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Seliciclib (also known as Roscovitine, CYC 202; CYC-202; CYC202) is a novel, potent, selective and orally bioavailable small-molecule CDK inhibitor with potential anticancer activity. In cell-free assays, it inhibits Cdc2, CDK2, and CDK5 with IC50 values of 0.65 μM, 0.7 μM, and 0.16 μM. An experimental medication candidate called seliciclib inhibits cyclin-dependent kinase 2/7/9. Furthermore, it seems that this agent obstructs CDK-mediated phosphorylation of RNA polymerase II's carboxy-terminal domain, thereby suppressing transcription dependent on RNA polymerase II. This could potentially lead to the down-regulation of antiapoptotic proteins like the induced myeloid leukemia cell differentiation protein Mcl-1.
Physicochemical Properties
| Molecular Formula | C19H26N6O | |
| Molecular Weight | 354.45 | |
| Exact Mass | 354.216 | |
| Elemental Analysis | C, 64.38; H, 7.39; N, 23.71; O, 4.51 | |
| CAS # | 186692-46-6 | |
| Related CAS # |
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| PubChem CID | 160355 | |
| Appearance | White to off-white solid powder | |
| Density | 1.3±0.1 g/cm3 | |
| Boiling Point | 577.5±60.0 °C at 760 mmHg | |
| Flash Point | 303.1±32.9 °C | |
| Vapour Pressure | 0.0±1.7 mmHg at 25°C | |
| Index of Refraction | 1.643 | |
| LogP | 1.68 | |
| Hydrogen Bond Donor Count | 3 | |
| Hydrogen Bond Acceptor Count | 6 | |
| Rotatable Bond Count | 8 | |
| Heavy Atom Count | 26 | |
| Complexity | 417 | |
| Defined Atom Stereocenter Count | 1 | |
| SMILES | O([H])C([H])([H])[C@@]([H])(C([H])([H])C([H])([H])[H])N([H])C1=NC(=C2C(=N1)N(C([H])=N2)C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H] |
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| InChi Key | BTIHMVBBUGXLCJ-OAHLLOKOSA-N | |
| InChi Code | InChI=1S/C19H26N6O/c1-4-15(11-26)22-19-23-17(20-10-14-8-6-5-7-9-14)16-18(24-19)25(12-21-16)13(2)3/h5-9,12-13,15,26H,4,10-11H2,1-3H3,(H2,20,22,23,24)/t15-/m1/s1 | |
| Chemical Name | (2R)-2-[[6-(benzylamino)-9-propan-2-ylpurin-2-yl]amino]butan-1-ol | |
| Synonyms | Seliciclib; R-Roscovitine; CYC-202; roscovitine; Seliciclib; 186692-46-6; R-Roscovitine; (R)-roscovitine; Roscovitin; CYC202; Roscovitin; Roscovitine; CYC202; CYC 202 | |
| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | cdc2/cyclin B (IC50 = 0.65 μM); cdk2/cyclin A (IC50 = 0.7 μM); Cdk2/cyclin E2 (IC50 = 0.7 μM); CDK5/p35 (IC50 = 0.16 μM); GST-erk1 (IC50 = 30 μM); erk1 (IC50 = 34 μM); erk2 (IC50 = 14 μM); IR tyrosine kinase (IC50 = 70 μM) |
| ln Vitro | Roscovitine displays high efficiency and high selectivity towards some cyclin-dependent kinases with IC50 of 0.65, 0.7, 0.7 and 0.16 μM for cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p53, respectively. [1] In vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts are inhibited by Roscovitine, which also reversibly inhibits the prophaselmetaphase transition in the micromolar range of starfish oocytes and sea urchin embryos. As an average IC50 of 16 μM, Roscovitine also suppresses molecular line proliferation. (Source: ) At doses of 7.5, 12.5, and 25 mM, roscovitine causes a 25, 50%, and 100% decrease in CDK2 activity in mesangial cells, respectively. This reduction in CDK2 activity is dose-dependent.[2] In Dictyostelium discoideum, a recent study demonstrates that roscovitine inhibits cdk5 kinase activity, cell proliferation, multicellular development, and cdk5 nuclear translocation without influencing the expression of cdk5 protein during axenic growth.[3] |
| ln Vivo |
Seliciclib (Roscovitine) significantly inhibits the growth of xenografts of The Ewing's Sarcoma Family of Tumors (ESFT) at a dose of 50 mg/kg.[4] In nude mice with established MCF7 xenografts, roscovitine increases the antitumor effect of doxorubicin without increasing toxicity through a mechanism involving cell cycle arrest rather than apoptosis.[5] Researchers subsequently investigated the effect of Seliciclib (Roscovitine) in vivo by evaluating the effect of drug treatment on tumor growth using nude mice xenografts of A4573 ESFT cells generated as described under Materials and Methods. When tumors reached a volume of ∼130 mm3, animals were injected i.p. with roscovitine or with the carrier solution alone, following different schedules, and tumor growth was measured over a period of up to several weeks. As can be seen in Fig. 5A, tumor growth was significantly slower in roscovitine-treated mice than in control animals, as a reflection of the markedly smaller size of individual tumors observed after excision (Fig. 5A,, inset). One day after completion of the first 5-day treatment series, tumors in roscovitine-treated animals had grown only ∼1.25-fold relative to their size at the time of treatment initiation, whereas tumors in untreated mice had already attained a volume ∼14.5-fold their original size. These values represented a difference of ∼11.5-fold in tumor volume and, although tumors in roscovitine-treated animals continued to grow very slowly, a significant difference (∼7.5-fold) in tumor size was still evident at the time (day 13; Fig. 5A) when control animals, whose tumors had grown to ∼15-fold their initial size, had to be sacrificed following Institutional Animal Care and Use guidelines. Counting from day 1 of roscovitine treatment, tumors in control animals reached a volume thrice the original in ∼2 days, whereas it took ∼10 days for the tumors in treated animals to triplicate their initial volume (Fig. 5A). Overall, this difference indicated that roscovitine treatment resulted in an ∼5-fold reduction in tumor growth. [4] Furthermore, and most importantly, treatment for 5 consecutive days with only one i.p. injection at 50 mg/kg/d (total Seliciclib (Roscovitine) dose of 250 mg/kg) reduced tumor size, relative to untreated control animals, by ∼85% (days 5-8; Fig. 5A), whereas treatment schedules including three daily i.p. injections at 100 mg/kg/d for 5 days (total dose of 1,500 mg/kg) were reported to reduce by only 45% and 62%, respectively, the growth of tumors induced in nude mice with human colon (LoVo) and uterine (MESSA-DX5) tumor cell lines (19). The fact that our treatment achieved a better antitumor response with 6-fold lower total doses strongly indicates that roscovitine is substantially more efficient against ESFT than against other human tumor cells. These results showed that roscovitine efficiently inhibited ESFT cell growth in vivo as well as in culture. To further elucidate the mechanism of roscovitine action in vivo, we examined whether tumor tissues showed any evidence of apoptosis. As shown in Fig. 5B, results from both TUNEL assays (Fig. 5B,, middle) and immunohistochemical detection of cleaved caspase-3 (Fig. 5B,, right) showed that roscovitine also induced apoptosis of ESFT tumors in vivo by a caspase-dependent mechanism. In contrast, negligible signs of apoptosis were detectable in tumors from control animals (Fig. 5B , top) injected with carrier solution alone. [4] Efficacy of Seliciclib (Roscovitine) + doxorubicin compared to doxorubicin as a single agent in a MCF7 xenograft model [5] Figure 3 illustrates the growth of MCF7 control tumors (untreated or treated with vehicle alone), tumors treated with doxorubicin or Seliciclib (Roscovitine) as a single agent and tumors treated with seliciclib + doxorubicin. Mean relative sizes of tumors treated with a single agent (doxorubicin or seliciclib) compared with seliciclib + doxorubicin were 304 mm3 and 180 mm3, respectively at the end of treatment. These correspond to statistically significant inhibition of tumor growth of 48 and 70%, respectively relative to the vehicle control group (Student's t-test p < 0.05). At the end of the treatment, the tumor volume of the seliciclib + doxorubicin treated animals was significantly lower than that of the vehicle + doxorubicin treated group (p < 0.05). The doubling time for the tumor size was 7 days for the untreated and vehicle treated groups, 11 days for doxorubicin or seliciclib treated groups and 23 days for the seliciclib + doxorubicin treated group. There was no weight loss or behavior change in the treated groups. |
| Enzyme Assay | The kinase activities in buffer C are measured at 30 °C. The data are stripped of blank values, and activities are computed as the molar amount of phosphate incorporated in the protein acceptor over the course of a 10-minute incubation. The proper DMSO dilutions are used for the controls. After SDS/PAGE, autoradiography is sometimes used to evaluate the substrate's phosphorylation. By using affinity chromatography, p34cdc2/cyclin B is isolated from M-phase starfish (M. glacialis) oocytes. In a final volume of 30 μL, 1 mg histone Hl/mL is used in the assay along with 15 μM [γ-32P]ATP (3000 Ci/mmol; 1 mCi/mL). 25-μL aliquots of supernatant are spotted onto Whatman P81 phosphocellulose paper after a 10-minute incubation period at 30 °C. The filters are then washed five times (for a minimum of five minutes each time) in a solution of 10mL phosphoric acid/L water after 20 seconds. After transferring the wet filters into 6-mL plastic scintillation vials, 5 mL of ACS scintillation fluid is added, and a Packard counter is used to measure the radioactivity. The kinase activity is reported as a percentage of maximal activity or as the molar amount of phosphate incorporated in histone H1 after 10 minutes of incubation. Reconstituted p33cdk2/cyclin A and p33cdk2/cyclin E are made from extracts of baculovirus-infected sf9 insect cells. Glutathione S-transferase fusion proteins, cyclins A and E, are purified on glutathione-agarose beads. As with p34cdk2/cyclin B kinase, kinase activities are measured using 1 mg/mL histone H1 and 15 μM [γ-32P]ATP over the course of 10 minutes in a final volume of 30 μL. Bovine brain is used to purify p33cdk5/p35; the Mono S-chromatographic step is not included. The Superose 12 column's active fractions are combined and concentrated until they reach a final concentration of about 25 μg enzyme/mL. As with the p34cdk2/cyclin B kinase, the kinase is assayed using 1 mg/mL histone HI in the presence of 15 μM [γ-32P]ATP, over the course of 10 minutes in a final volume of 30 μL. The source of p33cdk5/cyclin D1 is insect cell lysates. Glutathione-S-transferase and Cdk4 form a fusion protein, and the active complex is purified using glutathione-agarose beads. In a final volume of 30 μL, its kinase activity is measured using purified retinoblastoma protein (complexed with glutathione-S-transferase) in the presence of 15 μM [γ-32P]ATP. After the incubation period of 15 minutes, 30 μL of Laemmli sample buffer is added. The substrate that has been phosphorylated is separated using 10% SDS/PAGE and examined using autoradiography, densitometry, and an overnight exposure to Hyperfilm MP. The source of p33cdk4/cyclinD 2 is insect cell lysates. In a final volume of 30 μL, it is tested using purified retinoblastoma protein (complexed with glutathione-S-transferase) and 15 μM [γ-32P]ATP. After the incubation period of 30 minutes, 30 μL of Laemmli sample buffer is added. The phosphorylated substrate is separated using 10% SDS/PAGE and examined using densitometry and autoradiography after being exposed to Hyperfilm MP for an entire night. Purified on glutathione-agarose beads and assayed with 1 mg myelin basic protein/ml in the presence of 15 μM [γ-32P]ATP, MAP kinase erkl (tagged with glutathione-S-transferase) is produced in bacteria, as previously mentioned for p34cdc2cyclin B kinase. In vitro, mitogen-activated protein kinase kinase activates His-tagged erkl and erk2, which are then purified using Ni-affinity and Mono Q. The assay is conducted over ten minutes in a final volume of thirty microliters, following the previously mentioned protocol. Infected sf9 insect cells are used to isolate protein kinase C isoforms, which are then tested for 10 minutes at 30 °C in a final volume of 30 μL using 1 mg/mL protamine sulfate and 15 μM [γ-32P]ATP. The Whatman P81 phosphocellulose paper is used to recover phosphorylated protamine sulfate, just like it is for CDC2 kinase. Purified from the heart of cows, the catalytic subunit of cAMP-dependent protein kinase is measured using 1 mg of histone Hl/ml and 15 μM [γ-32P]ATP, just like p34cdc2/cyclin B kinase. After being homogenized and purified from cow tracheal smooth muscle, cGMP-dependent protein kinase is measured using 1 mg of histone Hl/mL and 15 μM [γ-32P]ATP, just like p34cdc2/cyclin B kinase. Rat liver cytosol is used to isolate casein kinase 2, which is then tested using 1 mg casein/mL and 15 μM [γ-32P]ATP. After being spotted on Whatmann 3MM filters, the substrate is cleaned with 10% (mass/vol.) trichloroacetic acid. A synthetic peptide based on the smooth-muscle myosin light-chain phosphorylation site is used to assay myosin light chain kinase that has been purified from chicken gizzard. The final volume of the assay is 50 μL, and the conditions include 100 nM calmodulin, 100 μM CaCl2, 50 mM Hepes, 5 mM MgCI, 1 mM dithiothreitol, and 0.1 mg BSA/ml at pH 7.5. As previously mentioned, radioactive phosphate incorporation is tracked on phosphocellulose filters. Plant homolog of GSK-3, ASK-γ, is purified on glutathione-agarose after being expressed in Escherichia coli as a glutathione-S-transferase fusion protein. For 10 minutes at 30°C, 5 μg of myelin basic protein is added to a final volume of 30 μL of 15 μM [γ-32P]ATP to test ASK-γ kinase. On Whatman P81 phosphocellulose paper, the phosphorylated myelin basic protein is recovered in the same manner as the p34cdc2/cyclin B kinase. In a baculovirus system, the insulin receptor tyrosine kinase domain (CIRK-41) is overexpressed and homogeneously purified. Its kinase activity is measured in a final volume of 30 μL, for 10 minutes at 30 °C, using 5 μg of Raytide and 15 μM [γ-32P]ATP. As stated for the p34cdc2/cyclin B kinase, the phosphorylated Raytide is recovered on Whatman P81 phosphocellulose paper. From Sf9 cells that are infected, c-src kinase is isolated. After being expressed in E. Coli, the v-abl kinase is affinity purified using IgG Affigel 10. The assay is conducted for 10 minutes at 30°C, using 5 μg of Raytide, 15 μM [γ-32P]ATP, and a final volume of 30 μL. As stated for the p34cdc2/cyclin B kinase, the phosphorylated Raytide is recovered on Whatman P81 phosphocellulose paper. |
| Cell Assay |
The cells used are rat kidney tubular epithelial cells (NRK52E). Treatment for NRK52E cells involves the use of CDK5 inhibitor (R)-Roscovitine (Seliciclib) (Ros.; 10 μM) and activator p35 (15 μM), PPARγ agonist BRL 49653 (Rosi.; 50 nM), and ERK1/2 inhibitor U0126 (50 nM). Following a 72-hour treatment period, cells are extracted from each group for additional analysis. Apoptosis and cell cycle assays. [4] Apoptosis was evaluated by viable cell counting and/or terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) assays. Cell viability was determined by the trypan blue exclusion method: Cells were suspended in 0.04% trypan blue in PBS, placed on a hemocytometer, and counted under the microscope. TUNEL assays were done for the in situ detection of apoptotic cells using the red-based TMR In situ Death Detection kit. Cells were cultured in chamber slides to a population density of 5 × 104 cells. Sixteen hours after Seliciclib (Roscovitine) exposure, cells were washed with PBS, fixed in freshly prepared paraformaldehyde (4% in PBS) for 30 minutes at room temperature, rinsed thrice in PBS, permeabilized with 0.2% Triton X-100 in PBS for 30 minutes, and incubated with the TUNEL reaction mixture for 1 hour at 37°C in a humidified atmosphere in the dark. TUNEL-positive cells were visualized with a Nikon E600 fluorescence microscope. For cell cycle analysis, cells were harvested 24 hours after exposure to Seliciclib (Roscovitine), washed once in PBS, fixed in citrate buffer (pH 7.6), resuspended in PBS containing 20 μg/mL of propidium iodide, and incubated for 30 minutes at 37°C before flow cytometric analysis on a FACScan instrument, done at the Flow Cytometry/Cell Sorting Shared Resource of the Vincent T. Lombardi Comprehensive Cancer Center. The same in situ death detection kit was used for TUNEL assays done on deparaffinated 5 μm tumor sections. Caspase assays. [4] Cultures of TC-71 and A4573 cells were established by plating either 2 × 104 cells per well in 96-well tissue culture plates (for caspase activity determinations) or 2 × 105 cells per well in six-well plates (for apoptosis assays). After overnight incubation, cells were treated for 24 hours with either 10 μmol/L Seliciclib (Roscovitine), 5 μg/mL cisplatin (as a positive inducer of caspase-3–dependent apoptosis), or DMSO vehicle (as the negative control), each in the presence or absence of the Ac-DEVD-CHO caspase-3/7 inhibitor at a 20 μmol/L final concentration. All treatments were done in triplicate. Following treatment, the extent of apoptosis induction was determined as described above, and caspase-3/7 activity determinations were carried out using the Apo-ONE Homogeneous Caspase-3/7 Assay following the manufacturer's protocol. Briefly, once the reagent and the cell culture plates had been equilibrated to room temperature, an equal volume of reagent was added directly to the cell cultures, the plates were shaken at 500 rpm, and the fluorescent output was determined 8 hours after adding the reagent in a fluorescent plate reader with a 485/535 excitation/emission filter and a gain setting of 25. Cytotoxicity assays [5] Subconfluent cells were trypsinized and seeded into 96-well tissue culture plates in 100 μl of medium. After overnight incubation, the medium was aspirated from the adherent cells, and fresh medium with predetermined drug concentrations, made fresh from stock solutions, was added. Doxorubicin stock solution was in sterile distilled water at 10 mM. Seliciclib (Roscovitine) was prepared in DMSO. Doxorubicin at 500 nM and seliciclib at 20 μM were used as single agents, or given sequentially at 24-hr intervals or given in combination. Cells were exposed to drugs for 72 hr. Cell survival was determined in quadruple wells for each drug concentration using the MTT assay as follows: to each well was added 50 μl of a 2 mg/ml solution of MTT in PBS. The plates were returned to 37°C, 5% CO2 for 4 hr. The media was carefully removed from each well and 50 μl DMSO added and the OD540 were determined using a microplate reader. Cells treated with media only served as the control for 100% cell survival. Cell cycle analysis [5] For cell cycle profile analysis, cells were seeded into 150-mm plates and grown under standard conditions. Subconfluent cultures were exposed to doxorubicin at 500 nM and Seliciclib (Roscovitine) at 20 μM as single agents or given sequentially (Seliciclib (Roscovitine) followed by doxorubicin) at a 24-hr interval. Cells were harvested after 48, 72 or 96 hr treatment and analyzed through the incorporation of BrdUrd (bromodeoxyuridine) followed by propidium iodide staining. At each time point, cells were labeled with 30 uM BrdUrd diluted in DMEM (10% FCS, 1% P/S) medium for 15–20 min at 37°C. Extracted media was retained, cells were washed and supernatant was also retained. Cells were trypsinized (5% trypsin, 2% EDTA) and added together with retained media and wash. PBS was added and cells were pelleted at 1,200 rpm for 5 min. These MCF7 were resuspended in 1-ml PBS and 3-ml ETOH, which was added dropwise while vortexing and incubated overnight at 4°C. Cells were pelleted by centrifugation at 2,500 rpm for 5 min, a 2 ml of a freshly made pepsin solution (1 mg/ml in 30 mM HCl pH 1.5) added and cells mixed for 30 min at 37°C. The cells were again pelleted by centrifugation, and 1-ml of 2M HCl was added to each sample and incubated for 20 min at room temperature. The MCF7 were resuspended in 200 ul of Becton Dickinson anti-BrdUrd antibody diluted 1:50 in antibody buffer (PBS, 0.5% BSA, 0.5% Tween 20) and incubated for 1 hr at room temperature. After washing in PBS, cells were incubated for 30 min in the dark at room temperature in 200-ul of FITC-conjugated anti-mouse antibody diluted to 20 ug/ml in antibody buffer. Finally, cells were washed in PBS, resuspended in 500 ul PBS containing 25 ug/ml propidium iodide counter stain and kept on ice in the dark until analyzed. |
| Animal Protocol |
Rats: Male Sprague Dawley rats (6–8 weeks old) receive a single intraperitoneal injection of either citrate buffer (non-diabetic) or 0.1 M citrate buffer pH 4.5 (diabetic) diluted with streptozotocin (65 mg/kg). Three days following the injection, the glucose oxidase method is used on a glucose analyzer to measure plasma glucose concentrations. The study includes rats that are classified as diabetics if their blood glucose level is greater than 16.7 mM. The level of plasma glucose is measured once a week. Seliciclib (Roscovitine) (25 mg/kg) is injected intraperitoneally into diabetic rats once a day until they are sacrificed in order to study the impact of CDK5 inhibition on renal tubulointerstitial fibrosis. As controls, DMSO is used. Mice: Subcutaneous injections of exponentially growing UMSCC47 cells are made into the sacral region of female NUDE mice. Each mouse is inoculated with 2×105 cells in 50% matrigel and 50% PBS at a volume of 100 μL. The mice receive intraperitoneal injections of either vehicle or Seliciclib (Roscovitine) at a dose of 16.5 mg/kg once the tumors have grown to a detectable size. General behavior, tumor growth, and body weight are tracked. Every three days, tumor volumes are measured. Once the tumor grows larger than 0.5 cm3, the mice are killed. In vivo studies. [4] Mice were inoculated s.c. into the right posterior flank with 4 × 106 A4573 cells in 100 μL of Matrigel basement membrane matrix. Xenografts were grown to a mean tumor volume of 129 ± 30 mm3. Seliciclib (Roscovitine) was first dissolved in either absolute methanol or DMSO (1 volume). A carrier solution was produced by using a diluent containing 10% Tween 80, 20% N-N-dimethylacetamide, and 70% polyethylene glycol 400. Mice were randomized into two groups (six animals per group) and treatment was initiated. One group was treated with Seliciclib (Roscovitine), administered as a single daily i.p. injection, at a dose of 50 mg/kg, for either 5 days or two 5-day series with a 2-day break in between. The control group received i.p. injections of the carrier solution following identical schedules. All mice were sacrificed by asphyxiation with CO2. Seliciclib (Roscovitine)-treated mice were euthanized either 7 days after the first injection or up to 4 weeks after completion of the treatment. At those times, tumors were removed, measured, and prepared for TUNEL assays. Primary tumor volumes were calculated by the formula V = (1/2)a × b2, where a is the longest tumor axis and b is the shortest tumor axis. Data are given as mean values ± SE in quantitative experiments. Statistical analysis of differences between groups was done by a one-way ANOVA followed by an unpaired Student's t test.[4] MCF7 xenografts [5] Xenograft studies with MCF7 were carried out under license 60/3045 in accordance with the guidelines of the UKCCCR. Female nude (nu/nu) mice were implanted with 17β-estradiol pellets (0.72 mg/pellet) at least 2 days before injection of the estrogen receptor positive MCF7 cells. Mice were injected subcutaneously in both flanks with 1 × 108 MCF7 cells in DMEM and matrigel suspension. The mice were housed under aseptic conditions in individually ventilated cages in a temperature (24°C) and light-controlled (12 hr/day) environment. Doxorubicin and Seliciclib (Roscovitine) preparation for xenograft studies [5] Doxorubicin was prepared in H2O and kept at 4°C for up to 1 month. Seliciclib (Roscovitine) was dissolved in PEG400:DMSO at 90:10, sonicated for 30 min and kept at 4°C. Fresh Seliciclib (Roscovitine) preparations were made each week. Treatment regime [5] Based on previous tests, Seliciclib (Roscovitine) at a concentration of 400 mg/kg was selected for this study. When tumors were in the range 50–150 mm3, mice were divided into 4 groups of 10 animals, and the combination of Seliciclib (Roscovitine) at 400 mg/kg (administered via orogastric intubation) and doxorubicin at 1.5 mg/kg (equivalent to the clinical dose; intraperitoneal injection) was tested by 1 schedule per group (Table I) repeated every week for 3 weeks. |
| ADME/Pharmacokinetics |
Pharmacokinetics [6] Plasma samples were collected in all participants at various times during day 1, up to 12 h, and at day 5 (120 h), and stored at -80°C until LC-MS/MS determination of the concentrations of roscovitine and its M3 metabolite. Global roscovitine and M3 pharmacokinetics data are presented in Fig. 4 and Fig. 5 Large variations were observed among subjects treated with the same amounts of roscovitine. A more detailed analysis, taking cytochrome P450 polymorphism and other factors into account has been published [20]. The pharmacokinetics analysis was carried out as non-compartmental analysis using WinNonlin® software. The following parameters were determined for both roscovitine (Table S19) and M3 metabolite (Table S20): Area Under Curve (AUCt and AUCInf), maximum concentration (Cmax), time to reach Cmax (Tmax) and half-life (t1/2). Pharmacokinetics parameters are summarized by number of observations, means, standard deviation (SD) and standard deviation of the mean (SEM), median, minimum and maximum for each treatment group (Table S21). The maximum roscovitine concentrations were 10.6-344 ng/mL, 54.2-1,533 ng/mL and 307-3,783 ng/mL, for the 200, 400 and 800 mg groups, respectively. Peak concentrations were observed between 1 and 4 h for the two first groups and between 2 and 6 h for the 800 mg group. Exposures were 43.5-3,385 ng.h/mL, 344-20,210 ng.h/mL and 1,767-60,437 ng.h/mL, respectively for the three groups (Fig. 4, Table S19). The maximum M3 concentrations were 87.6-1,600 ng.h/mL, 62.2-2,811 ng.h/mL and 754-4,190 ng/mL, respectively, for the 200, 400 and 800 mg groups. Peak concentrations were observed between 1 and 2 h for the first groups and between 1 and 4 h for the 400 and 800 mg group. Exposures were 270-8,294 ng.h/mL, 262-35,114 ng.h/mL and 5,881-116,512 ng.h/mL, respectively for the three groups (Fig. 4, Table S20). For both roscovitine and M3, the increases in AUCt and Cmax strongly correlated with the administered doses (Fig. 4, Tables S19 and S20). The M3/ roscovitine ratios for median AUCt and Cmax were getting close to 1 as the dose was increased (Fig. 4, Table S21). |
| Toxicity/Toxicokinetics |
Safety evaluation [6] The primary outcome was safety evaluation (Tables 1, 2, S6–S8). All subjects presented at least one adverse event (AE) during the ROSCO-CF study. Sixty AEs were reported among the 11 subjects receiving placebo and 132 AEs for the 23 subjects receiving roscovitine (Tables 1–3, S6–S11). Median numbers of AEs were 5 (placebo), 2 (200 mg), 8 (400 mg) and 5 (800 mg). The overall AE rate was 5.46 AE/subject in the placebo group and 5.74 AE/subject in the roscovitine groups. The distribution of the highest-grade AEs per subject is presented in Table S9A. No significant difference in the prevalence of AEs between the two experimental groups was observed. Among 34 subjects, 5 presented an SAE: 0/11 (placebo), 1/8 (200 mg), 1/8 (400 mg) and 3/7 (800 mg) (Table S9B). Adverse events (serious and non-serious) [6] AEs were grouped according to clinical reaction type using the MedDRA 21.1 classification dictionary (Table 1). Gastrointestinal disorders, infections & infestations and respiratory, thoracic and mediastinal disorders were the three most frequently reported AEs. Cardiac, eye, hepatobiliary and musculoskeletal disorders were reported more frequently in participants receiving roscovitine versus placebo. No “cardiac events” were reported in the placebo group but one in roscovitine group 2 and two in roscovitine group 3. One of them, “sinus tachycardia”, was reported as an SAE. Tachycardia and sinus tachycardia are frequent clinical reactions seen in many clinical diseases. For “eye disorders”, there was only one “non-serious AE” reported in roscovitine group 2. There were six reported “hepatobiliary disorders” events in roscovitine group 2 and three in roscovitine group 3. The observed “musculoskeletal disorders” reactions were non-specific, like myalgia, pain and arthralgia. One “renal disorder” and one dysmenorrheal (in reproductive disorders) were reported, only in roscovitine group 3. Severity of adverse events [6] The level of severity of all AEs was qualified by investigators with specific severity scales (Tables S6–S8). In the placebo group, 46 grade 1 AEs and 14 grade 2 AEs were reported. In the roscovitine groups, 95 grade 1 AEs, 35 grade 2 AEs and 2 grade 3 AEs were reported. The distribution of AEs relative to severity in the three roscovitine groups is presented in details in Tables S6-S8 and summarized in Table S9A. Expectedness of adverse events [6] The latest Seliciclib investigator brochure was used to qualify the expectedness of AEs. Some unlisted AEs were reported in the roscovitine groups: one photophobia and one visual impairment, reported as a non-serious AE. The CF status can be considered as a confounding factor for infectious diseases such as gastroenteritis, infective pulmonary exacerbation of CF, nasopharyngitis, oral herpes, pharyngitis, rhinitis tonsillitis, and viral infection. The electrocardiogram T wave inversion data was sent to health authorities as a Suspected Unexpected Serious Adverse Reaction (SUSAR). Cardiologic evaluation of pre-dose and post-dose ECG provided other episodes of T wave inversion in this subject. The case of “blood creatine phosphokinase increased” could be drawn close to unspecific musculoskeletal disorders as myalgia and arthralgia. Serious adverse reactions [6] All participants who presented at least one SAE were roscovitine-treated. Globally, 5 subjects presented a total of 8 SAEs (Table 2). Three of 5 subjects have presented 2 SAEs at the same date. The distribution of SAEs among clinical type of reaction showed that hepatobiliary events and infectious diseases were presented by more than one subject (Table 2). The distribution among roscovitine dose-escalating groups showed that subjects treated with high dosage presented more SAEs (Table 2). The independent data safety monitoring board considered that 3 of the SAEs on hepatobiliary disorders were possibly related to roscovitine treatment (2 at 400 mg and 1 at 800 mg), 1 on cardiac disorders and 1 in renal and urinary disorders were also related to roscovitine treatment (800 mg) (Table S10). |
| References |
[1]. Eur J Biochem. 1997 Jan 15;243(1-2):527-36. [2]. J Clin Invest. 1997 Nov 15;100(10):2512-20. [3]. J Cell Biochem. 2012 Mar;113(3):868-76. [4]. Cancer Res. 2005 Oct 15;65(20):9320-7. [5]. Int J Cancer. 2009 Jan 15;124(2):465-72. [6]. J Cyst Fibros. 2022 May;21(3):529-536. |
| Additional Infomation |
Seliciclib is 2,6-Diaminopurine carrying benzylamino, (2R)-1-hydroxybutan-2-yl and isopropyl substituents at C-6, C-2-N and N-9 respectively. It is an experimental drug candidate in the family of pharmacological cyclin-dependent kinase (CDK) inhibitors. It has a role as an EC 2.7.11.22 (cyclin-dependent kinase) inhibitor and an antiviral drug. R-roscovitine (Seliciclib or CYC202) is a cyclin-dependent kinase (CDK) inhibitor that preferentially inhibits multiple enzyme targets including CDK2, CDK7 and CDK9, which alter the growth phase of treated cells. Developed by Cyclacel, seliciclib is being researched for the treatment of non-small cell lung cancer (NSCLC), leukemia, HIV infection, herpes simplex infection, and the mechanisms of chronic inflammation disorders. Seliciclib has been reported in Ophioparma ventosa with data available. Seliciclib is an orally available small molecule and cyclin-dependent kinase (CDK) inhibitor with potential apoptotic and antineoplastic activity. CDKs, serine/threonine kinases that play an important role in cell cycle regulation, are overexpressed in various malignancies. Seliciclib primarily inhibits CDK 2, 7, and 9 by competing for the ATP binding sites on these kinases, leading to a disruption of cell cycle progression. In addition, this agent seems to interfere with CDK-mediated phosphorylation of the carboxy-terminal domain of RNA polymerase II, thereby inhibiting RNA polymerase II-dependent transcription. This may lead to the down-regulation of anti-apoptotic factors, such as myeloid cell leukemia sequence 1 (Mcl-1), a protein crucial for the survival of a range of tumor cell types. The down-regulation of anti-apoptotic factors may lead to an induction of apoptosis, thereby further contributing to seliciclib's antiproliferative effects. A purine derivative and competitive inhibitor of CYCLIN-DEPENDENT KINASES that has therapeutic potential as an antineoplastic and antiviral agent. Drug Indication Investigated for use/treatment in breast cancer, lung cancer, lymphoma (unspecified), multiple myeloma, leukemia (lymphoid), and cancer/tumors (unspecified). Cyclin-dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin-dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N6, N9-substituted adenines tested on purified cdc2/cyclin B, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine) displays high efficiency and high selectivity towards some cyclin-dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light-chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c-src, v-abl). Most kinases are not significantly inhibited by roscovitine. cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35 only are substantially inhibited (IC50 values of 0.65, 0.7, 0.7 and 0.2 microM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC50 > 100 microM). Extracellular regulated kinases erk1 and erk2 are inhibited with an IC50 of 34 microM and 14 microM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late prophase. Roscovitine inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts. It blocks progesterone-induced oocyte maturation of Xenopus oocytes and in vivo phosphorylation of the elongation factor eEF-1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC50 of 16 microM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2. In vivo phosphorylation of vimentin on Ser55 by cdc2/cyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin-dependent kinases, roscovitine provides a useful antimitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.[1] Glomerular injury is characterized by mesangial cell (MC) proliferation and matrix formation. We sought to determine if reducing the activity of cyclin-dependent kinase 2 (CDK2) with the purine analogue, Roscovitine, decreased MC proliferation in vitro and in vivo. Roscovitine (25 microM) inhibited FCS-induced proliferation (P < 0.0001) in cultured MC. Rats with experimental mesangial proliferative glomerulonephritis (Thy1 model) were divided into two groups. A prevention group received daily intraperitoneal injections of Roscovitine in DMSO (2.8 mg/kg) starting at day 1. A treatment group received daily Roscovitine starting at day 3, when MC proliferation was established. Control Thy1 rats received DMSO alone. MC proliferation (PCNA +/OX7 + double immunostaining) was reduced by > 50% at days 5 and 10 in the Roscovitine prevention group, and at day 5 in the treatment group (P < 0.0001). Early administration of Roscovitine reduced immunostaining for collagen type IV, laminin, and fibronectin at days 5 and 10 (r = 0.984; P < 0.001), which was associated with improved renal function (urinary protein/creatinine, blood urea nitrogen, P < 0.05). We conclude that reducing the activity of CDK2 with Roscovitine in experimental glomerulonephritis decreases cell proliferation and matrix production, resulting in improved renal function, and may be a useful therapeutic intervention in disease characterized by proliferation. [2] Roscovitine, a cyclin-dependent kinase (Cdk) inhibitor, inhibited kinase activity and the axenic growth of Dictyostelium discoideum at micromolar concentrations. Growth was almost fully rescued in 50 µM and ≈ 50% rescued in 100 µM roscovitine-treated cultures by the over-expression of Cdk5-GFP. This supports the importance of Cdk5 function during cell proliferation in Dictyostelium and indicates that Cdk5 is a primary target of the drug. Roscovitine did not affect the expression of Cdk5 protein during axenic growth but did inhibit its nuclear translocation. This novel result suggests that the effects of roscovitine could be due in part to altering Cdk5 translocation in other systems as well. Kinase activity was inhibited by roscovitine in assays using AX3 whole cell lysates, but not in assays using lysates from Cdk5-GFP over-expressing cells. At higher concentrations, roscovitine impaired slug and fruiting body formation. Fruiting bodies that did form were small and produced relatively fewer spores many of which were round. However, roscovitine did not affect stalk cell differentiation. Together with previous findings, these data reveal that roscovitine inhibits Cdk5 during growth and as yet undefined Cdks during mid-late development.[3] The Ewing's sarcoma family of tumors (ESFT) comprises several well-characterized malignant neoplasms with particularly aggressive behavior. Despite recent progress in the use of multimodal therapeutic approaches and aggressive local control measures, a substantial proportion of patients die because of disease progression. Furthermore, this outcome has not changed significantly over the last 15 to 20 years. Consequently, new, more effective therapeutic options are sorely needed for the treatment of ESFT. Because ESFT cells overexpress several cyclin-dependent kinases (CDK), we explored the efficacy against ESFT of roscovitine, a CDK inhibitor shown to be surprisingly safe for humans in clinical trials of their anticancer activity. Results showed that ESFT cell lines are uniformly sensitive to roscovitine. In addition to exerting comparatively minor cell cycle effects, roscovitine treatment concomitantly caused the up-regulation of the expression of the proapoptotic protein BAX and the down-regulation of both survivin and XIAP, thus resulting in caspase-dependent apoptosis. Furthermore, in vivo experiments showed that s.c. growth of ESFT xenografts was also significantly slowed by i.p. injection of roscovitine. These results strongly suggest that roscovitine may be an effective therapeutic agent against ESFT and recommend its evaluation against ESFT in clinical trials and its inclusion in future treatment protocols.[4] We sought to determine whether seliciclib (CYC202, R-roscovitine) could increase the antitumor effects of doxorubicin, with no increase in toxicity, in an MCF7 breast cancer xenograft model. The efficacy of seliciclib combined with doxorubicin was compared with single agent doxorubicin or seliciclib administered to MCF7 cells and to nude mice bearing established MCF7 xenografts. Post-treatment cells and tumors were examined by cell cycle analysis, immunohistochemistry and real-time PCR. Seliciclib significantly enhanced the antitumor effect of doxorubicin without additional murine toxicity. MIB1 (ki67) immunohistochemistry demonstrated reduced proliferation with treatment. The levels of p21 and p27 increased after treatment with doxorubicin or seliciclib alone or in combination, compared to untreated controls. However, no changes in p53 protein (DO1, CM1), survivin or p53 phosphorylation (SER15) were observed in treated tumors compared with controls. In conclusion, the CDK inhibitor seliciclib (R-roscovitine) enhances the antitumor effect of doxorubicin in MCF7 tumors without increased toxicity with a mechanism that involves cell cycle arrest rather than apoptosis.[5] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.05 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.05 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.5 mg/mL (7.05 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. Solubility in Formulation 4: ≥ 2.5 mg/mL (7.05 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 5: ≥ 2.5 mg/mL (7.05 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. 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. Solubility in Formulation 6: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8213 mL | 14.1064 mL | 28.2127 mL | |
| 5 mM | 0.5643 mL | 2.8213 mL | 5.6425 mL | |
| 10 mM | 0.2821 mL | 1.4106 mL | 2.8213 mL |