Fosnetupitant is a prodrug that is converted to its active metabolite netupitant, which targets the neurokinin-1 (NK1) receptor. NK1 receptors are G protein-coupled receptors that bind substance P, a neuropeptide involved in emesis and pain transmission. By antagonizing NK1 receptors, netupitant blocks substance P-mediated signaling, preventing emesis. The active metabolite is a potent and selective NK1 receptor antagonist.

Fosnetupitant (Pronetupitant) does not bind to all other proteins investigated, but it has micromolar affinity for L-type Ca2+ channels (pKi ∼ 5.7) and 5-HT6 receptors (pKi ∼ 5.2) [1].

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In vitro, fosnetupitant is a prodrug that is converted to the active NK1 receptor antagonist netupitant. The active metabolite netupitant demonstrates potent antagonistic activity at the NK1 receptor. Specific IC50 values for receptor binding or functional inhibition are not detailed in the available sources. The prodrug approach improves the solubility and formulation characteristics of the compound for intravenous administration.

Rats quickly and extensively convert Pronetupitant to Netupitant following an intravenous injection[1].

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In vivo, fosnetupitant is administered intravenously and is rapidly converted to netupitant, achieving effective plasma concentrations of the active metabolite. The compound is used in combination with palonosetron for the prevention of chemotherapy-induced nausea and vomiting (CINV). It has demonstrated efficacy in clinical trials and is approved for this indication. Specific animal model data are not detailed in the available sources.

The NK1 receptor binding assay for netupitant (the active metabolite) involves incubating the compound with membrane preparations from cells expressing the human NK1 receptor and a radiolabeled substance P ligand. After incubation, bound and free ligands are separated by filtration, and the radioactivity is counted. The IC50 for displacement of the radioligand is calculated. Functional antagonism is assessed using calcium mobilization assays in cells expressing NK1.

To evaluate the cellular activity of netupitant, cells expressing the human NK1 receptor are seeded in 96-well plates and loaded with a calcium-sensitive fluorescent dye. Cells are pre-incubated with varying concentrations of netupitant and then stimulated with substance P. The intracellular calcium flux is measured using a fluorescence plate reader. The IC50 for inhibition of substance P-induced calcium flux is calculated. The prodrug fosnetupitant can be assessed for its conversion to the active metabolite in cellular systems.

The in vivo efficacy of fosnetupitant is evaluated in animal models of emesis, such as ferret or dog models. Animals are treated with a chemotherapeutic agent (such as cisplatin) to induce emesis. Fosnetupitant is administered intravenously at various doses, and the number of emetic episodes is recorded over a specified period. The compound's ability to prevent emesis is expressed as the percentage inhibition of emesis compared to vehicle-treated controls.

Absorption, Distribution and Excretion
Following a single intravenous injection of Akynzeo (235 mg netutipant and 0.25 mg palonosetron, infused over 30 minutes) in patients or a single intravenous injection of netutipant (235 mg netutipant, infused over 30 minutes) in healthy subjects, the maximum concentration of netutipant was reached at the end of the 30-minute infusion. Oral bioavailability varied significantly among species, with 42–105% in rats, 34–83% in dogs, and 37–62% in monkeys. This large difference is likely due to the small number of animals used in the studies. Approximately half of the administered radioactivity following a single oral administration of [14C] netutipant was excreted in urine and feces within 120 hours. 3.95% and 70.7% of the radioactive dose were measured in urine and feces collected within 336 hours, respectively. The mean fraction of netutopeptide excreted unchanged in urine after an oral dose was less than 1%, indicating that renal clearance is not a significant elimination pathway for netutopeptide-related substances. It is estimated that approximately 86.5% of the radioactive material and 4.7% of the radioactive material are excreted in feces and urine, respectively, within 30 days after administration. The mean volume of distribution (standard deviation) of netutopeptide in healthy subjects and patients was 124 ± 76 L and 296 ± 535 L, respectively. The mean estimated systemic clearance of netutopeptide after a single oral dose of Akynzeo was 0.3 ± 9.2 L/h (mean ± standard deviation). Metabolites/Metabolites: Netutopeptide is a prodrug of netutopeptide. Netutopeptide is a moderate inhibitor and substrate of CYP3A4. Akynzeo should be used with caution in patients concurrently taking medications primarily metabolized via the CYP3A4 system. A single dose of 300 mg netupitant significantly inhibits CYP3A4 for approximately 6 days. It is recommended to avoid concurrent use of CYP3A4 substrate drugs within one week. If this cannot be avoided, a dose reduction of the CYP3A4 substrate drug should be considered. In liver microsomal incubation experiments in humans, rats, dogs, miniature pigs, and marmosets, two major metabolites were detected in all species except for the hydroxylation product (M3): an N-demethylation product (M1) and an N-oxidation product (M2). The study found that CYP3A4 is responsible for oxidizing netupitant to the same metabolites observed in human liver microsomal incubation experiments. Metabolism is widespread, and metabolites typically reach concentrations higher than the parent drug within 24 hours. M1 and M2 exposures were similar in rats and humans, but higher in dogs; however, M3 exposures were lower in both animals than in humans.

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Biological Half-Life
Netupitan is eliminated from the body in a multi-exponential manner, with an apparent elimination half-life of 80 ± 29 hours (mean ± standard deviation) in cancer patients.

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Fosnetupitant is administered intravenously and is rapidly converted to the active metabolite netupitant. Specific pharmacokinetic parameters (e.g., Cmax, Tmax, half-life, AUC) for both the prodrug and the active metabolite are not detailed in the available sources. The compound has a molecular weight of 676.6 and a molecular formula of C30H35F6N4O5P. It is formulated as an intravenous solution.

Hepatotoxicity
In a pre-registration clinical trial of a fixed combination of netupitant and palonosetron, the proportion of patients receiving the treatment was similar to that in the control group receiving cancer chemotherapy. The elevations were transient, mild to moderate, and unasymptomatic or without jaundice. The elevations were more likely due to cancer chemotherapy than to antiemetic prophylaxis. No definitive cases of clinically significant liver injury caused by netupitant in combination with palonosetron have been reported in the literature; therefore, even if it occurs, severe liver injury is extremely rare. Probability score: E (unlikely to be the cause of clinically significant liver injury). Protein Binding Netupitant has high plasma protein binding rates (>99%) in all species.

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Fosnetupitant is generally well-tolerated, with common adverse effects including headache, fatigue, and constipation. As an NK1 receptor antagonist, its safety profile is consistent with other drugs in this class. The compound is approved for use in combination with palonosetron for the prevention of CINV. Specific toxicity data from preclinical studies are not detailed in the available sources.

[1]. In vitro and in vivo pharmacological characterization of Pronetupitant, a prodrug of the neurokinin 1 receptor antagonist Netupitant. Peptides. 2015 Jul;69:26-32.

In April 2018, the U.S. Food and Drug Administration (FDA) and the Swiss company Helsinn approved the intravenous formulation of AKYNZEO® (NEPA, a fixed-dose antiemetic combination containing 235 mg of netupitant and 0.25 mg of palonosetron) as an alternative treatment for patients experiencing nausea and vomiting due to chemotherapy. Netupitant is a prodrug of netupitant. Typically, 25% to 30% of cancer patients receive chemotherapy, and 70% to 80% of these patients may experience major side effects such as nausea and vomiting. Nausea and vomiting are considered among the most distressing side effects of chemotherapy, significantly impacting the quality of life for patients undergoing certain anti-cancer treatments. Netupitant is an antiemetic often used in combination with palonosetron and dexamethasone to prevent nausea and vomiting caused by cancer chemotherapy. No cases of elevated liver enzymes or clinical liver injury with jaundice have been observed during treatment with netupitant in combination with palonosetron.
Fonetupitant is the prodrug of netupitant, a selective neurokinin 1 receptor (NK1R; TACR1) antagonist with potential antiemetic activity. After intravenous injection, netupitant is converted to its active form, netupitant, by phosphatase. Netupitant competitively binds to and blocks the activity of the NK1 receptor by inhibiting the binding of endogenous tachykinin-derived neuropeptide substance P (SP) to the NK1 receptor in the central nervous system (CNS). This prevents delayed vomiting associated with SP secretion. This may help prevent chemotherapy-induced nausea and vomiting (CINV).
Drug Indications

Netupitant, in combination with palonosetron (brand name: Akynzeo) and dexamethasone, is used in adults for the prevention of acute and delayed nausea and vomiting associated with cancer chemotherapy (including, but not limited to, highly emetogenic chemotherapy). The following is the indication listed on the EMA label: Prevention of acute and delayed nausea and vomiting associated with highly emetogenic cisplatin-based cancer chemotherapy. Prevention of acute and delayed nausea and vomiting induced by moderately emetogenic chemotherapy for cancer.
FDA Label

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Mechanism of Action
Fnetupitan, a component of this drug combination, is a selective P/neurokine-1 (NK-1) receptor antagonist. [Netupitan] is the active ingredient of netupitan, a selective neurokinin 1 (NK1) receptor antagonist with antiemetic activity. Netupitan competitively binds to and blocks the activity of human substance P/NK1 receptors in the central nervous system (CNS), inhibiting the binding of endogenous tachykinin neuropeptide substance P (SP) to NK1 receptors, thereby preventing chemotherapy-induced nausea and vomiting (CINV). Substance P is present in neurons innervating vagal afferent fibers of the brainstem innervating the nucleus solitarius and the area retrieval (containing the chemoreceptor trigger zone (CTZ)), and its levels may increase after chemotherapy. The NK receptor is a G protein-coupled receptor coupled to the inositol phosphate signaling pathway and is present in the nucleus solitarius and the area retrieval. Netutopeptide showed an NK1 receptor occupancy of 92.5% at 6 hours and 76% at 96 hours.
Pharmacodynamics
In the combination drug Akynzeo, palonosetron prevents acute nausea and vomiting after cancer chemotherapy, while netutopeptide prevents both acute and delayed nausea and vomiting after cancer chemotherapy. Neurokinin-1 (NK-1) inhibitors, such as netutopeptide, possess unique anxiolytic, antidepressant, and antiemetic properties.

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Fosnetupitant is a phosphate prodrug of netupitant, a potent and selective NK1 receptor antagonist. It is used in combination with palonosetron for the prevention of chemotherapy-induced nausea and vomiting (CINV). The prodrug is designed to improve the solubility and formulation characteristics of netupitant for intravenous administration.

C31H35F6N4O5P

688.61

688.224

1703748-89-3

Fosnetupitant chloride monohydrochloride;1643757-72-5

71544786

White to off-white solid powder

1.86

1

13

8

47

1100

0

HZIYEEMJNBKMJH-UHFFFAOYSA-N

InChI=1S/C31H35F6N4O5P/c1-20-8-6-7-9-24(20)25-17-27(40-10-12-41(5,13-11-40)19-46-47(43,44)45)38-18-26(25)39(4)28(42)29(2,3)21-14-22(30(32,33)34)16-23(15-21)31(35,36)37/h6-9,14-18H,10-13,19H2,1-5H3,(H-,43,44,45)

(4-(5-(2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethylpropanamido)-4-(o-tolyl)pyridin-2-yl)-1-methylpiperazin-1-ium-1-yl)methyl hydrogen phosphate

07PNET; 07-PNET; Fosnetupitant chloride; Akynzeo; trade name: Akynzeo

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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