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 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.

[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-Chlorotaurine is an inhibitor of inducible nitric oxide synthase and IkappaB kinase.

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Drug Indication
Investigated for use/treatment in eye disorders/infections.

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TauCl is produced endogenously in neutrophils under inflammatory conditions. The generation and release of TauCl by activated neutrophils confers important impacts on many nearby cells in several respects.
First, TauCl is formed upon elimination of toxic hypochlorite and thus its formation protects neutrophils from the toxic effects of hypochlorite.
Second, TauCl is microbicidal by transferring its Cl− to amine components of bacteria, fungi, and viruses (Gottardi and Nagl 2010) and thus, may contribute to intracellular killing of pathogens by neutrophils.
Third, TauCl inhibits production of pro-inflammatory mediators, such as NO, TNF-α, PGs, and pro-inflammatory interleukins in those inflammatory cells that infiltrate into the inflamed tissues, and it prevents the development of chronic inflammation.
Forth, TauCl also inhibits further overproduction of O2 −, thus diminishing additional oxidative stress in cells at the inflammatory site.
Fifth, TauCl promotes recovery of redox balance in diverse cells at the inflammatory site by increasing the expression of many antioxidant enzymes, such as HO-1, Prx, Trx, GPx, and catalase and thus protects these cells from the cytotoxicity of reactive oxygen metabolites. These diverse properties of TauCl could provide important physiological anti-inflammatory effects and prevent the development of pathogenic symptoms of chronic inflammatory diseases.[1]

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Dental plaque bacteria play an important role in the pathogenicity of periodontitis and peri-implantitis. Therefore, antimicrobial agents are one means of treatment. N-Chlorotaurine (NCT) as an endogenous well-tolerated topical antiseptic could be of advantage for this purpose. Accordingly, its microbicidal activity against some dental plaque bacteria was investigated at therapeutic concentrations in vitro. In quantitative killing assays, the activity of NCT against planktonic bacteria and against biofilms grown for 48 h on implantation screws was tested. Electron microscopy was used to demonstrate the formation of biofilm and its morphological changes. The killing of planktonic bacteria of all tested species, namely Streptococcus sanguinis, Streptococcus salivarius, Streptococcus oralis, Streptococcus cristatus, Rothia aeria, and Capnocytophaga ochracea, was shown within 10–20 min by 1% NCT in 0.01 M phosphate-buffered saline at 37 °C. Bacteria grown on screws for 24 h were inactivated by 1% NCT after 15–20 min as well, but the formation of biofilm on the screws was visible in electron microscopy not before 48 h. The killing of biofilms by 1% NCT was demonstrated after 30 min (streptococci) and 40 min (R. aeria). As expected, NCT has broad activity against dental plaque bacteria as well and should be further investigated on its clinical efficacy in periodontitis and peri-implantitis.[2]

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Background
Currently, povidone-iodine (PVP-I) and hydrogen peroxide (H2O2) are frequently used antiseptics in joint infections, but the cytotoxic effects of these solutions are already reported. N-Chlorotaurine (NCT) shows a broad-spectrum bactericidal activity and is well tolerated in various tissues, but its effect on human chondrocytes is unknown. The purpose of this study was to assess the cytotoxic effect of NCT, PVP-I, and H2O2 on human chondrocytes compared to a control group in an in vitro setting to get first indications if NCT might be a promising antiseptic in the treatment of septic joint infections for the future.

Material and methods
Chondrocytes extracted from human cartilage were incubated with various concentrations of NCT, PVP-I, and H2O2 for 5 and 30 min respectively. EZ4U cell viability kit was used according to the manufacturer’s recommendations determining cell viability. To assess cell viability based on their nuclear morphology, cells were stained with acridine-orange and identified under the fluorescence microscope.

Results
EZ4U kit showed after 5 and 30 min of incubation a significant decrease in cell viability at NCT 1%, NCT 0.1%, PVP-I, and H2O2, but not for NCT 0.001% and NCT 0.01%. Acridine-orange staining likewise presented a significant decrease in vital cells for all tested solutions except NCT 0.001% and NCT 0.01% after 5 and 30 min of incubation.

Conclusion
Our results demonstrate that N-Chlorotaurine/NCT is well tolerated by chondrocytes in vitro at the tested lower NCT concentrations 0.01% and 0.001% in contrast to the higher NCT concentrations 1% and 0.1%, PVP-I (1.1%), and H2O2 (3%), for which a significant decrease in cell viability was detected. Considering that the in vivo tolerability is usually significantly higher, our findings could be an indication that cartilage tissue in vivo would tolerate the already clinically used 1% NCT solution. In combination with the broad-spectrum bactericidal activity, NCT may be a promising antiseptic for the treatment of septic 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.)