1. Lexicon Pharmaceuticals, The Woodlands, TX
Normal Anabolic Skeletal Responses to Teriparatide Treatment with Inhibition of the WNT-Inactivating Lipase NOTUM
Robert Brommage, Andrea Y. Thompson, Melanie K. Shadoan, Jeff Liu, Sabrina Jeter-Jones, Jie Cui, Faika Mseeh, Jennifer P. Bardenhagen, Gwenn M. Hansen, James E. Tarver, Brian Zambrowicz, Qingyun Liu and David R. Powell
Lexicon Pharmaceuticals, The Woodlands, TX

2. Abstract
The secreted lipase NOTUM inactivates WNTs by removing palmitoleate essential for Frizzled receptor binding and subsequent WNT signaling. Inhibiting NOTUM by gene knockout (KO), orally active small molecule inhibitors and neutralizing antibodies each increase endocortical bone formation without influencing trabecular bone mass in rodents (Brommage, 2015 ASBMR). Since the anabolic skeletal actions of teriparatide (TPTD) are believed to involve LRP6-mediated WNT signaling, NOTUM inhibition might enhance TPTD action by enhancing WNT signaling. Consistent with this hypothesis, in two mouse studies 7 days of daily TPTD treatment (80 µg/kg) increased cortical bone Notum expression (measured by RT-PCR) greater than 10-fold.
Two pharmacology studies evaluated the effects of NOTUM inhibition on teriparatide efficacy. Mouse midshaft femur cortical thickness increased 7% with TPTD (20 µg/kg) and 10% with NOTUM inhibitor LP (3 mg/kg) treatments for 30 days. Both treatment doses were chosen to produce modest bone gains and show synergistic effects with cotreatment if NOTUM inhibition enhanced TPTD action. However, cotreatment with TPTD and LP produced an additive effect, increasing cortical thickness by 17%. By two-factor ANOVA, P < for TPTD and P < for LP treatments, with an interaction P = Dynamic bone histomorphometry at the distal tibia showed endocortical bone formation was increased 5-fold with TPTD and 7-fold with LP treatments and 12-fold with cotreatment. By two-factor ANOVA, P < for TPTD and P < for LP treatments, with an interaction P = 0.67.
Similarly, TPTD treatment (80 µg/kg) for 17 days increased midshaft femur cortical thickness 7% and 8% in littermate wild-type and Notum KO mice, respectively. Cortical thickness in untreated KO mice was elevated 19% compared to untreated wild-type mice. By two-factor ANOVA, P < for Notum KO, P = 0.01 for TPTD treatment, with an interaction P = TPTD treatment increased trabecular BV/TV in the LV5 vertebral body by 23% and 30% in wild-type and Notum KO mice, respectively. By two-factor ANOVA, P = 0.81 for Notum KO and P = 0.02 for TPTD treatment, with an interaction P = In a separate study, LP treatment was ineffective in Notum KO mice.
Both pharmacology studies demonstrated that inhibiting NOTUM results in elevated cortical bone thickness but does not reduce nor enhance the anabolic skeletal actions of TPTD, despite dramatic stimulation of cortical bone Notum expression. The actual WNTs and Frizzled receptors involved in the skeletal actions of TPTD remain to be identified, but LRP5 is not required (Sawakami 2006, Iwaniec 2007, Arantes 2011).

3. Introduction
NOTUM is a secreted lipase that inactivates WNTs by removing the palmitoleate essential for Frizzled receptor binding and WNT signaling
Notum KO mice (identified in Lexicon’s KO mouse phenotyping campaign examining ≈4,650 genes) have high cortical bone thickness with normal trabecular bone mass
Except for dentin dysplasia, Notum KO mice are normal
Lexicon developed neutralizing antibodies and orally-active NOTUM small molecule inhibitors that stimulate modeling-dependent endocortical bone formation in ovariectomized mice and rats
Since the anabolic skeletal actions of teriparatide (TPTD) are believed to involve LRP6-mediated WNT signaling, NOTUM inhibition might enhance TPTD action by enhancing WNT signaling
To explore possible synergistic actions of teriparatide treatment and NOTUM inhibition on cortical bone, TPTD was administered to 1) WT mice co-treated NOTUM inhibitor LP , and 2) Notum KO mice

4. 10-Fold Stimulation of Cortical Bone Notum Expression with Teriparatide Treatment
Male mice (14 weeks of age) were dosed with vehicle or TPTD (80 μg/kg) for 7 days
Femur shaft and serum were collected 6 hours after the 7th dose
Treatment increased serum PINP 2.3-fold and ALP 1.6-fold
Cortical bone Notum and Sost expression were determined by RT-PCR

5. Serum Markers of Bone Formation Are Additively Increased by Teriparatide Treatment and NOTUM Inhibition
Male WT mice at 12 weeks of age were dosed for 30 days with vehicle, LP (3 mg/kg via diet), teriparatide (20 μg/kg) or both LP and teriparatide
Serum was collected for PINP and ALP measurements on Day 7

6. Teriparatide Treatment and NOTUM Inhibition Produce Additive Skeletal Actions
Femur shaft cortical thickness was determined using a Scanco μCT40
Calcein injections for histomorphometry were given on Days 7 and 17
Cortical bone histomorphometry was performed at the distal tibia (halfway between the tibia-fibula junction and the distal end of the tibia)

7. Teriparatide Treatment Increases Serum PINP Levels in Notum KO Mice - Notum KO Mice Have Normal Trabecular Bone Mass
Male Notum KO and WT littermates/cagemates at 20 weeks of age were dosed with teriparatide (80 μg/kg) or vehicle daily for 17 days
Serum collected for PINP measurement on Day 7
Calcein injections for histomorphometry on Days 4 and 14

8. Teriparatide Treatment Increases Cortical Bone Thickness and Endocortical Bone Formation in Notum KO Mice
Femur shaft cortical thickness was determined using a Scanco μCT40
Cortical bone histomorphometry was performed at the midshaft femur

9. Lack of Skeletal Action of NOTUM Inhibitor in Notum KO Mice
Female Notum KO and WT littermates/cagemates at 16 to 19 weeks of age were dosed with LP (30 mg/kg) via diet for 28 days

10. Summary and Conclusions
TPTD treatment stimulates cortical bone Notum expression 10-fold
Inhibiting NOTUM results in elevated cortical bone thickness but does not reduce nor enhance the anabolic skeletal actions of TPTD
NOTUM inhibition achieved by both gene KO and pharmacology
Modeling-dependent endocortical bone formation is activated with NOTUM inhibition
Serum PINP is good marker for endocortical bone formation
NOTUM inhibition does not influence trabecular bone mass
Separate studies (2015 ASBMR, 2016 WCO-IOF-ESCEO) show:
Increased bone strength with NOTUM inhibition in multiple studies
Increased cortical bone thickness in OVX mice treated with NOTUM neutralizing antibody
Increased cortical bone thickness and strength in OVX rats treated with NOTUM small molecule inhibitor LP

11. References NOTUM Mechanism of Action Lexicon NOTUM Publications
Zhang X et al. Notum is required for neural and head induction via Wnt deacylation, oxidation, and inactivation. Dev Cell. 2015; 32:
Kakugawa S et al. Notum deacylates Wnt proteins to suppress signalling activity. Nature. 2015; 519:
Lexicon NOTUM Publications
Vogel P et al. Dentin dysplasia in Notum knockout mice. Vet Pathol [Epub ahead of print]
Tarver JE et al. Stimulation of cortical bone formation with thienopyrimidine based inhibitors of Notum Pectinacetylesterase. Bioorg Med Chem Lett. 2016; 15:
LRP5 Is Not Essential for Anabolic Skeletal Actions of Teriparatide
Sawakami K et al. The Wnt co-receptor LRP5 is essential for skeletal mechanotransduction but not for the anabolic bone response to parathyroid hormone treatment. J Biol Chem. 2006; 281:
Iwaniec UT et al. PTH stimulates bone formation in mice deficient in Lrp5. J Bone Miner Res. 2007;22:
Arantes HP et al. Teriparatide increases bone mineral density in a man with osteoporosis pseudoglioma. J Bone Miner Res. 2011; 26: