Adarotene (ST1926) is a novel retinoid-related molecule (RRM) and an analog of the prototype compound CD437. While its primary molecular target in myeloid leukemia cells is unknown, it is likely to have a conformation similar to that of the RARγ ligand-binding domain, as there is a strict correlation between the RARγ-transactivating and apoptotic activities of the RRM series [2].

Adarotene, with an IC50 ranging from 0.1 to 0.3 µM, inhibits the development of numerous human tumor cell lines in a dose-dependent manner. Depending on the tumor cells IGROV-1 and DU145, adarotene induces cells to accumulate in the G1/S or S phase of the cell cycle [1]. Adarotene exhibits apoptotic and cytotoxic properties against a range of leukemia and cancerous cells, including recently isolated AML blasts in primary culture. The ligand-binding domain of RARγ is similar to the molecular target of ST1926's apoptotic action in myeloid leukemia cells. When cells are treated with alpha-adarotene, intracellular calcium accumulates quickly [2].

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Apoptotic Activity in Myeloid Leukemia Cells: ST1926 induces potent and rapid apoptosis in various myeloid leukemia cell lines, including NB4 (acute promyelocytic leukemia), HL-60, and KG1 cells. It is more potent than the prototype RRM, CD437. In NB4 cells, the EC50 for apoptosis is 0.2 μM (confidence interval 0.08-0.67), which is significantly lower than that of CD437 (1.1 μM, confidence interval 0.82-1.51). The EC50 for cytotoxicity in NB4 cells is 0.1 μM (confidence interval 0.05-0.19), also significantly lower than CD437 (0.9 μM, confidence interval 0.43-2.07) [2].
Structure-Activity Relationship: A series of ST1926 congeners were tested for apoptotic activity in NB4 cells. Modifications to the adamantyl ring, hydroxyl residue, or carboxylic function significantly reduce apoptotic potential. Introduction of a heterocyclic ring in the center of the molecule (e.g., ST2060, ST2062) suppresses apoptotic activity. One such molecule, ST2065, acts as an antagonist, inhibiting ST1926-induced apoptosis when used in molar excess [2].
Gene Expression Profiling: Microarray analysis of NB4 cells treated with ST1926 (0.2 μM for 4h) shows down-regulation of at least 184 genes, many of which are also down-regulated by CD437. A high proportion of down-regulated genes code for mitochondrial, ribosomal, and translation-related proteins. Only a few genes are up-regulated [2].
MAP Kinase Activation: ST1926 induces phosphorylation of p38 and JNK, but only at fully apoptotic concentrations (1 μM). At a lower apoptotic concentration (0.2 μM), no significant phosphorylation is observed. Inhibition of p38 or JNK does not significantly affect ST1926-induced apoptosis, suggesting these kinases are not directly involved in the cell death pathway [2].
Intracellular Calcium Mobilization: ST1926 causes an immediate, dose-dependent, and long-lasting increase in cytosolic calcium levels in NB4 cells, rising from ~20 nM to 600-700 nM. This calcium-mobilizing effect correlates with the apoptotic potency across the RRM series. It is not observed with other apoptotic agents like doxorubicin, etoposide, or fenretinide. The effect is suppressed by the antagonist ST2065, the intracellular calcium chelator BAPTA, and by high concentrations of dihydropyridine-type calcium blockers (e.g., nicardipine, nitrendipine) [2].
Source of Calcium: The calcium rise is not from extracellular influx, as it occurs in calcium-free medium and is unaffected by nickel or verapamil. It is not from the endoplasmic reticulum, as SERCA inhibitors (thapsigargin, TBHQ) have no effect. The rise appears to result primarily from inhibition of mitochondrial calcium reuptake, as uncouplers of oxidative phosphorylation (antimycin A/oligomycin, FCCP) partially reduce the ST1926-induced calcium signal [2].
Role in Apoptosis: The calcium mobilization is necessary for apoptosis. The dihydropyridine nicardipine (100 μM) and the intracellular calcium chelator BAPTA (50-100 μM) inhibit both ST1926-induced calcium rise and apoptosis. Nicardipine also blocks ST1926-induced caspase-3 activation and the release of cytochrome c from mitochondria into the cytosol [2].
Resistant Cell Lines: The NB4.437r cell line, which is selectively resistant to CD437, is also resistant to ST1926-induced apoptosis. However, these cells still show a robust calcium rise upon ST1926 treatment, indicating that the resistance mechanism lies downstream of calcium mobilization [2].

In nude mice, adarotene (15, 20 mg/kg, orally administered) can considerably suppress the formation of tumors in human ovarian cancer A2780/DX and human melanoma MeWo [1]. Oral administration of adarotene (30, 40 mg/kg) has been shown to significantly and dose-dependently prolong the longevity of SCID mice harboring NB4 without evident harm [2].

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Antileukemic Activity in a Murine Model: In SCID mice intraperitoneally inoculated with NB4 human APL cells, intraperitoneal administration of ST1926 (50 mg/kg, 5 times/week for 3 weeks) significantly increases median survival time (55.5 days vs. 35.5 days for vehicle), with an increased life span (ILS) of 56%. This effect is comparable to ATRA (40 mg/kg, ILS 55%). The combination of ST1926 and ATRA shows an additive effect (ILS 89%) [2].
Oral Activity: ST1926 is also orally active. Oral administration at 30, 40, and 50 mg/kg (5 times/week for 3 weeks) results in a dose-dependent increase in survival (ILS of 21%, 35%, and 44%, respectively) compared to vehicle-treated mice [2].

Apoptosis Assay (DAPI Staining): Cells were fixed in methanol and stained with DAPI. The apoptotic index was determined by counting the percentage of cells with morphological features of nuclear fragmentation under a fluorescence microscope (minimum 300 nuclei/field) [2].
Apoptosis Assay (Annexin-V/PI Flow Cytometry): Cells were stained with Annexin-V-FLUOS and propidium iodide (PI) according to the manufacturer's instructions. Stained cells were analyzed by flow cytometry to quantify viable (PI⁻/AX⁻), apoptotic (PI⁻/AX⁺), necrotic (PI⁺/AX⁻), and necrotic/apoptotic (PI⁺/AX⁺) cells [2].
Caspase-3 Activity Assay: Cell extracts were incubated with the fluorogenic peptide substrate DEVD-amc. Caspase-3 activation was measured fluorometrically [2].
Cell Viability: Cell number and viability were determined following staining with erythrosin [2].
Western Blot Analysis: Cellular extracts were subjected to Western blot using antibodies directed against cytochrome c, actin, ERK-1/2, JNK, p38, and their corresponding phosphorylated forms. For cytochrome c release studies, cells were fractionated into mitochondrial and cytosolic fractions [2].
JNK Kinase Activity Assay: JNK was immunoprecipitated from cell extracts, and its activity was determined using c-JUN as a substrate. Phosphorylated c-JUN was detected by Western blot [2].
Microarray Gene Profiling: Poly(A)+ RNA was extracted from NB4 cells treated with vehicle, ST1926 (0.2 μM), or CD437 (1 μM) for 4h. ³²P-labeled cDNA probes were synthesized and hybridized to 12K human ATLAS plastic filters. Data were quantitated with ATLAS IMAGE 2.7 software [2].
Northern Blot Analysis: Total RNA was extracted and subjected to Northern blot analysis using specific human cDNA probes for selected genes to validate microarray results [2].
Intracellular Calcium Measurement: Cells were loaded with the fluorescent probe FURA-2 AM (1 μM) for 30 min at 37°C. After washing, cells were resuspended in PBS with or without calcium. Changes in fluorescence were measured continuously using a spectrophotofluorometer with dual excitation at 340 nm and 380 nm and emission fixed at 480 nm. Cytosolic calcium concentrations were calibrated from the FURA-2 fluorescence signals [2].
RAR Transactivation Assay: COS-7 cells were co-transfected with RARα, RARβ, or RARγ plasmids, a retinoic acid-dependent reporter construct (DR5-tk-CAT), and a β-galactosidase normalization plasmid (pCH110). Twenty-four hours after transfection, cells were treated with test compounds for an additional 24h. CAT and β-galactosidase activities were determined. Results were expressed as fold induction of CAT activity after normalization for β-galactosidase [2].

SCID Mouse Model of APL:** Female SCID mice were inoculated intraperitoneally with 3 × 10⁶ NB4 cells on day 0 (8 mice per group). Treatment started the day after cell inoculation and continued 5 times per week for 3 weeks. ST1926 was dissolved in a cremophor/ethanol (1:1) solution and diluted 1:10 in PBS to the final concentrations (30, 40, or 50 mg/kg). ATRA (40 mg/kg) was prepared similarly. Compounds were administered either intraperitoneally or orally (by gavage) in a volume of 10 mL/kg. For combination treatment, ST1926 (50 mg/kg) and ATRA (40 mg/kg) were co-administered intraperitoneally. Body weight and lethality were monitored throughout the study. Median survival time (MST) and increased life span (ILS) were calculated [2].

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SCID Mouse Model of APL: Female SCID mice were inoculated intraperitoneally with 3 × 10⁶ NB4 cells on day 0 (8 mice per group). Treatment started the day after cell inoculation and continued 5 times per week for 3 weeks. ST1926 was dissolved in a cremophor/ethanol (1:1) solution and diluted 1:10 in PBS to the final concentrations (30, 40, or 50 mg/kg). ATRA (40 mg/kg) was prepared similarly. Compounds were administered either intraperitoneally or orally (by gavage) in a volume of 10 mL/kg. For combination treatment, ST1926 (50 mg/kg) and ATRA (40 mg/kg) were co-administered intraperitoneally. Body weight and lethality were monitored throughout the study. Median survival time (MST) and increased life span (ILS) were calculated [2].

In the in vivo SCID mouse study, ST1926 treatment (30-50 mg/kg, oral or i.p.) was accompanied by a modest, dose-dependent decrease in body weight (maximal body weight loss of 6-17%). No other signs of overt toxicity were reported. The compound has a favorable toxicity profile at the doses and routes tested [2].

[1]. A novel atypical retinoid endowed with proapoptotic and antitumor activity. J Med Chem. 2003 Mar 13;46(6):909-12.

[2]. ST1926, a novel and orally active retinoid-related molecule inducing apoptosis in myeloid leukemia cells: modulation of intracellular calcium homeostasis. Blood. 2004 Jan 1;103(1):194-207.

Adalodine is a synthetic phenolic hydroxy retinoid with pro-apoptotic activity. As an atypical retinoid, adalodine can induce apoptosis, and this process is independent of the retinoid receptor signaling pathway.

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Adarotene (ST1926) is a novel, orally active retinoid-related molecule (RRM) designed to overcome the limitations of the prototype RRM, CD437, which has a narrow therapeutic window and unfavorable pharmacokinetics. ST1926 is a more potent apoptotic agent than CD437 in various myeloid leukemia cell lines in vitro and demonstrates significant antileukemic activity in a murine model of acute promyelocytic leukemia (APL), both intraperitoneally and orally. Its mechanism of action is distinct from that of all-trans-retinoic acid (ATRA) and other classic retinoids, as it does not require gene expression and shows additive effects with ATRA. A key early event in its apoptotic pathway is a rapid increase in cytosolic calcium, primarily due to inhibition of mitochondrial calcium uptake. This calcium mobilization is necessary for subsequent mitochondrial permeability transition pore opening, cytochrome c release, caspase activation, and apoptosis. ST1926 has a favorable toxicity profile in vivo and was under clinical development at the time of this publication [2].

C25H26O3

374.4721

374.188

496868-77-0

9864378

White to green solid powder

5.624

2

3

4

28

574

0

C1C2CC3CC1CC(C2)(C3)C4=C(C=CC(=C4)C5=CC=C(C=C5)/C=C/C(=O)O)O

QAWBIEIZDDIEMW-RXJOCBMKSA-N

InChI=1S/C25H26O3/c26-23-7-6-21(20-4-1-16(2-5-20)3-8-24(27)28)12-22(23)25-13-17-9-18(14-25)11-19(10-17)15-25/h1-8,12,17-19,26H,9-11,13-15H2,(H,27,28)/b8-3+/t17-,18+,19-,25?

(E)-3-(3'-((3r,5r,7r)-adamantan-1-yl)-4'-hydroxy-[1,1'-biphenyl]-4-yl)acrylic acid

ST1926 ST 1926 ST-1926

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 : ~25 mg/mL (~66.76 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.68 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 (6.68 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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 (Please use freshly prepared in vivo formulations for optimal results.)