1. Lexicon Pharmaceuticals, The Woodlands, Texas
2015 Annual Meeting of the American Society for Bone and Mineral Research
Neutralizing Antibody and Orally Active Small Molecule Inhibitors of the Secreted WNT Inactivating Lipase NOTUM Stimulate Cortical Bone Formation in Ovariectomized Rodents
Robert Brommage, Andrea Y. Thompson, Melanie K. Shadoan, Jeff Liu, Sabrina Jeter-Jones, Jie Cui, David G. Potter, Dawn Bright, Faika Mseeh, Jennifer P. Bardenhagan, Gwenn M. Hansen, Peter Vogel, James E. Tarver, Victoria K. Lombardo, David R. Powell, Qingyun Liu and Brian Zambrowicz
Lexicon Pharmaceuticals, The Woodlands, Texas

2. High-Throughput Screening of Mouse Gene Knockouts Identifies Established and Novel Skeletal Phenotypes
Brommage et al., Bone Research 2:14034; 2014
Elevated femoral cortical bone thickness, averaging 21%, was observed in 12 separate cohorts of male and female Notum KO mice, with a total of 242 WT and 220 KO mice examined from 2004 through 2011
No trabecular bone phenotypes
KOs show 32% reduction in viability and 26% of survivors have a single kidney

3. Midshaft Femur Cortical Thickness and Strength
Intermediate phenotypes in heterozygous mice suggest
pharmaceutical inhibition need not be 100% for efficacy

4. NOTUM – Secreted Enzyme That Inactivates WNTs by Removing Essential Palmitoleate
WNT Binding to Frizzled
WNT lipase activity of NOTUM identified by Lexicon during 2006
Independent confirmation of this mechanism of action by two groups published during early 2015
Kakugawa et al., Nature 519: ; 2015
Zhang et al., Dev Cell 32: ; 2015
Robotic enzymatic screening and cell-based WNT signaling assays established
These assays were employed to identify inhibitors of NOTUM activity
Orally active small molecules
Neutralizing antibodies
Brown = WNT Ligand
Green = Frizzled Receptor
Red = Essential Palmitoleate
Janda et al., Science
337:59-64; 2012

5. Mineralization Studies in MC3T3 Cells
Mineralization inhibited by NOTUM conditioned medium
This inhibition is overcome by addition of NOTUM small molecule inhibitor LP

6. Femur Cortical Thickness
NOTUM Antibody Treatment Increases Bone Mass in Both Intact and OVX Mice
Mice treated once weekly for 4 weeks starting 4 weeks after OVX at 16 weeks of age
Dose = 10 mg/kg; Enzyme IC50 = 37 nM; Cell-based EC50 = 4 nM
Treatment increased midshaft femur cortical thickness, cortical (but not trabecular) bone mass in LV5 vertebral body, and total BV/TV in femoral neck (Scanco μCT40)
Two-Factor ANOVA
Femur Cortical Thickness
LV5 Cortical Bone BV/TV
Femoral Neck BV/TV
Effect of OVX
P < 0.001
P = 0.002
Effect of NOTUM Ab
Interaction
P = 0.37
P = 0.32
P = 0.29

7. Transient Stimulation of Midshaft Femur Endocortical Bone Formation
Modeling-dependent
bone formation
OVX Control
OVX Treated
OVX Treated

8. Treatment with Orally-Active Small Molecule NOTUM Inhibitor Increases Bone Mass in OVX Rats
OVX Fischer 344 rats treated daily by oral gavage for 12 weeks starting at 80 weeks of age
OVX at 17 weeks of age
LP examined at doses of 1 and 5 mg/kg
Plus untreated OVX and Sham-surgery groups
Enzyme IC50 = 0.4 nM; Cell-based EC50 = 21 nM
Two courses of double fluorochrome labeling
Alizarin at 1 and 3 weeks
Calcein at 9 and 11 weeks
Multiple bones examined by standard DEXA, microCT and breaking strength analyses

9. LP-935001 Treatment Has Beneficial Cortical Bone Actions at Multiple Skeletal Sites
Parameter
Treatment Effect (5 mg/kg)
Statistics
Midshaft Tibia Cortical Thickness 6%
P = 0.02
Midshaft Tibia Strength 7%
Spine LV5 Cortical BV/TV
P = 0.002
Femoral Neck BV/TV
P < 0.001
Femoral Neck Strength
15%
P < 0.02
Midshaft Femur Cortical Thickness
Midshaft Femur Strength
13%
Pelvis Shaft Cortical Thickness
12%
Distal Tibia Cortical Thickness 9%
Distal Tibia BFR (Weeks 1 to 3)
3-fold
Distal Tibia BFR (Weeks 9 to 11)
Treatment effects are compared to OVX control values. Ns = 13 to 17
Statistical evaluation involved ANOVA followed by Dunnett’s test
for multiple comparisons to the OVX control group

10. Additional Studies (Data Not Shown)
Teriparatide treatment for 7 days stimulated Notum expression in femur shaft 10-fold
Notum KO mice responded normally to teriparatide treatment but LP treatment was ineffective
LP was effective when dosed twice per week
LP treatment increased serum total alkaline phosphatase and PINP levels within 4 days
Bone gained during LP treatment was lost after treatment stopped, but this gain was maintained with subsequent zoledronate treatment
Notum KO mice have defective dentin mineralization but enamel and all soft tissues show normal histology

11. Summary
NOTUM is a secreted enzyme that inactivates WNTs by cleaving a palmitoleate group essential for binding to Frizzled receptors
Inhibiting NOTUM by gene KO, neutralizing antibodies or orally-active small molecules stimulates modeling-dependent endocortical bone formation, resulting in greater bone strength
Trabecular bone is unaffected
NOTUM is a novel osteoporosis anabolic drug target
Genes that differentially affect cortical and trabecular bone include leptin, Klotho, Src, Wnt16, Sfrp4, ENU mutation
Lrp5 A214V and G171V activating mutations have site-specific actions
WNT signaling in bone involves both canonical and non-canonical pathways
Prx1 affects the appendicular but not the axial skeleton
Different activities of visceral, marrow and subcutaneous fat