Revisiting the Therapeutic Potential of Leptin
David Litzinger, PhD
Director, Pharmaceutical Sciences
Amylin Pharmaceuticals, Inc.
2011 AAPS National Biotechnology Conference
San Francisco, CA
Leptin 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.
2
Multi-Hormonal Control of Body Weight
Vagal afferents
Hypothalamus
GI tract
Adipose tissue
Ghrelin
Hindbrain
Leptin
CCK
PYY3-36
Insulin
Amylin
Resistin
GLP-1
Visfatin
OXM
Adiponectin
GIP
PP
Pancreatic
Amylinislets
Adapted from Badman M.K. and Flier J.S. Science 2005; 307: 1909-1914.
Question: Why aren’t anti-obesity drugs more effective?
3
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
4
Pramlintide: Clinical Studies in Obesity
Placebo (n = 17)
Pramlintide 360 µg BID (n = 21)
DoubleBlind Study
D in Body Weight (%)
0
In obese patients, pramlintide elicits
Single-Blind
Extension
Reduced food intake
-2
-4
6.8%
•
•
•
•
•
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
-6
Sustained weight loss
-8
-10
• With proportionate reduction in leptin
Weight Loss
0
2
Weight Maintenance
4
6
8
Time (mo)
10
Data are for evaluable population; N = 38; Mean ± SE
Smith SR, et al Diabetes Care. 2008; 31: 1816-1823
12
5
Metreleptin (r-metHuLeptin)
• 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
6
Leptin Replacement Elicits Profound Weight
Loss in Leptin-Deficient Mice and Humans
ob/ob Mice
Before
After
(Leptin Deficiency) (Leptin Replacement)
Congenital Leptin Deficiency*
Before
After
(Leptin Deficiency) (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.
7
Leptin Does Not Cause Weight Loss in Dietinduced (Non-leptin deficient) Obesity
DIO Rats
Obese Humans
Placebo BID (n = 25)
Metreleptin 10 mg BID (n = 71)
Vehicle
Leptin 500 µg/kg per day
0
D Body Weight (kg)
DBody Weight (%)
(Vehicle Corrected)
2
-5
-10
1
0
-1
-2
-3
-4
-5
-15
0
1
2
Time (wk)
3
4
Dietary
Lead-In
-3
Randomized Treatment
0
3
6
9
Time (wk)
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
12
8
Amylin+Leptin Synergy for Weight Loss is not
Explained by the Anorexigenic Effect of Amylin
250
Food Intake
% Change in Body Weight
(Vehicle-Corrected)
Cumulative Food Intake (grams)
Vehicle
Leptin 500 µg/kg/d
Amylin 100 µg/kg/d
(Pairfed-Amylin)+Leptin
Amylin+Leptin
200
150
100
*
50
2
Body Weight
0
-2
-4
-6
-8
-10
*
-12
-14
0
0
3
6
9
12
Time (Days)
0
3
6
9
12
Time (Days)
*p<0.05 compared to all groups
Diet-induced obesity prone rats (CRL; N=7/group).
Roth JD, et al. Proc Natl Acad Sci USA. 2008;105:7257–7262.
9
Amylin/Leptin Induced Greater Fat Loss and
Prevented Counter Regulatory Adaptations
• Amylin + Leptin treated DIO rats lost nearly 2-fold greater fat mass than
pair-fed control*
Veh
PF
A+L
Adipose
Tissue
(H&E)
• 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
Trevaskis JL et al. Endocrinology. 2008; 149(11):5679-87
10
Plasma Pharmacology of Synergy: Requires
Leptin Replacement, Pharmacological Amylin
Plasma Leptin
Plasma Amylin
600
25
500
Amylin (pM)
15
10
50
125
50
+
125
A+L
10
Leptin
Vehicle
0
Amylin
5
Amylin
Leptin (ng/mL)
20
400
300
200
100
0
Veh/Veh
Amylin (10)
Amylin (50)
Diet induced obesity prone rats (CRL; N=5/group).
Plasma leptin determined at Day 28
Selected doses (µg/kg/d) shown
Trevaskis JL et al. Endocrinology. 2008; 149(11):5679-87
11
Amylin Upregulated pSTAT3 Signaling in
the Ventromedial Hypothalamus in DIO Rats
Vehicle Controls
Leptin 15 mg/kg i.p.
Ventromedial Hypothalamus
pSTAT3-Activated Cells (n)
500
400
*, **
300
Lean
DIO–Vehicle
DIO–PF
DIO–Amylin
200
100
0
Lean
Vehicle
PF
Amylin
Leptin-Stimulated pSTAT3 Shown
DIO Rats
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
12
Pramlintide/Metreleptin: Phase 2 Clinical
Proof-of-Concept Study
– 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
20% kcal deficit
Pramlintide
360 µg BID
Pramlintide
180 µg BID
40% kcal
deficit
Placebo-P + Metreleptin 5 mg BID
Pramlintide 360 µg BID + Placebo-M
Pramlintide 360 µg BID + Metreleptin 5 mg BID
Lead-in
Screen -4
-2
Randomized Treatment
Day
1
1
4
8
12
16
20
Time (wk)
Roth JD et al Proc Natl Acad Sci USA. 2008; 105: 7257-7262.
13
Amylin Agonism Restores Leptin
Responsiveness in DIO Rats . . .and in Humans
DIO Rats
Obese and Overweight Humans
0
Mean (SE) % Change
in Body Weight
D Body Weight (%)
(Vehicle Corrected)
0
-5
-10
-15
-20
0
1
2
3
4
Time (Week)
Vehicle
Leptin (250 µg/kg/d)
Amylin (100 µg/kg/d
Amylin + Leptin
5
6
-5
-10
-15
-4
0
4
8
12
16
20
Time (Week)
Leptin (n=19)
Pramlintide (n=38)
Pramlintide + Leptin (n=36)
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Lipodystrophy (LD)
Pathophysiology
Metabolic Abnormalities
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
•
Rare set of syndromes characterized by
loss of adipose tissue (primarily
subcutaneous)
- Inability to store fat in normal depots
- Ectopic fat deposition (liver, muscle)
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
LFT, liver function test; Oral. Rev Endo Metab Disord 2003, 4:61-77. Chan et al. Endocr Pract 2010 ; 16:310-323.
15
Metreleptin Improved Hyperglycemia in
Lipodystrophy Patients
HbA1c
200
Mean (SE) FPG (mg/dL)
Mean (SE) HbA1c (%)
10
9
8
7
6
5
0
54
4
8
Time (months)
40
41
Number of Patients
12
38
Fasting Plasma Glucose
180
160
140
120
100
80
0
55
4
8
Time (months)
40
43
Number of Patients
12
39
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.)
16
Metreleptin Improved Hypertriglyceridemia in
Lipodystrophy Patients
Mean Triglycerides
Median Triglycerides
500
Median Triglycerides (mg/dL)
Mean (SE) Triglycerides (mg/dL)
1750
1500
1250
1000
750
500
250
400
300
200
100
0
0
0
54
4
8
Time (months)
40
42
Number of Patients
12
39
0
4
8
Time (months)
12
54
40
42
Number of Patients
39
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 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.
18
Glycemic Volatility of Insulin Monotherapy:
Why?
700
Insulin Dose 0.2 U b.i.d.
600
500
mg/dl
400
300
200
100
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Days
19
Normal Roles of Endogenous Insulin:
Paracrine (Local) and Endocrine (Systemic)
GLUCAGON
Paracrine
(Local)
a
b
INSULIN
PERIPHERAL
GLUCOSE
UPTAKE
+
Endocrine
(Systemic)
GLYCOGENOLYSIS
GLUCONEOGENESIS
KETOGENESIS
Fuels
20
STRATEGY: Since injected insulin cannot approach paracrine
levels in normal islets a-cells, let leptin suppress glucagon.
GLUCAGON
a
X
b
+
INSULIN
X
PERIPHERAL
GLUCOSE
UPTAKE
GLYCOGENOLYSIS
GLUCONEOGENESIS
KETOGENESIS
Fuels
21
Glycemic Volatility of Insulin Monotherapy is
Eliminated by Leptin
700
Insulin Dose 0.2 U b.i.d.
600
500
mg/dl
400
300
200
100
0
Leptin + Low Dose Insulin
(0.02 U b.i.d.)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Days
22
Adenoviral Leptin Normalizes Hyperglucagonemia in Insulin-deficient T1DM Rodents
L
Plasma Glucagon
p<0.01
800
pg/mL
600
400
200
0
Nondiabetic
N=4
Untreated
diabetic
N=4
Ad Leptin
diabetic
N=5
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Product Development
Product 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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Summary
• 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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Acknowledgements
•
In Vivo Pharmacology
–
–
–
–
–
–
–
–
–
•
Jonathan Roth
James Trevaskis
Victoria Turek
Christine Mack
Carrie Wittmer
Chunli Lei
Calvin Vu
Pete Griffin
David Parkes
•
–
–
–
–
–
•
• Research
Alain Baron
Christian Weyer
Joy Koda
Amy Halseth
Larry Shen
Biometrics
– Todd Coffey
– Colleen Burns
• R&D Strategic Relations
– Elaine Chiquette
Functional Imaging
– Barbara Roland
– Rebecca Cole
– Guibao Gu
Clinical Research
•
Collaborators
– Barry Levin
• Roger Unger and Group
– Soumitra Ghosh
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