Anti-inflammatory agent; endogenous metabolite

TauCl reacts with GSH and depletes cellular GSH. TauCl has microbicidal activity. TauCl inhibits superoxide production in activated neutrophils. TauCl inhibits production of pro-inflammatory mediators. TauCl inhibits NF-κB activation. TauCl increases nuclear translocation of Nrf2. TauCl protects cells from oxidative stresses via induction of anti-oxidant enzyme, HO-1. TauCl prevents cell death caused by oxidative stresses. TauCl prevents the development of chronic inflammatory diseases [1].

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Activity of N-Chlorotaurine (NCT) against Planktonic Dental Plaque Bacteria in PBS Solution [2]
There was a clear bactericidal activity of NCT against all test bacteria, R. aeria, C. ochracea, S. oralis, S. sanguinis, S. salivarius, and S. cristatus. As shown in Figure 1, the killing curves were dependent on the concentration of NCT and on the temperature. At 37 °C, 1% (55 mM) NCT reduced the CFU count to the detection limit within 10 min with all test strains; only R. aeria needed 15 min for the same effect. At 20 °C, the killing by 1% was slightly slower as expected. Reduction in the concentration to 0.1% NCT had a similar effect but still caused marked inactivation of bacteria within 20–30 min. A direct comparison of all bactericidal activities of NCT derived from the whole killing curves (BA values, log10 reduction in CFU per min) is shown in Table 1.

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Activity of N-Chlorotaurine/NCT against Planktonic Dental Plaque Bacteria on Dental Implant Screws [2]
Incubation of the screws, which were from four different companies as listed in Table 2, in the bacterial suspension for 24 h yielded only some small scattered areas of streptococci with a visible biofilm as evaluated by electron microscopy (see below). With R. aeria, no biofilm was found. Therefore, this series of experiments was regarded more as the activity of NCT against bacteria attached to screws than against biofilm. An incubation time of 15 min proved as sufficient for the standard concentration of 1% NCT at 37 °C and was chosen for all test streptococci, while it was 20 min for R. aeria. C. ochracea did not grow well in this setting over 24 h so it was not evaluated in these tests. NCT demonstrated a highly significant bactericidal activity against all tested strains cultured in the presence of the implant screws and attached to them (Figure 2). The log10 reduction in CFU compared to the controls reached between 3.00 and 4.55 for S. sanguinis, S. salivarius, and S. oralis with one exception of 1.98 for Profile 1 and S. salivarius. Due to significantly lower CFU counts in the controls, the log10 reduction came only to 0.96 to 2.63 in S. cristatus, although the detection limit was largely reached in the NCT samples similar to the other strains (Figure 2). High reduction values could be achieved with R. aeria after 20 min incubation in 1% NCT (Figure 2).

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Activity of N-Chlorotaurine/NCT against Biofilm of Dental Plaque Bacteria on Dental Implant Screws [2]
An incubation of the screws in the bacterial suspension for 48 h yielded biofilms on the screws with streptococci (see below electron microscopy). After removing the planktonic bacteria by three washing steps in PBS, 1% NCT at 37 °C killed the streptococcal biofilms to the detection limit after 30 min incubation. The log10 reduction in CFU/mL ranged between ≥3.80 and ≥5.01 (Figure 3). In three experiments with R. aeria and C. ochracea also, NCT led to complete killing. The controls of R. aeria, however, grew to 3.09 to 4.95 log10 CFU/mL only in 1–2 experiments in the presence of different screws with zero growth in the other experiments. With C. ochracea, only in one experiment, CFU/mL counts of up to 3.78 log10 were found in the controls. Therefore, no reliable and significant results could be obtained with these two strains.

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Scanning Electron Microscopy of Biofilm on Implant Screws [2]
With streptococci, scattered areas of biofilm could be produced on the implant screws after 24 h incubation time, while larger areas of more compact biofilm became visible after 48 h incubation in the presence of bacteria in nutrient broth. In Figure 5, a typical 48 h biofilm of S. sanguinis is shown after incubation in N-Chlorotaurine/NCT or PBS control for 30 min. No clear visible difference could be detected between test and control bacteria by electron microscopy, but there was the impression of less extracellular matrix in NCT samples (Figure 5). With R. aeria, no biofilm could be detected after 24 h and only very few spots of biofilm after 48 h so that no reliable evaluation was possible with this pathogen.

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Dose- and time-dependent decrease in cell viability [3]
Cell viability was measured using the EZ4U kit after 5 min respectively 30 min of incubation. After 5 min of incubation the Kruskal -Wallis test showed a significant decrease in cell viability (H = 125.1, p < 0.0001, N1–N7 = 24). The Dunn’s post-hoc test revealed a significant decrease in cell viability at N-Chlorotaurine/NCT 1% (p < 0.0001), NCT 0.1% (p < 0.0070), PVP-I (1.1%) (p < 0.0001), and H2O2 (3%) (p < 0.0001), in contrast for NCT 0.001% (p > 0.8102) and NCT 0.01% (p = 0.9999) chondrocytes showed no significant decrease in cell viability compared to the control group (Fig. 1).

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Assessment of nuclear morphology by acridine-orange staining [3]
After 5 min of incubation, the Kruskal–Wallis test presented a significant number of cells with altered nuclei (H = 97.76, p < 0.0001, N1–N7 = 16). In the Dunn’s post-hoc test a significant decrease in vital cells was presented for NCT 1% (p < 0.0001), N-Chlorotaurine/NCT 0.1% (p < 0.0052), PVP-I (1.1%) (p < 0.0001), and H2O2 (3%) (p < 0.0001) the detected decrease in vital cells was significant. (Fig. 3). Only NCT 0.001% (p > 0.9999) and NCT 0.01% (p > 0.9999) showed no significant decrease in the level of vital cells compared to the control group.
In accordance with the findings after 5 min of incubation, after 30 min of incubation the Kruskal–Wallis test demonstrated a significant decrease in vital cells (H = 100.1, p < 0.0001, N1–N7 = 16). The Dunn’s post-hoc test detected again a significant decrease in vital cells for N-Chlorotaurine/NCT 1% (p < 0.0001), NCT 0.1% (p = 0.0069), PVP-I (1.1%) (p < 0.0001), and H2O2 (3%) (p < 0.0001), but not for NCT 0.001% (p > 0.9999) and NCT 0.01% (p > 0.9999) (Fig. 4).

Taurine chloramine (TauCl) exhibits significant anti-inflammatory and cytoprotective effects across multiple disease models. In a collagen-induced arthritis (CIA) mouse model, daily subcutaneous injections of TauCl significantly attenuated paw swelling severity and reduced arthritis scores. TauCl-treated CIA mice showed significant reductions in synovial inflammation, cartilage damage, and bone erosion, with fewer TRAP-positive cells in the joints . In a septic arthritis model, intra-articular injection of TauCl with S. aureus significantly reduced arthritic lesions, although systemic intraperitoneal administration showed no obvious differences compared to controls . In an LPS-induced acute lung injury mouse model, intraperitoneal TauCl pretreatment attenuated LPS-induced lung weight gain and reduced IL-6 expression . In a UVB-induced skin damage model, topically applied TauCl reduced oxidative damage and apoptotic cell death in murine epidermis, evidenced by lower levels of 4-hydroxynonenal-modified protein, reduced TUNEL-positive epidermal cells, and suppression of caspase-3 cleavage. TauCl also significantly reduced expression of inflammatory enzymes COX-2 and iNOS, as well as pro-inflammatory cytokines Tnf, Il6, Il1b, and Il10 . In a rat middle cerebral artery occlusion (MCAO) stroke model, intranasal administration of TauCl (0.5 mg/kg) significantly reduced infarct volume, ameliorated neurological deficits, and promoted motor function .

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Taurine is one of the most abundant non-essential amino acid in mammals and has many physiological functions in the nervous, cardiovascular, renal, endocrine, and immune systems. Upon inflammation, taurine undergoes halogenation in phagocytes and is converted to taurine chloramine (TauCl) and taurine bromamine. In the activated neutrophils, TauCl is produced by reaction with hypochlorite (HOCl) generated by the halide-dependent myeloperoxidase system. TauCl is released from activated neutrophils following their apoptosis and inhibits the production of inflammatory mediators such as, superoxide anion, nitric oxide, tumor necrosis factor-α, interleukins, and prostaglandins in inflammatory cells at inflammatory tissues. Furthermore, TauCl increases the expressions of antioxidant proteins, such as heme oxygenase 1, peroxiredoxin, thioredoxin, glutathione peroxidase, and catalase in macrophages. Thus, a central role of TauCl produced by activated neutrophils is to trigger the resolution of inflammation and protect macrophages and surrounding tissues from being damaged by cytotoxic reactive oxygen metabolites overproduced during inflammation. This is achieved by attenuating further production of proinflammatory cytokines and reactive oxygen metabolites and also by increasing the levels of antioxidant proteins that are able to scavenge and diminish the production of cytotoxic oxygen metabolites. These findings suggest that TauCl released from activated neutrophils may be involved in the recovery processes of cells affected by inflammatory oxidative stresses and thus TauCl could be used as a potential physiological agent to control pathogenic symptoms of chronic inflammatory diseases [1].

Quantitative Killing Assays of Planktonic Bacteria [2]
Solutions of 1% and 0.1% N-Chlorotaurine/NCT in PBS, each 3.96 mL, at a pH of 7.1 were prepared in glass tubes and kept at room temperature (about 20 °C) or were pre-warmed to 37 °C in a water bath. Controls were prepared in 3.96 mL PBS without NCT. At time point zero, 40 µL of the bacterial cultures were added separately to the NCT or control solutions. After different adjusted incubation times, aliquots of 100 µL were removed and mixed with 900 µL of 1% methionine/1% histidine in distilled water to inactivate NCT. Aliquots of this suspension were spread onto Mueller–Hinton agar plates in duplicate (50 µL each) using an automatic spiral plater. The detection limit was 100 CFU/mL, taking into account both plates and the previous 10-fold dilution in the inactivating solution. Plates were grown for 24 h at 37 °C before colonies were counted. Plates with no growth or only a low CFU count were grown for up to five days to detect bacteria attenuated but not killed by the treatment.

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Quantitative Killing Assays of Planktonic Bacteria on Dental Implant Screws [2]
Bacteria from overnight cultures (10 µL) as described above in ‘test bacteria’ were added to 3 mL of tryptic soy broth in 12-well microtitre plates before the addition of screws. Dental implant screws comprised samples from 4 different companies as listed in Table 2. One screw each was placed subsequently in one well and incubated at 37 °C for 24 h in an incubator without shaking. Then, the screws were removed and placed in tubes containing pre-warmed 1% NCT/N-Chlorotaurine in PBS and incubated in the water bath at 37 °C for 10 (streptococci) or 20 (R. aeria) min. Afterwards, they were removed and placed in 1 mL of 1%methionine/1%histidine solution to immediately inactivate NCT. Controls were prepared in PBS without NCT and run in parallel. The tubes containing the screws in the inactivation solution were vortexed three times for 5 s, sonicated for 1 min in an ultrasound water bath, and vortexed again three times to detach the remaining live bacteria from the screws. Test samples were processed undiluted, and controls were 10-fold diluted in 0.9% sodium chloride (100 µL to 900 µL NaCl). Quantitative cultures were performed as detailed above.

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Quantitative Killing Assays of Biofilm Bacteria on Dental Implant Screws [2]
Again, 10 µL of overnight bacterial cultures were placed in 3 mL of tryptic soy broth in 12-well plates, followed by the screws. Incubation was performed at 37 °C under continuous shaking for 48 h. Subsequently, the screws were washed 3 times in 3 mL PBS in 12-well microtitre plates to remove planktonic bacteria before incubation in 1% NCT/N-Chlorotaurine in PBS for 20 min (controls in plain PBS). This was followed by transfer into inactivation solution, vortexing, ultrasonication, and quantitative cultures as described above.

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Electron Microscopy of Biofilm on Dental Implant Screws [2]
A similar procedure of scanning electron microscopy was used as in previous studies on the impact of NCT/N-Chlorotaurine on biofilms. After incubation in NCT or control PBS, the washed implant screws were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). After a brief wash in phosphate buffer, the samples were gradually dehydrated with 50%, 70%, 80%, and 99% ethanol. After the last step, the screws were incubated at room temperature for drying out. The dried screws were placed on aluminum pins and fixed with Leit-C. The pins were sputtered with Au 10 nm for 1 min and analyzed by scanning electron microscopy.

N-Chlorotaurine (NCT) was used as a crystalline sodium salt and stored at minus 20 °C. Use solutions of 1% and 0.1% (55 and 5.5 mM) were freshly prepared in 0.01 M PBS.

EZ4U cell viability assay [3]
The EZ4U cell viability kit was used according to the manufacturer’s recommendations. Each well of a flat-bottom 96-well plate was filled with 1 × 104 chondrocytes in 200 µl cell culture medium, which were allowed to adhere for 48 h. Subsequently, the cells were rinsed twice with PBS and incubated at 37 °C in humidified air with various concentrations of NCT/N-Chlorotaurine (1–0.001%), PVP-I (1.1%) and H2O2 (3%), each solution in triplets for 5 and 30 min, respectively. The control group was incubated with HBSS. Afterward, cells were rinsed again two times with PBS, each well was filled with 20 µl of EZ4U-substrate-solution and 200 µl HBSS and incubated for two hours. The measurement of the absorbance was performed with a micro-plate reader at 450 nm wavelength and 620 nm as reference.

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Acridine-orange fluorescence microscopy [3]
Nuclear morphology was assessed by acridine-orange staining. For this purpose, 5 × 104 cells in 1 ml cell culture medium per well were plated in 24-well plates and let to attach for 48 h. Thereafter, cells were washed two times with PBS and incubated for 5 and 30 min, respectively, with the different concentrations of NCT/N-Chlorotaurine (1–0.001%), PVP-I (1.1%), H2O2 (3%), and HBSS in the control group. Following incubation chondrocytes were washed twice with PBS and incubated with 1 ml 1.5% acridine-orange solution for 20 min under protection from direct light exposure. Subsequently, cells were rinsed as mentioned above and covered with 1 ml HBSS. For the calculation of the decrease in vital cells, 2 × 100 chondrocytes were counted under the fluorescence microscope differentiating between vital and dead cells based on their nuclear morphology.

NCT/N-Chlorotaurine was used in the form of a crystalline sodium salt. The molecular weight of the stock solution is 181.57 g/Mol. For the preparation of the test solutions, it was dissolved in Hank’s Balanced Salt Solution (“HBSS”). The tested concentrations in this study were 1%, 0.1%, 0.01%, and 0.001% (55 mM–55 µM), which were stored in the refrigerator at + 4 to + 8 °C.

Collagen-induced arthritis (CIA) mouse model: Seven-week-old male DBA/1J mice were immunized with bovine type II collagen to induce arthritis. TauCl was synthesized freshly by adding equimolar amounts of sodium hypochlorite to taurine dissolved in 1.8% NaCl. Mice were given daily subcutaneous injections of TauCl (0.5 mM or 1.0 mM) starting from the day of first collagen immunization. Arthritis severity was assessed three times per week by paw swelling measurement and arthritis scoring. At the 8th week, mice were sacrificed for histological examination using hematoxylin and eosin, safranin-O, and tartrate-resistant acid phosphatase (TRAP) staining . Septic arthritis mouse model: The murine model of hematogenous septic arthritis involved intravenous injection of a single dose of Staphylococcus aureus. TauCl was administered by daily intraperitoneal injections. In a separate experiment, S. aureus and TauCl were injected intra-articularly. Arthritis was evaluated clinically and histologically . LPS-induced acute lung injury mouse model: Mice were pretreated with TauCl intraperitoneally before intratracheal administration of LPS. Two days after LPS injection, body weight and lung weight were measured. Cytokine/chemokine mRNA expression including IL-1β, IL-6, IL-17, TNF-α, and MCP-1 was assessed . UVB-induced skin damage mouse model: Mice were exposed to UVB irradiation at 180 mJ/cm² intensity. TauCl was applied topically to the skin. Markers of oxidative damage (4-hydroxynonenal-modified protein), apoptosis (TUNEL-positive cells, caspase-3 cleavage), and inflammation (COX-2, iNOS, cytokine expression) were evaluated . Rat MCAO stroke model: Rats underwent middle cerebral artery occlusion to induce ischemic stroke. TauCl (0.5 mg/kg) was administered intranasally. Infarct volume, neurological deficits, and motor function were assessed .

Taurine chloramine exhibits limited systemic bioavailability due to its instability in biological fluids. In a pig inhalation model, there was no detectable systemic resorption of N-chlorotaurine, and its local inactivation occurred within 30 minutes. The concentration of N-chlorotaurine tolerated by lung epithelial cells in vitro was 0.25–0.5 mM . TauCl is produced endogenously by activated neutrophils in inflamed tissues, where it serves as a transient mediator rather than a systemically circulating drug . Due to its rapid decomposition upon systemic delivery, taurine chloramine is primarily investigated for topical clinical applications rather than systemic administration .

Taurine chloramine is generally well-tolerated at therapeutic concentrations. In a pig inhalation model, 1% (55 mM) N-chlorotaurine was well tolerated, with histological and ultrastructural investigations revealing no differences between test and control groups. Surfactant function remained intact. However, 1% N-chlorotaurine plus 1% ammonium chloride led to significantly lower arterial oxygen pressure (PaO₂) at the endpoint (62 ± 9.6 mmHg vs. 76 ± 9.2 mmHg for saline, p = 0.014) with a corresponding increase in alveolo-arterial oxygen difference (p = 0.004). The increase in pulmonary artery pressure was smallest with 1% N-chlorotaurine (p = 0.91 vs. controls) and higher with 5% N-chlorotaurine (p = 0.02) and NCT + NH₄Cl (p = 0.05) . In the pig model, the concentration of N-chlorotaurine tolerated by A549 lung epithelial cells in vitro was similar to that known from other body cells (0.25–0.5 mM) . In the collagen-induced arthritis mouse model, daily subcutaneous injections of 1.0 mM TauCl were well tolerated without reported mortality . TauCl is an endogenously produced metabolite, and its anti-inflammatory effects are achieved through physiological mechanisms rather than cytotoxic actions at therapeutic concentrations .

[1]. Taurine chloramine produced from taurine under inflammation provides anti-inflammatory and cytoprotective effects. Amino Acids. 2014;46(1):89-100.

[2]. Activity of N-Chlorotaurine against Periodontal Pathogens. Int J Mol Sci. 2024 Jul 30;25(15):8357.

[3]. Tolerability of N-chlorotaurine in comparison with routinely used antiseptics: an in vitro study on chondrocytes. Pharmacol Rep. 2024 May 17;76(4):878–886.

N-Chlorotaurate (N-Chlorotaurate) is an inhibitor of inducible nitric oxide synthase and IκB kinase.

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Drug Indications
It has been studied for the treatment of eye diseases/infections. Under inflammatory conditions, neutrophils endogenously produce N-Chlorotaurate. The TauCl produced and released by activated neutrophils has important effects on neighboring cells in several ways.
First, TauCl is formed after the clearance of toxic hypochlorite, thus its production can protect neutrophils from the toxic effects of hypochlorite.
Second, TauCl has bactericidal activity; it can transfer Cl⁻ to the amine components of bacteria, fungi, and viruses (Gottardi and Nagl, 2010), which may help neutrophils kill intracellular pathogens.
Third, TauCl inhibits the production of pro-inflammatory mediators (such as NO, TNF-α, PGs, and pro-inflammatory interleukins) in inflammatory cells that infiltrate inflamed tissues, thereby preventing the development of chronic inflammation. Fourth, TauCl can also inhibit further excessive production of O₂⁻, thereby reducing oxidative stress in cells at the site of inflammation. Fifth, TauCl promotes the restoration of redox balance in various cells at the site of inflammation by increasing the expression of various antioxidant enzymes (such as HO-1, Prx, Trx, GPx and catalase), thereby protecting these cells from cytotoxic damage by reactive oxygen metabolites. These diverse properties of TauCl may have important physiological anti-inflammatory effects and can prevent the occurrence of pathological symptoms of chronic inflammatory diseases. [1] Dental plaque bacteria play an important role in the pathogenesis of periodontitis and peri-implantitis. Therefore, antibacterial agents are one of the treatment methods. N-chlorotaurine (NCT) is a well-tolerated endogenous local antibacterial agent that may be suitable for this purpose. Therefore, this study investigated the bactericidal activity of N-chlorotaurine against certain dental plaque bacteria at in vitro therapeutic concentrations. In the quantitative bactericidal assay, we tested the activity of NCT against planktonic bacteria and biofilms grown on implanted screws for 48 hours. Electron microscopy was used to observe the formation and morphological changes of biofilms. The results showed that at 37°C, 1% NCT (dissolved in 0.01 M phosphate buffer) could kill all tested bacteria in their airborne state within 10-20 minutes, including Streptococcus sanguinis, Streptococcus salivarius, Streptococcus oralis, Streptococcus cristatus, Rothia aeria, and Capnocytophaga ochracea. Bacteria that had grown on screws for 24 hours could also be inactivated by 1% NCT within 15-20 minutes, but biofilm formation on the screws was not observed under electron microscopy; it required at least 48 hours. 1% NCT could kill biofilms after 30 minutes (streptococci) and 40 minutes (erythropoiesis-Pseudomonas). As expected, NCT also exhibits broad-spectrum activity against dental plaque bacteria, and its clinical efficacy in periodontitis and peri-implantitis warrants further investigation. [2]

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Background
Currently, povidone-iodine (PVP-I) and hydrogen peroxide (H2O2) are commonly used disinfectants in joint infections, but these solutions have been reported to be cytotoxic. N-chlorotaurine (NCT) has broad-spectrum bactericidal activity and is well tolerated in various tissues, but its effects on human chondrocytes are unclear. This study aimed to evaluate the cytotoxic effects of NCT, PVP-I, and H2O2 on human chondrocytes in vitro and compare them with a control group to preliminarily determine whether NCT has the potential to become an effective disinfectant for the future treatment of purulent joint infections.
Materials and Methods
Chondroblastocytes extracted from human cartilage were incubated with different concentrations of NCT, PVP-I, and H2O2 for 5 minutes and 30 minutes, respectively. Cell viability was determined using the EZ4U cell viability assay kit according to the manufacturer's instructions. To assess cell viability based on nuclear morphology, cells were stained with acridine orange and identified under a fluorescence microscope.
Results
The EZ4U kit showed that after 5 and 30 minutes of incubation, cell viability significantly decreased in the NCT 1%, NCT 0.1%, PVP-I, and H2O2 groups, while no decrease was observed in the NCT 0.001% and NCT 0.01% groups. Acridine orange staining results showed that, except for the 0.001% NCT and 0.01% NCT solutions, the number of viable cells significantly decreased in all test solutions after 5 and 30 minutes of incubation.
Conclusion
Our results indicate that N-chlorotaurine/NCT was well tolerated by chondrocytes at lower NCT concentrations (0.01% and 0.001%) tested in vitro, while higher NCT concentrations (1% and 0.1%), PVP-I (1.1%), and H2O2 (3%) resulted in a significant decrease in cell viability. Given that in vivo tolerance is generally significantly higher, our findings may suggest that in vivo cartilage tissue can tolerate 1% NCT solutions already used clinically. Combined with its broad-spectrum bactericidal activity, NCT may be a promising disinfectant for the treatment of purulent joint infections. [3]

C2H6CLNO3S

159.59

158.976

51036-13-6

108018

Typically exists as solid at room temperature

1.591g/cm3

1.512

1.089

2

4

3

8

136

0

C(CS(=O)(=O)O)NCl

NMMHHSLZJLPMEG-UHFFFAOYSA-N

InChI=1S/C2H6ClNO3S/c3-4-1-2-8(5,6)7/h4H,1-2H2,(H,5,6,7)

2-(chloroamino)ethanesulfonic acid

N-Chlorotaurine; N-Chlorotaurine; Taurine chloramine; 2-(Chloroamino)ethanesulfonic acid; Taurochloramine; N-Monochlorotaurine; 51036-13-6; Taurine monochloramine; Ethanesulfonic acid, 2-(chloroamino)-;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)