hematocrit

Duration of treatment of normal animals:

• Mouse = up to 6 months
• Rat = up to 2 years
• Dog = up to 9 months

In mouse, rat and dog, vadadustat had a relatively short half- life and did not accumulate after repeat dosing

			Gender	Gender
	Dose Level		Combined	Accumulation
Species	Day	Combined
(mg/kg)	AUClast	Ratio
		T1/2 (h)
			(µg*h/mL)
Mouse	100	1	2.40	234	NA
56	1.90	197	0.84
Rat	120	1	2.09	993	NA
28	2.05	902	0.90
Dog	120	1	2.86	740	NA
28	3.59	776	1.05

NA = Not Applicable

Conclusions

• In the preclinical setting, vadadustat
• • inhibited recombinant human PHD1, PHD2 and PHD3 isoenzymes at equivalent nanomolar concentrations
  • stabilized both HIF-1α and HIF-2α in vitro
  • stimulated EPO production in vitro and in vivo
  • increased hemoglobin and hematocrit in multiple species
  • did not stimulate VEGF production in vitro

• The pharmacology of vadadustat support development for anemia of CKD and ESRD

END

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Possible backup slides

Background and Mechanism of Action

No VADADUSTAT

+ VADADUSTAT

	HIFα
HIFβ
	O2	PHD
HIFβ	HIFα
				HIFβ
VADADUSTAT	PHD	PHD	HIFα	HIFβ
	O2

Hb and RBC	•	EPO
•	Iron Utilization/Absorption
production
•	Hepcidin

• Vadadustat is an orally bioavailable hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) in development for the potential treatment of anemia due to chronic kidney disease
• The HIF-PH enzymes are also referred to as EGLN proteins or prolyl 4-hydroxylase domains (PHDs)
• Pharmacological inhibition of PHD enzymes lead to the stabilization of hypoxia-inducible factor (HIF), a transcription factor that activates
  target genes to improve the O2 carrying capacity of the blood

EPO, erythropoietin; Hb, hemoglobin; HIF, hypoxia-inducible factor, HIF-PHD, HIF prolyl-hydroxylase domain; RBC, red blood cell.

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