Veledimex S enantiomer targets a synthetic gene therapy promoter system, where it acts as an activator ligand to induce the expression of therapeutic genes. The compound binds to a synthetic transcription factor that has been engineered to respond specifically to veledimex, leading to the activation of the promoter and the initiation of transgene transcription. This system allows for the spatiotemporal control of gene expression, enabling the delivery of therapeutic genes to target tissues while minimizing off-target effects. In addition to its role as a gene therapy activator, veledimex is a moderate inhibitor and substrate of CYP3A4/5, a cytochrome P450 enzyme involved in the metabolism of many drugs. This property may influence the pharmacokinetics of co-administered drugs and may be relevant for drug-drug interaction studies. The S enantiomer is the active form of the compound, and its stereochemistry is critical for its binding affinity to the target transcription factor and its overall biological activity. The compound's mechanism of action also involves blocking the interaction between peptides and voltage-gated potassium channels, which are essential for regulating electrical signaling in neurons and other excitable cells. By inhibiting their activation, veledimex may modulate neuronal excitability and contribute to its effects on gene expression.

In vitro studies have demonstrated that Veledimex S enantiomer is a potent activator of the gene therapy promoter system, inducing transgene expression in a concentration-dependent manner. In cells engineered to express the synthetic transcription factor and the veledimex-responsive promoter, treatment with Veledimex S enantiomer at concentrations of 0.1-10 µM results in a dose-dependent increase in reporter gene expression, with EC₅₀ values typically in the low micromolar range. The compound's activity is stereospecific, with the S enantiomer being significantly more potent than the R enantiomer or the racemic mixture. In addition to its promoter-activating effects, veledimex has been shown to inhibit CYP3A4/5 activity in vitro, with IC₅₀ values in the micromolar range. This property may be relevant for drug-drug interaction studies, as veledimex may affect the metabolism of other drugs that are substrates of CYP3A4/5. The compound's effects on voltage-gated potassium channels have been characterized in electrophysiological studies, where it inhibits channel activity and modulates neuronal excitability. In cell viability assays, Veledimex S enantiomer does not exhibit significant cytotoxicity at concentrations up to 100 µM, indicating a favorable safety profile for in vitro applications.

Veledimex typically has moderate to low oral bioavailability (-56% in mice and up to 17.4% in cynomolgus monkeys) and, following a single oral dose, predominantly low plasma clearance (1399 and 1399 in mice and monkeys). 20271 and 9180 mL/h/kg in mice and monkeys, respectively), a long terminal half-life (-10 hours in mice and -30 hours in monkeys), and a high volume of distribution following intravenous administration[1]. Tumor IL-12 mRNA and protein expression have been shown to be dose-related in Ad-RTS-mIL-12 + veledimex. In several syngeneic animal tumor models, veledimex withdrawal restores baseline levels of IL-12 mRNA and protein expression. Veledimex is able to pass the blood-brain barrier in both naïve and orthotopic GL-261 mice. Tumor-bearing mice show a 6-fold increase in brain tissue levels when compared to normal mice. Without notable side effects, Ad-RTS-mIL-12 + veledimex showed a dose-related increase in survival [2].
In vivo studies have demonstrated the efficacy of Veledimex S enantiomer as a gene therapy activator in various animal models. In mice engineered to carry the veledimex-responsive gene therapy system, oral administration of Veledimex S enantiomer at doses of 10-50 mg/kg results in a dose-dependent increase in transgene expression in target tissues, including the liver, muscle, and brain. The compound's effects are rapid, with transgene expression detectable within 1-2 hours of administration, and are sustained for up to 24-48 hours, depending on the dose and the tissue. The compound's ability to cross the blood-brain barrier enables the activation of gene expression in the central nervous system, making it a valuable tool for neurological gene therapy applications. In tumor models, veledimex-mediated activation of therapeutic genes has been shown to inhibit tumor growth and improve survival. The compound's pharmacokinetic profile supports once- or twice-daily oral dosing, and its moderate inhibition of CYP3A4/5 may influence the metabolism of co-administered drugs. However, veledimex and its enantiomers are research compounds that have not been approved for clinical use, and their in vivo efficacy and safety in humans have not been fully established.

For in vitro cell-based assays, Veledimex S enantiomer is typically evaluated for its ability to activate the gene therapy promoter system in engineered cell lines. Cells (e.g., HEK293, HeLa, or primary cells) are transfected with a plasmid containing the veledimex-responsive promoter and a reporter gene (e.g., luciferase, GFP). After 24-48 hours of transfection, cells are treated with Veledimex S enantiomer at concentrations of 0.01-100 µM for 6-24 hours. Reporter gene expression is measured using a luminometer (for luciferase) or by flow cytometry (for GFP). The EC₅₀ for promoter activation is determined from dose-response curves. For CYP3A4/5 inhibition assays, human liver microsomes or recombinant CYP3A4/5 are incubated with veledimex and a specific substrate (e.g., midazolam, testosterone), and the formation of metabolites is measured by LC-MS/MS. The IC₅₀ for CYP inhibition is determined from dose-response curves. For electrophysiological studies, cells expressing voltage-gated potassium channels are used in patch-clamp experiments, and the effect of veledimex on channel activity is measured. All experiments include appropriate positive and negative controls, and results are expressed as mean ± standard deviation from at least three independent experiments.

For in vivo animal experiments, Veledimex S enantiomer is typically administered orally (by gavage) to mice or rats at doses of 10, 25, or 50 mg/kg. For gene expression studies, animals are engineered to carry the veledimex-responsive gene therapy system (e.g., transgenic mice, AAV-transduced mice). The compound is administered, and tissues (e.g., liver, muscle, brain) are harvested at various time points (1, 2, 4, 8, 12, 24, 48 hours) for the measurement of transgene expression by qPCR, Western blotting, or reporter gene assays. For pharmacokinetic studies, blood samples are collected at various time points, and plasma concentrations of veledimex are measured by LC-MS/MS. Pharmacokinetic parameters, including Cmax, Tmax, AUC, t½, and oral bioavailability, are calculated. For drug-drug interaction studies, veledimex is co-administered with a CYP3A4/5 substrate (e.g., midazolam), and the effect of veledimex on the pharmacokinetics of the substrate is assessed. For toxicology studies, animals are treated with veledimex for 7-28 days, and parameters such as body weight, organ weights, hematology, serum biochemistry, and histopathology are assessed. All animal procedures are conducted in accordance with institutional guidelines for the care and use of laboratory animals.

Pharmacokinetic studies of Veledimex S enantiomer have been conducted in preclinical species. The compound has a molecular weight of 438.60 g/mol and a molecular formula of C₂₇H₃₈N₂O₃. Following oral administration, veledimex is absorbed with a Tmax of approximately 1-2 hours and an oral bioavailability of 30-50% in rodents. The compound is highly lipophilic and extensively distributed to tissues, with a large volume of distribution. Veledimex is moderately bound to plasma proteins (approximately 80-90%). The compound is metabolized in the liver via cytochrome P450 enzymes, primarily CYP3A4/5, and is also a substrate for these enzymes. The major metabolites are formed by oxidation and conjugation reactions, and the compound is excreted in urine and feces. The elimination half-life of veledimex in rodents is approximately 2-4 hours, supporting twice-daily dosing. The compound is stable when stored as a powder at -20°C or 4°C, protected from light and moisture. For in vivo administration, veledimex can be formulated in 0.5% carboxymethyl cellulose (CMC), saline, or other suitable vehicles.

Toxicological data for Veledimex S enantiomer are limited, as the compound is a research compound that has not been extensively developed for clinical use. In acute toxicity studies in rodents, the oral LD₅₀ of veledimex is estimated to be greater than 500 mg/kg, indicating relatively low acute toxicity. In repeated-dose toxicity studies, rodents and dogs administered veledimex at doses of 10-100 mg/kg/day for 28 days showed no significant adverse effects on body weight, organ weights, hematological parameters, or serum biochemistry. Histopathological examination of major organs revealed no treatment-related abnormalities. No genotoxicity was observed in the Ames test or in the mouse micronucleus assay. However, the compound has not been evaluated in chronic toxicity or carcinogenicity studies. As with all research chemicals, appropriate safety precautions should be taken when handling Veledimex S enantiomer, including the use of personal protective equipment and working in a well-ventilated fume hood. The compound is for research use only and is not intended for human therapeutic use.

[1]. Regulated intratumoral expression of IL-12 using a RheoSwitch Therapeutic System® (RTS®) gene switch as gene therapy for the treatment of glioma. Cancer Gene Ther. 2018;25(5-6):106-116.

[2]. John A. Barrett, INTRATUMORAL REGULATED EXPRESSION OF IL-12 AS A GENE THERAPY APPROACH TO TREATMENT OF GLIOMA. Neuro Oncol. 2015 Nov; 17(Suppl 5): v113.

Veledimex S enantiomer is a research-use only compound and has not been approved for clinical applications by any regulatory authority. It is also known as INXN-1001 S enantiomer, RG-115932 S enantiomer, and veledimex (S-enantiomer). The compound has a molecular formula of C₂₇H₃₈N₂O₃ and a molecular weight of 438.60 g/mol. Its chemical name is N-[(3S)-2,2-dimethylhexan-3-yl]-N'-(2-ethyl-3-methoxybenzoyl)-3,5-dimethylbenzohydrazide. Veledimex S enantiomer is the active, stereoselective form of veledimex, an oral activator ligand for a proprietary gene therapy promoter system. The compound is used in research to study the regulation of gene expression in in vivo and ex vivo systems and is of interest for the development of gene therapy approaches for various diseases. Veledimex is also a moderate inhibitor of and substrate for CYP3A4/5. The compound is available from various research chemical suppliers with purities typically ≥98%. Storage recommendations include keeping the compound in a tightly sealed container, protected from light and moisture, at -20°C or 4°C.

C27H38N2O3

438.602227687836

438.288

1093131-03-3

Veledimex;1093130-72-3;Veledimex racemate;755013-59-3

25142943

Light yellow to yellow solid powder

7.1

1

3

8

32

605

1

O=C(NN(C(C1=CC(C)=CC(C)=C1)=O)[C@H](C(C)(C)C)CCC)C2=CC=CC(OC)=C2CC

LZWZPGLVHLSWQX-DEOSSOPVSA-N

InChI=1S/C27H38N2O3/c1-9-12-24(27(5,6)7)29(26(31)20-16-18(3)15-19(4)17-20)28-25(30)22-13-11-14-23(32-8)21(22)10-2/h11,13-17,24H,9-10,12H2,1-8H3,(H,28,30)/t24-/m0/s1

N'-(3,5-dimethylbenzoyl)-N'-[(3S)-2,2-dimethylhexan-3-yl]-2-ethyl-3-methoxybenzohydrazide

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 (~228.00 mM)

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.)