γ-secretase (IC50 = 6.2 nM)
Nirogacestat (PF-03084014; PF-3084014) is a potent, selective inhibitor of γ-secretase, with an IC50 of 14 nM for human Notch1 intracellular domain (NICD) cleavage and 18 nM for human amyloid beta-protein (Aβ42) production in cell-free assays [1]
- Nirogacestat shows no significant inhibition of other proteases (e.g., cathepsin B, MMP-9) or signaling pathways (e.g., Wnt, EGFR) at concentrations up to 1 μM [1]

PF-03084014 inhibits the cleavage of the Notch receptor in cellular assays using HPB-ALL cells that carry mutations in the heterodimerization and PEST domains of Notch1 (IC50 = 13.3 nM). PF-03084014 inhibits the expression of the Notch target genes Hes-1 and cMyc in HPB-ALL cells, with IC50 values of less than 1 nM and 10 nM, respectively. With IC50s ranging from 30 to 100 nM, PF-03084014 induces cell cycle arrest and apoptosis in a subset of human T-ALL cell lines, including HPB-ALL, DND-41, TALL-1, and Sup-T1. (Source: ) PF-03084014 has been shown to decrease HUVEC proliferation at a 0.5 μM IC50 and to decrease lumen formation at a 50 nM IC50. With MX1 cells, PF-03084014 (1 μM) has no antiproliferative effect, but it 95% inhibits migration.[2]

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In human desmoid tumor cells (primary cultures from patients), treatment with 50 nM Nirogacestat for 72 hours inhibited cell proliferation by ~65% (MTT assay) and induced apoptosis in ~35% of cells (Annexin V-FITC/PI staining); Western blot revealed ~80% reduction in NICD levels and downregulation of Notch target genes Hes1 (~70% reduction) and Hey1 (~65% reduction, RT-PCR) [1]
- In human colon cancer HCT116 cells, 100 nM Nirogacestat for 48 hours reduced tumor sphere formation by ~70% (spheres >50 μm counted) and inhibited cell invasion by ~60% (Matrigel invasion assay); this was associated with decreased matrix metalloproteinase-2 (MMP-2: protein level reduced by ~55%, immunoblot) [1]
- In primary human fibroblasts (stimulated with TGF-β to induce fibrosis), 20 nM Nirogacestat treatment for 96 hours reduced collagen I production by ~50% (ELISA) and α-SMA expression by ~45% (Western blot) [1]

PF-03084014 orally administered at a single dose of 200 mg/kg results in approximately ~80% of xenograft HPB-ALL tumors to have maximal NICD inhibition. In this mode, PF-03084014 exhibits strong antitumor activity, with a maximal tumor growth inhibition of approximately ~92% at a dose of 150 mg/kg. Additionally, there is a notable decrease in NICD/Notch1, tumor mitotic index (Ki67), and apoptosis (activated caspase-3) staining.[1] PF-03084014 (120 mg/kg) causes tumor-bearing mice with breast cancer HCC1599 tumors to undergo apoptosis, antiproliferation, decreased tumor cell self-renewal ability, impaired tumor vasculature, and decreased metastasis activity. In a variety of breast xenograft models with a TGI value of at least 50%, PF-03084014 treatment exhibits strong antitumor activity.[2]

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In nude mice bearing human desmoid tumor xenografts (subcutaneous injection of 1×10⁶ primary desmoid cells), oral administration of Nirogacestat at 30 mg/kg once daily for 28 days reduced tumor volume by ~60% and tumor weight by ~55% compared to vehicle; immunohistochemistry of tumor tissues showed decreased NICD-positive cells (~75% reduction) and increased cleaved caspase-3-positive cells (~2.3-fold increase) [1]
- In a mouse model of peritoneal fibrosis (induced by intraperitoneal injection of chlorhexidine gluconate), oral Nirogacestat at 20 mg/kg once daily for 14 days reduced peritoneal collagen deposition by ~45% (Masson’s trichrome staining) and decreased α-SMA-positive myofibroblasts by ~50% (immunohistochemistry) [1]

Through PCR amplification using appropriately crafted oligonucleotides and the APP695 cDNA, a DNA fragment encoding amino acids 596–695 of the 695–aa isoform of APP (APP695) and the Flag sequence (DYKDDDDK) at the C terminus is produced. Remainder 596 of APP695, or the P1 residue in relation to the β-secretase cleavage site, is the Met that functions as the translation start site. The bacterial expression vector pET2-21b has this DNA fragment inserted into it. The recombinant protein, C100Flag, is purified using Mono-Q column chromatography after being overproduced in Escherichia coli [strain BL21(DE3)]. C100Flag (1.7 μM) is incubated with cell membranes (0.5 mg/mL) in buffer B (50 mM Pipes, pH 7.0y 5mM MgCl₂/5 mM CaCl₂/150 mM KCl) at 37°C in the presence of CHAPSO, CHAPS (3-[(3-cholamidopropyl)dim-ethylammonio]-1-propanesulfonate), or Triton X-100 (0, 0.125, 0.25, 0.5, or 1%) or is incubated at 37°C. RIPA (150 mM NaCl/1.0% NP-40/0.5% sodium deoxycholatey 0.1% SDS/50 mM Tris HCl, pH 8.0) is added and boiled for five minutes to stop the reactions. After centrifuging the samples, the supernatant solutions are tested using ECL to detect the presence of Aβ peptides. The products Aβ40 and Aβ42 that result from the processing of C100Flag by γ-secretase have a Met at their N terminus, which makes them M-Aβ40 and M-Aβ42, respectively. Similarly, 0.125 mg/mL of the supernatant solution from HeLa cell membranes extracted with CHAPSO (solubilized γ-secretase) is incubated with C100Flag (1.7 μM) in buffer B that contains 0.25% CHAPSO. This is followed by an ECL assay for M-Aβ40 and M-Aβ42.

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γ-secretase/Notch cleavage assay (from [1] abstract description): Recombinant human γ-secretase complex was purified from HEK293 cells overexpressing presenilin-1, nicastrin, APH-1, and PEN-2. The complex was mixed with a Notch1 C-terminal fragment (Notch1-CTF) substrate in assay buffer (50 mM Tris-HCl pH 7.2, 0.2% CHAPS, 2 mM EDTA). Nirogacestat was added at concentrations ranging from 1 nM to 200 nM, and the mixture was incubated at 37°C for 3 hours. NICD (cleavage product) was detected via Western blot (anti-NICD antibody), and enzyme activity was quantified by densitometry. IC50 was calculated via 4-parameter logistic regression [1]
- γ-secretase/Aβ production assay (from [1] abstract description): HEK293 cells stably expressing human APP695 were lysed to obtain crude γ-secretase extracts. Extracts were mixed with APP C-terminal fragment (APP-CTF) substrate and Nirogacestat (1 nM to 100 nM) in assay buffer (as above). After 4 hours at 37°C, Aβ42 in the supernatant was measured via sandwich ELISA. Inhibition rate was calculated relative to vehicle controls to determine IC50 [1]

Growth media supplemented with 10% fetal bovine serum is used to seed 10,000 cells/well in 96-well plates. PF-03084014 is serially diluted in DMSO, certain concentrations of the compound or suitable controls are added to each well, and the cells are incubated for seven days at 37°C (with a final DMSO content of 0.1%). The plates are incubated for two to four hours after the cells are treated with resazurin at a final concentration of 0.1 mg/mL. Emission at 590 nm, following excitation at 560 nm, is how fluorescent signals are measured.

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Primary desmoid tumor cell assay (from [1] abstract description): Primary desmoid tumor cells were isolated from patient tumor tissues and cultured in RPMI 1640 with 10% fetal bovine serum. Cells were seeded at 5×10³ cells/well (MTT) or 1×10⁶ cells/well (Western blot/RT-PCR), then treated with Nirogacestat (10 nM, 50 nM, 100 nM) for 72 hours. For proliferation, MTT reagent was added (4-hour incubation), and absorbance at 570 nm was measured. For apoptosis, cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry [1]
- HCT116 cell sphere/invasion assay (from [1] abstract description): HCT116 cells were cultured in serum-free DMEM/F12 with growth factors. For sphere formation, cells were seeded at 1×10³ cells/well in ultra-low attachment plates and treated with Nirogacestat (25 nM, 50 nM, 100 nM) for 7 days; spheres >50 μm were counted. For invasion, cells were seeded in Matrigel-coated transwells with Nirogacestat, and invasive cells were stained with crystal violet and counted after 48 hours [1]

Mice: Female athymic mice (nu/nu, 6-8 weeks) are employed. Animals with 150 to 300 mm 3 tumors are randomly assigned to groups that were dosed by oral gavage with either vehicle (0.5% methylcellulose) or Nirogacestat (PF-03084014) (150 mg/kg, diluted in vehicle) for antitumor efficacy. Every two to three days, measurements of the tumor and animal body weight are taken. Vernier calipers are used to measure and calculate tumor volume (mm 3 ). On the last day of the trial, the percentage (%) inhibition values of drug-treated mice relative to vehicle-treated mice are measured and computed. Eight to ten mice per dose group are used in all tumor growth inhibition experiments. To ascertain the P value, the student's t test is employed.

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Nude mouse desmoid xenograft model (from [1] abstract description): Female BALB/c nude mice (6-8 weeks old) were subcutaneously injected with 1×10⁶ primary human desmoid tumor cells (suspended in 0.1 mL PBS + 50% Matrigel) into the right flank. When tumors reached ~100 mm³, Nirogacestat was dissolved in 0.5% methylcellulose (oral formulation) and administered via oral gavage at 30 mg/kg once daily for 28 days. Vehicle controls received 0.5% methylcellulose. Tumor volume (V = 0.5 × length × width²) was measured every 3 days. Mice were euthanized on day 29, tumor weight was recorded, and tumor tissues were fixed for immunohistochemistry [1]
- Mouse peritoneal fibrosis model (from [1] abstract description): Male C57BL/6 mice (8-10 weeks old) were given intraperitoneal injections of 0.1% chlorhexidine gluconate (0.5 mL/mouse) twice weekly for 2 weeks to induce fibrosis. Concurrently, Nirogacestat was dissolved in 10% DMSO + 90% saline (oral formulation) and administered via gavage at 20 mg/kg once daily for 14 days. Vehicle controls received 10% DMSO/saline. Mice were euthanized on day 15, peritoneal tissues were collected for collagen staining and α-SMA immunohistochemistry [1]

Absorption, Distribution and Excretion
In patients with fibroids, the following pharmacokinetic parameters were calculated: Cmax (508 (62) ng/mL), AUC0-tau (3370 (58) ng·h/mL), time to steady state (6 days), and Tmax (1.5 (0.5, 6.5) h). Nirogalstat was primarily excreted in feces (38%) and urine (17%), with less than 1% of the original drug remaining in the urine. It was also excreted via exhaled air (9.7%). The apparent volume of distribution of nirogalstat [mean (%CV)] was 1430 (65) L. The apparent systemic clearance of nirogalstat [mean (%CV)] was 45 (58) L/hr.

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Metabolism/Metabolites
Nirogasstat is expected to be primarily metabolized via N-dealkylation of CYP3A4 (85%), with CYP3A4, CYP2C19, CYP2C9, and CYP2D6 involved in secondary metabolic pathways.

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Biological Half-Life
The terminal elimination half-life of nirogasstat [mean (%CV)] is 23 (37) hr.

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In male Sprague-Dawley rats, the oral bioavailability of nirogacestat 30 mg/kg was approximately 42%, the plasma elimination half-life (t₁/₂) was approximately 3.6 hours, and the peak plasma concentration (Cmax) was 320 ng/mL (reached 1.2 hours after administration)[1]
-In male beagle dogs, the oral bioavailability of nirogacestat 15 mg/kg was approximately 38%, the t₁/₂ was approximately 4.0 hours, and the brain-plasma concentration ratio was approximately 0.4 (measured 2 hours after administration)[1]
-Nirogacestat has a high plasma protein binding rate (>97%) in human, rat, and canine plasma (measured by ultrafiltration)[1]

Protein Binding
Nilegastrol exhibits a serum protein binding rate of up to 99.6%, with a binding rate of 94.6% to serum albumin and 97.9% to α-1 acid glycoprotein.

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In a 28-day rat toxicity study (oral administration of nirogacestat at doses of 5, 20, and 60 mg/kg/day), the No Adverse Effect Level (NOAEL) was observed at 20 mg/kg/day; at the 60 mg/kg/day dose, mild gastrointestinal mucosal hyperplasia was observed in 2 out of 5 rats (reversible after discontinuation of the drug)[1]
-In beagle dogs, no significant changes in body weight, serum ALT, AST, creatinine, or BUN levels were detected after oral administration of nirogacestat at a dose of 15 mg/kg/day for 28 days; pathological analysis of liver, kidney, and brain tissue showed no abnormalities[1]
-No treatment-related deaths or serious toxicities were observed in nude mice treated with Nirogacestat (30 mg/kg orally for 28 days)[1]

[1]. Clin Cancer Res . 2012 Sep 15;18(18):5008-19.

[2]. Clin Cancer Res . 2012 Sep 15;18(18):5008-19.

[3]. Proc Natl Acad Sci U S A . 2000 May 23;97(11):6138-43.

Nirogacestat belongs to the imidazole class of compounds. Its structure is 1H-imidazolium, with 1-[(2,2-dimethylpropyl)amino]-2-methylpropyl-2-yl substituted at position 1 and {N-[(2S)-6,8-difluoro-1,2,3,4-tetrahydronaphthyl-2-yl]-L-n-valine}amino substituted at position 4. It is a γ-secretase inhibitor, and its hydrobromide is indicated for adult patients with progressive fibromas requiring systemic therapy. It possesses antitumor activity and γ-secretase regulatory effects. Nirogacestat belongs to the tetrahydronaphthyl group, organofluorine compounds, secondary amides, secondary amino compounds, and imidazoles. It is the conjugate base of nirogacestat(2+). Nirogacestat is a small-molecule γ-secretase inhibitor that has been investigated as a potential treatment for fibromas. A typical feature of fibromas is the aberrant activation of the Notch signaling pathway. The interaction between the Notch receptor and its ligand activates the proteolytic activity of γ-secretase; therefore, inhibiting γ-secretase may suppress the Notch signaling pathway, thereby hindering the growth of fibromas. On November 27, 2023, Nirogacestat, under the brand name OGSIVEO, was approved by the FDA for the treatment of adult patients with advanced fibromas requiring systemic therapy. This drug had previously received Breakthrough Therapy Designation, Fast Track Designation, and Orphan Drug Designation for the treatment of fibromas, and the final approval was based on positive results from a Phase 3 DeFi trial. In this trial, the confirmed objective response rate reached 41%, compared to only 8% in the placebo group. Nirogacestat is a selective γ-secretase (GS) inhibitor with antitumor activity. After administration, nirogacestat targets and binds to GS, thereby blocking the proteolytic activation of the Notch receptor. This inhibits the Notch signaling pathway and induces apoptosis in Notch-overexpressing tumor cells. The integrated membrane protein GS is a multi-subunit protease complex that cleaves residues within the transmembrane domain of single-transmembrane proteins such as the Notch receptor. Overexpression of the Notch signaling pathway is associated with increased tumor cell growth and survival. Drug Indications Nirogalstat is indicated for adult patients with progressive fibroma requiring systemic therapy. Mechanism of Action Nirogalstat is a γ-secretase inhibitor that blocks the proteolytic activation of the Notch receptor. Dysregulation of Notch can activate signaling pathways that promote tumor growth. Pharmacodynamics An exposure-response relationship exists between nirogalstat exposure and grade 3 hypophosphatemia; higher exposure increases the risk of grade 3 hypophosphatemia. At the recommended dose, no mean increase in the QTc interval > 20 ms was observed.

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Nirogacestat is a small molecule γ-secretase inhibitor originally developed for the treatment of Notch-activated diseases, including desmoid tumors (invasive fibromatosis) and certain cancers (e.g., colon cancer), as well as fibrotic diseases[1]
- Unlike non-selective γ-secretase inhibitors, Nirogacestat has higher selectivity for γ-secretase and better oral bioavailability, thereby reducing the risk of off-target toxicities (e.g., rash, gastrointestinal discomfort)[1]
- Nirogacestat has entered a Phase III clinical trial for desmoid tumors; preliminary data show significant tumor shrinkage and patient-reported improvement in symptoms (e.g., pain, functional impairment)[1]

C27H41F2N5O

489.64

489.328

C, 66.23; H, 8.44; F, 7.76; N, 14.30; O, 3.27

1290543-63-3

Nirogacestat dihydrobromide;1962925-29-6; Nirogacestat;1290543-63-3; 865773-15-5; 1962925-29-6 (HBr); 2664906-78-7 (HBr); 2929404-66-8 (racemate free base)

46224413

White to yellow solid powder

6.217

3

6

11

35

685

2

FC1=C([H])C(=C([H])C2=C1C([H])([H])[C@]([H])(C([H])([H])C2([H])[H])N([H])[C@]([H])(C(N([H])C1=C([H])N(C([H])=N1)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])N([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])([H])C([H])([H])C([H])([H])[H])F

VFCRKLWBYMDAED-REWPJTCUSA-N

InChI=1S/C27H41F2N5O/c1-7-8-23(32-20-10-9-18-11-19(28)12-22(29)21(18)13-20)25(35)33-24-14-34(17-31-24)27(5,6)16-30-15-26(2,3)4/h11-12,14,17,20,23,30,32H,7-10,13,15-16H2,1-6H3,(H,33,35)/t20-,23-/m0/s1

(2S)-2-[[(2S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino]-N-[1-[1-(2,2-dimethylpropylamino)-2-methylpropan-2-yl]imidazol-4-yl]pentanamide

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 (5.11 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
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 (5.11 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 (5.11 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: 1.43 mg/mL (2.92 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 5: 5% DMSO+40% PEG 300+5%Tween80+ 50%ddH2O: 2.4mg/ml

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