PARP-2 ( IC50 = 2.9 nM ); PARP-1 ( IC50 = 5.2 nM ); Autophagy
Veliparib (ABT-888) is a potent and selective inhibitor of poly(ADP-ribose) polymerases (PARP), with highest activity against PARP1 (IC50 = 5.2 nM) and PARP2 (IC50 = 2.9 nM) in recombinant enzyme assays. It exhibits minimal inhibition of other PARP family members (e.g., PARP3, PARP6) with IC50 values >1000 nM [1]
- Veliparib (ABT-888) does not inhibit c-Met kinase activity (IC50 >10 μM) but blocks c-Met-mediated phosphorylation of PARP1 at Ser177, which enhances its PARP inhibitory efficacy in c-Met-overexpressing cancer cells [2]

ABT-888 is not active at SIRT2 (>5 μM).[1] ABT-888 inhibits PARP activity with an EC50 of 2 nM in C41 cells.[2] ABT-888 could decrease the PAR in H460 cells that have been exposed to radiation and those that have not. In H460 cells, ABT-888 also lowers clonogenic survival and suppresses PARP-1 to prevent DNA repair. When ABT-888 and radiation are combined, H460 cells undergo increased autophagy and apoptosis.[3] In H1299, DU145, and 22RV1 cells, ABT-888 also suppresses PARP activity.This suppression occurs independently of p53 activity. In the clonogenic H1299 cells, ABT-888 (10 μM) suppresses the surviving fraction (SF) by 43%. In oxic H1299 cells, ABT-888 exhibits effective radiosensitivity. Moreover, ABT-888 may reduce the survival factor (SF) of hypoxic-irradiated cells, such as H1299, DU145, and 22RV1.[4]

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Antiproliferative activity in HR-deficient cancer cells: Veliparib (ABT-888) shows preferential cytotoxicity to homologous recombination (HR)-deficient cells (e.g., BRCA1/2-mutant). IC50 values (72 h, MTT assay): BRCA1-mutant MDA-MB-436 (breast cancer, 0.8 μM), BRCA2-mutant CAPAN-1 (pancreatic cancer, 0.6 μM); vs. HR-proficient MCF-7 (breast cancer, IC50 = 12 μM), HCT116 (colorectal cancer, IC50 = 15 μM) [1]
- PARP inhibition and DNA damage accumulation: In MDA-MB-436 cells, Veliparib (ABT-888) (0.1–2 μM) dose-dependently reduced poly(ADP-ribose) (PAR) polymer levels (western blot): 1 μM reduced PAR by 85% vs. control. It also increased γ-H2AX foci (DNA double-strand break marker) by 4.2-fold (immunofluorescence) and prolonged G2/M cell cycle arrest (35% G2/M cells vs. 18% control) at 0.5 μM [1]
- Synergy with DNA-damaging agents: Combination of Veliparib (ABT-888) (0.2 μM) with pegylated liposomal doxorubicin (PLD, 0.1 μg/mL) in triple-negative breast cancer (TNBC) MDA-MB-231 cells enhanced cytotoxicity: cell viability reduced to 22% (combination) vs. 58% (Veliparib alone) and 65% (PLD alone). The combination index (CI) was 0.45, indicating strong synergy [4]
- Enhanced efficacy with c-Met inhibition: In c-Met-overexpressing NSCLC H1975 cells, Veliparib (ABT-888) (1 μM) combined with the c-Met inhibitor crizotinib (0.5 μM) increased apoptotic cells to 45% vs. 18% (Veliparib alone) and 12% (crizotinib alone). This was associated with reduced p-PARP1 (Ser177) levels (60% reduction vs. Veliparib alone) and increased γ-H2AX (3.8-fold vs. Veliparib alone) [2]
- Protection against sulfur mustard-induced cutaneous injury: In human dermal fibroblasts (HDFs) exposed to sulfur mustard (100 μM), Veliparib (ABT-888) (5 μM) reduced cell death by 40% (MTT assay) and decreased PARP overactivation (PAR levels reduced by 55%) and pro-inflammatory cytokine release (IL-6 reduced by 60%, TNF-α reduced by 50%) [3]

In mice, Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys, the oral bioavailability of ABT-888 ranges from 56% to 92%.[1] ABT-888 (25 mg/kg i.p.) could enhance tumor growth delay in a well-tolerated NCI-H460 xenograft model. When combined with radiation therapy, ABT-888 inhibits the growth of new tumor vessels.[3] ABT-888 decreases intratumor PAR levels in A375 and Colo829 xenograft models by more than 95% at doses of 3 and 12.5 mg/kg, respectively, and the suppression may be sustained over time.

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\n\nVeliparib (ABT-888) potentiates temozolomide in a syngeneic melanoma model. [1]
\nTemozolomide is a newer generation of cytotoxic alkylating agent that is currently used to treat central nervous system malignancies and melanoma. The pharmacokinetic profile of temozolomide is similar between mice and humans, and this allows studies in mice at similar exposures to those achieved in humans. This is important because preclinical models best predict clinical outcomes when plasma drug concentrations of the cytotoxic agents are similar to those seen in humans. Consequently, a dose of 50 to 62.5 mg/kg/d temozolomide was used, and this dose closely mimics human exposure at the clinically relevant dose of 200 mg/m2 (oral, q.d.×5) when measured by either area under the concentration curve (AUC) or Cmax. At this dose range, no overt toxicity (e.g., excessive weight loss, ruffled coats, dehydration, etc.) was observed in mice. [1]
\n\nThe B16 model, which is relatively resistant to most chemotherapeutics, is moderately sensitive to temozolomide and its sensitivity can be enhanced with PARP inhibitors. Veliparib (ABT-888), administered orally, significantly potentiated the temozolomide efficacy in a dose-dependent manner (Fig. 2A). Maximum potentiation was seen at day 19 with % T/C values (versus temozolomide) of 10 (P = 0.0003), 16 (P < 0.0001), and 23 (P < 0.0001) for the 25, 12.5, and 3.1 mg/kg/d Veliparib (ABT-888) combination groups, respectively. The combinations were well tolerated with maximum body weight loss of 11% for the 25 mg/kg/d ABT-888 and temozolomide combination compared with 7% for temozolomide and 2% for ABT-888. The mice rapidly regained weight once the dosing period ended. [1]
\n\nTo establish the steady-state concentration necessary for in vivo activity, Veliparib (ABT-888) was administered as a continuous infusion in combination with 50 mg/kg/d temozolomide. ABT-888 at doses of 25 to 1 mg/kg/d all significantly potentiated the temozolomide monotherapy (Fig. 2B). Maximum potentiation was seen at day 17 with % T/C values (versus temozolomide) of 13 (P < 0.0001), 12 (P < 0.0001), 16 (P < 0.0001), 39 (P = 0.0033), and 63 (not significant) for the 25, 12.5, 5, 1, and 0.3 mg/kg/d ABT-888 combination groups, respectively. A higher dose of 50 mg/kg/d ABT-888 could not be evaluated with temozolomide because this combination resulted in skin toxicity at the OMP implantation site. The 25 and 12.5 mg/kg/d ABT-888 combination treatments were equivalent in activity, thereby defining the maximally efficacious dose as 12.5 mg/kg/d in this model. Maximum weight loss for the combination groups was 1% compared with a 5% gain for temozolomide. [1]

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\n\nIn vivo inhibition of PAR. [1]
\nActivation of PARP in response to DNA damage results in ribosylation of various substrate proteins. To show inhibition of PARP activity in vivo, tumors from mice treated with Veliparib (ABT-888) were analyzed for levels of PAR by Western blot. B16F10 tumor-bearing mice were treated with either vehicle, temozolomide alone, or in combination with ABT-888 (Fig. 2C), similar to doses used in the efficacy study (Fig. 2A). A decrease in PAR proteins in tumors from animals treated with either ABT-888 alone or in combination with temozolomide was observed, indicating the ability of ABT-888 to inhibit PARP activity in vivo.\n [1]
\nVeliparib (ABT-888) potentiates temozolomide in a syngeneic glioma model. ABT-888 also potentiates temozolomide in a 9L orthotopic rat glioma model. Maximum efficacy was seen at day 14 using magnetic resonance imaging (Fig. 3A and C). ABT-888 at 50 mg/kg/d in combination with temozolomide reduced tumor volume by 63%, which was 44% better than temozolomide alone (P < 0.005). Tumor growth inhibition with ABT-888 was dose dependent (Fig. 3A and B). The combination of 50 mg/kg/d dose of ABT-888 with temozolomide significantly prolonged animal survival versus temozolomide with a median survival of 19 and 22 days, respectively (P < 0.0132, log-rank test; Fig. 3B). ABT-888 as a single agent at 50 mg/kg/d was not efficacious in this model (data not shown). The combinations were well tolerated with maximum weight losses of only 9% for the combination compared with 8% for temozolomide.\n

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\nVeliparib (ABT-888) potentiates platinum agents. [1]
\nThe ability of ABT-888 to potentiate the efficacy of platinum-based agents was investigated in the MX-1 breast carcinoma xenograft model. This line was derived from a 29-year-old female with a poorly differentiated mammary carcinoma. Internal sequencing efforts at Abbott have determined that MX-1 has BRCA1 deletions. A novel BRCA1 variant (BRCA1 33636delGAAA) was detected that would result in a frameshift mutation predicted to introduce a chain terminator and truncate the protein at residue 999. Two previously described nonsynomous single-nucleotide polymorphisms were also detected in BRCA2 (BRCA2 16864A>C, Asn289His, and BRCA2 221847A>G, Asn991Asp), both of which have been described in Chinese Breast Cancer families.\n
\nVeliparib (ABT-888) induced a pronounced potentiation of cisplatin activity (Fig. 4A). At day 68 when all mice were still present in each combination group, no significant differences were noted between the 5, 25, and 50 mg/kg/d combinations with cisplatin. However, at the end of the trial, ABT-888 at 5, 25, and 50 mg/kg/d in combination with cisplatin showed an increase in cures (8/9, 8/9, and 6/9 animals, respectively), whereas the cisplatin monotherapy had only 3/9 cures (no measurable tumors at end of the trial). Both the 5 and 25 mg/kg/d ABT-888 plus cisplatin were significantly different than cisplatin alone (P = 0.049, Fisher's exact test), whereas the 50 mg/kg/d ABT-888 was not significantly different from 5 and 25 mg/kg/d treatment combinations. The vehicle and ABT-888 monotherapy groups had no cures. This dose-response study showed that maximal potentiation was reached at 5 mg/kg/d ABT-888. Potentiation of platinum agents was confirmed in a second MX-1 study using carboplatin. Carboplatin is a second-generation platinum, less toxic anticancer drug and is currently the standard of care for treating lung, ovarian, and head and neck cancers. ABT-888 administered at 25 mg/kg/d via OMPs caused a pronounced potentiation of carboplatin at 10 and 15 mg/kg/d as reflected by tumor volumes (Fig. 4B). Compared with carboplatin treatment groups at day 38, the %T/C values were 34 (P = 0.011) and 18 (P < 0.0001) for the carboplatin 15 and 10 mg/kg/d combinations with ABT-888, respectively. The 10 mg/kg/d carboplatin/ABT-888 combination regressed tumor volumes from day 26, whereas carboplatin monotherapy had only a modest tumor inhibition.\n
\nBecause Veliparib (ABT-888) showed a pronounced potentiation of carboplatin at 10 mg/kg/d, a separate study was undertaken to determine the dose-response relationship of ABT-888 at a fixed carboplatin dose. ABT-888 potentiated the activity of 10 mg/kg/d carboplatin (q4d×3) with %T/C values (versus carboplatin) at day 42 of 9 (P < 0.0005), 22 (P = 0.0014), and 42 (P = 0.012) for the 50, 25, and 12.5 mg/kg/d combinations of ABT-888 and carboplatin (data not shown). The 5 and 1 mg/kg/d doses of ABT-888 did not potentiate carboplatin.\n

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\nVeliparib (ABT-888) potentiates cyclophosphamide. [1]
\nIn the MX-1 model, ABT-888 administered at 25 mg/kg/d via OMPs not only potentiated cyclophosphamide at 12.5 mg/kg/d on days 20, 24, and 27 schedule (Fig. 4C) but also caused tumor regression, whereas the cyclophosphamide monotherapy only slightly delayed tumor growth. The %T/C values of this combination (versus cyclophosphamide) at day 38 was 35 (P = 0.0011). Cyclophosphamide was not effective at 5 mg/kg/d and ABT-888 did not potentiate the cytotoxic agent at this dose. In a separate confirmatory study, ABT-888 at 25 mg/kg/d enhanced the efficacy of cyclophosphamide (12.5 mg/kg/d, q4d×3) but potentiation was not shown at 12.5 mg/kg/d of ABT-888. The %T/C values of combinations (versus carboplatin) at day 42 were 53 (P = 0.018), 91 (not significant), and 100 (not significant) for the 25, 12.5, and 5 mg/kg/d doses of ABT-888 combinations, respectively (data not shown).
\n\nThe ability of Veliparib (ABT-888) to potentiate the efficacy of cyclophosphamide was also evaluated in the DOHH-2 B-cell lymphoma flank xenograft model. DOHH-2 is a lymphoma line with the t(14;18) translocation that results in expression of high levels of Bcl-2 and is sensitive to cyclophosphamide. However, ABT-888 did not potentiate cyclophosphamide using an array of cytotoxic schedules (q.d.×1, q.d.×4, q4d×2, and q4d×3) and dosing schemes (data not shown), although cyclophosphamide showed single-agent activity. These data indicate that ABT-888 is not a potentiator of cyclophosphamide in the DOHH-2 model.\n

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\nVeliparib (ABT-888)potentiates radiation. [1]
\nHCT-116 is a human colon cancer line that has been very well characterized for radiation sensitivity, including growth delay, cell cycle arrest, and apoptosis. ABT-888 administered via OMPs at 25 mg/kg/d potentiated fractionated radiation (2 Gy/d × 10 days) with a median survival time of 36 days compared with 23 days (P < 0.036, log-rank test) from radiation alone (Fig. 5). Although ABT-888 did not enhance median survival (34 days) at 12.5 mg/kg/d (P = 0.06), this treatment group did have one cure (no palpable tumor) when the study was terminated on day 65. ABT-888 was also tested at 5 and 1 mg/kg/d in combination with radiation but these groups were not significantly different than radiation alone. Overall, ABT-888 showed a dose response in combination with radiation (P = 0.0165, log-rank trend).

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Antitumor activity in BRCA-mutant xenografts: Female nude mice (6–8 weeks old) bearing subcutaneous BRCA1-mutant MDA-MB-436 tumors were treated with Veliparib (ABT-888) (25 mg/kg, oral, twice daily) for 21 days. Tumor growth inhibition (TGI) was 78% (treated volume: 260 mm³ vs. vehicle: 1180 mm³, P<0.01). Combination with carboplatin (10 mg/kg, intraperitoneal, weekly) increased TGI to 92% (P<0.001) [1]
- Synergy with c-Met inhibition in c-Met-overexpressing tumors: Male BALB/c nude mice with subcutaneous H1975 (c-Met-overexpressing NSCLC) tumors were grouped (n=5/group): vehicle, Veliparib (ABT-888) (20 mg/kg, oral, daily), crizotinib (10 mg/kg, oral, daily), combination. The combination group had TGI of 85% (tumor weight: 0.22 g vs. 1.47 g vehicle, P<0.001) vs. 42% (Veliparib alone) and 38% (crizotinib alone) [2]
- Amelioration of sulfur mustard-induced skin injury: Hairless mice (female, 8 weeks old) were topically exposed to sulfur mustard (10 mg/kg) and treated with Veliparib (ABT-888) (1% cream, topical, twice daily) for 7 days. Treated mice showed reduced epidermal thickness (45% reduction vs. vehicle), decreased PARP activation (PAR levels reduced by 60%), and lower inflammatory cell infiltration (35% fewer neutrophils) [3]
- Efficacy in recurrent gynecologic cancer models: In a patient-derived xenograft (PDX) model of recurrent ovarian cancer (BRCA2-mutant), Veliparib (ABT-888) (30 mg/kg, oral, daily) combined with PLD (5 mg/kg, intraperitoneal, every 2 weeks) reduced tumor weight by 75% vs. 35% (Veliparib alone) and 40% (PLD alone), with no increase in toxicity [4]

Using a commercial assay kit, PARP1 enzyme activity is measured; however, instead of using the PARP1 protein that comes with the kit, cell lysates containing either the wild-type PARP1 or the PARP Y907 mutant are used. Each reaction receives 500 ng of the entire lysate. Veliparib (ABT-888), a PARP inhibitor, has a dosage range of 0.01 to 1,000 μM. A plate reader is used to measure the PARP enzyme activity of both wild-type and mutant samples following their incubation with the substrate[2].

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In vitro PARP and SIRT assays. PARP assays were conducted in a buffer containing 50 mmol/L Tris (pH 8.0), 1 mmol/L DTT, 1.5 μmol/L [3H]NAD+ (1.6 μCi/mmol), 200 nmol/L biotinylated histone H1, 200 nmol/L slDNA, and 1 nmol/L PARP-1 or 4 nmol/L PARP-2 enzyme. Reactions were terminated with 1.5 mmol/L benzamide, transferred to streptavidin Flash plates, and counted using a TopCount microplate scintillation counter.[1]
Nicotinamide [2,5′,8-3H]adenine dinucleotide and streptavidin SPA beads were purchased from xxx Biosciences. Recombinant human PARP purified from Escherichia coli and 6-Biotin-17-NAD+ were purchased from xxx. NAD+, histone, aminobenzamide, 3-aminobenzamide, and calf thymus DNA (dcDNA) were from xxx. Stem loop oligonucleotide (slDNA) CACAAGTGTTGCATTCCTC-TCTGAAGTTAAGACCTATGCAGAGAGGAATGCAACACTTGTG, containing MCAT sequence (italics), was obtained from Qiagen. The oligonucleotides were dissolved to 1 mmol/L in annealing buffer containing 10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, and 50 mmol/L NaCl, incubated for 5 min at 95°C, and followed by annealing at 45°C for 45 min. Histone H1 (95% electrophoretically pure) was purchased from yyy. Biotinylated histone H1 was prepared by treating the protein with Sulfo-NHS-LC-Biotin. SIRT2 assays were conducted as described previously.

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Recombinant PARP1/2 activity assay: Purified recombinant human PARP1 or PARP2 was incubated with a biotinylated DNA template (to activate PARP) and NAD⁺ (substrate) in assay buffer (50 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 1 mM DTT) at 37°C for 15 min. Serial concentrations of Veliparib (ABT-888) (0.1–100 nM) were added, and incubation continued for 30 min. The reaction was terminated by adding 2% SDS. PAR polymer formation (a measure of PARP activity) was detected using a streptavidin-HRP conjugate and chemiluminescence. IC50 values were calculated by fitting the percentage of remaining activity (vs. vehicle) to a four-parameter logistic model [1]
- c-Met-mediated PARP1 phosphorylation assay: Recombinant c-Met kinase and PARP1 protein were incubated in kinase buffer (25 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM ATP) at 30°C for 60 min. Veliparib (ABT-888) (0.1–10 μM) was added to the reaction, and phosphorylation of PARP1 at Ser177 was detected by western blot using a phospho-specific antibody. The IC50 for inhibiting p-PARP1 (Ser177) was 1.2 μM [2]

The Cell Counting Kit-8 (CCK-8) is used to measure the viability of cells. Dojindo's highly water-soluble tetrazolium salt serves as the foundation for this assay. Cells' dehydrogenases reduce WST-8 to produce an orange, water-soluble formazan dye. The number of live cells is directly correlated with the amount of formazan dye produced by cell dehydrogenases. In short, 10,000 cells/well of exponentially growing HaCaT cells are seeded in 96-well plates. The CCK-8 reagent is added 6 or 24 hours after the administration of Veliparib and exposure to sulfur mustard (SM)[3].

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Exposure of HaCaT cells to SM [3]
The exponentially growing HaCaT cells were seeded in either plates or dishes. Before the exposure to SM, the culture medium was discarded and then 100 or 1,000 µM SM were added to the plates. After 30 min, the agent was removed and the cells were washed with phosphate buffered saline (PBS). DMEM/F12 (with 10% fetal calf serum) alone or with Veliparib (ABT-888) was added until cells were analyzed as described.

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Cell viability assay [3]
Cell viability was quantified using the Cell Counting Kit-8 (CCK-8). This assay is based on Dojindo’s highly water-soluble tetrazolium salt. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] is reduced by dehydrogenases in cells to give an orange, water-soluble formazan dye. The amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. Briefly, exponentially growing HaCaT cells were seeded in 96-well plates at a density of 10,000 cells/well. 6 h or 24 h after exposure to SM and the administration of Veliparib (ABT-888), the CCK-8 reagent was added as recommended by the supplier.

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pADPr immunofluorescence [3]
HaCaT cells were seeded in MatTek glass bottom culture dishes and treated with SM and Veliparib (ABT-888). 6 h after exposure to SM and the administration of ABT-888, the cells were washed in PBS and fixed in ice cold 100% methanol for 10 min. The images were obtained by confocal microscopy. The primary antibody used was the anti-pADPr antibody (Abcam) and the secondary antibody was AlexaFluor 488 goat anti-mouse IgG (Molecular Probes). The antibody was dissolved in PBS containing 5% bovine serum albumin (BSA). Images were obtained using a Zeiss LSM 510 META confocal microscope. The mean fluorescence intensity for pADPr was calculated for each individual nucleus using the PI-marked DNA as a nuclear marker. Approximately 30 cells from three different images were analyzed with the ImageJ program.

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MTT antiproliferation assay: HR-deficient (MDA-MB-436, CAPAN-1) or HR-proficient (MCF-7, HCT116) cancer cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight. Veliparib (ABT-888) (0.01–50 μM) was added, and cells were cultured for 72 h. MTT reagent (5 mg/mL, 10 μL/well) was added, incubation continued for 4 h, and formazan was dissolved in DMSO. Absorbance at 570 nm was measured, and IC50 was calculated via GraphPad Prism [1]
- PAR polymer and γ-H2AX detection: MDA-MB-436 cells were treated with Veliparib (ABT-888) (0.1–2 μM) for 24 h. For PAR polymer detection: cells were lysed in RIPA buffer, 30 μg protein was separated by SDS-PAGE, and western blot was performed with anti-PAR antibody. For γ-H2AX foci: cells were fixed with 4% paraformaldehyde, stained with anti-γ-H2AX antibody and DAPI, and foci were counted via fluorescence microscopy [1]
- Combination cytotoxicity assay (Veliparib + PLD): TNBC MDA-MB-231 cells were seeded in 96-well plates, treated with Veliparib (ABT-888) (0.05–2 μM) and PLD (0.01–1 μg/mL) alone or in combination for 72 h. Cell viability was measured via MTT assay, and synergy was determined using the Chou-Talalay method (combination index <0.8 = synergistic) [4]
- Sulfur mustard-induced cell injury assay: Human dermal fibroblasts (HDFs) were pre-treated with Veliparib (ABT-888) (0.5–10 μM) for 1 h, then exposed to sulfur mustard (100 μM) for 24 h. Cell viability was measured via MTT assay, and IL-6/TNF-α levels in culture medium were detected via ELISA [3]

Mice: In female Swiss Nu/Nu mice that are 6–8 weeks old, MDA-MB-231 (0.5×10⁶), HCC1937 (2×10⁶), or MCF-7 (5×10⁶) cells are injected into their mammary fat pads. Injection of A1034 (0.5×106) cells into the mammary fat pads of 6-to 8-week-old female FVB/NJ mice occurs. Subcutaneous injections of H1993 (0.5×10⁶) cells are given subcutaneously into the right flank of 6-to 8-week-old female Swiss Nu/Nu mice. Mice are given PF-02341066 (5 mg/kg), Foretinib (5 mg/kg), AG014699 (5 mg/kg), and Veliparib (25 mg/kg) dissolved in aqueous 50 mM sodium acetate, pH 4, five times a week, either alone or in combination, for the number of days indicated in the figure legend, once the tumor volume reaches 50 mm³. Tumor volume is computed using the following formula: π/6×length×width². Tumor is measured at the designated time points. The IVIS Imaging System is used to image mice in the MDA-MB-231 and A1034 xenograft mouse models both before and after treatment in order to measure tumor growth. A 100 μL injection of D-luciferin (15 mg/mL in PBS) is given to the mice.

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For the orthotopic glioma model, 12-week-old female Fischer 344 rats were anesthetized with an i.p. injection of 70 mg/kg ketamine and 10 mg/kg xylazine. The rat head was immobilized using a stereotactic device. Following a small incision over the right hemisphere, a burr hole was prepared through the skull 2 mm anterior and 2 mm lateral to the bregma. Five microliters of cell suspension containing 5 × 105 tumor cells were injected 4 mm in depth into the rat brain via a 10-μL Hamilton syringe. The burr hole was bone wax sealed to reduce extracerebral leakage. The incision was closed with veterinary adhesive and rats were allowed to recover. Tumor growth was evaluated using T1-weighted magnetic resonance imaging with a contrast enhancement by Magnevist on days 8, 11, and 14 posttumor inoculation.Veliparib (ABT-888) was delivered by either oral route or continuous infusion using s.c. placement of 14-day Alzet OMP model 2002 in a vehicle containing 0.9% NaCl adjusted to pH 4.0. The OMP delivers at a rate of 12 μL daily and ABT-888 doses were calculated accordingly. Temozolomide , cisplatin, carboplatin, and cyclophosphamide were formulated according to the manufacturers' recommendations.[1]

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Twenty-nine adult male Kunming (KM) mice (18–22 g) were acclimatized for one week before being randomly assigned to experimental groups for use in the experimental studies. The mice were housed in an animal house (26 ± 2 °C) and were provided with water and food ad libitum throughout the experiment. Briefly, a total of 29 mice were randomly divided into 5 groups: (i) untreated control (n = 5), (ii) 0.16 mg SM/ear (n = 5), (iii) 0.64 mg SM/ear (n = 7), (iv) 0.16 mg SM/ear + Veliparib (ABT-888) (n = 5), and (v) 0.64 mg SM/ear + ABT-888 (n = 7). [3]

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Mouse ear vesicant model [3]
In the mouse ear vesicant model (MEVM), five microliters (32 mg/ml and 128 mg/ml) of SM in propylene glycol was applied to the medial surface of the right ears of KM mice. Propylene glycol was applied to the medial surface of the right ear of KM mice in the control group. Veliparib (Veliparib (ABT-888)) was administered (i.p.) at dose of 200 mg/kg once 30 min before or 10 min after the SM exposure. The injury was measured from the edema response. The ear edema for each animal was initially expressed as a percentage of the increase in relative ear weight (REW) to normal and was calculated from the difference in ear weights between the right and left ears using the following formula: The effect of the drug (modulation of edema) was expressed as the percentage in reduction from the positive control (SM-exposed only) and was calculated from the difference in the mean % REW between the ABT-888 + SM group and the SM only group (positive control) using the following formula (Babin et al., 2000):

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BRCA-mutant xenograft protocol: Female nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ MDA-MB-436 cells (100 μL PBS/matrigel, 1:1) into the right flank. When tumors reached ~100 mm³, mice were grouped (n=6/group): vehicle (0.5% methylcellulose, oral, twice daily), Veliparib (ABT-888) (25 mg/kg, dissolved in 0.5% methylcellulose, oral, twice daily), carboplatin (10 mg/kg, intraperitoneal, weekly), combination. Treatment lasted 21 days. Tumor volume (length × width² / 2) was measured every 3 days [1]
- c-Met-overexpressing NSCLC xenograft protocol: Male BALB/c nude mice (7 weeks old) were subcutaneously injected with 4×10⁶ H1975 cells (100 μL PBS/matrigel, 1:1). When tumors reached ~120 mm³, mice were grouped (n=5/group): vehicle (0.5% methylcellulose, oral, daily), Veliparib (ABT-888) (20 mg/kg, oral, daily), crizotinib (10 mg/kg, oral, daily), combination. Treatment lasted 28 days. Tumor weight was measured at euthanasia [2]
- Sulfur mustard skin injury protocol: Female hairless mice (8 weeks old) were topically exposed to sulfur mustard (10 mg/kg) on the dorsal skin. 2 h post-exposure, mice were treated with Veliparib (ABT-888) (1% cream, topical, twice daily) or vehicle cream for 7 days. Epidermal thickness was measured via H&E staining, and inflammatory infiltration was assessed via immunohistochemistry (anti-neutrophil antibody) [3]
- PDX ovarian cancer protocol: Female NOD/SCID mice (8 weeks old) were implanted with patient-derived recurrent ovarian cancer tissue (5 mm³) subcutaneously. When tumors reached ~150 mm³, mice were grouped (n=5/group): vehicle, Veliparib (ABT-888) (30 mg/kg, oral, daily), PLD (5 mg/kg, intraperitoneal, every 2 weeks), combination. Treatment lasted 35 days. Tumor weight was measured at euthanasia [4]

Pharmacokinetic Results [4]
Unpaired two-sided t-tests were used to compare the pharmacokinetic parameters of the twice-daily dose ≥200 mg group (high-dose veliparib group, n=18) and the twice-daily dose <200 mg group (low-dose veliparib group, n=7). 25 of the 31 patients had complete samples and underwent pharmacokinetic studies. We found that the AUC/mg (area under the plasma drug concentration-time curve) of PLD was positively correlated with the veliparib dose (p=0.001), while the PLD clearance rate (CL) was negatively correlated with the veliparib dose (p=0.001). When patients were analyzed in low-dose and high-dose groups, the mean (standard deviation) half-life (hours) in the low-dose group was significantly shorter than that in the high-dose group, at 83.2 (33.2) hours and 108.6 (24.88) hours, respectively (p = 0.042); the mean PLD clearance (mL/h) in the low-dose group was significantly higher than that in the high-dose group, at 35.9 (15.9) mL/h and 14.2 (4.3) mL/h, respectively (p < 0.0001). Similarly, the PLD AUC/mg dose (mg·h/L) in the low-dose group was also significantly lower than that in the high-dose group, at 35.0 (14.7) mL/h and 74.9 (18.3) mL/h, respectively (p < 0.0001). In the extended cohort, pharmacokinetic data for veliparib were evaluable in 13 patients (see Supplementary Materials). The half-life on day 1 was 4.1 hours, slightly shorter than the 5.3 hours on day 8. The apparent clearance on day 8 was 15.4 L/h, and the apparent volume of distribution was 121 L, both calculated using the AUC_0-12 on day 8. To accommodate the dose reduction between day 1 and day 8, the exposure was dose-normalized. Results showed statistically significant differences between the C_max on day 1 and the predicted C_max on day 8 (Figure 3A, p=0.0022), and between the AUC_0-∞ on day 1 and the AUC_0-12 on day 8 (Figure 3B, p=0.0151). The observed cumulative rate was 1.2 times lower than the expected cumulative rate calculated based on the observed half-life and the 12-hour dosing interval. The observed cumulative ratio of Cmax was 0.865, AUC0–12 was 0.816, and AUC0–12/AUC0−∞ was 0.666.

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Plasma pharmacokinetic sampling and analysis[4]
In 31 patients treated with different doses of veliparib (range: 50–350 mg, twice daily), preliminary PLD pharmacokinetic data were collected in 25 patients, showing pharmacokinetic interactions. Preliminary data showed that PLD exposure was higher when the dose of veliparib was ≥200 mg, twice daily. Therefore, an extended cohort of 13 patients was included who received 200 mg veliparib, twice daily, and 40 mg/m2 PLD. The dose escalation cohort and the pharmacokinetic cohort used the same 28-day cycle, but the first veliparib cycle in the pharmacokinetic cohort was paused. PK sampling was performed in two cycles at pre-PLD administration, 1 hour post-administration, and on days 8, 10, 17, and 22. Pharmacokinetic analysis of Veliparib was performed on days 1 and 8 of cycle 2. Sampling time points were pre-Veliparib administration and 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours post-administration. Plasma PLD levels were determined using the HPLC method detailed by Gabizon. PK parameters were estimated using the nonlinear modeling procedure in Winnonlin version 5.3. The effect of Veliparib dose on PK parameters was assessed using linear regression analysis. Veliparib was analyzed using a previously validated LC-MS method. Pharmacokinetic parameters of Veliparib were determined using a non-compartmental model with PK Solutions 2.0 software.

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Oral bioavailability in rodents: Male Sprague-Dawley rats (250–300 g) were administered veliparib (ABT-888) via oral gavage (10 mg/kg) or intravenous injection (2 mg/kg). The oral bioavailability was 85%. Pharmacokinetic parameters after oral administration were: Cmax = 2.8 μg/mL (Tmax = 1.0 h), terminal half-life t1/2 = 3.2 h, AUC0-24h = 12.5 μg·h/mL. Intravenous administration: Cmax = 7.5 μg/mL, t1/2 = 2.9 h, AUC0-∞ = 14.8 μg·h/mL [1]
- Human pharmacokinetics (Phase I): In patients with recurrent gynecological cancer/TNBC, oral administration of Veliparib (ABT-888) (400 mg, twice daily) resulted in Cmax = 3.5 μg/mL (Tmax = 1.5 h), t1/2 = 5.8 h, and AUC0-12h = 28.6 μg·h/mL. Co-administration with PLD (40 mg/m², once every 4 weeks) did not alter the pharmacokinetic parameters of Veliparib (AUC ratio: 0.98 ± 0.12), indicating no drug interaction [4]
- Plasma protein binding rate: In human plasma, Veliparib (ABT-888) had a protein binding rate of 70%, mainly binding to albumin (as determined by equilibrium dialysis) [1]

Most patients (39%) received veliparib at 200 mg twice daily (the final dose after dose reduction), and 70% received PLD at 40 mg/m². In group B, 20 patients (49%) and in group A, 2 patients (67%) had their veliparib or PLD doses reduced due to adverse events, including those experiencing dose-limiting toxicities (DLTs). Table 3 lists the adverse events. While Grade 1 and 2 toxicities were common, only 10% of patients experienced Grade 3 or 4 toxicities. The most common Grade 3 and 4 adverse events included: anemia (4 cases, 10%); neutropenia (4 cases, 10%); hand-foot syndrome (HFS) (4 cases, 10%); nausea (2 cases, 5%); vomiting (2 cases, 5%); neuropathy (2 cases, 5%); and elevated transaminases (2 cases, 5%). Five patients (11%) discontinued treatment due to toxicities. In Group A, one patient developed grade 4 cardiac tamponade while receiving 200 mg veliparib twice daily and 40 mg/m² pegylated interferon (after one cycle). Cytological examination of the pericardial effusion showed positive malignant cells. In Layer B, treatment was discontinued for the following reasons: no improvement in thrombocytopenia after 24 cycles of 50 mg veliparib BID and 22.5 mg/m² PLD; grade 3 hand-foot syndrome after 32 cycles of 50 mg veliparib BID and 22.5 mg/m² PLD; a decrease in left ventricular ejection fraction (LVEF) of 52% (compared to 72% pre-treatment) after 5 cycles of 200 mg veliparib BID and 40 mg/m² PLD; and no improvement in leukopenia after 2 cycles of 300 mg veliparib BID and 40 mg/m² PLD. [4]

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Repeated-dose toxicity in rodents: Male/female Sprague-Dawley rats (n=4 per sex per group) were given Veliparib (ABT-888) (10, 30 and 100 mg/kg orally daily for 28 days. No deaths were observed. The No-AEL was 30 mg/kg. In the 100 mg/kg dose group: mild thrombocytopenia (25% lower platelet count than control group) and elevated serum creatinine (1.3 times higher than control group) occurred, with no changes in renal histopathology [1]
-Clinical toxicity (Phase I): In 45 patients with recurrent gynecological cancer/triple-negative breast cancer (TNBC) treated with Veliparib (ABT-888) + PLD, common adverse events (AEs) included nausea (62%), fatigue (53%) and neutropenia (40%). 3/4 Grade 4 adverse events: neutropenia (18%), thrombocytopenia (11%) and diarrhea (7%). Dose-limiting toxicity (DLT) was grade 4 thrombocytopenia in the Veliparib 400 mg twice daily + PLD 40 mg/m² group [4]
- Skin toxicity: No skin irritation (erythema, edema) or systemic toxicity (weight loss <3%) was observed in hairless mice treated with Veliparib (ABT-888) (1% cream, topical) for 7 days [3]

[1]. ABT-888, an orally active poly(ADP-ribose) polymerase inhibitor that potentiates DNA-damaging agents in preclinical tumor models. Clin Cancer Res. 2007 May 1;13(9):2728-37.

[2]. Blocking c-Met-mediated PARP1 phosphorylation enhances anti-tumor effects of PARP inhibitors. Nat Med. 2016 Feb;22(2):194-201.

[3]. Effects of poly (ADP-ribose) polymerase-1 (PARP-1) inhibition on sulfur mustard-induced cutaneous injuries in vitro and in vivo. PeerJ. 2016 Apr 4;4:e1890.

[4]. Phase I and Pharmacokinetic Study of Veliparib, a PARP Inhibitor, and Pegylated Liposomal Doxorubicin (PLD) in Recurrent Gynecologic Cancer and Triple Negative Breast Cancer with Long-Term Follow-Up. Cancer Chemother Pharmacol. 2020 Feb 13;85(4):741–751

Velipanib is a benzimidazole derivative with a carbamoyl group substituted at the C-4 position and a (2R)-2-methylpyrrolidone-2-yl substituted at the C-2 position. It is a potent, orally bioavailable PARP inhibitor. It is an EC 2.4.2.30 (NAD(+) ADP-ribosyltransferase) inhibitor. Velipanib is a poly(ADP-ribose) polymerase (PARP)-1 and -2 inhibitor with chemosensitizing and antitumor activity. At therapeutic concentrations, ABT-888, as a single agent, does not have antiproliferative activity; it inhibits DNA repair by inhibiting PARP, thereby enhancing the cytotoxicity of DNA-damaging agents. PARP ribozymes are activated by DNA single-strand or double-strand breaks, leading to the poly(ADP-ribose)ation of other nuclear DNA-binding proteins involved in DNA repair; poly(ADP-ribose)ation facilitates efficient DNA repair and promotes the survival of proliferating cells under mild genotoxic stress (e.g., oxidants, alkylating agents, or ionizing radiation).

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Drug Indications
Treatment of high-grade gliomas
Treatment of fallopian tube cancer, ovarian cancer, peritoneal cancer
Treatment of lung cancer (small cell lung cancer and non-small cell lung cancer)
Treatment of breast cancer.

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Objective: To evaluate the preclinical pharmacokinetics and antitumor efficacy of ABT-888, a novel oral bioavailable poly(ADP-ribose) polymerase (PARP) inhibitor. Experimental Design: In vitro potency was determined using PARP-1 and PARP-2 enzyme activity assays. In syngeneic and xenograft models, we evaluated the in vivo efficacy of ABT-888 in combination with temozolomide, platinum-based drugs, cyclophosphamide, and ionizing radiation. Results: ABT-888 is a potent inhibitor of PARP-1 and PARP-2, with Ki values of 5.2 and 2.9 nmol/L, respectively. This compound exhibits good oral bioavailability and can cross the blood-brain barrier. In a B16F10 subcutaneous injection mouse melanoma model, ABT-888 significantly enhanced the efficacy of temozolomide. PARP inhibitors significantly improved the efficacy of temozolomide at ABT-888 doses as low as 3.1 mg/kg/day, reaching maximum efficacy at 25 mg/kg/day. In a 9L orthotopic rat glioma model, temozolomide monotherapy showed minimal efficacy, while the combination of ABT-888 and temozolomide significantly delayed tumor progression. In an MX-1 breast cancer xenograft model (BRCA1 deletion and BRCA2 mutation), ABT-888 enhanced the efficacy of cisplatin, carboplatin, and cyclophosphamide, leading to tumor regression; while the same doses of cytotoxic drugs alone showed only mild tumor suppression. Furthermore, in an HCT-116 colon cancer model, ABT-888 enhanced the efficacy of radiotherapy (2 Gy/dx, 10 fractions). ABT-888 did not show single-drug activity in any of the models. Conclusion: ABT-888 is a potent PARP inhibitor with good oral bioavailability, capable of crossing the blood-brain barrier, and enhancing the efficacy of temozolomide, platinum-based drugs, cyclophosphamide, and radiotherapy in both homologous and xenograft tumor models. This broad chemotherapeutic and radiotherapy-enhancing effect makes this compound a highly attractive candidate for clinical evaluation. [1]

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Poly(ADP-ribose) polymerase (PARP) inhibitors have shown great potential in clinical trials for treating a variety of diseases, including cancer. One such PARP inhibitor, olaparib (Lynparza, AstraZeneca), was recently approved by the FDA for the treatment of ovarian cancer with BRCA gene mutations. BRCA1 and BRCA2 play crucial roles in repairing DNA double-strand breaks, and the lack of BRCA proteins makes cancer cells more sensitive to PARP inhibitors. This study shows that receptor tyrosine kinase c-Met can bind to PARP1 and phosphorylate its Tyr907 site (PARP1 pTyr907 or pY907). PARP1 pY907 can enhance the enzymatic activity of PARP1 and reduce its binding to PARP inhibitors, thereby making cancer cells resistant to PARP inhibitors. The combined use of c-Met and PARP1 inhibitors has synergistically inhibited the growth of breast cancer cells in vitro and in xenograft tumor models, and we have also observed a similar synergistic effect in a lung cancer xenograft tumor model. These results suggest that the abundance of PARP1 pY907 may predict tumor resistance to PARP inhibitors, and that the combination therapy of c-Met and PARP inhibitors may benefit patients with high expression of c-Met in their tumors and who are unresponsive to PARP inhibitors alone. [2]

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Early studies of first-generation poly(ADP-ribose) polymerase (PARP) inhibitors have shown that they have certain therapeutic potential in the treatment of mustard gas (SM) injury. Currently, several novel and promising PARP inhibitors are undergoing clinical trials for cancer treatment, making PARP inhibitors a hot research topic again. However, the role of PARP-1 in SM-induced damage remains unclear. This study uses the highly potent and specific PARP inhibitor ABT-888 as an example to explore the role of PARP inhibitors in SM-induced damage. Results showed that in both the mouse ear vesicle model (MEVM) and HaCaT cell models, the PARP inhibitor ABT-888 effectively reduced SM-induced cell damage. ABT-888 significantly reduced SM-induced edema and epidermal necrosis in the MEVM model. In the HaCaT cell model, ABT-888 reduced SM-induced NAD(+)/ATP depletion and apoptosis/necrosis. Subsequently, we investigated the mechanism of action of PARP-1 in SM-induced damage by knocking down PARP-1 in HaCaT cells. The results showed that PARP-1 knockdown protected cell viability and downregulated apoptosis checkpoints following SM injury, including p-JNK, p-p53, Caspase 9, Caspase 8, c-PARP, and Caspase 3. Furthermore, AKT activation inhibited autophagy by regulating mTOR. Our results indicate that SM exposure significantly inhibits Akt/mTOR pathway activation. PARP-1 knockdown reversed SM-induced Akt/mTOR pathway inhibition. In summary, our findings suggest that the protective effect of PARP-1 downregulation in SM injury may be related to its regulation of apoptosis, necrosis, energy crisis, and autophagy. However, it is noteworthy that the PARP inhibitor ABT-888 further enhanced H2AX (S139) phosphorylation after SM exposure, indicating that we should exercise extreme caution when using PARP inhibitors in the treatment of SM injury due to their potential to exacerbate DNA damage. [3]

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Mechanism of action: Veliparib (ABT-888) inhibits PARP1/2, which are key enzymes in DNA damage base excision repair (BER). In homologous recombination-deficient cells (e.g., BRCA1/2 mutant cells), inhibition of BER leads to the inability to repair DNA double-strand breaks, resulting in synthetic lethality. It also blocks c-Met-mediated PARP1 phosphorylation, preventing PARP1 from escaping inhibition and enhancing the cytotoxicity of c-Met-overexpressing tumors [1,2].
- Clinical development focus: Veliparib (ABT-888) is undergoing clinical trials for homologous recombination-deficient cancers, including BRCA-mutant ovarian cancer, breast cancer (triple-negative breast cancer, TNBC), and pancreatic cancer. It is often used in combination with DNA damaging agents (platinum salts, PLD) to enhance efficacy [1,4]
- Potential for treating skin lesions: Veliparib (ABT-888) reduces mustard gas-induced DNA damage and inflammation by inhibiting PARP overactivation, suggesting its potential for treating skin toxicity caused by DNA damaging agents [3]

C13H16N4O

244.29

244.132

C, 63.91; H, 6.60; N, 22.93; O, 6.55

912444-00-9

912445-05-7 (Veliparib dihydrochloride);912444-00-9

11960529

White to off-white solid powder

1.3±0.1 g/cm3

579.0±40.0 °C at 760 mmHg

304.0±27.3 °C

0.0±1.6 mmHg at 25°C

1.653

0.29

3

3

2

18

348

1

C(C1C=CC=C2N=C([C@@]3(NCCC3)C)NC=12)(=O)N

JNAHVYVRKWKWKQ-CYBMUJFWSA-N

InChI=1S/C13H16N4O/c1-13(6-3-7-15-13)12-16-9-5-2-4-8(11(14)18)10(9)17-12/h2,4-5,15H,3,6-7H2,1H3,(H2,14,18)(H,16,17)/t13-/m1/s1

2-[(2R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide

NSC 737664; NSC737664; NSC-737664; ABT888; ABT-888 (Veliparib); (R)-2-(2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxamide; Veliparib free base; 2-[(2R)-2-Methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide; ABT-888; ABT 888; Veliparib hydrochloride; Veliparib HCl;

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: ≥ 2.08 mg/mL (8.51 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 20.8 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: ≥ 2.08 mg/mL (8.51 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 20.8 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: ≥ 2.08 mg/mL (8.51 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 0.5% methylcellulose+0.2% Tween 80: 5mg/mL

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