IL-12/IL-23

Apilimod has an IC50 of about 20 nM, which suppresses IFN-γ/SAC or SAC-induced IFN-γ production in human PBMC. On TNF-α generated by IFN-γ/SAC and IL-5 caused by ConA in high-concentration human PBMC, Apimod exhibits a certain amount of inhibitory action; nevertheless, in all cultures examined, it exhibits a certain amount of inhibitory action on IL-1β, IL-2, IL-4, IL-8, and IL-18. IFN-γ/LPS or IFN-γ/SAC stimulation greatly increased luciferase activity fueled by the p35 and p40 promoters, whereas 100 nM Apilimod totally blocked it [1].

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Potent inhibition of IL-12 and IL-23 production by Apilimod (STA-5326) [1]
IL-12 plays an integral role in both innate and adaptive immune responses. IFN-γ is a strong and selective enhancer of IL-12 production, and the effect is evident only after extended treatment with IFN-γ for at least 8 hours prior to stimulation with LPS or SAC.31 This suggests that in Th1-mediated chronic autoimmune or immunologic disorders that are characterized by recurrently high levels of IFN-γ, production of the IL-12 family is mediated by IFN-γ. In order to discover effective IL-12 inhibitors, we carried out a phenotypic screening assay using IFN-γ/LPS–stimulated human PBMCs, and screened an 80 000-compound library. A novel compound, a 1,3,5-triazine derivative, was discovered through this screening. The lead optimization process produced and evaluated approximately 500 compounds and led to the discovery of STA-5326, a unique morpholinopyrimidine derivative. IL-12 production in cultures of IFN-γ/LPS–stimulated human PBMCs was strongly inhibited by STA-5326 with an IC50 of 10 nM (Table 1). The inhibitory activity was more pronounced in IFN-γ/SAC–stimulated human PBMCs, where STA-5326 completely inhibited IL-12 with an IC50 of 1 nM (Figure 1; Table 1). No decrease in cell viability was observed, even at a concentration of 10 μM.

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Selectivity of Apilimod (STA-5326) in inhibition of cytokine production [1]
The selectivity of cytokine inhibition is important particularly for the understanding of the mechanism of action and in vivo activity. STA-5326 inhibited IFN-γ production induced by either IFN-γ/SAC or SAC in human PBMCs, with an IC50 of approximately 20 nM, while the compound had no significant effect on the production when T cells were stimulated using anti-CD3 and anti-CD28 antibodies, suggesting that the T-cell–receptor–dependent production of IFN-γ that occurs independently of suppression of Th1 cells via inhibition of IL-12 is not directly inhibited by the compound (Table 2). A significant reduction in IL-6 production was observed in IFN-γ/SAC–stimulated human PBMCs, but not in IFN-γ/LPS–stimulated human PBMCs, IFN-γ/SAC-stimulated THP-1, and mouse spleen cells (Table 2; Y. Wada, unpublished data, 2001-2002). Similarly, IFN-γ/SAC–specific inhibition was observed in IL-10 production. STA-5326 showed some inhibition against IFN-γ/SAC–induced TNF-α and ConA-induced IL-5 from human PBMCs at high concentrations, but no suppressive effect against IL-1β, IL-2, IL-4, IL-8, and IL-18 in all cultures tested.

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Effects of Apilimod (STA-5326) on IL-12 p35 and p40 promoter activities [1]
Having shown that STA-5326 reduces production of both IL-12 p70 and p40 protein, a study of the p35 and p40 promoter activities was then undertaken to obtain more insight into the mechanism of action of this compound. The murine macrophage cell line RAW264.7 was transiently transfected with DNA constructs in which the p35 and p40 promoters directed expression of the luciferase reporter gene. Cells were then stimulated with murine recombinant IFN-γ, followed by LPS or SAC in the presence or absence of STA-5326 or STA-5392 (an inactive compound that is structurally related to STA-5326). The p35 and p40 promoter-driven luciferase activities were significantly induced after stimulation with IFN-γ/LPS or IFN-γ/SAC, and were completely suppressed by 100 nM STA-5326 (Figure 2). The closely structurally related inactive compound STA-5392 had no effect, even at 1 μM (Figure 2B). The inhibition of p35 and p40 promoter-mediated luciferase expression is in good correlation with the observed inhibition of p70 protein production. This result indicates that the suppression of IL-12 and IL-23 by STA-5326 occurs at a transcriptional level, and that STA-5326 inhibits the transcription of both IL-12 p35 and IL-12/IL-23 p40 genes.

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The activity of Apilimod (STA-5326) does not require de novo protein synthesis [1]
Corticosteroids,33-36 cyclic AMP,37 IFN-β,38 and IL-1039 exert their suppressive effects on cytokine production, including IL-12, by a mechanism that requires de novo protein synthesis. To determine whether de novo protein synthesis is involved in the inhibitory mechanism of STA-5326, the effect of a protein synthesis inhibitor, CHX, on the inhibition of IL-12 production by STA-5326 was assessed in comparison with the effect of CHX on IL-12 inhibition by IL-10. CHX at a concentration of 5 μg/mL reduced IL-12 production by more than half. STA-5326 remained potent in inhibiting IL-12 production in the presence of CHX and reduced IL-12 production to a greater degree than CHX treatment alone, indicating that CHX does not abrogate the inhibitory activity of STA-5326 (Figure 3). It is noted that the estimated IC50 of STA-5326 in the presence of CHX was reproducibly 2- to 3-fold lower than the IC50 in cultures without CHX. In the same experiment, IL-10 at a concentration of 10 ng/mL significantly reduced IL-12 production in the absence of CHX, but completely lost the inhibitory activity in the presence of CHX.

Not only did apimod (10 mg/kg, po) work well when given continuously during the trial, but it also worked well when started on day 30, when the disease was manifestly detectable but not at its worst. Only in the Th1 model did Apimod significantly reduce the number of cells; on average, 51% ± 8% less cells were inhibited compared to the vehicle control. Patients with Th2 do not respond to apimod therapy [1]. In EAU mice, apilimimod (5 or 20 mg/kg) decreases blood IL-12 p40 levels without affecting body weight. Clinical and histological investigation shows that oral apimod lowers the severity of experimental autoimmune uveoretinitis (EAU) [2].

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Selective suppression of the in vivo Th1 response by Apilimod (STA-5326) [1]
The potent and selective inhibition of in vitro IL-12 and IL-23 production by STA-5326 suggests that this compound should significantly suppress the Th1 response in vivo, but not the Th2 response. We evaluated the effect of STA-5326 on in vivo Th1 and Th2 mouse models that were induced by Mycobacterium tuberculosis with Freund adjuvant in C57BL/6 mice and Ascaris/aluminum hydroxide with incomplete Freund adjuvant in BALB/c mice, respectively. STA-5326, the steroid prednisolone as a positive control, or vehicle were orally given to mice from the day of immunization, and lymph node cells from each group were harvested for assessment of the effect on the induction of Th1 and Th2 responses in vivo. Figure 4 shows the average percentage of cell numbers relative to the naive control from 4 Th1 and 6 Th2 experiments, respectively. After immunization, lymph node cells from the vehicle control groups in these Th1 and Th2 models increased approximately 3-fold over the corresponding naive controls. A reduction was observed in animals treated with prednisolone in both models, indicating the indiscriminate inhibition of both Th1 and Th2 responses. In contrast, treatment with STA-5326 caused a significant reduction in cell number only in the Th1 model, with an average percentage of inhibition of 51% ± 8% relative to the vehicle control. STA-5326 treatment had no effect in the Th2 setting (Figure 4). [1]
To examine the differentiation of T cells in mice, lymph node cells were then plated at equal cell numbers per well, and evaluated for anti-CD3/CD28 antibody-stimulated production of IFN-γ and IL-4, the cytokines that represent Th1 and Th2, respectively. Immunization of C57BL/6 mice with M tuberculosis effectively drove an in vivo Th1 response in the vehicle control with a marked increase in the production of IFN-γ (Figure 5). Alternatively, immunization of BALB/c mice with Ascaris/aluminum hydroxide gave rise to an in vivo Th2 response, and an increase in IL-4 was observed in the vehicle-treated group. Treatment with prednisolone blocked not only the elevation of IFN-γ in the Th1 model, but also IL-4 in the Th2 model moderately. In contrast, Apilimod (STA-5326) only inhibited IFN-γ in the Th1 model with an average 84% ± 10% reduction relative to the vehicle control group from 4 individual Th1 experiments. Interestingly, the production of IL-4, the Th2 cytokine, tended to be increased in the STA-5326–treated group, with an average 232% ± 91% increase relative to the vehicle control group from 6 individual Th2 experiments. These results clearly indicate that the effect of STA-5326 is stimulus dependent and selective against the Th1 response.

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In vivo suppressive activity of Apilimod (STA-5326) in an inflammatory bowel disease animal model [1]
Recent human and animal studies suggest that the local immune response in Crohn disease is predominantly Th1, and IL-12 plays a critical role in the initiation and progression of the disease.40-42 To examine the potential of STA-5326 in the treatment of CD, we tested oral administration of this compound in the CD4+CD45Rbhigh T-cell transfer model. Histologic analysis showed inflammatory histopathologic changes in colons from vehicle-treated animals, such as overt epithelial hyperplasia, marked increase in crypt length, prominent infiltration of chronic inflammatory cells in mucosa and LP, including crypt microabscesses, marked reactive atypia, and various extents of depletion of goblet cells. These changes were significantly reduced in animals treated with oral administration of Apilimod (STA-5326), and the colons maintained a normal structure (Figure 6A-B). The histologic score clearly differentiated animals receiving STA-5326 from vehicle-treated animals, and the suppressive effect was dose dependent, with a substantial reduction at a dose of 4 mg/kg and stronger suppression at 10 mg/kg (Figure 6C). The calculated colon–to–body-weight ratio was consistent with the histologic score, showing dose-dependent attenuation by treatment with STA-5326 (Figure 6D). STA-5326 was effective not only when administered throughout the entire experiment, but also when administration was initiated on day 30 when disease was clearly measurable but not maximal (D.Z., J.C., unpublished data, January 2002). Furthermore, ex vivo analysis of cytokines from LPMCs harvested from mice in this study demonstrated that LP cells from the vehicle control produced an augmented level of IFN-γ and an undetectable level of IL-4. The production of IFN-γ was greatly diminished in animals with STA-5326, indicating that oral administration of STA-5326 down-regulated the in vivo Th1 response in this inflammatory bowel disease animal model (Figure 6E).

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The level of IL-12 p40 in serum was decreased in mice treated with Apilimod (STA-5326). Oral administration of either 5 mg/kg or 20 mg/kg Apilimod (STA-5326) reduced the severity of EAU on day 14 and 18. In addition, mice treated with 20 mg/kg STA-5326 showed significantly decreased severity of EAU by histopathological analysis. Although IFN-gamma production of draining lymph node cells was increased in STA-5326-treated mice by ELISA analysis, the proportion of IFN-gamma-producing cells was not significantly altered. However, IL-17 production and the proportion of IL-17-producing cells were significantly reduced in STA-5326-treated mice. Furthermore, oral administration of STA-5326 during the effector phase reduced the severity of EAU. Conclusions: These results indicate that oral administration of the IL-12/IL-23 inhibitor Apilimod (STA-5326) is effective in suppressing inflammation in the EAU model, and reduces the expansion of IL-17-producing cells. STA-5326 may represent a new therapeutic modality for human refractory uveitis [2].

Isolation of LPMCs and cytokine measurement [1]
Freshly obtained colon was washed in Ca/Mg-free HBSS, and incubated twice in HBSS containing EDTA (0.75 mM), DTT (1 mM), and antibiotics (2.5 μg/mL amphotericin, 50 μg/mL gentamicin) at 37°C for 15 minutes. The tissue was digested in RPMI containing 0.5 mg/mL collagenase D, 0.01 mg/mL DNase I, and antibiotics at 37°C. Lamina propria (LP) cells were then layered on a 40% to 100% Percoll gradient, and lymphocyte-enriched populations were isolated at the 40% to 100% interface as lamina propia mononuclear cells (LPMCs). Cells were incubated with anti-CD3 and anti-CD28 antibody as described above, under “In vivo Th1 and Th2 response.” Supernatants were removed after 24 hours and assessed for IFN-γ using an ELISA kit.

In vitro assays [1]
Human and monkey PBMCs were isolated using NycoPrep. Human monocytes were purified using RosetteSep, and dendritic cells were differentiated from monocytes by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 ng/mL) and IL-4 (20 ng/mL) for 9 days. Purity of monocytes and dendritic cells was more than 70% CD14+ and CD1a+, respectively, by phenotypic analysis. Mouse PBMCs were isolated using OptiPrep, and spleen cells were prepared using ACK lysing buffer. Human PBMCs, THP-1 cells, and monkey PBMCs were primed with human IFN-γ (400 U/mL) for 22 hours and then stimulated with 1 μg/mL of LPS or 0.025% of SAC for 18 hours. To investigate requirement of de novo synthesis, cycloheximide (CHX) was added 2 hours or immediately prior to SAC. Human PBMCs were also stimulated by anti-CD3 (0.2 μg/mL) and anti-CD28 (1 μg/mL) antibodies, or ConA (1 μg/mL). Mouse cells were stimulated with a combination of mouse IFN-γ (100 ng/mL) with SAC (0.05%) for 22 hours. Apilimod (STA-5326) was prepared in DMSO; the final DMSO concentration was adjusted to 0.25% in all cultures, including the compound-free control.
IL-23 was detected using anti–IL-23 p19 polyclonal antibody and biotinylated goat anti–human p40 antibody, and calculated using recombinant IL-23. Total p40 proteins were measured using p40 ELISA kit. Monkey IL-12 p70 was measured using the human IL-12 p70 enzyme-linked immunosorbent assay (ELISA) kit. IL-12 induced by SAC alone was measured using Quantikine HS ELISA Kit. Other cytokines were measured using ELISA kits, or Bio-Plex assays.

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IL-12 promoter-driven luciferase assay [1]
To construct the human IL-12 p35 and p40 promoter/luciferase reporter constructs, we generated p35 (−1.5 kb/+3 bp) and p40 (−1.3 kb/+56 bp) promoter fragments by polymerase chain reaction (PCR) of genomic DNA obtained from human PBMCs. The resulting PCR products were ligated upstream of the luciferase gene in pGL3-Basic vector. All constructs were verified by DNA sequencing. RAW267.4 cells were transiently transfected using SuperFect Transfection Reagent. The cells were stimulated with IFN-γ (100 ng/mL) for 10 hours followed by LPS (1 μg/mL) in the presence or absence of test compound in duplicate for an additional 16 hours. Cells were cotransfected with a pCMVβ vector for monitoring transfection efficiency. Luciferase and β-galactosidase activity were determined according to luciferase assay system and luminescent β-gal detection system. Luciferase activity was then normalized using the β-galactosidase value.

CD4+CD45Rbhigh T-cell transfer SCID mouse inflammatory bowel disease [1]
CD4+ T cells in spleen cells from female BALB/c mice were negatively selected using antibodies against B220 (RA3-6B2), CD11b (M1/70), and CD8α (53-6.72), and labeled with FITC-conjugated anti-CD45RB (16A) and PE-conjugated anti-CD4. CD4+ CD45RBhigh cells were defined as the upper 40% of CD45Rb-staining CD4+ cells and were sorted by flow cytometry. Harvested cells were intraperitoneally injected into female C.B-17 SCID mice with 4 × 105 cells per mouse. Apilimod (STA-5326) and vehicle were orally administered once a day, 5 days per week, starting the day following the transfer.
Colon tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections (4 μm) from the ascending, transverse, and descending colon were stained with hematoxylin and eosin. Digital photomicrographs magnified at 40× and 200× of the original from the most affected areas were used for analysis. The extent of colonic inflammation was graded in a blind fashion on a scale of 0 to 3 in each of 4 histologic criteria: crypt elongation, inflammatory-cell infiltration, the number of crypt abscesses, and goblet-cell depletion. The controls were considered to be at baseline with a score of 0. Scores of 1, 2, and 3 represented the following: 2- to 3-fold, 4- to 6-fold, and more than 6-fold increase for crypt elongation; mild, moderate, and severe for inflammatory-cell infiltration; 1 to 2, 3 to 5, and more than 5 colonic crypt abscesses per section; and mild, moderate, and severe for goblet-cell depletion associated with epithelial hyperplasia or inflammatory infiltration. The total score for each animal was the sum of the average scores of ascending, transverse, and descending colon sections in all 4 categories.

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Oral administration of Apilimod (STA-5326) [2]
In most experiments, 5 mg/kg or 20 mg/kg Apilimod (STA-5326) or vehicle only (0.5% carboxyl methyl cellulose) was orally administered once a day for six days a week from day 0 to day 14 after immunization. In the effector phase experiments, 20 mg/kg Apilimod (STA-5326) or vehicle was orally administered once a day, from day 9 to day 14 after immunization.

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IL-12 production in the serum of Apilimod (STA-5326)-treated or vehicle-treated mice after immunization [2]
Mice were immunized as described above, and 5 mg/kg or 20 mg/kg Apilimod (STA-5326) or vehicle alone was orally administered once a day from day 0 to day 14 after immunization. STA-5326-treated or vehicle-treated mice were euthanased on day 18 after immunization, and serum from individual mice were collected for IL-12 p40 measurement using quantikine ELISA kits

Patient characteristics and safety and tolerability of Apilimod (STA-5326).
Twenty-nine eligible patients were included in this trial. The disease characteristics of the study patients were not significantly different between the apilimod- and placebo-treated groups (see Supplementary Table 1, available on the Arthritis & Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131), with the groups receiving MTX at an average weekly dosage of 21.1 mg and 22.5 mg, respectively. Since we observed favorable safety and pharmacokinetic (MTX and apilimod) profiles in stage 1 (data not shown), the study was continued to stage 2 and subsequently stage 3. In stage 1, 8 of 9 apilimod-treated patients completed the study, while 1 patient who developed side effects (severe headache), withdrew from the study on day 29. The patient refused to undergo the second arthroscopy, although safety and clinical evaluations were completed. All patients treated in stage 2 completed the study. In stage 3 (100 mg twice a day), 3 of 5 apilimod-treated patients continued until day 57, and 1 of these 3 decided to extend the treatment until day 85. Two patients withdrew prior to day 57 due to side effects. In stages 1 and 2, only mild adverse events (mainly gastrointestinal) were observed, in 15 of the 17 patients treated with apilimod (88%). In stage 3, side effects were experienced in all apilimod-treated patients and in patients receiving placebo (Table 1). A detailed listing of the adverse events is provided in Supplementary Table 2, available on the Arthritis & Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131. [Arthritis Rheum . 2012 Jun;64(6):1750-5]

Objective: To investigate the safety, tolerability, pharmacokinetics, and efficacy of Apilimod (STA-5326) mesylate, an oral interleukin-12 (IL-12)/IL-23 inhibitor, in patients with rheumatoid arthritis (RA).
Methods: We performed a phase IIa, randomized, double-blind, placebo-controlled proof-of-concept study of Apilimod (STA-5326), in combination with methotrexate, in 29 patients with active RA (3:1 ratio of apilimod-treated to placebo-treated patients) in 3 stages. Patients received apilimod 100 mg/day or placebo for 4 weeks (stage 1) or 8 weeks (stage 2). In stage 3, patients received apilimod 100 mg twice a day or placebo for 8 weeks, with an optional extension of 4 weeks. Clinical response (Disease Activity Score in 28 joints [DAS28] and American College of Rheumatology [ACR] criteria) was assessed throughout; synovial tissue samples collected at baseline and on day 29 (stages 1 and 2) or day 57 (stage 3) were stained for cellular markers and cytokines for immunohistochemistry analysis.
Results: While only mild adverse events were observed in stages 1 and 2, in stage 3, all patients experienced headache and/or nausea. Among apilimod-treated patients (100 mg/day), there was a small, but significant, reduction in the DAS28 on day 29 and day 57 compared with baseline. ACR20 response was reached in only 6% of patients on day 29 and 25% of patients on day 57, similar to the percentage of responders in the placebo group. Increasing the dosage (100 mg twice a day) did not improve clinical efficacy. Consistent with clinical results, apilimod did not have an effect on expression of synovial biomarkers. Of importance, we also did not observe an effect of apilimod on synovial IL-12 and IL-23 expression. Conclusion: Our results do not support the notion that IL-12/IL-23 inhibition by apilimod is able to induce robust clinical improvement in RA.
https://pubmed.ncbi.nlm.nih.gov/22170479/

[1]. Selective abrogation of Th1 response by STA-5326, a potent IL-12/IL-23 inhibitor. Blood. 2007 Feb 1;109(3):1156-64.

[3]. Therapeutic effect of the potent IL-12/IL-23 inhibitor STA-5326 on experimental autoimmune uveoretinitis. Arthritis Res Ther. 2008;10(5):R122.

[3]. Billich A. Drug evaluation: apilimod, an oral IL-12/IL-23 inhibitor for the treatment of autoimmune diseases and common variable immunodeficiency.IDrugs. 2007 Jan;10(1):53-9.

Apilimod (STA 5326) is a potent IL-12/IL-23 inhibitor, and strongly inhibits IL-12 in IFNg/SAC-stimulated human PBMCs and SAC-treated monkey PBMCs. Apilimod is a potent and highly selective PIKfyve inhibitor.

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Drug Indication
Investigated for use/treatment in crohn's disease and psoriasis and psoriatic disorders.

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The interleukin-12 (IL-12) cytokine induces the differentiation of naive T cells to the T helper cell type 1 (Th1) phenotype and is integral to the pathogenesis of Th1-mediated immunologic disorders. A more recently discovered IL-12 family member, IL-23, shares the p40 protein subunit with IL-12 and plays a critical role in the generation of effector memory T cells and IL-17-producing T cells. We introduce a novel compound, STA-5326, that down-regulates both IL-12 p35 and IL-12/IL-23 p40 at the transcriptional level, and inhibits the production of both IL-12 and IL-23 cytokines. Oral administration of STA-5326 led to a suppression of the Th1 but not Th2 immune response in mice. In vivo studies using a CD4+CD45Rbhigh T-cell transfer severe combined immunodeficiency (SCID) mouse inflammatory bowel disease model demonstrated that oral administration of STA-5326 markedly reduced inflammatory histopathologic changes in the colon. A striking decrease in interferon-gamma (IFN-gamma) production was observed in ex vivo culture of lamina propria cells harvested from animals treated with STA-5326, indicating a down-regulation of the Th1 response by STA-5326. These results suggest that STA-5326 has potential for use in the treatment of Th1-related autoimmune or immunologic disorders. STA-5326 currently is being evaluated in phase 2 clinical trials in patients with Crohn disease and rheumatoid arthritis. [1]

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STA-5326 is the first potent and selective IL-12/IL-23 inhibitor discovered through screening of a compound library. The unique action of the compound together with the in vivo efficacy in Th1-mediated animal disease models suggest that STA-5326 holds therapeutic promise for the treatment of chronic inflammatory diseases. Injection of monoclonal antibodies recognizing the p40 subunit shared by IL-12 and IL-23 has shown positive results in clinical trials in both patients with psoriasis and patients with Crohn disease. While the antibody acts by neutralizing the IL-12 and IL-23 proteins that already have been produced, STA-5326 acts by selectively shutting off transcription of the p35 and p40 genes. The investigation of the precise mechanism of transcriptional inhibition by STA-5326 is ongoing. STA-5326 now is in clinical evaluation in multiple studies. The compound was shown to be safe in healthy volunteers, and the inhibition of IL-12 production was demonstrated in an ex vivo assay (J.C., unpublished data, 2004). We have completed a phase 2 open-label clinical trial testing STA-5326 in patients with Crohn disease that shows preliminary signs of clinical response by this oral compound. STA-5326 is currently being tested in a blinded and placebo-controlled phase 2b Crohn disease study, and is being evaluated in a phase 2a study in rheumatoid arthritis and common variable immunodeficiency disease (CVID), a disease characterized by elevated IL-12 levels. [1]

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Introduction: The purpose of this study was to determine if oral administration of the interleukin (IL) 12/IL-23 inhibitor, STA-5326, is effective in experimental autoimmune uveoretinitis (EAU). Methods: C57BL/6J mice were immunised with human interphotoreceptor retinoid binding protein peptide (IRBP 1-20). STA-5326 at a dose of either 5 mg/kg or 20 mg/kg, or vehicle alone, was orally administered once a day for six days a week from day 0 to day 14. Fundus examination was performed on day 14 and day 18 after immunisation. Mice were euthanased on day 18 and the eyes were enucleated for histopathological examination. In vivo-primed draining lymph node cells were stimulated with IRBP 1-20 and culture supernatant was harvested for assay of interferon (IFN)-gamma and IL-17 by ELISA. Intracellular expression of IFN-gamma and IL-17 in CD4+ T cells of cultured draining lymph node cells was assessed by flow cytometry. The level of IL-12 p40 in serum was examined in STA-5326-treated or vehicle-treated mice receiving immunisation. [2]
Oral administration of STA-5326 was effective in suppressing inflammation in the EAU model, and reduced the serum level of IL-12/IL-23 p40 and the expansion of IL-17-producing cells. STA-5326 represents a new promising therapeutic modality for refractory uveitis in humans. [2]

C25H34N6O8S2

610.7029

610.187

870087-36-8

Apilimod;541550-19-0; Apilimod mesylate;870087-36-8; 870151-86-3; 1383916-59-3; 870087-37-9 (HCl); 870087-41-5 (besylate)

11527330

White to off-white solid powder

3

14

8

41

637

0

CC1=CC(=CC=C1)/C=N/NC2=CC(=NC(=N2)OCCC3=CC=CC=N3)N4CCOCC4.CS(=O)(=O)O.CS(=O)(=O)O

GAJWNIKZLYZYSY-OKUPSQOASA-N

InChI=1S/C23H26N6O2.2CH4O3S/c1-18-5-4-6-19(15-18)17-25-28-21-16-22(29-10-13-30-14-11-29)27-23(26-21)31-12-8-20-7-2-3-9-24-20;2*1-5(2,3)4/h2-7,9,15-17H,8,10-14H2,1H3,(H,26,27,28);2*1H3,(H,2,3,4)/b25-17+;;

(E)-4-(6-(2-(3-methylbenzylidene)hydrazinyl)-2-(2-(pyridin-2-yl)ethoxy)pyrimidin-4-yl)morpholine dimethanesulfonate

LAM-002; LAM 002; STA-5326; STA5326; Apilimod mesylate; 870087-36-8; Apilimod dimesylate; STA 5326 mesylate; STA-5326 mesylate; STA 5326; LAM002; Apilimod mesylate.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~163.75 mM)
DMSO : ~12.5 mg/mL (~20.47 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.05 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 12.5 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: ≥ 1.25 mg/mL (2.05 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 12.5 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: ≥ 1.25 mg/mL (2.05 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (163.75 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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