In mouse L1210 leukemia cells, trichomsalen (200 nM) combined with 365 nm light causes DNA cross-links but not breaks [4]. A synergistic effect of treating HaCaT keratinocytes with trioxsalen and UVA is the induction of NF-kappaB binding activity [5]. In vitro, trichsalen has demonstrated anti-inflammatory properties [5].

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Treatment of mouse L1210 leukemia cells with Trioxsalen followed by 365 nm ultraviolet light produced DNA interstrand crosslinks. This was demonstrated by the alkaline elution technique, where the crosslinks caused a significant reduction in the elution of DNA from cells that had been pre-exposed to 300 rad of X-ray (to introduce DNA single-strand breaks). The crosslinking effect was not observed in cells treated with Trioxsalen alone or light alone, indicating that both are required for the effect and that the treatment itself does not introduce DNA strand breaks. [4]
The frequency of DNA crosslinking, quantified by a crosslinking coefficient, was found to be a first-order linear function of Trioxsalen concentration. At a fixed light dose (9240 J/m²), increasing concentrations of Trioxsalen (from approximately 1·10⁻⁹ M to 2·10⁻⁸ M) resulted in a proportional increase in the crosslinking coefficient. [4]
The kinetics of crosslink formation were found to be a second-order function of the 365 nm light exposure time. At a fixed Trioxsalen concentration (2·10⁻⁸ M), the crosslinking coefficient was linearly dependent on the square of the light exposure time (ranging from 0 to 120 seconds). This supports the proposed two-photon mechanism for the formation of diadducts (crosslinks). [4]
In contrast to other DNA crosslinking agents like nitrogen mustard, the DNA crosslinks induced by Trioxsalen plus UVA were resistant to proteolytic digestion with proteinase-K. This resistance was confirmed using three different deproteinization methods, indicating that the crosslinks are exclusively of the interstrand type and do not involve DNA-protein crosslinks. [4]

In rat oral mucosa (OMM), doxsalen induces dose-dependent depletion of ATPase-positive LC using PUVA (UVA dose of 1-8 J/cm2), with a maximum depletion of 80% [3].

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The efficacy of hospital-administered bath Trioxsalen-UV-A (PUVA) was compared with home-based oral methoxsalen PUVA in a randomized controlled trial of 158 patients with chronic hand eczema. The hospital group (n=80) received 20 bi-weekly treatments over 10 weeks. Each treatment consisted of a 15-minute hand soak in a bath containing Trioxsalen at a concentration of 0.2 mg/L, followed by UV-A irradiation. The primary outcome, measured by a hand eczema score (0-21 scale evaluating desquamation, erythema, vesiculation, infiltration, fissures, itch, and pain), showed a statistically significant clinical improvement. At the end of the 10-week treatment period, the mean hand eczema score for the hospital group decreased from a baseline of 8.1 to 5.6, representing a mean reduction of 2.5 points (31% improvement; 95% CI, 1.7-3.2; P < .001). This improvement was maintained during an 8-week follow-up period, with a mean score of 5.4 at the end of follow-up. The study concluded that the efficacy of hospital-administered bath Trioxsalen PUVA was clinically comparable to that of the home-based oral PUVA regimen. [1]

Alkaline Elution Assay for DNA Crosslinks in Mouse L1210 Leukemia Cells: Mouse L1210 leukemia cells were grown in suspension culture and labeled with [¹⁴C]-thymidine. For experimental treatment, cells were washed and resuspended in an irradiation medium. Trioxsalen, dissolved in ethanol (final ethanol concentration < 0.5% v/v), was added to the cell suspension at desired concentrations (ranging from 1·10⁻⁹ M to 4·10⁻⁷ M). After a 60-second preincubation period, the cell suspension was exposed to monochromatic 365 nm light from a mercury-xenon arc lamp at an intensity of 77 W/m² for varying durations (0-120 seconds) at 22-24°C. Following light exposure, cells were immediately cooled to 0-4°C and then exposed to 300 rad of X-ray at 0°C to introduce a known frequency of DNA single-strand breaks. Alkaline elution was then performed: cells were layered onto a poly(vinyl chloride) filter, lysed with a solution containing sarcosyl, NaCl, and EDTA, and then DNA was eluted with a tetrapropylammonium hydroxide-EDTA solution (pH 12.1) pumped at ~0.04 ml/min for 15 hours. Fractions were collected, and the radioactivity was measured to determine the fraction of DNA retained on the filter. A higher retention of DNA from drug-treated cells compared to control cells indicated the presence of DNA crosslinks. A crosslinking coefficient (Kc) was calculated to quantify crosslink frequency. [4]
Proteolytic Digestion Assay to Distinguish Crosslink Type: To determine whether the observed crosslinks were DNA-protein or DNA-interstrand, a proteolytic digestion step was incorporated into the alkaline elution procedure using three different methods. In Method 1, after routine cell lysis on a poly(vinyl chloride) filter, the filter was incubated for 1 hour at room temperature with lysis solution containing 0.5 mg/ml proteinase-K before elution. In Method 2, the proteinase-K solution was layered on top of the filter after lysis, and elution was begun. In Method 3, cells were lysed on a polycarbonate filter with a 2% SDS/EDTA solution containing proteinase-K before elution. The crosslinking coefficient for Trioxsalen-treated cells was then compared with and without proteinase-K treatment. A reduction in the crosslinking coefficient after proteinase-K treatment would indicate the presence of DNA-protein crosslinks. [4]

In the clinical trial, one patient in the hospital group (bath Trioxsalen PUVA) discontinued treatment due to skin burning, a known side effect of PUVA therapy. Mild stinging was also reported as a side effect in this group. [1]

[1]. Effectiveness of psoralen photochemotherapy for vitiligo. J Formos Med Assoc. 1999 May;98(5):335-40.

[2]. Comparison of oral psoralen-UV-A with a portable tanning unit at home vs hospital-administered bath psoralen-UV-A in patients with chronic hand eczema: an open-label randomized controlled trial of efficacy. Arch Dermatol. 200.

[3]. The effect of PUVA on langerhans cells in rat oral epithelium photosensitized with systemic methoxsalen or topical trioxsalen. Photodermatol Photoimmunol Photomed. 2000 Jun;16(3):129-33.

[4]. Interstrand DNA crosslinking by 4,5'8-trimethylpsoralen plus monochromatic ultraviolet light. Studies by alkaline elution in mouse L1210 leukemia cells. Biochim Biophys Acta. 1980 Nov 14;610(1):56-63.

[5]. Trioxsalen in the presence of UVA is able to induce nuclear factor kappa B binding activity in HaCaT keratinocytes. Skin Pharmacol Appl Skin Physiol. Sep-Oct 2002;15(5):335-41.

Trioxsalen is a 7H-furan[3,2-g]chromen-7-one with methyl groups at positions 2, 5, and 9. Like other psoralen compounds, trioxsalen causes photosensitivity in the skin. It is used orally in combination with UV-A for phototherapy of vitiligo. Upon photoactivation, it forms interstrand crosslinks in DNA, inhibiting DNA synthesis and cell division, and may cause cell damage; after cell damage heals, epidermal melanin deposition may increase. It is both a photosensitizer and a dermatological drug. Trioxsalen (trimethylpsoralen, trioxsalen, or tripsoralen) is a furanocoumarin compound and a psoralen derivative extracted from various plants (primarily psoralen). Like other psoralen compounds, it causes photosensitivity in the skin. It can be administered topically or orally and in combination with UV-A (the least damaging form of ultraviolet radiation) for phototherapy of vitiligo and hand eczema. The photoactivated form creates interstrand crosslinks in DNA, leading to apoptosis. In studies, it can be combined with confocal microscopy dyes for visualization of DNA damage sites. [3] This compound has been used to develop antisense oligonucleotides that can specifically crosslink with mutant mRNA sequences without affecting normal transcripts even if there is only one base pair different.
Trioxalin is a psoralen.
Trioxalin has been reported to be found in celery (Apium graveolens), and there is relevant data.
A pigment photosensitizer extracted from a variety of plants, primarily Psoralea corylifolia. It can be administered topically or orally and in combination with ultraviolet light for the treatment of vitiligo.

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Drug Indications
Trioxalin is a pigment photosensitizer used in combination with ultraviolet light for the treatment of vitiligo.

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Mechanism of Action
Upon photoactivation, it forms interstrand crosslinks in DNA, leading to programmed cell death.

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Pharmacodynamics
Trioxazone itself has no pharmacological activity, but when exposed to ultraviolet light or sunlight, it is converted into its active metabolites, which can have a beneficial effect on diseased tissues.

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Trioxsalen is a psoralen derivative used in photochemotherapy (PUVA) for skin conditions like hand eczema. Its therapeutic effect results from the inhibition of DNA synthesis due to the formation of photoadducts between the psoralen and pyrimidine bases in DNA upon subsequent irradiation with UV-A light. [1]
In this study, Trioxsalen was administered via a bath (0.2 mg/L) for 15 minutes prior to UV-A exposure. This method of application is a form of local, topical PUVA treatment specifically for hand eczema. [1]
Hospital bath PUVA was a commonly used treatment in the Netherlands at the time of the study, utilized in 76% of surveyed dermatology centers, which justified its selection as the comparator against the home-based oral PUVA regimen. [1]

228.078

3902-71-4

5585

White to off-white solid powder

1.2±0.1 g/cm3

389.0±30.0 °C at 760 mmHg

229-231ºC(lit.)

189.1±24.6 °C

0.0±0.9 mmHg at 25°C

1.613

3.8

0

3

0

17

374

0

O1C(C([H])([H])[H])=C([H])C2=C([H])C3C(C([H])([H])[H])=C([H])C(=O)OC=3C(C([H])([H])[H])=C12

FMHHVULEAZTJMA-UHFFFAOYSA-N

InChI=1S/C14H12O3/c1-7-4-12(15)17-14-9(3)13-10(6-11(7)14)5-8(2)16-13/h4-6H,1-3H3

2,5,9-trimethylfuro[3,2-g]chromen-7-one

NSC71047 NSC 71047 NSC-71047

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 : ~6.6 mg/mL (~28.92 mM)
H2O : ~1 mg/mL (~4.38 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.)