Drugs in Clinical Pipeline: Perifosine | Orphan Drug | AKT Inhibitor | Treatment for Cancer | Signal Transduction Pathway Inhibitor | Kinase Inhibitor

Perifosine [1,1-Dimethylpiperidinium-4-yl octadecyl phosphate] is a synthetic oral alkylphospholipid (APL) bearing a piperidine head group. APLs were shown to have selectivity for neoplastic versus normal hematologic cells in vitro. 

Perifosine is an oral Akt inhibitor which unlike most kinase inhibitors that target the adenosine triphosphate (ATP)-binding region, prefers binding to the Pleckstrin Homology (PH) domain of Akt, thereby preventing Akt's translocation to the plasma membrane. It also modulates a number of other key signal transduction pathways, including the JNK and MAPK pathways, all of which are pathways associated with programmed cell death, cell growth, cell differentiation and cell survival. The effects of Perifosine on Akt are of particular interest because of the importance of this pathway in the development of most cancers, with evidence that it is often activated in tumors that are resistant to other forms of anticancer therapy, and the difficulty encountered thus far in the discovery of drugs that will inhibit this pathway without causing excessive toxicity [1, 2].

Perifosine : 2D and 3D Structure

Perifosine is based on the structure of lysophosphatidylcholine and belongs to lysolecithin analogues class of antitumor APLs that also includes Edelfosine and Miltefosine.

The antitumor activity of lysolecithin analogues has been known for almost three decades; but it was the discovery of Miltefosine (hexadecylphosphocholine), as the minimal chemical structure required to exert antitumour activity led to a renewal of scientific interest in this whole class of compounds. Miltefosine represents the first anticancer agent specifically formulated as a solution for topical use, and it has become a valuable tool in the clinical management of cutaneous breast cancer and other malignant lesions. The development of a systemic treatment with an oral miltefosine formulation was hampered by the gastrointestinaltract (GIT) toxicity of the compound, which prevented an adequate dosing in initial clinical studies.

Perifosine was the first heterocyclic alkylphospholipid which experimentally showed a significantly improved GIT tolerance.

In vivo, Perifosine induced thrombocytosis and leukocytosis and increased myelopoiesis in murine marrow and spleen, whereas it caused apoptosis in myeloma xenografts.

Perifosine has orphan drug status in the U.S. for the treatment of multiple myeloma and neuroblastoma, and for multiple myeloma in the EU. Presently, it is under clinical trials for various cancers as monotherapy or in combination with other agents.

AKT: A Mystery in Itself

The phosphoinositide-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is an important signal transduction pathway that controls processes integral in cancer development, including protein translation, growth, metabolism, and survival. Akt is a nodal regulator of cellular survival pathways and an attractive target in cancer therapy [2].

Akt is a serine/threonine kinase that lies upstream of mTOR in the pathway because Akt phosphorylates TSC2, which de-represses Rheb to interact with FKBP38 and allow mTOR activation. Akt also can be downstream of mTOR because the TORC2 complex consisting of mTOR and rictor (rapamycin-insensitive companion of mTOR) can phosphorylate Akt.

Aberrant Akt activation may occur through overexpression or mutation of receptor tyrosine kinases, activation of oncogenes such as Ras, inactivation of the tumor suppressor PTEN (phosphatidylinositol phosphate 3’-phosphatase) through epigenetic silencing or mutations, activating mutations or amplifi cation of isoforms of PI3K, or mutations in the pleckstrin homology (PH) domain or genomic amplification of Akt itself.

Akt is activated following activation of class I PI3K, which generates PI(3,4)P2 (PIP2) and PI(3,4,5)P3 (PIP3). Once synthesized, PIP2 and PIP3 interact with the PH domain of Akt and cause its translocation to the plasma membrane, where it is phosphorylated on T308 by PDK1 and on S473 by the rictor/mTOR complex, as well as possibly by other proteins. Phosphorylation of both T308 and S473 is required for full kinase activity. Once phosphorylated, Akt dissociates from the plasma membrane and moves to various cellular compartments, where it can phosphorylate downstream substrates such as VEGF (for angiogenesis), TSC2 (Tuberous sclerosis complex 2), FOXO (Forkhead box), p21, p27, GSK3ß (for glucose metabolism), BAD, XIAP, and MDM2, etc.

To make this riddle a bit more confusing, the researchers found that low levels of AKT activity is associated with elevated levels of FOXOs required to maintain the function and immature state of leukemia-initiating cells (LICs). FOXOs are active, implying reduced Akt activity, in ~40% of acute myeloid leukemia (AML) patient samples regardless of genetic subtype; and either activation of Akt or compound deletion of FoxO1/3/4 reduced leukemic cell growth in a mouse model.

The PI3K/Akt/mTOR pathway is frequently activated in tumors and promotes therapeutic resistance, providing a strong rationale to target it in cancer therapy. Validation of this approach came with recognition that the mTOR inhibitor temsirolimus prolonged survival in renal cell cancer patients, which led to its US Food and Drug Administration approval. However, a possible mechanism of resistance to mTOR inhibitors is feedback activation of Akt, highlighting the need to develop agents that target other pathway components.


Mechanism of Action in Perifosine:

In vitro, Perifosine inhibits Akt at low micromolar concentrations. Moreover, it  causes inhibition of PC-3 prostate carcinoma cell growth (GI₅₀ by 5 µM in 24 h) associated with rapidly decreased Akt activation, as assessed by Thr308 and Ser473 phosphorylation, and assay of enzymatic activity. Perifosine inhibits recruitment of Akt to the cell membrane but without significant inhibition of PI3K as well as PDK1, ILK, and Src. Inhibition has been measured in many xenograft models in vivo.

Perifosine targets the Pleckstrin Homology (PH) domain of Akt, thereby preventing its translocation to the plasma membrane. Perifosine-mediated inhibition of Akt phosphorylation is substantially relieved by introduction of MYR-Akt, which bypasses the requirement for PH domain-mediated membrane recruitment, particularly in the context of coexpression of the PI3K catalytic subunit. These experiments point to the Akt pathway as a prominent participant in the cellular effects of Perifosine. Yet it remains unclear whether Perifosine does so by disrupting membrane microdomains crucial to Akt activation or whether it binds directly to the PH domain of Akt, thereby displacing the natural PIP2 and PIP3 ligands.

Although there is strong evidence that Perifosine inhibits Akt in preclinical models of cancer, Perifosine possesses Akt-independent activities as well. Death receptors, lipid rafts, and JNK also are important in the death caused by Perifosine, depending on the cell type.

No wonder in absence of a reliable assays that measure Akt, the researchers communities are unable to put an answer to the question, is Perifosine Akt inhibitor or does it have other mechanism of action? Till that happens, this unusual molecule is cited as an Akt inhibitor.


Dosages and Approvals:

Perifosine (Tradename: -) is discovered and developed by Aeterna Zentaris; and they have licensed it out to Keryx for commercialization in the United States, Canada and Mexico.

Perifosine has been granted Fast Track designation (Colorectal cancer; Multiple myeloma), together with the SPA and Orphan Drug status by the US-FDA for Neuroblastoma and Multiple myeloma. Perifosine has also been granted orphan medicinal product designation from the European Medicines Agency (EMA) in multiple myeloma. Furthermore, Perifosine has received positive Scientific Advice from the EMA for both the multiple myeloma and advanced colorectal cancer programs.


Reported Activities for Perifosine:

Perifosine selectively inhibits the PH domain of Akt, but the researchers note that the binding of Perifosine to the PH domain occurs with relatively low affinity and that direct binding by titration calorimetry could not be detected. In the stated work, Perifosine inhibited binding of the Akt PH domain to artificial membranes containing 3% PIP2 with (50% inhibitory concentration) IC₅₀ greater than 10 µM. In contrast, it did not inhibit binding of two other PH domain–containing proteins to membranes containing phosphoinositides [DAPP1 PH domain binding to PIP2 or PLC-d-1 PH domain binding to PI(4,5)P2]  with IC₅₀ values more than100 µM [2]. 

Akt inhibition by perifosine has been documented in vivo against myeloma, Waldenström macroglobulinemia (WM), prostate cancer, and glioma xenografts in immune-compromised mice. In PC3 and Du145 xenografts, there was significant correlation among the cumulative dose of Perifosine, tumor growth inhibition, and decreases in phosphorylation of Akt and other pathway components in the tumors. In A431 and BT474 xenografts, Perifosine was ineffective in inhibiting tumor growth and did not inhibit Akt. Thus, the ability of Perifosine to inhibit Akt in tumors correlated with growth inhibition in vivo. In addition, cell lines with activating PI3K/ Akt genomic alterations (in PI3K, PTEN, and epidermal growth factor receptor [EGFR] overexpression) were more sensitive to Perifosine in vitro than those without. 

A recent publication reports the IC₅₀ value of Perifosine against Akt as 5.3 uM [3].

IC₅₀ (Inhibition of Akt Kinase Activity) = 5.3 ± 0.8 uM

Summary

Common name: AEZS-104; AEZS104; AEZS 104; D-21266; D21266; D 21266; KRX-0401; KRX0401; KRX 0401; YHI-1003; YHI 1003; YHI1003; NSC-639966; Perifosin; Perifosine

Trademarks: -

Molecular Formula: C25H52NO4P

CAS Registry Number: 157716-52-4

CAS Name: 1,1-dimethylpiperidin-1-ium-4-yl octadecyl phosphate

Molecular Weight: 461.66

SMILES: O=P(OC1CC[N+](C)(C)CC1)([O-])OCCCCCCCCCCCCCCCCCC

InChI Key: SZFPYBIJACMNJV-UHFFFAOYSA-N

InChI: 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

Mechanism of Action: Kinase Inhibitor; AKT Inhibitor; JNK Inhibitor; MAPK14 Inhibitor; Signal Transduction Pathway Inhibitors

Activity: Antineoplastics; Treatment for Cancers

Status: Under Phase Trials

Chemical Class: Small molecules; Quaternary ammonium compounds; Trimethyl ammonium compounds; Long chain alkyl groups; Phosphorus containing; Piperidine derivatives; Phosphate containing

Originator: AEterna Zentaris Inc / Yakult Honsha; Keryx

Perifosine Synthesis

US6172050: The patent reports first known synthesis for Perifosine and various derivatives. The techniques are scalable and yields are quantitative.

Perifosine Synthesis

Step1: 20.1 ml (0.22 mol) of phosphorus oxychloride are placed in 100 ml of methylene chloride and cooled to 5 - 10° C. and a solution of 54.1 g (0.20 mol) of octadecanol in 400 ml of methylene chloride and 70.5 ml of pyridine is added over 30 min, with stirring. After subsequent stirring for one hour, 29.9 g (0.26 mol) of 4-hydroxy-1-methylpiperidine in 80 ml of pyridine are added dropwise. After stirring for 3 hours at 10° C., the mixture is hydrolyzed with 30 ml of water while being cooled with ice and is subsequently stirred for one hour. The organic phase is washed with 200 ml each of water/methanol (1:1), 3 percent hydrochloric acid/ methanol (1:1) and water/methanol (1:1). The organic phase is dried over Na₂SO₄ and concentrated until turbidity appears, and 1 liter of methyl ethyl ketone is added. The crystals are recrystallized from 1 liter of methyl ethyl ketone, filtered off with suction and dried under vacuum over P₂O₅. Yield: 54.1 g (60%) of octadecyl 1-methylpiperidinio-4-yl phosphate.

Step 2: 98.1 g (0.22 mol) of octadecyl 1-methylpiperidinio-4-yl phosphate are suspended in 500 ml of absolute ethanol and heated to boiling. Under reflux, a total of 71.8 g (0.39 mol) of methyl p-toluenesulphonate and 26.5 g (0.19 mol) of potassium carbonate are added alternately in eight portions over 2 hours. When the addition is complete, the mixture is refluxed for a further hour. After cooling, it is filtered, the filtrate is concentrated to half and 150 g of moist Amberlite MB3 ion exchanger are added to the solution. After stirring for two hours, the mixture is filtered with suction over kieselguhr/activated charcoal and the filtrate is concentrated and crystallized with acetone. The crystal cake is recrystallized from methyl ethyl ketone and dried under vacuum over P₂O₅. Yield: 46.1 g (46%) of Perifosine (octadecyl 1,1-dimethylpiperidinio-4-yl phosphate).

Identifications:

1H NMR (Estimated) for Perifosine

Experimental: To be updated.

Sideeffects: Perifosine’s safety profile is distinctly different from that of most cytotoxic agents. It does not appear to cause myelosuppression (depression of the immune system that may lead to life threatening infections), thrombocytopenia (a decrease in platelets that may result in bleeding), skin rash, flu-like symptoms or alopecia (hair loss); all of these toxicities occur frequently with many of the currently available treatments for cancer. The main adverse events (AEs) of Perifosine treatment are gastrointestinal (GI) in nature with nausea, vomiting, diarrhea and fatigue being the commonly reported ones. These are either mild or non-existent in doses that are known to induce tumor regression. Others: 1. Discovery of Perifosine: Alkylphospholipids (APLs) are reported to interfere with phospholipid metabolism and survival signaling, induce apoptosis, inhibit neovascularization, prevent invasion, and induce tumor cell differentiation but the precise mechanism of action is not yet fully elucidated. Early clinical trials were limited because of dose-limiting gastrointestinal (GI) toxicity, and parenteral dosing of this class of agents is not possible because of their hemolytic properties; therefore, related compounds with an improved therapeutic index were developed. Analog research to produce compounds with a better systemic therapeutic index than Miltefosine yielded Perifosine, in which the choline head group has been substituted by a cyclic aliphatic piperidyl residue. The major metabolite of Miltefosine, phosphocholine, has a structure resembling that of acetylcholine, which may be the reason for the severe GI disturbances observed upon oral treatment. Its failure as an oral agent was caplitalize when Miltefosine was launched as a topical treatment against cancers. In contrast, Perifosine is not able to generate phosphocholine, and hence may be better tolerated, leading to its selection as an orally available clinical candidate. 2. Perifosine has been evaluated in clinical trials across a wide range of tumor types. With the exception of renal cell carcinoma, sarcoma, and Waldenstrom’s macroglobulinemia (WM), results as a single agent have been limited. However, appreciable clinical activity has been observed in combination with other agents in advanced refractory colorectal cancer (CRC) and relapsed/refractory multiple myeloma (MM). 3. Akt is a serine-threonine kinase activated through PI3K to promote cell growth and survival. Akt/PI3K activation is also associated with resistance to irradiation drug resistance. Antitumor effect of Perifosine was also beneficial in combination with radiation, suggesting a favorable profile of Perifosine in combination therapies. 4. In vivo, Perifosine inhibits both constitutive and inducible phosphorylation of Akt in a dose-dependent manner, thereby blocking Akt activity. In vitro, Perifosine showed antitumor effects against melanoma, nervous system, lung, prostate, colon, and breast cancer models, with an activity similar to or stronger than Miltefosine. Furthermore, Perifosine triggers apoptosis in human leukemia cells, in a dose-dependent manner. References: 1. Hilgard, P.; et. al. D-21266, a new heterocyclic alkylphospholipid with antitumour activity. Eur J Cancer 1997, 33(3), 442 - 446. (FMO) 2. Gills, J. J.; et. al. Perifosine: update on a novel Akt inhibitor. Curr Oncol Rep 2009, 11(2), 102 - 110. (FMO) 3. Alam, M. M.; et. al. Synthesis, characterization and Akt phosphorylation inhibitory activity of cyclopentanecarboxylate-substituted alkylphosphocholines. Bioorg Med Chem 2013, 21(7), 2018 - 2024. (FMO) 4. Richardson, P. G.; et. al. Perifosine , an oral, anti-cancer agent and inhibitor of the Akt pathway: mechanistic actions, pharmacodynamics, pharmacokinetics, and clinical activity. Expert Opin Drug Metab Toxicol 2012, 8(5), 623 - 633. (free article) 5. Nossner, G.; et. al. Phospholipid derivatives. US6172050B1

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