Coccidia; antibiotic; Wnt/β-catenin

Salinomycin is a strong Wnt signaling cascade agent. With an average IC50 of 230 nM, salinomycin oxazoline can be produced in cells in 48 hours. Another antibacterial potassium ionophore is salinomycin. According to recent reports, it is a novel and potent anti-cancer medication for breast cancer stem cells. The SW620 cells and Cisp-resistant SW620 cells are inhibited by salinomycin, with IC50 values of 1.54±0.23 μM and 0.32±0.05 μM, respectively. It was discovered that salinomycin had the ability to destroy cancer stem cells (CSC) and their carrying capacity. Following a 48-hour period of continuous salinomycin treatment, the stained cells were examined under a microscope, and a minimum of 100 cells were randomly counted within each field of view. The amount of Hoechst33342-stained cells in Cisp-resistant SW620 cells (20.20±3.72) revealed a significant difference from 9.40±2.07)/100 cells in SW620 cells (p<0.05). Both Cisp-resistant and SW620 cells were found using flow cytometric analysis 48 hours after the cells were treated with salinomycin. Compared to SW620 cells (16.78±2.56%), the disinfection rate of Cisp (37.82±3.63%) was much greater (p<0.05).[2].

After receiving 4 mg/kg of salinomycin (Sal), 8 mg/kg of salinomycin, and 10 μL/g of saline, the mice were sacrificed six weeks later. When compared to the control group, the liver tumor size was smaller in the salinomycin-treated group. There was a significant drop in the average tumor diameter (from 12.17 mm to 3.67 mm; p<0.05) and average tumor volume (V=length×width2×0.5) from 819 mm3 to 25.25 mm3. To assess salinomycin's antitumor activity, tumors were then removed and put through immunohistochemistry, TUNEL assay, and HE staining. The tissue structure of liver cancer is revealed by HE staining, which displays nuclei of varying sizes as well as the destroyed liver cord structure. Following salinomycin treatment, immunohistochemistry revealed decreased PCNA expression. The cell apoptosis rate was higher in the salinomycin-treated group than in the control group, as demonstrated by HE staining and the TUNEL assay. Moreover, immunohistochemistry demonstrated that the treatment with salinomycin increased the Bax/Bcl-2 ratio. The group treated with salinomycin had lower levels of β-catenin protein expression than the control group [4]. Streptomyces albicans fermentation results in the production of salinomycin, a monocarboxylic acid polyether antibiotic. Its unique ring structure enables it to form complexes with the extracellular cations of coccidia and pathogenic microorganisms, particularly K+, Na+, and Rb+, which alters the ion concentration both inside and outside of the cell [5].

Salinomycin, an antibiotic potassium ionophore, has been reported recently to act as a selective breast cancer stem cell inhibitor, but the biochemical basis for its anticancer effects is not clear. The Wnt/β-catenin signal transduction pathway plays a central role in stem cell development, and its aberrant activation can cause cancer. In this study, we identified salinomycin as a potent inhibitor of the Wnt signaling cascade. In Wnt-transfected HEK293 cells, salinomycin blocked the phosphorylation of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and induced its degradation. Nigericin, another potassium ionophore with activity against cancer stem cells, exerted similar effects. In otherwise unmanipulated chronic lymphocytic leukemia cells with constitutive Wnt activation nanomolar concentrations of salinomycin down-regulated the expression of Wnt target genes such as LEF1, cyclin D1, and fibronectin, depressed LRP6 levels, and limited cell survival. Normal human peripheral blood lymphocytes resisted salinomycin toxicity. These results indicate that ionic changes induced by salinomycin and related drugs inhibit proximal Wnt signaling by interfering with LPR6 phosphorylation, and thus impair the survival of cells that depend on Wnt signaling at the plasma membrane[1].

Postoperative chemotherapy for Colorectal cancer (CRC) patients is not all effective and the main reason might lie in cancer stem cells (CSCs). Emerging studies showed that CSCs overexpress some drug-resistance related proteins, which efficiently transport the chemotherapeutics out of cancer cells. Salinomycin, which considered as a novel and an effective anticancer drug, is found to have the ability to kill both CSCs and therapy-resistant cancer cells. To explore the potential mechanisms that salinomycin could specifically target on therapy-resistant cancer cells in colorectal cancers, we firstly obtained cisplatin-resistant (Cisp-resistant) SW620 cells by repeated exposure to 5 μmol/l of cisplatin from an original colorectal cancer cell line. These Cisp-resistant SW620 cells, which maintained a relative quiescent state (G0/G1 arrest) and displayed stem-like signatures (up-regulations of Sox2, Oct4, Nanog, Klf4, Hes1, CD24, CD26, CD44, CD133, CD166, Lgr5, ALDH1A1 and ALDH1A3 mRNA expressions) (p < 0.05), were sensitive to salinomycin (p < 0.05). Salinomycin did not show the influence on the cell cycle of Cisp-resistant SW620 cells (p > 0.05), but could induce cell death process (p < 0.05), with increased levels of LDH release and MDA contents as well as down-regulations of SOD and GSH-PX activities (p < 0.05). Our data also showed that the pro-apoptotic genes (Caspase-3, Caspase-8, Caspase-9 and Bax) were up-regulated and the anti-apoptotic gene Bcl-2 were down-regulated in Cisp-resistant SW620 cells (p < 0.05). Accumulated reactive oxygen species and dysregulation of some apoptosis-related genes might ultimately lead to apoptosis in Cisp-resistant SW620 cells. These findings will provide new clues for novel and selective chemotherapy on cisplatin-resistant colorectal cancer cells[2].

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The bladder cancer cell line T24 was cultured in vitro. The rat bladder tumor model was established in vivo. The rats were randomized into two groups, among which the rats in the experiment group were given intraperitoneal injection of salinomycin, while the rats in the control group were given intraperitoneal injection of normal saline. The change of tumor cells in the two groups was observed. Transwell was used to detect the cell migration and invasion abilities, Real-time PCR was used to detect the expression of mRNA, while Western-blot was utilized for the determination of the expressions of E-cadherin and vimentin proteins. Results: The metastasis and invasion abilities of serum bladder cancer cell line T24 after salinomycin treatment in the experiment group were significantly reduced when compared with those in the control group, and the tumor metastasis lesions were decreased from an average of 1.59 to 0.6 (P < 0.05). T24 cell proliferation in the experiment group was gradually decreasing. T24 cell proliferation at 48 h was significantly lower than that at 12 h and 24 h (P < 0.05). T24 cell proliferation at 24 h was significantly lower than that at 12 h (P < 0.05). T24 cell proliferation at each timing point in the experiment group was significantly lower than that in the control group (P < 0.05). The serum mRNA level and E-cadherin expression in the tumor tissues in the experiment group were significantly higher than those in the control group, while vimentin expression level was significantly lower than that in the control group (P < 0.05). Conclusions: Salinomycin can suppress the metastasis and invasion of bladder cancer cells, of which the mechanism is probably associated with the inhibition of EMT of tumor cells.[5]

Absorption, Distribution and Excretion
Salinomycin was administered to chickens orally and intravenously to determine blood concentration, kinetic behavior, bioavailability and tissue residues. The drug was given by intracrop and intravenous routes in a single dose of 20 mg kg-1 body-weight. The highest serum concentrations of salinomycin were reached half an hour after oral dosage with an absorption half-life (t0.5(ab)) of 3.64 hours and elimination half-life (t0.5(beta)) of 1.96 hours. The systemic bioavailability percentage was 73.02 per cent after intracrop administration, indicating the high extent of salinomycin absorption from this route in chickens. Following intravenous injection the kinetics of salinomycin can be described by a two-compartment open model with a t1/2(alpha) of 0.48 hours, Vd ss (volume of distribution) of 3.28 litre kg-1 and Cl(beta) (total body clearance) of 27.39 ml kg-1 min-1. The serum protein-binding tendency of salinomycin as calculated in vitro was 19.78 per cent. Salinomycin concentrations in the serum and tissues of birds administered salinomycin premix (60 ppm) for two weeks were lower than those after administration of a single intracrop dose of pure salinomycin (20 mg kg-1 bodyweight). The highest concentration of salinomycin residues were present in the liver followed by the kidneys, muscles, fat, heart and skin. No salinomycin residues were detected in tissues after 48 hours except in the liver and these had disappeared completely by 72 hours.

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Metabolism / Metabolites
... Salinomycin (SAL), a broad spectrum antibiotic and a coccidiostat has been found to counter tumour resistance and kill cancer stem cells with better efficacy than the existing chemotherapeutic agents; paclitaxel and doxorubicin. This refocused its importance for treatment of human cancers. In this study, we studied the in vitro drug metabolism and pharmacokinetic parameters of SAL. SAL undergoes rapid metabolism in liver microsomes and has a high intrinsic clearance. SAL metabolism is mainly mediated by CYP enzymes; CYP3A4 the major enzyme metabolising SAL. The percent plasma protein binding of SAL in human was significantly lower as compared to mouse and rat plasma. CYP inhibition was carried out by chemical inhibition and recombinant enzyme studies. SAL was found to be a moderate inhibitor of CYP2D6 as well as CYP3A4. As CYP3A4 was the major enzyme responsible for metabolism of SAL, in vivo pharmacokinetic study in rats was done to check the effect of concomitant administration of Ketoconazole (KTC) on SAL pharmacokinetics. KTC, being a selective CYP3A4 inhibitor increased the systemic exposure of SAL significantly to 7-fold in AUC0-a and 3-fold increase in Cmax of SAL in rats with concomitant KTC administration.

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Biological Half-Life
... The drug was given by intracrop and intravenous routes in a single dose of 20 mg kg-1 body-weight. The highest serum concentrations of salinomycin were reached half an hour after oral dosage with an absorption half-life (t0.5(ab)) of 3.64 hours and elimination half-life (t0.5(beta)) of 1.96 hours. ...

Toxicity Summary
IDENTIFICATION AND USE: Salinomycin is a veterinary drug used for the prevention of coccidiosis in broiler, roaster and replacement chickens caused by Eimeria tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti and E. mivati. It is also used for the prevention of coccidiosis in quail caused by Eimeria dispersa and E. lettyae. HUMAN EXPOSURE AND TOXICITY: The cytotoxic and genotoxic effects of salinomycin were investigated in human non-malignant cells. Primary human nasal mucosa cells (monolayer and mini organ cultures) and peripheral blood lymphocytes from 10 individuals were used to study the cytotoxic effects of salinomycin (0.1-175 uM) by annexin-propidiumiodide- and MTT-test. The comet assay was performed to evaluate DNA damage. Additionally, the secretion of interleukin-8 was analyzed by ELISA. Flow cytometry and MTT assay revealed significant cytotoxic effects in nasal mucosa cells and lymphocytes at low salinomycin concentrations of 10-20 uM. No genotoxic effects could be observed. IL-8 secretion was elevated at 5 uM. Salinomycin-induced cytotoxic and pro-inflammatory effects were seen at concentrations relevant for anti-cancer treatment. ANIMAL STUDIES: There are numerous reports of fatal outcomes when salinomycin is accidently fed to various animals. A sudden outbreak of mortality in one house of 600 48-week-old male breeder turkeys on a five-house turkey breeder farm was suspected to be feed-related. The turkeys gasped and became recumbent; 21.7% of affected turkeys died. Histological lesions, limited to skeletal muscle, consisted of degeneration and necrosis and were judged compatible with ionophore toxicosis. Feed samples from the affected house were analyzed and shown to contain 13.4 to 18.4 g of salinomycin per ton of feed. To further study the effects of salinomycin on turkeys, five 7-day trials using 336, 24, 24, 40, and 40 male turkeys when 7, 11, 15, 27, and 32 weeks of age, respectively. Salinomycin became more toxic as the age of the turkeys increased. When 7-week-old turkeys were fed diets containing 44 or 66 ppm salinomycin, only 1 of 84 died; when turkeys 27 or 32 weeks of age were fed those amounts, 13 of 20 died. Salinomycin at 22 ppm tended to depress rate of growth at young ages and to prevent or decrease growth and to increase mortality at older ages. Accidental poisonings were also reported in six horses fed salinomycin. The range of signs, including anorexia, colic, weakness and ataxia bore similarities to those described in horses poisoned with the related ionophore monensin. In another poisoning, horses were fed a concentrate containing 61 mg/kg salinomycin as faulty prepared by the manufacturer. All horses developed severe clinical signs of intoxication. Despite therapy eight horses died within three to six days. Ten others became recumbent and had to be euthanized. Only six horses survived. The dominating laboratory results were very high enzyme levels and alkalosis. The most characteristic clinical change appeared as paralysis of the hindlimbs. An outbreak of toxic polyneuropathy in cats that had ingested dry cat food contaminated with salinomycin has also been reported. Epidemiologic and clinical data were collected from 823 cats, or about 1% of the cats at risk. In 21 affected cats, postmortem examination was performed. The affected cats had acute onset of lameness and paralysis of the hindlimbs followed by the forelimbs. Clinical and pathologic examination indicated a distal polyneuropathy involving both the sensory and motor nerves. The clinical signs and pathology in an outbreak of toxicity in feedlot cattle attributed to the ingestion of toxic levels of salinomycin over an extended period of 11 weeks have also been reported. Thirty-nine out of 380 cattle developed signs consistent with cardiac failure and 8 of these died. Clinical signs included dyspnea, tachypnea, tachycardia, and exercise intolerance. Two cattle were necropsied and in one there were macroscopic lesions suggestive of congestive heart failure, namely pulmonary edema, hydrothorax and hepatomegaly. Histopathology revealed a chronic cardiomyopathy characterized principally by extensive myocardial fiber atrophy with multifocal hypertrophy and interstitial and replacement fibrosis. Hepatic and pulmonary lesions were consistent with those of congestive cardiac failure. Finally, 100% mortality was reported in a herd of sheep that were given feed containing salinomycin. The morning after the feeding, 78 sheep were found dead and one of them showed convulsive seizures. Postmortem examination revealed pulmonary congestion and edema, hemorrhages in abomasum, large pale kidney and white streak lines in myocardium.

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Interactions
Hepatocellular carcinoma (HCC) is one of the few cancers in which a continuous increase in incidence has been observed over several years. Drug resistance is a major problem in the treatment of HCC. In the present study, we used salinomycin (Sal) and 5-fluorouracil (5-FU) combination therapy on HCC cell lines Huh7, LM3 and SMMC-7721 and nude mice subcutaneously tumor model to study whether Sal could increase the sensitivity of hepatoma cells to the traditional chemotherapeutic agent such as 5-FU. The combination of Sal and 5-FU resulted in a synergistic antitumor effect against liver tumors both in vitro and in vivo. Sal reversed the 5-FU-induced increase in CD133(+) EPCAM(+) cells, epithelial-mesenchymal transition and activation of the Wnt/beta-catenin signaling pathway. The combination of Sal and 5-FU may provide us with a new approach to reverse drug resistant for the treatment of patients with HCC.
Chemotherapy for soft tissue sarcomas remains unsatisfactory due to their low chemosensitivity. Even the first line chemotherapeutic agent doxorubicin only yields a response rate of 18-29%. The antibiotic salinomycin, a potassium ionophore, has recently been shown to be a potent compound to deplete chemoresistant cells like cancer stem like cells (CSC) in adenocarcinomas. Here, we evaluated the effect of salinomycin on sarcoma cell lines, whereby salinomycin mono- and combination treatment with doxorubicin regimens were analyzed. To evaluate the effect of salinomycin on fibrosarcoma, rhabdomyosarcoma and liposarcoma cell lines, cells were drug exposed in single and combined treatments, respectively. The effects of the corresponding treatments were monitored by cell viability assays, cell cycle analysis, caspase 3/7 and 9 activity assays. Further we analyzed NF-kappaB activity; p53, p21 and PUMA transcription levels, together with p53 expression and serine 15 phosphorylation. The combination of salinomycin with doxorubicin enhanced caspase activation and increased the sub-G1 fraction. The combined treatment yielded higher NF-kappaB activity, and p53, p21 and PUMA transcription, whereas the salinomycin monotreatment did not cause any significant changes. Salinomycin increases the chemosensitivity of sarcoma cell lines - even at sub-lethal concentrations - to the cytostatic drug doxorubicin. These findings support a strategy to decrease the doxorubicin concentration in combination with salinomycin in order to reduce toxic side effects.
A factorial design (2 by 3) was used to evaluate the interaction between aflatoxin (0, 2.5, & 5 mg per kg) & salinomycin (1, 60 g per ton (909 kg)). There were four replicates of 10 chicks per treatment. ... No significant interaction was observed between aflatoxin & salinomycin on any of the parameters measured.
This study was aimed to investigate the effect of salinomycin combined with vincristine on the proliferation and apoptosis of Jurkat cells and its possible mechanisms. The proliferation of Jurkat cells was examined by CKK-8 assay. Flow cytometry was used to assess cellular apoptosis. Levels of BCL-2, caspase-3, and caspase- 8 were measured by Western blot. The salinomycin or vincristine, either alone or in combination, inhibited the proliferation of Jurkat cells in a dose-dependent manner. Salinomycin combined with vincristine produced more obvious inhibition of cell proliferation than either compound used alone (P<0.05). Western blot analysis showed that the combined use of Sal and VCR reduced the expression of BCL-2 protein, and increased expression of caspase 3 and caspase 8 protein, more significantly. Furthermore, combination of Sal and VCR synergistally promoted apoptosis of the Jurkat cells (P<0.05). The combination of salinomycin and vincristine synergistically inhibits proliferation and promotes apoptosis of T-cell acute lymphoblastic leukemia Jurkat cells.

[1]. Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells. Proc Natl Acad Sci U S A. 2011 Aug 9;108(32):13253-7.

[2]. Salinomycin induces apoptosis in cisplatin-resistant colorectal cancer cells by accumulation of reactiveoxygen species. Toxicol Lett. 2013 Oct 24;222(2):139-45.

[3]. Salinomycin: Anti-tumor activity in a pre-clinical colorectal cancer model. PLoS One. 2019 Feb 14;14(2):e0211916.

[4]. Salinomycin Inhibits Proliferation and Induces Apoptosis of Human Hepatocellular Carcinoma Cells In Vitro and In Vivo. PLoS One. 2012; 7(12): e50638.

[5]. Effect of salinomycin on metastasis and invasion of bladder cancer cell line T24. Asian Pac J Trop Med. 2015 Jul;8(7):578-82.

[6]. Salinomycin as a drug for targeting human cancer stem cells. J Biomed Biotechnol. 2012;2012:950658.

Therapeutic Uses
Anti-Bacterial Agents; Coccidiostats
EXPL: The drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages of Plasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin on Plasmodium falciparum asexual and sexual erythrocytic stages and on the development of the Plasmodium berghei and P. falciparum mosquito stages is reported here. Gametocytogenesis of the P. falciparum strain 3D7 was induced in vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads.
EXPL: Salinomycin has been introduced as a novel alternative to traditional anti-cancer drugs. The aim of this study was to test a strategy designed to deliver salinomycin to glioblastoma cells in vitro. Salinomycin-encapsulated polysorbate 80-coated poly(lactic-co-glycolic acid) nanoparticles (P80-SAL-PLGA) were prepared and characterized with respect to particle size, morphology, thermal properties, drug encapsulation efficiency and controlled salinomycin-release behaviour. The in vitro cellular uptake of P80-SAL-PLGA (5 and 10 uM) or uncoated nanoparticles was assessed in T98G human glioblastoma cells, and the cell viability was investigated with respect to anti-growth activities. SAL, which was successfully transported to T98G glioblastoma cells via P80 coated nanoparticles (~14% within 60 min), greatly decreased (p < 0.01) the cellular viability of T98G cells. Substantial morphological changes were observed in the T98G cells with damaged actin cytoskeleton. Thus, P80-SAL-PLGA nanoparticles induced cell death, suggesting a potential therapeutic role for this salinomycin delivery system in the treatment of human glioblastoma.
MEDICATION (VET): Use Sacox 60 ... for the prevention of coccidiosis in quail caused by Eimeria dispersa and E. lettyae.
MEDICATION (VET): Use Sacox 60 ... for the prevention of coccidiosis in broiler, roaster and replacement chickens caused by Eimeria tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti and E. mivati.

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Drug Warnings
Do not feed to laying hens producing eggs for human consumption.
May be fatal if accidentally fed to adult turkeys or to horses.

C42H70O11

751.00

750.49

C, 67.17; H, 9.40; O, 23.43

53003-10-4

Salinomycin sodium salt;55721-31-8

3085092

brown solid powder

1.2±0.1 g/cm3

839.2±65.0 °C at 760 mmHg

112.5-113.5 °C(lit.)

243.2±27.8 °C

0.0±0.6 mmHg at 25°C

1.547

6.1

4

11

12

53

1320

18

O1[C@@]2([C@@]([H])(C([H])=C([H])[C@@]3([C@]([H])(C([H])([H])[H])C([H])([H])[C@]([H])(C([H])([H])[H])[C@@]([H])([C@@]([H])(C([H])([H])C([H])([H])[H])C([C@@]([H])(C([H])([H])[H])[C@]([H])([C@]([H])(C([H])([H])[H])[C@@]4([H])[C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[C@]([H])([C@]([H])(C(=O)O[H])C([H])([H])C([H])([H])[H])O4)O[H])=O)O3)O2)O[H])C([H])([H])C([H])([H])[C@@]1(C([H])([H])[H])[C@@]1([H])C([H])([H])C([H])([H])[C@](C([H])([H])C([H])([H])[H])([C@]([H])(C([H])([H])[H])O1)O[H]

KQXDHUJYNAXLNZ-XQSDOZFQSA-N

InChI=1S/C42H70O11/c1-11-29(38(46)47)31-15-14-23(4)36(50-31)27(8)34(44)26(7)35(45)30(12-2)37-24(5)22-25(6)41(51-37)19-16-32(43)42(53-41)21-20-39(10,52-42)33-17-18-40(48,13-3)28(9)49-33/h16,19,23-34,36-37,43-44,48H,11-15,17-18,20-22H2,1-10H3,(H,46,47)/t23-,24-,25+,26-,27-,28-,29+,30-,31+,32+,33+,34+,36+,37-,39-,40+,41-,42-/m0/s1

(2R)-2-[(2R,5S,6R)-6-[(2S,3S,4S,6R)-6-[(3S,5S,7R,9S,10S,12R,15R)-3-[(2R,5R,6S)-5-ethyl-5-hydroxy-6-methyloxan-2-yl]-15-hydroxy-3,10,12-trimethyl-4,6,8-trioxadispiro[4.1.57.35]pentadec-13-en-9-yl]-3-hydroxy-4-methyl-5-oxooctan-2-yl]-5-methyloxan-2-yl]butanoic acid

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.33 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.33 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 2.5 mg/mL (3.33 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)