Glasdegib (formerly known as PF-04449913; PF4449913; PF04449913; PF-4449913; Daurismo) is a novel, potent, and orally bioavailable small-molecule inhibitor of Smoothened (Smo) in the Hedgehog (Hh) signaling pathway with potential antitumor activity. It is a drug that was authorized in 2018 to treat acute myeloid leukemia that has just been discovered. Smoothened (Smo) is inhibited by Glasdegib at a 5 nM IC50. Cellular development, differentiation, and repair are all significantly impacted by the Hh signaling pathway. Many forms of cancer have been shown to exhibit constitutive activation of the Hh pathway signaling. In a Ptch1^+/-p53^+/- allograft model, Glasdegib dramatically inhibits the growth of medulloblastoma at doses that also reduce the expression of the murine Shh target gene. When PF-04449913 is used in stromal co-culture experiments, FACS analysis shows a significant decrease in BC LSC relative to normal progenitors.

Physicochemical Properties

Molecular Formula	C21H22N6O
Molecular Weight	374.44
Exact Mass	374.185
Elemental Analysis	C, 67.36; H, 5.92; N, 22.44; O, 4.27
CAS #	1095173-27-5
Related CAS #	1095173-27-5; 2030410-25-2; 1095173-64-0 (HCl); 1352568-48-9 (2HCl)
PubChem CID	25166913
Appearance	White solid powder
Density	1.3±0.1 g/cm3
Boiling Point	633.4±55.0 °C at 760 mmHg
Flash Point	336.9±31.5 °C
Vapour Pressure	0.0±1.9 mmHg at 25°C
Index of Refraction	1.686
LogP	2.77
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	28
Complexity	595
Defined Atom Stereocenter Count	2
SMILES	O=C(NC1=CC=C(C#N)C=C1)N[C@H]2C[C@H](C3=NC4=CC=CC=C4N3)N(C)CC2
InChi Key	SFNSLLSYNZWZQG-VQIMIIECSA-N
InChi Code	InChI=1S/C21H22N6O/c1-27-11-10-16(24-21(28)23-15-8-6-14(13-22)7-9-15)12-19(27)20-25-17-4-2-3-5-18(17)26-20/h2-9,16,19H,10-12H2,1H3,(H,25,26)(H2,23,24,28)/t16-,19-/m1/s1
Chemical Name	1-[(2R,4R)-2-(1H-benzimidazol-2-yl)-1-methylpiperidin-4-yl]-3-(4-cyanophenyl)urea
Synonyms	PF 04449913; PF-4449913; PF-04449913; PF4449913; PF04449913; PF 4449913; daurismo
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Smo ( IC50 = 4 nM )
ln Vitro	In vitro activity: Glasdegib (PF-04449913) at doses that decrease murine Shh target gene expression significantly reduces the growth of medulloblastoma in a Ptch1+/-p53+/- allograft model and inhibits sonic hedgehog (Shh) stimulated luciferase expression in mouse embryonic fibroblasts with an IC50 of 6.8 nM. When compared to normal progenitors, FACS analysis shows a significant reduction in BC LSC by Glasdegib (PF-04449913) in stromal co-culture experiments. Significantly, the spleen weight of human BC LSC engrafted RAG2-/-p53-/-mice treated daily with Glasdegib (PF-04449913) was significantly lower than that of vehicle-treated controls (p=0.006). This reduction in leukemic burden corresponded with decreased GLI2 protein expression, as determined by both nanoproteomic analysis of FACS purified human progenitors and GLI2 confocal fluorescence microscopic analysis of splenic sections[1].
ln Vivo	Glasdegib (PF-04449913)-treated human BC LSC engrafted RAG2-/-p53-/- mice showed a significant reduction in spleen weight (p=0.006) when compared to vehicle-treated controls. Contrary to LSC, normal human HSC cell fate decisions are Hh pathway independent. This is demonstrated by the fact that when CD34+cord blood engrafted NSG mice are treated with Glasdegib (PF-04449913), the frequency of human CD45+cells, progenitors, and both myeloid and lymphoid cell fate commitment remained comparable to vehicle treated controls. These findings underscore the significant context- and cell-specific niche-dependent effects of selective SMO inhibition, which cause GLI2 downregulation[1].
Enzyme Assay	Glasdegib, a novel potent small-molecule inhibitor with an IC50 of 5 nM, is an oral bioavailable inhibitor of Smoothened (Smo) in the Hedgehog (Hh) signaling pathway. It was previously known as PF-04449913. In cells, the Hh signaling pathway is essential for growth, differentiation, and repair. In many different kinds of cancer, constitutive activation of the Hh pathway signaling has been reported. In an allograft model with Ptch1+/-p53+/-, Glasdegib dramatically inhibits the growth of medulloblastoma at doses that also reduce the expression of the murine Shh target gene. When compared to normal progenitors, FACS analysis shows a significant reduction in BC LSC by PF-04449913 in stromal co-culture experiments.
Cell Assay	BMS-354825 (50 nM), Glasdegib (PF-04449913) (1 μM), or a combination of these treatments are used to treat CD34+cells in BC CML or normal cells. The M2-10B4 (M2) and SL/SL (SL) mouse bone marrow stromal cell lines are plated 1:1 mixture at a final concentration of 100,000 cells/mL one day before being co-cultured with 10,000–20,000 CD34+BC CML or normal progenitors. At 14 days, progenitors are scored and FACS sorted into hematopoietic progenitor assays following a week of culture. Normal human hematopoietic stem and progenitor cells are evaluated for survival using the following methods: irradiated (20 Gray) OP9 (M2 clone) stromal cells are co-cultured with 50,000 human CD34+cord blood cells, vehicle, or Glasdegib (PF-04449913) in AlphaMEM with 20% Hyclone FBS, 1% pen strep glutamine, and supplemented with 50 ng/mL SCF, 10 ng/mL thrombopoietin, and 10 ng/mL Flt3. The results are recorded using weekly FACS analysis[1].
Animal Protocol	Mice: Equal amounts of BC LSC or normal progenitors are intrahepatically transplanted into RAG2-/-c-/- mice. After the identification of human CD45+cell peripheral blood engraftment, mice are given oral gavage every day for 14 days with either Glasdegib (100 mg/kg), BMS-354825 (50 mg/kg), or a combination of these drugs. The amount of human engraftment in hematopoietic niches is then measured using FACS. Eight weeks after sublethally irradiating seven to ten week old NOD Cg-PrkdcSCID IL2R1Wjl/SzJ mice, 100,000 CD34+human cord blood cells are transplanted retro-orbitally, and the mice are treated with vehicle or Glasdegib (100 mg/kg) for 14 days, followed by FACS engraftment analysis, in order to evaluate the effects on normal HSC function.
ADME/Pharmacokinetics	Absorption, Distribution and Excretion Glasdegib presents a dose-proportional pharmacokinetic profile which is observed by the presence of a broad dose-proportional maximum plasma concentration. In this study and on a dose of 50 mg, the median time to reach a maximum concentration of 321 ng/ml was of 4 hours with an AUC of 9587 ng.h/ml. The oral bioavailability of glasdegib is reported to be of 55%. In a multiple dose study of 50 mg, the Cmax, tmax and AUC was reported to be 542 ng/ml, 4 h and 9310 ng.h/ml respectively. In this same study, the average concentration at a steady state was of 388 ng/ml. The absorption rates of glasdegib can be modified by the concomitant consumption of a high-fat, high-calorie meal. From a single oral dose of 100 mg radiolabeled glasdegib, 49% is eliminated in the urine from which 17% is excreted as the unchanged form while 42% is eliminated in feces where 20% represents the unchanged form. Glasdegib reported volume of distribution in a dose of 50 mg is 225 L. The geometric mean (%CV) apparent volume of distribution (Vz/F) was 188 L (20%) in patients with hematologic malignancies. The clearance rate of 50 mg of glasdegib is reported to be of 5.22 L/h. The geometric mean (%CV) apparent clearance of 6.45 L/h (25%) following 100 mg once daily dosing in patients with hematologic malignancies. Metabolism / Metabolites After oral administration, glasdegib was primarily metabolized by CYP3A4 with minor contributions of CYP2C8 and UGT1A9. The amount of unchanged glasdegib in plasma accounts only for 69% of the administered dose. Biological Half-Life The reported half-life of glasdegib is of 17.4 hours.
Toxicity/Toxicokinetics	Hepatotoxicity Elevations in serum ALT levels are common during glasdegib therapy, occurring in 31% of patients and rising above 5 times the upper limit of the normal range in 11%. Glasdegib has had limited clinical use but has not been linked to instances of acute liver injury with symptoms or jaundice. Because of the limited clinical experience with the use of hedgehog inhibitors, their potential for causing liver injury is not well defined. Likelihood score: E* (unproved but suspected cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on glasdegib during breastfeeding. It is 91% bound to plasma proteins, so amounts in milk are likely to be low. With a half-life of 17.4 hours, it is likely to be eliminated from milk by 4 to 7 days after the last dose. However, the manufacturer recommends that breastfeeding be discontinued during glasdegib therapy and for at least 30 days after the last dose. ◉ 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. Protein Binding Glasdegib is reported to be 91% protein bounded which is explained due to its high lipophilic profile.
References	[1]. GLI2 inhibition abrogates human leukemia stem cell dormancy. J Transl Med. 2015 Mar 21;13:98.
Additional Infomation	Pharmacodynamics In preclinical studies, glasdegib achieved a significant reduction in leukemic stem cell burden in xenograft models and a reduction in cell population expressing leukemic stem cell markers. In clinical trials, glasdegib demonstrated a marked downregulation of more than 80% of the expression of glioma-associated transcriptional regulator GL11 in skin. In this same study 8% of the studied individuals with acute myeloid leukemia achieved morphological complete remission while 31% achieved stable disease state. The latest clinical trial proved glasdegib to generate an overall survival of 8.3 months which was almost double to what has been observed in patients under low-dose cytarabine treatment. As well, there have been reports of dose-dependent QTc prolongation in patients administered with glasdegib.

Solubility Data

Solubility (In Vitro)

DMSO: 47~74 mg/mL (125.5~197.6 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.55 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.55 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 20.8 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (5.55 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6707 mL	13.3533 mL	26.7065 mL
	5 mM	0.5341 mL	2.6707 mL	5.3413 mL
	10 mM	0.2671 mL	1.3353 mL	2.6707 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.