mSmo ( IC50 = 1.3 nM ); hSmo ( IC50 = 2.5 nM )

Sonidegib (NVP-LDE225) has a pKa of 4.2, making it a weak base with comparatively low solubility in water. Sonidegib exhibits dose-related antitumor activity in the subcutaneous Ptch+/-p53-/- medulloblastoma allograft mouse model following ten days of oral administration of a suspension of the diphosphate salt. Sonidegib exhibits a significant tumor growth inhibition at a dose of 5 mg/kg/day qd, with a corresponding T/C value of 33% (p<0.05) in comparison to vehicle controls. Sonidegib provides 51 and 83% regression when administered at doses of 10 and 20 mg/kg/day qd, respectively[1]. Secondary recipient mice are transplanted with bone marrow and spleen cells from a subset of treated mice. In comparison to Sonidegib or Nilotinib alone, transplanting bone marrow (BM) or spleen cells from mice treated with Sonidegib (LDE225)+Nilotinib reduces leukaemia development and the number of white blood cells (WCC) in secondary recipients[2].

Fluorescence binding assays using BODIPY-cyclopamine [1]
Fluorescence binding assays using BODIPY FL or BODIPY® 558/568 labeled binding assays were conducted as described. Briefly, binding assays were conducted in 384-well plates using fixed CHO cells stably expressing mouse or human Smo. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed, covered in PBS buffer containing 0.5% fetal bovine serum, and incubated with fluorescence labeled BODIPY-cyclopamine (20 nM) and the test compounds [e.g. Sonidegib (Erismodegib; LDE225; NVP-LDE225)] for 4 h at 37 °C. The treated cells then were washed with PBS, stained with Hoechst 33258, and analyzed by ImageXpress® Ultra imaging system.

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TM3-Gli-Luc reporter gene assay [1]
Test compounds [e.g. Sonidegib (Erismodegib; LDE225; NVP-LDE225)] were prepared for assay by serial dilution in DMSO and then added to empty assay plates. TM3Hh12 cells (TM3 cells containing Hh-responsive reporter gene construct pTA8xGli-Luc) were cultured in F12 Ham’s/DMEM (1:1) containing 5% horse serum, 2.5% fetal bovine serum (FBS), and 15 mM HEPES, pH 7.3. Cells were harvested by trypsin treatment, resuspended in F12 Ham’s/DMEM (1:1) containing 5% horse serum and 15 mM HEPES, pH 7.3, added to assay plates, and incubated with test compounds for approximately 30 min at 37 °C in 5% CO2. Then 1 or 25 nM Ag1.5 was added to assay plates and incubated at 37 °C in the presence of 5% CO2. After 48 h, either Bright-Glo (Promega E2650) or MTS reagent was added to the assay plates and luminescence or absorbance at 492 nm was determined. IC50 values, defined as the inflection point of the logistic curve, were determined by nonlinear regression of the Gli-driven luciferase luminescence or absorbance signal from MTS assay vs log10 (concentration) of test compounds using the R statistical software package. [1]
LLDE225 blocks the TM3 luciferized cell line with 0.6 nM and 8 nM of Hh agonist Ag1.5 present, respectively.

Proliferation/apoptosis/cell cycle analysis[2]
CD34+ CP-CML cells were seeded in SFM alone ± Sonidegib (Erismodegib; LDE225; NVP-LDE225) ± nilotinib and cultured for 24–72 h prior to assessment. Proliferation was measured using colorimetric assessment of BrDU incorporation. Proportion of viable cells versus those in early and late apoptosis was assessed by flow cytometry using annexin V–FITC and 7-amino-actinomycin D (7-AAD, Via-Probe solution) according to the manufacturer’s instructions. Cell cycle status was assessed as previously described using Ki67 (FITC) expression and 7-AAD incorporation55.

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CFC assay/re-plating assay[2]
CD34+ CP-CML cells were seeded in SFM ± Sonidegib (Erismodegib; LDE225; NVP-LDE225) ± nilotinib and cultured for 72 h then washed three times, inoculated at a concentration of 4 × 103/ml into methylcellulose supplemented with growth factors and cultured in duplicate for 14d prior to colony assessment. Following assessment, at least 20 colonies (granulocyte-erythroid-megakaryocyte-macrophage [GEMM] or granulocyte macrophage [GM]) colonies were plucked from each experimental arm and serially re-dispersed in Methocult with secondary and tertiary colony formation assessed after 7d intervals.

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LTC-IC assay[2]
Primary CD34+ normal and CP-CML cells were cultured in SFM ±Sonidegib (Erismodegib; LDE225; NVP-LDE225) ± nilotinib for 72 h. Following this, they were thoroughly washed and inoculated into pre-prepared long term cultures comprising a stromal feeder layer (a 1:1 mix of irradiated (80 Gy) SL/SL and M210B4 murine fibroblasts) in long term myeloid culture medium (MyeloCult supplemented with hydrocortisone) as previously described35. These cultures were maintained for 5 weeks with 50% media changes performed weekly. Following this, the contents of the wells were harvested and cells counted prior to seeding into Methocult to perform CFC assays as described above.

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Long term stromal co-culture[2]
CD34+ CP-CML cells were inoculated directly into pre-prepared stromal co-cultures, as described above, in the presence of Sonidegib (Erismodegib; LDE225; NVP-LDE225) ± nilotinib. Cultures were maintained for 5 weeks with 80% media changes and addition of fresh drug weekly. Co-cultures were examined weekly by microscopy to ensure that the stromal layer remained morphologically normal and adherent. After 5 weeks, CFC assays were performed as described.

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Prior to assessment, CD34⁺ CP-CML cells are cultured for 24-72 hours in SFM alone±Sonidegib±Nilotinib. BrDU incorporation colorimetric assessment is used to quantify proliferation. Utilizing annexin V-FITC and 7-amino-actinomycin D (7-AAD, Via-Probe solution), flow cytometry is used to determine the ratio of viable cells to those in early and late apoptosis. Ki67 (FITC) expression and 7-AAD incorporation are used to determine the cell cycle status.

Subcutaneous Ptch+/-p53-/- medulloblastoma allograft model. [1]
Mouse Ptch+/-p53-/- medulloblastoma cells ((1.0-5.0) × 106 ), dissociated directly from tumor fragments, were inoculated subcutaneously into the right flank of Harlan nu/nu mice. Treatment was initiated approximately 7 days after implantation. Animals were randomized into treatment groups with similar mean tumor volumes of 271 mm3 with individual tumor sizes ranging from approximately 200 to 340 mm3 . Tumor volumes (mm3 ) and body weights (g) were recorded two or three times per week from all groups for analysis. Dose was body weight adjusted at time of dosing. Comparisons between treatment groups was performed using ANOVA rank sum test.

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Orthotopic Ptch+/-p53-/- medulloblastoma allograft model. [1]
Twenty four athymic nude mice (age 6 week, body weight 21.31 ± 1.52 g) were implanted with 100,000 tumor cells 17 days prior to the intiation of dosing. Tumor cells were stereotactically implanted subcortically at a depth of 3 mm and at 1.5 mm posterior to and 2.5 mm right of bregma. MRI was performed on day 4 prior to initiation of treatment for randomization into treatment group (baseline measurement). Nine animals were excluded from the study based on tumor size. The remaining 16 mice were sorted into a vehicle-treated group and a 5m-treated group so that the mean and SEM were similar. One animal in the5m -treatment group was subsequently excluded from the analysis because the tumor volume did not change over the observation period, and the finding was confirmed by histological evaluation. The mean (± SEM) tumor volume of the 5m-treated group was 3.39 ± 0.26 mm3 , and the mean (± SEM) tumor volume of the vehicle-treated group was 3.19 ± 0.39 mm3 . Treatment (vehicle or 5m at 40 mg/kg/day p.o. b.i.d) was initiated on day 0 (17 days following tumor implantation). Doses are provided as free base equivalents started on day 0. MRI scans were performed on days -4, 0 and +4 In reference to initiation of dosing) Mice were euthanized when they exhibited signs of morbidity.

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Demonstration of an intact blood-brain barrier in the orthotopic Ptch+/-p53-/- medulloblastoma allograft model. [1]
Animals (8 total; 4 groups of 2 each) were implanted with 50,000 or 100,000 tumor cells, and treated with either with 40 mg/kg/day po bid 5m or vehicle. MRI was performed at day 9 post implantation. Images were acquired before and after intraperitoneal administration of 0.4 ml/kg of the contrast agent gadopentetate dimeglumine (Gd-DTPA). In 7 out of 8 animals, the brain was unenhanced after contrast injection, while surrounding cranial muscles indicating the integrity of the blood-brain barrier (Figure 1). No difference was observed between the treatment groups. The remaining animal was in the vehicle-treated group implanted with 100,000 cells. In this case, the tumor grew along the great cerebral vein of Galen, and disrupting the blood-brain barrier, resulting in a hyperintense tumor.

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Imaging of orthotopic Ptch+/-p53-/- medulloblastoma allograft model. [1]
MRI was performed in a Bruker BioSpec 7.0 T scanner, using a 35 mm innerdiameter birdcage resonator for transmission and reception. The mice were anaesthetized with 1.2% – 1.5% isoflurane in oxygen. The head of animal was fixed by a tooth bar and a facemask to minimize motion. Respiration rate and body temperature were monitored continuously and temperature maintained between 32 – 35°C by heated airThe T2-weighted anatomical images were acquired in the coronal view to image the whole mouse brain with a multislice multi-spinecho sequence. The following parameters including: repetition time of 3000 ms, echo train length of 8, echo spacing of 11.5 ms, effective echo time of 51.75 ms, 160×128 matrix, field of view of 20×20 mm2 , spatial resolution of 0.125×0.156 mm2 /pixel, bandwidth of 50000 Hz, 2×2 oversampling, 2 averages, 30 slices, slice thickness 0.5 mm, and a total scan time of 25 min 36 sec were used. These images were segmented to quantify tumor volume using ITK-SNAP [Yushkevich, P. A., Piven, J., Hazlett, H. C., Smith, R. G., Ho, S., Gee, J. C. and Gerig, G. Neuroimage 2006, 31, 1116-1128.] For assessment of blood-brain-barrier integrity, T1- weighted images were acquired with a gradient-echo sequence using the following parameters: repetition time of 200 ms, echo time of 2.7 ms, 128×128 matrix, field of view of 20×20 mm2, spatial resolution of 0.156×0.156 mm2/pixel, 2×1 oversampling, flip angle of 90°, 8 averages, bandwidth of 50505.1 Hz, echo position at 40%, 30 slices, slice thickness 0.5 mm, and a total scan time of 3 min 25 sec.

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Mice: The impact of sonidegib treatment on CML LSC is examined in vivo using the transgenic EGFP+/SCLtTA/TRE-BCR-ABL mouse model. Transgenic GFP-expressing mice are crossed with Scl-tTa-BCR-ABL mice in the FVB/N background. After 4 weeks of induction, bone marrow cells are extracted. GFP+ cells are then identified using flow cytometry and injected into the tail veins of wild-type FVB/N recipient mice at a density of 106 cells per mouse. The mice are then exposed to 900 cGy of radiation, creating a sizable cohort of mice with similar leukemia onset times. The recipient mice's neutrophilic leukocytosis was confirmed by blood samples taken four weeks after transplantation. Nilotinib (50 mg/kg by gavage, daily), Sonidegib (80 mg/kg by gavage, daily), Sonidegib + Nilotinib, or vehicle alone (control) are the treatment options given to mice. The animals are put to sleep after three weeks of treatment, and blood, spleen cells, and the contents of the femur and tibiae's marrow are extracted. Using flow cytometry, the total white cell count (WCC), GFP-expressing WCC, myeloid cells, and GFP+ progenitors and stem cells are quantified. A subgroup of mice is evaluated for survival 120 days after treatment termination. After combining sperm and bone marrow cells from a subgroup of mice in each arm, 5x106 cells/mouse (eight mice per condition) are injected into wild-type FVB/N recipient mice that have been exposed to 900 cGy of radiation. Peripheral blood (PB) is drawn every four weeks to monitor engraftment. Flow cytometry is used to determine the proportion of GFP+ cells in PB.

Absorption, Distribution and Excretion
Sonidegib is rapidly absorbed in the fasted state with peak concentrations occurring 2-4 hours after administration. (2) However, the total absorption of Sonidegib is low (roughly 6-7%). (1)
Around 70% of Sonidegib is eliminated in the feces, while 30% is eliminated in the urine. (2)
Estimated volume of distribution = 9166 L (2)

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Metabolism / Metabolites
Sonidegib is primarily metabolized via oxidation and amide hydrolysis. (1) The enzyme responsible for the majority of metabolism is the cytochrome P450 (CYP) 3A4 enzyme. (2)

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Biological Half-Life
Half-life ~ 28 days (2)

Hepatotoxicity
Most clinical trials of sonidegib included few patients and rates of liver tests abnormalities were often not reported. In isolated trials, serum ALT elevations were reported in 15% to 27% of patients and to rise above 5 times the upper limit of normal (ULN) in 1% to 6%. Rates of serum enzyme elevations were greater with higher doses, and all were apparently transient and resolved either spontaneously or with dose reductions or discontinuation. In these trials, there were no cases of clinically apparent liver injury, hepatitis with jaundice or death from liver failure. The product label for sonidegib mentions serum enzyme elevations as a possible adverse event, but does not mention liver injury with jaundice or hepatic failure. Since its approval and more widespread use, there have been no published cases of hepatotoxicity attributed to sonidegib, but it is an uncommonly used antineoplastic agent. Serum enzyme elevations were also rare with the initial hedgehog inhibitor, vismodegib, which has been implicated in causing at least one case of acute, self-limited cholestatic hepatitis (Case 1 in Vismodegib).
Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of sonidegib during breastfeeding. Because sonidegib is 97% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 28 days and it might accumulate in the infant. The manufacturer recommends that breastfeeding be discontinued during sonidegib therapy.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Sonidegib is over 97% bound to plasma proteins, and binding is independent of concentration. (2)

[1]. Discovery of NVP-LDE225, a Potent and Selective Smoothened Antagonist. ACS Med Chem Lett. 2010 Mar 16;1(3):130-4.

[2]. Deregulated hedgehog pathway signaling is inhibited by the smoothened antagonist LDE225 (Sonidegib) in chronic phase chronic myeloid leukaemia. Sci Rep. 2016 May 9;6:25476.

Pharmacodynamics
Sonidegib has been shown to inhibit a transmembrane protein called SMO which plays a role in Hh signal transduction. This has resulted in inhibition of Hh signaling as well as antitumour activity in various animal models. In a transgenic mouse model of islet cell neoplasms, tumour volume was reduce by 95% in mice treated with sonidegib when compared with untreated mice. (2)

C26H26F3N3O3

485.5

485.192

C, 64.32; H, 5.40; F, 11.74; N, 8.66; O, 9.89

956697-53-3

Sonidegib diphosphate; 1218778-77-8

24775005

White to light yellow solid powder

1.3±0.1 g/cm3

544.5±50.0 °C at 760 mmHg

283.1±30.1 °C

0.0±1.5 mmHg at 25°C

1.569

5.43

1

8

5

35

691

2

CC1C(C(=O)NC2C=NC(N3C[C@H](C)O[C@H](C)C3)=CC=2)=CC=CC=1C1C=CC(OC(F)(F)F)=CC=1

VZZJRYRQSPEMTK-CALCHBBNSA-N

InChI=1S/C26H26F3N3O3/c1-16-14-32(15-17(2)34-16)24-12-9-20(13-30-24)31-25(33)23-6-4-5-22(18(23)3)19-7-10-21(11-8-19)35-26(27,28)29/h4-13,16-17H,14-15H2,1-3H3,(H,31,33)/t16-,17+

N-[6-[(2S,6R)-2,6-dimethylmorpholin-4-yl]pyridin-3-yl]-2-methyl-3-[4-(trifluoromethoxy)phenyl]benzamide

Sonidegib; LDE 225; NVP-LDE225; LDE-225; NVP LDE-225; LDE225; NVP LDE225; Erismodegib; trade name of Odomzo

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (5.15 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 2% DMSO+corn oil: 10 mg/mL

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Solubility in Formulation 5: 2 mg/mL (4.12 mM) in 75% PEG 300 25% (5% dextrose in water) (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)