Pharmacological control

of energy expenditure and obesity

with PI3K inhibitors

in mice and monkeys

Spanish National Cancer Research Center (CNIO)

Reducing the IIS pathway:

Akt

PI3K

insulin/IGF1

--

Foxo mTORC1

S6K

IRS

Raptor

Lts8

Pten

The insulin/IGF1 signaling (IIS) pathway in healthspan and lifespan

anabolism

CR

RAPA

# increases longevity

• in mice, flies, worms

• in humans (FOXO3 alleles

are linked to longevity)

# improves glucose homeost.

• upon aging or high fat

# increases energy expenditure

• lower nutritional storage

EDITORIAL

September 13, 2012

Humans with PTEN germline mutations are obese

Genetic evidence in humans also links PTEN with the control of obesity

Rationale

anabolism ins. res.metab. synd.

longevityobesityIIS PI3Kovernutrition

PI3Ki

• CNIO-PI3Ki (a, d)

• GDC-0941 (a, b, g, d)

• BYL-719 (a)

• obese mice after 8 months of HFD

• 13-25% body weight gain

-5 -4 0 1 2 3 4 5 6 7 8 9 11 1210 13

CNIO-PI3Ki 10 mg/kg

15 mg/kgCNIO-PI3Ki

GDC-0941 10 mg/kg

GDC-0941 75 mg/kg

vehicle

-3 -2 -1

70

80

90

100

110

0 2 4 6 8 10

time (days)

body

weig

ht

change (

%)

body weight change

** * * * *** ** * * * ** ** ***

*** ** * * * *

0

50

100

150

200

glu

cose (

mg/d

l)

body weight change

at day 10

60

70

80

90

100

110

body

weig

ht

change (

%)

*

***

*

std.

diet

glycemia at day 13

0

average food intake

g/d

ay/

mouse

1

2

3

4

Weight loss after 2 weeks of PI3Ki treatment

Ortega-Molina et al., Cell Metab. 2015

Decreased adiposity after 2 weeks of PI3Ki treatment

vehicle

day -7 day 7 day 14

CNIO-

PI3Ki

15mg/kg

adiposity by DXA

14.2%6.5%

CNIO-PI3Ki GDC-0941

0

10

20

30

40

vehicle 10mg/kg 15mg/kg 10mg/kg 75mg/kg

fat (%

)

0

10

20

30

40

fat (%

)

**

0.06

SD

SD0

5

10

15

20

25

30

35

lea

n (

g)

0

5

10

15

20

25

30

35

lea

n (

g)

lean mass

CNIO-PI3Ki GDC-0941

vehicle 10mg/kg 15mg/kg 10mg/kg 75mg/kg

D -7

D 7

D 14

fat mass

Ortega-Molina et al., Cell Metab. 2015

vehicleCNIO-PI3Ki 10

mg/kg

GDC-0941

10mg/kg GDC-0941 75

mg/kg

liver

CNIO-PI3Ki 15

mg/kg

heart/lung

Reduced liver steatosis and pericardial fat after 2 weeks of PI3Ki treatment

Ortega-Molina et al., Cell Metab. 2015

Mechanisms of action of PI3Ki

H&E Ucp1

vehicle

PI3Ki

BAT

0

2

4

6

8

10

fold

change

** **

*

* *

Ortega-Molina et al., Cell Metab. 2012

brown

*

- - -

fold

change

forsk: + + +

100

150

200

250

****

**

Ucp1

pre-brown

- - -+ + +

PI3Kicontrol

20

***

** **

pre-brown brown

3T3-L1 Cebpβ

3T3-L1 Cebpβ

40

30

10

0

50

25

0

Pgc1a

**

PI3Kicontrol

Mechanisms of action of PI3Ki

April 2015

Figure adapted from:

Long-term PI3Ki treatment reduces high fat diet-induced obesity

body weight during PI3Ki treatment

(drinking water)

20

30

40

50

60

0 10 20 30 40 50 60 70

time (days)

bo

dy w

eig

ht (g

) HFD: vehicle

HFD: CNIO-PI3Ki

SD: vehicle

SD: CNIO-PI3Ki

24%

Ortega-Molina et al., Cell Metab. 2015

# Body weight reduction is not continuous, but reaches an equilibrium

# Body weight reduction is only observed under overnutrition

Note: Dosing is 0.1 mg/ml in the drinking water, producing an average of ~60 ng/ml serum concentration, which is ~40x lower that the concentration achieved by gavage of 15 mg/kg.

HFD: high fat dietSD: standard diet

Long-term treatment with CNIO-PI3Ki protects from steatosis and reduces lipid stores

F4

/80

H&

E

1

0

x

SD-vehicle SD-CNIO-PI3Ki HFD-vehicle HFD-CNIO-PI3KiH

&E

Oil

Red

O

live

reW

AT

Ortega-Molina et al., Cell Metab. 2015

Mechanisms of action of PI3Ki

Ortega-Molina et al., Cell Metab. 2015

Long-term PI3Ki treatment reverts high fat diet-induced obesity

0 20 40 60 140 160 180 200

80

100

120

140

time (days)

body

weig

ht

change (

%)

vehicle

CNIO-PI3Ki

no treatment

no treatment

obese mice under continuous HFD

Ortega-Molina et al., Cell Metab. 2015

# No evidence for the emergence of resistance

# No evidence for irreversible metabolic changes

# No evidence for toxicities

Long-term PI3Ki treatment reverts high fat diet-induced obesity

vehicle

CNIO-Pi3Ki

Ob/Ob mice

Ortega-Molina et al., Cell Metab. 2015

# PI3Ki is effective under overnutrition conditions

(even if the diet is “balanced”)

Long-term PI3Ki treatment reverts high fat diet-induced obesity

Ortega-Molina et al., Cell Metab. 2015

# Overnutrition (even in the absence of pre-existent obesity) is

sufficient for the activity of PI3Ki

# Lab-to-lab reproducibility

Other properties of CNIO-PI3Ki

Ortega-Molina et al., Cell Metab. 2015

• The drug does not cross the blood-brain barrier

• No alterations on the arcuate nucleus of the hypothalamus

(expression of orexigenic and anorexigenic neuropeptides)

Preliminary dosing study in Rhesus monkeys at the NIA-NIH (Rafael de Cabo)

Ortega-Molina et al., Cell Metab. 2015

The highest dose tested of 2.1 mg/kg i.v. was considered safe.

Note. the following assay using obese macaques was done with the same dose

but per os and this gave a serum concentration of drug at 2h of ~30 ng/ml.

Assay in Rhesus monkeys at the NIA-NIH (Rafael de Cabo)

Ortega-Molina et al., Cell Metab. 2015

Note: Dosing is 2 mg/kg p.o. which produced a peak at 2h of ~30 ng/ml serum concentration.

This is ~80x lower than the peak achieved in mice with 15 mg/kg p.o. or ~2x lower than the continuous concentration achieved in mice with 0.1 mg/ml in the drinking water.

Summary of the data in macaques

Ortega-Molina et al., Cell Metab. 2015

Treatment of obese macaques with low and chronic doses of CNIO-PI3Ki results in:

• Reduction of adiposity

• A tendency to correct fasting glycemia

• No evidence of toxicities, morbidities, or discomfort

The dose used in the macaques is extremely low, in the order of ~80x lower than

the one used in mice under similar administration conditions.

• Mice: 15 mg/kg p.o., which results in a serum peak of drug at 2h of ~2 µg/ml

• Macaques: 2 mg/kg p.o., which results in a serum peak of drug at 2h of ~30 ng/ml

Based on the preliminary escalation assay, we anticipate that the dose could be

increased at least by 10x.

Tumor Suppression Group

Assays with monkeys:

Rafael de Cabo, NIA-NIH, Baltimore

NIH Animal Center, Poolesville

Experimental Therapeutics

Joaquin Pastor

Sonia MartinezAna Ortega-Molina (now at MSKCC, NY)

Elena Lopez-Guadamillas

Analysis of the hypothalamus:

Miguel Lopez

CIMUS, Santiago de Compostela

Spanish National Cancer Research Centre (CNIO)