γ-secretase (IC50 = 6.2 nM)

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]

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]

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.

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.

Mice: Female athymic mice (nu/nu, 6-8 weeks) are employed. Animals with 150 to 300 mm³ 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 (mm³). 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.

Absorption, Distribution and Excretion
The following pharmacokinetics parameters in patients with desmoid tumors were calculated as follows: Cmax (508 (62) ng/mL), AUC0-tau (u 3370 (58) ng·h/mL), time to steady state (6 days), and Tmax (1.5 (0.5, 6.5) hours).
Nirogacestat is excreted mostly in feces (38%) and urine 17%, with less than 1% of the unchanged drug remains in the urine. It can also be eliminated through expired air (9.7%).
The apparent volume of distribution [Mean (%CV)] of nirogacestat is 1430 (65) L.
The apparent systemic clearance [Mean (%CV)] of nirogacestat is 45 (58) L/hr.

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Metabolism / Metabolites
Nirogacestat is expected to be metabolized primarily through the N-dealkylation via CYP3A4 (85%), with the involvement of CYP3A4, CYP2C19, CYP2C9, and CYP2D6 in a minor secondary pathway.

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

Protein Binding
Nirogacestat has a high level of serum protein binding of 99.6%, with 94.6% to serum albumin and 97.9% to alpha-1 acid glycoprotein.

[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 is a member of the class of imidazoles that is 1H-imidazole substituted by a 1-[(2,2-dimethylpropyl)amino]-2-methylpropan-2-yl group at position 1 and a {N-[(2S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]-L-norvalyl}amino group at position 4. It is a gamma-secretase inhibitor whose hydrobromide salt is indicated for adult patients with progressing desmoid tumours who require systemic treatment. It has a role as an antineoplastic agent and a gamma-secretase modulator. It is a member of tetralins, an organofluorine compound, a secondary carboxamide, a secondary amino compound and a member of imidazoles. It is a conjugate base of a nirogacestat(2+).
Nirogacestat is a small-molecule gamma-secretase inhibitor that was investigated as a potential treatment for desmoid tumors. Desmoid tumors are typically characterized by aberrant activation in Notch signaling. Interaction between the notch receptors and their ligands activates proteolytic cleavage by gamma-secretase; therefore, the inhibition of gamma-secretase can potentially inhibit Notch signaling and thus impede the growth of desmoid tumors. Nirogacestat was approved under the brand name OGSIVEO on November 27, 2023, by the FDA for the treatment of adult patients with progressing desmoid tumors who require systemic treatment. It was previously granted breakthrough therapy, fast track, and orphan drug designations for the treatment of desmoid tumors, and the final approval was based on positive results obtained in the Phase 3 DeFi trial, where a confirmed objective response rate was observed to be 41% compared to 8% with the placebo.
Nirogacestat is a selective gamma secretase (GS) inhibitor with antitumor activity. Upon administration, nirogacestat targets and binds to GS, thereby blocking the proteolytic activation of Notch receptors. This inhibits the Notch signaling pathway and results in the induction of apoptosis in tumor cells that overexpress Notch. The integral membrane protein GS is a multi-subunit protease complex that cleaves single-pass transmembrane proteins, such as Notch receptors, at residues within their transmembrane domains. Overexpression of the Notch signaling pathway has been correlated with increased tumor cell growth and survival.

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Drug Indication
Nirogacestat is indicated for adult patients with progressing desmoid tumors who require systemic treatment.

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Mechanism of Action
Nirogacestat is a gamma secretase inhibitor that blocks proteolytic activation of the Notch receptor. When dysregulated, Notch can activate pathways that contribute to tumor growth.

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Pharmacodynamics
There is an exposure-response relationship between nirogacestat exposure and Grade 3 hypophosphatemia with a higher risk of Grade 3 hypophosphatemia at higher exposure. At the recommended dosage, a mean increase in the QTc interval > 20 ms was not observed.

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

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.)