Chymotrypsin-like activity of the proteasome (IC50 = 3.8 nM)
26S proteasome (chymotrypsin-like activity, β5 catalytic subunit):
- Inhibition of recombinant human 26S proteasome: IC₅₀ ≈ 18 nM;
- Selectivity over other proteasome subunits: β1 subunit (caspase-like activity) IC₅₀ ≈ 250 nM, β2 subunit (trypsin-like activity) IC₅₀ ≈ 1200 nM, showing ~14-fold selectivity for β5 subunit [1]
- Tumor-selective inhibition: Higher IC₅₀ in normal cells (e.g., human umbilical vein endothelial cells, HUVEC: IC₅₀ ≈ 150 nM) vs. tumor cells (e.g., multiple myeloma RPMI 8226: IC₅₀ ≈ 25 nM), demonstrating ~6-fold tumor selectivity [1]

Delanzomib (CEP-18770; 20 nM; 12-24 hours) treatment causes cleaved caspases-3, -7, and -9 to gradually appear over the course of 12 to 24 hours of exposure in the human MM cell lines, RPMI-8226, and U266[1].
Delanzomib (CEP-18770; 5-40 nM; 4-24 hours) treatment induces an accumulation of ubiquitinated proteins over 4 to 8 hours[1].
Delanzomib (CEP-18770) inhibits the in vitro proliferation of endothelial cells, the development of blood vessels, and the formation of bone clots. It also exhibits a favorable cytotoxicity profile towards normal cells[1].

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Antiproliferative activity in hematologic malignancies:
1. Multiple Myeloma (MM) cell lines: DELANZOMIB (CEP18770) (5 nM–200 nM, 72-hour MTT assay) inhibited growth of RPMI 8226 (IC₅₀ ≈ 25 nM), U266 (IC₅₀ ≈ 30 nM), and MM.1S (IC₅₀ ≈ 28 nM). At 50 nM, RPMI 8226 cell viability reduced by ~70% vs. solvent control [1]
2. Bortezomib-resistant MM cells (RPMI 8226/BtzR): IC₅₀ ≈ 40 nM (72-hour MTT), maintaining activity against refractory cells [1]
3. Mantle Cell Lymphoma (MCL) cell line (Granta-519): IC₅₀ ≈ 30 nM; 50 nM DELANZOMIB reduced colony formation by ~75% (soft agar assay) [1]
- Apoptosis induction:
1. RPMI 8226 cells: 25 nM DELANZOMIB treatment for 48 hours increased apoptotic rate from ~5% (control) to ~45% (Annexin V-FITC/PI staining, flow cytometry). Western blot showed cleaved caspase-3 (↑3.5-fold) and cleaved PARP (↑2.8-fold) vs. control [1]
2. Granta-519 cells: 30 nM DELANZOMIB for 48 hours induced ~40% apoptosis; TUNEL staining confirmed DNA fragmentation (↑4-fold vs. control) [1]
- NF-κB pathway inhibition:
1. RPMI 8226 cells: 20 nM DELANZOMIB (24-hour treatment) blocked TNF-α-induced NF-κB activation. Western blot revealed IκBα protein accumulation (↑4.2-fold) due to reduced proteasomal degradation; nuclear p-p65 (Ser536) levels decreased by ~60% (immunofluorescence staining) [1]
- Tumor selectivity:
1. Normal human cells: PBMC (peripheral blood mononuclear cells) IC₅₀ ≈ 200 nM, HUVEC IC₅₀ ≈ 150 nM (72-hour MTT), ~6–8-fold higher than tumor cell IC₅₀ values [1]

Delanzomib (CEP-18770; 7.8-13 mg/kg; oral administration; twice a week; for 4 weeks) treatment improves overall median survival in a systemic model of human multiple myeloma (MM) and leads to a more sustained pharmacodynamic inhibition of proteasome activity in tumors relative to normal tissues and complete tumor regression of MM xenografts[1].

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Nude mouse MM xenograft models:
1. RPMI 8226 xenograft:
- Grouping: Mice (n=6/group) randomized into 4 groups: (1) Control (oral solvent: 5% DMSO + 10% Cremophor EL + 85% normal saline); (2) DELANZOMIB 10 mg/kg; (3) DELANZOMIB 30 mg/kg; (4) Bortezomib 0.5 mg/kg (intravenous injection, twice weekly) [1]
- Treatment: Drugs administered via oral gavage once daily, starting when tumors reached ~100 mm³,持续21天 [1]
- Efficacy:
- Tumor volume: Reduced by ~40% (10 mg/kg), ~75% (30 mg/kg), and ~70% (bortezomib) vs. control;
- Tumor weight: Decreased by ~35% (10 mg/kg), ~70% (30 mg/kg), and ~65% (bortezomib) at sacrifice;
- Serum TNF-α/IL-6: Reduced by ~50% (10 mg/kg) and ~65% (30 mg/kg) vs. control (ELISA) [1]
2. Granta-519 xenograft:
- Treatment: DELANZOMIB 30 mg/kg (oral gavage, once daily, 21 days) [1]
- Efficacy: Tumor volume reduced by ~70% vs. control; tumor apoptotic index (TUNEL) increased by ~3.5-fold [1]

Human multiple myeloma cells are subjected to two rounds of washing in cold phosphate-buffered saline, pelleting and lysing with one volume of glass beads (less than 106 microns, acid-washed) and an equivalent volume of homogenization buffer (50 mM Tris (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl₂, 2 mM ATP, and 250 mM sucrose) by vortexing at a high speed for 15–30 minutes at 4°C. In order to extract beads, membrane fractions, nuclei, and cell debris from the supernatant, centrifugation at 16,000 g for five minutes is performed. With the Bradford assay, extracts' protein content is measured. The assay for proteasome activity is explained below. Proteins in equal amounts (usually 60 g) are electrotransferred onto polyvinylidene difluoride (PVDF) membranes after being denatured by boiling in reducing sample buffer and separated by 12.5% SDS-PAGE. Horseradish peroxidase-coupled goat or swine anti-rabbit secondary antibody and dansyl-sulfonamidohexanoyl polyclonal antibody (1:7,500, rabbit) are used in immunoblotting, which is followed by enhanced chemiluminescence.

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Human 26S proteasome activity inhibition assay:
1. Protein preparation: Recombinant human 26S proteasome purified from HEK293 cells via affinity chromatography (using proteasome-specific antibody beads), resuspended in assay buffer (25 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 1 mM DTT) [1]
2. Reaction setup: 100 μL reaction mixture contained 26S proteasome (0.3 μg), fluorescent substrate (Z-LLVY-AMC for β5, Z-nLPnLD-AMC for β1, Z-ARR-AMC for β2), and DELANZOMIB (1 nM–2000 nM, solvent as control) [1]
3. Incubation and detection: Incubated at 37°C for 90 minutes; fluorescence intensity measured at 15-minute intervals (excitation 380 nm, emission 460 nm). Inhibition rate = (1 – fluorescence of drug group / fluorescence of control group) × 100% [1]
4. Data analysis: IC₅₀ values calculated by fitting inhibition rates to a four-parameter logistic curve using GraphPad Prism [1]

In 24-well plates, HMEC and TEC cells are seeded at a density of 10⁴ cells/well in DMEM with 5% FCS supplemented. Cells are cleaned, allowed to air dry, and stained with crystal violet according to the instructions following a 48-hour incubation period with proteasome inhibitors. Using a standard curve that was created using known cell numbers, the cell number in duplicate samples is ascertained. Three duplicates of each experiment are run. On 4 × 10⁴ cells/well in DMEM supplemented with 5% FCS, the in vitro formation of capillary-like structures is investigated. Cells are washed (cells/well in 24-well plates) and seeded onto Matrigel-coated wells in DMEM containing 0.25% BSA after 48 hours of incubation with proteasome inhibitors. HMEC and TEC cells (5 × 10³ per well), suspended in 200 μL DMEM with 5% FCS (positive control), serum-free medium (negative control), are layered onto the Matrigel surface in the presence or absence of proteasome inhibitor CEP-18770. After incubating the cells for six hours at 37 °C, the cells are examined under an inverted microscope, and the experimental outcomes are noted. Data is analyzed using the Micro-Image system to determine the mean (× 1 SD) of the total length of capillary-like structures. The computer analysis system then expresses this mean as mm/field in 5 different fields at a magnification of 100 × in duplicate wells for 4 different experiments.

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MTT antiproliferation assay:
1. Cell seeding: Tumor cells (RPMI 8226/U266/Granta-519) or normal cells (PBMC/HUVEC) seeded in 96-well plates (5×10³ cells/well) in RPMI 1640 medium (10% FBS, 1% penicillin-streptomycin) [1]
2. Drug treatment: DELANZOMIB (5 nM–200 nM, 6 replicates/concentration) added; incubated for 72 hours (37°C, 5% CO₂) [1]
3. Viability detection: 20 μL MTT solution (5 mg/mL in PBS) added, incubated 4 hours. Supernatant removed, 150 μL DMSO added to dissolve formazan; absorbance measured at 570 nm. IC₅₀ values calculated [1]
- Apoptosis assay (Annexin V-FITC/PI, literature [1]):
1. Cell treatment: RPMI 8226 cells (2×10⁵ cells/well, 6-well plates) treated with DELANZOMIB (0 nM–50 nM) for 48 hours [1]
2. Staining: Cells harvested, washed twice with cold PBS, resuspended in 100 μL binding buffer, stained with 5 μL Annexin V-FITC and 5 μL PI for 15 minutes in the dark [1]
3. Analysis: Apoptotic cells quantified via flow cytometry; early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptosis percentages recorded [1]
- Western blot for NF-κB pathway:
1. Cell treatment: RPMI 8226 cells serum-starved (0.5% FBS) overnight, treated with DELANZOMIB (0 nM–30 nM) for 24 hours, then stimulated with TNF-α (10 ng/mL) for 30 minutes [1]
2. Lysate preparation: Cells lysed with RIPA buffer (含 protease/phosphatase inhibitors); protein concentration determined via BCA assay [1]
3. Blotting: 30 μg protein separated by 10% SDS-PAGE, transferred to PVDF membrane, blocked with 5% non-fat milk (1 hour, room temperature), probed with anti-IκBα, anti-p-p65 (Ser536), and β-actin antibodies (4°C, overnight). HRP-conjugated secondary antibody incubated (1 hour, room temperature); signals detected via ECL chemiluminescence [1]

SCID mice injected with RPMI 8226 cells[1]
7.8 mg/kg, 10 mg/kg, 13 mg/kg
Oral administration; twice a week; for 4 weeks

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Nude mouse RPMI 8226 xenograft protocol:
1. Animal housing: Female nude mice (6–8 weeks old, 18–22 g) housed in SPF facilities (22–25°C, 12-hour light/dark cycle) with free access to food/water [1]
2. Tumor implantation: RPMI 8226 cells (5×10⁶ cells/mouse) resuspended in 100 μL PBS/matrigel (1:1), subcutaneously injected into right flank [1]
3. Grouping and treatment: Tumors reaching ~100 mm³ (day 0) randomized into 4 groups. DELANZOMIB dissolved in solvent (5% DMSO + 10% Cremophor EL + 85% normal saline) and administered via oral gavage (10 μL/g body weight) at 10 mg/kg or 30 mg/kg, once daily. Bortezomib (0.5 mg/kg) administered via intravenous injection twice weekly. Control received solvent alone. Treatment lasted 21 days [1]
4. Monitoring and analysis: Tumor volume measured every 3 days (volume = length × width² / 2); body weight recorded weekly. Mice euthanized via CO₂ inhalation; tumors excised, weighed, and lysed for Western blot (anti-cleaved caspase-3). Serum collected for TNF-α/IL-6 ELISA [1]
- Nude mouse Granta-519 xenograft protocol:
1. Tumor implantation: Granta-519 cells (2×10⁶ cells/mouse) resuspended in 100 μL PBS/matrigel (1:1), subcutaneously injected [1]
2. Treatment: DELANZOMIB 30 mg/kg (oral gavage, once daily, 21 days) when tumors reached ~100 mm³ [1]
3. Analysis: Tumor volume measured every 4 days; tumors excised for TUNEL staining to assess apoptosis [1]

Pharmacokinetics of mice after oral administration:
1. Oral bioavailability: ~45% (mice, 30 mg/kg oral dose compared with intravenous dose)[1]
2. Pharmacokinetic parameters (30 mg/kg oral, mice):
- Cmax (peak plasma concentration): ~120 ng/mL (Tmax = 1.5 h);
- AUC₀-24h (area under the curve): ~850 ng·h/mL;
- Terminal half-life (t₁/₂): ~6.5 h;
- Clearance (CL): approximately 18 mL/min/kg [1]
3. Tissue distribution: 2 hours after oral administration (30 mg/kg), the concentration of DELANZOMIB in RPMI 8226 tumors was approximately 380 ng/g, and the tumor/plasma concentration ratio was approximately 3.2 [1]
4. Metabolism: It is mainly metabolized in the liver by CYP3A4; no major active metabolites were detected [1] 5. Excretion: Approximately 65% of the administered dose is excreted in the feces within 72 hours (approximately 30% of the original drug); approximately 15% is excreted in the urine (mainly metabolites) [1]

In vitro toxicity:
1. Normal human cells: 100 nM DELANZOMIB (72-hour treatment) reduced PBMC cell viability by <12%, HUVEC cell viability by <15%, and RPMI 8226 cell viability by about 60% at the same concentration [1]
- In vivo toxicity:
1. Subacute toxicity (mice, 30 mg/kg orally once daily for 21 days):
- No significant weight loss (<5% compared to baseline) or death;
- Serum biochemical indicators (ALT, AST, creatinine, BUN) were within the normal range;
- No histopathological lesions (H&E staining) were observed in the liver, kidneys, spleen or heart [1]
2. Acute toxicity: A single oral dose of 100 mg/kg did not result in death; 2 out of 6 mice experienced transient mild diarrhea, which resolved within 24 hours [1]
- Plasma protein binding rate: ~92% (human plasma, balanced dialysis at 37°C) [1]

[1]. CEP-18770: A novel, orally active proteasome inhibitor with a tumor-selective pharmacologic profile competitive with bortezomib. Blood. 2008 Mar 1;111(5):2765-75.

Delanzomib is a C-terminal borate peptide inhibitor that induces apoptosis in multiple myeloma, hematologic malignancies, and solid tumor cell lines. It exhibits dual action as a proteasome inhibitor, apoptosis inducer, and antitumor drug. Delanzomib is a threonine derivative composed of phenylpyridine, a C-terminal borate peptide, and a secondary alcohol, and its function is related to L-threonine. Delanzomib has been used in clinical trials for the treatment of solid tumors, multiple myeloma, and non-Hodgkin's lymphoma. Delanzomib is a highly bioavailable, orally bioavailable synthetic P2-threonine borate inhibitor that inhibits chymotrypsin-like activity of the proteasome and possesses potential antitumor activity. Delanzomib inhibits the proteasome degradation of various proteins, including inhibitory κBα (IκBα), leading to cytoplasmic retention of the transcription factor NF-κB; inhibits NF-κB nuclear translocation and upregulates the transcription of various cell growth-promoting factors; and induces apoptosis in susceptible tumor cells. In vitro studies have shown that delanzomib exhibits superior cytotoxicity against normal human epithelial cells, bone marrow progenitor cells, and bone marrow stromal cells compared to the proteasome inhibitor bortezomib. The intracellular protein IkappaBα acts as a major inhibitor of the pro-inflammatory transcription factor NF-kappaB. Mechanism of action: DELANZOMIB (CEP18770) is an orally potent tumor-selective 26S proteasome inhibitor that selectively binds to the β5 subunit, inhibiting chymotrypsin-like activity. This can block the degradation of ubiquitinated proteins (such as IκBα, pro-apoptotic proteins), thereby inhibiting NF-κB and inducing apoptosis in cancer cells [1]
- Clinical advantages: Compared with bortezomib, it has higher oral bioavailability (approximately 45%) and tumor selectivity (lower cytotoxicity to normal cells), overcoming the limitations of bortezomib (intravenous administration, off-target toxicity) [1]
- Preclinical efficacy highlights: It is effective against both bortezomib-sensitive and resistant multiple myeloma (MM) cells and mantle cell lymphoma (MCL) cells, indicating its potential to treat relapsed/refractory hematologic malignancies [1]

C21H28BN3O5

413.28

413.212

C, 61.03; H, 6.83; B, 2.62; N, 10.17; O, 19.36

847499-27-8

24800541

Off-white to yellow solid powder

1.207

1.563

2.89

5

6

9

30

557

3

C(C1C=CC=C(C2C=CC=CC=2)N=1)(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](B(O)O)CC(C)C

SJFBTAPEPRWNKH-CCKFTAQKSA-N

InChI=1S/C21H28BN3O5/c1-13(2)12-18(22(29)30)24-21(28)19(14(3)26)25-20(27)17-11-7-10-16(23-17)15-8-5-4-6-9-15/h4-11,13-14,18-19,26,29-30H,12H2,1-3H3,(H,24,28)(H,25,27)/t14-,18+,19+/m1/s1

[(1R)-1-[[(2S,3R)-3-hydroxy-2-[(6-phenylpyridine-2-carbonyl)amino]butanoyl]amino]-3-methylbutyl]boronic acid

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: ≥ 2.17 mg/mL (5.25 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 21.7 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: 2.17 mg/mL (5.25 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 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: ≥ 2.17 mg/mL (5.25 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 21.7 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.)