CRM1/chromosome region maintenance 1

KPT-330, a clinical candidate counterpart of KPT-185, causes a fast apoptotic response and has comparable effects on T-ALL cell survival. With IC50 values ranging from 34 to 203 nM, KPT-330 also inhibits the proliferation of MOLT-4, Jurkat, HBP-ALL, KOPTK-1, SKW-3, and DND-41 cell lines [1].

Selinexor (KPT-330) has no negative effects on healthy hematopoietic cells while dramatically suppressing the proliferation of AML (MV4-11) and T-ALL (MOLT-4) cells in vivo [1]. In SCID mice exhibiting diffuse human MM bone lesions, KPT-330 prolongs survival by inhibiting MM-induced osteolysis. Furthermore, by inhibiting RANKL-induced NF-κB and NFATc1, KPT-330 directly reduces osteoclastogenesis and bone resorption while having no effect on osteoblasts and BMSCs [2].

NF-κB p65 DNA-binding activity[2]
MM cells and CD14 + OC precursor (OCP) cells were pretreated with KPT-185 or KPT-330 for 2 h and stimulated with a proliferation-inducing ligand (APRIL, 400 ng/ml) and RANKL (100 ng/ml), respectively. Nuclear protein was then extracted for NF-κB activity using TransAM NF-κB p65 ELISA Kit.

Cell lines and cell viability assay[1]
T-ALL cell lines (HPB-ALL, DU528, Jurkat, MOLT-4, SKW-3, KARPAS-45, HSB-2, KOPTK1, PF-382, CCRF-CEM, SUPT7, MOLT-16, P12-ICHIKAWA, LOUCY) were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cell Titer Glo assay was used to assess cell viability upon treatment with either dimethyl sulfoxide (DMSO) or KPT-185. Cells were plated at a density of 10,000 cells per well in a 96-well plate and incubated with DMSO or increasing concentrations of KPT-185. The cell viability was measured after 72 h exposure to KPT-185 and reported as a percentage of DMSO control cells. Jurkat cells that overexpress BCL2 were generated using MSCV-IRES-GFP retroviral expression system. Jurkat cells infected with BCL2 or control vector viruses were sorted by flow cytometry and the expression of BCL2 confirmed by Western blot analysis using BCL2 antibody.

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Apoptosis Analysis[1]
Jurkat and MOLT-4 cells were incubated with either DMSO control or KPT-185 for 6 h or 13 h, washed with phosphate-buffered saline (PBS), and co-incubated with Annexin V- fluorescein isothiocynate (FITC) and propidium iodide (PI) from MEBCYTO Apoptosis Kit. Cells were analysed by two-colour FACS cytometry and the percentage of Annexin V and PI positive cells was determined based on the dot plots of FITC vs. PI.

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Mitochondrial Sensitivity in permeabilized whole cells[1]
2 × 104 cells/well of Jurkat cells were used. 15 μl of 100 μM peptide in T-EB (300 mM Trehalose, 10 mM HEPES-KOH pH 7.7, 80 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1% bovine serum albumin, 5 mM succinate) were deposited per well in a black 384-well plate. One volume of the 4x single cell suspension was added to one volume of a 4x dye solution (4 μM JC-1, 40 μg/ml oligomycin, 0.02% digitonin, 20 mM 2-mercaptoethanol) in T-EB. This 2x cell/dye solution was incubated for 5–10 min at room temperature to allow permeabilization and dye equilibration. 15 μl of the cell/dye mix was then added to each treatment well of the plate and the fluorescence at 590 nm monitored every 5 min at room temperature. Percentage loss of Ψm was calculated by normalization to the solvent only control DMSO (0%) and the positive control FCCP (Ryan, et al 2010).

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Cell cycle analysis[1]
Jurkat and MOLT-4 cells were incubated with serial dilutions of KPT-185 for 24 h, washed with PBS, fixed with 70% ethanol, and incubated overnight at −20°C. The cells were then washed with PBS, stained with PI/RNase staining buffer, and analysed by flow cytometry using BD FACS Canto. The DNA histograms of Jurkat and MOLT-4 cells were analysed using FCS Express 4 Flow Cytometry cell cycle analysis software and ModFit LT cell cycle analysis software

Orthograft mouse models[1]
T-ALL orthograft mouse model [1]
MOLT-4 cells (3 × 106) expressing luciferase were injected into 7-week-old female NOD-SCID-IL2Rcγnull (NSG) mice via tail-vein injections. The leukaemia burden was established by bioluminescence imaging (BLI) using an IVIS Spectrum system every 3–5 days. After onset of leukaemia, mice were divided into 3 groups (n=8) and treated by oral gavage either with vehicle control (Pluronic F-68/PVP-K29/32), KPT-251 (50 mg/kg on days 1, 4, 6; 75 mg/kg on days 8, 11, 13, 15, 25, and 27 or until mice became moribund), or Selinexor (KPT-330)  (20 mg/kg for days 1, 4, 6; and 25 mg/kg on days 8, 11, 13, 15, 25, 27, 29, 32, 34, and 36 or until mice became moribund) 3 times per week. [1]

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AML orthograft mouse model [1]
Luciferase-expressing MV4-11 cells (2×106) were intravenously injected into 7-week-old female NSG mice. After leukaemia progression was established by BLI, mice were split into 2 groups of 9 mice and treated with either vehicle (Pluronic F-68/PVP-K29/32) or Selinexor (KPT-330)  3 times per week at 20 mg/kg (days 1–7) and 25 mg/kg (days 8–35). Following 5 weeks of treatment, femur from one mouse from the treatment group was fixed in 10% formalin, sectioned, and paraffin-embedded. Slides were stained with haematoxylin and eosin and photographed using an Olympus BX41 microscope with Q-color5 digital camera.

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Formulated in Pluronic F-68/PVP-K29/32; 20 -25 mg/kg; oral gavage

T-ALL and AML orthograft mouse model

Absorption, Distribution and Excretion
A single 80 mg dose of selinexor produces a mean Cmax of 680 ng/mL and a mean AUC of 5386 ng*h/mL. This relationship is dose proportion over the range of 3-85 mg/m2 which encompasses the range of 0.06-1.8 times the approved dosage. The official FDA labeling reports the Tmax as 4 hours but phase 1 studies have found a range of 2-4 hours. Administering selinexor with food, either a high or low fat meal, results in an increase in the AUC of approximately 15-20% but this is not expected to be clinically significant.
The mean apparent volume of distribution is 125 L. A phase 1 study reported mean apparent volumes of distribution ranging from 1.9-2.9 L/kg in their investigation of food and formulation effects.
Selinexor has a mean apparent clearance of 17.9 L/h.

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Metabolism / Metabolites
Selinexor is known to be metabolized through CYP3A4, UDP‐glucuronosyltransferases, and glutathione S-transferases although the metabolite profile has yet to be characterized in published literature. The primary metabolites found in urine and plasma are glucuronide conjugates.

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Biological Half-Life
Selinexor has a mean half-life of elimination of 6-8 hours.

Hepatotoxicity
In prelicensure open label trials of selinexor in a total of 202 patients with advanced, refractory or relapsed multiple myeloma, serum ALT elevations arose in 8.4% of treated subjects and were above 5 times the ULN in 2.5%. The timing and character of the elevations were not described, but no patient developed raised serum enzymes with jaundice or symptoms. Since approval and general availability of selinexor, there have been no published reports of clinically apparent liver injury attributed to its use.
Likelihood score: E* (unproven, but possible rare 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 use of selinexor during breastfeeding. Most sources consider breastfeeding to be contraindicated during maternal antineoplastic drug therapy. The manufacturer recommends that mothers should not breastfeed during treatment with selinexor and for one week after the last dose. Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk. Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant.
◉ 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
Selinexor is 95% bound to plasma proteins.

[1]. KPT-330 inhibitor of CRM1 (XPO1)-mediated nuclear export has selective anti-leukaemic activity in preclinical models of T-cell acute lymphoblastic leukaemia and acute myeloid leukaemia. Br J Haematol. 2013 Apr;161(1):117-27.

[2]. CRM1 inhibition induces tumor cell cytotoxicity and impairs osteoclastogenesis in multiple myeloma: molecular mechanisms and therapeutic implications. Leukemia. 2014 Jan;28(1):155-65.

Pharmacodynamics
Selinexor causes cell cycle arrest and apoptosis in cancer cells.

C17H11F6N7O

443.31

443.092

C, 46.06; H, 2.50; F, 25.71; N, 22.12; O, 3.61

1393477-72-9

1393477-72-9; 1421923-86-5 (E-isomer); 1621865-82-4 (Z-isomer)

71481097

White to light yellow solid powder

1.6±0.1 g/cm3

1.594

3.62

2

12

5

31

621

0

C1=CN=C(C=N1)NNC(=O)/C=C\N2C=NC(=N2)C3=CC(=CC(=C3)C(F)(F)F)C(F)(F)F

DEVSOMFAQLZNKR-RJRFIUFISA-N

InChI=1S/C17H11F6N7O/c18-16(19,20)11-5-10(6-12(7-11)17(21,22)23)15-26-9-30(29-15)4-1-14(31)28-27-13-8-24-2-3-25-13/h1-9H,(H,25,27)(H,28,31)/b4-1-

(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyrazin-2-yl)acrylohydrazide

KPT-330; KPT 330; 1393477-72-9; Xpovio; Selinexor (KPT-330); KPT 330; (Z)-3-(3-(3,5-Bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyrazin-2-yl)acrylohydrazide; Selinexor free base; KPT330

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 (5.64 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 2: ≥ 2.08 mg/mL (4.69 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.

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Solubility in Formulation 3: 2.08 mg/mL (4.69 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 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.

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Solubility in Formulation 4: 2% DMSO +49% PEG 300 +dd H2O: 5mg/mL

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