Frank Stappenbeck

Fragment-based discovery of JAK-2 inhibitors.

Fragment-based hit identification coupled with crystallographically enabled structure-based drug design was used to design potent inhibitors of JAK-2. After two iterations from fragment 1, we were able to increase potency by greater than 500-fold to provide sulfonamide 13, a 78-nM JAK-2 inhibitor.

A Novel Osteogenic Oxysterol Compound for Therapeutic Development to Promote Bone Growth: Activation of Hedgehog Signaling and Osteogenesis through Smoothened Binding

Osteogenic factors are often used in orthopedics to promote bone growth, improve fracture healing, and induce spine fusion. Osteogenic oxysterols are naturally occurring molecules that were shown to induce osteogenic differentiation in vitro and promote spine fusion in vivo. The purpose of this study was to identify an osteogenic oxysterol more suitable for clinical development than those previously reported, and evaluate its ability to promote osteogenesis in vitro and spine fusion in rats in vivo. Among more than 100 oxysterol analogues synthesized, Oxy133 induced significant expression of osteogenic markers Runx2, osterix (OSX), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN) in C3H10T1/2 mouse embryonic fibroblasts and in M2‐10B4 mouse marrow stromal cells. Oxy133‐induced activation of an 8X‐Gli luciferase reporter, its direct binding to Smoothened, and the inhibition of Oxy133‐induced osteogenic effects by the Hedgehog (Hh) pathway inhibitor, cyclopamine, demonstrated the role of Hh pathway in mediating osteogenic responses to Oxy133. Oxy133 did not stimulate osteogenesis via BMP or Wnt signaling. Oxy133 induced the expression of OSX, BSP, and OCN, and stimulated robust mineralization in primary human mesenchymal stem cells. In vivo, bilateral spine fusion occurred through endochondral ossification and was observed in animals treated with Oxy133 at the fusion site on X‐ray after 4 weeks and confirmed with manual assessment, micro‐CT (µCT), and histology after 8 weeks, with equal efficiency to recombinant human bone morphogenetic protein‐2 (rhBMP‐2). Unlike rhBMP‐2, Oxy133 did not induce adipogenesis in the fusion mass and resulted in denser bone evidenced by greater bone volume/tissue volume (BV/TV) ratio and smaller trabecular separation. Findings here suggest that Oxy133 has significant potential as an osteogenic molecule with greater ease of synthesis and improved time to fusion compared to previously studied oxysterols. Small molecule osteogenic oxysterols may serve as the next generation of bone anabolic agents for therapeutic development.

Novel oxysterols activate the Hedgehog pathway and induce osteogenesis

Localized induction of bone formation is essential during orthopedic procedures that involve skeletal repair, such as surgical treatment of non-union bone fractures and degenerative disk disease. Herein we disclose the synthesis and biological evaluation of novel oxysterol derivatives designed as anabolic bone growth agents. Structure-activity relationship studies of oxysterol 4 have identified analogues such as 18, 21 and 30. These new analogues are characterized by higher potency in an osteoblast differentiation assay and/or by increased metabolic stability in human liver microsomes. Oxysterols 4, 18 and 21 were evaluated in vivo in a rat spinal fusion model.

Comparison of a novel oxysterol molecule and rhBMP2 fusion rates in a rabbit posterolateral lumbar spine model

BACKGROUND CONTEXT The nonunion rate after lumbar spinal fusion is as high as 25%. Recombinant human bone morphogenetic protein 2 (rhBMP2) has been used as a biological adjunct to promote bony fusion. However, recently there have been concerns about BMP2. Oxysterol 133 (Oxy133) has been shown to promote excellent fusion rates in rodent lumbar spine models and offers a potential alternative to rhBMP2. PURPOSE The purpose of this study was to compare the fusion rate of rhBMP2 and Oxy133 in a randomized controlled trial using a posterolateral lumbar rabbit spinal fusion model. STUDY DESIGN This was a randomized control animal study. METHODS Twenty-four male adult white New Zealand rabbits (3-3.5 kg) underwent bilateral posterolateral lumbar spinal fusion at L4-L5. Rabbits were divided into four groups: control (A), 30-μg rhBMP2 (B), 20-mg Oxy133 (C), and 60-mg Oxy133 (D). At 4 weeks, fusion was evaluated by fluoroscopy, and at 8 weeks, the rabbits were sacrificed and fusion was evaluated radiographically, by manual palpation, and with microcomputed tomography. RESULTS Fusion rates by radiographic analysis at 8 weeks were Group A, 40.0%; Group B, 91.7%; Group C, 91.7%; and Group D, 100%. Evaluation of fusion masses by manual palpation of excised spines after sacrifice showed the following fusion rates: Group A, 0%; Group B, 83.3%; Group C, 83.3%; and Group D, 90%. Microcomputed tomography scanning confirmed these findings. CONCLUSIONS These findings in a rabbit model demonstrate that both 20- and 60-mg Oxy133 doses promote fusion that is equivalent to fusion induced by 30-μg rhBMP2 and significantly greater than the control group. The present findings confirm that Oxy133 is a promising candidate for therapeutic development as an alternative to rhBMP2 to promote spinal fusion.

Arylsulfonamide pyrimidines as VLA-4 antagonists

A series of (S)-2-(2-(diethylamino)-5-(N-alkyl-N-sulfonamido)pyrimidin-4-ylamino)-3-(4-(carbamoyloxy)phenyl)propanoic acid is discovered as orally available VLA-4 antagonists. Representative compounds 11b and 11p showed efficacy in multiple in vivo animal models. The in vitro selectivity of 11p is also described.

Oxy133, a novel osteogenic agent, promotes bone regeneration in an intramembranous bone‐healing model

Current reconstructive techniques for complex craniofacial osseous defects are challenging and are associated with significant morbidity. Oxysterols are naturally occurring cholesterol oxidation products with osteogenic potential. In this study, we investigated the effects of a novel semi‐synthetic oxysterol, Oxy133, on in vitro osteogenesis and an in vivo intramembranous bone‐healing model. Rabbit bone marrow stromal cells (BMSCs) were treated with either Oxy133 or BMP‐2. Alkaline phosphatase (ALP) activity, expression of osteogenic gene markers and in vitro mineralization were all examined. Next, collagen sponges carrying either Oxy133 or BMP‐2 were used to reconstruct critical‐sized cranial defects in mature rabbits and bone regeneration was assessed. To determine the mechanism of action of Oxy133 both in vitro and in vivo, rabbit BMSCs cultures and collagen sponge/Oxy133 implants were treated with the Hedgehog signalling pathway inhibitor, cyclopamine, and similar outcomes were measured. ALP activity in rabbit BMSCs treated with 1 μm Oxy133 was induced and was significantly higher than in control cells. These results were mitigated in cultures treated with cyclopamine. Expression of osteogenic gene markers and mineralization in BMSCs treated with 1 μm Oxy133 was significantly higher than in control groups. Complete bone regeneration was noted in vivo when cranial defects were treated with Oxy133; healing was incomplete, however, when cyclopamine was added. Collectively, these results demonstrate that Oxy133 has the ability to induce osteogenic differentiation in vitro in rabbit BMSCs and to promote robust bone regeneration in vivo in an animal model of intramembranous bone healing. Copyright

Abstract P13. Oxy133, A Novel Osteogenic Agent, Promotes Bone Regeneration In The Rat Alveolar Cleft Model

RESULTS: Of the 24 patients identified, 75% were male, mean age was 8.6, mean ISS 23.7 and mean GCS 9.7. The two most common causes of these fractures patterns were MVA (67%) and pedestrian (25%). 60% were managed operatively. 58% patients had skull fractures, 45.8% intracranial trauma, and 17% developed meningitis. One patient had a C-Spine injury. Group 1 had 8, group 2 had 4 and group 3 had 12 patients. Significant differences were age 5.9 vs 10.4 between group 1 vs group 3 (p=0.004). No differences in ISS, operative repair, complications, LOS, presence of skull fractures or intracranial trauma were found between these groups.

Inhibition of Pancreatic Cancer Cell‐Induced Paracrine Hedgehog Signaling by Liver X Receptor Agonists and Oxy16, a Naturally Occurring Oxysterol

The widespread involvement of the Hedgehog (Hh) signaling pathway in human malignancies has driven efforts to develop Hh pathway inhibitors as anti‐cancer agents. The majority of these agents antagonize Smoothened (Smo), a plasma membrane‐associated signal transducer molecule. However, several such Smo antagonists have failed in clinical trials to benefit patients with cancers that arise from aberrant Hh signaling (which often bypasses Smo). In this study, we report that a naturally occurring oxysterol, 20α, 22(R)‐dihydroxycholesterol (Oxy16), a known metabolite in the biosynthesis of steroid hormones, strongly inhibits Hh signaling induced in C3H10T1/2 embryonic fibroblasts and NIH3T3‐E1 fibroblasts through a mechanism that is independent of liver X receptor (LXR) activation. We demonstrate that Oxy16 inhibits Hh signaling in Suppressor of Fused (Sufu) null mouse embryonic fibroblast (MEF) cells, indicating that its inhibitory effect on Hh signaling is epistatic to Sufu. We further demonstrate that Oxy16 inhibits Gli1 transcriptional activity in NIH3T3‐E1 cells overexpressing Gli1 and a Gli‐dependent reporter construct. Altogether, data presented here suggest that Oxy16 may be a suitable starting point for the development of new drugs that inhibit Hh signaling downstream of Smo. By targeting aberrant Hh signaling, such novel Hh pathway inhibitors could significantly broaden the range of clinical applications compared to existing Smo antagonists. Furthermore, the present study adds a new facet to the spectrum of Hh pathway modulation that naturally occurring oxysterol derivatives are capable of, ranging from allosteric activation of the pathway via Smo binding to inhibition of the pathway downstream of Smo. J. Cell. Biochem. 118: 499–509, 2017.