Tubulin protein/microtubule

In a dose- and time-dependent manner, VERU-111 (2.5-80 nM; 24-48 hours) suppresses the development of Panc-1, AsPC-1, and HPAF-II cells (24 hours: IC50 of 25, 35, and 35 nM, respectively; 48 hours: IC50 of 11.8, 15.5, and 25 nM, respectively) [4]. VERU-111 (5-20 nM; 24 hours) arrests Panc-1 and AsPC-1 cells in the G2/M phase in a dose-dependent manner [4]. VERU-111 (5-20 nM; 24 hours) demonstrates dose-dependent suppression of pro-caspase 3 and 9 and activation of caspase-3 and 9, promotes the production of Bax and Bad, and inhibits the expression of Bcl-2 AsPC-1 and Bcl-xl protein in Panc-1 cells [4].

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Compound II/Sabizabulin and IAT Exhibit Potent Cytotoxicity in Cancer Cells, Including Multidrug-Resistant Cells The ability of compound II/Sabizabulin and IAT to inhibit the growth of cancer cell lines was evaluated using SRB assay (Table I). Both compounds inhibited the growth of several human cancer cell lines, including one glioma and five prostate cancer cell lines, with IC50 values in the low nanomolar range. Compound II exhibited 1.7~4.3 fold higher potency than compound IAT in these cell lines. The PC-3/TxR cell line that over-expressed P-glycoprotein was used to examine the ability of compounds II and IAT to maintain activity in paclitaxel-refractory cells in vitro (Table I). Compounds II and IAT were equipotent against parents PC-3 and drug resistant PC-3/TxR cells, whereas paclitaxel and docetaxel exhibited relative resistance value of 85- and 15-fold, respectively. These data indicate that both compounds II and IAT circumvent P-glycoprotein-mediated drug resistance consistent with our previous lead compound, 4-(3,4,5-trimethoxybenzoyl)- 2-phenyl-thiazole (SMART-H).[7]

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Compounds II/Sabizabulin and IAT Bind to the Colchicine-Binding Site on Tubulin, Inhibit Tubulin Polymerization, and Induce Cancer Cell Apoptosis A competitive mass spectrometry binding assay was developed to study the interactions of small molecule inhibitors with tubulin, and was applied to examine our lead compound, SMART-H, which selectively binds to the colchicine site on tubulin. Compounds II/Sabizabulin and IAT compounds competed effectively with colchicine for tubulin binding (Fig. 1a), with potency similar to podophyllotoxin. Vinblastine, the negative control, did not inhibit colchicine binding to tubulin, successfully demonstrating the specificity of this competitive mass spectrometry binding assay. Porcine brain tubulin (> 97% pure) was incubated with II or IAT (5 μM) to test their effects on tubulin polymerization (Fig. 1b). Compounds II and IAT inhibited tubulin polymerization by 47 and 40% at 15 min, respectively. Colchicine at 5 μM was used as a positive control and inhibited tubulin polymerization by 32%. These data suggested that both II and IAT inhibit tubulin polymerization better than colchicine and indicated that these compounds bind the same site as SMART compounds. PC-3 and PC-3/TxR cells were exposed to 0.8–600 nmol/ L of compound II, IAT or docetaxel for 24 h. Both compound II/Sabizabulin and IAT were equally potent in their ability to induce cell apoptosis in PC-3 (Fig. 1c) and PC-3/TxR (Fig. 1d) within 24 h as measured by DNA histone complex formation. Although docetaxel potently induced cell apoptosis in PC-3 cells, it was substantially weaker in PC-3/TxR cells due to the overexpression of P-glycoprotein [7].

Comparing the vehicle-treated group with VERU-111 (50 μg/mouse; intratumoral; three times a week for three weeks) demonstrated a significant reduction in tumor growth. Mice given VERU-111 did not exhibit any overt toxicity, even though they kept gaining weight [4].

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Compounds II/Sabizabulin an IAT Inhibit Paclitaxel-Resistant Prostate (PC-3/TxR) Xenograft Growth Parental PC-3 and paclitaxel-resistant prostate cancer PC-3 (PC-3/TxR) cells were inoculated into nude mice and the tumor volumes were allowed to reach about 150~300 mm3. Docetaxel (10 or 20 mg/kg), an anticancer drug approved for clinical use in advanced prostate cancer, was used for comparison. PC-3/TxR tumor xenografts grew rapidly and reached tumor volumes of 1500–2500 mm3 by the end of the study. Although intravenously administered docetaxel (10 and 20mg/kg) demonstrated in vivo anticancer activity in both models (Fig. 2a, b), the tumor growth inhibition (TGI) effect decreased from 84% TGI in PC-3 tumors to 14% TGI in PC-3/TxR tumors when intravenously dosed at 10 mg/kg (Table V). At the higher dose (20 mg/kg), docetaxel elicited partial regression (>100% TGI) of PC-3 tumors, but only 56% TGI in PC-3/TxR tumors. The effectiveness of docetaxel in PC-3/TxR tumors was decreased when compared to that in PC-3 tumors, suggesting that the efficacy was impaired by P-glycoprotein-mediated drug resistance. These results were consistent with our in vitro cytotoxicity and apoptosis data. In contrast to the lack of efficacy of docetaxel in PC-3/ TxR tumors, compound II/Sabizabulin (6.7 mg/kg) was dosed orally and demonstrated more than 100% TGI without an effect on body weight (Fig. 2b and Table V). In addition, 2 out of 4 nude mice bearing PC-3/TxR tumors treated with compound II were tumor free on day 19. The PC-3/TxR xenograft model was further utilized to evaluate the efficacies of compound II and IAT when administered using different dosing schedules. The maximal tolerated doses (body weight loss >20%) of II was 10 mg/kg when orally dosed once daily for 4 days; or at 3.3 mg/kg when administered twice daily (b.i.d.) for 5 days (data not shown). As shown in Fig. 2c, 3.3 mg/kg of II/Sabizabulin was administered b.i.d. for the first four consecutive days in the first week, and the schedule was then changed to once daily on week 2 and 4. Partial regression was obtained during days 4–19. The TGI was 97% and one of the seven mice was tumor free on day 26. A higher dose (10 mg/kg) with lower dosing frequency (q2d) of compound II (Fig. 2d) elicited partial tumor regression during days 13–29, suggesting that alternative dosing regimens successfully inhibit PC-3/TxR xenograft growth. Compound IAT was orally administered to nude mice at a dose of 10 or 30 mg/kg b.i.d., five times a week between weeks 1 and 4 (Fig. 2c). The TGI value was 59% for mice that received 10 mg/kg IAT, while partial regression (>100% TGI) from day 19 to the termination of the study (day 26) was observed in animals treated with the higher dose (30 mg/kg) of IAT. Some mice in vehicle group had lower body weights at the endpoint, in part, due to muscle wasting and/or cancer cachexia. On the contrary, mice treated with Compound II (3.3 mg/kg) or IAT (30 mg/kg) gained weight (Table V), suggesting that these optimized doses of II or IAT were well-tolerated [7].

In Vitro Tubulin Polymerization Assay[5,6]
According to the method described by Wang et al.,porcine brain tubulins (>97% pure) were mixed with general tubulin buffer (80 mM PIPES, 2.0 mM MgCl2, 0.5 mM EGTA, and 1 mM GTP) to reach a final concentration of 3 mg/mL at 4 °C. The tubulin polymerization assay was incubated at 37 °C in a SYNERGY 4 Microplate Reader immediately after mixing tubulin protein solution and the test compounds in a 96-well plate and monitored every 30 s for 65 min at 340 nm. The experiment was performed in duplicates with paclitaxel as a positive control for tubulin polymerization, and colchicine and ABI-274 as positive controls for tubulin depolymerization.

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SPR for Affinity Assay[5,6]
Binding affinity with tubulin was analyzed using SPR technology in a Reichert4SPR system equipped with a dextran SPR sensor chip (Reichert Polycarboxylate Hydrogel Chip P/N 13206067). Then, 50 μg/mL tubulin was immobilized to the sensor chip surface to attain 12 000 μRIU. One of the four flow cells on the chip was left free as a negative control. 4v or colchicine at different concentrations was injected over the sensor chip surface for association analysis, followed by dissociation analysis. The experiment data were obtained at 25 °C with a running buffer PBST (8 mM Na2HPO4, 136 mM NaCl, 2 mM KH2PO4, 2.6 mM KCl, and 0.05% (v/v) Tween 20, pH 7.4). The equilibrium dissociation constant (KD) was calculated by a steady-state fitting mode with TraceDrawer software.

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Competitive Mass Spectrometry Binding Assay [7]
Competitive mass spectrometry binding studies were conducted as previously described. Colchicine (1.2 μM) was incubated with porcine brain tubulin (1.0 mg/mL) in the incubation buffer [80 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 2.0 mM magnesium chloride (MgCl2), 0.5 mM ethylene glycol tetraacetic acid (EGTA), pH 6.9] at 37°C for 1 h. Varying concentrations (0.2–200 μM) of podophyllotoxin (positive control), compound II/Sabizabulin, compound IAT, and vinblastine (negative control) were used to compete with the binding of colchicine to tubulin. After 1 h incubation, the filtrate was obtained using an ultrafiltration method (microconcentrator) with Li et al. Author's personal copy a molecular cutoff size of 30 kDa. The ability of the compounds of interest to inhibit the binding of each ligand was expressed as a percentage of control binding in the absence of any competitor. Each experiment was performed in triplicate.

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In Vitro Microtubule Polymerization Assay [7]
Porcine brain tubulin (0.4 mg) was mixed with 5 μM of the compounds of interest or vehicle [Dimethyl sulfoxide (DMSO)] and incubated in 100 μL of buffer [80 mM PIPES, 2.0 mM MgCl2, 0.5 mM EGTA, pH 6.9 and 1 mM guanosine-5′-triphosphate (GTP)]. The absorbance at 340 nm wavelength was monitored every min for 15 min. The spectrophotometer was maintained at 37 °C for tubulin polymerization.

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Metabolic Stability [7]
Metabolic stability studies were conducted by incubating the compounds of interest (0.5 μM) in a total reaction volume of 1 mL containing 1 mg/mL microsomal protein in reaction buffer [0.2 M of phosphate buffer solution (pH 7.4), 1.3 mM nicotinamide adenine dinucleotide phosphate (NADP+), 3.3 mM glucose-6-phosphate, and 0.4 U/mL glucose-6- phosphate dehydrogenase] at 37°C in a shaking water bath. Pooled human liver microsomes were utilized to examine metabolic stability. The NADPH regenerating system (solution A and B) was obtained from BD Biosciences (Bedford, MA). The total DMSO concentration in the reaction solution was approximately 0.5% (v/v). Aliquots (100 μL) from the reaction mixtures used to determine metabolic stability were sampled at 5, 10, 20, 30, 60, and 90 min. Acetonitrile (150 μL) containing 200 nM of the internal standard (23) was added to quench the reaction and to precipitate the proteins. Samples were then centrifuged at 4,000g for 15 min at room temperature, and the supernatant was analyzed directly by liquid chromatography tandem mass spectrometry (LC-MS/MS).

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CYP Enzyme Specific Assays [7]
Five CYP enzyme inhibition assays were performed, and analyzed by LC-MS/MS. Briefly, for CYP2D6, CYP2C9, CYP1A2, and CYP2C19, 0.1 mg/mL microsomal protein was incubated with their specific substrates in 0.1 M potassium phosphate buffer (pH 7.4) at 37°C, while 0.05 mg/mL microsomal protein was used for CYP3A4 assays. The substrates, testosterone (50 μM), dextromethorphan (7 μM), (S)- mephenytoin (80 μM), diclofenac (7 μM), and phenacetin (100 μM), were used for CYP 3A4, 2D6, 2 C19, 2 C9, 1A2 inhibition assays, respectively. Various concentrations ranging 0.04–50 μM of compound II/Sabizabulin or IAT were examined in these assays. Positive controls used for CYP 3A4, 2D6, 2 C19, 2 C9, and 1A2 inhibition assays were ketoconazole (0.0009–1 μM), quinindine (0.0009–1 μM), ticlopidine (0.019–20 μM), sulfaphenazole (0.019–20 μM), and furafylline (0.04–50 μM), respectively.

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Aqueous Solubility [7]
The solubility of compounds II/Sabizabulin ad IAT was determined by Multiscreen Solubility Filter Plate coupled with LC-MS/MS. Briefly, 198 μL of phosphate buffered saline (PBS) buffer (pH 7.4) was loaded into a 96-well plate, and 2 μL of 10 mM test compounds (in DMSO) was dispensed and mixed with gentle shaking (200–300 rpm) for 1.5 h at room temperature (N0 3). The plate was centrifuged at 800g for 10 min, and the filtrate was used to determine its concentration and solubility of test compound by LC-MS/MS methods.

Cell proliferation assay [4]
Cell Types: Panc-1, AsPC-1, HPAF-II Cell
Tested Concentrations: 2.5, 5, 10, 20, 40, 80 nM
Incubation Duration: 24, 48 hrs (hours)
Experimental Results: Inhibited the growth of PanCa cells and dose- and time-dependent manner. After 24 hrs (hours) of treatment, the IC50 of VERU-111 in Panc-1, AsPC-1, and HPAF-II were 25, 35, and 35 nM, respectively, and after 48 hrs (hours) of treatment they were 11.8, 15.5, and 25 nM, respectively.

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Apoptosis analysis [4]
Cell Types: Panc-1, AsPC-1 Cell
Tested Concentrations: 5, 10, 20 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: Panc-1 and AsPC-1 cells arrested at G2/ M period method.

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Western Blot Analysis[4]
Cell Types: AsPC-1 and Panc-1 Cell
Tested Concentrations: 5, 10, 20 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: Dose-dependent inhibition of Caspase 3 and 9 precursors and Caspase-3 and 9 9 in both activated AsPC-1 and Panc-1 cells. Induces the expression of Bax and Bad and inhibits the expression of Bcl-2 and Bcl-xl proteins.

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Cell Culture and Cytotoxicity Assays of Prostate Carcinoma and Glioma Cell Lines [7]
The prostate cancer cell lines (LNCaP, PC-3, DU145, PPC- 1) and glioma cell line (U87MG) were obtained from ATCC. Prostate cancer cells were selected for these studies due to the prevalence of prostate cancer and the common use of docetaxel to treat this disease. The glioma cell line (U87MG) was used to examine the activity of the compounds in another type of cancer (i.e., glioma) and coordinate with the brain–blood barrier studies. Paclitaxelresistant PC-3 (PC-3/TxR; a prostate cancer cell line that over-expresses P-glycoprotein) was a gift from Dr. Evan T. Keller at Department of Pathology, University of Michigan, Ann Arbor, Michigan. PC-3/TxR was employed as a MDR model. All cell lines were tested and authenticated by ATCC, and were immediately expanded and frozen such that they could be restarted every 2~3 months from a frozen vial of the same batch of cells. Cell culture supplies were purchased from Cellgro Mediatech. Prostate cancer cell lines were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), while the glioma cancer cell line was maintained in Eagle’s MEM media with 2 mML-glutamine and 10% FBS. The antiproliferative activity of the compounds of interest, paclitaxel, and docetaxel were tested in cell lines by sulforhodamine B (SRB) assay as previous described.

Animal/Disease Models: Sixweeks old female athymic nude mice (carrying AsPC-1 cells)
Doses: 50 μg/animal
Route of Administration: intratumoral injection; 3 times a week for 3 weeks
Experimental Results: Effectively inhibited tumor growth.

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Pharmacokinetic Study [7]
Male ICR mice (N03 or 4 per group) 6–8 weeks of age were used to examine the pharmacokinetics (PK) of compound II/Sabizabulin or IAT. Both chemicals were formulated in DMSO/Polyethylene glycol 300 (PEG300), 1/9, v/v, and Tween80/DMSO/ H2O, 2/2/6, v/v/v for intravenous bolus (i.v., 10 mg/kg) and oral (p.o., 20 mg/kg) administration, respectively. Dosing volume for i.v. was 50 μL via tail vein, while the volume Orally Active Tubulin Antagonists Author's personal copy for p.o. was 200 μL through oral gavage. For i.v. administration, blood samples were collected at 2, 5, 15, and 30 min, 1, 2, 4, 8, 16, and 24 h after administration. For p.o. administration, blood samples were collected at 0.5, 1, 1.5, 2, 3, 4, 8, 16, and 24 h after administration. Plasma samples were prepared by centrifuging the blood samples at 8,000g for 5 min. All plasma samples were stored immediately at −80°C until analyzed. Female Sprague–Dawley rats (N03 or 4 per group) were used. Rat thoracic jugular vein catheters were purchased from Braintree Scientific Inc. (Braintree,MA). All animals were fed prior to dosing. Dosing volumes for intravenous bolus (i.v.) and oral (p.o.) solutions were 2 and 4 mL/kg, respectively. Compound II/Sabizabulin or IAT was administered i.v. into the thoracic jugular vein at a dose of 5 mg/kg (in DMSO/PEG300, 1/9, v/v). The dose in rats was chosen to be one-half of the dose in mice based onbody surface area. Catheters were flushed with 1 mL of heparinized saline after i.v. bolus. An equal volume of heparinized saline was injected to replace the removed blood, and blood samples (250 μL) were collected via the jugular vein catheter at 10, 20, 30 min, and 1, 2, 4, 8, 12, 24 h. Compounds II/Sabizabulin and IAT were also given (p.o.) by oral gavage at 10mg/kg (in Tween80/DMSO/H2O, 2/2/6, v/v/v) to evaluate their oral bioavailability. All blood samples (250 μL) after oral administration were collected via the jugular vein catheter at 30, 60, 90 min, 120 min, 150 min, 180 min, 210 min, 240 min, and 8, 12, 24 h. Heparinized syringes and vials were prepared prior to blood collection. Female beagle dogs weighing about 10 kg were used in this study. The dogs (N04) were given a single intravenous dose of compound II/Sabizabulin or IAT (2 mg/kg, in DMSO/PFG300, 1/9, v/v), in a dosing volume of 0.2 mL/kg. Blood was drawn at 10, 20, 30 min, and 1, 2, 4, 8, 12, 24, 48, 96 h. For p.o. administration (N04), the dogs (N04) were given a single oral dose of compound IAT (5 mg/kg, in Tween80/ DMSO/H2O, 2/2/6, v/v/v) in a dosing volume of 1 mL/ kg. We selected an oral dose level (5 mg/kg) in dogs that was one-fourth of the dose in mice and slightly higher than would be needed to correct for differences in body surface area (i.e., one sixth of the dose in mice) due to in vitro studies with liver microsomes from these species indicating less metabolic stability of the compounds in dogs (data not shown). Blood was drawn at 20, 40, 60, 80, 100, 120, 150, 180, 210 min and 4, 8, 12, 24, 48, 96 h. A protein precipitation method was used for sample preparation. An aliquot (200 μL) of acetonitrile (ACN) containing the internal standard was added to 100 μL of plasma and then was thoroughly vortexed for 15 s. After centrifugation, the supernatant was analyzed by LC-MS/MS. The pharmacokinetic parameters were determined using noncompartmental analysis.

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Brain Penetration Study [7]
Plasma and brain tissue were collected after a single dose oral administration (20 mg/kg) of compounds II/Sabizabulin and IAT, and single intraperitoneal administration (10 mg/kg) of docetaxel from nude mice. All three chemicals were formulated in Tween80/DMSO/H2O, 2/2/6, v/v/v. At the indicated time points (1 h and 4 h) after dosing, blood and brain tissue was collected from nude mice. Plasma was prepared as previously described and stored at −80°C until analyzed. Brain tissue samples were individually ground to a powder with a Bessman tissue pulverizer. The pulverizer was pre-cooled for 1 min in liquid nitrogen. Approximately 50 mg of tissue was placed on the pulverizer, and the whole apparatus was cooled in liquid nitrogen for 1 min and then the tissue was ground to a fine powder. The powder was immediately transferred to a sample vial, vortexed with 4 volumes of water, and then 10 volumes of acetonitrile containing the internal standard were added for extraction. After centrifugation, the supernatant was analyzed by LC-MS/MS to determine their brain and plasma concentrations.

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PC-3 and Paclitaxel-Resistant PC-3 (PC-3/TxR) Tumor Xenograft Studies [7]
PC-3 or PC-3/TxR cells (108 per mL) were prepared in growth media containing 10% FBS and mixed with high concentration, phenol red-free Matrigel at 1:1 ratio. Tumors were established by injecting 100 μL of the mixture (5×106 cells per animal) subcutaneously (s.c.) into the flank of 6–8-week-old male athymic nude mice. The length and width of tumors were measured and the tumor volume (mm3) was calculated by the formula, π/6 × L × W2, where length (L) and width (W) were determined in mm. When the tumor volumes reached about 150–300 mm3, the animals were treated with an intravenous formulation [Tween80/ethanol/saline (7.5/ 7.5/85)] or oral formulation [Tween80/DMSO/H2O (2/ 2/6)]. Docetaxel (10 or 20 mg/kg) was intravenously dosed on day 1 and day 9 in both PC-3 and PC-3/TxR xenograft models while compound II/Sabizabulin (6.7 mg/kg) was dosed orally (qd, five times a week) in PC-3/TxR xenograft model. In another PC-3/TxR xenograft study, compound II/Sabizabulin (3.3 mg/kg) was dosed twice a day (b.i.d.) for the first four days in the first week, and then the schedule was changed to once daily, five days a week during week 2–4 due to toxicity. While compound IAT (10 and 30 mg/kg) was orally dosed b.i.d. on mice, five times a week for four weeks, a higher dose of compound II (10 mg/kg) was also examined in PC-3/TxR xenografts, with every other day (q2d) treatments.

Compounds II/Sabizabulin and IAT Exhibited Favorable Drug-Like Properties [7]
The drug-like properties of II and IAT, such as metabolic stability, permeability, aqueous solubility, and drug-drug interactions, were examined (Table II). Compound II/Sabizabulin exhibited greater metabolic stability and aqueous solubility than IAT. Both compounds exhibited more than adequate permeability values, suggesting that they would be amenable to oral administration. In addition, both compounds showed high IC50 values (in the micromolar range) during CYP enzyme inhibition assays, suggesting that they will not cause CYP-mediated drug-drug interactions.

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Pharmacokinetic Studies in Mice, Rats and Dogs [7]
The pharmacokinetic parameters of compounds II/Sabizabulin and IAT after single intravenous or oral doses in ICR mice, Sprague–Dawley rats, and Beagle dogs are summarized in Table III. Compound II exhibited low clearance in mice and rats, suggesting that it exhibited prolonged metabolic stability and minimal first-pass hepatic metabolism in these species. In addition, II had a moderate volume of distribution in mice and rats, suggesting that it is widely distributed in tissues. Surprisingly, the total clearance of compound II in dogs was high. Two abundant metabolites in dog plasma, a hydroxylated metabolite and an unknown metabolite with +34 m/z than the parent (data not shown), were observed, which were consistent with those found in dog liver microsomes. In addition, abundant metabolites were observed when compound II was incubated with dog liver microsomes, but not in mouse, rat or human liver microsomes (data not shown). Nevertheless, compound II/Sabizabulin showed acceptable oral bioavailability of 21%, 36%, and 50% in rats, mice, and dogs, respectively. Compound IAT had low systemic clearance in rats and moderate clearance inmice and dogs. Similar to II, compound IAT exhibited moderate volume of distribution in these species. Compound IAT had comparable oral bioavailability among the three species (24%~36%).

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Brain Penetration of Compounds II/Sabizabulin and IAT in Nude Mice The ratios of whole brain to plasma concentrations of compound II and IAT were determined and compared to docetaxel in the nude mice (Table IV). Compound IAT exhibited a greater brain penetration than compound II and docetaxel. Compound II achieved slightly greater brain/plasma concentration ratios than docetaxel at both 1 and 4 h, while the IAT concentrations in brain reached 14–19% of plasma concentrations at 1 h and 4 h, respectively, showing a 3.2-fold higher brain/plasma ratio at both 1 h and 4 h compared to docetaxel.

[1]. Structure-Guided Design, Synthesis, and Biological Evaluation of (2-(1H-Indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl) Methanone (ABI-231) Analogues Targeting the Colchicine Binding Site in Tubulin. J Med Chem . 2019 Jul 25;62(14):6734-6750.

[2]. Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI-III) analogues targeting tubulin polymerization as antiproliferative agents. J Med Chem . 2012 Aug 23;55(16):7285-9.

[3]. ABI-231: A novel small molecule suppresses tumor growth and metastatic phenotypes of cervical cancer cells via targeting Human papilloma virus (HPV) E6 and E7. Cancer Research 78(13 Supplement):679-679.

[4]. Therapeutic efficacy of a novel βIII/βIV-tubulin inhibitor (VERU-111) in pancreatic cancer. J Exp Clin Cancer Res. 2019 Jan 23;38(1):29.

[5]. Structure-Activity Relationship Study of Novel 6-Aryl-2-benzoyl-pyridines as Tubulin Polymerization Inhibitors with Potent Antiproliferative Properties. J Med Chem. 2020 Jan 23;63(2):827-846.

[6]. Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI-III) analogues targeting tubulin polymerization as antiproliferative agents. J Med Chem . 2012 Aug 23;55(16):7285-9.

[6]. Orally bioavailable tubulin antagonists for paclitaxel-refractory cancer. Pharm Res. 2012 Nov;29(11):3053-63.

Sabizabulin is an orally bioavailable, small molecule tubulin inhibitor, with potential antineoplastic, antiviral and anti-inflammatory activities. Upon oral administration, sabizabulin binds to the colchicine-binding site of alpha- and beta-tubulin subunits of microtubules and crosslinks the microtubules, thereby inhibiting microtubule polymerization in tumor blood vessel endothelial cells and tumor cells. This blocks the formation of the mitotic spindle and leads to cell cycle arrest at the G2/M phase. As a result, this agent disrupts the tumor vasculature, tumor blood flow, deprives tumor cells of nutrients, and induces apoptosis. In addition, as microtubules plays an important role in intracellular transport, the inhibition of its polymerization may disrupt the transport of the androgen receptor (AR) into the cell nucleus, as well as virus trafficking around the cell. This may decrease viral replication and assembly. Inhibition of tubulin polymerization may also inhibit the release of pro-inflammatory cytokines and disrupt inflammatory cell activities. Sabizabulin is not a substrate of P-glycoprotein (Pgp), an efflux pump that when overexpressed, may confer resistance to taxane agents.

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VERU-111, an investigational drug intended for various therapeutic uses, was under investigation in clinical trials NCT04842747, NCT03752099, NCT04388826, NCT04844749, NCT05008510, and NCT05079360. These trials aimed to evaluate its efficacy, safety, and tolerability in conditions such as SARS-CoV-2 infection, metastatic castration-resistant prostate cancer (mCRPC), respiratory distress syndrome in adults, and metastatic triple-negative breast cancer.
SABIZABULIN is a small molecule drug with a maximum clinical trial phase of III (across all indications) and has 5 investigational indications.

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Mechanism of Action
Veru-111 is a selective tubulin inhibitor currently being tested for the treatment of pancreatic cancer. Veru-111 represses alpha- and beta-tublin subunits through enhanced expression of miR-200C. In both melanoma and prostate cancer cell lines, it has displayed strong antiproliferative activity. It also prevents microtubule polymerization and causes cell cycle arrest in the G2/M phase, which suggests anti-tumor properties.

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Background: The management of pancreatic cancer (PanCa) is exceptionally difficult due to poor response to available therapeutic modalities. Tubulins play a major role in cell dynamics, thus are important molecular targets for cancer therapy. Among various tubulins, βIII and βIV-tubulin isoforms have been primarily implicated in PanCa progression, metastasis and chemo-resistance. However, specific inhibitors of these isoforms that have potent anti-cancer activity with low toxicity are not readily available. Methods: We determined anti-cancer molecular mechanisms and therapeutic efficacy of a novel small molecule inhibitor (VERU-111) using in vitro (MTS, wound healing, Boyden chamber and real-time xCELLigence assays) and in vivo (xenograft studies) models of PanCa. The effects of VERU-111 treatment on the expression of β-tubulin isoforms, apoptosis, cancer markers and microRNAs were determined by Western blot, immunohistochemistry (IHC), confocal microscopy, qRT-PCR and in situ hybridization (ISH) analyses. Results: We have identified a novel small molecule inhibitor (VERU-111), which preferentially represses clinically important, βIII and βIV tubulin isoforms via restoring the expression of miR-200c. As a result, VERU-111 efficiently inhibited tumorigenic and metastatic characteristics of PanCa cells. VERU-111 arrested the cell cycle in the G2/M phase and induced apoptosis in PanCa cell lines via modulation of cell cycle regulatory (Cdc2, Cdc25c, and Cyclin B1) and apoptosis - associated (Bax, Bad, Bcl-2, and Bcl-xl) proteins. VERU-111 treatment also inhibited tumor growth (P < 0.01) in a PanCa xenograft mouse model. Conclusions: This study has identified an inhibitor of βIII/βIV tubulins, which appears to have excellent potential as monotherapy or in combination with conventional therapeutic regimens for PanCa treatment.[4]

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We recently reported the crystal structure of tubulin in complex with a colchicine binding site inhibitor (CBSI), ABI-231, having 2-aryl-4-benzoyl-imidazole (ABI). Based on this and additional crystal structures, here we report the structure-activity relationship study of a novel series of pyridine analogues of ABI-231, with compound 4v being the most potent one (average IC50 ∼ 1.8 nM) against a panel of cancer cell lines. We determined the crystal structures of another potent CBSI ABI-274 and 4v in complex with tubulin and confirmed their direct binding at the colchicine site. 4v inhibited tubulin polymerization, strongly suppressed A375 melanoma tumor growth, induced tumor necrosis, disrupted tumor angiogenesis, and led to tumor cell apoptosis in vivo. Collectively, these studies suggest that 4v represents a promising new generation of tubulin inhibitors. [5]

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Novel ABI-III compounds were designed and synthesized based on our previously reported ABI-I and ABI-II analogues. ABI-III compounds are highly potent against a panel of melanoma and prostate cancer cell lines, with the best compound having an average IC(50) value of 3.8 nM. They are not substrate of Pgp and thus may effectively overcome Pgp-mediated multidrug resistance. ABI-III analogues maintain their mechanisms of action by inhibition of tubulin polymerization.[6]

C21H19N3O4

377.393265008926

377.14

C, 66.83; H, 5.07; N, 11.13; O, 16.96

1332881-26-1

2635953-17-0 (HCl);1332881-26-1;

53379371

Light yellow to yellow solid powder

3.4

2

5

6

28

534

0

O(C)C1C(=C(C=C(C=1)C(C1=CN=C(C2=CNC3C=CC=CC2=3)N1)=O)OC)OC

WQGVHOVEXMOLOK-UHFFFAOYSA-N

InChI=1S/C21H19N3O4/c1-26-17-8-12(9-18(27-2)20(17)28-3)19(25)16-11-23-21(24-16)14-10-22-15-7-5-4-6-13(14)15/h4-11,22H,1-3H3,(H,23,24)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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 mg/mL (5.30 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 20.0 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 mg/mL (5.30 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 20.0 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.)