HIF prolyl hydroxylase

inhibitors

11th May 2017

Erythropoiesis stimulating agents in renal anemia

Anaemia is a cardinal feature of chronic kidney disease

Primarily due to impaired erythropoietin production

Until late 1980’s

Regular transfusion

Immune sensitisation, iron overload, Hepatitis B & C

For most patients - chronic anaemia

Effect on life, and on combustion

1977

1987 Used in humans with kidney disease

2003 $10bn per annum

Problems with ESA’s in renal anemia

Require injection

Difficult to manufacture, store

Many patients require iv iron

Primary red cell aplasia (~1 in 10,000 years)

Targeting normal haematocrit associated with increased CV events

FDA Black box warning

Revised targets

Besarab et al New Engl J Med 1998

Normal HCT trial – 42% vs 30%

Risk ratio 1.3 (0.9 – 1.9)

Pfeffer et al New Engl J Med 2009

TREAT: CKD + DIABETES Hb rescue at 9 g/dl vs 13 g/dl

CHOIR: 11.3 vs 13.5 Higher rate of CV events

Singh et al New Engl J Med 2006

Why is treatment to a higher haematocrit associated

with increased cardiovascular events?

1. An effect of supraphysiological erythropoietin levels

2. Related to iron supplementation

3. Subnormal haematocrit may be beneficial in CKD

Ben Foster as Lance Armstrong in The Program, 2015

Less oxygen

More erythropoietin

1% O2

ONHREErythropoietin gene

OFF

21% O2

HREErythropoietin gene

HIF-1bHIF-a

HRE

HIF-2a

Wiesener et al, Blood 1998

Hypoxia Inducible Factor (HIF)

Transcription factor regulated via destruction of a subunit

von Hippel-Lindau disease

Autosomal dominant

1 in 36,000

Retinal angiomas

Renal cell carcinoma

Gene cloned in 1993

mutated / inactivated in ~ 80% of sporadic clear cell kidney cancer

Knudson’s two hit model

Recessive at a cellular level

Mutated / inactivated in ~ 80% of sporadic clear cell renal carcinomas

Maxwell et al, Nature 1999

O2HIFa Pro564

HIFa Hyp564

UbUb

Ub

Rbx1

Cul2Elongin

BC

E2/E1

HIFa

VHL

Hyp564

UbUb

Ub

HIFa

C. elegans - evolutionary conservation of the HIF system

Epstein et al, Science 2001

Three closely related isoforms are

mammalian homologues of EGL-9

O2

Fe

HIFa Pro564

2oxoglutarate

PHD

CO2

Fe

HIFa Hyp564

succinate

PHD

UbUb

Ub

Rbx1

Cul2Elongin

BC

E2/E1

HIFa

VHL

Hyp564

UbUb

Ub

HIFa

Three PHD enzymes

Two HIFa subunits

Vasomotor

NOS isoforms

Endothelins

Adrenoreceptors

Tyrosine hydroxylase

Blood oxygen

Erythropoietin

Metal transport

Transferrin

Caeruloplasmin

Angiogenesis

VEGF

PLGF

PDGF

Matrix metabolism

Collagens/prolyl hydroxylases

Transglutaminase

Cell proliferation/survival

IGF/IGF-BPs

Cyclin G2

Nip/Nix

Energy metabolism

Glucose transporters

Glycolytic enzymes

TranscriptionHIF-1bHIF-1a

coactivators

HRE

HIF regulates hundreds of genes

Small molecule PHD inhibitors activate HIF

Jaakkola et al, Science

HIF has many targets

PHD’s may regulate other pathways

HIF-1 other PHD targets HIF-2

Many structurally related enzymes

Autosomal recessive - Hypomorphic VHL allele. Chuvash polycythaemia

Autosomal dominant - Haploinsufficiency. PHD2 erythrocytosis

Autosomal dominant - Gain of function. HIF2A erythrocytosis

Modest HIF activation / PHD2 inactivation is sufficient to increase haematocrit

Validates HIF2 activation and PHD2 inhibition as a therapeutic strategy

Genetic HIF activation in humans

A small molecule PHD inhibitor, FG-2216

increases EPO in humans

Normal volunteers

Dialysis patients

with kidneys in situ

Dialysis patients

Without kidneys

Bernhardt et al J Am Soc Nephrol 2010

On target effects – mouse studies

HIF-1 other PHD targets HIF-2

+/- -/-+/+

Inheritance in PHD-deficient mice

PHD-1 (135) 22% 55% 23%

PHD-2 (331) 38% 62% 0%

PHD-3 (186) 31% 51% 18%

On target effects of PHD enzyme removal

Aragones et al Nature Genetics

Wild typephd2 -/-

PHD2 (and HIF-2a) major regulators of

haematocrit

Loss of PHD1 protects against ischemic muscle

necrosis

WT gastrocnemius muscle PHD1-/- gastrocnemius

muscle

Loss of PHD1 protects ischaemic skeletal muscle

Aragones et al Nature Genetics 2008

Hill et al, J Am Soc Nephrol 2008

PHD inhibitors protect from renal IRI

HIF is not fully activated in complete ischemia

HIF1 or HIF2 heterozygosity exacerbates renal IRI

DMOG, L-mimosine activate HIF-a in IRI

DMOG Saline

Hill et al, J Am Soc Nephrol 2008

PHD inhibitors protect from renal IRI

Casts Tubular Dilatation

Treated TreatedUntreated Untreated

Casts

Treated U ntr ea te d0

1

2

3

4Treated

Untreated

Score

P<0.0001

Score

Score

Tubular Dilatation

Trea ted Unt re a te d0

1

2

3

4Treated

Untreated

P<0.0001

Score

Hill et al J Am Soc Nephrol 2008

PHD inhibitors from renal IRI

Necrosis

PHD-1 WT0

1

2

3P<0.001

Sco

re

Brush Border

PHD-1 WT0

1

2

3

4P<0.001

Sco

re

Casts

PHD-1 WT0

1

2

3

4P<0.001

Sco

re

Dilated tubules

PHD-1 WT0

1

2

3

4P<0.001

Sco

re

Loss of PHD1 protects from renal IRI

WTPHD1-/- WTPHD1-/-

WTPHD1-/- WTPHD1-/-

Peter Hill unpublished data

Casts

0

1

2

3

Brush Border

0

1

2

3

Dilated Tubules

0

1

2

3

4

Necrosis

0

1

2

3

PHD3 -/- WT PHD3 -/- WT

PHD3 -/- WT PHD3 -/- WT

Loss of PHD3 exacerbates renal IRI

Peter Hill unpublished data

Mouse knockout: myocardial damage is reduced

after cardiac ischemia/reperfusion in PHD3-/- mice

PHD WT PHD3-/-

PHD

WT

PHD3-/-

40

30

20

10

0Infa

rcte

d /

to

tal a

rea

x 1

00

P < 0.01

Peter Carmeliet unpublished data

Autosomal dominant - Cellular recessive / 2 hit. Von Hippel Lindau disease

Cerebellar, retinal haemangioblastoma; renal cysts, tumors:

pheochromocytoma

Not inherited - 70% of clear cell renal cancer. Two somatic VHL hits.

Autosomal recessive - Hypomorphic VHL allele. Chuvash polycythaemia

Autosomal dominant - Haploinsufficiency. PHD2 erythrocytosis

Autosomal dominant - Gain of function. HIF2A erythrocytosis

Valuable for defining physiological role

Chuvash: haemangiomas, thromboses, varicose veins, shortened lifespan,

altered cytokines

On target effects in humans

Very large numbers of foci of HIF activation

Proximal tubule

Distal tubule

Minimal proliferation

HIF2α

Gale et al, Blood 2008

“An activating mutation in HIF2A would be too good to be true”

*

Associated with severe pulmonary hypertension

Confirmed in humans and mice with Chuvash

polycythaemia & VHL compound heterozygotes

• key component of the classical complement pathway

• Is involved in several other immunological processes

maintenance of immune tolerance via clearance of apoptotic cells,

phagocytosis of bacteria

neutralization of retroviruses,

cell adhesion,

modulation of dendritic cells (DCs), B cells and fibroblasts

• C1q can engage a broad range of ligands

Immunoglobulins

envelope proteins of certain retroviruses,

lipopolysaccharides (LPS), porins from Gram-negative bacteria,

phospholipids (PL), apoptotic cells.

Example off target effect - complement C1q

Structural organization of the C1q molecule

460 kd

U

IFN

/DM

OG

IFN

0

10

20

30

Untreated

IFN

IFN/DMOG

***

C1

q (

ng

/ml)

20% O2 1% O2

IFN - + + - + +

DMOG - - + - - +

HIF-1a

a-tubulin

C1q expression by THP-1 derived macrophages is sensitive to DMOG

Kiriakidis et al, Kidney Intl in press

N N+IFN H H+IFN0

25

50

75

100

C1q

(U

/ml)

C1q expression by THP-1 derived macrophages is resistant to hypoxia

P4HA1 is present in macrophages and necessary for C1q secretion

Small molecule PHD inhibitors reduce C1q through an off target effect

on P4HA1

Phase I + II Tolerated, safe and effective

Phase III in progress

PHD inhibitors 2017

AKB-6548/vadadustat

PHD inhibitors in renal anemia

Orally active

Manufacturing, stability

Less intravenous iron required

Critical question in Phase 3 trials

Cardiovascular endpoints / mortality compared with ESA

Effects on blood pressure, lipids, inflammation, cancer….?

Unusual target for pharma: on-target + off-target effects

Activating a pathway rather than inhibiting

“Tickling the target”

What is your prediction?

PHD inhibitors in ischemia

Major unmet medical need – stroke, claudication, ulcers

PHD1 looks like a good choice

Inhibition of C1q may be advantageous

Track record of translation from animal models is very poor

Dose, timing etc may be critical

Route to market is unclear

Market size is unclear

Summary and conclusions

1. Many processes, including erythropoiesis, regulated by oxygen

tension

2. Underpinned by PHD enzymes, which are druggable

3. PHD inhibitors are effective in increasing haematocrit, and are well

tolerated

4. Currently being tested vs ESA’s as a treatment for renal anaemia.

Included in doping controls. Benefits vs risks likely to vary.

5. Likely to be attractive in other settings

Acknowledgements

Chris Pugh, Peter Ratcliffe, Chris Schofield, David Mole, Peter Robbins

Peter Carmeliet, Dominic Withers, James Cantley, Colin Selman, Melanie Percy,

Mary-Francis McMullin, Ted Tuddenham, John Chambers, Jaspal Kooner,

Roberto Mayor, Margaret Ashcroft

Frauke Forstreuter, Sarah Harten, Tapan Bhattacharyya, Reiko Ueki, Maxine

Tran, Peter Hill, Ravi Barod, Daniel Gale, Deepa Shukla, Daz Khan, Serafim

Kiriakidis, Simon Hoer, Natalie Burrows

With financial support from the MRC, EU, Wellcome Trust, CRUK, Imperial and

the BHF.

In kidney Epo-producing cells are cortical / OM fibroblasts

Maxwell et al, KI, 1993