in vitro and in vivo pharmacokinetic-pharmacodynamic ... · analogues into the in vivo animal model...

In vitro and In vivo Pharmacokinetic-PharmacodynamicRelationships for the Trisubstituted AminopurineCyclin-Dependent Kinase Inhibitors Olomoucine,Bohemine and CYC202Florence I. Raynaud,1Steven R.Whittaker,1PeterM. Fischer,2 StevenMcClue,2 Michael I.Walton,1

S. Elaine Barrie,1Michelle D. Garrett,1Paul Rogers,1SimonJ. Clarke,1Lloyd R. Kelland,1

MelanieValenti,1Lisa Brunton,1Suzanne Eccles,1David P. Lane,2 and PaulWorkman1

Abstract Purpose: To investigate pharmacokinetic-pharmacodynamic relationships for the trisubstitutedaminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine, and CYC202 (R-roscovitine; seliciclib) in the HCT116 human colon carcinoma model.Experimental Design: The in vitro activity of the agents was determined in a human tumorpanelusing the sulforhodamineBassay.The concentration and time dependencewas establishedin HCT116 cells.Molecular biomarkers, including RB phosphorylation and cyclin expression, wereassessed byWestern blotting. Pharmacokinetic properties were characterized in mice followinganalysis by liquid chromatography-tandemmass spectrometry. Based on these studies, a dosingregimen was developed for CYC202 that allowed therapeutic exposures in the HCT116 tumorxenograft.Results: The antitumor potency of the agents in vitro was in the order olomoucine (IC50,56 Amol/L) < bohemine (IC50, 27 Amol/L) < CYC202 (IC50, 15 Amol/L), corresponding to theiractivities as cyclin-dependent kinase inhibitors. Antitumor activity increased with exposure timeup to 16 hours. The agents caused inhibition of RB and RNA polymerase II phosphorylation anddepletion of cyclins. They exhibited relatively rapid clearance following administration to mice.CYC202 displayed the slowest clearance from plasma and the highest tumor uptake, with oralbioavailability of 86%. Oral dosing of CYC202 gave active concentrations in the tumor, modula-tion of pharmacodynamic markers, and inhibition of tumor growth.Conclusions: CYC202 showed therapeutic activity on human cancer cell lines in vitro andon xenografts. Pharmacodynamic markers are altered in vitro and in vivo, consistent with theinhibition of cyclin-dependent kinases. Such markers may be potentially useful in the clinicaldevelopment of CYC202 and other cyclin-dependent kinase inhibitors.

The cell cycle is coordinated through the activities of the cyclin-dependent kinases (CDKs; refs. 1, 2). The CDKs require theirpartner cyclins for activity and these are expressed in a cellcycle–dependent manner. In addition, regulatory phosphory-lation events and binding of the cyclin-dependent kinase

inhibitors (CDKI) ensure timely activation or inhibition ofthe CDK complexes (1, 2). CDKs are deregulated in numerousways in cancer. Overexpression of cyclin E has been observed inhuman tumors and is known to result in a poor prognosis inbreast cancer (3). Loss of the CDKI proteins such as p16INK4A isfound in many malignancies and can predispose to melanoma(4). Therefore, small-molecule pharmacologic CDK inhibitorsare being developed to block cell cycle progression and henceto inhibit tumor growth (5, 6). Although the validity ofinhibiting CDK2 alone as a cancer drug target has beenquestioned by recent data (7), other studies showing selectivekilling of transformed cells by a peptide inhibitor of CDK2/cyclin A binding to E2F-1 have suggested the possibility thatCDK2 inhibitors may not only block tumor cell growth butmight also preferentially induce apoptosis in tumor cells (8, 9).Furthermore, a number of small-molecule CDK2 inhibitors thatare in development also show inhibitory activity towards CDK1as well as the transcriptional kinases CDK7 and CDK9 (6).Combinatorial inhibition of more than one CDK may providegreater antitumor activity and overcome resistance that may beassociated with blockade of, for example, CDK2 alone.

Cancer Therapy: Preclinical

Authors’Affiliations: 1Cancer Research UKCentre for CancerTherapeutics atTheInstitute of Cancer Research, Haddow Laboratories, Belmont, Sutton, UnitedKingdom and 2Cyclacel Ltd., James Lindsay Place, Dundee, United KingdomReceived11/9/04; revised 3/21/05; accepted 4/13/05.Grant support: Cancer Research UK grant C309/A2187 (P.Workman), CyclacelLtd. (P. Workman and S.R. Whittaker), the Sir Samuel Scott of Yews Truststudentship (S.R. Whittaker), Cancer Research UK Gibb Fellowship (D.P. Lane),and Cancer Research UKLife Fellowship (P. Workman).The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Note: F.I. Raynaud and S.R. Whittaker, contributed equally to this work.Requests for reprints: PaulWorkman, Cancer Research UK Centre for CancerTherapeutics, The Institute of Cancer Research, Haddow Laboratories, 15Cotswold Road, Sutton, Surrey, SM2 5NG, United Kingdom. Phone: 44-208-722-4301; Fax: 44-208-722-4324; E-mail: [email protected].

F2005 American Association for Cancer Research.

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Through an initial screen for CDK1/cyclin B inhibitors,olomoucine was discovered to be a relatively specific andpotent inhibitor compared with the structurally similar butmore general kinase inhibitor N6-dimethyl-aminopurine (10).Further exploration of the trisubstituted aminopurine structureled to the more potent and selective roscovitine (11). Potencyand selectivity for CDK2 kinase inhibition was furtherimproved by synthesis and purification of the R-enantiomerof roscovitine (CYC202; refs. 10, 12–15). Roscovitine andother closely related trisubstituted aminopurine analogues caninduce cell cycle arrest and apoptosis in a range of humantumor cell lines (13–16).

The objective of the present work was to extend our previousmechanistic studies (15) and in particular to translate ourinvestigations on the in vitro properties of CYC202 and itsanalogues into the in vivo animal model setting. We describethe comparative in vitro and in vivo properties of olomoucine,bohemine, and CYC202 (Fig. 1) with particular regard topharmacokinetic-pharmacodynamic relationships and howthis information may be used to support the development ofCDK inhibitors. Initially, relative potencies of the analoguesagainst a panel of human cancer cell lines in vitro weredetermined and the influence of length of compound exposureon in vitro cancer cell proliferation was defined. To show thatthe compounds were acting in a manner consistent with theproposed mechanism of CDK inhibition, RB phosphorylationwas assessed in tumor cells in vitro and the cell cycle effectswere determined. In view of the potential importance of the

depletion of cyclin D1 and other cyclins (15), the effects ofcompound treatment on these regulatory proteins was alsodetermined. In parallel, comparative effects on cell cycle andcell death were determined by flow cytometry. Following thisin vitro characterization, the pharmacokinetic properties of thethree compounds were characterized following administrationby the i.v., i.p., and oral routes, and the data were reviewed inrelation to the exposures required for in vitro antitumoractivity. Comparing the in vitro results and the in vivopharmacokinetic behavior, CYC202 emerged as the analogueexhibiting the best profile in both respects. Based on theaforementioned properties, a dosing regimen was developedfor CYC202, which led to the achievement of antitumoractivity in the HCT116 human colon cancer xenograft model.Inhibition of RB phosphorylation and depletion of cyclin D1was shown in the tumor xenografts, revealing in vivopharmacokinetic-pharmacodynamic relationships. These phar-macokinetic-pharmacodynamic relationships and other thera-peutic studies (13) provide support for the development ofCYC202, which is now undergoing phase II clinical trials incancer patients.

Materials andMethods

Materials. Unless otherwise stated, materials were from SigmaChemical Ltd. (Poole, Dorset, United Kingdom).

Test compounds. 2-(6-Benzylamino-9-methyl-9H-purin-2-ylamino)-ethanol (olomoucine), (R)-2-(6-benzylamino-9-isopropyl-9H-purin-2-ylamino)butan-1-ol (R-roscovitine; seleciclib; CYC202), and 3-(6-benzylamino-9-isopropyl-9H -purin-2-ylamino)-propan-1-ol (bohe-mine) were obtained from Cyclacel Ltd. (Dundee, United Kingdom).For structures, see Fig. 1.

In vitro kinase assays. These were carried out by measurement ofincorporation of radioactive phosphate from ATP into appropriatepolypeptide substrates by purified recombinant human protein kinasesand kinase complexes, as described (17). Assays were done using 96-well plates and appropriate assay buffers [typically 25 mmol/L h-glycerophosphate, 20 mmol/L MOPS, 5 mmol/L EGTA, 1 mmol/L DTT,1 mmol/L Na3VO3 (pH 7.4)], into which were added 2 to 4 Ag of activeenzyme with appropriate substrates. The reactions were initiated byaddition of Mg/ATP mix (15 mmol/L MgCl2 and 100 Amol/L ATP with30-50 kBq per well of [g-32P]-ATP) and mixtures were incubated asrequired at 30jC. Reactions were stopped on ice followed by filtrationthrough p81 filterplates or GF/C filterplates (Whatman Polyfiltronics,Kent, United Kingdom). After washing thrice with 75 mmol/L aqueousorthophosphoric acid, plates were dried, scintillant added, andincorporated radioactivity measured in a scintillation counter (Top-Count, Packard Instruments, Pangbourne, Berks, United Kingdom).Compounds for kinase assay were made up as 10 mmol/L stocks inDMSO and diluted into 10% DMSO in assay buffer. Data were analyzedusing curve-fitting software (GraphPad Prism version 3.00 forWindows, GraphPad Software, Inc., San Diego, CA) to determine IC50

values (concentration of test compound which inhibits kinase activityby 50%).

Cell culture

Human tumor cell lines, including the HCT116 (NationalCancer Institute, Bethesda, MD) human colon carcinoma, weregrown in DMEM (Invitrogen, Paisley, United Kingdom)supplemented with 10% fetal bovine serum (Invitrogen,Paisley, United Kingdom) in an atmosphere of 5% CO2. Amatched pair of HCT116 cells isogenic for TP53 was kindlyprovided by Professor Bert Vogelstein, The Johns HopkinsFig. 1. Chemical structures of olomoucine, bohemine, and CYC202.


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University School of Medicine, Baltimore MD (18). For drugtreatment, 2 � 106 cells were seeded into a T75 flask (Corning,Acton, MA) and left to attach for 36 hours. Compounds weredissolved in DMSO and diluted directly into the culture mediawhen required. The total concentration of DMSO in themedium did not exceed 0.4% (v/v) during treatments.

Sulforhodamine B assay. Drugs were made up at 20 to50 mmol/L in DMSO. Growth inhibition assays were done in96-well microtiter plates using the sulforhodamine B assay asdescribed previously (19). Briefly, cells were seeded at 3 to 8 �103 cells per well (dependent upon doubling time) in 160 ALgrowth medium and allowed to attach overnight. Agents werethen added at 10 different concentrations (typically from2.5 nmol/L to 100 Amol/L) in quadruplicate wells andincubated at 37jC for 96 hours. Where the effect of durationof exposure was investigated, compounds were added for thetimes indicated and washed off and the cells incubated in freshgrowth medium for the remainder of the 96-hour period. Fiftymicroliters of sulforhodamine B (0.4%, w/v) dissolved in 1%acetic acid were then added and the absorbance at 492 nm wasdetermined using a Multiscan MCC/340 MKII (Titertek, Hunts-ville, AL). Absorbance of treated wells was expressed as apercentage of control wells. IC50 levels were calculatedgraphically and mean IC50 levels derived. Results are the meanof at least three determinations.

Animals. Female BALB/c� mice were maintained on SDAExpanded Rodent diet (Harlan UK Ltd., Bicester, Oxon,United Kingdom) and water ad libitum . They were housed inan Individual Ventilated Caging System manufactured byThoren. Female CD1 nude mice were supplied by CharlesRiver UK Ltd. (Martgate, United Kingdom) and maintained onHarlan Teklad 9607 R&M diet and water ad libitum. All animalprocedures complied with local and national animal welfareguidelines (20).

Maximum tolerated dose. BALB/c� mice were given increas-ing doses of 50, 100, and 200 mg/kg of the three compoundsi.v. in 50 mmol/L HCl/saline. Animals were checked daily onseveral occasions and monitored for a period of 5 days. For oraladministration BALB/c� mice were given a single dose of 500or 2,000 mg/kg orally in 50 mmol/L HCl/saline.

Pharmacokinetic experiments: Pharmacokinetics of olomoucine,bohemine, and CYC202 iv. Compounds were given i.v. in avolume of 0.1 mL/10 g bodyweight in 50 mmol/L HCl/saline at50 mg/kg to BALB/c� mice bearing the s.c. colon 26 murinetumor. A 1-mm3 brei was implanted under anesthetic using aBashford syringe into the flank of 100 BALB/c� female mice.Fourteen days post-implantation, mice bearing comparablysized tumors (diameters of f6 mm) were randomized intoeither a control group of six mice or treatment groups of threemice per time point. Plasma, liver, kidney, spleen, and tumorwere collected at 0.25, 0.5, 1, 3, 6, and 24 hours afteradministration.

Pharmacokinetics of CYC202 i.p., i.v., and orally. CYC202at a dose of 50 mg/kg (0.1 mL/10 g, 5 mg/mL) in 9% (w/v)Lutrol (polyethylene glycol 660-12 hydroxystearate), 3.5% (w/v)Solutol HS15 (BASF), and 87% water, was given i.v., i.p., andorally to BALB/c� mice. Solutol was weighed and warmed at70jC until liquid. Lutrol was weighed and added to the Solutol.CYC202 was added and the solution kept at 70jC untildissolved. The appropriate volume of sterile water was thenadded to the mixture. Blood was collected after 5, 15, 30

minutes, 1, 2, 4, 6, and 24 hours after administration. TheLutrol/Solutol vehicle was used because HCl/saline wasunsuitable for the i.p. route and because a drug solution wasrequired for i.v. administration. CYC202 (50, 500, and 2,000mg/kg) was given orally in 50 mmol/L HCl/saline in a volumeof 0.1 mL/10 g body weight to female BALB/c� mice. Bloodwas collected at 5, 15, 30 minutes, 1, 2, 4, 6, and 24 hours afteradministration.

Pharmacokinetic calculations and simulation experiments.Pharmacokinetic variables were calculated using WinnonlinProfessional software (21), version 3.2 (Pharsight, MountainView, CA). Variables presented were derived from noncom-partmental analysis. Model 200 (extravascular administration)was used for plasma following i.p. and oral administration andfor tissues following i.v. administration. Model 201 (i.v. bolusmodel) was used for plasma following i.v. administration. Areaunder the concentration versus time curve to the last time point(AUC), Cmax (maximum concentration observed), Cl (clear-ance), t1/2 (half-life), and Vz (volume of distribution based onthe terminal phase) were evaluated. For simulation studies,variables derived from one-compartment analysis followingoral administration of 50 mg/kg were used.

Protein binding experiments. Fresh human or BALB/c�mouse plasma was incubated at 37jC for 5 and 30 minutes,2 and 6 hours with 2 and 20 Amol/L CYC202. Plasma andstandard curve samples in PBS were then ultrafiltered bycentrifugation for 40 minutes at 4jC at 1,500 � g using 10,000MW exclusion membranes (Amicon, Dorset, United Kingdom).Samples were subsequently frozen at �20jC until analysis.Control experiments were carried out to show that testcompound did not bind to the filter membranes.

Analytic method. Drug measurements were done by liquidchromatography-tandem mass spectrometry (LC-MS/MS). Ini-tially, a 150 mm � 2.1 mm zwitterionic ABZ+ column (Supelco,Poole, Dorset, United Kingdom) was used and drugs eluted with10 mmol/L ammonium acetate and 70% methanol at a flowrate of 0.2 mL/min; total run time was generally 3 minutes.Tissue homogenates and plasma were treated with 3 volumes ofmethanol to precipitate protein followed by dichloromethaneextraction. Standard curves were made in the appropriate matrixand analyzed at the level of 1, 10, 100, 1,000, 10,000, and100,000 ng/mL. Samples were reconstituted in 200 AL mobilephase and 10 AL injected onto the column. Although thisanalytic method was specific, sensitive, and reproducible, it wasfound that the column could not withstand a large number ofextracts. Therefore, a second method was used for subsequentroutine CYC202 analysis, as follows. In this method, analyseswere done on a 50 mm � 4.6 mm zwitterionic ABZ+ column(Supelco) and the drugs eluted with a gradient of 80% to 0.1%formic acid, 20% to 100% methanol over 5 minutes. Standardcurves were made in mouse plasma at the level of 10, 100,1,000, 10,000, and 100,000 nmol/L. Quality controls weremade in control mouse plasma. The assay was validated inmouse plasma by running three precision batches of fivereplicates at the levels of 25, 90, 500, and 50,000 nmol/L onthree separate days. Plasma (100 AL aliquots) was added to30 Al of internal standard (500 ng/mL olomoucine inmethanol) incubated for 30 minutes and treated with 3 volumesof methanol. Following centrifugation, the supernatant wastransferred to high-performance liquid chromatography vialsand 10 AL injected onto the column. For ultrafiltrate analysis,

Pharmacology of Olomoucine, Bohemine, and CYC202




standard curve and quality controls were made in plasmaultrafiltrates.

Detection was achieved by multiple reaction monitoring on atriple sector mass spectrometer (TSQ700, Thermoquest Ltd.,Hemel Hempstead, Herts, United Kingdom). Multiple reactionmonitoring of the sum of two daughter ions of the pseudo-molecular ion [M+H]+ 299 (99 and 177 AMU) for olomoucine,355 (99 and 233 AMU) for CYC202, and 341 (91 and 206) forbohemine. Peak areas were monitored and plotted againstconcentration (GraphPad Software). When internal standardwas used, relative areas were monitored.

Pharmacokinetic-pharmacodynamic relationships in humantumor xenografts. Nude mice bearing established (f130mm3) HCT116 human colon tumors as a s.c. xenograft in theflank were given 500 mg/kg CYC202 orally, twice daily for5 days. This regimen was based on the pharmacokineticsimulation models. CYC202 was dissolved in 10% DMSO,5% Tween 20, and 85% of 50 mmol/L HCl/saline. Forpharmacodynamic profiling, three mice were sacrificed pertime point. Time points comprised 3 days vehicle control, 1 dayof treatment (harvested 4 hours after the second dose), 3 daystreated (again 4 hours after the second dose), and 5 days treated(also 4 hours after the second dose). Plasma and tumor wererecovered from each animal and immediately either homoge-nized in cold lysis buffer or snap-frozen in liquid nitrogen andstored at �80jC. For antitumor therapy studies, 10 mice weretreated with vehicle control alone and eight mice were treatedwith CYC202 for 5 days, using the same protocol as describedabove. Tumor volume was determined from measurement oftwo orthodiagonal diameters and antitumor activity wasdetermined by the comparison of volumes for treated andcontrol groups. The difference in the means of paired sampleswas determined using a two-tailed Student’s t test and Prismsoftware (GraphPad Software) and Ps < 0.05 were consideredstatistically significant.

Western blotting. To harvest cells, the medium was removedand cells were incubated with 5 mL trypsin for 5 minutes at37jC to detach them from the plastic. The cells were thenpelleted, washed in ice-cold PBS, and resuspended in ice-coldlysis buffer containing 50 mmol/L HEPES (pH 7.4), 250 mmol/LNaCl, 0.1% NP40, 1 mmol/L DTT, 1 mmol/L EDTA, 1 mmol/LNaF, 10 mmol/L h-glycerophosphate, 0.1 mmol/L sodiumorthovanadate, and one complete protease inhibitor cocktailtablet (Roche, East Sussex, United Kingdom) per 10 mL of lysisbuffer for 30 minutes on ice. Lysates were centrifuged atf18,000 � g for 10 minutes at 4jC to remove cellular debris.The supernatant was stored at �80jC before use. The proteinconcentration of lysates was determined using the protein assayreagent; bicinchoninic acid protein assay (Pierce, Rockford, IL).Proteins were separated by SDS-PAGE using Novex precast tris-glycine gels (Invitrogen, Groningen, the Netherlands) andtransferred to Immobilon-P membranes (Millipore, Bedford,MA). Membranes were blocked for 1 hour in TBSTM [50 mmol/LTris (pH 7.5), 150 mmol/L NaCl, 0.1% Tween 20 (SigmaChemical)] and 3% milk. Immunoblotting with primaryantibodies diluted in TBSTM was done at 4jC overnightfollowed by a 1-hour incubation with horseradish peroxidase–conjugated secondary antibodies at room temperature. Mem-branes were washed with enhanced chemiluminescencereagents and exposed to Hyperfilm (Amersham PharmaciaBiotech, Buckinghamshire, United Kingdom). Antibodies used

were phospho-RB Ser780 1:5,000, phospho-ERK1/2 1:1,000,phospho-CDK2 Thr160 1:1,000 (Cell Signaling Technologies,Beverly, MA); total RB SC-50 1:2,000 (Santa Cruz Biotech-nology, Santa Cruz, CA); phospho-RB Ser608 (22) 1:2,000 (Dr.Sibylle Mittnacht, Institute of Cancer Research, London, UnitedKingdom); nonphosphorylated RB Ser608 (underphosphory-lated RB) 1:1,000 (PharMingen, San Diego, CA); phospho-RBSer807/811 1:5,000 (Sigma Chemical); phospho-RB Thr821

1:1,000 (Biosource, Nivelles, Belgium); cyclin B1 Ab-1 1:200,cyclin D1 Ab-1 1:200, cyclin A Ab-6 1:200, cyclin E Ab-1 1:200,CDK1 Ab-1 1:200, CDK2 Ab-4 1:200, CDK4 1:200 Ab-1(Neomarkers, Fremont, CA); poly(ADP-ribose) polymerase C-2-10 1:1000 (BD Biosciences, Oxford, United Kingdom); totalRNA polymerase II AB817 1:5000 (Abcam, Cambridge, UnitedKingdom); total glyceraldehyde-3-phosphate dehydrogenase1:5000 (Chemicon, Temecula, CA); goat anti-rabbit and goatanti-mouse horseradish peroxidase–conjugated secondary anti-bodies 1:5,000 (Bio-Rad, Hercules, CA). Western blots werequantified by densitometry with Image Quant (AmershamBiosciences). Using a high-throughput 96 well plate-based‘‘in-cell western’’ approach, RB phosphorylation was assessed inintact cells as described previously (23).

Flow cytometry. HCT116 cells (2.5 � 105) were seeded intoa T25 flask and left for 36 hours to attach to the plastic. Drugtreatments were done as above. Cells were harvested in 1 mLtrypsin-versene by incubating at 37jC for 5 minutes to form acell suspension, gently pelleted and resuspended in 100 ALice-cold PBS, and fixed by slow addition of 1 mL ice-cold70% ethanol while vortexing. Cells were resuspended in 200AL of 0.02 mg/mL propidium iodide (Molecular Probes,Cambridge, United Kingdom)/0.25 mg/mL RNase A (SigmaChemical) and incubated at 37jC for 30 minutes. Sampleswere analyzed on a Beckman Coulter Elite ESP (BeckmanCoulter, High Wycombe, United Kingdom) and cell cycleanalysis was done with WinMidi2.8 software (ScrippsResearch Institute, La Jolla, CA).

Results

Activity against recombinant kinases in vitro. The ability ofthe trisubstituted aminopurine CDK inhibitors olomoucine,bohemine, and CYC202 to inhibit recombinant kinases wasassessed and the results are reported in Table 1. CYC202 wasclearly the most potent inhibitor against all kinases tested. Thekinase most potently inhibited by CYC202 was CDK2/cyclin E(IC50, 0.13 Amol/L), closely followed by CDK7/cyclin H (IC50,0.46 Amol/L) and CDK9/cyclin T1 (IC50, 0.78 Amol/L). Theinhibition of CDK1, CDK4, CDK6, and extracellular signal–related kinase 2 (ERK2) was much less.

Activity against human tumor lines in cell culture. Table 2shows the activities of the three analogues against a range ofhuman tumor cell lines. Mean IC50 values across the panel were56 Amol/L for olomoucine, 27 Amol/L for bohemine, and15 Amol/L for CYC202. The mean value for CYC202 wasidentical to that of 15 Amol/L found in an independent panel(13). These relative potencies for the three aminopurinescorrelated with the increased potency of CYC202 overbohemine and olomoucine observed in CDK inhibition assays(Table 1). Bohemine and CYC202 showed a broad spectrum ofin vitro anticancer activity, which was not directed towards anyparticular tumor cell type.





Included within the cell line panel were sublines possessingacquired drug resistance to cisplatin (CisR lines; ref. 24) anddoxorubicin (DoxR line, with resistance mediated by over-expression of P-glycoprotein; ref. 25). In these paired cell lines,no cross-resistance was observed to the CDK inhibitors. Also ofinterest is the possible influence of RB and TP53 status indetermining cellular sensitivity to CDK inhibitors. A comparisonof sensitivity of the widely used pair of human osteosarcomalines (SAOS-2, RB negative and U2-OS, RB positive; ref. 26)suggests little or no effect of RB status on sensitivity. In contrast,when comparing two breast cancer cell lines which differ in TP53status (MCF-7, wild-type TP53 and MDA-MB-231, mutant TP53;ref. 27) the mutant cell line was f2-fold more resistant than thewild-type line. Previous studies in a human cancer cell line panelshowed a similar modest difference (13). However, thesensitivity to CYC202 of an isogenic variant of the HCT116human colon carcinoma cell line in which TP53 was knockedout by homologous recombination (HCT116 TP53�/�; ref. 18)was essentially no different from that of the vector control(HCT116 TP53+/+) or parental cell line (Fig. 2A).

Time of exposure required for in vitro activity. The effect ofvarying times of exposure to CYC202 and bohemine wasinvestigated using the HCT116 parental human colon cancercell line in monolayer culture. Following the times of exposureshown in Fig. 2B, the compounds were washed off and cellgrowth inhibition determined at 96 hours using the sulforhod-amine B assay. Between 8 and 16 hours of drug exposure wasrequired to achieve the maximum growth inhibitory effect.Similar data were obtained for the human ovarian carcinomacell line A2780 when exposed to olomoucine, bohemine, andCYC202 (data not shown).

Molecular pharmacodynamic markers in vitro. We investi-gated potential molecular pharmacodynamic markers ofresponse to the three aminopurines in the HCT116 cell line(Fig. 3B). This line was selected because it was among the mostsensitive of those tested (Table 2) and was suitable for use as axenograft in subsequent in vivo studies. Figure 3B shows theeffect of CYC202, bohemine, and olomoucine on the phos-phorylation status of RB following treatment of cells for24 hours with 3 � IC50 of these agents as measured by thesulforhodamine B assay at 96 hours (Table 2). Cell counts were

done at this time in the same samples from which molecularanalysis was carried out. Cell number was reduced by 60% to70%, confirming that similarly effective concentrations of thethree agents were being used (Fig. 3A). It is clear that RBphosphorylation was reduced by pharmacologically activeconcentrations of the three aminopurines. This was shownboth by a gel mobility shift to the hypophosphorylated formand also by a major reduction (z70%) in phosphorylation atall sites tested, including the proposed CDK2-preferred site atThr821 (Fig. 3B). The decrease in RB phosphorylation wasshown not to be due to a loss of total RB protein asdemonstrated by the use of the antibody that recognized theSer608 site in the nonphosphorylated state (Fig. 3B), the signalfor which increased as phosphorylation was lost. Concentra-tions of the three inhibitors that elicited a similar inhibition ofcell growth exhibited comparable inhibition of RB phosphor-ylation (Fig. 3C). Inhibition of RB phosphorylation in HCT116cells by CYC202 was confirmed and quantified using a DELFIA-based assay measuring RB phosphorylation at Ser608 followinga 24-hour exposure (Fig. 3D). The IC50 for inhibition of RBphosphorylation by CYC202 was 18 Amol/L. This correlateswell with the 50% to 60% inhibition of proliferation seen atthis concentration, as determined by cell counts.

Table 1. Inhibition of recombinant or purified kinasesin vitro by olomoucine, bohemine, and CYC202

Kinase Compound IC50F SD (Mmol/L)

Olomoucine Bohemine CYC202

CDK2/E 0.94F 0.35 4.6F1.8 0.13F 0.07CDK2/A 8.8F 1.0 83F 9 2.2F 0.7CDK1/B >100 >100 14.1F2.7CDK4/D1 45F1 >100 14.7F 3.6CDK6/D3 136F 29 >200 50F 6CDK7/H 0.60F 0.10 >20 0.46F 0.09CDK9/T1 2.03F 0.38 2.7F1.3 0.78F 0.15ERK2 60F 13 52F 5 36F 4

NOTE: IC50 values are expressed as Amol/L and are the mean (FSD) of atleast two independent determinations.

Table 2. In vitro growth inhibitory activityofolomoucine,bohemine, and CYC202 in a panel of cancer cell lines

Tumor type Cell line 96-h sulforhodamine B IC50(Mmol/L)

Olomoucine Bohemine CYC202

Ovarian A2780 30 12.5 4.9A2780CisR 45 17 8.4CH1 48 21.5 7.7CH1CisR 78* 22 9.3CH1DoxR 86* 19 7.4SKOV-3 >50 81* 31

Colon BE >50 25 17.5HT29 58 27.7 20.3Mawi >50 28.5 18Lovo >50 25 20SW620 >50 27 23HCT116 52* 17 6.9COLO-205 41 21 8.5KM12 90 32 15

Osteosarcoma SA-OS2 78 38 16.5U2-0S 101.5* 36 15

Breast MCF 7 64 21.5 7.8MBMDA231 101.5* 40 15

Lung A549 54.5 20 9.3MOR 66 36 12.5HX147 106.5* 38 19CORL23 54 29.5 10.5

Testicular GCT27 47 14.5 5.2Murine colon Colon 26 >100 54 42.5Mean 56.29 27.11 14.63

NOTE: IC50 values (Amol/L)were obtainedusing the 96-hour sulforhodamineB assay. Results shown aremean values of at least three determinations.*Results obtained by extrapolation.





In addition to decreases in RB phosphorylation, all threeaminopurine compounds reduced the expression of cyclin D1(77-100% decrease; Fig. 3B-C). Cyclins A and B1 also showeddecreased expression but to a lesser extent (8-75% reduction).Cyclin E was least sensitive to inhibition by the three compoundsbut was also reduced (4-52% loss). The expression of CDK1 andCDK2 showed a modest decrease in response to the compounds(16-40% reduction). However, the expression of CDK4 wasgreatly reduced following exposure to all three inhibitors(f80% loss). The expression of phosphorylated RNA polymer-ase II was also reduced by all three compounds, as shown by aswitch to the lower, hypophosphorylated form. This is consistentwith inhibition of CDKs 7 and 9. In support of inhibition ofCDK7 (28) Thr160 phosphorylation on CDK2 was reducedfollowing 8 to 24 hours exposure to 3� IC50 CYC202 (data notshown). In addition, a partial cleavage of poly(ADP-ribose)polymerase was observed following a 24-hour exposure to thesecompounds, indicative of apoptosis (Fig. 3B-C).

In contrast to previous results in HT29 and KM12 humancolon cancer cells (15), no increase in phosphorylated ERK1/2was observed in response to the three compounds (Fig. 3B-C),possibly because the HCT116 cell line has a Kirsten-RASmutation (29).

Treatment of HCT116 cells with CYC202, bohemine andolomoucine resulted in a 11% to 15% loss of cells from the Sphase of the cell cycle with a 6% to 17% increase in theproportion of cells in the G2-M phase after 24 hours oftreatment, as determined by propidium iodide staining andanalysis by flow cytometry (Fig. 3E). Cells in the S phase wereconfirmed to have ceased DNA replication as determined bybromodeoxyuridine incorporation (data not shown). A sub-G1

fraction was also seen with all three agents, indicative ofapoptosis. Taken together, the above results are consistent withthe agents acting as CDK inhibitors although other possibilitiescannot be ruled out.

Maximum tolerated dose in BALB/c� mice. Given i.v., thesingle dose maximum tolerated dose for both bohemine andCYC202 was 100 mg/kg. For olomoucine, the maximumtolerated dose was higher at 200 mg/kg. Subsequent i.v. studieswere done with 50 mg/kg of the agents. At 150 mg/kg, CYC202was well tolerated when given i.p. Following a single oraladministration, CYC202 was well tolerated up to 2,000 mg/kg.

Analytic results and pharmacokinetic variables. LC-MS/MSproved to be specific, sensitive, and reproducible in bothanalytic systems described in Materials and Methods. Whereasonly 100 ng/mL of each trisubstituted aminopurine could bedetected with UV, the limit of quantification with LC-MS/MSwas 3.5 ng/mL or 10 nmol/L. The interbatch precision andaccuracy at 25, 90, 5,000, and 50,000 nmol/L were below 15%.These values are in accordance with the established guidelinesfor assay validation.

Tables 3 and 4 show the pharmacokinetic variables forCYC202, olomoucine, and bohemine in plasma, liver, kidney,and colon 26 tumor in BALB/c� mice following a dose of50 mg/kg i.v. Variables were obtained using noncompartmentalanalysis. Plasma levels decayed in a biexponential fashion in allthree cases (Fig. 4). All compounds cleared rapidly fromplasma. CYC202 had the highest plasma concentrations andexhibited the longest half-life (1.19 hours) and the slowestclearance of 43 mL/h. Drug uptake from the general circulationwas very rapid for all three compounds with Cmax observed intissues at the sampling time of 0.25 minutes (Figs. 4 and 5). Thetumor to plasma AUC ratios were 0.71, 0.45, and 0.18 forCYC202, olomoucine, and bohemine, respectively. These ratioswere higher than those observed for kidney and spleen butlower than the corresponding liver to plasma ratios. Theterminal half-life for the elimination of CYC202 from tissueswas greater than that observed for the other two analogues(Table 4). Overall, CYC202 exhibited the highest AUC, thelongest plasma t1/2, and the highest tissue to plasma ratio of thethree analogues.

Figure 5 shows the plasma concentration versus time curvesfollowing i.p., i.v., and oral administration of 50 mg/kgCYC202. The pharmacokinetic data in Table 5 show that rapidabsorption occurred from the peritoneal cavity, with maximalplasma concentrations observed 5 minutes after administra-tion. The concentrations obtained by this route of administra-tion were very similar to those observed by the i.v. route withan overall i.p. bioavailability of 67% (Table 5). There was no

Fig. 2. A , effect ofTP53 status on sensitivity to CYC202 in HCT116 human coloncarcinoma cells. HCT116 parental cells (PAR,WT TP53), vector control (WT,WTTP53), orTP53 knock out (NULL,TP53 null) were exposed to increasing doses ofCYC202 for 96 hours. Cell growthwas assayed using the sulforhodamine B assayand IC50 values were calculated graphically. Columns, mean (n = 3); bars, FSD.Statistical analysis of the mean IC50 values by a one-wayANOVA test showedno significant differences (P > 0.05). B, effect of time of exposure on response toCDKIs. HCT116 cells in logarithmic growthwere exposed to increasingconcentrations of either bohemine or CYC202 for 4, 8,16, 24, and 96 hours, thecompounds were washed off and cell growth assayed by sulforhodamine B after atotal of 96 hours. Points, mean IC50 values (n = 3); bars, FSD.The IC50 following a4-hour exposure to bohemine was >200 Amol/L, as indicated by the vertical arrow.





Fig. 3. Cellular and molecular responses to CDK inhibitors.HCT116 cells were exposed to equiactive doses of CYC202,bohemine or olomoucine (3� 96 hours IC50) for 24 hours.A , attached cell number following exposure to the CDKIs(n = 6 from two independent experiments).B,Western blotsfor the indicated proteins following analysis of cell lysates(50 Ag protein). GAPDHwas used as loading control.C, densitometric analysis of theWestern blots in (B) ascarried out in Image Quant. Columns , mean (n = 3);bars,FSE.D, inhibition of RB phosphorylation at Ser608 inHCT116 cells using a 96-well plate-based DELFIA assay.Points , mean from one of two independent experiments;bars,FSD. E, cell cycle distribution of fixed cells usingpropidium iodide and flow cytometry. Columns, mean from2 independent experiments (n = 6); bars,F SD.





increase in terminal half-life by this route of administrationsuggesting that delayed absorption did not occur.

CYC202 concentrations peaked 15 minutes after oraladministration, and the terminal half-life increased comparedwith that measured after i.v. or i.p. administration (2.77 hoursfor oral dosing versus 1.38 hours by the i.v. route). The oralbioavailability was 86%. Following increasing oral doses ofCYC202, the half-life was extended from 2.88 to 6.67 hoursand 11.9 hours. Plasma concentrations were maintained above15 Amol/L (the mean IC50 level across the human tumor cellline panel) for 4, 12, and 24 hours following 50, 500, and2,000 mg/kg, respectively (Table 6; Fig. 6). Following increasedoral doses, the AUC increased 10-fold from 50 to 500 mg/kgbut only a further 2-fold from 500 to 2,000 mg/kg. Thisdifference was also reflected in the Cmax , which did not increaselinearly with dose. It should be noted that the plasma AUCmeasured following 50 mg/kg CYC202 in the Solutol/Lutrolvehicle was 35% lower than that observed using the HCl salinevehicle at the same dose level. This could be explained bydifferences in Cmax as the t1/2 values were identical. The resultssuggest the possibility that acid conditions may favor oralabsorption of CYC202.

Computer simulation experiments derived from variables inTable 5 showed that to maintain a concentration of 15 Amol/Lover 24 hours (mean IC50 level), CYC202 would need to begiven orally thrice a day at 200 mg/kg or twice a day at 500 mg/kg (Fig. 7). Protein binding was similar at 2 and 20 Amol/L. Inmouse plasma 97.2% of CYC202 was bound to proteins as

early as 5 minutes after incubation and the binding was thesame 6 hours after incubation. In human plasma, the proteinbinding increased slightly but gradually from 92% to 96% overa period of 5 minutes to 6 hours.

Pharmacokinetic-pharmacodynamic analysis. To explore thepharmacokinetic-pharmacodynamic relationships in vivo , theHCT116 human colon cancer xenograft model in nude micewas used to determine the antitumor effect, tumor drugconcentrations, and alterations in molecular biomarkers. Giventhe high oral bioavailability for CYC202 reported here and theattractiveness of oral dosing for clinical application, this routewas selected for the pharmacokinetic-pharmacodynamic stud-ies. Previous studies showed that CYC202 was tolerated intumor-bearing nude mice at a dose of 500 mg/kg thrice a day(13). Based on the pharmacokinetic simulation (Fig. 7), micewere treated with 500 mg/kg orally CYC202 twice per day for 5days. In the experiment shown in Fig. 8, the mean body weights(expressed as percentage of the starting weight on day 0) were102%, 101%, 101%, 101%, and 95% on days 1 to 5 comparedwith 99%, 98%, 98%, 98%, and 98% for the vehicle controls.One of the eight animals died in the treated group during thisperiod (day 2). CYC202 caused a reduction in the growth ofestablished tumors (f130 mm3) compared with controls forthe duration of treatment (Fig. 8A). %T/C values were 79%,75%, 72%, and 65% at days 2, 3, 4, and 5, respectively (P <0.05). This is comparable with the T/C value of 47% that wasreported in the MESSA-DX5 human uterine carcinoma xeno-graft using a comparable schedule of 500 mg/kg orally thrice a

Table 4. Tissue pharmacokinetic variables for kidney, liver, and colon 26 tumor derived from Winnonlin non-compartmental analysis following administration of 50 mg/kg i.v. olomoucine, bohemine, and CYC202 toBALB/c� mice

Tissue Compound Cmax

(nmol/L)Tmax(h)

AUClast(h nmol/L)

AUCINF(observed,h nmol/L)

Cl (predicted)/F (L/h)

Vz (observed)/F (L)

t1/2�z(h)

Kidney Olomoucine 15,536 0.50 8,079 ND ND ND NDBohemine 5,936 0.25 2,363 2,395 1.23 2.16 1.23CYC202 29,063 0.25 43,475 43,904 0.06 0.08 0.90

Liver Olomoucine 21,986 0.25 18,027 18,051 0.19 0.16 0.60Bohemine 8,626 0.25 6,524 6,670 0.44 0.60 0.94CYC202 25,773 0.25 61,099 62,175 0.05 0.31 4.81

Tumor Olomoucine 39,124 0.25 18,913 18,941 0.18 0.15 0.57Bohemine 6,386 0.25 2,369 2,461 1.19 1.40 0.82CYC202 24,548 0.25 46,742 47,946 0.06 0.33 3.86

Abbreviation: ND, not determined.

Table 3. Plasma pharmacokinetic variables derived from Winnonlin noncompartmental analysis followingadministration of 50 mg/kg i.v. olomoucine, bohemine, and CYC202 to BALB/c�mice

Compound Cmax

(nmol/L)AUClast(h nmol/L)


Cl (observed, L/h) Vz (observed, L) t1/2�z (h)

Olomoucine 59,300 41,059 41,075 0.07 0.06 0.58Bohemine 72,308 12,901 12,931 0.23 0.45 1.39CYC202 40,457 65,596 67,570 0.04 0.08 1.20





day for 4 days (13). To determine if the inhibition of tumorgrowth was related to and consistent with the proposedmechanism of action of CYC202, tumor lysates were preparedand subjected to analysis by Western blotting for RBphosphorylation (Fig. 8B). Vehicle-treated tumor lysatesexhibited readily detectable levels of total and phosphorylatedRB. Mice treated with CYC202 on the above schedule for 1 dayshowed a decrease in total RB expression when measured at4 hours after the second of the two daily doses (30% reductionas determined by densitometry). After 5 days of treatment, RBprotein expression decreased further (55% reduction). Inhibi-

tion of RB phosphorylation at specific phosphorylation siteswas determined using phospho-specific antibodies. Whereasthe signal for all sites was markedly decreased after 3 to 5 daysof treatment, the proposed CDK2-preferred Thr821 site was themost sensitive (95% inhibition on day 5). In addition, cyclinD1 levels were decreased by 40% to 77% after 3 to 5 days oftreatment. Both the RB and cyclin D1 biomarker changes weresimilar to those described earlier for HCT116 cells treatedin vitro. Also in agreement with the in vitro data, no increase inERK phosphorylation was observed in vivo in response toCYC202.

To relate the molecular marker changes to drug exposure,CYC202 concentrations in the HCT116 tumor and plasmawere determined by LC-MS/MS using samples from the samegroup of mice used for the biomarker measurements (Fig. 8C).Plasma concentrations were in accordance with predicted levelsfrom the computer simulation described earlier. Tumorconcentrations measured at 4 hours after the second of thetwice daily doses were greater than those required to inhibit thegrowth of HCT116 cells in cell culture (IC50 of 6.9 Amol/L;Table 2) on days 1 and 3. However, the concentration ofCYC202 was markedly reduced on day 5 (4 hours after the lastdose) in both plasma and tumor. In a single oral doseexperiment at 500 mg/kg, tumor concentrations were abovecell proliferation IC50 levels for >12 hours and the final half-life was 3 hours (data not shown).

Discussion

The purpose of this study was to investigate the propertiesof three trisubstituted aminopurine CDK inhibitors, olomou-cine, bohemine, and CYC202, with particular emphasison pharmacokinetic-pharmacodynamic relationships forCYC202. A detailed understanding of the pharmacokinetic-pharmacodynamic properties should facilitate rational selec-tion of a dosing schedule that would support therapeuticactivity in tumor xenograft models (30). Identification ofappropriate pharmacokinetic-pharmacodynamic variablesallows construction of a ‘‘pharmacologic audit trail’’ (31, 32)to aid interpretation of preclinical data and for use insubsequent clinical trials.

Fig. 4. Plasma (A), colon 26 tumor (B), liver (C), and kidney (D) concentrationversus time curves following administration of 50 mg/kg olomoucine (5), CYC202(w ), and bohemine (o), i.v. in 50 mmol/LHCl saline to BALB/c mice.

Fig. 5. CYC202 plasma concentration versus time curves following 50 mg/kgi.v. (5), i.p. (o), oral administration (w ) in Solutrol Lutrol vehicle to femaleBALB/c�mice.





CYC202 was the most potent of the three aminopurineanalogues in terms of in vitro CDK2 inhibition and growthinhibition in a human cancer cell line panel. Interestingly, it wasactive in cell lines resistant to doxorubicin and cisplatin. Also ofnote is the lack of dependence on RB status as exemplified bysimilar IC50 values in the SAOS-2 versus U2-OS osteosarcomacell lines. Similarly, TP53 status had little effect, if any, on thegrowth inhibition. Comparison of the HCT116 isogenic cell pair,differing only in TP53 status, showed near-identical IC50 valuesfor CYC202. Together with previous data (13), these resultssuggest that the aminopurine analogues may exhibit broad-spectrum antitumor activity. The differences in the CDKinhibitory potency among the three purine analogues werereflected in their relative cancer cell growth inhibitory activities,CYC202 being more potent than bohemine which was in turnmore active than olomoucine. This is consistent with CDKinhibition being a major contributor to the antiproliferativeeffects but does not exclude other possible cellular effects.

We showed that olomoucine, bohemine, and CYC202 allreduced RB phosphorylation at pharmacologically relevantconcentrations that inhibit cell growth in cell culture. Althoughfunctional RB was not required for an antiproliferative effect,RB phosphorylation was nevertheless a valuable indicator ofCDK activity and could be useful in those tumors that arepositive for RB. Loss of RB phosphorylation in HCT116 cellswas concomitant with cell growth inhibition, as seen withCYC202 in HT29 and KM12 human colon cancer cells (15).Assessing all the combined data, we concluded that inhibitionof RB phosphorylation is related to in vitro potency in RB-positive cancer cell lines, whether comparing between thedifferent analogues in a given cell line or comparing the effectsof CYC202 across different lines.

Inhibition of RB phosphorylation in HCT116 cells wasshown by the gel mobility shift seen with the hypophos-phorylated form of RB and also by antibodies to specificphosphorylation sites. Consistent with inhibition of CDK2,

phosphorylation at Thr821 (a CDK2-preferred RB phosphoryla-tion site) was most affected by CYC202. There was some loss oftotal RB protein, but this was not responsible for reducedphosphorylation, as shown by the increase in nonphosphory-lated Ser608. Over the same concentration range and timecourse as the effects on RB phosphorylation were shown, theanalogues increased cell number in G2-M phase and decreasedthe S-phase fraction. Use of a quantitative DELFIA assayshowed that RB phosphorylation at Ser608 was inhibited by50% following 24 hours exposure to 18 Amol/L CYC202,correlating well with the 50% to 60% decrease in cell number,compared with controls, observed with this treatment.

Inhibition of RB phosphorylation and the above cell cyclechanges could be occurring through inhibition of CDK1/cyclin Bor of CDK2. Introduction of dominant-negative CDK2 intocancer cell lines has been shown to cause an arrest in G2-M (33).We also showed that CYC202, bohemine, and olomoucinecaused a marked decrease in cyclin D1 expression and a smallerloss of cyclins A and B1, with cyclin E affected much less.Decreased expression of cyclin D1 and other cyclins in HCT116cells also occurred in HT29 and KM12 cells (15). In addition,CYC202 decreased CDK4 expression in HCT116 cells, an effectnot seen in HT29 or KM12 cells previously (15). The data areconsistent with the analogues acting directly or indirectly (orboth) as inhibitors of CDKs, although other cellular mecha-nisms are not excluded. The compounds can inhibit activity ofthe transcription-related CDKs, CDK7 and CDK9 (Table 1),which are involved in phosphorylation of RNA polymerase II.Reduced phosphorylation of RNA polymerase II was observed inHCT116 cells in vitro. Inhibition of T160 phosphorylation onCDK2, mediated by CDK7, was also shown. Therefore, thereduction in cyclin expression may occur through transcriptionalinhibition due to reduced CDK7 and CDK9 activity (15). Geneexpression profiling can be used to evaluate such effects (34).The relative decreases in the individual cyclin proteins mayreflect differential protein half-lives following an inhibition of

Table 5. Plasma pharmacokinetic variables derived from Winnonlin noncompartmental analysis followingadministration of 50 mg/kg CYC202, i.p., i.v., and orally to BALB/c�mice

Route Cmax

(nmol/L)Tmax(h)


AUClast(h nmol/L)

F Cl (observed)/F (L/h)

Vz (observed)/F (L)

t1/2�z(h)

MRTlast(h)

i.p. 72,756 0.083 75,455 75,397 0.67 0.037 0.14 2.67 2.01i.v. 130,473 0.083 111,900 111,892 1 0.03 0.10 1.97 1.56oral 30,831 0.25 95,348 94,816 0.86 0.033 0.13 3.27 3.70

Table 6. Plasma pharmacokinetic variables derived from Winnonlin noncompartmental analysis followingadministration of 50, 500, and 2,000 mg/kg CYC202 orally to BALB/c�mice

Dose(mg/kg)

Cmax

(nmol/L)Tmax(h)

t1/2�z(h)

MRTINF(observed, h)

AUClast(h nmol/L)

Vz (observed)/F (L)

Cl (predicted)/F (L/h)


50 25,628 0.25 2.9 3.5 95,199 0.12 0.03 95,428500 80,269 0.5 6.7 7.7 906,880 0.28 0.03 966,1952,000 108,266 2 11.9 16.2 1,723,508 0.85 0.05 2,282,182





mRNA expression downstream of CDK7 and CDK9 inhibition.Interestingly, the most marked effect occurred with the G1 phase-active cyclin D1, whereas the cells predominantly arrest in theG2-M phase of the cell cycle. Thus, CDK inhibition (see above)and/or loss of other proteins is more likely to contribute to theG2-M phase arrest. Loss of several cyclins may inhibit multipleCDK activities and may therefore block cell proliferationindependent of CDK2 activity, which has been reported asdispensable for colon cancer cell proliferation (7). It should beemphasized that CDKs phosphorylate important cellular sub-strates other than RB (35–48). As noted previously (15), thismay explain the activity of the aminopurine CDK inhibitors suchas CYC202 on cells lacking RB.

A sub-G1 peak was seen in HCT116 cells after treatment withCYC202 and its analogues. An increase in poly(ADP-ribose)polymerase cleavage provided biochemical evidence of apo-ptosis, consistent with positivity in the terminal deoxynucleo-tidyl transferase–mediated nick-end labeling assay reportedpreviously for CYC202 (13). Thus, over the concentration rangein which olomoucine, bohemine, and CYC202 reduced RBphosphorylation and cyclin expression, these compoundscaused not only a G2-M arrest and a decrease in the S-phasefraction but also the induction of apoptosis. Transformed andnormal cells may differ in their requirements for CDK2 (8).Phosphorylation of E2F by CDK2/cyclin A is a majortranscriptional control point, and modulation by CDK2inhibitors such as CYC202, might cause deregulation of E2Fand induction of apoptosis (8, 9).

Our previous studies have shown that ERK1/2 phosphoryla-tion is induced by CYC202 in HT29, KM12, and NIH3T3 cellsin a mitogen-activated protein kinase kinase 1/2–dependentmanner (15). Interestingly, the effect was not seen here inHCT116 cells. This may be because the Kirsten-RAS mutationin HCT116 cells (29) causes constitutive activation of ERK1/2,reducing the opportunity for further activation by CYC202.

Pharmacokinetic properties are frequently critical in thetranslation of in vitro anticancer properties into therapeuticactivity in an animal model, in the selection of a clinicaldevelopment candidate and in the subsequent clinical evalu-ation. The growth inhibitory IC50 for bohemine and CYC202decreased markedly with increased exposure time. Maximumgrowth inhibitory effect was seen after 8 to 16 hours. These

results indicate that pharmacokinetic behavior is likely to becritical for in vivo activity. Here LC-MS/MS provided rapid,specific, and sensitive analysis of olomoucine, CYC202, andbohemine in biological tissues and fluids. As mentioned, thetime dependence of the effects of CYC202 and boheminein vitro emphasized the likely importance of pharmacokineticbehavior in vivo. All three compounds distributed quickly intomouse liver, kidney, and tumor tissue but also cleared quiterapidly from plasma and tissues. Bohemine cleared the fastestand CYC202 the slowest with olomoucine intermediate.Although CYC202 plasma protein binding was rapid andextensive (97%), it is important to note that this did not limitdrug distribution, which was equally fast. Uptake into tumortissue, as exemplified by the colon 26 transplantable syngeneicmouse tumor, was quite different for the three agents, withAUC values ranging from 18% of plasma for olomoucine to71% for CYC202. Tissue clearance rates were comparable tothose of plasma. CYC202 clearance is via oxidative metabolismof the side chain hydroxyl group to form the carboxylic acid,subsequently excreted in urine (49). The pharmacokineticsbehaviour described here was predictive of that in healthy malesubjects receiving single doses up to 800 mg CYC202 (50). Ofthe three analogues, CYC202 showed the best pharmacokineticprofile with the slowest clearance and highest tissue distribu-tion. The nanomolar potency towards CDK2/cyclin E takentogether with the high cellular potency and the better tissuedistribution and clearance data, indicated that CYC202 had themost suitable overall properties for further therapeutic evalu-ation. It was therefore selected for pharmacokinetic-pharmaco-dynamic studies in the same HCT116 human colon cancermodel as used in vitro.

Despite rapid distribution and high micromolar peak levels,tumor concentrations at the maximum tolerated single dose ofCYC202 were not sustained above the in vitro IC50. Thisindicated the need to lengthen drug exposure by continuousinfusion or repeated administration. The high bioavailability,both i.p. (67%) and orally (86%), suggested both routes would

Fig. 6. CYC202 plasma concentrations versus time curves following administrationof 50 (o), 500 (5), and 2,000 mg/kg (w ) CYC202 orally to BALB/c�mice.

Fig. 7. Pharmacokinetic simulations for CYC202 following administration of (A)500 mg/kg orally twice daily and (B) 200 mg/kg orally thrice daily.





be suitable for multiple dosing in tumor xenografts. Thereseemed to be saturation in oral absorption from 500 to 2,000mg/kg, but the AUC increased 10-fold with a 10-fold increase indose from 50 to 500 mg/kg. Based on pharmacokineticvariables obtained with single doses, our multiple dosepharmacokinetic simulation study indicated that CYC202should be given thrice a day at 200 mg/kg or twice a day at500 mg/kg to sustain therapeutic exposures. Concentrationsmeasured after 500 mg/kg orally twice daily showed that thesimulation was quite predictive for days 1 to 4 of dosing.However, the decrease in plasma and tumor levels after 5 dayssuggests metabolic induction or decreased absorption. Impor-tantly, tumor concentrations were well above the growthinhibitory concentrations before day 5.

In the pharmacokinetic-pharmacodynamic studies, RB phos-phorylation was measured to determine whether CDKs werelikely to have been inhibited by CYC202 and to assess whetherthis was a valid PD biomarker in the xenograft tumor model. Aspredicted from the in vitro sensitivity of HCT116 cells and thesimulated and measured tumor pharmacokinetics, CYC202showed antitumor activity against HCT116 tumor xenograft forthe duration of the 5-day treatment with T/C values in the rangeof 79% to 65%. A comparable T/C value of 47% was reportedfor the MESS-DX5 human uterine carcinoma xenograft using asimilar regimen of 500 mg/kg orally thrice a day for 4 days (13)and the same schedule has been used in a Phase I clinical trialof CYC202 (51, 52). More prolonged drug administration atthe dose level used was not well tolerated. Progressive reduction

Fig. 8. Pharmacokinetic-pharmacodynamic relationships for CYC202 in the HCT116 human colon cancer carcinoma xenograft model.A , effect of 500mg/kg orally CYC202twice daily on the growth of established HCT116 human colon carcinoma tumor xenografts (treated atf130mm3) growing s.c. in nude mice. Points, mean relative tumorvolume; bars, FSE. %T/C values are indicated. P < 0.05 for treated versus control tumors. B, changes in various molecular biomarkers in the HCT116 tumor xenograft inresponse to CYC202 treatment as assessed byWestern blotting. GAPDHwas used as the loading control. C, tumor and plasma concentrations of CYC202 corresponding tosamples used in (A) and (B) as determined by LC-MS/MS.The in vitro IC50 of 6.9 Amol/L for HCT116 cell growth inhibition is marked by a horizontal line.D, densitometricanalysis of theWestern blots in (C), as carried out in Image Quant.





of tumor RB phosphorylation and cyclin D1 expressionoccurred over the 5 days of treatment. As mentioned, aconcentration of 18 Amol/L CYC202 is required to inhibit RBphosphorylation by 50% in cell culture after a 24-hourexposure. Tumor concentrations measured 4 hours after thesecond dose exceeded 18 Amol/L on days 1 and 3. Given thatthe exposure to CYC202 is not continuous in the mouse butrather is intermittent due to repeated dosing and subsequentclearance of the compound, it is probably not surprising that>24 hours is needed to achieve a measurable effect on RBphosphorylation in the HCT116 xenograft. The sustained PDeffects at 5 days, which occur despite the lower CYC202 tumorconcentrations at this time, remain unexplained. Differences intumor microenvironment for in vivo tumor xenografts com-pared with in vitro cell culture may play a role.

It is important to stress that although the inhibition of RBphosphorylation in the human tumor xenograft is consistentwith CYC202 acting as a CDK inhibitor in vivo, whether this isdirect via inhibition of CDK catalytic activity or indirect viadepletion of cyclins and CDK4 is unclear. Regardless of theprecise mechanism, our results indicate that RB phosphoryla-tion may be a useful pharmacodynamic marker that generallycorrelates with the antitumor activity of CYC202, and suggestthat cyclin D1 may be an additional biomarker. Cyclin D1 maybe especially useful in situations where RB phosphorylation isnot detectable, as in RB-negative tumors. With respect toclinical application, these measurements require suitable tumoror surrogate tissue to be available before and after treatment.

We have shown that a detailed understanding of the in vitroCDK inhibitory potency and cellular activity of this group oftrisubstituted aminopurines, coupled to a similarly detailedanalysis of pharmacokinetic and pharmacodynamic propertiesin vivo , led to the rational selection of CYC202 for tumorxenograft studies. Based on the knowledge of the drug exposuresrequired for antiproliferative activity and of the PK properties ofCYC202, computer simulation of drug concentrations washelpful in developing an oral dosing schedule that allowedtherapeutic activity to be shown at exposures causing inhibitionof RB phosphorylation and cyclin D1 depletion in tumor tissue.The data presented constitute a ‘‘pharmacologic audit trail,’’particularly in relation to pharmacokinetic-pharmacodynamicrelationships (31, 32). The construction of such an audit trail canimprove the quality of decision-making in preclinical drugdevelopment. Together with the antitumor activity in otherxenograft models reported elsewhere using similar schedules(13), these results support the ongoing development of CYC202,which is now undergoing Phase II clinical trial (51).

Acknowledgments

We thank the members of the Signal Transduction and Molecular PharmacologyTeam and the Cell Cycle ControlTeam in the Cancer Research UKCentre for CancerTherapeutics, together with colleagues at Cyclacel Ltd., for valuable discussions;JennyTitley of the Cell Cycle ControlTeam for flow cytometry; and the protein bio-chemistry and the assay development/screening teams at Cyclacel for kinaseassays.

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2005;11:4875-4887. Clin Cancer Res Florence I. Raynaud, Steven R. Whittaker, Peter M. Fischer, et al. and CYC202Cyclin-Dependent Kinase Inhibitors Olomoucine, BohemineRelationships for the Trisubstituted Aminopurine

Pharmacokinetic-PharmacodynamicIn vivo and In vitro

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