SQU Med J, May 2011, Vol. 11, Iss. 2, pp. 165-178, Epub. 15th May 11
Submitted 13th Jul 10
Revision ReQ. 5th Oct 10, Revision recd. 2nd Nov 10
Accepted 24th Nov 10

1Experimental Cancer Medicine (ECM), Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; 2Clinical 
Research Centrum, Karolinska University Hospital, Huddinge, Stockholm, Sweden.
*Corresponding Author email: moustapha.hassan@ki.se 

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

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2 15



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

aBstract: Pharmacokinetics, pharmacodynamics and pharmacogenetics play an important role in drug 
discovery and contribute to treatment success. This is an essential issue in cancer treatment due to its high 
toxicity. During the last decade, cyclin-dependent kinase inhibitors were recognised as a new class of compounds 
that was introduced for the treatment of several diseases including cancer.  Cyclin-dependent kinases (Cdks) 
play a key role in the regulation of cell cycle progression and ribonucleic acid transcription. Deregulation of 
Cdks has been associated with several malignancies, neurodegenerative disorders, viral and protozoa infections, 
glomerulonephritis and inflammatory diseases. (R)-roscovitine is a synthetic tri-substituted purine that inhibits 
selectively Cdk1, 2, 5, 7 and 9. Roscovitine has shown promising cytotoxicity in cell lines and tumor xenografts. In 
this paper, we present several aspects of pharmacokinetics (PK) and pharmacodynamics (PD) of roscovitine. We 
present also some of our investigations including bioanalysis, haematotoxicity, age dependent kinetics, PK and 
effects on Cdks in the brain. Unfavourable kinetic parameters in combination with poor distribution to the bone 
marrow compartment could explain the absence of myelosuppression in vivo despite the efficacy in vitro.Higher 
plasma and brain exposure and longer elimination half-life found in rat pups compared to adult rats may indicate 
that roscovitine can be a potential candidate for the treatment of brain tumours in children. Cdk5 inhibition and 
Erk1/2 activation that was detected in brain of rat pups may suggest the use of roscovitine in neurodegenerative 

REVIEW

The Role of Pharmacokinetics and 
Pharmacodynamics in Early Drug 

Development with reference to the 
Cyclin-dependent Kinase (Cdk) 

Inhibitor - Roscovitine
*Moustapha Hassan,1,2 Hatem Sallam,1 Zuzana Hassan1,2



The Role of Pharmacokinetics and Pharmacodynamics in Early Drug Development with reference to the Cyclin-dependent Kinase 
(Cdk) Inhibitor - Roscovitine

166 | SQU Medical Journal, May 2011, Volume 11, Issue 2

Clinical approval of new drugs is preceded by intensive research consisting of two steps, drug discovery and drug 
development. In the drug discovery stage, target 
enzymes and/or receptors for a particular disease 
are identified, and new molecules are designed and 
screened for their biological activities. Promising 
drug candidates are evaluated for their toxicity and 
efficacy in the development stage.1 

Studies on the metabolism and pharmacokinetics 
(DMPK) of candidate drugs have become an 
essential part in drug discovery and development 
programmes and start usually concomitantly with 
the screening for biological activity. It is estimated 
that approximately 10–40 % of drug candidates fail 
due improper pharmacokinetic properties. Further, 
DMPK studies provide vital information about the 
PK/PD (pharmacokinetics/pharmacodynamics) 
relationship.2 This knowledge is prerequisite for 
safety phase I clinical trials3 and prediction of a 
clinical effective dose.

Moreover, PK is essential for further investigation 
of drugs in phase II and III clinical trials. PK/PD 
also play important roles in dose optimisation, 
personalised treatment and prevention of side 
effects. This in turn determines the clinical 
outcome. These aspects are especially important 
in anticancer drugs due to their toxicities and the 
narrow therapeutic window. 

Age is one of the important factors affecting 
the PK and efficacy of drugs. Variability in drug 
exposure and efficacy occurs among the different 
age populations, i.e. children, adults and elderly. 
It is essential to investigate the PK parameters in 
the age population that is treated with the drug. In 
the younger aged population, doses of many drugs 
are still not optimised due to lack of knowledge 
about drug disposition and PK in this particular 
population. Several factors such as the ontogeny of 
the metabolising enzymes and drug transporters 
are responsible for the variability of DMPK.4 
Continuous efforts are being made to develop 
accurate PK models to predict the PK parameters 

in this population without conducting a large 
scale investigation which might be difficult due to 
technical and ethical restraints.5 Age-dependent 
pharmacokinetics in young animals at different 
stages of development should be considered before 
clinical use.6

Cyclin-Dependent Kinases 
Cyclin-dependent kinases (Cdks) are a family of 
serine/threonine kinases that are activated through 
binding to regulatory subunits called cyclins.7 Cdk 
enzymes are homologues and highly conserved in 
their cyclin binding domain. Despite the fact that 
the human sequencing programme has successfully 
indentified 20 Cdks and 25 cyclins, their functions 
are still not fully understood and a limited number 
of active Cdk/cyclin complexes have been identified 
so far.7 

Cyclin-dependent kinases are regulated by 
several mechanisms7 including, transcription and 
translation of their subunits, heterodimerisation 
with cyclins, post-translational modification by 
phosphorylation and dephosphorylation and 
interactions with the natural inhibitors. The natural 
inhibitors CIP/KIP (p21, p27, p57) suppress the 
Cdk/cyclin complexes and INK4 proteins (p15, p16, 
p18 and p19) inhibit the Cdk4 and Cdk6 monomers.

Cdk/cyclin complexes play an essential role in 
the regulation of the cell cycle progression. Cyclins 
transcription and degradation varies during the 
different phases of the cell cycle and lead to the 
activation or inactivation of the corresponding 
Cdks. 

During the last decade, the roles of Cdks in 
several diseases including cancer were extensively 
studied. Over expression of cyclin B1 and 
hyperactivation of Cdk1 has been observed in a 
number of primary tumours including breast-, 
colon- and prostate carcinoma. Inactivation of 
Cip–Kip inhibitors and over expression of cyclin 
E and/or cyclin A lead to deregulation of Cdk2 in 
various malignancies, including melanoma, ovarian 

diseases. Early pharmacokinetic/pharmacodynamic studies are important issues in drug discovery and may affect 
further development of promising drug candidates.

Key words: Pharmacokinetics; Pharmacodynamics; Roscovitine; Cdk inhibitor; Anticancer drugs; Toxicity; age-
dependent kinetics



Moustapha Hassan, Hatem Sallam and Zuzana Hassan

review | 167

with adenosine triphosphate (ATP) for binding in 
the kinase ATP-binding site, and 4) Cdkis bind 
mostly by hydrophobic interactions and hydrogen 
bonds with the kinase.

Cdki have been classified according to their 
selectivity into three groups: 1) Pan-Cdki that 
inhibit Cdk1, 2, 4, 5, 6, 7 and 9 with almost similar 
potency like flavopiridol; 2) Selective Cdki for Cdks 
1, 2, 5, 7 and 9 such as the 2, 6, 9 tri-substituted 
purines (olomoucine, roscovitine and purvalanol), 
and 3) Selective Cdki for Cdk4 and 6 (PD-0332991 
or P-276–00).

(R)-Roscovitine (CYC202)
Roscovitine belongs to the 2, 6, 9 tri-substituted 
purines [Figure 1].22,23 Roscovitine was found to 
be a selective inhibitor for Cdk1, 2, 5, 7 and 9. In 
the kinase inhibitory assay, roscovitine has been 
shown to inhibit these kinases with the IC50 at the 
nanomolar range.24,25 Roscovitine was also found to 
inhibit several other kinases such as CaM Kinase 2, 
CK1α, CK1δ, DYRK1A, EPHB2, ERK1, ERK2, FAK, 
and IRAK4 at the micromolar range (1-40 µM). 
However, other kinases including Cdk4, Cdk6 and 
Cdk8 were not sensitive to roscovitine.24,25 

Since Cdks have an important role in a wide 
range of cellular functions, roscovitine has been 
suggested as a potential treatment for several 
pathophysiologically different diseases. The effects 
of roscovitine have been studied in vitro in cell lines 
and in vivo in animal models. The in vitro effects 
of roscovitine have been studied in more than 100 
cell lines. Several studies have reported the IC50 
required to inhibit cell proliferation including the 
NCI 60 cell line panel (average IC50 = 16 μM),23 
the McClue et al. panel (19 cell lines; average IC50 
= 15.2 μM),26 and the Raynaud et al. panel (24 cell 
lines; average IC50 = 14.6 μM).27 The IC50 average 
required for inhibition of cell proliferation in 
cancer cell lines does not exceed 17 μM;  moreover, 
roscovitine was shown to be cell cycle phase non-
specific. Direct inhibition of several Cdks results in 
inhibition of the exit from G0 (Cdk3/cyclin C), G1/S 
transition (Cdk2/cyclin E), S phase progression 
(Cdk2/ cyclin A), G2 phase (Cdk1/Cyclin A) and 
G2/M transition (Cdk1/Cyclin B). Depending on 
the cycling status of the cells, the antimitotic effects 
of roscovitine may comprise combinations of these 
mechanisms 

carcinoma, lung carcinoma and osteosarcoma.8 
Cdk5 has been found to modulate the metastatic 
potential of some malignancies including breast 
and prostate carcinomas.9,10

Cdk2 and Cdk5 have been shown to play 
important roles in apoptosis in various tissues.11 
Cdk2 was found to regulate apoptosis in 
thymocytes,12 while subcellular localisation of Cdk2 
have been found as a determinant of the apoptotic 
or proliferative fate of mesangial cells.13

Cdk5 has an essential role in the central and 
peripheral nervous system.14 Cdk5 regulates the 
cytoarchitecture of the developing brain15 and 
mediates the neuronal migration in the post 
mitotic neurons. Cdk5 has also many important 
functions in the neuronal cytoskeleton dynamics, 
synaptic plasticity,16 drug addiction,17 synaptic 
endocytosis18 and neurotransmitter release.19 Cdk5 
may be involved in the pathogenesis of several 
neurodegenerative disorders such as Alzheimer’s 
and Parkinson’s disease.14 It has been shown that 
Cdk9 induces the differentiation in distinct tissues 
and the degree of expression of Cdk9 correlates 
with differentiation of primary neuroectodermal 
and neuroblastoma tumours.20 

Due to the important role of Cdks in several 
diseases including cancer, intensive efforts to find 
selective cyclin-dependent kinase inhibitors (Cdki) 
have been made during the last 15 years. Several 
classes of different Cdki have been identified. In 
spite of their chemical diversity they share common 
characteristics:21 1) Cdkis have low molecular 
weights (about 600); 2) Cdkis are flat hydrophobic 
heterocyclic compounds; 3) Cdkis act by competing 

HN

N
N

NNN
H

HO

Figure 1: The structure of roscovitine and other 
members of the 2, 6, 9 trisubstituted cyclin-dependent 
kinase inhibitors (Cdki)



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168 | SQU Medical Journal, May 2011, Volume 11, Issue 2

Indirect inhibition of the cell cycle by roscovitine 
is mediated through the inhibition of the activity 
of Cdk7/ cyclin H/ MAT1 (CAK) resulting in 
prevention of the phosphorylation of the T loop 
threonine of various Cdks. This finally decreases the 
activity of Cdk1, 2 and 4. Also phosphorylation of the 
natural inhibitor p27 by Cdk2 will be diminished28 
28 leading to its stabilisation and more inhibition of 
the cell cycle.29 In addition, roscovitine was shown 
to inhibit the initiation of DNA synthesis,30 the 
formation of centrosomes31 and the formation of 
the nucleolus.32

Roscovitine has been shown to induce apoptosis 
in several cell lines regardless of the p53 status; 
however, roscovitine has a higher potency to 
induce apoptosis in wild type p53 cells compared 
to p53 null cells [23, 26, 33-34]. Cell death has 
been detected in all phases of the cell cycle and 
different mechanisms may be involved including 
inhibition of the cell cycle due to p53 activation and 
inhibition of Cdk7/Cdk9-dependent transcription 
inhibiting RNA polymerase II enzyme.33,34 Effects 
of roscovitine on global transcription have been 
shown to be limited and only few proteins such 
as Mcl-1, XIAP, and survivin have been found 
to be severely reduced. Induction of cell death by 
roscovitine, thus, seems to correlate rather well 
with inhibition of transcription of essential cell 
survival factors.35,36 Down regulation of survivin 
and XIAP by roscovitine was shown to contribute 
to the activation of caspases in glioma cell.37 Alvi et 
al. have reported that roscovitine induced apoptotic 
cell death in chronic lymphocytic leukaemia 
B-lymphocytes at significantly higher level than 
in normal blood mononuclear cells, purified B- or 
T-lymphocytes. Apoptosis was caspase-dependent 
but p53- independent and was accompanied with 
down regulation of Mcl-1 and XIA.38

Anti-proliferative and pro-apoptotic effects 
of roscovitine have been implicated in cancer 
treatment and used in studies on the antitumour 
effects of roscovitine. So far, no cell line resistant to 
roscovitine has been reported until now.39

Interestingly, tumour cells are more dependent 
on the short-lived survival factors compared to 
normal cells, and thus, down regulation of these 
factors by roscovitine treatment has a higher impact 
on tumour cells.40 Synergistic effects of roscovitine 
in combination with other chemotherapeutic 
agents such as camptothecin in MCF-7 breast 

tumour,41  irinotecan in p53-mutated colon cancer,42 
histone deacetylase (LAQ824) in HL60 and Jurkat 
leukaemic cells and doxorubicin in sarcoma cell 
lines 43 have been shown in vitro. 

Antitumour effects of roscovitine as a single 
treatment, or in combination with conventional 
cytostatics, have been studied in vivo in various 
tumour xenografts models. Nude mice bearing 
human colorectal cancer or human uterine cancer 
xenografts were treated with roscovitine at different 
dosing schedules. Roscovitine inhibited the tumour 
growth rate and reduced tumour volumes and 
weights.26,44 Roscovitine was also shown to be 
effective in reducing the growth of A4573 (Ewing’s 
sarcoma) and PC3 prostate tumour xenografts.45.46

The efficacy of roscovitine in non-nude 
BDF1 male mice bearing Glasgow osteosarcoma 
xenografts was investigated in relation to biological 
circadian rhythm. Roscovitine was administered 
orally (300 mg/kg x1 daily) for 5 days Zeitgeber 
time 3 (ZT3, 3 hours after light onset) or at ZT11 
or ZT19. Roscovitine reduced the tumour growth 
by 35% when administered in the active time of the 
mice (ZT19) and 55% when administered during 
their rest span (ZT3 or ZT11).47 

Roscovitine showed higher antitumour activity 
when combined with other antitumour treatments.  
Maggiorella et al. have reported better reduction in 
tumour volume from 54% to 72% when a single dose 
of 100 mg/kg was given intraperitoneal (i.p.) and 
combined with radiation therapy in mice bearing 
MDA-MB 231 (breast cancer).48 Roscovitine was 
shown to have a synergistic effect in inhibiting 
HT29 colon cancer xenografts when combined 
with irinotecan.42

Pharmacokinetics and 
Metabolism of Roscovitine
The PK of roscovitine have been reported in mice, 
rats and human. Vita et al. reported the PK and 
biodistribution of roscovitine in rats after a dose of 
25 mg/kg. Roscovitine PK was described by a two-
compartment open model and short elimination 
half-life (<30 min). The highest distribution of 
roscovitine was observed in lungs followed by liver, 
fat and kidney, while exposure to roscovitine in 
brain was 30% of that observed in plasma. Three 
major metabolites were detected in plasma, but no 
metabolites were detected in brain.49 50



Moustapha Hassan, Hatem Sallam and Zuzana Hassan

review | 169

PK/PD of Roscovitine in 
the Bone Marrow in Mice
Myelosuppression is one of the most frequent 
complications and a dose limiting factor for the 
majority of conventional chemotherapeutic agents. 
Depending on the dose, several cytostatics may 
induce complete myeloablation of the bone marrow. 
Studies on haematotoxicity in vitro and in animal 
models help to predict the possible side effects prior 
to clinical trials. 

In order to investigate the myelosuppressive 
potential of roscovitine we studied the effect of 
roscovitine on bone marrow cells in vitro and in 
vivo in Balb/c mouse. Crude bone marrow was 
incubated in vitro with roscovitine at concentrations 
of 25–250 µM for 4 hrs and viability was studied 
using resazurin assay. The viability of bone 
marrow cells was decreased in a concentration-
dependent manner. Concentration of 250 µM 
significantly reduced the viability of the cells to 
70% compared to controls (P = 0.015) while lower 
concentrations did not have a significant effect. 
Our results were in agreement with the findings 
that roscovitine induced apoptosis of mature 
neutrophils,58 eosinophils59 and proliferating 
T-cells60 in a concentration and exposure-time 
dependent manner. The myelosuppressive effect 
of roscovitine on haematopoietic progenitors was 
studied using a clonogeneic assay. Bone marrow 
cells were exposed to roscovitine at different 
concentrations (25–100 µM) for up to 24 hrs in 
suspension cultures. After washing, the capacity 
of haematopoietic progenitors to form colony-
forming unit granulocyte/macrophade (CFU-
GM), burst-forming unit erythroid (BFU-E)  
and colony-forming unit granulocyte/ 
erythrocyte/macrophage/megakaryocyte (CFU-
GEMM) colonies was studied using semisolid 
media. The clonogenic capacity of the bone marrow 
decreased in a dose- and time-dependent manner. 
BFU-E colonies were more sensitive than CFU-
GM and completely blocked after 12 and 24 hr 
incubation with both 50 and 100 µM of roscovitine. 
Since a decrease in the colony formation in controls 
after 12 and 24 hrs was observed, which most 
probably is due to lack of the growth factors in the 
suspension media, bone marrow cells were exposed 
to roscovitine (1 - 100 µM) in semisolid MethoCult 
media containing growth factors. Suppression of 

The PK of roscovitine was investigated in 
BALB/c and Tg26 mice. These studies showed 
rapid and biphasic clearance of roscovitine from 
plasma following intravenous (i.v.), i.p. or oral 
administration.44,51,52 Roscovitine had rapid tissue 
distribution and rapid elimination with a half-life 
of 1.19 hr. Plasma concentrations above 15 μM (the 
average IC50 values obtained with various tumour 
cell lines) were observed for 4, 12, and 24 h following 
oral administration of 50, 500, and 2000 mg/kg, 
respectively.44 

The PK of roscovitine in humans were reported 
in two phaseI trials. Roscovitine was administered 
orally as a single dose (50 to 800 mg) to healthy 
volunteers and the concentrations of roscovitine and 
its carboxylated metabolite were followed in plasma 
and urine. Roscovitine was found to undergo slow 
absorption from the gastrointestinal tract, but food 
intake did not affect the bioavailability of the drug. 
Roscovitine was found to have rapid metabolism and 
non-saturated high protein binding.53  

In the second investigation, twenty-one patients 
with a median age of 62 years (range: 39–73 years) 
were treated with roscovitine in doses of 100, 200 
and 800 mg twice daily for 7 days.  The elimination 
half-life ranged between 2–5 hrs depending on 
the dose of roscovitine. Neither objective tumour 
responses, nor inhibition of retinoblastoma protein 
phosphorylation (suggested as a suitable PD 
endpoint) in peripheral blood mononuclear cells 
were observed.54 High protein binding of roscovitine 
(92% to 96%) was shown in human and mice 
plasma.44,55

In vitro and in vivo metabolism of roscovitine 
was reported recently.50,52,56,57 Several metabolites 
were indentified including the carboxylate 
metabolite (oxidation of the alcohol group at C2 of 
the purine ring).50 CYP3A4 and CYP2B6 enzymes 
have been shown to be the main enzymes in 
roscovitine metabolism. Roscovitine was found to 
undergo phase II metabolism through conjugation 
with glucoronic acid by the phase II UGT1A3, 1A9 
and 2B7. Moreover, roscovitine was able to inhibit 
its own metabolism in vitro through inhibition of 
CYP3A4 with the IC50 of 3.2 µM. Thus, possible 
drug-drug interactions should be considered in the 
clinic.57 



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170 | SQU Medical Journal, May 2011, Volume 11, Issue 2

the bone marrow compared to plasma. Thus, low 
distribution of roscovitine to bone marrow may 
explain the low haematotoxicity in vivo. This 
example illustrates the importance of PK/PD and 
biodistribution in preclinical studies. This may 
be also implicated in the fact that, despite a good 
cytotoxic effect of roscovitine in leukaemic cell lines 
in vitro, the therapeutic potential of roscovitine in 
haematological malignancies may be limited.

Age-Dependent Kinetics 
and Dynamics of 
Roscovitine in Rat Brains
Age-dependent PK is an important issue when the 
drug may be used in the treatment of paediatric 
patients and/or when the drug has a narrow 
therapeutic window. Unfortunately, scaling down 
the PK data from adults to paediatrics, has been 
proven not to be sufficiently predictive for many 
drugs.4 Roscovitine has been found to inhibit 
different solid and haematological tumour cell lines 
including acute lymphoblastic leukaemia (ALL)61 
which is frequent in children and is correlated with 
a high central nervous system (CNS) relapse rate.62

Recently, we have explored the effect of age on 
the PK of roscovitine and investigated the effect 
of roscovitine on two neuronal targets, Cdk5 and 
Erk1/2, in different brain regions.63 Fourteen day-
old pups and adult Sprague-Dawley rats were 

colony formation in a concentration- and cell type- 
dependent manner was observed. CFU-GEMM 
were most sensitive and were completely blocked 
at 25 µM concentration, followed by BFU-E which 
were also significantly inhibited at 25 µM while 
CFU-GM were least sensitive and were inhibited at 
100 µM only [Figure 2].

We further studied the myelosuppressive 
effect of roscovitine in vivo in female Balb/c mice. 
Roscovitine was administered to the mice and 
bone marrow cells were cultured in the MethoCult 
media and assessed for colonogenic growth. No 
myelosuppressive effect was detected after the 
administration of a single dose of roscovitine up 
to 250 mg/kg. Then, roscovitine was administered 
at a dose of 175 mg/kg twice daily for 4 days. Only 
transient inhibition of the BFU-E colonies occurred 
one day after the last dose of roscovitine. The colony 
formation capacity of bone marrow was recovered 5 
days after the last dose of roscovitine [Figure 3]. The 
lack of activity of roscovitine on haematopoietic 
progenitors in vivo was not expected after its 
proven inhibitory effect in vitro and the reported 
activity on different xenografts in vivo.26,44,46,47 
Therefore we decided to study the distribution and 
PK of roscovitine in Balb/c mice. Roscovitine was 
administered as a single i.p. injection in a dose of 
50 mg/kg.  As presented in Table 1, roscovitine 
had a short half-life (less than 1 hr) and only a 
small fraction of roscovitine (about 1.5%) reached 

Figure 2: Effect of roscovitine on haematopoietic progenitors in vitro. 20,000 nucleated bone marrow cells were cultured 
in semi solid media (MethoCult M3434) with roscovitine at final concentrations of 1,10, 25 or 100 µM or dimetylsulfoxid 
(DMSO) (control for solvent toxicity). Colony forming unit granulocyte-macrophage (CFU-GM), erythroid-brust 
froming units (BFU-E) and colony-forming unit granulocyte erythrocyte monocyte macrophage (CFU-GEMM) were 
counted at day 12. Results are expressed as mean ± standard deviation (SD) from 5 mice.



Moustapha Hassan, Hatem Sallam and Zuzana Hassan

review | 171

found in plasma (Table 2). The Cmax was significantly 
(P <0.05) higher (>22 µg/g) in pups brain compared 
to that found in plasma, while 4-fold higher Cmax 
was found in plasma compared to that observed the 
brain (17.7 µg/ml and about 4 µg/g, respectively) in 
adult rats. The high concentrations of roscovitine 
found in the pups’ brains indicate the free passage 
of roscovitine into the brain. 

This difference in exposure might be due to the 
immaturity of the CYP450 enzymes responsible 
for roscovitine metabolism64 or immaturity of BBB. 
Roscovitine is metabolised in humans mainly by 
CYP3A4 and CYP2B6 enzymes.57 Several CYP450 
enzymes are not fully matured at the age of 2 weeks 
in rats.65 A similar situation was also reported in 
humans and CYP3A4, for example, approaches the 
adult full capacity only after first year of life.66,67 

Most chemotherapeutic agents do not cross 
the BBB and do not reach the CNS in enough high 
concentrations to eliminate tumour cells despite 

treated with a single i.p. injection of roscovitine 
in a dose of 25 mg/kg and plasma and brain were 
sampled at different time points. Table 2 shows 
the pharmacokinetic parameters of roscovitine in 
plasma and in different brain regions in pups and 
adult rats. The PK of roscovitine was best described 
by a 2-compartment open model with distribution 
half-lives of 0.6 hrs in pups and 0.06 hr in adult 
rats. A significantly longer elimination half-life (7 
hrs) was observed in the plasma and brain of the 
rat pups compared to 30 and 20 min found in the 
plasma and brain in adult rats, respectively. 

The area under the concentration–time curve 
(AUC) of roscovitine was 22-fold higher in the 
pups’ plasma and 100-fold higher in the pups’ brains 
compared to that found in adult rats [Figure 4]. No 
significant difference between roscovitine AUC in 
plasma and AUCs in different brain regions in pups 
was found. On the contrary, in adult rats, the AUC 
of roscovitine in the brain was about 25% of that 

CFU-GM

BFU-E

CFU-GEMM

DMSO 5Rosco 5Rosco 1Control
0

10

20

30

40

50

60

70

80

90

100

DMSO 1

N
r 

of
 C

ol
on

ie
s 

x1
00

0/
fe

m
ur

Figure 3: Effect of roscovitine on haematopoietic progenitors in vivo. Mice were treated withroscovitine (350 mg/kg/
day) divided into two daily doses for 4 days. Mice treated with dimetylsulfoxid (DMSO) and untreated animals served as 
controls. Bone marrow was examined 1 and 5 days after the last dose of roscovitine. Colony forming unit granulocyte-
macrophage (CFU-GM), erythroid-brust froming units (BFU-E) and colony-forming unit granulocyte erythrocyte 
monocyte macrophage (CFU-GEMM) were counted on day 12. Each group consisted of 5 mice, control group of 8 mice. 
Results are expressed as mean ± standard deviation (SD).

Table 1:  Pharmacokinetic parameters in plasma and bone marrow following intraperitoneal administration of 
roscovitine (50 mg/kg)

AUC 
µmol/l .h

Cmax
µmol/l

Cl
l/h

Vd
l

T½
h

Plasma 275.8 202 0.05 0.015 0.82

BM 4.6 4.9 0.62 0.54 0.61

Legend: AUC = area under the concentration–time curve (AUC is derived using WinNonlin analysis); (Cmax) = estimated maximum 
concentrations; Cl = clearance; Vd = apparent volume of distribution; T½ = half-life; BM = Bone marrow.



The Role of Pharmacokinetics and Pharmacodynamics in Early Drug Development with reference to the Cyclin-dependent Kinase 
(Cdk) Inhibitor - Roscovitine

172 | SQU Medical Journal, May 2011, Volume 11, Issue 2

function of BBB. Butt et al. have shown that the 
BBB of the rat fully matures 3–4 weeks postnatal.68 
No roscovitine metabolites were found in the brains 
of both adult and young rats.

In pups, roscovitine concentrations in plasma 
and brain were higher than the reported IC50 (10-
15 µM) for cancer cell lines for more than 8 hours. 
However, this level of exposure was achieved for less 

the high systemic exposure. Roscovitine was highly 
distributed over the BBB in the pups and the brain 
exposure in all studied regions (e.g. hippocampus, 
cerebral cortex and cerebellum) was 100% of that 
found in plasma which can be compared to about 
25% that has been found in the brain of adult rats. 
The high distribution to the brain could be explained 
by an age-dependent variation in the maturity and 

Table 2: Pharmacokinetic parameters in plasma and brain of adult and pups rats. Results are presented as mean ± 
standard deviation (SD) (n = 3)

PK parameters Plasma Frontal Cortex Hippocampus Cerebellum

 AUC (h.μg/ml)/ 
(h.μg/g)

Pups 66.79 ± 7.15 69.57 ± 15 74.92 ± 12 78.72 ± 11.2

Adults 3.01 ± 0.21 0.71 ± 0.14 0.58 ± 0.03 0.62 ± 0.06

Tα (h) Pups 0.50 ± 0.09 0.48 ± 0.19 0.43 ± 0.1 0.59 ± 0.14 

Adults 0.081 ± 0.05 0.045 ± 0.02 0.062 ± 0.012 0.062 ± 0.018 

 Tβ (h) Pups 7.2 ± 1.4 6.8 ± 1.3 8.0 ± 1.7 7.7 ± 2.2

Adults 0.54 ± 0.26 0.35 ± 0.13 0.36 ± 0.15 0.42 ± 0.18

 Cmax (μg/ml)/ (μg/g) Pups 15.79 ± 0.38 24.9 ± 1.8 24.75 ± 1.9 23.69 ± 1.4

Adults 17.71 ± 4.42 4.47 ± 0.70 4.64 ± 0.81 3.81 ± 1.22 

Vss (ml) Pups 88 ± 15.3 90 ± 21 86 ± 20 102 ± 13 

Adults 650 ± 223 1095 ± 167 2056 ± 219 1909 ± 484 

Cl (ml/h) Pups 9.7 ± 1.2 10.2 ± 1.5 11.1 ± 2 11.3 ± 1.2 

Adults 1637 ± 118 7262 ± 1612 8737 ± 452 8139 ± 727 

Legend: AUC = Area under the concentration–time curve; Tα,Tβ = distribution and elimination half-lives; Cmax  = maximum 
concentration; Vss = volume of distribution; Cl = clearance.

Figure 5: Effect of roscovitine on cyclin-dependent kinase 5 – neuronal protein specific cyclin-dependent kinase (Cdk) 
Cdk5 regulator (Cdk5-p35) in different brain parts of 14 days old rat pups after single intraperitoneal (i.p.) injection of 
25 mg/kg. Pups were killed at different time points after injection, brains dissected, homogenised, and immunoblotted 
for active Cdk5-p35. The figure shows densitometric analysis of the Western blotting bands for both p35 in the frontal 
cortex, hippocampus and cerebellum until 48 hr after single i.p. injection of roscovitine. Data are presented as mean ± 
standard deviation (SD) of values expressed as percentage of control animals (*, P < 0.05 for analysis of p35 data; Analysis 
of variance (ANOVA) followed by all pairwise Fisher’s Protected Least Significant Difference (PLSD) testing were used.



Moustapha Hassan, Hatem Sallam and Zuzana Hassan

review | 173

than 30 minutes in plasma and brain of adult rats. 
These results may be implicated in the treatment of 
paediatric malignancies especially brain tumours.

Roscovitine is a potent inhibitor of Cdk5 which 
has important function in the developing brain such 
as neuronal migration.15 Moreover, the negative 
feedback regulation of mitogen activated protein 
kinases (MAPK) signalling by Cdk5 has been 
suggested to be important for neuronal survival.69 

High concentrations of roscovitine found in the 
brain of pups raised the question about the effects 
of roscovitine on target enzymes. We assessed the 
expression of p35 as an indicator of Cdk5 activity. 
Inhibition of p35 phosphorylation by Cdk5 stabilises 
it and delays its proteasomal degradation.70,71 
Roscovitine induced a transient and significant 
accumulation of p35 protein in all brain regions in 
rat pups that indicates the inhibition of the Cdk5 
enzyme. An increase in p35 was found in the 
frontal cortex 1–2 hrs post-administration (140% of 
controls, Figure 5, P < 0.05), in the hippocampus 
and in cerebellum at 2 hrs post-administration 
(150% and 200%, respectively, Figure 5). The levels 
of p35 were normalised at 6–15 h [Figure 5]. No 
change in p35 levels was observed in the adult brain 
which probably is due to the low concentration and 
the rapid elimination half-life.

Cdk5 was found to inhibit Erk1/2 
phosphorylation by a MEK1 and RasGRF2 

mediated mechanism and the inhibition of Cdk5 by 
roscovitine increased the levels of phosphorylated 
Erk1/2 (active form) in neuronal cells in vitro.69,72

At early time points after administration of 
roscovitine, the accumulation of p35 protein 
was accompanied by increased levels of the 
phosphorylated (activated) form of Erk1/2. In 
the frontal cortex and hippocampus, a transient 
activation of Erk1/2 was observed at 1 and 2 
hrs after injection [Figure 6]. In the cerebellum, 
significant increases of pErk1/2 levels at 2 hrs were 
followed by a significant decrease at 6 hrs after 
administration [Figure 6]. At later time points, 
levels of pErk1/2 returned to control levels in all 
brain regions [Figure 6]. Altogether, roscovitine 
was presented in the brain of rat pups in sufficient 
amounts to inhibit the Cdk5 resulting in increased 
phosphorylation of Erk1/2.

Discussion
Cyclin-dependent kinases (Cdks) are serine/
threonine kinases that play key roles in cell cycle 
progression and RNA transcription. Deregulation 
of Cdks has been shown in several diseases 
including several types of cancer in which increased 
activity of Cdks has been observed. Synthetic 
cyclin dependent kinase inhibitors (Cdkis) are 
small heterocyclic compounds which compete 

Figure 6: Effect of roscovitine on p-Erk in different brain parts of rat pups 14 days old after single intrapertoneal 
(i.p.) injection of 25 mg/kg. Pups were killed at different time points after injection, brains dissected, homogenized, 
and immunoblotted for active phosphorylated Erk1/2. Control animals were injected with vehicle.  The figure show 
densitometric analysis of the Western blotting bands for pErk1/2 in the frontal cortex, hippocampus and cerebellum until 
48 hr after single i.p. injection of roscovitine. Data are presented as mean ± standard deviation (SD) of values expressed as 
percentage of control animals (*, P < 0.05 is the significant level for analysis of p-ERK data; Analysis of variance (ANOVA) 
followed by all pairwise Fisher’s Protected Least Significant Difference (PLSD) post-hoc test).



The Role of Pharmacokinetics and Pharmacodynamics in Early Drug Development with reference to the Cyclin-dependent Kinase 
(Cdk) Inhibitor - Roscovitine

174 | SQU Medical Journal, May 2011, Volume 11, Issue 2

with ATP and inhibit the phosphorylation of the 
target substrates. Exposure of tumour cells to Cdkis 
results in both cell cycle arrest and apoptosis. 

The family of 2,6,9-trisubstituted purines 
are one of the first described Cdk inhibitors.73 
The (R)-stereoisomer of roscovitine is a member 
of this family and has now reached phaseII clinical 
trials for non-small cell lung (NSCL) cancer 
and nasopharyngeal cancers and phaseI trials 
for glomerulonephritis. Preclinical investigations 
of the role of roscovitine in the treatment of 
neurodegenerative disorders such as Alzheimer’s 
disease, viral infections, protozoal infections and 
inflammatory diseases are ongoing. Roscovitine has 
a rapid metabolism and short elimination half-life in 
rodents and man.44,50,52,53 The poor pharmacokinetic 
profile and the insufficient exposure to the drug in 
cancer patients may explain the modest success in 
the clinical trials.54 Current research is focusing on 
overcoming pharmacokinetic barriers that limit the 
clinical use of roscovitine. Moreover, a novel class 
of second generation analogues of roscovitine has 
been designed and is under development. Studies 
on the pan-Cdk inhibitor flavopiridol confirmed 
the importance of optimising the schedule of 
dosing according to the PK/PD relationship. By 
changing the dose schedule from 72 hrs infusion to 
30 minutes i.v. bolus followed by a 4-hrs infusion, 

a significant difference in the clinical outcome 
and final response of refractory CLL patients was 
achieved.74

No myelosuppression has been reported until 
now in the preclinical and clinical studies with 
roscovitine.51,54 However, clinically beneficial low 
haematotoxicity of roscovitine may reflect in 
reality poor distribution of roscovitine to the bone 
marrow. In vitro, the haematopoietic progenitors 
were inhibited by roscovitine within the same 
exposure range as the tumour cells when comparing 
the inhibitory AUC reported for tumour cell lines 
26,44 with the inhibitory AUC of the haematopoietic 
progenitors found in our study. 

Under certain circumstances the haema-
totoxicity of roscovitine may become more 
evident: 1) Changes in the form of administration, 
aiming to increase the half-life of the drug, may 
result in higher exposure to roscovitine and 
changes in biodistribution. This in turn may 
change the toxicity profile; 2) A combination of 
roscovitine with radiation therapy, which increases 
the permeability of blood-bone marrow barrier,75 
and thus the distribution of some drugs to the 
bone marrow, may increase the myelotoxicity of 
roscovitine, and 3) In pediatric patients where 
age-dependent longer elimination half-life is most 
likely leading to higher exposure of haematopoietic 
progenitors to roscovitine and thus toxicity risk.66

Figure 6: Effect of roscovitine on p-Erk in different brain parts of rat pups 14 days old after single intrapertoneal 
(i.p.) injection of 25 mg/kg. Pups were killed at different time points after injection, brains dissected, homogenized, 
and immunoblotted for active phosphorylated Erk1/2. Control animals were injected with vehicle.  The figure show 
densitometric analysis of the Western blotting bands for pErk1/2 in the frontal cortex, hippocampus and cerebellum until 
48 hr after single i.p. injection of roscovitine. Data are presented as mean ± standard deviation (SD) of values expressed as 
percentage of control animals (*, P < 0.05 is the significant level for analysis of p-ERK data; Analysis of variance (ANOVA) 
followed by all pairwise Fisher’s Protected Least Significant Difference (PLSD) post-hoc test).



Moustapha Hassan, Hatem Sallam and Zuzana Hassan

review | 175

Age dependent PK is an important issue 
concerning toxic drugs and drugs with a narrow 
therapeutic window such as anticancer drugs, where 
underdosing may lead to relapse while overdosing 
can cause severe side effects. Age dependent kinetics 
were reported for several drugs including cis platin, 
busulfan, thioguanine, etoposide, lamivudine and 
mycophenolate mofetil.76-81 Our studies showed that 
roscovitine elimination half-life was 14-fold higher 
in young rats compared to adults. Moreover, the 
exposure to the drug was 22-fold and 100-fold higher 
in the plasma and brain, respectively. These results 
indicate the importance of early determination of 
the PK-parameters in different age groups.   

Conclusion
Roscovitine inhibits mouse haematopoietic proge-
nitors in vitro within the same concentration range 
required to inhibit malignant cells; however, the 
cytotoxic effect of roscovitine on haematopoietic 
progenitors in vivo is transient due to a short half-
life in combination with low distribution to the 
arrow compartment.

Roscovitine demonstrates age-dependent 
PK. Prolonged systemic and brain exposure to 
roscovitine was found in pups compared to adult  
rats, which may be due to immature CYP450  
enzymes as well as the BBB. Moreover, roscovitine  
was able to induce a transient effect on critical  
neuronal targets and signalling pathways in the brain 
of young rats. These studies show the importance 
of early pharmacokinetic and phamacodynamic 
studies in drug development.

a c k n o w l e d g m e n t s 
The authors would like to express their gratitude to 
the Swedish Cancer Foundation (Cancer Fonden) 
and the Swedish Children Cancer Society (Barn 
Cancer Fonden) for their support for this work.

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