KSP (kinesin spindle protein) (Kiapp = 1.7 nM)

Having a Ki app of 1.7 nM, ispinesib is a strong and very selective inhibitor of KSP[1]. BT-474 and MDA-MB-468 cell lines are inhibited by ispinesib (150 nM), with GI50s of 45 and 19 nM, respectively[2]. Ispinesib (SB715992, 15 and 30 nM) causes prostate cancer cells to undergo apoptosis by 1094.88% and 1516.70%, respectively, and suppresses PC-3 prostate cancer cell proliferation by 48.65% and 52.16%. Genes involved in cell cycle arrest and apoptosis are upregulated by ispinenesib, whereas genes involved in cell survival and proliferation are downregulated. Genistein can augment ispinenesib's pro-apoptotic and anti-proliferation properties[3].

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Ispinesib (SB-715992) Is a Potent, Specific Inhibitor of KSP/human kinesin spindle protein;. Ispinesib Is an Allosteric, Reversible Inhibitor of KSP. Ispinesib Slows the Association of KSP to MT/microtubule. Ispinesib Accelerates ATP-Promoted Dissociation of KSP from MT/microtubule. Binding of Ispinesib Is Slowed in the Presence of MT. Ispinesib Inhibits ADP Release with and without MT. Ispinesib Does Not Perturb mantATP Binding to the KSP−MT Complex. Ispinesib Accelerates Phosphate Release from the KSP−MT Complex.[1]

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Sensitivity of human breast cancer cell lines to Ispinesib (SB-715992) in vitro [2]
Researchers investigated the possibility that specific breast cancer subtypes might exhibit particular sensitivity to ispinesib in a panel of 50 human breast tumor cell lines representative of diverse primary tumor histotypes and genetic backgrounds and in three normal mammary epithelial lines: MCF10A, MCF10F, and MCF12A (Fig. 1A; ref. 23). Cells were treated with increasing concentrations of ispinesib and ranked according to the concentration of drug required to reduce growth by 50% (GI50; Fig. 1A). All lines exhibited sensitivities between 7.4 and 600 nmol/L, with most falling within a 10-fold range, between 7.4 and 80 nmol/L. Three lines of luminal subtype exhibited sensitivities between 100 and 600 nmol/L. Across this relatively narrow range of sensitivity, we were unable to discern any obvious correlation with subtype, receptor expression, or mutational status.

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Ispinesib (SB-715992) inhibited cell proliferation and induced apoptosis in PC-3 cells. SB715992 was found to regulate the expression of genes related to the control of cell proliferation, cell cycle, cell signaling pathways, and apoptosis. In addition, our results showed that combination treatment with SB715992 and genistein caused significantly greater cell growth inhibition and induction of apoptosis compared to the effects of either agent alone. Conclusion: Our results clearly show that SB715992 is a potent anti-tumor agent whose therapeutic effects could be enhanced by genistein. Hence, we believe that SB715992 could be a novel agent for the treatment of prostate cancer with greater success when combined with a non-toxic natural agent like genistein [3].

In mice containing tumor xenografts of ER-positive (MCF7), HER2-positive (KPL4, HCC1954, and BT-474), and triple-negative (MDA-MB-468) breast cancer cells, iminetsinib (SCID, 8 mg/kg; nude, 10 mg/kg, q4d × 3) decreases tumor volume by an intraperitoneal injection given three times[2].

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Efficacy of Ispinesib (SB-715992) as a single agent in preclinical breast cancer models [2]
To determine the extent of ispinesib antitumor activity in breast cancer models in vivo, we chose cell lines that exhibited different in vitro sensitivity to ispinesib and represent different subtypes of human breast tumors. Their rank from most sensitive to less sensitive to ispinesib in vitro is as follows: MDA-MB-468 > HCC1954 = MCF7 > BT-474. MCF7 is a well-characterized ER-positive luminal breast cancer cell line. MDA-MB-468 is a model for basal triple-negative breast cancer. To represent HER2-overexpressing breast cancer, we chose BT-474, HCC1954, and KPL4, a breast tumor line of metastatic origin. The transcriptomic, genomic, and functional characteristics of these cell lines, except KPL4, have been characterized previously. Mice bearing tumor xenografts of the lines listed were treated i.p. with ispinesib at its MTD (SCID, 8 mg/kg; nude, 10 mg/kg) on its optimal q4d×3 schedule. Ispinesib was active in all models tested (Fig. 2; Table 1), producing regressions in each. However, the respective tumors differed in sensitivity as judged by the extent of tumor shrinkage, the number of regressions, and extent of tumor regrowth. The triple-negative xenograft model MDA-MB-468, among the most sensitive lines in vitro (Fig. 1A), exhibited the greatest ispinesib sensitivity in vivo. On ispinesib treatment, MDA-MB-468 tumors regressed completely in all mice, each scoring as TFS at the end of the study and 30 days beyond (data not shown). In the ER-positive model MCF7, ispinesib caused tumor regressions in five of nine mice (one PR and four CR, two of which were TFS at study end) and a TGI of 92%. Of the HER2-positive models, KPL4 showed the best response to ispinesib treatment. All 10 treated mice exhibited regressions (four PR, six CR, and four TFS). In the HCC1954 model, ispinesib caused regressions in four of the five treated mice. However, in both of these models, tumor regrowth began 35 days after treatment in the less responsive tumors. In the third HER2-positive model BT-474, ispinesib caused a CR in 2 of 8 mice, a lower TGI (61%) than that observed in the other models, and tumors had regrown in all mice by the end of the study (mean tumor volume, 875 mm3).

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MDA-MB-468 xenografts are hypersensitive to Ispinesib (SB-715992) [2]
To investigate further the hypersensitivity of the MDA-MB-468 tumors to ispinesib, we compared the antitumor activity of ispinesib with that of ixabepilone or paclitaxel, two antimitotic therapies approved for the treatment of breast cancer. We administered each agent to two cohorts of tumor-bearing animals, receiving either the MTD or a lower dose. Ispinesib antitumor activity was comparable with that of paclitaxel and ixabepilone in terms of TGI and regressions (Fig. 3A; Supplementary Data 2). One of nine mice treated with the higher dose of ixabepilone (5 mg/kg) developed limb paralysis and was sacrificed early. No such toxicity was observed with paclitaxel or ispinesib.

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Activity of Ispinesib (SB-715992) in combination with standards of care in breast cancer [2]
We sought to identify potentially beneficial combination regimens of ispinesib with agents commonly used to treat breast cancer: the HER2-targeted therapies, trastuzumab and lapatinib, doxorubicin (anthracycline), and capecitabine (antimetabolite). In all combination studies, we dosed the approved agent at MTD and optimal dosing schedule and adjusted the dose of ispinesib as necessary to achieve a tolerated combination regimen. We combined ispinesib with trastuzumab in two different tumor models overexpressing HER2: the luminal model BT-474 (Fig. 4A) and the metastasis-derived model KPL4 (Fig. 4B). In both models, the absence of trastuzumab toxicity allowed combination with the single-agent MTD of ispinesib. The combination proved superior to treatment with either single agent. In BT-474, the combined agents caused a TGI of 99% compared with 61% and 88% with ispinesib and trastuzumab, respectively (Table 2), and cured seven of eight mice. In KPL4, all 10 mice receiving the combination experienced PR or CR, 4 remained tumor-free at the end of the study, and TGI was 97%.

Steady-State Kinetic Analysis of Human KSP ATPase Activity and Inhibition by Ispinesib (SB-715992) [1]
Kinesin specificity analysis was carried out using a pyruvate kinase−lactate dehydrogenase detection system that couples the production of ADP to oxidation of NADH. Absorbance changes were monitored at 340 nm. Steady-state studies using nanomolar concentrations of KSP were performed using a sensitive fluorescence-based assay utilizing a pyruvate kinase, pyruvate oxidase, and horseradish peroxidase coupled detection system that couples the generation of ADP to oxidation of Amplex Red to fluorescent resorufin. Generation of resorufin was monitored by fluorescence (λexcitation = 520 nm and λemission= 580 nm). Steady-state biochemical experiments were performed in PEM25 buffer [25 mM Pipes-K+ (pH 6.8), 2 mM MgCl2, 1 mM EGTA] supplemented with 10 µM paclitaxel for experiments involving microtubules. The IC50 for steady-state inhibition was determined at 500 µM ATP, 5 µM MTs, and 1 nM KSP in PEM25 buffer. Ki app (apparent inhibitor dissociation constant) estimates of Ispinesib (SB-715992) were extracted from the concentration–response curves, with explicit correction for enzyme concentration by using the Morrison equation。

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Determination of the Residence Half-Life (t1/2) for Ispinesib (SB-715992) [1]
The residence half-life (t1/2) for Ispinesib (SB-715992) dissociation from KSP was determined by equilibrium dialysis. Measurements were performed in a final reaction volume of 1 mL consisting of 800 nM KSP, 800 nM ispinesib, and 5 µM MT. The solution was loaded into dialysis cassettes and dialyzed against PEM25 buffer supplemented with 2 mM DTT for 30 h. The dialysis buffer was changed three times during the first 2 h to ensure complete dialysis. Aliquots of reaction samples were collected at various time points and tested for activity. The activities of the reaction samples were normalized to the activities of a DMSO (no inhibitor) control. The release rates (koff) for ispinesib were determined by single-exponential fitting of the experimental data. The residence half-life (t1/2) was calculated using eq 2.

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Transient-State Experiments [1]
Stopped-flow (SF-61 DX2) experiments were conducted to determine the effect of Ispinesib (SB-715992) on mantATP binding, KSP association and disassociation from MTs, phosphate (Pi) release, and mantADP release. A nucleotide-free KSP−MT complex was prepared by incubating a 1:1 complex of KSP and MT with 1 unit/mL apyrase for 15 min at room temperature. Apyrase was removed through a 20% sucrose cushion by centrifugation (75000g) for 15 min. The pelleted KSP−MT complex was resuspended in PEM25 buffer supplemented with 10 µM paclitaxel. [1]
For each data point transient traces were collected in triplicate and averaged, before fitting. For mantATP or mantADP experiments, fluorescence emission was measured using a 400 nm cutoff filter with excitation at 360 nm. The increase in fluorescence upon KSP binding to mantATP or decrease in fluorescence following the release of mantADP from KSP was monitored as previously described. The rates of Pi release were measured using the bacterial phosphate binding protein (PBP) modified with 7-diethylamino-3-[[[2-(maleimidyl)ethyl]amino]carbonyl]coumarin (MDCC) dye. The nucleotide-free KSP−MT complex plus MDCC-PBP and Pi-mop (vide infra) was rapidly mixed with increasing concentrations of MgATP and Pi-mop. The Pi-mop consisted of a solution of PNPase (10 units/mL), 7-MEG (10 mM), MnCl2 (5 µM), glucose 1,6-bisphosphate (1 mM), and PDRM (1 µg/mL) in a ratio to eliminate competition with MDCC-PBP for Pi in solution. KSP−MT complex dissociation and association kinetics were monitored by the change in solution turbidity at 340 nm. Experiments measuring KSP dissociation rates from MTs were conducted using MgATP as previously described. Briefly, a complex of nucleotide-free KSP and microtubules was preformed in the absence and presence of Ispinesib (SB-715992) and rapidly mixed in the stopped-flow instrument with varying concentrations of MgATP (0–500 µM) and KCl (100 mM) in PEM25. In this experimental design, KSP binds and hydrolyzes ATP, which results in the detachment of KSP from the microtubule. The additional salt (KCl) weakens the interaction of the motor with MT, minimizing MT rebinding. [1]
To determine the binding rate of ispinesib to KSP, we monitored Trp127 fluorescence. The rates of ispinesib binding to KSP under its various physiological states (KSP−MT, KSP−AMPPMP−MT (ATP-like), KSP−ADP, and KSP−AMPPNP) were determined by monitoring fluorescence quenching of Trp127 within the loop 5 region. The excitation wavelength was 295 nm, and the emission filter cutoff was 320 nm. The stock solutions of ispinesib (0–100 µM) and KSP or KSP−MT (1 µM) in the presence of either 500 µM ADP or 500 µM AMP-PNP in PEM25 buffer were mixed by a stopped-flow apparatus. The fluorescence quenching traces were fitted to a double-exponential equation.

Western blot analyses [2]
Cells were treated with 150 nmol/L Ispinesib (SB-715992) and lysed in radioimmunoprecipitation assay buffer. Primary antibodies for Bax, Bid, xIAP, Bcl2, phospho-Bcl2 (Ser70), and Bcl-XL (54H6) were used. Other primary antibodies used were cyclin B and cyclin E (HE12). Secondary antibodies were IR 680/800CW LI-COR, and signal detection and analysis were done on a LI-COR Odyssey imaging system.

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DNA cell cycle analysis by flow cytometry [2]
Cells were treated with 150 nmol/L Ispinesib (SB-715992), fixed in 85% ice-cold ethanol, resuspended in PBS containing 10 μg/mL propidium iodide DNA stain and 250 μg/mL RNase A, and analyzed with a FACSCalibur flow cytometer. Cell cycle analyses were done with FlowJo.

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Cell inhibition assay [3]
PC-3 prostate cancer cells were seeded in 96 well plates at a density of 4 × 103 cells/well. PC-3 cells were incubated for 24 hours to allow attachment to the surface of each well of the tissue culture plate. Then, the cells were treated with varying concentration of reagents and incubated for 1 to 3 days. First, PC-3 cells were treated with 15 and 30 nM of Ispinesib (SB-715992), respectively. Second, PC-3 cells were subjected to combinational treatments with 7.5 or 10 nM of SB715992 plus 30 μM of genistein. Finally, PC-3 cells were pre-treated with 30 μM of genistein for 24 hours followed by treatment with 15 nM of SB715992. Control cells were treated with 0.3 mM Na2CO3 (vehicle control). After treatment, PC3 cells were incubated at 37°C with MTT (0.5 mg/ml, Sigma, St. Louis, MO, USA) for 2 hours and isopropyl alcohol at room temperature for 1 hour.

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DNA ladder analysis for detecting apoptosis [3]
PC-3 cells were seeded in 100 mm dishes at 3.5 × 105 cells/dish and allowed to adhere and grow for 36 hours. Following growth and attachment, PC-3 cells were treated with 15 nM of Ispinesib (SB-715992) for 48 and 72 hours. After treatment, cellular cytoplasmic DNA was extracted using 10 mM Tris (pH 8.0), 0.5 mM EDTA, and 0.2% Triton X-100. The lysate was then centrifuged at 4°C for 15 minutes at 13,800 × g to separate the cytoplasmic DNA fragments from the nuclear pellet. The supernatant was then collected and treated with 15 μl of RNase and incubated at 37°C for 1 hour. Following incubation, the supernatant was treated with 20 μl of 20% SDS, 8 μl of proteinase K (20 mg/ml), 25 μl of 5.0 M NaCl and allowed to incubate at 37°C for 30 minutes. Thereafter, phenol/chloroform/isoamyl-alcohol extraction and isopropyl alcohol precipitation were carried out. After precipitation, the DNA fragments were washed in 70% alcohol and separated through a 1.5% agarose gel at 100 volts for 80 minutes. After electrophoresis, running gels were stained with ethidium bromide and visualized by ultra-violet light.

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Microarray analysis for gene expression profiles [3]
PC-3 cells were treated with 10 nM of Ispinesib (SB-715992) for 6, 24, and 48 hours respectively. Total RNA was extracted from each sample by the use of Trizol following manufacture's protocol. The total RNA of each sample was then purified with RNeasy Mini Kit and RNase-free DNase Set following manufacturer's protocol. The purified RNA samples were subject to microarray anaylsis using Human Genome U133A Array, which contains 54,613 human gene probes. Gene expression was then quantified by using Microarray Suite, MicroDB™, and Data Mining Tool Software. Clustering and annotation of the gene expression were analyzed by using Cluster and TreeView, Onto-Express, and GenMAPP.

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Analysis of RNA expression by reverse transcription-polymerase chain reaction [3]
To verify the alterations of gene expression at the mRNA level, which appeared on the microarray, we chose representative genes (Table 1 and Table 2) with varying expression profiles for real-time RT-PCR analysis. PC-3 cells were treated with 10 nM of Ispinesib (SB-715992) for 6 and 48 hours and total RNA was isolated and purified as mentioned above. Two micrograms of total RNA from each sample were subjected to reverse transcription using the Superscript first strand cDNA synthesis kit according to the manufacturer's protocol. Real-time PCR reactions were then carried out in a total of 25 μL reaction mixture (2 μl of cDNA, 12.5 μl of 2× SYBR Green PCR Master Mix, 1.5 μl of each 5 μM forward and reverse primers, and 7.5 μl of H2O) in SmartCycler II. The PCR program was initiated by 10 min at 95°C before 40 thermal cycles, each of 15 s at 95°C and 1 min at 60°C. Data were analyzed according to the comparative Ct method and were normalized by actin expression in each sample. Melting curves for each PCR reaction were generated to ensure the purity of the amplification product.

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Western blot analysis[3]
PC-3 cells were seeded into 100 mm culture dishes at 3.0 × 105 cells per dish and allowed to attach overnight for 24 hours. The cells were treated with 10 nM of Ispinesib (SB-715992) for 24 and 48 hours. The cells were then lysed in 62.5 mM Tris-HCl and 2% SDS. The protein concentrations of each sample were measured using a BCA Protein Assay Kit. The cellular protein extracts were then subjected to 10 or 14% of SDS-PAGE and transferred to nitrocellulose membranes at 100 V for 2 hours at 4°C. The membranes were incubated with anti-EGFR, anti-p27 (1:100), and anti-p15 (1:200), and anti-β-actin (1:10000) primary antibodies, and subsequently incubated with secondary antibodies conjugated with peroxidase.

Xenograft studies [2]
Female mice (7–8 wk) obtained from Charles River were implanted on their flank with 107 cells in 100 μL of 1:1 PBS/Matrigel. nu/nu mice were used for all tumor models, except BT-474 and MDA-MB-468, which were established in Fox Chase severe combined immunodeficient (SCID) mice. BT-474 tumors were generated by s.c. implanting 30 mm3 tumor fragments from previously established xenografts. For MCF7 xenografts, mice were implanted s.c. at the base of the neck with 90-d release 0.36 mg 17β-estradiol pellets (Innovative Research of America) 3 d before tumor cell implantation. Tumor volume (length × width2)/2 and body weight were measured twice weekly. For efficacy studies, drug treatment started when tumor volume was ~100 mm3 and mice were sacrificed at 60 d after treatment or when tumor volume reached 1,500 mm3. Drug-treated mice were categorized as a partial regression (PR) if three consecutive tumor measurements were less than half the starting tumor volume on day 0 of treatment, a complete regression (CR) if tumor volume was <12.5 mm3 for three consecutive measurements, and a tumor-free survivor (TFS) if it had no measurable tumor or remained a CR at the end of the study. Tumor growth inhibition (TGI) is the percentage difference in tumor volume between vehicle- and drug-treated groups, determined on the final day when all tumor volumes in the vehicle group are <1,000 mm3. Statistical analyses of tumor volume differences between only two groups, such as single-agent Ispinesib (SB-715992) and vehicle control, were conducted using unpaired t tests of tumor volumes at the end of the study (day 60). Statistical analyses of multiple treatment groups were conducted using one-way ANOVA followed by Newman Keuls post hoc test to determine the significance of differences in tumor volumes on day 60 (unless otherwise noted) among treatment groups.

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Drugs [2]
All drugs were dosed at their maximum tolerated dose (MTD) unless otherwise stated, and drug volumes were 200 μL/25 g mouse. Ispinesib (SB-715992) was formulated in 10% ethanol, 10% cremophor, and 80% D5W (dextrose 5%) and dosed i.p. on a q4d×3 schedule (three doses, every 4 d) at 10 mg/kg in nu/nu mice or 8 mg/kg in SCID mice, unless otherwise stated. Trastuzumab was dosed i.p. twice weekly for 4 wk at 10 mg/kg. Doxorubicin was formulated in 0.9% saline and dosed q4d×3 at 3 mg/kg in nu/nu mice or on days 1, 7, and 21 at 2.5 mg/kg in SCID mice. Lapatinib was formulated in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 80 in water and dosed orally twice daily for 3 wk at 40 mg/kg. Capecitabine was formulated in 40 mmol/L citrate buffer (pH 6) in 0.5% methylcellulose and orally dosed daily at 450 mg/kg for 14 d. Paclitaxel and ixabepilone were formulated in 10% ethanol, 10% cremophor, and 80% D5W and dosed i.v. q4d×3 at their respective MTDs of 30 and 5 mg/kg. Vehicle-treated control mice were injected i.p. q4d×3 with a formulation of 10% ethanol, 10% cremophor, and 80% D5W.

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Immunohistochemistry [2]
Mice with a tumor volume of ~250 mm3 received a single dose of Ispinesib (SB-715992) (10 mg/kg). Tumors were dissected, fixed in 10% buffered formalin, and embedded in paraffin, and 5-μm tissue sections were prepared. Antigen retrieval was done by boiling in 50 mmol/L citrate buffer (pH 5.5), and sections were then incubated in 3% hydrogen peroxide, washed in PBS–0.1% Tween, and blocked in 10% goat serum. Phospho–histone H3 (PH3) antibody was detected using Alexa Fluor 488 secondary antibody. Images were taken with a Nikon Eclipse TE-2000U microscope at ×10 magnification and captured using MetaMorph software to quantify PH3 expression by computing the area ratio of PH3-positive cells per total cells. Ki67/cleaved caspase-3 staining was done according to the manufacturer’s guidelines. Nonfluorescent images were taken on an Olympus BX41 microscope at ×20 magnification.

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Dissolved in 10% ethanol, 10% cremophor, and 80% D5W (dextrose 5%); 10 mg/kg for nude mice or 8 mg/kg for SCID mice; i.p. injection on a q4d× schedule, 3 doses, every 4 day

Nude (nu/nu) mice models of MCF7, KPL4, and HCC1954 cells; severe combined immunodeficient (SCID) mice model of MDA-MB-468 cells;

Descriptive PK parameters were calculated using noncompartmental methods. Because of a sparse sampling scheme, the full PKs of ispinesib cannot be completely described. Available PK data (Table 4) suggest that systemic exposure of ispinesib was not dose proportional over the dose range of 10–14 mg/m2. The mean Cmax (C1 h/end of infusion) and AUClast on day 1 were 349±107 ng/ml (10 mg/m2), 223±48.5 ng/ml (12 mg/m2), and 213±109 ng/ml (14 mg/m2). On day 15, the mean Cmax and AUClast were 396±129 ng/ml (10 mg/m2), 248±136 ng/ml (12 mg/m2), and 230±68.7 ng/ml (14mg/m2). There was no significant difference in drug exposure between days 1 and 15. https://pubmed.ncbi.nlm.nih.gov/22123335/

Determination of maximum tolerated dose and safety results:
Two DLTs, both transient grade 3 aspartate aminotransferase (AST) and ALT increases, were observed at 14 mg/m2. Both patients remained in the study without dose reduction without further toxicity. These were the only two DLTs reported in the study. The MTD was determined to be 12 mg/m2. Table 3 summarizes AEs reported in one or more patients during the study. The most common AEs, reported in at least three patients, included neutropenia (n=14, 87.5%), increased ALT (n=9, 56.3%), anemia (n=6, 37.5%), increased AST and diarrhea (n=5, 31.3%), increased alkaline phosphatase (n=4, 25.0%), and leukopenia, thrombocythemia, nausea, vomiting, headache, and breast pain (n=3, 18.8%). No AEs of neuropathy, mucositis, or alopecia were reported. The only grade 3 and grade 4 AEs reported were neutropenia (grade 3, n=6, 37.5%; grade 4, n=7, 43.8%), ALT and AST increased (grade 3, n=2, 12.5%), and febrile neutropenia (grade 3), thrombocytopenia (grade 4; likely a laboratory error), pleural effusion (grade 3), and spontaneous abortion (grade 4), each n=1, 6.3%. The spontaneous abortion occurred in a patient who had a negative pregnancy test at study entry but was found to be pregnant following the completion of cycle 1 dosing. Because of the pregnancy, post-treatment imaging was not performed and the patient was excluded from the study. There were no grade 5 AEs. No notable changes in ECG measurements, including Bazett’s corrected Q–T interval, vital signs, or physical exam including neurological assessments, were observed. https://pubmed.ncbi.nlm.nih.gov/22123335/

[1]. Mechanism of inhibition of human KSP by ispinesib. Biochemistry. 2008 Mar 18;47(11):3576-85.

[2]. Activity of the kinesin spindle protein inhibitor ispinesib (SB-715992) in models of breast cancer. Clin Cancer Res. 2010 Jan 15;16(2):566-76.

[3]. Increased therapeutic potential of an experimental anti-mitotic inhibitor SB715992 by genistein in PC-3 human prostate cancer cell line. BMC Cancer. 2006 Jan 24;6:22.

N-(3-aminopropyl)-N-[(1R)-1-[7-chloro-4-oxo-3-(phenylmethyl)-2-quinazolinyl]-2-methylpropyl]-4-methylbenzamide is a member of benzamides.
Ispinesib is a synthetic small molecule, derived from quinazolinone, with antineoplastic properties. Ispinesib selectively inhibits the mitotic motor protein, kinesin spindle protein (KSP), resulting in inhibition of mitotic spindle assembly, induction of cell cycle arrest during the mitotic phase, and cell death in tumor cells that are actively dividing. Because KSP is not involved in nonmitotic processes, such as neuronal transport, ispinesib may be less likely to cause the peripheral neuropathy often associated with the tubulin-targeting agents.

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Drug Indication
Investigated for use/treatment in breast cancer, lung cancer, solid tumors, renal cell carcinoma, pediatric indications, ovarian cancer, and head and neck cancer.

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KSP, also known as HsEg5, is a kinesin that plays an essential role in the formation of a bipolar mitotic spindle and is required for cell cycle progression through mitosis. Ispinesib is the first potent, highly specific small-molecule inhibitor of KSP tested for the treatment of human disease. This novel anticancer agent causes mitotic arrest and growth inhibition in several human tumor cell lines and is currently being tested in multiple phase II clinical trials. In this study we have used steady-state and pre-steady-state kinetic assays to define the mechanism of KSP inhibition by ispinesib. Our data show that ispinesib alters the ability of KSP to bind to microtubules and inhibits its movement by preventing the release of ADP without preventing the release of the KSP-ADP complex from the microtubule. This type of inhibition is consistent with the physiological effect of ispinesib on cells, which is to prevent KSP-driven mitotic spindle pole separation. A comparison of ispinesib to monastrol, another small-molecule inhibitor of KSP, reveals that both inhibitors share a common mode of inhibition. [1]

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Purpose: Ispinesib (SB-715992) is a potent inhibitor of kinesin spindle protein, a kinesin motor protein essential for the formation of a bipolar mitotic spindle and cell cycle progression through mitosis. Clinical studies of ispinesib have shown a 9% response rate in patients with locally advanced or metastatic breast cancer and a favorable safety profile without significant neurotoxicities, gastrointestinal toxicities, or hair loss. To better understand the potential of ispinesib in the treatment of breast cancer, we explored the activity of ispinesib alone and in combination with several therapies approved for the treatment of breast cancer. Experimental design: We measured the ispinesib sensitivity and pharmacodynamic response of breast cancer cell lines representative of various subtypes in vitro and as xenografts in vivo and tested the ability of ispinesib to enhance the antitumor activity of approved therapies. Results: In vitro, ispinesib displayed broad antiproliferative activity against a panel of 53 breast cell lines. In vivo, ispinesib produced regressions in each of five breast cancer models and tumor-free survivors in three of these models. The effects of ispinesib treatment on pharmacodynamic markers of mitosis and apoptosis were examined in vitro and in vivo, revealing a greater increase in both mitotic and apoptotic markers in the MDA-MB-468 model than in the less sensitive BT-474 model. In vivo, ispinesib enhanced the antitumor activity of trastuzumab, lapatinib, doxorubicin, and capecitabine and exhibited activity comparable with paclitaxel and ixabepilone. Conclusions: These findings support further clinical exploration of kinesin spindle protein inhibitors for the treatment of breast cancer. [2]

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Background: Kinesin spindle proteins (KSP) are motor proteins that play an essential role in mitotic spindle formation. HsEg5, a KSP, is responsible for the formation of the bipolar spindle, which is critical for proper cell division during mitosis. The function of HsEg5 provides a novel target for the manipulation of the cell cycle and the induction of apoptosis. SB715992, an experimental KSP inhibitor, has been shown to perturb bipolar spindle formation, thus making it an excellent candidate for anti-cancer agent. Our major objective was a) to investigate the cell growth inhibitory effects of SB715992 on PC-3 human prostate cancer cell line, b) to investigate whether the growth inhibitory effects of SB715992 could be enhanced when combined with genistein, a naturally occurring isoflavone and, c) to determine gene expression profile to establish molecular mechanism of action of SB715992. Methods: PC-3 cells were treated with varying concentration of SB715992, 30 microM of genistein, and SB715992 plus 30 microM of genistein. After treatments, PC-3 cells were assayed for cell proliferation, induction of apoptosis, and alteration in gene and protein expression using cell inhibition assay, apoptosis assay, microarray analysis, real-time RT-PCR, and Western Blot analysis. Results: SB715992 inhibited cell proliferation and induced apoptosis in PC-3 cells. SB715992 was found to regulate the expression of genes related to the control of cell proliferation, cell cycle, cell signaling pathways, and apoptosis. In addition, our results showed that combination treatment with SB715992 and genistein caused significantly greater cell growth inhibition and induction of apoptosis compared to the effects of either agent alone. Conclusion: Our results clearly show that SB715992 is a potent anti-tumor agent whose therapeutic effects could be enhanced by genistein. Hence, we believe that SB715992 could be a novel agent for the treatment of prostate cancer with greater success when combined with a non-toxic natural agent like genistein. [3]

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The objective of the study was to evaluate the safety, pharmacokinetics, and antitumor activity of ispinesib, a kinesin spindle protein inhibitor. Patients with locally advanced or metastatic breast cancer who had received only prior neoadjuvant or adjuvant chemotherapy were treated with escalating doses of ispinesib administered as a 1-h infusion on days 1 and 15 every 28 days until toxicity or progression of disease. Doses were escalated until dose-limiting toxicity was observed in two out of six patients during cycle 1. A total of 16 patients were treated at three dose levels: 10 mg/m (n=3), 12 mg/m (n=6), and 14 mg/m (n=7). Forty-four percent of the patients had locally advanced disease and 56% had metastatic disease; 50% were estrogen receptor positive, 44% were progesterone receptor positive, 25% human epidermal growth factor 2 were positive, and 31% triple (estrogen receptor, progesterone receptor, human epidermal growth factor 2) negative. Sixty-nine percent of patients were chemo-naive. The maximum tolerated dose was 12 mg/m and dose-limiting toxicity was grade 3 increased aspartate aminotransferase and alanine aminotransferase. The most common toxicities included neutropenia (88%; 38% grade 3 and 44% grade 4), increased alanine aminotransferase (56%), anemia (38%), increased aspartate aminotransferase (31%), and diarrhea (31%). No neuropathy, mucositis, or alopecia was reported. Among the 15 patients evaluable for antitumor activity, there were three partial responses, one confirmed by the response evaluation criteria in solid tumors (7% response rate). Nine patients (60%) had stable disease lasting at least 42 days, with four (27%) lasting for at least 90 days. Disease stabilization (partial responses+stable disease) was observed in 11 (73.3%) patients. In conclusion, ispinesib was well tolerated when administered on days 1 and 15 every 28 days. Limited activity was observed with this schedule in patients with previously untreated advanced breast cancer. Anticancer Drugs . 2012 Mar;23(3):335-41.

C30H33CLN4O2

517.06

516.229

C, 69.69; H, 6.43; Cl, 6.86; N, 10.84; O, 6.19

336113-53-2

336113-53-2;514820-03-2 (mesylate);

6851740

White to light yellow solid powder

1.2±0.1 g/cm3

708.0±70.0 °C at 760 mmHg

382.0±35.7 °C

0.0±2.3 mmHg at 25°C

1.619

5.2

1

4

9

37

803

1

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InChI=1S/C30H33ClN4O2/c1-20(2)27(34(17-7-16-32)29(36)23-12-10-21(3)11-13-23)28-33-26-18-24(31)14-15-25(26)30(37)35(28)19-22-8-5-4-6-9-22/h4-6,8-15,18,20,27H,7,16-17,19,32H2,1-3H3/t27-/m1/s1

(R)-N-(3-aminopropyl)-N-(1-(3-benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylpropyl)-4-methylbenzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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: ≥ 2.5 mg/mL (4.84 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.84 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 25.0 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.)