p210Bcr/abl kinase [4]

Adaphostin inhibits granulocyte colony formation in specimens of chronic myelogenous leukemia (CML) at lower concentrations without increasing toxicity in normal progenitors and down-regulates p210^bcr/abl in K562 cells[1].
N-acetylcysteine (NAC) inhibits adipostin-induced p53 up-regulation, DNA strand breaks, and ROS production in ML-1 cells[4].

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Adaphostin showed IC50 values ranging from 17 to 216 nM in nine human leukemia cell lines (CEM/0, ara‑C‑resistant clones, ASNase‑resistant clones, Jurkat/E6‑1) after 48 h MTT assay. NSC 682492 (a congener) was less potent. Phase‑contrast microscopy revealed apoptotic morphology (nuclear condensation and apoptotic bodies) within 24 h at 0.1‑1 μM. Western blot showed caspase‑3 cleavage (11 and 21 kDa fragments) and ELISA demonstrated up to 5‑fold increase in caspase‑3 activity after 1 μM treatment for 24 h. Protein synthesis (³H‑leucine incorporation) was inhibited by 90‑97% at 24 h. VEGF secretion in supernatants was dose‑dependently inhibited (0.1 and 1 μM) in several cell lines, while cell lines 3,5,9 did not secrete VEGF. VEGF‑R1 protein levels decreased after treatment as shown by Western blot. U87 MG glioblastoma cells treated with Flt‑1/Fc chimera (VEGF inhibitor) showed 30‑43% cell kill; combination of adaphostin plus Flt‑1/Fc chimera exhibited 8‑fold synergism (reduction of IC50 of the chimera). [2]
Adaphostin inhibited growth of Ph‑positive (KBM5, KBM7) and Ph‑negative (OCI/AML2, OCI/AML3) cell lines with IC50 values of 0.5‑1 μM after 72 h MTS assay. K562 cells were less sensitive (IC50 ~13 μM). Imatinib‑resistant KBM5‑R and KBM7‑R showed cross‑resistance to adaphostin (IC50 ~1.3 μM at 72 h). Adaphostin induced apoptosis in a dose‑ and time‑dependent manner: caspase‑3/7 activity increased at 18 h and 48 h (example AML2 and KBM7). In KBM5 cells, 24 h treatment caused dose‑related loss of mitochondrial membrane potential (CMXRos/MTGreen), activation of caspase‑3 (PhiPhiLux/PI), and annexin V positivity. Imatinib induced apoptosis only in KBM7, whereas adaphostin induced apoptosis in both KBM5 and KBM7. Combination of adaphostin with imatinib (IC20 of imatinib) resulted in additive (not synergistic) effects in KBM5, KBM7, KBM5‑R, KBM7‑R; only K562 showed strong synergy. Primary CML and AML patient cells (including imatinib‑resistant chronic phase, blast phase, and relapsed AML) showed dose‑dependent reduction in colony formation (clonogenic assay) with adaphostin (0.5‑2 μM). Normal bone marrow colony growth was not significantly affected. Adaphostin also killed AML cells in short‑term MTS assay. Western blot with anti‑phosphotyrosine revealed time‑ and dose‑dependent modulation of tyrosine phosphorylation patterns in KBM5 and OCI/AML3 cells, which were cell‑line specific. Crk‑L and Cbl proteins were undetectable in KBM5 and OCI/AML3, suggesting adaphostin cytotoxicity is independent of these Bcr/Abl downstream effectors. Bcl‑2 expression did not change significantly. Adaphostin induced superoxide production in KBM5 and OCI/AML3 cells (dose‑ and time‑dependent), which was significantly reduced by the antioxidant N‑acetyl‑cysteine (NAC). [3]

In an orthotopic brain tumor model, athymic mice were inoculated with 10⁶ U87 MG glioblastoma cells into the caudate putamen. Starting on day 3, mice received i.p. Adaphostin at 8.33 mg/kg daily for three doses per week for 4 weeks alone or combined with a single s.c. dose of Flt‑1/Fc chimera (5 mg/kg). Control mice had large intra‑ and extracranial tumors and median survival of 23 days. Adaphostin alone increased median survival to 133% of control, with mice showing small intracranial tumors with necrotic centers. Combination treatment increased survival to 142% of control, with very small residual tumors around the needle track. Mice treated with adaphostin alone killed on day 29 had smaller brain tumors and no extracranial tumors; however, two mice kept until day 33 (6 days after last drug) showed average 1450 mg extracranial tumor regrowth. In contrast, combination‑treated mice had minimal tumors that did not grow between day 28 and day 33, indicating longer‑lasting inhibition. [2]

Caspase‑3 activity was quantified by a colorimetric assay using the CaspACE Assay System. Cell extracts (20 μl equivalent to 2×10⁶ cells) were incubated with provided substrate at 37°C for 4 h, and absorbance was read at 405 nm against a standard curve. [2]
Caspase‑3/7 activity was measured using a fluorogenic substrate (rhodamine 110, bis‑(N‑CBZ‑L‑aspartyl‑L‑glutamyl‑L‑valyl‑L‑aspartic acid amide, Z‑DEVD‑R110). Cells were seeded at 1.5×10⁶/mL, incubated with or without drug for 18 h or 48 h, then homogeneous caspase‑3/7 reagent was added, incubated for 2 h at room temperature, and fluorescence measured at excitation 499 nm and emission 521 nm. Results expressed as relative fluorescence light units. [3]
Superoxide production was assessed by dihydroethidium (DHE) staining. Cells were treated with adaphostin (2 or 5 μM) for indicated times, with or without 24 mM NAC. Twenty minutes before harvest, 10 μM DHE was added. Cells were washed in Ca²⁺‑free PBS and immediately analyzed for ethidium fluorescence intensity by flow cytometry. [3]

MTT cytotoxicity assay: Cells seeded at 2×10⁵/mL in 24‑well plates, treated with multiple drug concentrations for 48 h, then MTT added and OD₄₉₅ measured. IC₅₀ calculated using Drug Effect Analysis computer program. [2]
DNA, RNA, and protein synthesis inhibition: Cells treated with drug, then pulsed with 5 μCi ³H‑thymidine, ³H‑uridine, or ³H‑leucine for 30 min. Cells were centrifuged, washed, lysed, macromolecules extracted, and radioactivity counted. [2]
Apoptosis microphotography: Phase‑contrast images taken with Nikon inverted microscope and digital camera; apoptotic cells identified by nuclear condensation and apoptotic bodies, counted per field. [2]
Caspase‑3 ELISA: Cells (approx. 10⁷) lysed in provided buffer, protein extracts collected after centrifugation, 20 μl extract (equivalent to 2×10⁶ cells) incubated with substrate at 37°C for 4 h, OD₄₀₅ measured. [2]
VEGF ELISA: Cell culture supernatants (with 10% FBS) analyzed using Quantikine Human VEGF ELISA kit per manufacturer’s instructions. [2]
Western blot: Protein (50 μg) separated by SDS‑PAGE (10% resolving gel), transferred to nitrocellulose, blocked with 5% BLOTTO, hybridized overnight with primary antibody (caspase‑3 mouse monoclonal IgG2a 1:1000; VEGF‑R1 rabbit 1:100; actin goat 1:1000), then secondary antibody (anti‑mouse/anti‑goat IgG‑HRP 1:1000), visualized by ECL. [2]
MTS assay (cell viability): Cells seeded at 0.4×10⁶/mL (cell lines) or 1.0×10⁶/mL (primary cells) in 96‑well plates. After drug exposure for 24, 48 or 72 h, 20 μL MTS solution added, incubated 3 h at 37°C, absorbance measured at 490 nm. [3]
Clonogenic assay: Mononuclear cells plated at 1×10⁵/mL in methylcellulose medium with recombinant cytokines. Adaphostin added at increasing concentrations. Colonies (>50 cells) counted after 8 days at 37°C, 5% CO₂. IC₅₀ determined relative to control wells. [3]
Apoptosis by flow cytometry:
- Annexin V/PI: Cells washed, incubated with FITC‑conjugated annexin V (1:100) for 15 min in dark, then PI added and analyzed.
- Mitochondrial membrane potential: Cells stained with MitoTracker Red CMXRos and MitoTracker Green FM (submicromolar) for 1 h at 37°C, then flow cytometry.

- Caspase‑3 like activity: Cells incubated with PhiPhiLux G1D2 substrate for 1 h at 37°C, then PI added before flow cytometry.

- Subdiploid population: Cells labeled only with PI for DNA fragmentation quantification. [3]
Superoxide detection: Cells treated with adaphostin (2 or 5 μM) for indicated times, with/without 24 mM NAC. 20 min before harvest, 10 μM dihydroethidium added, then washed and analyzed for ethidium fluorescence by flow cytometry. [3]
Western blot and immunoprecipitation: Cells lysed in TENN buffer with protease inhibitors. Lysates centrifuged at 20,000 g for 50 min at 4°C. Protein (200 μg) separated on 9.5% SDS‑PAGE, transferred to nitrocellulose, blocked with 5% non‑fat milk, probed with mouse anti‑phosphotyrosine (1:2000) or anti‑β‑actin, then HRP‑conjugated secondary antibody, detected by ECL. For immunoprecipitation, lysate mixed with agarose‑A/G and mouse anti‑phosphotyrosine (clone 4G) for 16 h at 4°C, washed, eluted, separated by SDS‑PAGE, probed with rabbit anti‑Crk‑L (0.5 μg/mL) or anti‑Cbl. [3]

Female athymic mice were orthotopically inoculated with 10⁶ U87 MG cells (expressing GFP) in 10 μL PBS into the caudate putamen (1.8 mm right of bregma, 0.4 mm anterior to coronal suture, 3.0 mm from skull). Treatment started on day 3. Adaphostin was dissolved in 50% DMSO then sequentially diluted with sterile PBS to 1% DMSO. Mice received i.p. injections of adaphostin at 8.33 mg/kg daily for three doses per week for 4 weeks (total 4 weeks treatment, ending day 28). One group additionally received a single s.c. injection of Flt‑1/Fc chimera (5 mg/kg) on day 3. Control group received vehicle i.p. (PBS with 1% DMSO). Body weight recorded daily. Control animals were killed by day 17 due to tumor progression; treated animals killed between day 29 and 34. Brains and tumors fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin‑eosin. [2]

Adaphostin has a longer serum half-life compared to its parent compound AG957. [4]

Adaphostin treatment (8.33 mg/kg i.p., three doses/week for 4 weeks) produced no weight changes in athymic mice. [2]
Adaphostin at concentrations up to 2 μM had no significant effect on colony growth of normal human bone marrow cells in clonogenic assays. [3]

[1]. Effects of the bcr/abl kinase inhibitors AG957 and NSC 680410 on chronic myelogenous leukemia cells in vitro. Clin Cancer Res. 2000;6(1):237-249.

[2]. In vitro and in vivo evaluations of the tyrosine kinase inhibitor NSC 680410 against human leukemia and glioblastoma cell lines. Cancer Chemother Pharmacol. 2002;50(6):479-489.

[3]. Adaphostin has significant and selective activity against chronic and acute myeloid leukemia cells. Cancer Sci. 2006;97(9):952-960.

[4]. Involvement of reactive oxygen species in adaphostin-induced cytotoxicity in human leukemia cells. Blood. 2003;102(13):4512-4519.

Adaphostin is the adamantyl ester of AG957. It has approximately 2‑fold lower IC₅₀ against cell lines than AG957 but 5‑fold poorer Km for p210bcr/abl autokinase, suggesting additional targets. It inhibits VEGF secretion and VEGF‑R1 expression. The combination with Flt‑1/Fc chimera showed 8‑fold synergism against U87 MG cells in vitro and increased survival in an orthotopic brain tumor model. The drug induces apoptosis via caspase‑3 activation and is active against p53‑null, drug‑resistant leukemia clones. [2]
Adaphostin induces superoxide production in leukemic cells, which is critical for its cytotoxicity; this effect is selective for leukemic cells versus normal progenitors. Imatinib‑resistant cell lines (KBM5‑R with T315I mutation, KBM7‑R with BCR/ABL amplification) showed cross‑resistance to adaphostin. Combination with imatinib was additive in most cell lines except K562. Crk‑L and Cbl are not required for adaphostin activity. Adaphostin has demonstrated synergy with proteasome inhibitors (e.g., bortezomib) via ROS‑dependent mechanisms in other studies. It warrants clinical development for CML and AML, including imatinib‑resistant disease. [3]

C24H27NO4

393.47500

393.194

C, 73.26; H, 6.92; N, 3.56; O, 16.26

241127-58-2

241127-58-2

387042

Off-white to light brown solid powder

1.33g/cm3

606.2ºC at 760mmHg

320.4ºC

2.73E-15mmHg at 25°C

1.661

4.991

3

5

6

29

558

0

OC1=CC=C(O)C(CNC2=CC=C(C(OC34CC5CC(C3)CC(C5)C4)=O)C=C2)=C1

YJZSUCFGHXQWDM-UHFFFAOYSA-N

InChI=1S/C24H27NO4/c26-21-5-6-22(27)19(10-21)14-25-20-3-1-18(2-4-20)23(28)29-24-11-15-7-16(12-24)9-17(8-15)13-24/h1-6,10,15-17,25-27H,7-9,11-14H2

1-adamantyl 4-[(2,5-dihydroxyphenyl)methylamino]benzoate

Adaphostin

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: ~50 mg/mL (~127.1 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.35 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 (6.35 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.)