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)

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]

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]

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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]

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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]

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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.

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, p34^cdc2/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 p33^cdk2/cyclin A and p33^cdk2/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 p34^cdk2/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 p33^cdk5/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 p34^cdk2/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 p33^cdk5/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 p33^cdk4/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 p34^cdc2cyclin 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 p34^cdc2/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 p34^cdc2/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 p34^cdc2/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 p34^cdc2/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 p34^cdc2/cyclin B kinase, the phosphorylated Raytide is recovered on Whatman P81 phosphocellulose paper.

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.

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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.

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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.

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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.

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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.

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.

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\nMice: 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.\n

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\n\nIn vivo studies. [4]
\nMice 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]

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\n\nMCF7 xenografts [5]
\nXenograft 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.

\n\nDoxorubicin and Seliciclib (Roscovitine) preparation for xenograft studies [5]
\nDoxorubicin 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.\n

\nTreatment regime [5]
\nBased 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.\n\n

Pharmacokinetics [6] Plasma samples were collected from all subjects at different time points on day 1 (up to 12 hours) and on day 5 (120 hours) and stored at -80°C until the concentrations of roscovitine and its M3 metabolite were determined by LC-MS/MS. Overall pharmacokinetic data for roscovitine and M3 are shown in Figures 4 and 5, respectively. Significant differences were observed among subjects even when treated with the same dose of roscovitine. A more detailed analysis considering cytochrome P450 polymorphism and other factors has been published [20]. Pharmacokinetic analysis was performed using a non-compartmental model using WinNonlin® software. The following parameters were determined for roscovitine (Table S19) and its metabolite M3 (Table S20): area under the curve (AUCt and AUCInf), maximum concentration (Cmax), time to peak concentration (Tmax), and half-life (t1/2). The pharmacokinetic parameters are summarized below: number of observations, mean, standard deviation (SD), standard deviation of mean (SEM), median, minimum, and maximum values for each treatment group (Table S21). The maximum concentrations of roscovetin in the 200 mg, 400 mg, and 800 mg groups were 10.6–344 ng/mL, 54.2–1,533 ng/mL, and 307–3,783 ng/mL, respectively. Peak concentrations in the first two groups occurred between 1 and 4 hours, while the peak concentration in the 800 mg group occurred between 2 and 6 hours. The exposures for the three groups were 43.5–3,385 ng·h/mL, 344–20,210 ng·h/mL, and 1,767–60,437 ng·h/mL, respectively (Figure 4, Table S19). The maximum concentrations of M3 in the 200 mg, 400 mg, and 800 mg groups were 87.6–1,600 ng·h/mL, 62.2–2,811 ng·h/mL, and 754–4,190 ng/mL, respectively. Peak concentrations in the first two groups occurred between 1 and 2 hours, while peak concentrations in the 400 mg and 800 mg groups occurred between 1 and 4 hours. The exposures for the three groups were 270–8,294 ng·h/mL, 262–35,114 ng·h/mL, and 5,881–116,512 ng·h/mL, respectively (Figure 4, Table S20). For both roscovitine and M3, increases in AUCt and Cmax were significantly positively correlated with the administered dose (Figure 4, Tables S19 and S20). As the dose increases, the median AUCt and Cmax ratio of M3/roscovetin approaches 1 (Figure 4, Table S21).

Safety assessment [6]
The primary endpoint was safety assessment (Tables 1, 2, S6-S8). All subjects experienced at least one adverse event (AE) during the ROSCO-CF study. A total of 60 AEs were reported in the 11 subjects receiving placebo, and a total of 132 AEs were reported in the 23 subjects receiving roscovitine (Tables 1-3, S6-S11). The median number of AEs in each group was 5 in the placebo group, 2 in the 200 mg group, 8 in the 400 mg group, and 5 in the 800 mg group. The overall AE rate was 5.46 per subject in the placebo group and 5.74 per subject in the roscovitine group. The distribution of the highest grade AE for each subject is shown in Table S9A. There was no significant difference in the incidence of AEs between the two experimental groups. Of the 34 participants, 5 experienced serious adverse events (SAEs): 0/11 in the placebo group, 1/8 in the 200 mg group, 1/8 in the 400 mg group, and 3/7 in the 800 mg group (Table S9B).

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Adverse events (serious and non-serious)[6]
Adverse events were grouped according to the type of clinical response using the MedDRA 21.1 classification dictionary (Table 1). Gastrointestinal disorders, infections and invasiveness, and respiratory, thoracic, and mediastinal disorders were the three most frequently reported adverse events. Participants receiving roscovetin reported cardiac, ocular, hepatobiliary, and musculoskeletal disorders more frequently than those in the placebo group. No “cardiac events” were reported in the placebo group, 1 in roscovetin group 2, and 2 in roscovetin group 3. One of these “sinus tachycardia” was reported as a serious adverse event. Tachycardia and sinus tachycardia are common clinical responses in many clinical disorders. Regarding “ocular disorders,” only one “non-serious adverse event” was reported in roscovetin group 2. Six “hepatobiliary disease” events were reported in Roskovitin group 2 and three in Roskovitin group 3. The observed “musculoskeletal disease” reactions were nonspecific reactions, such as myalgia, pain and arthralgia. Only one “kidney disease” and one dysmenorrhea (reproductive system disease) were reported in Roskovitin group 3.

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Adverse event severity[6]
The severity of all adverse events was assessed by the investigator using specific severity grading criteria (Tables S6-S8). 46 Grade 1 adverse events and 14 Grade 2 adverse events were reported in the placebo group. In the Roskovitin group, 95 Grade 1 adverse events, 35 Grade 2 adverse events and 2 Grade 3 adverse events were reported. The distribution of adverse event severity in the three Roskovitin groups is detailed in Tables S6-S8 and summarized in Table S9A.

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Expectancy of adverse events[6]
Expectancy of adverse events was assessed using the latest Celicillin Investigator’s Manual. Several unlisted adverse events were reported in the Roskovitin group: one case of photophobia and one case of visual impairment, both reported as non-serious adverse events. Cystic fibrosis (CF) status can be considered a confounding factor for infectious diseases such as gastroenteritis, acute exacerbation of CF-infected lung, nasopharyngitis, oral herpes, pharyngitis, rhinitis, tonsillitis, and viral infections. ECG T-wave inversion data were reported to the health department as suspected unexpected serious adverse reactions (SUSAR). ECG evaluations of the subject before and after administration revealed other T-wave inversions. Cases of “elevated serum creatine phosphokinase” may be associated with nonspecific musculoskeletal disorders such as myalgia and arthralgia. Serious adverse reactions [6] All subjects who experienced at least one serious adverse reaction received Roskovitin. Overall, a total of 8 serious adverse reactions occurred in 5 subjects (Table 2). Three of these subjects experienced two serious adverse reactions on the same day. The clinical distribution of serious adverse events showed that the incidence of hepatobiliary events and infectious diseases was higher in subjects than in other subjects (Table 2). The distribution of roscovetin dose escalation groups showed that subjects in the high-dose group experienced more serious adverse events (Table 2). The independent data safety monitoring committee considered that 3 serious adverse events of hepatobiliary disease may have been associated with roscovetin treatment (2 at 400 mg and 1 at 800 mg), and 1 case of cardiac disease and 1 case of renal and urinary system disease were also associated with roscovetin treatment (800 mg) (Table S10).

[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.

Seliciclib is a 2,6-diaminopurine with benzylamino, (2R)-1-hydroxybutyl-2-yl, and isopropyl substituents at C-6, C-2-N, and N-9, respectively. It is an experimental drug candidate in the cyclin-dependent kinase (CDK) inhibitor family. It is both 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, thereby altering the cell cycle. Developed by Cyclacel, seliciclib is being investigated for its mechanism of action in treating non-small cell lung cancer (NSCLC), leukemia, HIV infection, herpes simplex virus infection, and chronic inflammatory diseases.
Seliciclib has been reported to exist in Ophioparma ventosa, and relevant data exist.
Seliciclib is an orally administered small-molecule cyclin-dependent kinase (CDK) inhibitor with potential pro-apoptotic and anti-tumor activities. CDKs are serine/threonine kinases that play an important role in cell cycle regulation and are overexpressed in various malignant tumors. Seliciclib primarily inhibits these kinases by competing with CDK 2, 7, and 9 for ATP binding sites, thereby blocking cell cycle progression. Furthermore, the drug appears to interfere with CDK-mediated phosphorylation of the C-terminal domain of RNA polymerase II, thereby inhibiting RNA polymerase II-dependent transcription. This may lead to the downregulation of anti-apoptotic factors such as myeloid leukemia sequence 1 (Mcl-1), a protein crucial for the survival of various tumor cell types. The downregulation of anti-apoptotic factors may induce apoptosis, further enhancing the anti-proliferative effect of seliciclib.
A purine derivative, a competitive inhibitor of cyclin-dependent kinases (CDKs), with therapeutic potential as an antitumor and antiviral drug.

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Drug Indications
Investigated for the treatment of breast cancer, lung cancer, lymphoma (not specified), multiple myeloma, lymphocytic leukemia, and cancer/tumor (not specified).

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Cycin-dependent kinases (CDKs) play a crucial role in the intracellular regulation of the cell cycle (CDC). These kinases and their regulators are frequently dysregulated in human tumors. Enzyme screening has recently identified several specific inhibitors of CDKs, such as butyrolactone I, flavonoid pyridinol, and the purine compound olomoxin. Among a series of C2, N6, N9-substituted adenine compounds targeting purified cdc2/cyclin B, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roskovitin) exhibits high efficiency and selectivity against certain CDKs. The kinase specificity of roskovitin was investigated using 25 high-purity kinases, including protein kinases A, G, and C isoforms, myosin light chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c-src, and v-abl. The results showed that roskovitin did not significantly inhibit most kinases. Only cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E, and cdk5/p35 were significantly inhibited (IC50 values of 0.65, 0.7, 0.7, and 0.2 μM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 were weakly inhibited by roskovitin (IC50 > 100 μM). Extracellular signal-regulated kinases erk1 and erk2 were inhibited, with IC50 values of 34 μM and 14 μM, respectively. Roskovitin reversibly arrested starfish oocytes and sea urchin embryos in the pre-late stage. Roskovine inhibits the activity of M phase promoting factors in vitro and DNA synthesis in Xenopus oocyte extract. It blocks progesterone-induced maturation of Xenopus oocytes and phosphorylation of eEF-1 in vivo. Roskovine inhibits the proliferation of mammalian cell lines with a mean IC50 of 16 μM. In the presence of roskovine, L1210 cells arrest in the G1 phase and accumulate in the G2 phase. Roskovine inhibits in vivo phosphorylation of vimentin Ser55 by cdc2/cyclin B. Due to its unique selectivity for certain cyclin-dependent kinases, roskovine could be a useful antimitotic agent for cell cycle studies and may have potential value in controlling cells with dysregulated cdc2, cdk2 or cdk5 kinase activity. [1]

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Glomerular injury is characterized by mesangial cell (MC) proliferation and matrix formation. We aimed to determine whether the use of the purine analogue roscovitine to reduce cyclin-dependent kinase 2 (CDK2) activity could decrease mesangial cell proliferation in vitro and in vivo. Roscovitine (25 μM) inhibited fetal bovine serum (FCS)-induced proliferation of cultured mesangial cells (P < 0.0001). Experimental mesangial proliferative glomerulonephritis (Thy1 model) rats were divided into two groups. The prevention group received daily intraperitoneal injections of roscovitine (2.8 mg/kg) dissolved in dimethyl sulfoxide (DMSO) starting from day 1. The treatment group received daily roscovitine injections starting from day 3, when mesangial cell proliferation was established. The control group of Thy1 rats received DMSO injections only. In the roscovitine prevention group, MC proliferation (PCNA+/OX7+ double immunostaining) was reduced by more than 50% on days 5 and 10; in the treatment group, MC proliferation was also reduced by more than 50% on day 5 (P < 0.0001). Early administration of Roscovitine reduced the immunostaining intensity of type IV collagen, laminin, and fibronectin on days 5 and 10 (r = 0.984; P < 0.001), which was associated with improved renal function (urine protein/creatinine ratio, blood urea nitrogen, P < 0.05). We conclude that in experimental glomerulonephritis, the use of Roscovitine to reduce CDK2 activity can reduce cell proliferation and matrix production, thereby improving renal function, which may be an effective treatment for diseases characterized by proliferation. [2]

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Roscovitine is a cyclin-dependent kinase (Cdk) inhibitor that inhibits kinase activity and aseptic culture growth of Dendrobium distichum at micromolar concentrations. In cultures treated with 50 µM Roscovitine, cell growth was almost completely restored by overexpression of Cdk5-GFP; in cultures treated with 100 µM Roscovitine, cell growth was restored by about 50%. This supports the view that Cdk5 plays an important role in the proliferation of Dendrobium distichum cells and indicates that Cdk5 is the primary target of this drug. Roscovitine does not affect the expression of Cdk5 protein during aseptic culture, but it inhibits its nuclear translocation. This new result suggests that the effect of roscovitine may be partly attributed to its alteration of Cdk5 nuclear translocation in other systems. In experiments using AX3 whole-cell lysates, roscovitine inhibited kinase activity, but this was not observed in experiments using lysates of Cdk5-GFP overexpressing cells. At high concentrations, roscovitine inhibits fruiting bodies and their formation. The formed fruiting bodies are small, produce relatively few spores, and many spores are round. However, roscovitine does not affect the differentiation of stalk cells. Combined with previous findings, these data suggest that roscovitine inhibits Cdk5 during growth and suppresses the undefined Cdks in the later stages of development. [3]

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Ewing's sarcoma family tumors (ESFT) comprise several well-defined malignancies that are particularly aggressive. Despite recent advances in multimodal treatments and aggressive local control measures, a significant proportion of patients still die from disease progression. Moreover, this outcome has not significantly improved over the past 15 to 20 years. Therefore, there is an urgent need for new and more effective treatment options for Ewing's sarcoma/primary osteosarcoma (ESFT). Since ESFT cells overexpress multiple cyclin-dependent kinases (CDKs), we explored the efficacy of roscovitine for ESFT. Roscovitine is a CDK inhibitor that has shown unexpected safety in humans in clinical trials of its anticancer activity. The results showed that all ESFT cell lines were sensitive to roscovitine. Roscovitine treatment, in addition to having a relatively small effect on the cell cycle, also led to upregulation of the pro-apoptotic protein BAX and downregulation of survivin and XIAP, thereby inducing caspase-dependent apoptosis. In addition, in vivo experiments showed that intraperitoneal injection of roskovetin also significantly slowed the subcutaneous growth of ESFT xenografts. These results strongly suggest that roskovetin may be an effective treatment for ESFT and recommend that its efficacy for ESFT be evaluated in clinical trials and included in future treatment regimens. [4]

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We aimed to determine whether celicillin (CYC202, R-roskovetin) could enhance the antitumor effect of doxorubicin in an MCF7 breast cancer xenograft model without increasing toxicity. We compared the efficacy of celicillin in combination with doxorubicin versus doxorubicin alone or celicillin in treating MCF7 cells and nude mice carrying established MCF7 xenografts. Cell cycle analysis, immunohistochemistry, and real-time PCR were used to detect cells and tumors after treatment. Celicillin significantly enhanced the antitumor effect of doxorubicin without increasing toxicity in mice. MIB1 (Ki67) immunohistochemical staining showed reduced cell proliferation after treatment. Compared with the untreated control group, the levels of p21 and p27 were increased after treatment with doxorubicin alone or seliciclib, or in combination. However, compared with the control group, there were no changes in p53 protein (DO1, CM1), survivin, or p53 phosphorylation (SER15) in the tumors of the treatment group. In conclusion, the CDK inhibitor seliciclib (R-roscovitine) enhances the antitumor effect of doxorubicin in MCF7 tumors through cell cycle arrest rather than apoptosis, without increasing toxicity. [5]

C19H26N6O

354.45

354.216

C, 64.38; H, 7.39; N, 23.71; O, 4.51

186692-46-6

160355

White to off-white solid powder

1.3±0.1 g/cm3

577.5±60.0 °C at 760 mmHg

303.1±32.9 °C

0.0±1.7 mmHg at 25°C

1.643

1.68

3

6

8

26

417

1

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]

BTIHMVBBUGXLCJ-OAHLLOKOSA-N

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

(2R)-2-[[6-(benzylamino)-9-propan-2-ylpurin-2-yl]amino]butan-1-ol

Seliciclib; R-Roscovitine; CYC-202; roscovitine; Seliciclib; 186692-46-6; R-Roscovitine; (R)-roscovitine; Roscovitin; CYC202; Roscovitin; Roscovitine; CYC202; CYC 202

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 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.

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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.

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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.

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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.

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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.

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Solubility in Formulation 6: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30 mg/mL

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