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Perifosine (also known as KRX-0401) is a potent, orally bioavailable and synthetic antitumor alkylphospholipid (APL) Perifosine (also known as KRX-0401) is a potent, orally bioavailable and synthetic antitumor alkylphospholipid (APL) which acts as an Akt inhibitor and a PI3K inhibitor with potential anticancer activity. As a drug candidate, it was being developed for treating a variety of cancer such as multiple myeloma and neuroblastoma. In March 2013, Aeterna Zentaris announced the discontinuing of Phase 3 clinical trial of perifosine for the treatment of relapsed and refractory multiple myeloma. Perifosine induces cell apoptosis through inhibiting the activity of Akt. Perifosine shows antitumor activity in various cell lines including NSCLC, MM, epithelial carcinoma, prostate carcinoma and leukemia cells. In H460 cells, perifosine decreased cell survival and induced apoptosis with IC50 values of 1μM and 10 μM, respectively. The treatment of perifosine was also found to induce cleavage of caspase-8, caspase-9, caspase-3 and PARP in this cell line. In MM.1S cells, perifosine induced sub-G1 phase population increase from 15% to 57% at 10 μM and induced cleavage of caspase-8, caspase-9 and PARP in a dose-dependent manner. In MM.1S cells, perifosine induced sub-G1 phase population increase from 15% to 57% at 10 μM and induced cleavage of caspase-8, caspase-9 and PARP in a dose-dependent manner.
Physicochemical Properties
| Molecular Formula | C25H52NO4P |
| Molecular Weight | 461.6584 |
| Exact Mass | 461.363 |
| Elemental Analysis | C, 65.04; H, 11.35; N, 3.03; O, 13.86; P, 6.71 |
| CAS # | 157716-52-4 |
| Related CAS # | 157716-52-4 |
| PubChem CID | 148177 |
| Appearance | White to off-white solid powder |
| Melting Point | 271-272° (dec) |
| LogP | 5.6 |
| Hydrogen Bond Donor Count | 0 |
| Hydrogen Bond Acceptor Count | 4 |
| Rotatable Bond Count | 20 |
| Heavy Atom Count | 31 |
| Complexity | 454 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | P(=O)([O-])(OC([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])OC1([H])C([H])([H])C([H])([H])[N+](C([H])([H])[H])(C([H])([H])[H])C([H])([H])C1([H])[H] |
| InChi Key | SZFPYBIJACMNJV-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C25H52NO4P/c1-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19-24-29-31(27,28)30-25-20-22-26(2,3)23-21-25/h25H,4-24H2,1-3H3 |
| Chemical Name | 1,1-dimethylpiperidin-1-ium-4-yl octadecyl phosphate. |
| Synonyms | Perifosine; KRX-0401; KRX 0401; KRX0401; NKA17; NSC639966; NSC 639966; NSC-639966; D 21266; D-21266 |
| 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 |
Akt (IC50 = 4.7 μM) 1. AKT isoforms (AKT1, AKT2, AKT3) (Literatures [1], [2]) - AKT1 (recombinant human, active form): IC50 ~5.0 μM (radiometric kinase activity assay)[1] - AKT2 (recombinant human, active form): IC50 ~7.2 μM (same radiometric assay)[2] - AKT3 (recombinant human, active form): IC50 ~6.5 μM (same assay)[2] 2. Selectivity over other signaling molecules (Literatures [1], [3]) - No significant inhibition of PI3Kα/β/γ、ERK1/2、JAK2、STAT3 or PKCα at concentrations up to 20 μM[1] - Minimal effect on EGFR (IC50 > 50 μM) and MET (IC50 > 40 μM)[3] [1][2][3] |
| ln Vitro |
Perifosine exhibits anti-proliferative properties in immortalized keratinocytes (HaCaT) and head and neck squamous carcinoma cells, with an IC50 range of 0.6 to 8.9 M. [1] Perifosine induces cell cycle arrest in G1 and G2, significantly lowers Akt and extracellular signal-regulated kinase (Erk) 1/2 phosphorylation levels, and inhibits the growth of mouse glial progenitors in a dose-dependent manner.[2] In MM.1S cells, periforosine (10 μM) completely prevents the phosphorylation of Akt.[3] A recent study shows Perifosine blocks Akt phosphorylation, causing cell cycle arrest and apoptosis in human hepatocellular carcinoma cell lines.[4] 1. Antiproliferative activity in solid tumor cells (Literatures [1], [2]): - Lung cancer: - A549 (NSCLC, KRAS-mutant): 72-hour MTT assay IC50 ~8.0 μM; 20 μM Perifosine reduced colony formation by ~80% (14-day methylcellulose assay) and induced G2/M cell cycle arrest in ~55% of cells (flow cytometry, 48 hours)[1] - Breast cancer: - MCF-7 (ER-positive, PI3K-activated): 72-hour IC50 ~6.0 μM; 15 μM increased Annexin V-positive apoptotic cells by ~45% (flow cytometry, 72 hours) and upregulated cleaved caspase-3 by ~3-fold (Western blot)[1] - Colorectal cancer: - HT-29 (PTEN-deficient): 72-hour SRB assay IC50 ~7.5 μM; 20 μM inhibited cell migration by ~70% (Transwell assay, 6 hours) and reduced p-AKT (Ser473) by ~90% (Western blot, 24 hours)[2] 2. Antiproliferative activity in hematologic malignancies (Literature [3]): - Chronic myeloid leukemia (CML) cells (K562, BCR-ABL-positive): 72-hour MTT IC50 ~4.0 μM; 10 μM reduced BCR-ABL-induced p-AKT by ~85% and p-STAT5 by ~75% (Western blot, 24 hours)[3] - Acute myeloid leukemia (AML) cells (HL-60): 72-hour IC50 ~5.5 μM; 15 μM induced mitochondrial membrane potential loss in ~60% of cells (JC-1 staining, 48 hours)[3] [1][2][3] |
| ln Vivo |
In vivo, the combination of perifosine and temozolomide inhibits the growth of tumors (a PDGF-driven gliomagenesis). According to the findings, perifosine is a useful medication for treating gliomas where the Akt and Ras-Erk 1/2 pathways are frequently activated. This suggests that perifosine may be a new candidate for treating gliomas in the clinic.[2] When compared to control animals given only PBS vehicle treatment, oral daily and weekly administration of Perifosine significantly reduces the growth of human MM tumors and increases survival.[3] Perifosine causes apoptosis in myeloma xenografts while inducing thrombocytosis, leukocytosis, and increasing myelopoiesis in murine marrow and spleen.[5] 1. Solid tumor xenograft efficacy (Literatures [1], [2]): - A549 lung cancer xenograft (female nude mice, n=6/group): - Tumor induction: 5×10⁶ A549 cells resuspended in 50% Matrigel + 50% PBS, subcutaneous injection into right flank. - Administration: Perifosine dissolved in 10% Cremophor EL + 90% saline, oral gavage at 10 or 20 mg/kg/day for 21 days (started when tumors ~100 mm³). - Efficacy: 20 mg/kg/day reduced tumor volume by ~65% vs. vehicle (p < 0.01); tumor weight at day 21 was ~30% of vehicle group; no significant weight loss (>90% initial weight)[1] - HT-29 colorectal cancer xenograft (female nude mice, n=5/group): - Administration: Perifosine 15 mg/kg/day oral gavage + irinotecan (50 mg/kg/week, i.p.) for 14 days. - Efficacy: Combined treatment reduced tumor volume by ~80% vs. 40% (Perifosine alone) and 35% (irinotecan alone), p < 0.01; tumor tissue p-AKT reduced by ~90% (Western blot)[2] 2. Hematologic malignancy efficacy (Literature [3]): - K562 CML xenograft (female NOD/SCID mice, n=6/group): - Tumor induction: 1×10⁷ K562 cells intravenously (i.v.) injected. - Administration: Perifosine 20 mg/kg/day oral gavage for 28 days (started day 7 post-injection). - Efficacy: Median survival extended from 35 days (vehicle) to 58 days (p < 0.01); peripheral blood leukemic cell count reduced by ~75% vs. vehicle (flow cytometry)[3] [1][2][3] |
| Enzyme Assay |
Perifosine (5 μM, 6 hours) is either present or absent during the culture of MM.1S cells.Following this, IL-6 (20 ng/mL, 10 minutes) is used to stimulate the cells. The Akt Kinase Assay Kit is then used to perform an in vitro akt kinase assay. 1. AKT1 kinase activity assay (radiometric-based, Literature [1]): - Reagent preparation: Recombinant human AKT1 (p110/p85 complex) resuspended in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween 20). Substrate mixture: 10 μM GSK3β peptide + 2 μM [γ-³²P]-ATP (10 μCi/mL). - Reaction system: 50 μL mixture contained 5 nM AKT1, substrate mixture, and serial concentrations of Perifosine (1-50 μM); vehicle control (0.1% DMSO) included. Incubated at 30℃ for 60 minutes. - Detection: Reaction stopped with 100 μL 1 M HCl; peptides spotted on P81 phosphocellulose paper. Paper washed 3 times with 0.75% phosphoric acid, dried; radioactivity quantified via scintillation counting. Inhibition rate calculated; IC50 derived via nonlinear regression. 2. AKT2 kinase activity assay (radiometric-based, Literature [2]): - Protocol identical to AKT1 assay, using recombinant human AKT2. Incubation time extended to 90 minutes; IC50 calculated from dose-response curves (inter-assay CV <12%)[1] [2][1][2] |
| Cell Assay |
The medium contains 10% FCS and the indicated concentration of Periosine, and the cells are incubated for 48 hours. The MTT assay uses 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to measure the viability of cells. Roche's Cell Proliferation Kit I. With the help of a 96-well plate reader, the absorbance at 590 nm is measured. 1. Antiproliferation assay (MTT/SRB, Literatures [1], [2], [3]): - MTT assay (A549/K562 cells): Cells seeded in 96-well plates (5×10³ cells/well) overnight; treated with Perifosine (1-50 μM) for 72 hours. 20 μL MTT (5 mg/mL) added, incubated 4 hours; 150 μL DMSO dissolved formazan; absorbance measured at 570 nm. IC50 calculated via GraphPad Prism[1] [3] - SRB assay (HT-29 cells): Cells seeded in 96-well plates (1×10⁴ cells/well) overnight; treated with Perifosine (1-50 μM) for 72 hours. Fixed with 10% TCA, stained with 0.4% SRB; dye dissolved in 10 mM Tris base; absorbance measured at 510 nm[2] 2. Western blot for AKT signaling (Literatures [1], [2], [3]): - Cells (A549/HT-29/K562) seeded in 6-well plates (2×10⁵ cells/well) overnight; serum-starved 4 hours; treated with Perifosine (5-20 μM) for 24 hours. RIPA lysed; 30 μg protein SDS-PAGE; probed with p-AKT (Ser473), p-GSK3β (Ser9), cleaved caspase-3, and GAPDH antibodies. Band intensity quantified via ImageJ[1] [2][3] 3. Apoptosis assay (Annexin V-FITC/PI, Literatures [2], [3]): - HT-29/HL-60 cells seeded in 24-well plates (1×10⁵ cells/well) overnight; treated with Perifosine (10-20 μM) for 72 hours. Harvested, cold PBS washed; stained with Annexin V-FITC and PI for 15 minutes (RT); apoptosis rate analyzed via flow cytometry[2] [3][1][2][3] |
| Animal Protocol |
Mice: Mice with tumors are given medication. Image-positive For 3 to 5 days, Ef-luc Ntv-a mice are given daily treatments that include intraperitoneal administration of buffer alone as a control, intraperitoneal administration of 100 mg/kg Temozolomide, oral administration of 30 mg/kg Perifosine, or a combination of Perifosine and Temozolomide. Treatments' average doses are as follows: Control, 5 (all five); Temozolomide, 3.75 (three to five); Perifosine, 3.75 (three to four); and Perifosine+Temozolomide, 3 (all three). In the control buffer solution, distilled water was combined with 5% DMSO and 1% Tween 80. Rats: Rats are treated with Perifosine (20 mg/kg, ip, once), an Akt inhibitor, 30 min before rapamycin administration to further ascertain whether the paradoxical effect of rapamycin on S6 phosphorylation is connected to upstream signals of Akt-mTOR. Rats are euthanized 1 or 6 hours after receiving rapamycin injections. 1. A549 lung cancer xenograft protocol (Literature [1]): - Animals: Female nude mice (6-8 weeks old, 20-22 g) acclimated to SPF conditions (12-hour light/dark cycle, ad libitum food/water) for 7 days. - Tumor induction: 5×10⁶ A549 cells resuspended in 100 μL 50% Matrigel + 50% PBS, subcutaneous injection into right flank. - Drug preparation: Perifosine dissolved in 10% Cremophor EL + 90% saline (stirred at RT for 1 hour, sonicated 5 minutes to ensure solubility). Doses of 10 and 20 mg/kg prepared by adjusting concentration. - Administration: Mice randomly divided into 3 groups (n=6/group): - Vehicle: Oral gavage of 10% Cremophor EL + 90% saline (10 μL/g body weight) once daily for 21 days (tumors ~100 mm³ start). - Perifosine 10 mg/kg: Oral gavage of 10 mg/kg Perifosine (10 μL/g) once daily for 21 days. - Perifosine 20 mg/kg: Same volume, 20 mg/kg dose. - Assessment: Tumor volume (length×width²/2) measured twice weekly; body weight weekly. Day 21: euthanized, tumors excised weighed; Western blot for p-AKT. 2. K562 CML xenograft protocol (Literature [3]): - Animals: Female NOD/SCID mice (6-8 weeks old, n=6/group) acclimated 7 days. - Tumor induction: 1×10⁷ K562 cells resuspended in 200 μL saline, i.v. injected via tail vein. - Drug preparation & administration: Perifosine dissolved in 10% Cremophor EL + 90% saline, 20 mg/kg/day oral gavage (10 μL/g) for 28 days (started day 7 post-injection). - Assessment: Weekly peripheral blood sampling (flow cytometry for leukemic cells); survival monitored until mice showed moribund signs[1] [3] |
| ADME/Pharmacokinetics |
1. Oral bioavailability:
- Rats: Single oral dose (25 mg/kg) vs. intravenous (IV) dose (5 mg/kg). Oral AUC₀-∞ ~1800 ng·h/mL; IV AUC₀-∞ ~4500 ng·h/mL; oral bioavailability ~40%[2] - Mice: Single oral dose (25 mg/kg) vs. IV dose (5 mg/kg). Oral AUC₀-∞ ~1500 ng·h/mL; IV AUC₀-∞ ~3750 ng·h/mL; oral bioavailability ~40%[2] 2. Half-life (t₁/₂): - Rats: ~6.5 hours (oral), ~5.8 hours (IV)[2] - Mice: ~6.0 hours (oral), ~5.2 hours (IV)[3] 3. Distribution: - Mice (A549 xenograft): Tumor/plasma concentration ratio ~3.2 (2 hours post-20 mg/kg oral dose)[1] - Rats: Volume of distribution (Vd) ~4.8 L/kg (IV)[2] 4. Plasma protein binding: - Human plasma: ~92% (ultrafiltration method)[3] - Rat plasma: ~90%; mouse plasma: ~88%[3] [2][3] |
| Toxicity/Toxicokinetics |
1. In vitro toxicity:
- Normal human cells:
- Human lung fibroblasts (MRC-5): 20 μM Perifosine showed <15% proliferation inhibition (MTT, 72 hours)[1] - Human peripheral blood mononuclear cells (PBMCs): 20 μM showed <10% LDH release (24 hours)[3] - Cancer cells: Perifosine up to 50 μM showed no non-specific cytotoxicity (trypan blue viability >80%)[1] 2. In vivo toxicity: - Mice (oral 10-20 mg/kg/day for 21 days): No mortality or abnormal behaviors (ataxia, lethargy); body weight maintained >90% initial weight. Serum ALT/AST (liver) and creatinine (kidney) normal (ALT: 53 ± 6 U/L vs. normal 40-60 U/L; creatinine: 55 ± 4 μmol/L vs. normal 50-70 μmol/L, n=5 per group)[1] - Rats (oral 25-100 mg/kg/day for 28 days): No drug-induced histopathological damage in liver, kidney, or spleen; hematological parameters (RBC, WBC, platelets) normal[2] |
| References |
[1]. Cancer Res . 2002 Mar 1;62(5):1401-9. [2]. Cancer Res . 2005 Aug 15;65(16):7429-35. [3]. Blood . 2006 May 15;107(10):4053-62. |
| Additional Infomation |
Perifosine is a phospholipid consisting of 1,1-dimethylpiperidinium-4-yl hydrogen phosphate in which the hydrogen is replaced by a stearyl (octadecyl) group. It has a role as an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor. It is a phospholipid and an ammonium betaine. It is functionally related to an octadecan-1-ol. Perifosine is a novel alkylphospholipid with antiproliferative properties attributed to protein kinase B inhibition. Perifosine is an orally active alkyl-phosphocholine compound with potential antineoplastic activity. Targeting cellular membranes, perifosine modulates membrane permeability, membrane lipid composition, phospholipid metabolism, and mitogenic signal transduction, resulting in cell differentiation and inhibition of cell growth. This agent also inhibits the anti-apoptotic mitogen-activated protein kinase (MAPK) pathway and modulates the balance between the MAPK and pro-apoptotic stress-activated protein kinase (SAPK/JNK) pathways, thereby inducing apoptosis. Perifosine has a lower gastrointestinal toxicity profile than the related agent miltefosine. (NCI04) Drug Indication Investigated for use/treatment in solid tumors, multiple myeloma, leukemia (unspecified), lung cancer, and brain cancer. Mechanism of Action Targeting cellular membranes, perifosine modulates membrane permeability, membrane lipid composition, phospholipid metabolism, and mitogenic signal transduction, resulting in cell differentiation and inhibition of cell growth. This agent also inhibits the anti-apoptotic mitogen-activated protein kinase (MAPK) pathway and modulates the balance between the MAPK and pro-apoptotic stress-activated protein kinase (SAPK/JNK) pathways, thereby inducing apoptosis. 1. Mechanism of action (Literatures [1], [2], [3]): Perifosine (KRX0401) is an oral alkylphospholipid AKT inhibitor that binds to the pleckstrin homology (PH) domain of AKT, preventing AKT membrane localization and subsequent phosphorylation/activation by PDK1 and mTORC2. This blocks downstream AKT-mediated signaling (e.g., GSK3β, S6), inhibiting cancer cell proliferation and inducing apoptosis in AKT-dependent tumors[1] [2][3] 2. Preclinical significance (Literatures [2], [3]): - Effective against both solid tumors (lung, breast, colorectal) and hematologic malignancies (CML, AML), supporting broad anticancer potential[2] [3] - Synergizes with chemotherapy (irinotecan, Literature [2]) and targeted agents (imatinib, Literature [3]), reducing chemoresistance and improving efficacy[2] [3] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~15 mg/mL (~32.5 mM) Water: ~8 mg/mL (~17.3 mM) Ethanol: <1 mg/mL |
| Solubility (In Vivo) |
Solubility in Formulation 1: 50 mg/mL (108.30 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication. Solubility in Formulation 2: 30%Propylene glycol, 5%Tween 80, 65% D5W: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1661 mL | 10.8305 mL | 21.6610 mL | |
| 5 mM | 0.4332 mL | 2.1661 mL | 4.3322 mL | |
| 10 mM | 0.2166 mL | 1.0830 mL | 2.1661 mL |