RARα（IC50 = 8 nM; EC50 = 0.36 nM）

In the presence of G-CSF, AM580 (at 10-8 M) significantly increased the expression of LAP mRNA in NB4 cells. When combined with G-CSF, AM580 and ATRA induced almost the same amounts of LAP transcripts at a concentration of 10-5 M. In NB4 cells, AM580 (at 10-8 M) increases steady-state levels of transcripts encoding G-CSF receptors by about six times [1]. In all colonies, AM580 (50 nM) elevates caspase-3 expression, and in 30% of colonies, it causes acinar cavitation [2]. Higher levels of CRBP1 expression were observed following RARη1 knockdown in primary Myc cells using shRARη1 and Am580 treatment, suggesting that RARη has an inhibitory effect on the RARα target gene CRBP1 in these cells. The antiproliferative effects of RARγ knockdown in MCF-10A and MCF-7 cell lines are enhanced by Am580 (200 nM), whereas MDA-MB-231 cells are not affected [3].

---

We treated the three human cell lines and Myc-expressing mouse mammary cells with RARγ agonist BMS961, RARγ/β agonist CD347 and RARγ antagonist SR11253 alone, or in combination with ATRA or AM580. Similarly to the results obtained using RARγ knockdown (Figure 3), co-treatment of these cell lines, including the mouse Myc cells, with the RARγ antagonist SR11253 and RARα agonist Am580, resulted in strong growth inhibition (Figure 4). As expected, Am580-inhibited MCF-10A- and MCF-7-cell growth (Figure 4, upper panels) were growth-inhibited by Am580, whereas RARγ agonist BMS961 increased their proliferation. Antagonist SR11253 inhibited the proliferation of MDA-MB-231 cells (Figure 4, lower left panel), which was not further inhibited by combination with AM580, as was expected due to their low expression of RARα. The general pattern of responses of the Myc mouse cells to treatment was similar to the human cells, except that the CD437/AHPN (RARγ/β agonist) and its combination with Am580 impaired cell proliferation most effectively (Figure 4, lower right panel). These results show that the pro-proliferative effect of RARγ is ligand-dependent and more importantly, that it can be targeted by RARγ-specific antagonists such as SR11253, alone or in combination with Am580. [3]

---

All-trans retinoic acid (ATRA) is successfully used in the cyto-differentiating treatment of acute promyelocytic leukemia (APL). Paradoxically, APL cells express PML-RAR, an aberrant form of the retinoic acid receptor type alpha (RAR alpha) derived from the leukemia-specific t(15;17) chromosomal translocation. We show here that AM580, a stable retinobenzoic derivative originally synthesized as a RAR alpha agonist, is a powerful inducer of granulocytic maturation in NB4, an APL-derived cell line, and in freshly isolated APL blasts. After treatment of APL cells with AM580 either alone or in combination with granulocyte colony-stimulating factor (G-CSF), the compound induces granulocytic maturation, as assessed by determination of the levels of leukocyte alkaline phosphatase, CD11b, CD33, and G-CSF receptor mRNA, at concentrations that are 10- to 100-fold lower than those of ATRA necessary to produce similar effects. By contrast, AM580 is not effective as ATRA in modulating the expression of these differentiation markers in the HL-60 cell line and in freshly isolated granulocytes obtained from the peripheral blood of chronic myelogenous leukemia patients during the stable phase of the disease. In NB4 cells, two other synthetic nonselective RAR ligands are capable of inducing LAP as much as AM580, whereas RAR beta- or RAR gamma-specific ligands are totally ineffective. These results show that AM580 is more powerful than ATRA in modulating the expression of differentiation antigens only in cells in which PML-RAR is present. Binding experiments, using COS-7 cells transiently transfected with PML-RAR and the normal RAR alpha, show that AM580 has a lower affinity than ATRA for both receptors. However, in the presence of PML-RAR, the synthetic retinoid is a much better transactivator of retinoic acid-responsive element-containing promoters than the natural retinoid, whereas, in the presence of RAR alpha, AM580 and ATRA have similar activity. This may explain the strong cyto-differentiating potential of AM580 in PML-RAR-containing leukemic cells [1].

Treatment with AM580 (0.3 mg/kg/day) had a more significant impact on the tumor-free survival of MMTV-wnt1 mice; effects were seen in the liver, lung, kidney, and spleen as well as in early-emerging tumors. In both transgenic glands, Am580 treatment significantly and equally decreased the levels of hyperplasia [2]. When administered to MMTV-Myc mice, the RARα selective agonist Am580 can effectively suppress the growth of mammary tumors, lung metastases, and extend tumor latency in 63% of the mice [3].

---

We hypothesized that specific activation of a single retinoic acid receptor-alpha (RARalpha), without direct and concurrent activation of RARbeta and gamma, will inhibit mammary tumor oncogenesis in murine models relevant to human cancer. A total of 50 uniparous mouse mammary tumor virus (MMTV)-neu and 50 nuliparous MMTV-wnt1 transgenic mice were treated with RARalpha agonist (retinobenzoic acid, AM580) that was added to the diet for 40 (neu) and 35 weeks (wnt1), respectively. Among the shared antitumor effects was the inhibition of epithelial hyperplasia, a significant increase (P<0.05) in tumor-free survival and a reduction in tumor incidence and in the growth of established tumors. In both models, the mechanisms responsible for these effects involved inhibition of proliferation and survival pathways, and induction of apoptosis. The treatment was more effective in the MMTV-wnt1 model in which Am580 also induced differentiation, in both in vivo and three-dimensional (3D) cultures. In these tumors Am580 inhibited the wnt pathway, measured by loss of nuclear beta-catenin, suggesting partial oncogene dependence of therapy. Am580 treatment increased RARbeta and lowered the level of RARgamma, an isotype whose expression we linked with tumor proliferation. The anticancer effect of RARalpha, together with the newly discovered pro-proliferative role of RARgamma, suggests that specific activation of RARalpha and inhibition of RARgamma might be effective in breast cancer therapy.[2]

---

Modulation of the RARα/β to RARγ expression in mammary glands of normal mice, oncomice, and human mammary cell lines through the alteration of RAR-target gene expression affected cell proliferation, survival and tumor growth. Treatment of MMTV-Myc mice with the RARα-selective agonist AM580 led to significant inhibition of mammary tumor growth (~90%, P<0.001), lung metastasis (P<0.01) and extended tumor latency in 63% of mice. Immunocytochemical analysis showed that in these mice, RARα responsive genes such as Cyp26A1, E-cadherin, cellular retinol-binding protein 1 (CRBP1) and p27, were up-regulated. In contrast, the mammary gland tumors of mice that responded poorly to Am580 treatment (37%) expressed significantly higher levels of RARγ.[3]

---

Effects of RARα activation by AM580 on tumor latency, growth and lung metastasis [3]
Based on our prior work and the above results, we reasoned that use of a RARα-selective agonist might circumvent the pro-proliferative and pRb repressive effects induced by activation of RARγ and thereby prevent tumor development. To accomplish this goal, we selected the RARα agonist AM580, which was reported to have 10-fold higher binding affinity for RARα than RARβ and no detectable affinity for RARγ. Uniparous, 15-week-old MMTV-Myc female mice, 30 per group, were fed standard diet containing Am580 or the standard diet alone (Myc-Ctrl group) and palpated twice weekly for the appearance of tumors. Tumors were first noted at week 16 in both experimental groups (control diet and Am580 diet). At week 50, no significant difference in tumor development was observed between the two groups analyzed by Kaplan-Meier plot. At that time, 80% of the Am580-treated mice and 100% of the control mice had tumors (Figure 6A, Myc-Ctrl vs. Am580-treated; NR+R, where NR are Am580 non-responders, and R are Am580 responders). Analysis of the tumor growth rates after initial tumor detection revealed two distinct sub-populations in the Am580-treatment group, those with fast-growing tumors (Figure 6B, Am580 non-responders denoted by squares) and those with slow-growing tumors (Figure 6B, Am580-responders denoted by triangles). Of 27 treated mice available for evaluation at week 50, 17 (63%) responded to Am580 treatment with 90% reduction in tumor size relative to the untreated controls, while 10 (37%) did not show a tumor size reduction (non-responders) (Figure 6B). Comparison of the Am580 responders to the Myc-Ctrl group by Kaplan-Meier analysis showed that tumor latency in the responders was significantly extended (P = 0.0465, log-rank test, Am580 R vs. Myc-Ctrl), with 35% of the treated Am580-responding mice not developing tumors at 50 weeks (Figure 6B).

Metastatic dissemination of tumor cells is the ultimate cause of death in breast cancer with the lungs being one of the major target organs for metastasis to occur. Analysis of lung metastasis incidence and number of metastatic lesions per mouse revealed that, compared to untreated mice (Myc-Ctrl) of which 66.6% developed lung metastases, only 16.6% of the AM580-responsive mice had metastases. The incidence of metastases in Am580 non-responsive mice was 71.4%, whereas compared to this group, Am580 responders had 36.8% metastasis incidence. The number of metastases per responder mouse was also reduced by Am580 (Figure 6C). Together these results demonstrate the ability of Am580 to reduce tumor growth and aggressiveness in the MMTV-Myc tumor model.

---

Difference in AM580 response is related to the RARα/RARγ balance in tumors [3]
Because the MMTV-Myc mouse is an inbred population, we did not expect the difference in response to AM580 to be host-dependent. However, to rule it out formally, two tumors from each Am580 treatment group were excised, minced and then inoculated into the mammary fat pads of separate groups of syngenic FVB females, which have the same genetic background of the MMTV-Myc transgenic model. Inoculated mice were fed the diet containing Am580 for 20 days. The transplanted tumors recapitulated the individual patterns of response and non-response of the original tumor (Additional file 2, Figure S2) indicating that the response to Am580 was independent of the host and intrinsic to the tumor.

We, therefore, tested whether an imbalance in RAR isotype expression defined the response to AM580 treatment. Analysis of RAR isotype protein levels showed that Am580 NR tumors expressed higher RARγ protein levels than Am580 R tumors (Figure 7A). To confirm that high expression of RARγ correlated with lack of responsiveness to Am580, sections from untreated (control) and treated responsive and nonresponsive tumors were examined for protein levels of RARα target genes by western blotting and immunohistochemistry. As shown in Figure 7A, tumor lysates of Am580-responsive mice treated with AM580 exhibited increased levels of growth arrest (p27) and differentiation (E-cadherin) markers compared to the Am580-nonresponsive tumors. Tumor lysates of Am580-treated nonresponsive mice also had increased levels of p-Erk1/2 (Figure 7A), which also suggests an increased proliferative response. RARα protein levels were low in both Am580 treatment groups, whereas RARγ was much higher in the Am580 non-responsive tumors. Immunohistochemistry of tumor sections (Figure 7B) confirmed the western blot results by showing enhanced levels of E-cadherin and p27 in the Am580 responsive tumors and also revealed, that the RARα target gene involved in ATRA catabolism, cytosolic Cyp26A1, was strongly induced in these tumors compared to the Am580 nonresponsive and Myc-Ctrl tumors. Both immunohistochemistry (Figure 7C) and western blotting (Figure 8A) showed that Am580 treatment induced robust CRBP1 expression exclusively in the responsive tumors.

Preparation of nuclear extracts and binding experiments. [1]
In routine binding assays, nuclear extracts (200 to 400 μL) were incubated with 1 nmol/L [3H]CD367 (CIRD-Galderma; specific activity, 52.8 Ci/mmol) in the presence or absence of varying concentrations of CD367, AM580, or ATRA as cold competitors. The preparations were incubated for 18 hours at 4°C and directly loaded on a Superose 6HR 10/30. Chromatography was performed in isocratic conditions using PTG buffer containing 0.4 mol/L KC1 at room temperature at a flow rate of 0.4 mL/min. Fractions (0.4 mL each) were collected and the radioactivity in each fraction was determined by liquid scintillation counting after the addition of 5 mL Filtercount. Although most of the saturation or competition binding assays were performed with this methodology, we also performed experiments with a faster technique that gives similar results and allows the simultaneous handling of a large number of samples.37 Briefly, nuclear extracts were loaded over PD10 desalting columns previously washed consecutively with 15 mL phosphate-buffered saline and 10 mL PTG buffer containing 0.4 mol/L KC1 and eluted with the same buffer at room temperature. The first 2 mL of the eluted buffer were discarded, and the following 2.5 mL was collected and the radioactivity measured by liquid scintillation counting as above. Linear least square analysis of the scatchard plot was performed with the aid of the computer program BDATA-EMF. The retinoid concentrations that inhibit 50% of the total specific CD367 binding were calculated using the nonlinear least squares regression analysis program ALLFIT.

Determination of intracellular levels of AM580 and ATRA in NB4 and HL-60 cells. [1]
ATRA and AM580 were extracted from NB4 and HL-60 cells with acetonitrile, after adding the internal standard and quantified by high-performance liquid chromatography with UV detection as described.40,41 Drug-free cells and cells containing known amounts of ATRA or AM580 were analyzed concurrently with each set of samples. Standard calibration curves were constructed by linear least squares regression analysis of the plot of the peak-height ratios between the compounds and the internal standard versus their concentrations in biologic samples. The limit of detection, precision, and reproducibility were as previously described for plasma analysis.40 Area under the curve (AUC) values were calculated by computer-assisted integration of the intracellular levels of AM580 or ATRA measured at various time after the addition of the two compounds to the culture medium.

---

Transactivation experiments. [1]
Simian COS-7 fibroblasts were obtained from ATCC and routinely passaged in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS. Transient transfection experiments were performed according to a standard calcium-phosphate coprecipitation procedure. After leaving the DNA coprecipitate in contact with cells for 16 hours, fresh medium (10% charcoal stripped fetal calf serum to eliminate endogenous retinoids) alone or fresh medium containing ATRA or AM580 was added and cells were further incubated for 36 hours. At the end of the treatment, cells were harvested and processed for determination of CAT and β-galactosidase activity.

---

Proliferation assay [3]
Primary Myc cells (2 × 104) were seeded in triplicate in 6-well culture dishes and allowed to attach overnight. They were then washed with culture medium and treated with the RARγ antagonist SR11253 (2-(4-carboxyphenyl)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-1,3-dithiolane) at increasing doses (10, 50 and 250 nM) or with DMSO (0.001% final concentration) alone, and then detached with 0.05% trypsin and counted every 24 h for 4 days. Statistical significance was determined by t-test. Following the same protocol described above for Myc, MCF-10A, MCF-7 and MDA-MB-231 cells were treated with 1 μM ATRA (RAR pan-agonist), 200 nM AM580 (RARα agonist), 100 nM CD437 (the RARγ/β agonist 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid, AHPN), 30 nM BMS961 (the RARγ agonist 3-fluoro-4-[[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)acetyl]amino]benzoic acid) and SR11253 or DMSO (0.001% final concentration) alone as the control.

AM580 treatment [2]
Four months old uniparous (1 pregnancy/lactation cycle) MMTV-neu and 6 weeks old nulliparous MMTV-wnt1 female mice (50 mice/group) were treated with the RARα agonist AM580 (0.3 mg/kg body weight per mouse per day) in the diet, by adding 1.5mg AM580 per kg of Purina 5053 diet. Mice that developed tumors within the first month of treatment were removed from the study. Mice were palpated twice a week and tumor appearance was recorded. Once palpable, the size of the tumors was measured weekly. Tumor-free survival was calculated from Kaplan-Meier curves and statistical significance was determined by the Log-rank test for the survival studies and t-test for the tumor growth studies. Metastasis was evaluated by removing the lungs of all the anesthetized mice, selecting randomly 20 mice per group and inspecting the lung surface for lesions using a stereoscope.

---

In vivo studies [3]
Three-month-old uniparous MMTV-Myc female mice (30 mice/group) were fed with 0.3 mg/kg/day of the RARa agonist AM580 (4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid). AM580 was mixed into their regular diet. Food consumption was measured to calculate the amount of AM580 to be added to the diet to achieve the daily dose as described previously. Regular diet was used as the control. Because the objective was to study the effect of AM580 on tumor initiation and development, mice that developed tumors within the first month were removed from the study on the assumption that their tumors had developed before treatment began. Mice were palpated twice weekly and the onset of tumor development was recorded. Once palpable, the tumor sizes were measured weekly in two dimensions and volumes calculated using the equation Vol = Dxd2/2 (where D = major diameter and d = minor diameter). Tumor-free survival was calculated from Kaplan-Meier curves, and statistical significance was determined using the log-rank test for survival and the t-test for tumor growth. Metastasis dissemination was evaluated by dissecting the lungs from euthanized mice and inspecting the Bouin-fixed lung surface for lesions using a stereoscope. For xenograft experiments, 8-week-old syngenic FVB mice were used. Cells or tumor fragments were inoculated into the mammary fat pad of the inguinal mammary glands (gland numbers 4 and 8) under soft anesthesia and analgesia in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines.

[1]. AM580, a stable benzoic derivative of retinoic acid, has powerful and selective cyto-differentiating effects on acute promyelocytic leukemia cells. Blood. 1996 Feb 15;87(4):1520-31.

[2]. Mechanism of inhibition of MMTV-neu and MMTV-wnt1 induced mammary oncogenesis by RARalpha agonist AM580. Oncogene. 2010 Jun 24;29(25):3665-76.

[3]. Reversal by RARα agonist Am580 of c-Myc-induced imbalance in RARα/RARγ expression during MMTV-Myc tumorigenesis. Breast Cancer Res. 2012 Aug 24;14(4):R121.

4-{[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbonyl]amino}benzoic acid is an amidobenzoic acid obtained by formal condensation of the carboxy group of (5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)benzoic acid with the anilino group of 4-aminobenzoic acid. A selective RARalpha agonist. It has a role as a retinoic acid receptor alpha/beta agonist and an antineoplastic agent. It is an amidobenzoic acid and a member of tetralins.

---

Regardless of the mechanism of action, AM580 is much more interesting than CD367 and TTNPB from a perspective therapeutical point of view. In vivo, the retinoic acid-mimetic activity of the two latter compounds is very strong; however, both CD367 and TTNPB show significant systemic toxicity1,, probably as a consequence of their promiscous ability to interact with RARα, β, and γ. The toxicologic profile of AM580 in animals and humans is not yet completely known, although it looks favorable. This probably reflects the ability of the compound to significantly interact only with RARα in normal animals. The results obtained with AM580 show that it is possible to develop retinoic acid derivatives that preferentially interact with PML-RAR and are more effective than ATRA in causing granulocytic differentiation of APL cells. Such compounds may show lower toxicity and a higher therapeutic index than ATRA in the treatment of this type of leukemia. [1]

---

Taken together, our data document that two mammary carcinoma models, MMTV-neu and MMTV-wnt1 are responsive to AM580 treatment and that this response is only partially oncogene-dependent. The fact that AM580, a RARα agonist insensitive to P450 cytochrome degradation is effective in 2 cancer models relevant to the human disease and in a human breast cell line, has potentially important clinical implications. Based on these observations and the novel role of RARγ, we suggest that better understanding of the specific roles of individual RARs in cancer cell differentiation, proliferation and apoptosis will help develop rational chemopreventive and, possibly, chemotherapeutic retinoid-based approaches to breast cancer. Combinations of selective RARα agonists with RARγ antagonists may prove to be one such successful approach. [2]

---

Introduction: Retinoic acid signaling plays key roles in embryonic development and in maintaining the differentiated status of adult tissues. Recently, the nuclear retinoic acid receptor (RAR) isotypes α, β and γ were found to play specific functions in the expansion and differentiation of the stem compartments of various tissues. For instance, RARγ appears to be involved in stem cell compartment expansion, while RARα and RARβ are implicated in the subsequent cell differentiation. We found that over-expressing c-Myc in normal mouse mammary epithelium and in a c-Myc-driven transgenic model of mammary cancer, disrupts the balance between RARγ and RARα/β in favor of RARγ.
Methods: The effects of c-Myc on RAR isotype expression were evaluated in normal mouse mammary epithelium, mammary tumor cells obtained from the MMTV-Myc transgenic mouse model as well as human normal immortalized breast epithelial and breast cancer cell lines. The in vivo effect of the RARα-selective agonist 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid (AM580) was examined in the MMTV-Myc mouse model of mammary tumorigenesis.
Results: Modulation of the RARα/β to RARγ expression in mammary glands of normal mice, oncomice, and human mammary cell lines through the alteration of RAR-target gene expression affected cell proliferation, survival and tumor growth. Treatment of MMTV-Myc mice with the RARα-selective agonist AM580 led to significant inhibition of mammary tumor growth (~90%, P<0.001), lung metastasis (P<0.01) and extended tumor latency in 63% of mice. Immunocytochemical analysis showed that in these mice, RARα responsive genes such as Cyp26A1, E-cadherin, cellular retinol-binding protein 1 (CRBP1) and p27, were up-regulated. In contrast, the mammary gland tumors of mice that responded poorly to AM580 treatment (37%) expressed significantly higher levels of RARγ. In vitro experiments indicated that the rise in RARγ was functionally linked to promotion of tumor growth and inhibition of differentiation. Thus, activation of the RARα pathway is linked to tumor growth inhibition, differentiation and cell death.[3]

---

AM580: 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid; ATCC: American Type Culture Collection; ATRA: All-trans retinoic acid; BSA: bovine serum albumin; CRBP1: cellular retinol-binding protein 1; DAB: 3,3'-diaminobenzidine; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; EGF: epithelial growth factor; EMT: epithelial to mesenchymal transition; ER: estrogen receptor; FBS: fetal bovine serum; FVB: Friend Virus B wild type mice; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H & E: haematoxylin and eosin; IACUC: Institutional Animal Care and Use Committee; IgG: immunoglobulin G; MEC: mammary epithelial cells; MMTV: mouse mammary tumor virus; NR: nonresponders; PBS: phosphate buffered saline; PCR: polymerase chain reaction; R: responders; RA: retinoic acid; RAR: retinoic acid receptor; RARE: retinoic acid response element; RIPA: radioimmunoprecipitation assay; RXR: retinoid × receptor; SDS: sodium dodecyl sulfate. [3]

C22H25NO3

351.438806295395

351.183

C, 75.19; H, 7.17; N, 3.99; O, 13.66

102121-60-8

AM580;102121-60-8

2126

White to off-white solid powder

1.2±0.1 g/cm3

461.0±45.0 °C at 760 mmHg

232.6±28.7 °C

0.0±1.2 mmHg at 25°C

1.593

6.48

2

3

3

26

546

0

O=C(C1C=CC2=C(C=1)C(C)(C)CCC2(C)C)NC1C=CC(C(=O)O)=CC=1

SZWKGOZKRMMLAJ-UHFFFAOYSA-N

InChI=1S/C22H25NO3/c1-21(2)11-12-22(3,4)18-13-15(7-10-17(18)21)19(24)23-16-8-5-14(6-9-16)20(25)26/h5-10,13H,11-12H2,1-4H3,(H,23,24)(H,25,26)

4-[(5,5,8,8-tetramethyl-6,7-dihydronaphthalene-2-carbonyl)amino]benzoic acid

AM-580; AM 580; AM580; CD336; NSC608001; Ro406055; 102121-60-8; 4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carboxamido)benzoic acid; 4-{[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbonyl]amino}benzoic acid; Ro-406055; CD-336; CD 336; NSC 608001 Ro 40-6055; CD-336; NSC-608001 Ro-40-6055;

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 (7.11 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (7.11 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.

---

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