TRPV1

Nonivamide is a synthetic capsaicin derivative that has strong antifouling properties. For capsaicin, the 4d-EC50 values for Pseudomonas putida, Lake Erie bacteria, and Vibrio natrigeni were 5.5±0.5 mg/L, 23±2 mg/L, and 6.9±0.2 mg/L and 15.6±0.4 mg/L, and V. parahaemolyticus, respectively, in static toxicity experiments. The 1 mg/L Nonivamide treatment group showed significant growth inhibition for 4 days (p<0.01), with an EC50 value of 5.1 mg/L for the 4 day-EC50[1]. Treatment with nonivamide releases calcium from the endoplasmic reticulum (ER) and modifies transcription of growth arrest and DNA damage-induced transcript 3 (GADD153), GADD45α, GRP78/BiP, ATF3, CCND1, and CCNG2) in a way characteristic of usual ER stress. After pretreating cells with 2.5 μM thapsigargin for 5 min, ER calcium flow was measured by adding 2.5 μM Nonivamide. Calcium from ER reserves was released when 2.5 μM Nonivamide was given to TRPV1-overexpressing cells, leading to a notable rise in cytosolic calcium. After 24 hours, treating TRPV1-overexpressing cells with 1 μM Nonivamide led to a roughly 50% reduction in cell viability. Additionally, BEAS-2B cells treated with 100 and 200 μM Nonivamide displayed an increase in GADD153 mRNA and protein expression as well as alterations in the relative amount of EIF2α-P [2]. At every examined concentration, the effects of nonivamide treatment on lipid buildup were identical to those following CAP treatment in terms of reduction. Nonivamide treatment decreased lipid accumulation by 5.34±1.03% (P<0.05) at 0.01 µM and by 10.4±2.47% (P<0.001) at 1 µM in comparison to untreated control cells [3].

In this study, researchers investigated the effects of nonivamide (pelargonic acid vanillylamide, PAVA; 1 mg/kg) and rosuvastatin (RSV; 10 mg/kg) on hepatic steatosis induced by a high-fat diet (HFD). Male Sprague-Dawley rats were fed a HFD for 16 weeks then received PAVA or RSV for 4 additional weeks. We examined the metabolic parameters, function, fat content, histological alterations, reactive oxygen species production, and apoptotic cell death of the liver, in addition to the expression of the following important molecules: transient receptor potential cation channel subfamily V member 1 (TRPV1) phosphorylation of sterol regulatory element binding protein (pSREBP-1c/SREBP-1c), total and membrane glucose transporter 2 (GLUT2), 4-hydroxynonenal (4-HNE), and cleaved caspase-3. HFD-induced hepatic steatosis was associated with significantly increased morphological disorganization, injury markers, oxidative stress, lipid peroxidation, and apoptosis. However, metabolic dysfunction and hepatic injury were reduced by RSV and PAVA treatment. PAVA regulated lipid deposition, improved insulin resistance, and decreased oxidative stress and apoptotic cell death. Therefore, PAVA represents a promising therapeutic approach for treating metabolic disorders in patients with NAFLD[4].

SOD enzyme assay[1]
A sample supernatant of 0.1 ml was mixed with 0.4-ml solution 1, 0.04-ml solution 2, 0.04-ml solution 3, and 0.04-ml solution 4. For the control, 0.1-ml distilled water was mixed with 0.4-ml solution 1, 0.04-ml solution 2, 0.04-ml solution 3, and 0.04-ml solution 4. After preparation, both mixtures were incubated in 37°C for 40 min. Then 0.6-ml chromogenic reagent was added into them. After 10-min reaction, the OD550 values were recorded. The results were calculated by using the formula: SOD activity = (ODCONTROL – ODSAMPLE)/ODCONTROL.

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POD enzyme assay[1]
A sample supernatant of 0.1 ml was mixed with 2.4-ml solution 1, 0.3-ml solution 2, and 0.2-ml solution 3. For the control, solution 3 was replaced by distilled water of the same volume. After preparation, both the sample and the control were bathed in 37°C for 30 min. Then 0.1-ml solution 4 was separately added into two mixtures. Supernatants of the sample and control were obtained by centrifugation and the OD420 values were recorded. The results were calculated by using the formula: POD activity = ODSAMPLE – ODCONTROL.

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CAT enzyme assay[1]
The reaction solution supplied by the reagent kit was incubated in 28°C for 10 min. Then, 3 ml of this reaction solution was mixed with 0.5-ml sample supernatant. After the preparation, the OD240 value of the mixture was measured for two times with an interval time of 1 min recorded as OD1 and OD2, respectively. The results were calculated using the formula: CAT activity = 2.303 × log (OD1/OD2)/60.

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ALP enzyme assay[1]
A sample supernatant of 0.03 ml was mixed with 0.5-ml buffer solution and 0.5-ml substrate solution. For the standard, 0.03 ml was mixed with a 0.5-ml buffer solution and 0.5-ml substrate solution, which was supplied by the reagent kit. For the standard, 0.03-ml distilled water was mixed with 0.5-ml buffer solution and 0.5-ml substrate solution. After preparation, the mixtures were water-bathed in 37°C for 15 min. Then, 1.5-ml chromogenic reagent was added into each mixture and the OD520 value was measured. The results were calculated using the formula: ALP activity = ODSAMPLE/ODSTANDARD.

Cells were seeded in 96‐well plates and treated with 1 nM–10 µM CAP or nonivamide with or without addition of 25–100 µM BCH or the corresponding ethanol concentration (0.1–0.2% (v/v), solvent control) for 12 days after initiation of differentiation. Cell culture media was exchanged every second day. On Day 12, 100 µl of the MTT working reagent (0.83 mg/ml MTT diluted in PBS/serum‐free media (1:5)), was added to each well, and cells were incubated at 37°C for approximately 15 min. The MTT working solution was removed and the purple formazan formed during incubation was dissolved in 150 µl DMSO per well. Absorbance was measured at 550 nm with 690 nm as reference wavelength using multiwell plate reader. The number of metabolically active cells was calculated relative to untreated control cells or the corresponding solvent control (100%).[3]

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Transient receptor potential vanilloid 1 (TRPV1) is a calcium-selective ion channel expressed in human lung cells. We show that activation of the intracellular subpopulation of TRPV1 causes endoplasmic reticulum (ER) stress and cell death in human bronchial epithelial and alveolar cells. TRPV1 agonist (nonivamide) treatment caused calcium release from the ER and altered the transcription of growth arrest- and DNA damage-inducible transcript 3 (GADD153), GADD45alpha, GRP78/BiP, ATF3, CCND1, and CCNG2) in a manner comparable with prototypical ER stress-inducing agents. The TRPV1 antagonist N-(4-tert-butylbenzyl)-N'-(1-[3-fluoro-4-(methylsulfonylamino)-phenyl]ethyl)thiourea (LJO-328) inhibited mRNA responses and cytotoxicity. EGTA and ruthenium red inhibited cell surface TRPV1 activity, but they did not prevent ER stress gene responses or cytotoxicity. Cytotoxicity paralleled eukaryotic translation initiation factor 2, subunit 1 (EIF2alpha) phosphorylation and the induction of GADD153 mRNA and protein. Transient overexpression of GADD153 caused cell death independent of agonist treatment, and cells selected for stable overexpression of a GADD153 dominant-negative mutant exhibited reduced sensitivity. Salubrinal, an inhibitor of ER stress-induced cytotoxicity via the EIF2alphaK3/EIF2alpha pathway, or stable overexpression of the EIF2alpha-S52A dominant-negative mutant also inhibited cell death. Treatment of the TRPV1-null human embryonic kidney 293 cell line with TRPV1 agonists did not initiate ER stress responses. Likewise, n-benzylnonanamide, an inactive analog of nonivamide, failed to cause ER calcium release, an increase in GADD153 expression, and cytotoxicity. We conclude that activation of ER-bound TRPV1 and stimulation of GADD153 expression via the EIF2alphaK3/EIF2alpha pathway represents a common mechanism for cytotoxicity by cell-permeable TRPV1 agonists. These findings are significant within the context of lung inflammatory diseases where elevated concentrations of endogenous TRPV1 agonists are probably produced in sufficient quantities to cause TRPV1 activation and lung cell death.[2]

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Red pepper and its major pungent principle, capsaicin (CAP), have been shown to be effective anti-obesity agents by reducing energy intake, enhancing energy metabolism, decreasing serum triacylglycerol content, and inhibiting adipogenesis via activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1). However, the binding of CAP to the TRPV1 receptor is also responsible for its pungent sensation, strongly limiting its dietary intake. Here, the effects of a less pungent structural CAP-analog, nonivamide, on adipogenesis and underlying mechanisms in 3T3-L1 cells were studied. Nonivamide was found to reduce mean lipid accumulation, a marker of adipogenesis, to a similar extent as CAP, up to 10.4% (P < 0.001). Blockage of the TRPV1 receptor with the specific inhibitor trans-tert-butylcyclohexanol revealed that the anti-adipogenic activity of nonivamide depends, as with CAP, on TRPV1 receptor activation. In addition, in cells treated with nonivamide during adipogenesis, protein levels of the pro-adipogenic transcription factor peroxisome-proliferator activated receptor γ (PPARγ) decreased. Results from miRNA microarrays and digital droplet PCR analysis demonstrated an increase in the expression of the miRNA mmu-let-7d-5p, which has been associated with decreased PPARγ levels[3].

Male Sprague–Dawley rats (150–180 g) were were housed under a 12-h dark/light cycle with a constant temperature (25 ± 1 °C). After a 1-week acclimation period, the rats were randomly divided into normal chow diet (NCD) and high-fat diet (HFD) groups. The control group received standard rat chow, with fat representing 19.79% of total calories. This group was further divided into three groups (n = 6 per group): NCD, NCD plus 1 mg PAVA per kilogram body weight (BW) (N + PAVA), and NCD plus 10 mg rosuvastatin per kilogram BW (N + RSV). The HFD rats were fed a HFD for 20 weeks, with fat representing 65.26% of total calories. The HFD rats were divided into three groups (n = 6 per group): HFD, HFD plus PAVA (H + PAVA), and HFD plus rosuvastatin (H + RSV). After 16 weeks, the H + PAVA and H + RSV groups received PAVA or RSV for the final 4 weeks of the experimental period. Nonivamide (purity ≥ 96%) was dissolved in 10% Tween 80 and 10% ethanol in 0.9% normal saline solution. Animals in the PAVA groups were injected with 1 mg PAVA/kg/day via subcutaneous injection. Rosuvastatin was freshly dissolved in 0.5% carboxymethyl cellulose and administered by gastric gavage at a dose of 10 mg/kg/day for 4 weeks.21 The rats in the NCD and HFD control groups received the same vehicle as the treatment groups. The selected dose of PAVA was based on the results of a preliminary study, and the RSV dose was based on previous reports in rats which demonstrated its potential to improve metabolic parameters.22,23 The body weight and food and water intake of rats were recorded weekly. Metabolic parameters and oral glucose tolerance test (OGTT) were performed before and after the treatments. At the end of the study, the rats were euthanized and blood and liver tissue samples were collected for subsequent experiments.[4]

Metabolism / Metabolites
Limited information is available on pharmacokinetics and metabolism of nonivamide. For the closely related [DB06774] and other capsaicinoids, gastrointestinal absorption is rapid and almost entirely complete in studies of rats (oral dose of about 10 to 15 mg/kg of combined capsaicin and dihydrocapsaicin) with about 85% of the dose being absorbed within 3 hours. Both substances undergo first-pass metabolism in the liver and partly metabolized at the site of absorption.

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Biological Half-Life
About 7 minutes.

mouse LD50 intraperitoneal 8 mg/kg Journal of Medicinal Chemistry., 36(2595), 1993

[1]. Toxic effects of environment-friendly antifoulant Nonivamide on Phaeodactylum tricornutum. Environ Toxicol Chem. 2013 Apr;32(4):802-9.

[2]. Transient receptor potential vanilloid 1 agonists cause endoplasmic reticulum stress and cell death in human lung cells. J Pharmacol Exp Ther. 2007 Jun;321(3):830-8.

[3]. Nonivamide enhances miRNA let-7d expression and decreases adipogenesis PPARγ expression in 3T3-L1 cells. J Cell Biochem. 2015 Jun;116(6):1153-63.

[4]. Capsaicinoid nonivamide improves nonalcoholic fatty liver disease in rats fed a high-fat diet. J Pharmacol Sci . 2020 Jul;143(3):188-198.

Nonivamide is a capsaicinoid that is the carboxamide resulting from the formal condensation of the amino group of 4-hydroxy-3-methoxybenzylamine with the carboxy group of nonanoic acid. It is the active ingredient in many pepper sprays. It has a role as a lachrymator. It is a capsaicinoid and a member of phenols.
Nonivamide is found in herbs and spices. It is an alkaloid from the Capsicum species. The structures of [DB06774] and nonivamide differ only slightly with respect to the fatty acid moiety of the side chain (8-methyl nonenoic acid versus nonanoic acid). Nonivamide is a flavoring ingredient. Nonivamide is an organic compound and a capsaicinoid. It is an amide of pelargonic acid and vanillylamine. It is naturally found in chili peppers but manufactured to produce a synthetic form for various pharmacologic preparations. This drug has been studied in combination with Nicarboxil in the treatment of lower back pain. Nonivamide has also been studied for its anti-inflammatory properties, as well as in fat loss therapies and has demonstrated promising results,,,.
Nonivamide has been reported in Capsicum annuum with data available.
See also: Paprika (part of); Methyl salicylate; nonivamide (component of) ... View More ...

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Drug Indication
Nonivamide is used as a topical analgesic and is also used as a flavoring ingredient,,.

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Mechanism of Action
Nonivamide is a naturally occurring analog of [DB06774], isolated from peppers, described to produce effects similar to [DB06774]. It is an agonist of the VR1 (vanilloid/TRPV1 receptor). It serves as a transient agonist of these receptors, which are potentiated by pro-inflammatory drugs, a phenomenon that leads to thermal hyperalgesia, or increased heat sensation. Nonivamide has been shown to stimulate afferent neurons with about half the potency of [DB06774]. Agonism of the VR1 (TRPV1) (vanilloid) receptor by Nonivamide was demonstrated to induce the release of Ca2+ from the endoplasmic reticulum (ER) of human lung cells, producing ER stress and cell death [MSDS]. Nonivamide, like other capsaicinoids, acts on the vanilloid receptors located in the peripheral afferent nerve fibers, providing short-acting irritant and algesic properties. Applied dermally, these substances act by stimulating sensitive chemoreceptors of the skin and by reflex, hyperemia and a local elevation in temperature. After repetitive administration, capsaicinoids have been reported to lead to desensitization to nociceptive stimuli possibly by long-acting depletion of peptide neurotransmitters (substance P) from peripheral sensory neurons. Capsaicinoids can modulate muscle tone (in bladder, bronchus etc.). Intravenous injection of nonivamide to rats (10 μg/kg) has been found to lead to bradycardia. The cardiovascular effects are partly explained by substance P release. Nonivamide given to rats subcutaneously (1 mg/kg) was found to cause body temperature decrease, vasodilatation, and increased salivation. Capsaicinoids have shown to illicit bronchospastic effects in guinea pigs. Capsaicin and its analogs were reported to increase barbiturate sleeping time in rats by interacting with hepatic metabolizing enzymes.

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Pharmacodynamics
Relieves minor aches and pains of muscles and joints,.

C18H27NO3

305.4119

293.199

C, 66.43; H, 8.20; N, 4.56; O, 20.82

2444-46-4

2998

White to off-white solid powder

1.0±0.1 g/cm3

450.4±55.0 °C at 760 mmHg

54°C

226.2±31.5 °C

0.0±1.2 mmHg at 25°C

1.509

4.38

2

3

10

21

283

0

RGOVYLWUIBMPGK-UHFFFAOYSA-N

InChI=1S/C17H27NO3/c1-3-4-5-6-7-8-9-17(20)18-13-14-10-11-15(19)16(12-14)21-2/h10-12,19H,3-9,13H2,1-2H3,(H,18,20)

N-[(4-hydroxy-3-methoxyphenyl)methyl]nonanamide

NONIVAMIDE; 2444-46-4; N-Vanillylnonanamide; Pseudocapsaicin; N-Vanillylnonamide; Pelargonic acid vanillylamide; N-(4-Hydroxy-3-methoxybenzyl)nonanamide; N-Vanillylpelargonamide;

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)

DMSO : ~100 mg/mL (~340.83 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 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 (8.52 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 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 (8.52 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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 (Please use freshly prepared in vivo formulations for optimal results.)