p38α (IC50 = 9 nM); p38β (IC50 = 90 nM)

Talmapimod (SCIO-469) inhibits p38 MAPK phosphorylation in MM cells at concentrations of 100–200 nM for an hour[1].
Talmapimod reduces the amount of TNF-a that LPS causes to be produced in human whole blood[2].
Talmapimod decreases the 5T2MM and 5T33MM cells' constitutive phosphorylation of p38alpha MAPK[3].

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Although PS-341 (bortezomib) is a promising agent to improve multiple myeloma (MM) patient outcome, 65% of patients with relapsed and refractory disease do not respond. We have previously shown that heat shock protein (Hsp)27 is upregulated after PS-341 treatment, that overexpression of Hsp27 confers PS-341 resistance, and that inhibition of Hsp27 overcomes PS-341 resistance. Since Hsp27 is a downstream target of p38 mitogen-activated protein kinase (MAPK)/MAPK-mitogen-activated protein kinase-2 (MAPKAPK2), we hypothesized that inhibition of p38 MAPK activity could augment PS-341 cytotoxicity by downregulating Hsp27. Although p38 MAPK inhibitor Talmapimod/SCIO-469 alone did not induce significant growth inhibition, it blocked baseline and PS-341-triggered phosphorylation of p38 MAPK as well as upregulation of Hsp27, associated with enhanced cytotoxicity in MM.1S cells. Importantly, SCIO-469 enhanced phosphorylation of c-Jun NH2-terminal kinase (JNK) and augmented cleavage of caspase-8 and poly(ADP)-ribose polymerase. Moreover, SCIO-469 downregulated PS-341-induced increases in G2/M-phase cells, associated with downregulation of p21Cip1 expression. Importantly, SCIO-469 treatment augmented cytotoxicity of PS-341 even against PS-341-resistant cell lines and patient MM cells. These studies therefore provide the framework for clinical trials of SCIO-469 to enhance sensitivity and overcome resistance to PS-341, thereby improving patient outcome in MM. [1]

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We have previously shown that p38 MAPK is overactivated in MDS hematopoietic progenitors, which led to current clinical studies of the selective p38alpha inhibitor, Talmapimod/SCIO-469, in this disease. We now demonstrate that the myelosuppressive cytokines TNFalpha and IL-1beta are secreted by bone marrow (BM) cells in a p38 MAPK-dependent manner. Their secretion is stimulated by paracrine interactions between BM stromal and mononuclear cells and cytokine induction correlates with CD34+ stem cell apoptosis in an inflammation-simulated in vitro bone marrow microenvironment. Treatment with SCIO-469 inhibits TNF secretion in primary MDS bone marrow cells and protects cytogenetically normal progenitors from apoptosis ex vivo. [2]

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In this study, we determined whether Talmapimod/SCIO-469, a selective p38alpha MAPK inhibitor, inhibits multiple myeloma growth and prevents bone disease in the 5T2MM and 5T33MM models. SCIO-469 decreased constitutive p38alpha MAPK phosphorylation of both 5T2MM and 5T33MM cells in vitro. This was associated with decreased DNA synthesis and an induction of apoptosis when the cells were cultured with bone marrow stromal cells. Treatment of C57Bl/KaLwRij mice bearing 5T33MM cells with SCIO-469 inhibited p38alpha MAPK phosphorylation and was associated with a significant decrease in serum paraprotein, an almost complete reduction in tumor cells in the bone marrow, a decrease in angiogenesis, and a significant increase in disease-free survival. [3]

Talmapimod (SCIO-469) reduces the burden of myeloma while also preventing the emergence of myeloma bone disease[2].
Talmapimod suppresses multiple myeloma cell proliferation and guards against bone disease in the 5T2MM and 5T33MM models[3].
Talmapimod (10-90 mg/kg; p.o. ; twice daily orally for 14 days) decreased tumor weight at termination as well as tumor growth in a dose-dependent manner[4].

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To assess the effect of blocking the p38α MAPK pathway in vivo on the development of multiple myeloma disease, 5T33MM injected mice were treated with Talmapimod/SCIO-469 from the time of tumor cell injection. Pharmacokinetic analysis of the serum samples of these mice resulted in values of 1 and 3 μmol/L, respectively, which were similar to the values obtained in patients. Treatment was associated with a reduction in p38α MAPK phosphorylation, as assessed on bone marrow samples of treated animals (Fig. 2). This was also associated with a decrease in serum paraprotein (8.8 ± 1.4 g/dL to 0.04 ± 0.03 g/dL with 150 mg/kg and to 0.0 ± 0.0 g/dL with 450 mg/kg; P < 0.001) and a reduction in the proportion of tumor cells in the bone marrow (67.2 ± 8.1% to 1.09 ± 0.5% for the 150 mg/kg and to 0.0 ± 0.0% for the 450 mg/kg group; P < 0.001; Table 1). Microvessel density decreased from 25.4 ± 1.2 to 19.2 ± 0.7 and 19.2 ± 0.5, respectively, for the 150 mg/kg group and the 450 mg/kg group (P < 0.001), levels similar to that of the naïve controls. This decrease could be the result of either a direct effect on angiogenesis or an indirect effect via the reduction in tumor burden. Kaplan-Meier analysis showed an increase in disease-free survival following treatment of the mice with SCIO-469 (vehicle, 27.5 days, versus SCIO-469, 96 days; P < 0.0001; Fig. 2).[3]

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To address whether inhibiting the p38α MAPK pathway also affects the development of myeloma bone disease, studies were done in the 5T2MM model. Injection of 5T2MM murine myeloma cells into C57Bl/KaLwRij mice resulted in the growth of myeloma cells in the bone marrow and the development of bone disease characterized by increased osteoclast surface (P < 0.05), a reduction in cancellous bone (P < 0.01), and the presence of osteolytic bone lesions on X-ray (P < 0.01; Fig. 3). Treatment of 5T2MM-bearing mice with Talmapimod/SCIO-469 resulted in a 40% decrease in serum paraprotein (P < 0.1, for both the 150 mg/kg group and the 450 mg/kg group). Microvessel density was reduced from 25.5 ± 0.8 for the control group to 18.8 ± 0.7 for the zoledronic acid group, 20.3 ± 0.7 for the Talmapimod/SCIO-469 150 mg/kg group, and 18.7 ± 0.5 for the 450 mg/kg group (all values, P < 0.001), levels similar to that of the naïve controls. SCIO-469 treatment (both 150 and 450 mg/kg) also prevented the development of osteolytic lesions (P < 0.01; Fig. 3). This was also seen with the bisphosphonate zoledronic acid (P < 0.01), used as a positive control. Histologic analysis showed that zoledronic acid treatment significantly reduced the increase in bone surface covered by osteoclasts by 5T2MM cells (P < 0.01). This is consistent with our previous report showing that repeated dosing is effective in reducing osteoclast formation and the development of lytic bone lesions in this model (11). In contrast, SCIO-469 had no effect on osteoclast surface when compared with mice treated with vehicle. Indeed, osteoclast perimeter remained significantly increased when compared with mice treated with zoledronic acid (P < 0.05). The strong inhibition of lytic lesions yet the absence of an effect on the proportion of bone surface covered by osteoclasts is consistent with SCIO-469 inhibiting osteoclast activity and function rather than osteoclast formation. [3]

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Multiple myeloma (MM) is a clonal plasma cell malignancy, which is currently incurable. Therefore, new mono- or combined therapy treatment regimens in the early and advanced phases of MM are urgently needed to combat this disease. Recently, p38 mitogen-activated protein kinase (MAPK) has been implicated as playing an important role in MM. Therefore, the effect of a p38alpha-selective MAPK inhibitor, Talmapimod/SCIO-469 (indole-5-carboxamide, ATP-competitive inhibitor), or its structural analog, SD-282 (indole-5-carboxamide, ATP-competitive inhibitor) was examined in mouse xenograft models of MM using human RPMI-8226 or H-929 plasmacytoma inocula. Oral treatment with SCIO-469 (10, 30, 90 mg/kg) twice daily was initiated in mice with palpable tumors of RPMI-8226 origin, a condition that mimics early human myeloma disease. In mice with palpable tumors, 14 days of SCIO-469 treatment significantly reduced RPMI-8226 tumor growth in a dose-dependent manner. A significant dose-dependent reduction in RPMI-8226 tumor growth was also observed when SCIO-469 oral treatment at doses of 10, 30 and 90 mg/kg twice daily was initiated in mice with tumors of pronounced size, a condition that mimics advanced human myeloma disease. In a similar set of studies employing the SCIO-469 analogue SD-282 at 90 mg/kg/bid orally, histological assessment at the end of the study demonstrated a significant reduction in RPMI-8226 tumor growth and angiogenesis. SD-282 treatment was additionally shown to significantly reduced expression of heat-shock protein-27 (HSP-27) and phospho-p38 in the tumor cells. Furthermore, co-administration of SCIO-469 with dexamethasone elicited antitumor properties in dexamethasone-sensitive H-929 tumors at much lower than the typically effective doses of dexamethasone, suggesting its potential for combined therapy. In conclusion, p38 inhibitors reduced human myeloma cell growth in vivo both at early and advanced phases of the disease. The current study also provides evidence of potential for co-therapy with dexamethasone. [4]

MAPKAP kinase2 kinase assay [1]
MAPKAPK2 kinase assay was performed using MAPKAPK2 kinase assay kit. Briefly, MM.1S cells were pre-incubated with Talmapimod/SCIO469 for 30 min, and then treated with PS-341 for 1 h. Whole-cell lysates (1 mg) were subjected to MAPKAPK2 immunoprecipitation kinase assay, according to the manufacturer's protocol.

Talmapimod (SCIO-469) was pretreated with 5TMM cells (0.5 × 106/mL) in serum-free medium before being added to the lower compartment of a Transwell system. In the Transwell itself, stromal cells from synthetic bone marrow were seeded. According to the manufacturer's instructions, the 5TMM cells were taken from the lower compartment after 18 hours and stained for active caspase-3 using a FITC-labeled antibody.

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BMSC cultures and ELISA [1]
BM specimens were obtained from patients with MM. Mononuclear cells (MNCs) separated by Ficoll-Hypaque density sedimentation were used to establish long-term BM cultures, as previously described (Uchiyama et al., 1993). When an adherent cell monolayer had developed, cells were harvested in Hank's buffered saline solution containing 0.25% trypsin and 0.02% EDTA, washed and collected by centrifugation. IL-6 levels in culture supernatants from BMSCs cultured for 24 h, in the presence or absence of Talmapimod/SCIO-469, were quantitated by ELISA as previously described (Hideshima et al., 2001c).

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Growth inhibition assay [1]
The inhibitory effect of Talmapimod/SCIO-469 inhibitor on MM cell line growth was assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide dye absorbance, as previously described (Hideshima et al., 2000).

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Cell cycle analysis [1]
MM.1S cells were treated with either 200 nM Talmapimod or DMSO control prior to culture with 40 nM PS-341 for 12 h. Cell cycle analysis was performed, as in prior studies (Hideshima et al., 2003c, 2003d).

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Immunoblotting [1]
MM.1S cells were treated with Talmapimod for 12 h prior to culture with 40 nM PS-341; cells were harvested, washed, and lysed, as in prior studies (Hideshima et al., 2000, 2001c). Cell lysates were subjected to SDS–PAGE, transferred to PVDF membrane, and immunoblotted with anti-phospho-p38 MAPK, phospho-JNK, p38 MAPK, phospho-Hsp27, Hsp27, p53, and poly ADP-ribose polymerase (PARP) Abs, as well as with anti-JNK1, hsp70, p21Cip1, p27Kip1, and actin Abs.

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cDNA Microarray Analysis [2]
Details of microarray and data analysis have been described previously The data was normalized using the maNorm function in marray package of Bioconductor version 1.5.8. Differential expression values were expressed as the ratio of the median of background-subtracted fluorescence intensity of the experimental RNA to the median of background-subtracted fluorescence intensity of the control RNA. The total BMSC RNA was extracted from cells using the RNeasy kit. Arrays were probed in quadruplicate for a total of 16 hybridizations: control versus TNF□ (24 hours), TNFα̣ versus Talmapimod/SCIO-469 + TNFα (24 hours), control versus IL-1β (24 hours), IL-1β versus SCIO-469 + IL-1β (24 hours).

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Fluorescent In situ hybridization [2]
Primary MDS bone marrow aspirate cells were treated in the presence and absence of Talmapimod/SCIO-469 (500uM) for 48 hours and then cytospun on slides. Fluorescence in situ hybridization analysis (FISH) was performed on methanol-acetic acid fixed interphase nuclei using the manufacturer's protocol with slight modifications. Slides were denatured in 70% formamide/2X SSC at 72°C for 5 minutes and dehydrated in a cold ethanol series. Probes against EGR1 on chromosome 5q31 locus and centromeric controls were used to detect cells with chromosome 5q deletion. Probes were mixed with appropriate volumes of buffer/distilled water and denatured at 72° C for 5 minutes. Probe mixtures were applied to denatured chromosomes and placed in a moist chamber at 37° C overnight. Post-hybridization washes for all the probes were in a 0.4X SSC/0.3% NP-40 solution at 73° C for 2 minutes, and then in 2X SSC/0.1% NP-40 solution at room temperature. Air-dried slides were then counterstained with DAPI. FISH images were captured, enhanced and stored using the computerized image analysis system.

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Assessment of p38α MAPK inhibition by Western blot. [3]
5T33MM and 5T2MM cells were isolated from the tibiae of diseased mice and lysed as previously described. For assessing the effect of Talmapimod/SCIO-469 on in vitro p38α phosphorylation, 5T2MM and 5T33MM cells were preincubated with 0.5 μmol/L SCIO-469 for 1 h before being lysed. For assessing the effect of SCIO-469 on in vivo p38α phosphorylation, 5T33MM mice were treated twice a day with 90 mg/kg SCIO-469 p.o. Two hours after last treatment, bone marrow was collected for Western blotting. The cell debris was then removed by centrifugation (5 min, 13,000 × g) and sample buffer added. After boiling, the samples were separated on a 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked with PBS containing 5% low fat milk and 0.1% Tween 20 and probed with anti–phospho-Thr180/Tyr182 p38. For measuring total protein levels, the blots were stripped and reprobed with total p38 antibody. The bands were visualized using the enhanced chemiluminescence system.

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Effect of Talmapimod/SCIO-469 on thymidine incorporation. [3]
5TMM cells (1 × 106/mL) were pretreated with different concentrations of SCIO-469 in either serum-free medium or in 10% Fetal Clone I for (FCI) 1 h. The cells in serum-free medium were then incubated on irradiated (1,500 rad) syngeneic bone marrow stromal cells. Sixteen hours before harvest, cells were pulsed with 1 μCi [methyl-3H]thymidine. Cells were harvested using a cell harvester onto fiberglass filters. Filters were dried for 1 h in a 60°C oven and sealed in sample bags containing 4 mL of Optiscint Scintillation Liquid. Radioactivity was counted using a 1450 Microbeta Liquid Scintillation Counterl. Results are expressed as the relative DNA synthesis compared with untreated cells.

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Effect of Talmapimod/SCIO-469 on caspase-3 activity. [3]
5TMM cells (0.5 × 106/mL) were pretreated with different concentrations of SCIO-469 in serum-free medium and then placed in the lower compartment of a Transwell system. Syngeneic bone marrow stromal cells were seeded into the Transwell itself. After 18 h, the 5TMM cells were collected from the lower compartment and stained for active caspase-3 with a FITC-labeled antibody according to manufacturer's instructions.

Six-week-old male triple immune-deficient BNX mice (RPMI-8226 MM palpable tumors)[4]
P.o.; twice daily orally for 14 days
10, 30, 90 mg/kg

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Assessment of the effects of Talmapimod/SCIO-469 on the development of myeloma disease in vivo. For studies of the effect of SCIO-469 on myeloma development, three groups of male mice (n = 12) were injected i.v. with 0.5 × 106 5T33MM cells. Mice were left untreated (naive) or, if injected with tumor cells, treated from the time of tumor cells injection with either SCIO-469 (150 or 450 mg/kg powder diet continuously available for the mice) or a vehicle (PBS) until the first mice showed signs of morbidity (at 3.7 weeks). Serum paraprotein concentration was assessed using standard electrophoretic techniques (9), bone marrow tumor burden was assessed by determining plasmacytosis on cytosmears, and bone marrow angiogenesis was assessed by determining microvessel density (see below).
To determine the effect of Talmapimod/SCIO-469 on survival, an identical experiment to that described above was done, with the exception that treatment continued until each animal showed signs of morbidity (i.e., hind limb paralysis), at which point they were sacrificed. Kaplan-Meier analysis was done to determine the effect on time to morbidity. Tumor load was confirmed on bone marrow samples.
To determine the effect of Talmapimod/SCIO-469 on the development of myeloma-bone disease, studies were done in the 5T2MM model, which develops a characteristic myeloma bone disease (8, 10, 11). Mice were divided into the following groups: group 1 (n = 10) remained without tumor cells (naïve group) and groups 2 to 4 (n = 10 each) were injected via the tail vein with 2 × 106 5T2MM cells. At the time of tumor cell injection, mice were treated with either zoledronic acid (120 μg/kg, s.c., single dose at week 7) or SCIO-469 [150 or 450 mg/kg given in the diet throughout the experimental period (11 weeks)]. At 11 weeks, all mice were sacrificed and the effects of SCIO-469 and zoledronic acid on tumor burden, development of myeloma bone disease, and angiogenesis were assessed (see below). [3]

[1]. p38 MAPK inhibition enhances PS-341 (bortezomib)-induced cytotoxicity against multiple myeloma cells. Oncogene. 2004 Nov 18, 23(54), 8766-76.

[2]. Inhibition of p38alpha MAPK disrupts the pathological loop of proinflammatory factor production in the myelodysplastic syndrome bone marrow microenvironment. Leuk Lymphoma. 2008 Oct;49(10):1963-75.

[3]. Inhibition of p38alpha mitogen-activated protein kinase prevents the development of osteolytic bone disease,reduces tumor burden, and increases survival in murine models of multiple myeloma. Cancer Res. 2007 May 15;67(10):4572-7.

[4]. p38alpha-selective MAP kinase inhibitor reduces tumor growth in mouse xenograft models of multiple myeloma. Anticancer Res. 2008 Nov-Dec;28(6A):3827-33.

Talmapimod is an indolecarboxamide obtained by formal condensation of the carboxy group of 6-chloro-3-[(dimethylamino)(oxo)acetyl]-1-methylindole-5-carboxylic acid with the secondary amino group of (2S,5R)-1-[(4-fluorophenyl)methyl]-2,5-dimethylpiperazine. It is a potent inhibitor of MAPK and exhibits anti-cancer properties. It has a role as an EC 2.7.11.24 (mitogen-activated protein kinase) inhibitor, an apoptosis inducer and an antineoplastic agent. It is a N-acylpiperazine, a N-alkylpiperazine, an aromatic amide, a member of monofluorobenzenes, a chloroindole, an indolecarboxamide, a dicarboxylic acid diamide and an aromatic ketone.
Talmapimod is the first-generation oral p38 MAP kinase inhibitor developed by Scios. It has shown to be effective to cure inflammatory diseases such as Rheumatoid Arthritis.
Talmapimod is an orally bioavailable, small-molecule, p38 mitogen-activated protein kinase (MAPK) inhibitor with potential immunomodulating, anti-inflammatory, and antineoplastic activities. Talmapimod specifically binds to and inhibits the phosphorylation of p38 MAPK, which may result in the induction of tumor cell apoptosis, the inhibition of tumor cell proliferation, and the inhibition of tumor angiogenesis. This agent may also enhance proteasome inhibitor-induced apoptosis. p38 MAPK is a serine/threonine protein kinase involved in a MAPK signaling cascade that controls cellular responses to various environmental stresses, cytokines, and endotoxins.

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Drug Indication
Investigated for use/treatment in pain (acute or chronic) and rheumatoid arthritis.

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Mechanism of Action
SCIO-469 inhibits p38 kinase, a stimulatory modulator of pro-inflammatory factors including tumor necrosis factor-alpha (TNFa), interleukin-1 (IL-1), and cyclooxygenase-2 (COX-2), all of which are known to contribute to both symptoms and disease progression in patients with Rheumatoid Arthritis (RA). Existing protein-based products that antagonize TNFa have been shown to markedly relieve the symptoms and retard the progression of RA. It also has the potential for additional benefits associated with its inhibition of IL-1 and COX-2.

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We further studied whether SCIO-469 could also augment cytotoxicity of PS-341 in DHL-4 cells, which overexpress Hsp27 and are resistant to PS-341 (Chauhan et al., 2003c). Consistent with our previous studies, SCIO-469 enhanced cytotoxicity of PS-341 in DHL-4 cells, associated with inhibition of PS-341-triggered phosphorylation of Hsp27. Finally, we also examined the cytotoxicity of combined treatment with SCIO-469 and PS-341 in patient MM cells resistant to PS-341. SCIO-469 significantly augments cytotoxicity of PS-341 in these cells, and its effect on Hsp27 expression in patient cells is under investigation. Our results therefore provide the preclinical rationale for clinical trials for SCIO-469 in combination with PS-341 to either enhance the sensitivity or overcome resistance to PS-341, thereby improving patient outcome. [1]

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Altogether, our results demonstrate that in addition to its direct anti-apoptotic effects on CD34+ stem cells, SCIO-469 also inhibits the expression of various proinflammatory factors in the bone marrow and disrupts the inflammatory loop that leads to the pleiotropic production of such factors. SCIO-469 is presently being used in a Phase I/II clinical trial in low grade cases of MDS. Early results have shown some efficacy in this disease. Due to the multiple cytokine pathways implicated in MDS pathogenesis, strategies to selectively inhibit individual cytokines and their receptors have not yielded much success in this disease. Our data demonstrates that p38 MAPK may represent a common signaling pathway used by multiple cytokine pathways in MDS and thus may be an attractive therapeutic target in this disease. [2]

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Our results suggest that, in addition to the previous published role of SCIO-469 on suppression of soluble factors within the bone marrow microenvironment in vitro (2), SCIO-469 also reduces p38α phosphorylation in multiple myeloma cells, both in vitro and in vivo, resulting in a decreased tumor burden, angiogenesis, and bone disease, and therefore targets the multiple myeloma disease at multiple levels. This raises the possibility that targeting p38α MAPK may offer a novel therapeutic approach in the treatment of multiple myeloma. [3]

C27H30N4O3FCL

513.0035

512.199

C, 63.21; H, 5.89; Cl, 6.91; F, 3.70; N, 10.92; O, 9.36

309913-83-5

Talmapimod hydrochloride;309915-12-6

9871074

white solid powder

1.3±0.1 g/cm3

658.0±65.0 °C at 760 mmHg

351.7±34.3 °C

0.0±2.0 mmHg at 25°C

1.619

2.73

0

5

5

36

836

2

O=C(N(C)C)C(C1=CN(C)C2=C1C=C(C(N3[C@H](C)CN(CC4=CC=C(F)C=C4)[C@@H](C)C3)=O)C(Cl)=C2)=O

ZMELOYOKMZBMRB-DLBZAZTESA-N

InChI=1S/C27H30ClFN4O3/c1-16-13-33(17(2)12-32(16)14-18-6-8-19(29)9-7-18)26(35)21-10-20-22(25(34)27(36)30(3)4)15-31(5)24(20)11-23(21)28/h6-11,15-17H,12-14H2,1-5H3/t16-,17+/m0/s1

2-[6-chloro-5-[(2R,5S)-4-[(4-fluorophenyl)methyl]-2,5-dimethylpiperazine-1-carbonyl]-1-methylindol-3-yl]-N,N-dimethyl-2-oxoacetamide

Talmapimod; SD282; SD 282; SD-282; 309913-83-5; SCIO-469; SCIO 469; Scios 469; Talmapimod [USAN]; SCIO 469 hydrochloride; Talmapimod [USAN:INN]; SCI O282; SCI-O282; SCIO282

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)

DMSO: ≥ 100 mg/mL (~194.9 mM)

Solubility in Formulation 1: 2.5 mg/mL (4.87 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (4.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.87 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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 (Please use freshly prepared in vivo formulations for optimal results.)