Akt; PI3K; PKC (IC50 = ~7 μM)

Miltefosine is an alkylphosphocholine medication with demonstrated activity against various parasite species and cancer cells as well as some pathogenic bacteria and fungi. Miltefosine inhibits PKC from NIH3T3 cells in cell-free extracts with a IC50 of about 7 μM.[1] In vivo, macrophages that are HIV-infected serve as long-lived HIV-1 reservoirs, and miltefosine targets these cells. By blocking the PI3K/Akt pathway, miltefosine eliminates infected macrophages from circulation without harming healthy cells.[2] In carcinoma cell lines, miltefosine inhibits the PI3K/Akt survival pathway.[3] Miltefosine interferes with the insulin signaling pathway and prevents insulin-stimulated glucose uptake, which results in skeletal muscle insulin resistance in vitro. With 75% inhibition at 40 M and 98% inhibition at 60 μM, miltefosine inhibits insulin-stimulated Akt phosphorylation in a dose-dependent manner.[4]

Miltefosine inhibits anti-IgE induced histamine release from human skin mast cells. Miltefosine can significantly slow down the esterification of cholesterol as well as lower levels of the cytokines IL-1β, IL-4, and IL-6 in some skin tissue cells. [5]

The ApoAlert Caspase Fluorescent assay kit is used to measure the amounts of enzymatically active caspase-3. In a nutshell, 1 106 BC-1 PEL cells are exposed to vehicle controls, 50 M Miltefosine, 50 M Perifosine, or 20 nM NVP-BEZ235. After 12 hours, cells are collected and lysed. For each sample, an identical amount of cell lysate is incubated with a fluorogenic caspase-3 substrate (DEVD-AFC). With the excitation and emission filter wavelengths set to 400 and 505 nm, respectively, cleavage of DEVD by caspase-3 releases AFC, the fluorescence of which is measured using a FLUOstar OPTIMA fluorometer.

2 × 105 PEL cells are either treated with the therapeutic substances at the recommended doses or with the appropriate vehicle as a negative control. Trypan blue exclusion is performed in quadruplicate to assess cell viability after 96 hours of cell monitoring.

Mice: PEL cells are collected, counted, and diluted in 100 L of PBS combined with 100 L of Matrigel depleted of growth factors after being washed in ice-cold phosphate buffered saline. Subcutaneous injection of 1 105 to 7.5 105 BC-1 cells is made into the right flank of NOD. Alternatively, CB17-Prkdcscid/J mice. On alternate days, the mice are checked for the development of palpable tumors (2 mm3). If this occurs, drug or vehicle treatments are started, and the mice receive either intraperitoneal (Perifosine) or oral gavage (Rosiglitazone, NVP-BEZ235) treatments 5 days a week. PEL tumors are created using groups of 5–7 mice, and either a vehicle or drug cocktail is used to treat them. Multiple replications of every biological experiment are carried out. 30 mg/kg or 60 mg/kg of Rosiglitazone is suspended in 0.25% methylcellulose, which serves as the vehicle for the medication. PBS serves as a vehicle for the drugs Perifosine and Miltefosine, which are dissolved in the solution at a concentration of 50 mg/kg each. In order to dissolve NVP-BEZ235, the substance is combined with polyethylene glycol 300 in a 1:9 vol/vol ratio of 1-methyl-2-pyrrolidone. A dose of 40 mg/kg NVP-BEZ235 or an equivalent volume of the vehicle is given. Digital calipers are used to measure the tumor diameters, and tumor volume is computed. The tumors are removed and then fixed in formalin. With each animal treated as a random effect, statistical analyses are carried out using a linear model fit with the maximum likelihood.
Rats: There are five groups of male Sprague-Dawley rats (n=5), each weighing between 270 and 290 g. Miltefosine (MFS) is given to rats in the treatment groups as a single oral dose of 10 mg/kg as either an aqueous solution or MFS-LNCs dispersion by gastric gavage. This dosage, adjusted for rats, is equivalent to the 20 mg/kg Miltefosine dose given to mice in the preclinical study. Following administration, blood samples are taken through the orbital plexus while the patient is under anesthesia at intervals of 0.5, 1, 2, 4, 7, 10, 24, 48, 72, and 216 hours. The Eppendorf tubes contain EDTA. The next step is an immediate, 10-minute centrifugation of blood samples at 4000 rpm. While awaiting analysis, plasma samples are kept frozen and at -80°C.

Absorption, Distribution and Excretion
After oral administration, miltefosine is slowly absorbed from the gastrointestinal tract with an absolute bioavailability of 82% in rats and 94% in dogs. Absolute bioavailability has not been assessed in humans, however GI absorption rate in a two-compartment model is estimated to be 0.416 hr-1.
Miltefosine is almost completely eliminated by degradation via phospholipase D. Drug keeps accumulating until the end of treatment due to the extremely slow elimination, as seen by the long elimination half lives.
Radioactivity studies have found that miltefosine has a wide distribution with high levels in the kidney, intestinal mucosa, liver, and spleen.
Plasma clearance is very low and the terminal elimination half life was found to be 84 and 159 hours in rats and dogs respectively.

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Metabolism / Metabolites
Miltefosine is metabolized mainly by phospholipase D, releasing choline, choline-containing metabolites, and hexadecanol, which are likely to enter the intermediary metabolism. The metabolites produced by this reaction are all endogenous and are likely used for bio-synthesis of acetylcholine, cell membranes, and long-chain fatty acids.

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Biological Half-Life
The primary elimination half life is 7.05 days (range: 5.45-9.10 days) and the terminal half-life is 30.9 days (range: 30.8-31.2 days).

Hepatotoxicity
Serum aminotransferase levels are frequently elevated in patients with visceral leishmaniasis and miltefosine therapy regularly results in a decline in mean values into the normal range. In prospective studies of miltefosine therapy, however, as many as half of patients had mild-to-moderate ALT elevations during therapy, although values above 5 times ULN were rare (
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Protein Binding
Plasma protein binding ranges from 96% to 98%. Miltefosine binds to both serum albumin (97% bound) and low-density lipoprotein (3% bound).

[1]. Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy. Retrovirology. 2008 Jan 31;5:11.

[2]. Hexadecylphosphocholine inhibits inositol phosphate formation and protein kinase C activity. Cancer Res. 1991 Feb 1;51(3):807-12.

[3]. Dual inhibition of PI3K and mTOR inhibits autocrine and paracrine proliferative loops in PI3K/Akt/mTOR-addicted lymphomas. Blood. 2010 Jun 3;115(22):4455-63.

[4]. Miltefosine Lipid Nanocapsules for Single Dose Oral Treatment of Schistosomiasis Mansoni: A Preclinical Study. PLoS One. 2015 Nov 17;10(11):e0141788.

[5]. Effects of miltefosine on the proliferation, ultrastructure, and phospholipid composition of Angomonas deanei, a trypanosomatid protozoan that harbors a symbiotic bacterium. FEMS Microbiol Lett. 2012 Aug;333(2):129-37.

Miltefosine is a phospholipid that is the hexadecyl monoester of phosphocholine. It has a role as an antineoplastic agent, an antiprotozoal drug, an antifungal agent, an immunomodulator, an anti-inflammatory agent, an apoptosis inducer, a protein kinase inhibitor and an anticoronaviral agent. It is a member of phosphocholines and a phospholipid.
Miltefosine is an antiprotozoal prescription medicine approved by the U.S. Food and Drug Administration (FDA) for the treatment of leishmaniasis.
Leishmaniasis can be an opportunistic infection (OI) of HIV.
Miltefosine is a broad spectrum antimicrobial, anti-leishmanial, phospholipid drug that was originally developed in the 1980s as an anti-cancer agent. It is currently the only recognized oral agent used to treat visceral, cutaneous, and mucosal forms of leishmaniasis, a neglected tropical disease. It can be administered topically or orally and is only indicated in patients aged 12 years or older. The CDC has also recommended it as a first line treatment for free-living amebae (FLA) infections such as primary amebic meningoencephalitis and granulomatous amebic encephalitis.
Miltefosine is an Antileishmanial.
Miltefosine is an orally available, alkyl phospholipid that is used in the treatment of both cutaneous and visceral leishmaniasis. Miltefosine therapy is often accompanied by transient mild-to-moderate serum aminotransferase elevations during the first 1 or 2 weeks of treatment, but has not been implicated in cases of clinically apparent liver injury with jaundice.
Miltefosine has been reported in Carica papaya and Xenorhabdus nematophila with data available.
Miltefosine is an orally- and topically-active alkyl-phosphocholine compound with potential antineoplastic activity. Miltefosine targets cellular membranes, modulating cell 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. As an immunomodulator, miltefosine stimulates T-cells, macrophages and the expression of interleukin 3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF), and interferon gamma (INF-gamma). (NCI04)

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Drug Indication
For the treatment of mucosal (caused by Leishmania braziliensis), cutaneous (caused by L. braziliensis, L. guyanensis, and L. panamensis), and visceral leishmaniasis (caused by L. donovani). In comparing Leishmania drug susceptibility, it has been found that L. donovani is the most susceptible to miltefosine while L. major is the least susceptible. Off-label use includes treatment of free-living amebae (FLA) infections (unlabeled use; CDC, 2013).
FDA Label

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Mechanism of Action
Miltefosine has demonstrated activity against Leishmania parasites and neoplastic cells primarily due to its effects on apoptosis and disturbance of lipid-dependent cell signalling pathways. Several potential antileishmanial mechanisms of action have been proposed, however no mechanism has been identified definitely. Within the mitochondria, miltefosine inhibits cytochrome-c oxidase leading to mitochondrial dysfunction and apoptosis-like cell death. Antineoplastic mechanisms of action are related to antileishmanial targets and include inhibition of phosphatidylcholine biosynthesis and inhibition of Akt (also known as protein kinase B), which is a crucial protein within the PI3K/Akt/mTOR intracellular signalling pathway involved in regulating the cell cycle. Animal studies also suggest it may be effective against Trypanosome cruzi (the organism responsible for Chagas' disease), metronidazole-resistant strains of Trichonomas vaginalis, and it may have broad-spectrum anti-fungal activity.

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Pharmacodynamics
Little is known about the clinical pharmacodynamics of miltefosine and other antileishmanial drugs.

C21H46NO4P

407.57

407.316

C, 61.89; H, 11.38; N, 3.44; O, 15.70; P, 7.60

58066-85-6

58066-85-6

3599

White to off-white solid powder

232-234ºC

3.58

0

4

20

27

363

0

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])[H])OC([H])([H])C([H])([H])[N+](C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H]

PQLXHQMOHUQAKB-UHFFFAOYSA-N

InChI=1S/C21H46NO4P/c1-5-6-7-8-9-10-11-12-13-14-15-16-17-18-20-25-27(23,24)26-21-19-22(2,3)4/h5-21H2,1-4H3

hexadecyl (2-(trimethylammonio)ethyl) phosphate

HePC; Hexadecyl phosphocholine; Miltefosin C; HePC; Hexadecylphosphocholine; HDPC; Hexadecylphosphorylcholine; Miltefosinum; mpavido; Miltex; Choline Phosphate Hexadecyl Ester Hydroxide Inner Salt; hexadecylphosphocholine; Miltefosin; Miltefosina; Miltefosinum

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: 100 mg/mL (245.36 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: Saline: 30mg/mL

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