Revisiting the Therapeutic Potential of LeptinRevisiting the Therapeutic Potential of Leptin

David Litzinger, PhD

Director, Pharmaceutical Sciences

Amylin Pharmaceuticals, Inc.

2011 AAPS National Biotechnology Conference

San Francisco, CA

2

Leptin HistoryLeptin History

• 1950 Obese mice appear randomly in a colony at Jackson Laboratory.

• 1994 Dr. Jeffrey Friedman and colleagues at Rockefeller University discover the ob gene and protein, and explore their role in body weight regulation, appetite, and metabolism.

• 1997 Amgen begins clinical trials of leptin for obesity.

• 1999 Amgen publishes disappointing obesity trial results.

• 2007 Amylin conducts clinical POC test of leptin/pramlintide combination; reduces bodyweight on average by 12.7% over 24 weeks.

• 2009 A 28-week pramlintide/leptin clinical study, followed by a 52-week extension study, showed sustained and robust weight-loss.

• 2010 Amylin submits the initial sections of a rolling submission for a BLA for the use of leptin to treat patients with rare forms of

lipodystrophy.

• 2011 Amylin and JDRF announce that they entered into a research collaboration agreement to provide financial support for a clinical

POC study to investigate leptin in patients with type 1 diabetes.

3

Multi-Hormonal Control of Body WeightMulti-Hormonal Control of Body Weight

Amylin

Question: Why aren’t anti-obesity drugs more effective?

Adapted from Badman M.K. and Flier J.S. Science 2005; 307: 1909-1914.

GI tract

Adipose tissue

Pancreatic islets

Hypothalamus

Hindbrain

CCK

Adiponectin

Insulin

Amylin

Leptin

OXM

Ghrelin

GLP-1

PYY3-36

GIP PP

ResistinVisfatin

Vagal afferents

4

Pramlintide (25,28,29Pro-h-amylin)Pramlintide (25,28,29Pro-h-amylin)

• 3949.4 Da, 37 amino acids

• Isoelectric point 5.3

• Single disulfide bond, C-term amidated

• No free cysteines

• Limited solubility at neutral pH (<0.5 mg/mL), higher at lower pH

5

0 2 4 6 8 10 12-10

-8

-6

-4

-2

0

Pramlintide: Clinical Studies in ObesityPramlintide: Clinical Studies in Obesity

Time (mo)

D in

Bo

dy

Wei

gh

t (%

)

6.8%

Weight Loss Weight Maintenance

In obese patients, pramlintide elicits

Reduced food intake• Increased satiation and satiety• Reduced 24-h intake (~500-750 kcal/d)• Reduced fast food intake• Reduced binge eating score (~45%)• Effects independent of nausea

Sustained weight loss• With proportionate reduction in leptin

Placebo (n = 17)Pramlintide 360 µg BID (n = 21)

Data are for evaluable population; N = 38; Mean ± SESmith SR, et al Diabetes Care. 2008; 31: 1816-1823

Double-Blind Study

Single-BlindExtension

6

• 16.2 kDa, 147 amino acids, (native leptin 146 AA)

• Isoelectric point 6.1

• Single disulfide bond

• No free cysteines

• Limited solubility at neutral pH (2-3 mg/mL), higher at lower pH

• Four helix bundle tertiary structure

Metreleptin (r-metHuLeptin)Metreleptin (r-metHuLeptin)

7

Leptin Replacement Elicits Profound Weight Loss in Leptin-Deficient Mice and HumansLeptin Replacement Elicits Profound Weight Loss in Leptin-Deficient Mice and Humans

ob/ob Mice

Before (Leptin Deficiency)

After (Leptin Replacement)

Congenital Leptin Deficiency*

Before (Leptin Deficiency)

After (Leptin Replacement)

Zhang,Y, et al. Nature. 1994;372:425–432* Republished with permission of American Society for Clinical Investigation, from Farooqi IS, et al. J Clin Invest. 2002;110:1093–1103; permission conveyed through Copyright Clearance Center, Inc.

8

-3 0 3 6 9 12-5

-4

-3

-2

-1

0

1

2

Leptin Does Not Cause Weight Loss in Diet-induced (Non-leptin deficient) ObesityLeptin Does Not Cause Weight Loss in Diet-induced (Non-leptin deficient) Obesity

Obese Humans

Time (wk)

D B

od

y W

eig

ht

(kg

)

Obese humans, mean ± SE; Continuous infusion of peptide at full doses (osmotic minipump) Roth et al., Obesity. 2006: 14(9 Suppl): A57-8. Abstract 177-P

Placebo BID (n = 25)Metreleptin 10 mg BID (n = 71)

Dietary Lead-In Randomized Treatment

DIO Rats

0 1 2 3 4-15

-10

-5

0

DB

od

y W

eig

ht

(%)

(Veh

icle

Co

rrec

ted

)

Time (wk)

VehicleLeptin 500 µg/kg per day

9

0 3 6 9 120

50

100

150

200

250

Roth JD, et al. Proc Natl Acad Sci USA. 2008;105:7257–7262.

Food Intake

Cu

mu

lati

ve F

oo

d In

take

(g

ram

s)

*

*p<0.05 compared to all groupsDiet-induced obesity prone rats (CRL; N=7/group).

Amylin+Leptin Synergy for Weight Loss is not Explained by the Anorexigenic Effect of AmylinAmylin+Leptin Synergy for Weight Loss is not Explained by the Anorexigenic Effect of Amylin

Amylin+Leptin

VehicleLeptin 500 µg/kg/d

Amylin 100 µg/kg/d

(Pairfed-Amylin)+Leptin

Time (Days)

0 3 6 9 12-14

-12

-10

-8

-6

-4

-2

0

2 Body Weight

% C

han

ge

in B

od

y W

eig

ht

(Veh

icle

-Co

rrec

ted

)*

Time (Days)

10

Veh A+LPF

AdiposeTissue(H&E)

Amylin/Leptin Induced Greater Fat Loss and Prevented Counter Regulatory AdaptationsAmylin/Leptin Induced Greater Fat Loss and Prevented Counter Regulatory Adaptations

Trevaskis JL et al. Endocrinology. 2008; 149(11):5679-87

• Amylin + Leptin treated DIO rats lost nearly 2-fold greater fat mass than pair-fed control*

• Amylin + Leptin prevented metabolic counter-regulatory adaptations

− No decrease in energy expenditure as was seen in pair-fed controls (oxygen consumption results)

− Fat utilized throughout study whereas pair-fed controls initially showed fat followed by carbohydrate utilization (respiratory quotient results)

*Pair-fed control: no leptin or amylin

11

Plasma Pharmacology of Synergy: Requires Leptin Replacement, Pharmacological AmylinPlasma Pharmacology of Synergy: Requires Leptin Replacement, Pharmacological Amylin

0

5

10

15

20

25

Lep

tin

(n

g/m

L)

10 50 125

50+

125

Lep

tin

A+

L

Am

ylin

Am

ylin

Veh

icle

Diet induced obesity prone rats (CRL; N=5/group).Plasma leptin determined at Day 28Selected doses (µg/kg/d) shown

Trevaskis JL et al. Endocrinology. 2008; 149(11):5679-87

Plasma Leptin

Veh/Veh Amylin (10) Amylin (50)0

100

200

300

400

500

600

Am

ylin

(p

M)

Plasma Amylin

12

Amylin Upregulated pSTAT3 Signaling in the Ventromedial Hypothalamus in DIO RatsAmylin Upregulated pSTAT3 Signaling in the Ventromedial Hypothalamus in DIO Rats

Ventromedial Hypothalamus

Lean Vehicle PF Amylin0

100

200

300

400

500

*, **

DIO Rats

pS

TA

T3-

Act

ivat

ed C

ells

(n

)

Lean Harlan Sprague Dawley rats or DIO-prone rats; Mean ± SE; *P<0.05 vs vehicle controls; **P<0.05 vs PF(amylin) Roth JD, et al. Proc Natl Acad Sci U S A. 2008;105:7257–7262

Vehicle ControlsLeptin 15 mg/kg i.p.

Lean DIO–Vehicle

DIO–PF DIO–Amylin

Leptin-Stimulated pSTAT3 Shown

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– Design: Randomized, double-blind, controlled, multicenter– Study population: Overweight or obese subjects (BMI 27-35 kg/m2)– Treatment: 4-week lead-in requiring 2-8% weight loss followed by 20

weeks randomized treatment 2:2:1 pramlintide: pramlintide/metreleptin: metreleptin

– Primary efficacy endpoint: Weight loss in pramlintide vs pramlintide/metreleptin

Pramlintide/Metreleptin: Phase 2 Clinical Proof-of-Concept StudyPramlintide/Metreleptin: Phase 2 Clinical Proof-of-Concept Study

Placebo-P + Metreleptin 5 mg BID

Pramlintide 360 µg BID + Placebo-M

Pramlintide 360 µg BID + Metreleptin 5 mg BID36

0 µ

g B

IDP

ram

lin

tid

e

18

0 µ

g B

IDP

ram

lin

tid

e

40% kcal deficit 20% kcal deficit

Lead-in Randomized Treatment

Day 1Screen -4 161 4 8 12 20-2

Roth JD et al Proc Natl Acad Sci USA. 2008; 105: 7257-7262.

Time (wk)

14

Amylin Agonism Restores Leptin Responsiveness in DIO Rats . . .and in Humans

Obese and Overweight Humans DIO Rats

Time (Week)

-4 0 4 8 12 16 20-15

-10

-5

0

Time (Week)

Mea

n (

SE

) %

Ch

ang

e in

Bo

dy

Wei

gh

t

Leptin (n=19)Pramlintide (n=38)

Pramlintide + Leptin (n=36)

VehicleLeptin (250 µg/kg/d)Amylin (100 µg/kg/d

Amylin + Leptin

0 3 4 5 61 2-20

-15

-10

-5

0

D B

od

y W

eig

ht

(%)

(Veh

icle

Co

rrec

ted

)

15

Lipodystrophy (LD)Lipodystrophy (LD)

Pathophysiology

Rare set of syndromes characterized by loss of adipose tissue (primarily subcutaneous) - Inability to store fat in normal depots - Ectopic fat deposition (liver, muscle)

Often more severe than those associated with excess adiposity (i.e. obesity)

• Severe insulin resistance / diabetes:• Often on 100’s-1000’s IU insulin/day

• Severe hypertriglyceridemia: • Often several 100’s-1000’s mg/dL • Increased risk of acute pancreatitis

• Hepatic steatosis / steatohepatitis: • Hepatomegaly and elevated LFTs• Can lead to cirrhosis

• Multiple other co-morbidities

Metabolic Abnormalities

LFT, liver function test; Oral. Rev Endo Metab Disord 2003, 4:61-77. Chan et al. Endocr Pract 2010 ; 16:310-323.

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0 4 8 1280

100

120

140

160

180

200

55 40 43 39

Number of Patients

Time (months)

Me

an

(S

E)

FP

G (

mg

/dL

)

0 4 8 125

6

7

8

9

10

54 40 41 38

Number of Patients

Time (months)

Me

an

(S

E)

Hb

A1

c (

%)

Metreleptin Improved Hyperglycemia in Lipodystrophy PatientsMetreleptin Improved Hyperglycemia in Lipodystrophy Patients

HbA1c Fasting Plasma Glucose

National Institutes of Health (NIH) open label study of metreleptin treatment, n=55 patients with metreleptin exposure ranging from 3 months to 9 years (Data on File, Amylin Pharmaceuticals, Inc.)

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Metreleptin Improved Hypertriglyceridemia inLipodystrophy PatientsMetreleptin Improved Hypertriglyceridemia inLipodystrophy Patients

Mean Triglycerides Median Triglycerides

0 4 8 120

100

200

300

400

500

54 40 42 39

Number of Patients

Time (months)

Me

dia

n T

rig

lyc

eri

de

s (

mg

/dL

)

0 4 8 120

250

500

750

1000

1250

1500

1750

54 40 42 39

Number of Patients

Time (months)

Me

an

(S

E)

Tri

gly

ce

rid

es

(m

g/d

L)

National Institutes of Health (NIH) open label study of metreleptin treatment, n=55 patients with metreleptin exposure ranging from 3 months to 9 years (Data on File, Amylin Pharmaceuticals, Inc.)

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Effect of Metreleptin on LipodystrophyEffect of Metreleptin on Lipodystrophy

• Unique therapy that directly addresses underlying pathophysiology

• Corrects relative leptin deficiency and associated hyperphagia, which exacerbates metabolic abnormalities as patients are driven to consume more

• Stimulates breakdown of fat in liver and muscle and reduces high triglyceride levels

• Improves insulin sensitivity and reduces hyperglycemia

Oral et al. Endocr Pract 2010; 16:324-333.

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Glycemic Volatility of Insulin Monotherapy:Why?

Glycemic Volatility of Insulin Monotherapy:Why?

Insulin Dose 0.2 U b.i.d.

0

100

200

300

400

500

600

700

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

mg

/dl

Days

20

Normal Roles of Endogenous Insulin:Paracrine (Local) and Endocrine (Systemic) Normal Roles of Endogenous Insulin:Paracrine (Local) and Endocrine (Systemic)

GLUCAGON INSULIN

+

GLYCOGENOLYSISGLUCONEOGENESIS

KETOGENESIS

a b

PERIPHERALGLUCOSEUPTAKE

Fuels

Paracrine

Endocrine(Systemic)

(Local)

21

STRATEGY: Since injected insulin cannot approach paracrine levels in normal islets -cells, let leptin suppress glucagon.STRATEGY: Since injected insulin cannot approach paracrine levels in normal islets -cells, let leptin suppress glucagon.

GLUCAGON INSULIN

GLYCOGENOLYSISGLUCONEOGENESIS

KETOGENESIS

b

PERIPHERALGLUCOSEUPTAKE

Fuels

XInsulin

Suppressora X

+

22

Glycemic Volatility of Insulin Monotherapy is Eliminated by LeptinGlycemic Volatility of Insulin Monotherapy is Eliminated by Leptin

Insulin Dose 0.2 U b.i.d.

0

100

200

300

400

500

600

700

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

mg

/dl

Days

Leptin + Low Dose Insulin (0.02 U b.i.d.)

23

Adenoviral Leptin Normalizes Hyper-glucagonemia in Insulin-deficient T1DM RodentsAdenoviral Leptin Normalizes Hyper-glucagonemia in Insulin-deficient T1DM Rodents

L Plasma Glucagon

0

200

400

600

800

Untreateddiabetic

Ad Leptindiabetic

Nondiabetic

p<0.01

N=4N=4 N=5

pg

/mL

24

Product DevelopmentProduct Presentation/Patient ComplianceProduct DevelopmentProduct Presentation/Patient Compliance

• Co-Formulation

− Both API’s in single formulation for administration

• Sustained-Release Formulations

− Extend duration of release from injection site

− Extend duration of activity, reduce injection frequency

• Second Generation Compounds

− Reduce clearance and extend circulation

− Extended duration of activity, reduce injection frequency

− Improve pharmaceutical properties

• Other

− Devices

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SummarySummary

• Weight loss with leptin can be restored by amylin/pram – a potential drug for obesity

• Leptin markedly improves the metabolic pathology of LD - a potential drug for LD

• Leptin normalizes diabetes in preclinical models - a potential drug for Type 1 diabetes

• SR formulations, Second Generation molecules, and devices in development for optimal product presentation and patient compliance

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AcknowledgementsAcknowledgements

• In Vivo Pharmacology– Jonathan Roth– James Trevaskis– Victoria Turek– Christine Mack– Carrie Wittmer– Chunli Lei– Calvin Vu– Pete Griffin– David Parkes

• Functional Imaging– Barbara Roland– Rebecca Cole– Guibao Gu

•Research– Soumitra Ghosh

• Clinical Research– Alain Baron– Christian Weyer– Joy Koda– Amy Halseth– Larry Shen

• Biometrics– Todd Coffey– Colleen Burns

•R&D Strategic Relations– Elaine Chiquette

• Collaborators– Barry Levin

•Roger Unger and Group