CDK1 ≤1 μM (IC50) CDK2 ≤1 μM (IC50) CDK5 ≤1 μM (IC50) CDK7 ≤1 μM (IC50) CDK9 ≤1 μM (IC50)

Ischemic stroke is the second leading cause of death worldwide. Following ischemic stroke, Neurovascular Unit (NVU) inflammation and peripheral leucocytes infiltration are major contributors to the extension of brain lesions. For a long time restricted to neurons, the 10 past years have shown the emergence of an increasing number of studies focusing on the role of Cyclin-Dependent Kinases (CDKs) on the other cells of NVU, as well as on the leucocytes. The most widely used CDKs inhibitor, (R)-roscovitine, and its (S) isomer both decreased brain lesions in models of global and focal cerebral ischemia. We previously showed that (S)-roscovitine acted, at least, by modulating NVU response to ischemia. Interestingly, roscovitine was shown to decrease leucocytes-mediated inflammation in several inflammatory models. Specific inhibition of roscovitine majors target CDK 1, 2, 5, 7, and 9 showed that these CDKs played key roles in inflammatory processes of NVU cells and leucocytes after brain lesions, including ischemic stroke. The data summarized here support the investigation of roscovitine as a potential therapeutic agent for the treatment of ischemic stroke, and provide an overview of CDK 1, 2, 5, 7, and 9 functions in brain cells and leucocytes during cerebral ischemia [1].

(S)-Roscovitine (25 mg/kg; ip; 15 min before and 1 hr after pMCAo) showed neuroprotective efficacy in an adult mouse model of permanent middle cerebral artery occlusion [2].

Immunoprecipitation and protein kinase assays [2]
Brain lysates (500 µg) were incubated with 0.8 µg of p35 antibodies. The CDK5/p35 and CDK5/p25 immunocomplexes were washed 4 times with bead buffer (50 mM Tris pH 7.4, 5 mM NaF, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% NP-40, 10 µg/ml of leupeptin, aprotinin and soybean trypsin inhibitor and 100 µM benzamidine) and one time with buffer C (60 mM β-glycerophosphate, 30 mM p-nitrophenylphosphate, 25 mM Mops (pH 7.0), 5 mM EGTA, 15 mM MgCl2, 1 mM DTT, 0.1 mM sodium vanadate). The CDK5 kinase activity was assessed directly on the beads in buffer C, with 1 mg histone H1/ml, in the presence of 15 µM [γ-33P] ATP (3,000 Ci/mmol; 10 mCi/ml) in a final volume of 30 µl. After 20 min of incubation at 30°C, 20 µl aliquots of supernatant were spotted onto 2.5×3 cm pieces of Whatman P81 phosphocellulose paper, and, 20 seconds later, the filters were washed four times (for at least 5 min each time) in a solution of 10 ml phosphoric acid/liter of water. The wet filters were counted in the presence of 1 ml ACS scintillation fluid.

Animal/Disease Models: 20-25 g, P60 male C57 b/6 mice[2]
Doses: 25 mg/kg
Route of Administration: I.p.; at 15 min before and 1 hr after pMCAo
Experimental Results: Decreased of the total infarct volume.

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Blood brain barrier permeability [2]
SD adult rats received intravenous injection of (S)-roscovitine solution (HPbCD 30% pH 7.4) at 2 doses (20 and 30 mg/kg; n = 2 rats/dose). Dosed animals were sacrificed at 15 min after the end of the intravenous injection. Blood were collected through the retro-orbital sinus using a capillary tube. Animals were then perfused with saline solution into the heart to extract the maximum blood sample from the brain. Brain was collected, and homogenized. Both plasma and brain samples were analyzed using LC/MS/MS determination, according to ADME-Bioanalysis procedures. Schemes of the experimental procedures are shown in Figures 2A, C . For experiments done at Neurokin ( Figure 2A ), (S)-roscovitine (n = 16 rats) or vehicle (n = 18 rats) was injected into the animals through a IV bolus (25 mg/kg in HPbCD 30%, pH 7.4) followed by 3 successive SC injections (54 mg/kg in saline HCl 50 mM, pH 1.5) performed 15 min before and at 24 and 29 hrs post-occlusion. For experiments performed at MDS Pharma Services ( Figure 2C ), drug (n = 11–12 rats) or vehicle (n = 13 rats) was administered through a bolus IV (25 mg/kg in HPbCD 30%, pH 7.4) followed by immediate SC infusion for 48 hrs, and performed either at 15 min prior or 2 hrs 15 min after the occlusion. Several drug doses were tested for the SC infusion (10, 5, and 1 mg/kg in saline HCl 50 mM, pH 1.5). Animal treatments were performed in a blind manner in both tMCAo studies.

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E18 mixed hippocampal cells, pharmacological treatments and evaluation of neuronal death [2]
Mixed hippocampal cell cultures were prepared from embryonic day 18 (E18) Wistar rats as previously described and grown in vitro for 10 days. Treatments were done by directly adding KA (200 µM), drugs, and/or vehicle (DMSO 0.1%) to the medium and left for 5 hrs. No significant KA-induced toxic effect was ever detected on glial cells at this concentration (date not shown). The effects of roscovitine compounds [(R)-, (S)-, N6-methyl-(R)-, or O6-(R)-roscovitine] were determined by adding different concentrations of the molecules ranging from 0.05 to 50 µM to the medium at the same time as the KA addition. The cell death marker, propidium iodide (PI; 7.5 µM), was added to the medium at 4 hrs. Neuronal death was evaluated at 5 hrs by combining phase contrast and fluorescent microscopy observations. Neurons from random and representative fields were counted at low magnification (4x). At least 5 fields per condition (number total of neurons exceeding in general 150) were examined from 3 independent cultures. For every condition in every experiment, percentage of neuronal death was expressed as the ratio between PI-positive neurons and the total number of neurons visualized by phase contrast microscopy. To assess neuroprotection, relative neuronal death (RND) was calculated in 3 independent cultures and a neuroprotection index (NI) defined as: RND = (% of neuronal death with KA/roscovitine – % of neuronal death with roscovitine)/(% of neuronal death with KA - % of neuronal death with vehicle), and NI = 1-RND. By definition, relative percentage of neuronal death (RND) in KA-treated culture was 100% and neuroprotection index (NI) was 0%.

[1]. Le Roy L, et al. Cellular and Molecular Mechanisms of R/S-Roscovitine and CDKs Related Inhibition under Both Focal and Global Cerebral Ischemia: A Focus on Neurovascular Unit and Immune Cells. Cells. 2021 Jan 8;10(1):104.

[2]. Delayed treatment with systemic (S)-roscovitine provides neuroprotection and inhibits in vivo CDK5 activity increase in animal stroke models. PLoS One. 2010 Aug 12;5(8):e12117.

(S)-Roscovitine and Infarct Size in Focal Ischemia [1]
(S)-roscovitine ICV administration to mice 48 h before pMCAo and throughout the duration of pMCAo led to a 28% decrease of infarct volume compared to vehicle-treated animals at 3 h post-occlusion. Systemic administration of (S)-roscovitine by two successive IP injections at 15 min prior and 1 h after the occlusion led to a 31% decrease of the total infarct volume at 3 h post-occlusion, showing no loss of neuroprotective effect. For both administration modes, the hypometabolic zone volume, but not the infarct core, decreased in (S)-roscovitine-treated animals compared to vehicle. Interestingly, they observed that the increase of CDK5 activity post-pMCAo was prevented by (S)-roscovitine treatment, suggesting that the beneficial effect of (S)-roscovitine was at least partly due to CDK5 inhibition. (S)-roscovitine neuroprotective efficacy was also assessed on two independents blinded studies in a tMCAo rat model. In the first study, a 90 min tMCAo rat model, (S)-roscovitine was administered by IV bolus 15 min prior to ischemia followed by three successive SC injections at 15 min prior to and 24 h and 29 h after the occlusion. (S)-roscovitine significantly decreased the infarct volume by 30% 48 h after reperfusion. In the second study, a 120 min tMCAo rat model, (S)-roscovitine was administered by IV bolus followed by continuous SC infusion performed 135 min after (post-MCA) the occlusion, leading to a significant decrease by 27% of the infarct volume. Rousselet et al. studied (S)-roscovitine effect in a randomized blind study on a tMCAo rat model. (S)-roscovitine administration 15 min post-reperfusion by IV bolus followed by 48 h SC infusion decreased infarct volume by 21%, 48 h after reperfusion. [1]

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Background: Although quite challenging, neuroprotective therapies in ischemic stroke remain an interesting strategy to counter mechanisms of ischemic injury and reduce brain tissue damage. Among potential neuroprotective drug, cyclin-dependent kinases (CDK) inhibitors represent interesting therapeutic candidates. Increasing evidence indisputably links cell cycle CDKs and CDK5 to the pathogenesis of stroke. Although recent studies have demonstrated promising neuroprotective efficacies of pharmacological CDK inhibitors in related animal models, none of them were however clinically relevant to human treatment. Methodology/principal findings: In the present study, we report that systemic delivery of (S)-roscovitine, a well known inhibitor of mitotic CDKs and CDK5, was neuroprotective in a dose-dependent manner in two models of focal ischemia, as recommended by STAIR guidelines. We show that (S)-roscovitine was able to cross the blood brain barrier. (S)-roscovitine significant in vivo positive effect remained when the compound was systemically administered 2 hrs after the insult. Moreover, we validate one of (S)-roscovitine in vivo target after ischemia. Cerebral increase of CDK5/p25 activity was observed 3 hrs after the insult and prevented by systemic (S)-roscovitine administration. Our results show therefore that roscovitine protects in vivo neurons possibly through CDK5 dependent mechanisms. [2]

C19H26N6O

354.44934

354.216

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

186692-45-5

6603989

Typically exists as solids at room temperature

3.2

3

6

8

26

417

1

CC[C@H](NC1=NC(NCC2C=CC=CC=2)=C2N=CN(C(C)C)C2=N1)CO

BTIHMVBBUGXLCJ-HNNXBMFYSA-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-/m0/s1

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

(S)-Seliciclib; (S)-ROSCOVITINE; 186692-45-5; (S)-Seliciclib; Seliciclib, (S)-; Seliciclib (2S)-form [MI]; (2S)-2-[[6-(benzylamino)-9-propan-2-ylpurin-2-yl]amino]butan-1-ol; UNII-8C43G94891; Roscovitine, (S)-Isomer; (S)-CYC202

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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