New calcium-targeting therapies for acute pancreatitis

PancreasFest: 2019

Fred Gorelick

Yale School of Medicine/VA HealthCare CT

Conflicts of Interest: Dr. Gary Desir, a collaborator, has a financial interest in Bessor Pharmaceutical, a company developing renalase as a diagnostic and therapeutic agent

Funding: NIDDK, NCI, DOD, Veterans Administration Collaborators: Gary Desir (Yale), Larry Miller (Mayo), Markus Lerch (Greifswald)

An ideal therapy

Value given prophylactically or therapeutically

High therapeutic index

Low cost

Multiple targets in acute injury

Promotes recovery

Acute Pancreatitis

Phases and effectors

INJURY RESPONSES

Acute

-Immediate

-Intermediate

Stabilization

Recovery

EFFECTORS

Acinar cell

Inflammatory cells

Vascular

Acute Pancreatitis

• Risk DM independent of severity

• Other studies up to 40% -population dependent (male, etoh,hyperlipidemia)

• Rate up to 80% in recurrent acute

• Risk similar for pancreatic exocrine insufficiency (PEI) but severity dependent

• Implications: need to follow AP patients long-term for diabetes and PEI

Also: Das Gut 2014; 63(5):818-31; Tu BMC Gastro 2017; R. Hollemans: Pancreatology, 2018;

Petrov and Yadav, Nature Reviews, 2018; Huang DDS

Hsiu-Nien Shen, AJG, 2015 W Huang DDS Feb 2019

Both exocrine and endocrine insufficiency develop often

Acute Pancreatitis

Multiple cell types mediate disease

Ca2+ signaling

Zymogen activation

Inflammatory mediators

Mechanism of cell death

Paracellular leak

Thrombosis

Transient infiltration

Inflammatory

Anti-Inflammatory

Acinar cell Acinar cell

M2-inflammatory

Acute Pancreatitis

New/rationale therapies

Targets: 1• Store-operated calcium entry

• Mitochondrial transition pore

• Autophagy stimulation

• Unfolded protein response

• Inflammasome: GPR81/lactate

• Kynurenine inhibition

• Neurogenetic inflammation (alpha-2-adrenergic receptor agonist)

Lee and Papachristou: Nature Reviews, 2019; Faghih, Fan, Singh Curr Treat Options Gastro, 2019; ClinTrials.gov (June 2019)

Targets: 2• COX (prostaglandin) inhibition

• TNF synthesis and activity

• Angiopoietin inhibition

• Lipase inhibition/generation of FFA from TG

• IL6 receptor inhibitor

• Thrombin inhibition

• Microbiome (FMT)

Acute Pancreatitis

Calcium signaling: targeting for treatment

Calcium

Entry

Orai1

Piezo

Calcium

Extrusion

PMCA

RYR IP3R

Calcium

Calcineurin

• Husain S. PNAS 2005 and AJP 2012 (dantrolene)

• Orabi AI Cell Mol Gastroenterol Hepatol 2017 (Calciuneurin/NFATc)

• Gerasimenko PNAS 2013 and Wen L Gastroenterology 2015 (ORAI1)

• Mankad 2012 and Samad 2014 (JBC)

• Romac..Liddle, Nat Comm. 2018

Intracellular

calcium

stores

Acute Pancreatitis

Calcium signaling: targeting for treatment

Calcium

Entry

Orai1

PiezoRYR IP3R

Calcium

Calcineurin

Some calcium-targeting agents reduce both acinar cell

and inflammatory cell pancreatitis responses

Intracellular

calcium

stores

dantrolene

FK506Caloxin

Estradiol

GsMTx4

CM4620

caffeineRenalase

Insulin

Calcium

Extrusion

PMCA

Acute Pancreatitis

Calcium signaling drives activation of others pancreatitis responders

Neutrophil Macrophage Stellate cell

Hollander, Cancer Res 76: 3884, 2016

Waldron, J Physiol. 2019 Jun;597(12):3085-3105

M2-inflammatory

M1-Anti-

inflammatory

STIM1/Orai1

STIM1/Orai1 STIM1/Orai1 Renalase

Acute Pancreatitis

Renalase: (RNLS) a new survival factor

• 37 kD secretory protein (RNLS1; RNLS2 not secreted) made by the kidneys and other tissues

• NADH/FAD and catecholamine binding domains

• Pro-survival: activates plasma-membrane calcium ATPase 4B (PMCA4b) and calcium efflux; also activates ERK and AKT

• Pro-survival activity contained in 22 aa domain

• Pro-survival factor in pancreatic cancer and melanoma

• Reduced levels in renal failure

Acute Pancreatitis

In collaboration with G. Desir, Yale University

KOLODECIK J

Biol Chem 2017

Trypsin Edema TUNEL

Neutrophils Macrophages

Renalase deficiency worsens cerulein pancreatitis

Kolodecik J Biol

Chem 2017

Exogenous renalase reduces cerulein AP* severity when given after onset (2 hrs)

*Also effective in

arginine-induced

pancreatitis

Renalase reduces injury responses in acinar cells and macrophages

Renalase

PMCA4b

Acinar cell Macrophages

Zymogen activation

LDH release

Mitochondrial function

Inflammasome

activation

M1 specification

Cerulein

Carbachol

Bile salts

LPS

ATP

Cellular “pancreatitis”

Kolodecik. J Biol Chem 2017; Guo Cancer Research 2016

Acute Pancreatitis

RNLS-KO mice have delayed cerulein AP recovery

0 2 6 12 24 36 48

0.0

0.5

1.0

1.5

2.0

2.5

Hours after first CER injection

His

tolo

og

ic S

co

re E

dem

a

/40x f

ield

C57-WT

KO **

**

0 2 6 12 24 36 48

0.0

0.5

1.0

1.5

Hours after first CER injection

His

tolo

og

ic S

co

re V

acu

ole

s/4

0x f

ield

C57-WT

KO

*

0 2 6 12 24 36 48

0.0

0.5

1.0

1.5

2.0

Hours after first CER injection

His

tolo

og

ic S

co

re

pykn

oti

c n

ucle

i /4

0x f

ield

C57-WT

KO

*

*

Histologic Edema

VacuolesPyknotic Nuclea

0 2 6 12 24 36 48

60

65

70

75

80

85

Hours after first Cerulein injection

Ed

em

a %

wet

weig

ht

KO

C57-WT*

**

*

Edema (wet/dry)

Acute Pancreatitis

Serum renalase levels decrease with the onset of acute pancreatitis

Murine

induction recovery

Could serum renalase levels have prognostic value in human acute pancreatitis?

Kolodecik J Biol Chem 2017

K id n e y P a n c r e a s L iv e r S p le e n

0

2 0

4 0

6 0

8 0

RN

LS

to

To

tal

La

ne

Pro

tein

Ra

tio

C o n tro l

1 H C E R

n 3

R N L S

6 H C E R

2 4 H C E R* *

RNLS partitions to the kidney

kidney

Control

cerulein

Serum renalase levels fall in clinical AP and may predict severity

With M Lerch and G Desir

Plasma RNLS maybe dramatically reduced in

severe AP

Serum by ELISA

Plasma RNLS are decreased in acute pancreatitis

Summary of renalase data:

• Secretory protein with paracrine and hormonal actions

• A broadly active survival factor• Acute injury

• Select cancers (negative effects on survival- PDAC, melanoma)*

• Requires PMCA4b; possibly other co-receptors

• Blood levels decrease in experimental AP and in humans with AP – may correspond to severity

• May modulate AP recovery

• Exogenous RNLS reduces experimental AP severity

*Hollander, Cancer Res 2016; Guo, Sci Reptrs 2016

New and rationale calcium targeting therapies: Suggestions for the future

EFFECT ON:

SAP to MOF transition• Pulmonary• Renal• Vascular• Intestinal

• Recovery

• Short-term• Unresolved AP• Infections

• Long-term• Exocrine• Endocrine• Neoplasia• Infections

Acute Pancreatitis