DNA (intercalation) [1]
Enzymes (through degeneration) [1]

The growth and Kir3.2 activity of Kir3.2-transformed cells are inhibited in a concentration-dependent manner by proflavine (0.1-10 μM; 24 hours) [1]. The Kir3.2 mutant's current amplitude was progressively decreased to 27.7±4.3% of the control by proflavine (300 μM) [2].

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Proflavine Hemisulfate binds to DNA, with a preference for alternating purine-pyrimidine sequences. In neutral aqueous solutions, it exhibits a strong absorption maximum at 444 nm. Upon addition of DNA, a pronounced red shift to 460 nm occurs, indicative of intercalation. [1]
Studies using CD spectroscopy showed that Proflavine Hemisulfate relaxes the superhelical organization of DNA, leading to the formation of a B-like structure. Both major and minor grooves become wider with the addition of the drug. [1]
Proflavine Hemisulfate causes frameshift mutation during in vitro DNA replication. Using a single-stranded DNA template and the Klenow fragment of Escherichia coli DNA polymerase I, a novel inhibition of polymerization was found opposite to all pyrimidines in the template when proflavine-template complexes were exposed for 10 seconds to white light. [1]
Proflavine Hemisulfate was found to perform both classical and threading intercalation with the TAR RNA of HIV-1. [1]
The main mode of enzyme action for Proflavine Hemisulfate is by degeneration, and it also prevents the binding of enzyme-substrate complexes. Low concentrations of acriflavine (a related acridine) inhibit RNA synthesis in rat liver mitochondria. [1]
Proflavine Hemisulfate possesses potent activity against various groups of microorganisms, including Gram-positive and Gram-negative bacteria, fungi, parasites, and some viruses. It is used as a topical antiseptic and disinfectant. [1]
Proflavine Hemisulfate can be used as a fluorochrome for automated cell analysis. It can aid in the differentiation of leukocytes and the counting of reticulocytes without altering cell morphology. [1]
Proflavine Hemisulfate binds to ferrous human adult hemoglobin, causing the X-band in the EPR spectrum to display characteristics of the T-state of the ligated tetramer. Consequently, the oxygen affinity for the deoxygenated derivative of ferrous human adult hemoglobin decreases. [1]

Proflavin (20 mg/kg) concentration in whole blood decreased quickly after intravenous injection and stabilized around half an hour later [3].

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Proflavine Hemisulfate can penetrate beyond epidermal and dermal structures and accumulate in the nuclei of cells in vivo. Only cells of the central nervous system did not absorb any proflavine. [1]
A treatment using not more than 0.5g of powdered Proflavine Hemisulfate at a time gave good results in a number of intractable mixed infections. [1]
Proflavine Hemisulfate is still used for umbilical cord care, influencing umbilical cord separation time. [1]

The inhibition of RNA synthesis in rat liver mitochondria by low concentrations of acriflavine (a related acridine) revealed that mitochondrial DNA is involved in the process, indicating the toxic effect of acridines on enzymes. [1]

Cell viability assay [2]
Cell Types: Kir3.2* transformant BYT123 Cell
Tested Concentrations: 0.1, 1 and 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Dose-dependent inhibition of Kir3.2* transformant cell growth. Attenuates the growth of Kir3.2*-transformed cells without affecting the growth of control cells.

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In human cell culture, Proflavine Hemisulfate is known to be taken up by many kinds of cells and is concentrated in the nuclei. [1]

Animal/Disease Models: Adult male Sprague Dawley rats (body weight approximately 200 g) [3]
Doses: 20 mg/kg (pharmacokinetic/PK/PK analysis)
Route of Administration: intravenous (iv) (iv)injection; 20 mg/kg. Results at 2, 4, 5, 10, 15, 20, 25 and 30 minutes after
Route of Administration: Whole blood concentration diminished rapidly in the first 5 minutes after administration, and then diminished slowly.

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Absorption, Distribution and Excretion
Flow cytometry was used to determine the uptake of the fluorescent drug propofol in hepatocyte suspensions from normal rats and rats fed with a carcinogen (2-acetaminofluorouracil, AAF). Drug uptake by hepatocytes from carcinogen-fed rats was consistently lower than that from normal rats. Cell nuclei isolated from the livers of normal and AAF-fed rats showed similar propofol uptake. However, prior administration of a metabolic inhibitor increased drug uptake in AAF-fed rat hepatocytes. Therefore, the difference in drug uptake may reflect changes in the cell membrane and alterations in cellular metabolic integrity. Drug uptake was lower in AAF-fed rat hepatocytes in each cell preparation. However, differences in drug uptake were observed not only between tumors occurring in the livers of these animals but also within the same tumor cell preparation. This study demonstrates that flow cytometry can provide an effective method for analyzing drug uptake by cell populations during hepatocellular carcinoma development. 1. This study investigated the distribution of proxoflavone (PRO) and acridine fibrin (ACR) in channel catfish after intravenous injection (1 mg/kg) or water exposure (10 mg/L, lasting 4 hours). 2. Following intravenous injection, the plasma concentration-time curves of the parent drugs PRO and ACR best conformed to two-compartment and three-compartment pharmacokinetic models, respectively. The terminal elimination half-lives of PRO and ACR in plasma were 8.7 hours and 11.4 hours, respectively. 3. In animals administered 14C-PRO or 14C-ACR, the total drug equivalent concentrations were highest in excretory organs and lowest in muscle, fat, and plasma. In animals administered PRO, residual drug in the liver and trunk kidneys consisted primarily of PRO glucuronide and acetyl conjugates; residual drug in muscle consisted primarily of the parent drug (>95%). In animals treated with ACR, the parent compound accounted for over 90% of the total residues in all tested tissues. 4. During aquatic exposure, the absorption rates of PRO and ACR in catfish were very low. Four hours after exposure, the concentrations of parent PRO and ACR in muscle were 0.064 and 0.020 μg/g, respectively. The concentrations in muscle decreased to below the detection limit (0.005 μg/g) within 1–2 weeks.

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Metabolites/Metabolites
Probetazone (3,6-diaminoacridine) has potential as an anti-infective agent in fish, therefore its metabolism in rainbow trout was investigated. Fourteen hours after intra-arterial injection of 10 mg/kg propofol, three metabolites were detected in liver and bile, and one metabolite was detected in plasma by reversed-phase high-performance liquid chromatography (HPLC) at 262 nm UV detection. Hydrochloric acid treatment converted these three metabolites into propylene glycol, suggesting that these metabolites are propylene glycol conjugates. Treatment with β-glucuronidase and the specific β-glucuronidase inhibitor, gluconic acid 1,4-lactone, indicated that two of the metabolites were propylene glycol glucuronide. To determine UV-Vis absorption spectroscopy and mass spectrometry, the metabolites purified by HPLC were isolated from the liver. Experimental data showed that the propylene glycol metabolites were 3-N-glucuronylpropylene glycol (PG), 3-N-glucuronyl-6-N-acetylpropylene glycol (APG), and 3-N-acetylpropylene glycol (AP). The identity of the metabolites was verified by chemical synthesis. Comparison of the synthesized PG and AP with two metabolites isolated from trout revealed that they had the same molecular weight (determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry). Furthermore, they were co-eluted by high-performance liquid chromatography (HPLC) under different mobile phase conditions. Finally, in vitro incubation with liver subcellular preparations confirmed this property and provided evidence that APG can be formed via glucuronidation of AP or acetylation of PG. A liquid chromatography (LC) method was established for the determination of acridine yellow (ACR) and profenoflavin (PRO) residues in channel catfish muscle. Residues were extracted with acidified methanol solution, and the extract was purified using a C18 solid-phase extraction column. Residue concentrations were determined on an LC cyano column and detected spectrophotometrically at 454 nm. Catfish muscle was supplemented with ACR (purified from a commercially available product) and PRO at concentrations of 5, 10, 20, 40, and 80 ppb (each concentration level was repeated 5 times). At each concentration level, the mean recoveries of the fortified muscle ranged from 86% to 95%, with relative standard deviations (RSDs) ranging from 2.5% to 5.7%. This method was also used to determine the residual amounts of ACR and PRO in the muscle of catfish after exposure to commercially available acridine yellow (total dye concentration 10 ppm, exposure time 4 hours). The RSD of ACR and PRO residues in the muscle after exposure was in the same range as that of fortified muscle. Low residue concentrations (< 1% of the concentration in the exposed water) indicate that the catfish have a low absorption rate of ACR and PRO.

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Proflavine Hemisulfate can be absorbed by the human body through cutaneous, respiratory, and oral routes. [1]
It can penetrate beyond epidermal and dermal structures and accumulate in cell nuclei. [1]

Non-Human Toxicity Values
Intraperitoneal LD50 in mice: 50 mg/kg
Subcutaneous LD50 in mice: 140 mg/kg

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Due to its DNA intercalating property, Proflavine Hemisulfate affects host DNA, which has potential chances to induce skin cancer and other malignancies. After exposure in human skin, it alters the structure of epidermal DNA strands, leading to mutation. [1]
The key mechanism of action for Proflavine Hemisulfate is the generation of Reactive Oxygen Species (ROS), which plays a crucial role in denaturing host DNA. Photo-illumination or sunlight further induces this ROS generation. [1]
The binding of Proflavine Hemisulfate to DNA and subsequent irradiation with visible light can cause single-strand scission. This is consistent with the hypothesis that a free electron is ejected during the excitation of bound proflavine. [1]
Using 2'-deoxynucleotide-proflavine model systems, only guanosine-5'-monophosphate gives rise to substantial quenching of the fluorescence, indicating that guanine residues are responsible for this effect. An enhancement in proflavine fluorescence decay kinetics is correlated with the G-C content of the DNA. [1]
Proflavine Hemisulfate can cause frameshift mutation, potentially leading to various types of malignancies and tumors. [1]

[1]. Proflavine an acridine DNA intercalating agent and strong antimicrobial possessing potential properties of carcinogen. Karbala International Journal of Modern Science. 2017 Dec, 3(4): 272-278.

[2]. Isolation of proflavine as a blocker of G protein-gated inward rectifier potassium channels by a cell growth-based screening system. Neuropharmacology. 2016 Oct;109:18-28.

[3]. Determination of proflavine in rat whole blood without sample pretreatment by laser desorption postionization mass spectrometry. Anal Bioanal Chem. 2017 Apr;409(11):2813-2819.

3,6-Diaminoacrimidine is an aminoacrimidine with amino groups substituted at the 3 and 6 positions. It is a slow-acting antibacterial agent effective against a variety of Gram-positive bacteria (but ineffective against spores), and its salts have been used to treat burns and infected wounds. It has various uses, including as a disinfectant, carcinogen, antibacterial agent, chromophore, and intercalating agent. It is the conjugate base of 3,6-diaminoacrimidine (1+). 3,6-Diaminoacrimidine is primarily used as a topical disinfectant in wound dressings. Propylene hemisulfate is the hemisulfate form of propylene, an acrylidine fluorescent contrast agent and disinfectant with potential applications in cell imaging and disinfection. After topical application of propylene hemisulfate, the propylene diffuses into cells and intercalates into DNA, accumulating and staining the cell nucleus. During fluorescence imaging, the cell nucleus can be observed. This enables the measurement of nuclear morphology and the identification of cancer cells. Furthermore, propofol exerts its antibacterial effect by binding to bacterial DNA, thereby disrupting DNA synthesis and inhibiting bacterial cell growth. It is primarily used as a topical disinfectant in wound dressings. Drug Indications Propofol works by chelating (intercalating) DNA, thereby disrupting DNA synthesis and leading to high-level mutations in the replicating DNA strand. This can prevent bacterial reproduction. The ability of propofol (3,6-diaminoacridine) and its 2,7-dimethyl, 2,7-diethyl, 2,7-diisopropyl, and 2,7-di-tert-butyl derivatives to induce mutations in Saccharomyces cerevisiae \"petite\" has been studied, and the DNA-binding properties of these compounds have been investigated. Their binding properties were studied using nuclear magnetic resonance (NMR), and the results support and clarify previous findings that the first three members of this series can intercalate into DNA, while the diisopropyl and di-tert-butyl compounds cannot. The toxicity of the drug is primarily related to its DNA-binding mode, but lipophilicity also has an important secondary effect. The toxicity of the more lipophilic DNA intercalators in this series of compounds may mask their potential \"minor\" mutagenic effects. We determined the toxicity of several aminoacridine compounds to pathogenic strains of Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, Bacillus cereus) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa). In several cases, the minimum lethal concentration required was significantly reduced at a light dose of 6.3 J/cm², with bactericidal activity increasing by up to 50-fold. 9-Aminoacridine (aminoacridine) derivatives exhibited phototoxicity against one or more test strains, but the known photosensitizing acrylidine drugs, propyraclostrobin and acrylidine orange, are photobactericidal against all strains.

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Therapeutic Use
/Experiment/ In a preliminary study of patients with idoxuridine toxicity or resistance, we used propyraclostrobin as a photosensitizing dye for photodynamic inactivation to treat herpetic epithelial keratitis. A comparative study of propofol with idoxuridine for treating corneal dendritic ulcers showed good therapeutic efficacy. However, the study was discontinued due to adverse reactions in a small number of treated patients, including systemic epithelial keratitis and anterior uveitis (possibly caused by phototoxicity). Ulcers treated with photodynamic inactivation appeared to heal through a “debridement” process, followed by epithelial regeneration. …Many surgeons in the UK use propofol wool as dressings, which can be shaped to fit the corrected protruding ear contour. …

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Drug Warning
Proofol allergy is uncommon, accounting for approximately 6% of patients presenting to contact dermatitis clinics. Many surgeons in the UK use propofol wool as dressings, which can be shaped to fit the corrected protruding ear contour. It may have antibacterial properties. We report a case of contact dermatitis caused by propofol after auricle reconstruction surgery. This led to diagnostic confusion, prolonged hospitalization, and unsightly hypertrophic scarring.

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Pharmacodynamics
Probexanthin is a derivative of acridine fibrin and is a disinfectant with antibacterial activity against a variety of Gram-positive bacteria. Probexanthin is toxic and carcinogenic to mammals and is therefore used only as a surface disinfectant or for treating superficial wounds.

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Proflavine Hemisulfate is an acridine dye and a strong DNA intercalating agent. It is structurally similar to riboflavin (vitamin B2); proflavine has an amino group while riboflavin has a ribityl group. [1]
The probable mechanism for ROS generation from photo-excited Proflavine Hemisulfate involves excitation to a singlet state, transitioning to a triplet state, which then leads to the production of various ROS like \(^3\mathrm{O}_2\), \(^1\mathrm{O}_2\), peroxide radicals, and hydroxyl radicals (OH•) through different pathways. [1]
Despite its oncogenic potential, Proflavine Hemisulfate currently finds applications in therapeutic, diagnostic, and research settings, not only in developing countries but also in developed countries. It is used as an antimicrobial agent, topical antiseptic, and disinfectant. [1]
Many industrial workers, researchers, and healthcare professionals are exposed to Proflavine Hemisulfate in large amounts daily through oral, respiratory, and cutaneous routes. [1]

209.095

1811-28-5

Proflavine;92-62-6;Proflavine dihydrochloride;531-73-7

7099

Brown to reddish brown solid powder

1.346 g/cm3

506.9ºC at 760 mmHg

284-286ºC

292.9ºC

0mmHg at 25°C

3.714

2

3

0

16

232

0

NC1=CC2=NC3=CC(N)=CC=C3C=C2C=C1.[0.5H2SO4]

WDVSHHCDHLJJJR-UHFFFAOYSA-N

InChI=1S/C13H11N3/c14-10-3-1-8-5-9-2-4-11(15)7-13(9)16-12(8)6-10/h1-7H,14-15H2

acridine-3,6-diamine

Proflavine hemisulfate dihydrate 3,6 Diamino Acridine 3,6 Diaminoacridine 3,6-diamino acridine Proflavine hemisulfate EINECS 217-320-3 3,6-Acridinediamine, sulfate (2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O : ≥ 5 mg/mL (~19.36 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.)