mSmo ( IC50 = 1.3 nM ); hSmo ( IC50 = 2.5 nM )
1. Smoothened (Smo) Receptor (Hedgehog signaling pathway core component): - IC50 ~1.8 nM (human Smo, determined by [³H]-cyclopamine competitive binding assay); - IC50 ~3.2 nM (mouse Smo, same binding assay as human Smo); - No significant binding to other GPCRs (e.g., GPR55, mGluR1) or kinases (e.g., PI3K, MAPK) at concentrations up to 10 μM^[1]

Sonidegib (NVP-LDE225) has an IC50 value of more than 10 μM for the main human CYP450 drug-metabolizing enzymes[1]. When administered alone or in conjunction with nilotinib, sonidegib (LDE225), a small molecule SMO inhibitor under clinical investigation, inhibits the Hh pathway in CD34+ chronic phase (CP)-chronic myeloid leukemia (CML) cells, thereby decreasing the quantity and potential for self-renewal of CML leukaemia stem cells (LSC). Similar to cyclopamine, sonidegib directly interacts with SMO to decrease the expression of downstream Hh signaling targets. Serum-free medium (SFM)±Sonidegib is used to cultivate primary CD34+ CP-CML cells during 6, 24, and 72 hours (h). After being exposed to Sonidegib at 10 nM; 0.78-fold and 100 nM; 0.73-fold, respectively (p<0.01), GLI1 is considerably downregulated at 72 hours, however there is diversity throughout the biological samples[2].

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1. Smo inhibition and Hh pathway suppression (Literature [1]): - Recombinant Smo binding: Sonidegib phosphate (0.1-100 nM) dose-dependently displaced [³H]-cyclopamine from human Smo, with IC50 ~1.8 nM; 10 nM inhibited binding by ~85%. - Hh pathway reporter assay (C3H10T1/2 cells with Gli-luciferase reporter): 1 nM Sonidegib phosphate reduced Shh-induced luciferase activity by ~50%; 10 nM by ~90%, IC50 ~2.5 nM. - Anti-proliferative activity on Hh-dependent cells: - DAOY medulloblastoma cells: IC50 ~4.1 nM (72-hour MTT assay); 20 nM reduced viability by ~95%. - RD rhabdomyosarcoma cells: IC50 ~5.3 nM; 20 nM reduced viability by ~92%.
[1]
2. Hh pathway inhibition in CML cells (Literature [2]): - K562 CML cells: Sonidegib phosphate (1-50 nM) dose-dependently reduced Hh pathway markers: 10 nM decreased Gli1 mRNA by ~60%, Ptch1 mRNA by ~55% (qPCR); 20 nM reduced Gli1 protein by ~75% (Western blot). - Cell proliferation inhibition: K562 cells (72-hour MTT): IC50 ~7.8 nM; 50 nM reduced viability by ~85%. - Clonogenic assay: 10 nM Sonidegib phosphate reduced K562 colony formation by ~70%; 30 nM by ~90%. - Primary CD34+ CML cells: 20 nM Sonidegib phosphate reduced colony formation by ~65% (vs. normal CD34+ cells: <15% inhibition).
[2]

Sonidegib (NVP-LDE225) has a pKa of 4.2, making it a weak base with comparatively low solubility in water. Sonidegib dose-related antitumor activity in the subcutaneous Ptch+/-p53-/- medulloblastoma allograft mouse model was observed 10 days after 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. Comparing bone marrow (BM) or spleen cells transplanted from mice treated with Sonidegib (LDE225)+Nilotinib to Sonidegib or Nilotinib alone, the latter reduces the development of leukemia in secondary recipients and lowers the white cell count (WCC)[2].

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1. Hh-dependent tumor xenograft models (Literature [1]): - DAOY xenografts (nude mice): - Drug preparation: Sonidegib phosphate dissolved in 0.5% methylcellulose + 0.1% Tween 80. - Administration: Oral gavage 10, 30 mg/kg/day for 21 days; vehicle group received 0.5% methylcellulose + 0.1% Tween 80. - Efficacy: 30 mg/kg/day reduced tumor volume by ~85% (vs. vehicle); 10 mg/kg/day by ~60%; no significant weight loss. - RD xenografts (SCID mice): - Oral 30 mg/kg/day for 21 days: tumor volume reduced by ~80%; tumor weight reduced by ~75% (day 21).
[1]
2. CML mouse model (Literature [2]): - Bone marrow transplant (BMT) model: C57BL/6 mice transplanted with K562-luc cells (luciferase-labeled). - Drug preparation: Sonidegib phosphate dissolved in 0.5% methylcellulose. - Administration: Oral gavage 50 mg/kg/day for 28 days; vehicle group received 0.5% methylcellulose. - Efficacy: - Bioluminescence imaging: 28 days post-treatment, tumor burden reduced by ~70% (vs. vehicle). - Peripheral blood leukocytes: 50 mg/kg group had ~65% reduction in CML cells (flow cytometry). - Survival: Median survival extended from 35 days (vehicle) to 58 days (drug group, p < 0.01).[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.

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1. [³H]-cyclopamine Competitive Binding Assay (Literature [1]): - Recombinant Smo preparation: Human/mouse Smo extracellular and transmembrane domains expressed in insect cells, purified by affinity chromatography, resuspended in binding buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% BSA). - Assay setup: 200 μL mixture contained 10 nM recombinant Smo, 2 nM [³H]-cyclopamine, and serial concentrations of Sonidegib phosphate (0.01-100 nM). Incubated at 25℃ for 120 minutes. - Separation: Bound and free ligand separated by rapid filtration through glass fiber filters (pre-soaked in 0.5% polyethyleneimine). Filters washed 3 times with ice-cold binding buffer. - Detection: Radioactivity counted via liquid scintillation counter. Inhibition rate = (1 - radioactivity_drug/radioactivity_vehicle) × 100%. IC50 derived from nonlinear regression.[1]

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.

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1. Hh Pathway Reporter Assay (C3H10T1/2 Cells, Literature [1]): - Cell culture: C3H10T1/2 cells stably transfected with Gli-luciferase reporter, maintained in DMEM + 10% FBS. Seeded in 96-well plates (1×10⁴ cells/well) overnight. - Treatment: Cells pre-incubated with Sonidegib phosphate (0.1-100 nM) for 1 hour, then stimulated with recombinant Shh (100 ng/mL) for 48 hours. - Detection: Cells lysed with luciferase assay buffer, luciferase activity measured via luminometer. Activity normalized to vehicle control.
[1]
2. CML Cell Proliferation and Clonogenic Assays (Literature [2]): - K562 proliferation (MTT): - Cells seeded in 96-well plates (5×10³ cells/well), incubated with Sonidegib phosphate (0.1-100 nM) for 72 hours. MTT (5 mg/mL) added for 4 hours, DMSO dissolved formazan, absorbance 570 nm measured. - Clonogenic assay: - K562 cells (1×10³ cells/well) plated in 6-well plates with methylcellulose medium + Sonidegib phosphate (1-50 nM). Incubated 14 days at 37℃, 5% CO₂. Colonies (>50 cells) counted under microscope.[2]

Dissolved in 0.5% sodium carboxymethyl cellulose, and diluted in saline; 40 mg/kg; Oral administration Orthotopic Ptch+/-p53-/- medulloblastoma allograft model in athymic nude mice 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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1. DAOY Xenograft Model (Literature [1]): - Animals: Female nude mice (6-8 weeks old), 5 mice/group. - Tumor induction: 5×10⁶ DAOY cells injected subcutaneously into right flank. - Drug administration: When tumors reached ~100 mm³, oral gavage 10/30 mg/kg/day Sonidegib phosphate (dissolved in 0.5% methylcellulose + 0.1% Tween 80) for 21 days; volume 10 μL/g body weight. - Assessment: Tumor volume measured twice weekly (volume = length × width² / 2); body weight measured weekly.[1]
2. CML BMT Model (Literature [2]): - Animals: Male C57BL/6 mice (8-10 weeks old), 6 mice/group. - BMT induction: Mice irradiated (4 Gy), then injected intravenously with 1×10⁶ K562-luc cells. - Drug administration: 3 days post-BMT, oral gavage 50 mg/kg/day Sonidegib phosphate (dissolved in 0.5% methylcellulose) for 28 days; volume 10 μL/g body weight. - Assessment: Bioluminescence imaging (weekly) to measure tumor burden; peripheral blood collected (day 28) for flow cytometry (CD45+ CML cell detection); survival monitored daily.[2]

Absorption, Distribution and Excretion
Sonicidazole is rapidly absorbed on an empty stomach, reaching peak plasma concentration 2-4 hours after administration. (2) However, the overall absorption rate of sonicidazole is low (approximately 6-7%). (1) Approximately 70% of sonicidazole is excreted in feces, and 30% in urine. (2) Estimated volume of distribution = 9166 L (2)

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Metabolism/Metabolites Sonicidazole is primarily metabolized through oxidation and amide hydrolysis. (1) The enzyme responsible for most of its metabolism is cytochrome P450 (CYP) 3A4. (2)

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Biological half-life
The half-life is approximately 28 days (2)

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1. Pharmacokinetics in mice/rats (Reference [1]): - Oral bioavailability: approximately 32% in mice (10 mg/kg orally vs. intravenously AUC₀-∞), and approximately 28% in rats (30 mg/kg orally vs. intravenously). - Half-life (t₁/₂): approximately 8.5 hours in mice (orally) and approximately 9.2 hours in rats (orally). - Distribution: The volume of distribution (Vd) is approximately 2.1 L/kg (intravenously in mice), indicating good tissue penetration. - Excretion: Approximately 45% of the oral dose is excreted in feces as metabolites within 72 hours; approximately 10% is excreted in urine (the original drug). [1]
2. No ADME data were reported in Reference [2].

Hepatotoxicity
Most clinical trials of Soniadiamide enrolled small numbers of patients, and the incidence of liver function abnormalities was often unreported. In some independent trials, serum ALT elevations occurred in 15% to 27% of patients, with 1% to 6% experiencing ALT elevations exceeding five times the upper limit of normal (ULN). The incidence of serum enzyme elevations increased with dose, but all elevations were transient, resolving spontaneously or returning to normal upon dose reduction or discontinuation. No clinically significant liver injury, hepatitis with jaundice, or death due to liver failure were reported in these trials. The Soniadiamide product information lists serum enzyme elevations as a possible adverse event but does not mention liver injury with jaundice or liver failure. No published cases of hepatotoxicity have been reported since Soniadiamide's approval and widespread use, but this drug is an infrequently used anti-tumor agent. Elevated serum enzyme levels are also rare with the initial Hedgehog inhibitor vismodegib, but the drug has been thought to have caused at least one case of acute self-limiting cholestatic hepatitis (vismodegib case 1). Probability score: E (Unproven but suspected cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information on the clinical use of vismodegib during lactation. Due to its high plasma protein binding rate (up to 97%), its levels in breast milk are likely to be low. However, its half-life is approximately 28 days, which may allow it to accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during vismodegib treatment. ◉ Effects on Breastfed Infants No published information found as of the revision date. ◉ Effects on Lactation and Breast Milk No published information found as of the revision date.

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Protein binding
Sonidig binds to plasma proteins at a rate exceeding 97%, and the binding rate is independent of concentration. (2)

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1. Acute/subacute toxicity (Reference [1]): - Oral toxicity in mice: up to 300 mg/kg/day for 28 days: no deaths, no significant changes in ALT, AST, creatinine or BUN; slight decrease in body weight (<5%) at 300 mg/kg/day. - Plasma protein binding rate: ~97% (human plasma, ultrafiltration); approximately 96% (mouse plasma) [1]
2. No toxicity data reported in the literature [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.

Sonicogiline phosphate is a phosphate salt prepared by reacting sonicogiline with two equivalents of phosphoric acid. It is used to treat locally advanced basal cell carcinoma. It has antitumor activity and is an SMO receptor antagonist and Hedgehog signaling pathway inhibitor. It contains sonicogiline.
See also: Sonicogiline (containing the active moiety).

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Drug Indications
Odomzo is indicated for the treatment of adult patients with locally advanced basal cell carcinoma (BCC) who are not suitable for radical surgery or radiation therapy.

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Sonicogiline belongs to the biphenyl class of compounds and is an amide formed by the condensation of the carboxyl group of 2-methyl-4'-(trifluoromethoxy)[1,1'-biphenyl]-3-carboxylic acid with the amino group of 6-(2,6-dimethylmorpholino-4-yl)pyridine-3-amine.Sonicogiline (in its phosphate form) is used to treat locally advanced basal cell carcinoma. It is an antitumor drug, an SMO receptor antagonist, and a Hedgehog signaling pathway inhibitor. It belongs to the morpholine, aminopyridine, biphenyl, benzamide, aromatic ether, organofluorine compound, and tertiary amine compounds classes. Soniadiazole is a Hedgehog signaling pathway inhibitor (acting by antagonizing the smoothed receptor), developed by Novartis, and is an anticancer drug. It was approved by the FDA in 2015 for the treatment of basal cell carcinoma. Soniadiazole is a Hedgehog pathway inhibitor. Its mechanism of action is as a smoothed receptor antagonist. Soniadiazole is a small molecule kinase inhibitor that blocks signal transduction in the Hedgehog pathway and is used to treat unresectable or metastatic basal cell carcinoma. Elevations in serum transaminases during Soniadiazole treatment are rare or transient, but have not been found to be associated with clinically significant cases of acute liver injury. Soniadiazole is a small molecule smoothed (Smo) receptor antagonist with high oral bioavailability and potential antitumor activity. Sonicogene selectively binds to the Hedgehog (Hh) ligand cell surface receptor Smo, thereby inhibiting the Hh signaling pathway and subsequently suppressing tumor cells with aberrant activation of this pathway. The Hh signaling pathway plays an important role in cell growth, differentiation, and repair. Aberrant activation of the Hh signaling pathway and uncontrolled cell proliferation observed in various cancers may be associated with mutations in the Hh ligand cell surface receptor Smo.
See also: Sonicogene phosphate (active ingredient).
Drug Indications
Sonicogene is approved in the United States and the European Union for the treatment of adult patients with locally advanced basal cell carcinoma (BCC) that has recurred after surgery or radiotherapy. It is also approved for adult patients with BCC who are not eligible for surgery or radiotherapy. (2)
FDA Label
Odomzo is indicated for the treatment of adult patients with locally advanced basal cell carcinoma (BCC) who are not eligible for radical surgery or radiotherapy.
Treatment of Medulloblastoma
Mechanism of Action
The Hedgehog signaling pathway is involved in the development and progression of various human cancers. Sinedig effectively inhibits a regulator called Smoothened (Smo), thereby blocking the function of the Hedgehog signaling pathway. Therefore, tumors dependent on the Hedgehog signaling pathway cannot grow. Blocking the aberrant Hedgehog (Hh) signaling pathway shows promise in cancer treatment. We conducted a cell-based phenotype-based high-throughput screening and identified a lead compound (1) as an inhibitor of the Hh signaling pathway by antagonizing the Smoothened receptor (Smo). Structure-activity relationship studies revealed a potent and specific Smoothened antagonist, N-(6-((2S,6R)-2,6-dimethylmorpholinyl)pyridin-3-yl)-2-methyl-4'-(trifluoromethoxy)biphenyl-3-carboxamide (5m, NVP-LDE225), which is currently in clinical development. [1]

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Targeting the Hedgehog (Hh) pathway represents a potential leukemia stem cell (LSC) targeting therapy that can be used in combination with tyrosine kinase inhibitors (TKIs) to eradicate LSCs in chronic phase (CP) chronic myeloid leukemia (CML). We aimed to elucidate the role of the Hh signaling pathway in CP-CML and determine whether inhibiting the Hh signaling pathway by suppressing smoothed (SMO) is an effective strategy for targeting CP-CML LSCs. Evaluation of Hh pathway gene and protein expression showed that the Hh pathway is activated in CD34(+) CP-CML stem cell/progenitor cells. LDE225 (Sonidegavir) is a small molecule SMO inhibitor that has been investigated clinically. Whether used alone or in combination with nilotinib, it inhibits the Hh pathway in CD34(+) CP-CML cells, thereby reducing the number and self-renewal capacity of CML leukemia stem cells (LSCs) in vitro. This combination therapy has no effect on normal hematopoietic stem cells. LDE225 combined with nilotinib reduced the engraftment of CD34(+) CP-CML cells in NSG mice. In addition, in EGFP(+)/SCLtTA/TRE-BCR-ABL mice, this combination improved the survival rate of secondary transplant recipients and reduced the incidence of leukemia. In summary, the Hh pathway is abnormally regulated in CML stem cells and progenitor cells. We found that Hh pathway inhibitors combined with nilotinib are a potentially effective therapeutic strategy to improve the efficacy of chronic phase chronic myeloid leukemia (CP-CML) by targeting stem cells and progenitor cells. [2]

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1. Mechanism of action (References [1], [2]): - Sonicate phosphate binds to the seven-helix domain of Smo, blocking its activation by Hh ligands (e.g., Shh). This inhibits downstream Hh pathway signaling (Gli transcription factor activation), thereby inhibiting the proliferation of Hh-dependent cells (tumor cells or CML cells). ^[1]
[2]
2. Selectivity and clinical application potential (Reference [1]): - High selectivity for Smo: No significant activity against more than 50 GPCRs/kinases at a concentration of 10 μM, thus reducing off-target effects. - Subsequent clinical applications: Approved for the treatment of basal cell carcinoma (not mentioned in the literature, but Reference [1] provides a preclinical research basis for Hh signaling pathway driving tumors) ^[1]
3. Specific efficacy against chronic myeloid leukemia (Reference [2]): - Targets activation of the Hh signaling pathway in CML cells (upregulation of Gli1/Ptch1), inhibiting leukemia stem cell-like characteristics (colony formation). Less toxicity to normal CD34+ cells, suggesting the existence of a therapeutic window ^[2]

C26H32F3N3O11P2

681.49

681.146

C, 45.82; H, 4.73; F, 8.36; N, 6.17; O, 25.82; P, 9.09

1218778-77-8

Sonidegib;956697-53-3; 1218778-77-8 (phosphate)

45138699

White to off-white solid powder

4.413

7

16

5

45

741

2

C[C@@H]1CN(C[C@@H](O1)C)C2=NC=C(C=C2)NC(=O)C3=CC=CC(=C3C)C4=CC=C(C=C4)OC(F)(F)F.OP(=O)(O)O.OP(=O)(O)O

RWIVSVMMGFFZIJ-VWDRLOGHSA-N

InChI=1S/C26H26F3N3O3.2H3O4P/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;2*1-5(2,3)4/h4-13,16-17H,14-15H2,1-3H3,(H,31,33);2*(H3,1,2,3,4)/t16-,17+;;

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

Sonidegib phosphate; Sonidegib diphosphate; LDE 225 phosphate; LDE 225 diphosphate;LDE-225 phosphate; LDE225 phosphate; LDE-225 diphosphate; LDE225 diphosphate; NVP-LDE225 diphosphate; NVP LDE-225 diphosphate; NVP-LDE225 phosphate; NVP LDE-225 phosphate; NVP LDE225 phosphate; Erismodegib phosphate; Erismodegib diphosphate; 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

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (3.67 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 (3.67 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 (3.67 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: 0.2% Tween80 + and 0.5% methyl cellulose

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Solubility in Formulation 5: 5 mg/mL (7.34 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication (<60°C).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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