F. Stappenbeck et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5893–5897
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References and notes
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Figure 3. New bone formation at the site of implant. Comppound 21-treatment
induced more bone formation than the positive control, 4 (⁄p < 0.05, Dunnett’s
multiple comparison test, mean SD, n = 10/treatment group).
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the extent of fusion in freshly isolated samples by manual palpa-
tion, and scored the extent of fusion as described by Qiu, et al.29
In this scoring system, no detectable new bone is scored as 0,
and complete fusion is scored as 4, with grades of partial fusion
scored as 1, 2 or 3. After fixation in 10% neutral buffered formalin,
the excised tissue was assessed for new bone formation by mi-
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18. SHHLight2 assay: Activation of the Hedgehog pathway was measured using the
SHHLight2 reporter cell line. This reporter line contains a concatemer of 8 Gli
response elements upstream of a minimal promoter driving the expression of
firefly luciferase. The cells constitutively express Renilla luciferase for
normalization of the firefly luciferase signal. SHHLight2 cells were plated at
croCT. The samples were scanned at 18 lM resolution. In this
experiment, 21 and 18 were tested in parallel with 4 at 4 mg/kg.
We had previously observed that this dose of 4 is submaximal in
the rat spinal fusion model,16 and therefore provides a window
for measuring improved efficacy relative to this positive control.
As shown in Figure 2, mechanical stability testing of treated verte-
bral segments did not reveal a difference between 21 or 18 and 4. A
trend towards higher scores for the 21 and 18 groups compared to
4 was noted, but did not reach statistical significance when ana-
lyzed by Kruskal-Wallis testing,30 a nonparametric analysis of
variance.
10,000 cells/well in 384 well plates with 20
lL/well of DMEM/10% FCS. 24 h
after plating, cells were treated 20 L/well of test compound in DMEM/0.5%
l
FCS. Triplicate wells were used for each treatment. After 24 h of treatment,
firefly luciferase and Renilla luciferase were measured with Promega’s DualGlo
reagent. The firefly signal in each well was divided by the Renilla signal in that
well to normalize for cell number, and results are expressed relative to cells
treated with the DMSO solvent control.
19. C3H/10T1/2 Osteogenesis assay: The ability of test compounds to stimulate
osteogenesis was measured in vitro using the C3H/10T1/2 cell line. This
multipotential cell line is a model of mesenchymal stem cells, and can be
differentiated into osteoblasts, chondrocytes, myoblasts and adipocytes. C3H/
10T1/2 cells were plated in 24 well plates and cultured to confluence in
DMEM/10% FCS. After reaching confluence, duplicate wells of cells were
treated 3 times per week for 2 weeks with test compound in fresh DMEM/10%
FCS. Total RNA was prepared using the Qiagen RNeasy Plus kit, and cDNA was
created from total RNA using invitrogen superscript III kit. Quantitative RT-PCR
was performed on cDNA samples using ABI Taqman assays, with the DDCt
method, to measure the expression levels of osteoblast genes, BGLAP and IBSP.
Results were normalized to GAPDH and expressed relative to cells treated with
the DMSO solvent control: Dworetzky, S. I.; Fey, E. G.; Penman, S.; Lian, J. B.;
Stein, J. L.; Stein, G. S. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 4605.
MicroCT analysis revealed a larger bone volume in the callus of
21-treated rats compared to 4, while 18 treated animals were com-
parable to 4-treated rats (Figure 3).
In conclusion, a tentative correlation of in vitro potency (Hh-
activity and OCN induction, Table 1) and a single in vivo efficacy
outcome (Bone Volume, Figure 3) was observed with oxysterol
analogues 4 and 21, affirming the importance of potency optimiza-
tion. Dosed at 4 mg/kg in a rat spinal fusion model, deuterated ana-
logue 18 did not produce a significantly enhanced response
compared to parent compound 4, suggesting that in vivo efficacy
may not be exclusively limited by metabolic stability. These results
also suggest that in vivo efficacy of 4 may not be a consequence of
metabolic activation involving the side-chain at C-22 and C-23. We
will report additional data on compounds 2131 and 30 in due time.
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28. Testing in the rat spinal fusion model was performed by Ricerca, Bothell, WA.
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Acknowledgment
We wish to thank Dr. Eugene Thorsett for his wisdom and guid-
ance through this work.
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Supplementary data
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