Pharmacological and analytical studies of the cyclin dependent kinase inhibitors

Cyclin-dependent kinases (Cdks) play a key role in the regulation of cell cycle progression and RNA transcription. Deregulation of Cdks has been associated with several malignancies, neurodegenerative disorders, viral and protozoal infections, glomerulonephritis and inflammatory diseases. (R)-roscovitine (Rosco) is a synthetic tri-substituted purine that inhibits selectively Cdk1, 2, 5, 7 and 9. Rosco has shown promising cytotoxicity in cell lines and tumor xenografts. Rosco so far has only demonstrated modest antitumor activity in phase-II clinical trials, which is attributed mainly to the short elimination half-life and thus suboptimal exposure.

Within the frame of the present thesis we aimed to investigate several aspects of the pharmacokinetics (PK) and pharmacodynamics (PD) of Rosco and two recently discovered analogues namely N-&-N1 and CR8. Our studies included bioanalysis, hematotoxicity, chrono-biodistribution, age dependent kinetics, PK and effect on Cdks.

In vitro and in vivo studies of Rosco hematotoxicity were performed in Balb/c mice. Bone marrow cells were incubated with Rosco in semisolid methylcellulose media and assessed for their clonogenic capacity. Rosco inhibited the colony formation in cell type-, concentration- and exposure time-dependent manner. CFUGEMM were most sensitive, followed by BFU-E and the least sensitive progenitors were CFU-GM. In vivo studies showed low distribution of the drug to the bone marrow (AUCBM/AUCplasma 1.5%) and only transient inhibition of BFU-E formation was observed. These finding may explain the absence of myelosuppression in vivo.

Age-dependent PK of Rosco were investigated in 14-days rat pups and adult rats. Higher plasma and brain (22- and 100- fold, respectively) exposure was found in rat pups compared to adult rats. The elimination half-life in young rats was 7 hr compared to 30 min in adult rats. Brain exposure (AUC brain/AUC plasma) was 100% in rat pups compared to 20% found in adult rats. Moreover, transient Cdk5 inhibition and Erk1/2 activation was detected in brain of rat pups. The high brain exposure may indicate Rosco as s potential candidate for the treatment of brain tumors in children.

The chronopharmacokinetics of Rosco was investigated in BDF1 male mice. Rosco was administered orally at ZT3 or at ZT19. We found that exposure to roscovitine was 38% higher and elimination half-life was 100% longer when dosing at ZT3 compared to ZT19. Moreover the tissue AUC/plasma AUC was higher at ZT3 in kidney, adipose, testis and lungs. The opposite was found in liver. In vitro microsomal assays indicated higher intrinsic clearance at ZT19. From these results, dosing times of roscovitine should be carefully considered in clinical trials.

Analytical method for the detection of N-&-N1 and CR8 using high performance liquid chromatography with UV detection (HPLC-UV) were developed and validated. The PK of both drugs was investigated in Balb/c mice. N-&-N1 showed higher potency in tumor cell death induction compared to roscovitine; however, N-&N1 showed similar PK profile as roscovitine.

CR8 has 100% oral bioavailability, longer elimination half-life, rapid and extensive biodistribution. Systemic exposure higher than IC50 reported for cell death in tumor cell lines was achievable for more than 10 hr. These two analogues displayed favorable pharmacological properties, and thus are good candidates for further in vivo studies.

To conclude, these studies provide important knowledge about the PK, PD and PK/PD relationship of Rosco and its analogues. These studies may add more knowledge for treatment schedules and further preclinical and clinical development of Cdk inhibitors.

List of scientific papers

I. Song H, Vita M, Sallam H, Tehranchi R, Nilsson C, Sidén A, Hassan Z (2009). Effect of the Cdk-inhibitor roscovitine on mouse hematopoietic progenitors in vivo and in vitro. Cancer Chemother Pharmacol. 60(6): 841-9.
https://pubmed.ncbi.nlm.nih.gov/17318617

II. Sallam H, Jimenez P, Song H, Vita M, Cedazo-Minguez A, Hassan M (2008). Age-dependent pharmacokinetics and effect of roscovitine on Cdk5 and Erk1/2 in the rat brain. Pharmacol Res. 58(1): 32-7.
https://pubmed.ncbi.nlm.nih.gov/18588979

III. Sallam H, Filipski E, Terelius Y, Lévi F, Hassan M (2009). The effect of circadian rhythm on pharmacokinetics and metabolism of the Cdk inhibitor, roscovitine. [Manuscript]

IV. Bettayeb K, Sallam H, Ferandin Y, Popowycz F, Fournet G, Hassan M, Echalier A, Bernard P, Endicott J, Joseph B, Meijer L (2008). N-&-N, a new class of cell death-inducing kinase inhibitors derived from the purine roscovitine. Mol Cancer Ther. 7(9): 2713-24.
https://pubmed.ncbi.nlm.nih.gov/18790752

V. Sallam H, Meijer L, Hassan M (2009). Quantitative analysis, pharmacokinetics and biodistribution of CR8, a new, roscovitine-derived, CDK inhibitor. [Manuscript]