Cancer Therapy: Preclinical

In vitro and In vivo Pharmacokinetic-Pharmacodynamic Relationships for the Trisubstituted Aminopurine Cyclin-Dependent Kinase Inhibitors Olomoucine, Bohemine and CYC202 Florence I. Raynaud,1Steven R. Whittaker,1Peter M. Fischer,2 Steven McClue,2 Michael I. Walton,1 S. Elaine Barrie,1Michelle D. Garrett,1Paul Rogers,1Simon J. 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 trisubstituted aminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine, and CYC202 (R- roscovitine; seliciclib) in the HCT116 human colon carcinoma model. Experimental Design: The activity of the agents was determined in a human tumor panel using the sulforhodamine B assay.The concentration and time dependence was established in HCT116 cells. Molecular biomarkers, including RB phosphorylation and cyclin expression, were assessed by Western blotting. Pharmacokinetic properties were characterized in mice following analysis by liquid chromatography-tandem mass spectrometry. Based on these studies, a dosing regimen was developed for CYC202 that allowed therapeutic exposures in the HCT116 tumor xenograft. Results: The antitumor of the agents in vitro was in the order olomoucine (IC50, 56 Amol/L) < bohemine (IC50,27Amol/L) < CYC202 (IC50,15Amol/L), corresponding to their activities as cyclin-dependent kinase inhibitors. Antitumor activity increased with exposure time up to 16 hours. The agents caused inhibition of RB and RNA polymerase II phosphorylation and depletion of cyclins. They exhibited relatively rapid following administration to mice. CYC202 displayed the slowest clearance from plasma and the highest tumor uptake, with oral 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 lines in vitro and on xenografts. Pharmacodynamic markers are altered in vitro and in vivo, consistent with the inhibition of cyclin-dependent kinases. Such markers may be potentially useful in the clinical development of CYC202 and other cyclin-dependent kinase inhibitors.

The cell cycle is coordinated through the activities of the cyclin- inhibitors (CDKI) ensure timely activation or inhibition of dependent kinases (CDKs; refs. 1, 2). The CDKs require their the CDK complexes (1, 2). CDKs are deregulated in numerous partner cyclins for activity and these are expressed in a cell ways in cancer. Overexpression of cyclin E has been observed in cycle–dependent manner. In addition, regulatory phosphory- human tumors and is known to result in a poor prognosis in lation events and binding of the cyclin-dependent kinase breast cancer (3). Loss of the CDKI proteins such as p16INK4A is found in many malignancies and can predispose to melanoma (4). Therefore, small-molecule pharmacologic CDK inhibitors Authors’Affiliations: 1Cancer Research UKCentre for CancerTherapeutics atThe are being developed to block cell cycle progression and hence Institute of Cancer Research, Haddow Laboratories, Belmont, Sutton, United to inhibit tumor growth (5, 6). Although the validity of Kingdom and 2Cyclacel Ltd., James Lindsay Place, Dundee, United Kingdom Received 11/9/04; revised 3/21/05; accepted 4/13/05. inhibiting CDK2 alone as a cancer target has been Grant support: Cancer Research UK grant C309/A2187 (P.Workman), Cyclacel questioned by recent data (7), other studies showing selective Ltd. (P. Workman and S.R. Whittaker), the Sir Samuel Scott of Yews Trust killing of transformed cells by a peptide inhibitor of CDK2/ studentship (S.R. Whittaker), Cancer Research UK Gibb Fellowship (D.P. Lane), cyclin A binding to E2F-1 have suggested the possibility that and Cancer Research UK Life Fellowship (P. Workman). CDK2 inhibitors may not only block tumor cell growth but The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance might also preferentially induce apoptosis in tumor cells (8, 9). with 18 U.S.C. Section 1734 solely to indicate this fact. Furthermore, a number of small-molecule CDK2 inhibitors that Note: F.I. Raynaud and S.R. Whittaker, contributed equally to this work. are in development also show inhibitory activity towards CDK1 Requests for reprints: Paul Workman, Cancer Research UK Centre for Cancer as well as the transcriptional kinases CDK7 and CDK9 (6). Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, United Kingdom. Phone: 44-208-722- Combinatorial inhibition of more than one CDK may provide 4301;Fax: 44-208-722-4324; E-mail: [email protected]. greater antitumor activity and overcome resistance that may be F 2005 American Association for Cancer Research. associated with blockade of, for example, CDK2 alone.

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Through an initial screen for CDK1/cyclin B inhibitors, depletion of cyclin D1 and other cyclins (15), the effects of olomoucine was discovered to be a relatively specific and compound treatment on these regulatory proteins was also potent inhibitor compared with the structurally similar but determined. In parallel, comparative effects on cell cycle and more general kinase inhibitor N6-dimethyl-aminopurine (10). cell death were determined by flow cytometry. Following this Further exploration of the trisubstituted aminopurine structure in vitro characterization, the pharmacokinetic properties of the led to the more potent and selective roscovitine (11). Potency three compounds were characterized following administration and selectivity for CDK2 kinase inhibition was further by the i.v., i.p., and oral routes, and the data were reviewed in improved by synthesis and purification of the R-enantiomer relation to the exposures required for in vitro antitumor of roscovitine (CYC202; refs. 10, 12–15). Roscovitine and activity. Comparing the in vitro results and the in vivo other closely related trisubstituted aminopurine analogues can pharmacokinetic behavior, CYC202 emerged as the analogue induce cell cycle arrest and apoptosis in a range of human exhibiting the best profile in both respects. Based on the tumor cell lines (13–16). aforementioned properties, a dosing regimen was developed The objective of the present work was to extend our previous for CYC202, which led to the achievement of antitumor mechanistic studies (15) and in particular to translate our activity in the HCT116 human colon cancer xenograft model. investigations on the in vitro properties of CYC202 and its Inhibition of RB phosphorylation and depletion of cyclin D1 analogues into the in vivo animal model setting. We describe was shown in the tumor xenografts, revealing in vivo the comparative in vitro and in vivo properties of olomoucine, pharmacokinetic-pharmacodynamic relationships. These phar- bohemine, and CYC202 (Fig. 1) with particular regard to macokinetic-pharmacodynamic relationships and other thera- pharmacokinetic-pharmacodynamic relationships and how peutic studies (13) provide support for the development of this information may be used to support the development of CYC202, which is now undergoing phase II clinical trials in CDK inhibitors. Initially, relative potencies of the analogues cancer patients. against a panel of human cancer cell lines in vitro were determined and the influence of length of compound exposure Materials and Methods on in vitro cancer cell proliferation was defined. To show that the compounds were acting in a manner consistent with the Materials. Unless otherwise stated, materials were from Sigma proposed mechanism of CDK inhibition, RB phosphorylation Chemical Ltd. (Poole, Dorset, United Kingdom). was assessed in tumor cells in vitro and the cell cycle effects Test compounds. 2-(6-Benzylamino-9-methyl-9H-purin-2-ylamino)- were determined. In view of the potential importance of the 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 of incorporation of radioactive phosphate from ATP into appropriate polypeptide substrates by purified recombinant human protein kinases and 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 active with appropriate substrates. The reactions were initiated by

addition of Mg/ATP mix (15 mmol/L MgCl2 and 100 Amol/L ATP with 30-50 kBq per well of [g-32P]-ATP) and mixtures were incubated as required at 30jC. Reactions were stopped on ice followed by filtration through p81 filterplates or GF/C filterplates (Whatman Polyfiltronics, Kent, United Kingdom). After washing thrice with 75 mmol/L aqueous orthophosphoric acid, plates were dried, scintillant added, and incorporated 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 in DMSO and diluted into 10% DMSO in assay buffer. Data were analyzed using curve-fitting software (GraphPad Prism version 3.00 for

Windows, GraphPad Software, Inc., San Diego, CA) to determine IC50 values (concentration of test compound which inhibits kinase activity by 50%).

Cell culture

Human tumor cell lines, including the HCT116 (National Cancer Institute, Bethesda, MD) human colon carcinoma, were grown in DMEM (Invitrogen, Paisley, United Kingdom) supplemented with 10% fetal bovine serum (Invitrogen, Paisley, United Kingdom) in an atmosphere of 5% CO2.A matched pair of HCT116 cells isogenic for TP53 was kindly Fig. 1. Chemical structures of olomoucine, bohemine, and CYC202. provided by Professor Bert Vogelstein, The Johns Hopkins

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University School of , Baltimore MD (18). For drug minutes, 1, 2, 4, 6, and 24 hours after administration. The treatment, 2  106 cells were seeded into a T75 flask (Corning, Lutrol/Solutol vehicle was used because HCl/saline was Acton, MA) and left to attach for 36 hours. Compounds were unsuitable for the i.p. route and because a drug solution was dissolved in DMSO and diluted directly into the culture media required for i.v. administration. CYC202 (50, 500, and 2,000 when required. The total concentration of DMSO in the mg/kg) was given orally in 50 mmol/L HCl/saline in a volume medium did not exceed 0.4% (v/v) during treatments. of 0.1 mL/10 g body weight to female BALB/cÀ mice. Blood Sulforhodamine B assay. were made up at 20 to was collected at 5, 15, 30 minutes, 1, 2, 4, 6, and 24 hours after 50 mmol/L in DMSO. Growth inhibition assays were done in administration. 96-well microtiter plates using the sulforhodamine B assay as Pharmacokinetic calculations and simulation experiments. described previously (19). Briefly, cells were seeded at 3 to 8  Pharmacokinetic variables were calculated using Winnonlin 103 cells per well (dependent upon doubling time) in 160 AL Professional software (21), version 3.2 (Pharsight, Mountain growth medium and allowed to attach overnight. Agents were View, CA). Variables presented were derived from noncom- then added at 10 different concentrations (typically from partmental analysis. Model 200 (extravascular administration) 2.5 nmol/L to 100 Amol/L) in quadruplicate wells and was used for plasma following i.p. and oral administration and incubated at 37jC for 96 hours. Where the effect of duration for tissues following i.v. administration. Model 201 (i.v. bolus of exposure was investigated, compounds were added for the model) was used for plasma following i.v. administration. Area times indicated and washed off and the cells incubated in fresh under the concentration versus time curve to the last time point growth medium for the remainder of the 96-hour period. Fifty (AUC), Cmax (maximum concentration observed), Cl (clear- microliters of sulforhodamine B (0.4%, w/v) dissolved in 1% ance), t1/2 (half-life), and Vz ( based on acetic acid were then added and the absorbance at 492 nm was the terminal phase) were evaluated. For simulation studies, determined using a Multiscan MCC/340 MKII (Titertek, Hunts- variables derived from one-compartment analysis following ville, AL). Absorbance of treated wells was expressed as a oral administration of 50 mg/kg were used. percentage of control wells. IC50 levels were calculated Protein binding experiments. Fresh human or BALB/cÀ graphically and mean IC50 levels derived. Results are the mean mouse plasma was incubated at 37jC for 5 and 30 minutes, of at least three determinations. 2 and 6 hours with 2 and 20 Amol/L CYC202. Plasma and Animals. Female BALB/cÀ mice were maintained on SDA standard curve samples in PBS were then ultrafiltered by Expanded Rodent diet (Harlan UK Ltd., Bicester, Oxon, centrifugation for 40 minutes at 4jC at 1,500  g using 10,000 United Kingdom) and water ad libitum. They were housed in MW exclusion membranes (Amicon, Dorset, United Kingdom). an Individual Ventilated Caging System manufactured by Samples were subsequently frozen at À20jC until analysis. Thoren. Female CD1 nude mice were supplied by Charles Control experiments were carried out to show that test River UK Ltd. (Martgate, United Kingdom) and maintained on compound did not bind to the filter membranes. Harlan Teklad 9607 R&M diet and water ad libitum. All animal Analytic method. Drug measurements were done by liquid procedures complied with local and national animal welfare chromatography-tandem mass spectrometry (LC-MS/MS). Ini- guidelines (20). tially, a 150 mm  2.1 mm zwitterionic ABZ+ column (Supelco, Maximum tolerated dose. BALB/cÀ mice were given increas- Poole, Dorset, United Kingdom) was used and drugs eluted with ing doses of 50, 100, and 200 mg/kg of the three compounds 10 mmol/L ammonium acetate and 70% methanol at a flow i.v. in 50 mmol/L HCl/saline. Animals were checked daily on rate of 0.2 mL/min; total run time was generally 3 minutes. several occasions and monitored for a period of 5 days. For oral Tissue homogenates and plasma were treated with 3 volumes of administration BALB/cÀ mice were given a single dose of 500 methanol to precipitate protein followed by dichloromethane or 2,000 mg/kg orally in 50 mmol/L HCl/saline. extraction. Standard curves were made in the appropriate matrix Pharmacokinetic experiments: of olomoucine, and analyzed at the level of 1, 10, 100, 1,000, 10,000, and bohemine, and CYC202 iv. Compounds were given i.v. in a 100,000 ng/mL. Samples were reconstituted in 200 AL mobile volume of 0.1 mL/10 g bodyweight in 50 mmol/L HCl/saline at phase and 10 AL injected onto the column. Although this 50 mg/kg to BALB/cÀ mice bearing the s.c. colon 26 murine analytic method was specific, sensitive, and reproducible, it was tumor. A 1-mm3 brei was implanted under anesthetic using a found that the column could not withstand a large number of Bashford syringe into the flank of 100 BALB/cÀ female mice. extracts. Therefore, a second method was used for subsequent Fourteen days post-implantation, mice bearing comparably routine CYC202 analysis, as follows. In this method, analyses sized tumors (diameters of f6 mm) were randomized into were done on a 50 mm  4.6 mm zwitterionic ABZ+ column either a control group of six mice or treatment groups of three (Supelco) and the drugs eluted with a gradient of 80% to 0.1% mice per time point. Plasma, liver, kidney, spleen, and tumor formic acid, 20% to 100% methanol over 5 minutes. Standard were collected at 0.25, 0.5, 1, 3, 6, and 24 hours after curves were made in mouse plasma at the level of 10, 100, administration. 1,000, 10,000, and 100,000 nmol/L. Quality controls were Pharmacokinetics of CYC202 i.p., i.v., and orally. CYC202 made in control mouse plasma. The assay was validated in at a dose of 50 mg/kg (0.1 mL/10 g, 5 mg/mL) in 9% (w/v) mouse plasma by running three precision batches of five Lutrol (polyethylene glycol 660-12 hydroxystearate), 3.5% (w/v) replicates at the levels of 25, 90, 500, and 50,000 nmol/L on Solutol HS15 (BASF), and 87% water, was given i.v., i.p., and three separate days. Plasma (100 AL aliquots) was added to orally to BALB/cÀ mice. Solutol was weighed and warmed at 30 Al of internal standard (500 ng/mL olomoucine in 70jC until liquid. Lutrol was weighed and added to the Solutol. methanol) incubated for 30 minutes and treated with 3 volumes CYC202 was added and the solution kept at 70jC until of methanol. Following centrifugation, the supernatant was dissolved. The appropriate volume of sterile water was then transferred to high-performance liquid chromatography vials added to the mixture. Blood was collected after 5, 15, 30 and 10 AL injected onto the column. For ultrafiltrate analysis,

www.aacrjournals.org 4877 Clin Cancer Res 2005;11(13) July 1, 2005 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Cancer Therapy: Preclinical standard curve and quality controls were made in plasma were phospho-RB Ser780 1:5,000, phospho-ERK1/2 1:1,000, ultrafiltrates. phospho-CDK2 Thr160 1:1,000 (Cell Signaling Technologies, Detection was achieved by multiple reaction monitoring on a Beverly, MA); total RB SC-50 1:2,000 (Santa Cruz Biotech- triple sector mass spectrometer (TSQ700, Thermoquest Ltd., nology, Santa Cruz, CA); phospho-RB Ser608 (22) 1:2,000 (Dr. Hemel Hempstead, Herts, United Kingdom). Multiple reaction Sibylle Mittnacht, Institute of Cancer Research, London, United monitoring of the sum of two daughter ions of the pseudo- Kingdom); nonphosphorylated RB Ser608 (underphosphory- molecular ion [M+H]+ 299 (99 and 177 AMU) for olomoucine, lated RB) 1:1,000 (PharMingen, San Diego, CA); phospho-RB 355 (99 and 233 AMU) for CYC202, and 341 (91 and 206) for Ser807/811 1:5,000 (Sigma Chemical); phospho-RB Thr821 bohemine. Peak areas were monitored and plotted against 1:1,000 (Biosource, Nivelles, Belgium); cyclin B1 Ab-1 1:200, concentration (GraphPad Software). When internal standard cyclin D1 Ab-1 1:200, cyclin A Ab-6 1:200, cyclin E Ab-1 1:200, was used, relative areas were monitored. CDK1 Ab-1 1:200, CDK2 Ab-4 1:200, CDK4 1:200 Ab-1 Pharmacokinetic-pharmacodynamic relationships in human (Neomarkers, Fremont, CA); poly(ADP-ribose) polymerase C- tumor xenografts. Nude mice bearing established (f130 2-10 1:1000 (BD Biosciences, Oxford, United Kingdom); total mm3) HCT116 human colon tumors as a s.c. xenograft in the RNA polymerase II AB817 1:5000 (Abcam, Cambridge, United flank were given 500 mg/kg CYC202 orally, twice daily for Kingdom); total glyceraldehyde-3-phosphate dehydrogenase 5 days. This regimen was based on the pharmacokinetic 1:5000 (Chemicon, Temecula, CA); goat anti-rabbit and goat simulation models. CYC202 was dissolved in 10% DMSO, anti-mouse horseradish peroxidase–conjugated secondary anti- 5% Tween 20, and 85% of 50 mmol/L HCl/saline. For bodies 1:5,000 (Bio-Rad, Hercules, CA). Western blots were pharmacodynamic profiling, three mice were sacrificed per quantified by densitometry with Image Quant (Amersham time point. Time points comprised 3 days vehicle control, 1 day Biosciences). Using a high-throughput 96 well plate-based of treatment (harvested 4 hours after the second dose), 3 days ‘‘in-cell western’’ approach, RB phosphorylation was assessed in treated (again 4 hours after the second dose), and 5 days treated intact cells as described previously (23). (also 4 hours after the second dose). Plasma and tumor were Flow cytometry. HCT116 cells (2.5 Â 105) were seeded into recovered from each animal and immediately either homoge- a T25 flask and left for 36 hours to attach to the plastic. Drug nized in cold lysis buffer or snap-frozen in liquid nitrogen and treatments were done as above. Cells were harvested in 1 mL stored at À80jC. For antitumor therapy studies, 10 mice were trypsin-versene by incubating at 37jC for 5 minutes to form a treated with vehicle control alone and eight mice were treated cell suspension, gently pelleted and resuspended in 100 AL with CYC202 for 5 days, using the same protocol as described ice-cold PBS, and fixed by slow addition of 1 mL ice-cold above. Tumor volume was determined from measurement of 70% ethanol while vortexing. Cells were resuspended in 200 two orthodiagonal diameters and antitumor activity was AL of 0.02 mg/mL propidium iodide (Molecular Probes, determined by the comparison of volumes for treated and Cambridge, United Kingdom)/0.25 mg/mL RNase A (Sigma control groups. The difference in the means of paired samples Chemical) and incubated at 37jC for 30 minutes. Samples was determined using a two-tailed Student’s t test and Prism were analyzed on a Beckman Coulter Elite ESP (Beckman software (GraphPad Software) and Ps < 0.05 were considered Coulter, High Wycombe, United Kingdom) and cell cycle statistically significant. analysis was done with WinMidi2.8 software (Scripps Western blotting. To harvest cells, the medium was removed Research Institute, La Jolla, CA). and cells were incubated with 5 mL trypsin for 5 minutes at 37jC to detach them from the plastic. The cells were then Results pelleted, washed in ice-cold PBS, and resuspended in ice-cold lysis buffer containing 50 mmol/L HEPES (pH 7.4), 250 mmol/L Activity against recombinant kinases in vitro. The ability of NaCl, 0.1% NP40, 1 mmol/L DTT, 1 mmol/L EDTA, 1 mmol/L the trisubstituted aminopurine CDK inhibitors olomoucine, NaF, 10 mmol/L h-glycerophosphate, 0.1 mmol/L sodium bohemine, and CYC202 to inhibit recombinant kinases was orthovanadate, and one complete protease inhibitor cocktail assessed and the results are reported in Table 1. CYC202 was tablet (Roche, East Sussex, United Kingdom) per 10 mL of lysis clearly the most potent inhibitor against all kinases tested. The buffer for 30 minutes on ice. Lysates were centrifuged at kinase most potently inhibited by CYC202 was CDK2/cyclin E f18,000 Â g for 10 minutes at 4jC to remove cellular debris. (IC50, 0.13 Amol/L), closely followed by CDK7/cyclin H (IC50, The supernatant was stored at À80jC before use. The protein 0.46 Amol/L) and CDK9/cyclin T1 (IC50, 0.78 Amol/L). The concentration of lysates was determined using the protein assay inhibition of CDK1, CDK4, CDK6, and extracellular signal– reagent; bicinchoninic acid protein assay (Pierce, Rockford, IL). related kinase 2 (ERK2) was much less. Proteins were separated by SDS-PAGE using Novex precast tris- Activity against human tumor lines in cell culture. Table 2 glycine gels (Invitrogen, Groningen, the Netherlands) and shows the activities of the three analogues against a range of transferred to Immobilon-P membranes (Millipore, Bedford, human tumor cell lines. Mean IC50 values across the panel were MA). Membranes were blocked for 1 hour in TBSTM [50 mmol/L 56 Amol/L for olomoucine, 27 Amol/L for bohemine, and Tris (pH 7.5), 150 mmol/L NaCl, 0.1% Tween 20 (Sigma 15 Amol/L for CYC202. The mean value for CYC202 was Chemical)] and 3% milk. Immunoblotting with primary identical to that of 15 Amol/L found in an independent panel antibodies diluted in TBSTM was done at 4jC overnight (13). These relative potencies for the three aminopurines followed by a 1-hour incubation with horseradish peroxidase– correlated with the increased potency of CYC202 over conjugated secondary antibodies at room temperature. Mem- bohemine and olomoucine observed in CDK inhibition assays branes were washed with enhanced chemiluminescence (Table 1). Bohemine and CYC202 showed a broad spectrum of reagents and exposed to Hyperfilm (Amersham Pharmacia in vitro anticancer activity, which was not directed towards any Biotech, Buckinghamshire, United Kingdom). Antibodies used particular tumor cell type.

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done at this time in the same samples from which molecular Ta b l e 1. Inhibition of recombinant or purified kinases analysis was carried out. Cell number was reduced by 60% to in vitro by olomoucine, bohemine, and CYC202 70%, confirming that similarly effective concentrations of the three agents were being used (Fig. 3A). It is clear that RB F M Kinase Compound IC50 SD ( mol/L) phosphorylation was reduced by pharmacologically active Olomoucine Bohemine CYC202 concentrations of the three aminopurines. This was shown both by a gel mobility shift to the hypophosphorylated form F F F CDK2/E 0.94 0.35 4.6 1. 8 0.13 0.07 and also by a major reduction (z70%) in phosphorylation at F F F CDK2/A 8.8 1.0 8 3 9 2.2 0.7 all sites tested, including the proposed CDK2-preferred site at F CDK1/B >100 >100 14.1 2.7 Thr821 (Fig. 3B). The decrease in RB phosphorylation was F F CDK4/D1 45 1 >100 14.7 3.6 shown not to be due to a loss of total RB protein as F F CDK6/D3 136 29 >200 50 6 demonstrated by the use of the antibody that recognized the F F CDK7/H 0.60 0.10 >20 0.46 0.09 Ser608 site in the nonphosphorylated state (Fig. 3B), the signal F F F CDK9/T1 2.03 0.38 2.7 1. 3 0.78 0.15 for which increased as phosphorylation was lost. Concentra- F F F ERK2 60 13 5 2 5364 tions of the three inhibitors that elicited a similar inhibition of cell growth exhibited comparable inhibition of RB phosphor- F NOTE: IC50 values are expressed as Amol/L and are the mean ( SD) of at ylation (Fig. 3C). Inhibition of RB phosphorylation in HCT116 least two independent determinations. cells by CYC202 was confirmed and quantified using a DELFIA- based assay measuring RB phosphorylation at Ser608 following a 24-hour exposure (Fig. 3D). The IC50 for inhibition of RB Included within the cell line panel were sublines possessing phosphorylation by CYC202 was 18 Amol/L. This correlates acquired to cisplatin (CisR lines; ref. 24) and well with the 50% to 60% inhibition of proliferation seen at doxorubicin (DoxR line, with resistance mediated by over- this concentration, as determined by cell counts. expression of P-glycoprotein; ref. 25). In these paired cell lines, no cross-resistance was observed to the CDK inhibitors. Also of interest is the possible influence of RB and TP53 status in Ta b l e 2 . In vitro growth inhibitory activity of olomoucine, determining cellular sensitivity to CDK inhibitors. A comparison bohemine, and CYC202 in a panel of cancer cell lines of sensitivity of the widely used pair of human osteosarcoma lines (SAOS-2, RB negative and U2-OS, RB positive; ref. 26) Tumor type Cell line 96-h sulforhodamine B IC50 suggests little or no effect of RB status on sensitivity. In contrast, (Mmol/L) when comparing two breast cancer cell lines which differ in TP53 Olomoucine Bohemine CYC202 status (MCF-7, wild-type TP53 and MDA-MB-231, mutant TP53; ref. 27) the mutant cell line was f2-fold more resistant than the Ovarian A2780 30 12.5 4.9 R wild-type line. Previous studies in a human cancer cell line panel A2780Cis 45 17 8.4 showed a similar modest difference (13). However, the CH1 48 21.5 7.7 R sensitivity to CYC202 of an isogenic variant of the HCT116 CH1Cis 78* 22 9.3 R human colon carcinoma cell line in which TP53 was knocked CH1Dox 86* 19 7.4 out by homologous recombination (HCT116 TP53À/À; ref. 18) SKOV-3 >50 81* 31 was essentially no different from that of the vector control Colon BE >50 25 17.5 (HCT116 TP53+/+) or parental cell line (Fig. 2A). HT29 58 27.7 20.3 Time of exposure required for in vitro activity. The effect of Mawi >50 28.5 18 varying times of exposure to CYC202 and bohemine was Lovo >50 25 20 investigated using the HCT116 parental human colon cancer SW620 >50 27 23 HCT116 52* 17 6.9 cell line in monolayer culture. Following the times of exposure COLO-205 41 21 8.5 shown in Fig. 2B, the compounds were washed off and cell KM12 90 32 15 growth inhibition determined at 96 hours using the sulforhod- Osteosarcoma SA-OS2 78 38 16.5 amine B assay. Between 8 and 16 hours of drug exposure was U2-0S 101.5* 36 15 required to achieve the maximum growth inhibitory effect. Breast MCF 7 64 21.5 7.8 Similar data were obtained for the human ovarian carcinoma MBMDA231 101.5* 40 15 cell line A2780 when exposed to olomoucine, bohemine, and Lung A549 54.5 20 9.3 CYC202 (data not shown). MOR 66 36 12.5 Molecular pharmacodynamic markers in vitro. We investi- HX147 106.5* 38 19 gated potential molecular pharmacodynamic markers of CORL23 54 29.5 10.5 response to the three aminopurines in the HCT116 cell line Testicular GCT27 47 14.5 5.2 (Fig. 3B). This line was selected because it was among the most Murine colon Colon 26 >100 54 42.5 sensitive of those tested (Table 2) and was suitable for use as a Mean 56.29 27.11 14.63 xenograft in subsequent in vivo studies. Figure 3B shows the effect of CYC202, bohemine, and olomoucine on the phos- phorylation status of RB following treatment of cells for NOTE: IC50 values (Amol/L) were obtained using the 96-hour sulforhodamine B assay. Results shown are mean values of at least three determinations. 24 hours with 3 Â IC50 of these agents as measured by the *Results obtained by extrapolation. sulforhodamine B assay at 96 hours (Table 2). Cell counts were

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In addition to decreases in RB phosphorylation, all three In contrast to previous results in HT29 and KM12 human aminopurine compounds reduced the expression of cyclin D1 colon cancer cells (15), no increase in phosphorylated ERK1/2 (77-100% decrease; Fig. 3B-C). Cyclins A and B1 also showed was observed in response to the three compounds (Fig. 3B-C), decreased expression but to a lesser extent (8-75% reduction). possibly because the HCT116 cell line has a Kirsten-RAS Cyclin E was least sensitive to inhibition by the three compounds mutation (29). but was also reduced (4-52% loss). The expression of CDK1 and Treatment of HCT116 cells with CYC202, bohemine and CDK2 showed a modest decrease in response to the compounds olomoucine resulted in a 11% to 15% loss of cells from the S (16-40% reduction). However, the expression of CDK4 was phase of the cell cycle with a 6% to 17% increase in the greatly reduced following exposure to all three inhibitors proportion of cells in the G2-M phase after 24 hours of (f80% loss). The expression of phosphorylated RNA polymer- treatment, as determined by propidium iodide staining and ase II was also reduced by all three compounds, as shown by a analysis by flow cytometry (Fig. 3E). Cells in the S phase were switch to the lower, hypophosphorylated form. This is consistent confirmed to have ceased DNA replication as determined by with inhibition of CDKs 7 and 9. In support of inhibition of bromodeoxyuridine incorporation (data not shown). A sub-G1 CDK7 (28) Thr160 phosphorylation on CDK2 was reduced fraction was also seen with all three agents, indicative of following 8 to 24 hours exposure to 3Â IC50 CYC202 (data not apoptosis. Taken together, the above results are consistent with shown). In addition, a partial cleavage of poly(ADP-ribose) the agents acting as CDK inhibitors although other possibilities polymerase was observed following a 24-hour exposure to these cannot be ruled out. compounds, indicative of apoptosis (Fig. 3B-C). Maximum tolerated dose in BALB/cÀ mice. Given i.v., the single dose maximum tolerated dose for both bohemine and CYC202 was 100 mg/kg. For olomoucine, the maximum tolerated dose was higher at 200 mg/kg. Subsequent i.v. studies were done with 50 mg/kg of the agents. At 150 mg/kg, CYC202 was well tolerated when given i.p. Following a single oral administration, CYC202 was well tolerated up to 2,000 mg/kg. Analytic results and pharmacokinetic variables. LC-MS/MS proved to be specific, sensitive, and reproducible in both analytic systems described in Materials and Methods. Whereas only 100 ng/mL of each trisubstituted aminopurine could be detected with UV, the limit of quantification with LC-MS/MS was 3.5 ng/mL or 10 nmol/L. The interbatch precision and accuracy at 25, 90, 5,000, and 50,000 nmol/L were below 15%. These values are in accordance with the established guidelines for assay validation. Tables 3 and 4 show the pharmacokinetic variables for CYC202, olomoucine, and bohemine in plasma, liver, kidney, and colon 26 tumor in BALB/cÀ mice following a dose of 50 mg/kg i.v. Variables were obtained using noncompartmental analysis. Plasma levels decayed in a biexponential fashion in all three cases (Fig. 4). All compounds cleared rapidly from plasma. CYC202 had the highest plasma concentrations and exhibited the longest half-life (1.19 hours) and the slowest clearance of 43 mL/h. Drug uptake from the general circulation was very rapid for all three compounds with Cmax observed in tissues at the sampling time of 0.25 minutes (Figs. 4 and 5). The tumor to plasma AUC ratios were 0.71, 0.45, and 0.18 for CYC202, olomoucine, and bohemine, respectively. These ratios were higher than those observed for kidney and spleen but lower than the corresponding liver to plasma ratios. The terminal half-life for the elimination of CYC202 from tissues was greater than that observed for the other two analogues (Table 4). Overall, CYC202 exhibited the highest AUC, the longest plasma t1/2, and the highest tissue to plasma ratio of the Fig. 2. A, effect ofTP53 status on sensitivity to CYC202 in HCT116 human colon three analogues. carcinoma cells. HCT116 parental cells (PAR ,WT TP53 ), vector control (WT,WT Figure 5 shows the plasma concentration versus time curves TP53 ), or TP53 knock out (NULL,TP53 null) were exposed to increasing doses of CYC202 for 96 hours. Cell growth was assayed using the sulforhodamine B assay following i.p., i.v., and oral administration of 50 mg/kg F and IC50 values were calculated graphically. Columns, mean (n =3);bars, SD. CYC202. The pharmacokinetic data in Table 5 show that rapid Statistical analysis of the mean IC50 values by a one-wayANOVA test showed no significant differences (P > 0.05). B, effect of time of exposure on response to absorption occurred from the peritoneal cavity, with maximal CDKIs. HCT116 cells in logarithmic growth were exposed to increasing plasma concentrations observed 5 minutes after administra- concentrations of either bohemine or CYC202 for 4, 8, 16, 24, and 96 hours, the tion. The concentrations obtained by this route of administra- compounds were washed off and cell growth assayed by sulforhodamine B after a tion were very similar to those observed by the i.v. route with total of 96 hours. Points, mean IC50 values (n =3);bars, FSD. The IC50 following a 4-hour exposure to bohemine was >200 Amol/L, as indicated by the vertical arrow. an overall i.p. bioavailability of 67% (Table 5). There was no

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Fig. 3. Cellular and molecular responses to CDK inhibitors. HCT116 cells were exposed to equiactive doses of CYC202, bohemine or olomoucine (3 Â 96 hours IC50) for 24 hours. A, attached cell number following exposure to the CDKIs (n = 6 from two independent experiments). B,Western blots for the indicated proteins following analysis of cell lysates (50 Ag protein). GAPDH was used as loading control. C, densitometric analysis of theWestern blots in (B)as carried out in Image Quant. Columns ,mean(n =3); bars, FSE. D, inhibition of RB phosphorylation at Ser608 in HCT116 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 using propidium iodide and flow cytometry. Columns,meanfrom 2 independent experiments (n =6);bars, F SD.

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

Compound Cmax AUClast AUCINF Cl (observed, L/h) Vz (observed, L) t1/2Ãz (h) (nmol/L) (h nmol/L) (observed, hnmol/L) Olomoucine 59,300 41,059 41,075 0.07 0.06 0.58 Bohemine 72,308 12,901 12,931 0.23 0.45 1.39 CYC202 40,457 65,596 67,570 0.04 0.08 1.20 increase in terminal half-life by this route of administration early as 5 minutes after incubation and the binding was the suggesting that delayed absorption did not occur. same 6 hours after incubation. In human plasma, the protein CYC202 concentrations peaked 15 minutes after oral binding increased slightly but gradually from 92% to 96% over administration, and the terminal half-life increased compared a period of 5 minutes to 6 hours. with that measured after i.v. or i.p. administration (2.77 hours Pharmacokinetic-pharmacodynamic analysis. To explore the for oral dosing versus 1.38 hours by the i.v. route). The oral pharmacokinetic-pharmacodynamic relationships in vivo, the bioavailability was 86%. Following increasing oral doses of HCT116 human colon cancer xenograft model in nude mice CYC202, the half-life was extended from 2.88 to 6.67 hours was used to determine the antitumor effect, tumor drug and 11.9 hours. Plasma concentrations were maintained above concentrations, and alterations in molecular biomarkers. Given 15 Amol/L (the mean IC50 level across the human tumor cell the high oral bioavailability for CYC202 reported here and the line panel) for 4, 12, and 24 hours following 50, 500, and attractiveness of oral dosing for clinical application, this route 2,000 mg/kg, respectively (Table 6; Fig. 6). Following increased was selected for the pharmacokinetic-pharmacodynamic stud- oral doses, the AUC increased 10-fold from 50 to 500 mg/kg ies. Previous studies showed that CYC202 was tolerated in but only a further 2-fold from 500 to 2,000 mg/kg. This tumor-bearing nude mice at a dose of 500 mg/kg thrice a day difference was also reflected in the Cmax , which did not increase (13). Based on the pharmacokinetic simulation (Fig. 7), mice linearly with dose. It should be noted that the plasma AUC were treated with 500 mg/kg orally CYC202 twice per day for 5 measured following 50 mg/kg CYC202 in the Solutol/Lutrol days. In the experiment shown in Fig. 8, the mean body weights vehicle was 35% lower than that observed using the HCl saline (expressed as percentage of the starting weight on day 0) were vehicle at the same dose level. This could be explained by 102%, 101%, 101%, 101%, and 95% on days 1 to 5 compared differences in Cmax as the t1/2 values were identical. The results with 99%, 98%, 98%, 98%, and 98% for the vehicle controls. suggest the possibility that acid conditions may favor oral One of the eight animals died in the treated group during this absorption of CYC202. period (day 2). CYC202 caused a reduction in the growth of Computer simulation experiments derived from variables in established tumors (f130 mm3) compared with controls for Table 5 showed that to maintain a concentration of 15 Amol/L the duration of treatment (Fig. 8A). %T/C values were 79%, over 24 hours (mean IC50 level), CYC202 would need to be 75%, 72%, and 65% at days 2, 3, 4, and 5, respectively (P < given orally thrice a day at 200 mg/kg or twice a day at 500 mg/ 0.05). This is comparable with the T/C value of 47% that was kg (Fig. 7). Protein binding was similar at 2 and 20 Amol/L. In reported in the MESSA-DX5 human uterine carcinoma xeno- mouse plasma 97.2% of CYC202 was bound to proteins as graft using a comparable schedule of 500 mg/kg orally thrice a

Ta b l e 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 to BALB/cÀ mice

Tissue Compound Cmax Tmax AUClast AUCINF Cl (predicted)/ Vz (observed)/ t1/2Ãz (nmol/L) (h) (h nmol/L) (observed, F (L/h) F (L) (h) hnmol/L) Kidney Olomoucine 15,536 0.50 8,079 ND ND ND ND Bohemine 5,936 0.25 2,363 2,395 1.23 2.16 1.23 CYC202 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 0.60 Bohemine 8,626 0.25 6,524 6,670 0.44 0.60 0.94 CYC202 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 0.57 Bohemine 6,386 0.25 2,369 2,461 1.19 1.40 0.82 CYC202 24,548 0.25 46,742 47,946 0.06 0.33 3.86

Abbreviation: ND, not determined.

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Fig. 5. CYC202 plasma concentration versus time curves following 50 mg/kg i.v. (5), i.p. (o), oral administration ( w ) in Solutrol Lutrol vehicle to female BALB/cÀ mice.

tion of RB phosphorylation at specific phosphorylation sites was determined using phospho-specific antibodies. Whereas the signal for all sites was markedly decreased after 3 to 5 days of treatment, the proposed CDK2-preferred Thr821 site was the most sensitive (95% inhibition on day 5). In addition, cyclin D1 levels were decreased by 40% to 77% after 3 to 5 days of treatment. Both the RB and cyclin D1 biomarker changes were similar to those described earlier for HCT116 cells treated in vitro. Also in agreement with the in vitro data, no increase in ERK phosphorylation was observed in vivo in response to CYC202. To relate the molecular marker changes to drug exposure, CYC202 concentrations in the HCT116 tumor and plasma were determined by LC-MS/MS using samples from the same group of mice used for the biomarker measurements (Fig. 8C). Plasma concentrations were in accordance with predicted levels from the computer simulation described earlier. Tumor concentrations measured at 4 hours after the second of the twice daily doses were greater than those required to inhibit the growth of HCT116 cells in cell culture (IC50 of 6.9 Amol/L; Table 2) on days 1 and 3. However, the concentration of CYC202 was markedly reduced on day 5 (4 hours after the last dose) in both plasma and tumor. In a single oral dose experiment at 500 mg/kg, tumor concentrations were above cell 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 properties of three trisubstituted aminopurine CDK inhibitors, olomou- cine, bohemine, and CYC202, with particular emphasis on pharmacokinetic-pharmacodynamic relationships for CYC202. A detailed understanding of the pharmacokinetic- pharmacodynamic properties should facilitate rational selec- tion of a dosing schedule that would support therapeutic activity in tumor xenograft models (30). Identification of appropriate pharmacokinetic-pharmacodynamic variables allows construction of a ‘‘pharmacologic audit trail’’ (31, 32) Fig. 4. Plasma (A), colon 26 tumor (B), liver (C), and kidney (D) concentration versus time curves following administration of 50 mg/kg olomoucine (5), CYC202 to aid interpretation of preclinical data and for use in ( w ), and bohemine (o), i.v. in 50 mmol/L HCl saline to BALB/c mice. subsequent clinical trials.

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

Route Cmax Tmax AUCINF AUClast F Cl (observed)/ Vz (observed)/ t1/2Ãz MRTlast (nmol/L) (h) (observed, (h nmol/L) F (L/h) F (L) (h) (h) hnmol/L) i.p. 72,756 0.083 75,455 75,397 0.67 0.037 0.14 2.67 2.01 i.v. 130,473 0.083 111,900 111,892 1 0.03 0.10 1.97 1.56 oral 30,831 0.25 95,348 94,816 0.86 0.033 0.13 3.27 3.70

CYC202 was the most potent of the three aminopurine phosphorylation at Thr821 (a CDK2-preferred RB phosphoryla- analogues in terms of in vitro CDK2 inhibition and growth tion site) was most affected by CYC202. There was some loss of inhibition in a human cancer cell line panel. Interestingly, it was total RB protein, but this was not responsible for reduced active in cell lines resistant to doxorubicin and cisplatin. Also of phosphorylation, as shown by the increase in nonphosphory- note is the lack of dependence on RB status as exemplified by lated Ser608. Over the same concentration range and time similar IC50 values in the SAOS-2 versus U2-OS osteosarcoma course as the effects on RB phosphorylation were shown, the cell lines. Similarly, TP53 status had little effect, if any, on the analogues increased cell number in G2-M phase and decreased growth inhibition. Comparison of the HCT116 isogenic cell pair, the S-phase fraction. Use of a quantitative DELFIA assay 608 differing only in TP53 status, showed near-identical IC50 values showed that RB phosphorylation at Ser was inhibited by for CYC202. Together with previous data (13), these results 50% following 24 hours exposure to 18 Amol/L CYC202, suggest that the aminopurine analogues may exhibit broad- correlating well with the 50% to 60% decrease in cell number, spectrum antitumor activity. The differences in the CDK compared with controls, observed with this treatment. inhibitory potency among the three purine analogues were Inhibition of RB phosphorylation and the above cell cycle reflected in their relative cancer cell growth inhibitory activities, changes could be occurring through inhibition of CDK1/cyclin B CYC202 being more potent than bohemine which was in turn or of CDK2. Introduction of dominant-negative CDK2 into more active than olomoucine. This is consistent with CDK cancer cell lines has been shown to cause an arrest in G2-M (33). inhibition being a major contributor to the antiproliferative We also showed that CYC202, bohemine, and olomoucine effects but does not exclude other possible cellular effects. caused a marked decrease in cyclin D1 expression and a smaller We showed that olomoucine, bohemine, and CYC202 all loss of cyclins A and B1, with cyclin E affected much less. reduced RB phosphorylation at pharmacologically relevant Decreased expression of cyclin D1 and other cyclins in HCT116 concentrations that inhibit cell growth in cell culture. Although cells also occurred in HT29 and KM12 cells (15). In addition, functional RB was not required for an antiproliferative effect, CYC202 decreased CDK4 expression in HCT116 cells, an effect RB phosphorylation was nevertheless a valuable indicator of not seen in HT29 or KM12 cells previously (15). The data are CDK activity and could be useful in those tumors that are consistent with the analogues acting directly or indirectly (or positive for RB. Loss of RB phosphorylation in HCT116 cells both) as inhibitors of CDKs, although other cellular mecha- was concomitant with cell growth inhibition, as seen with nisms are not excluded. The compounds can inhibit activity of CYC202 in HT29 and KM12 human colon cancer cells (15). the transcription-related CDKs, CDK7 and CDK9 (Table 1), Assessing all the combined data, we concluded that inhibition which are involved in phosphorylation of RNA polymerase II. of RB phosphorylation is related to in vitro potency in RB- Reduced phosphorylation of RNA polymerase II was observed in positive cancer cell lines, whether comparing between the HCT116 cells in vitro. Inhibition of T160 phosphorylation on different analogues in a given cell line or comparing the effects CDK2, mediated by CDK7, was also shown. Therefore, the of CYC202 across different lines. reduction in cyclin expression may occur through transcriptional Inhibition of RB phosphorylation in HCT116 cells was inhibition due to reduced CDK7 and CDK9 activity (15). Gene shown by the gel mobility shift seen with the hypophos- expression profiling can be used to evaluate such effects (34). phorylated form of RB and also by antibodies to specific The relative decreases in the individual cyclin proteins may phosphorylation sites. Consistent with inhibition of CDK2, reflect differential protein half-lives following an inhibition of

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

Dose Cmax Tmax t1/2Ãz MRTINF AUClast Vz (observed)/ Cl (predicted)/ AUCINF (mg/kg) (nmol/L) (h) (h) (observed, h) (h nmol/L) F (L) F (L/h) (observed, hnmol/L) 50 25,628 0.25 2.9 3.5 95,199 0.12 0.03 95,428 500 80,269 0.5 6.7 7.7 906,880 0.28 0.03 966,195 2,000 108,266 2 11.9 16.2 1,723,508 0.85 0.05 2,282,182

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results indicate that pharmacokinetic behavior is likely to be critical for in vivo activity. Here LC-MS/MS provided rapid, specific, and sensitive analysis of olomoucine, CYC202, and bohemine in biological tissues and fluids. As mentioned, the time dependence of the effects of CYC202 and bohemine in vitro emphasized the likely importance of pharmacokinetic behavior in vivo. All three compounds distributed quickly into mouse liver, kidney, and tumor tissue but also cleared quite rapidly from plasma and tissues. Bohemine cleared the fastest and CYC202 the slowest with olomoucine intermediate. Although CYC202 was rapid and extensive (97%), it is important to note that this did not limit drug distribution, which was equally fast. Uptake into tumor tissue, as exemplified by the colon 26 transplantable syngeneic mouse tumor, was quite different for the three agents, with AUC values ranging from 18% of plasma for olomoucine to Fig. 6. CYC202 plasma concentrations versus time curves following administration 71% for CYC202. Tissue clearance rates were comparable to of 50 (o), 5 00 (5), and 2,000 mg/kg ( w )CYC202orallytoBALB/cÀ mice. those of plasma. CYC202 clearance is via oxidative metabolism of the side chain hydroxyl group to form the carboxylic acid, mRNA expression downstream of CDK7 and CDK9 inhibition. subsequently excreted in urine (49). The pharmacokinetics Interestingly, the most marked effect occurred with the G1 phase- behaviour described here was predictive of that in healthy male active cyclin D1, whereas the cells predominantly arrest in the subjects receiving single doses up to 800 mg CYC202 (50). Of G2-M phase of the cell cycle. Thus, CDK inhibition (see above) the three analogues, CYC202 showed the best pharmacokinetic and/or loss of other proteins is more likely to contribute to the profile with the slowest clearance and highest tissue distribu- G2-M phase arrest. Loss of several cyclins may inhibit multiple tion. The nanomolar potency towards CDK2/cyclin E taken CDK activities and may therefore block cell proliferation together with the high cellular potency and the better tissue independent of CDK2 activity, which has been reported as distribution and clearance data, indicated that CYC202 had the dispensable for colon cancer cell proliferation (7). It should be most suitable overall properties for further therapeutic evalu- emphasized that CDKs phosphorylate important cellular sub- ation. It was therefore selected for pharmacokinetic-pharmaco- strates other than RB (35–48). As noted previously (15), this dynamic studies in the same HCT116 human colon cancer may explain the activity of the aminopurine CDK inhibitors such model as used in vitro. as CYC202 on cells lacking RB. Despite rapid distribution and high micromolar peak levels, A sub-G1 peak was seen in HCT116 cells after treatment with tumor concentrations at the maximum tolerated single dose of CYC202 and its analogues. An increase in poly(ADP-ribose) CYC202 were not sustained above the in vitro IC50. This polymerase cleavage provided biochemical evidence of apo- indicated the need to lengthen drug exposure by continuous ptosis, consistent with positivity in the terminal deoxynucleo- infusion or repeated administration. The high bioavailability, tidyl transferase–mediated nick-end labeling assay reported both i.p. (67%) and orally (86%), suggested both routes would previously for CYC202 (13). Thus, over the concentration range in which olomoucine, bohemine, and CYC202 reduced RB phosphorylation and cyclin expression, these compounds caused not only a G2-M arrest and a decrease in the S-phase fraction but also the induction of apoptosis. Transformed and normal cells may differ in their requirements for CDK2 (8). Phosphorylation of E2F by CDK2/cyclin A is a major transcriptional control point, and modulation by CDK2 inhibitors such as CYC202, might cause deregulation of E2F and induction of apoptosis (8, 9). Our previous studies have shown that ERK1/2 phosphoryla- tion is induced by CYC202 in HT29, KM12, and NIH3T3 cells in a mitogen-activated protein kinase kinase 1/2–dependent manner (15). Interestingly, the effect was not seen here in HCT116 cells. This may be because the Kirsten-RAS mutation in HCT116 cells (29) causes constitutive activation of ERK1/2, reducing the opportunity for further activation by CYC202. Pharmacokinetic properties are frequently critical in the translation of in vitro anticancer properties into therapeutic activity in an animal model, in the selection of a clinical development candidate and in the subsequent clinical evalu- ation. The growth inhibitory IC50 for bohemine and CYC202 decreased markedly with increased exposure time. Maximum Fig. 7. Pharmacokinetic simulations for CYC202 following administration of (A) growth inhibitory effect was seen after 8 to 16 hours. These 500 mg/kg orally twice daily and (B) 200 mg/kg orally thrice daily.

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Fig. 8. Pharmacokinetic-pharmacodynamic relationships for CYC202 in the HCT116 human colon cancer carcinoma xenograft model. A, effect of 500 mg/kg orally CYC202 twice daily on the growth of established HCT116 human colon carcinoma tumor xenografts (treated at f130mm3) growing s.c. in nude mice. Points, mean relative tumor volume; 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 in response to CYC202 treatment as assessed byWestern blotting. GAPDH was used as the loading control. C, tumor and plasma concentrations of CYC202 corresponding to samples 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, densitometric analysis of theWestern blots in (C), as carried out in Image Quant. be suitable for multiple dosing in tumor xenografts. There In the pharmacokinetic-pharmacodynamic studies, RB phos- seemed to be saturation in oral absorption from 500 to 2,000 phorylation was measured to determine whether CDKs were mg/kg, but the AUC increased 10-fold with a 10-fold increase in likely to have been inhibited by CYC202 and to assess whether dose from 50 to 500 mg/kg. Based on pharmacokinetic this was a valid PD biomarker in the xenograft tumor model. As variables obtained with single doses, our multiple dose predicted from the in vitro sensitivity of HCT116 cells and the pharmacokinetic simulation study indicated that CYC202 simulated and measured tumor pharmacokinetics, CYC202 should be given thrice a day at 200 mg/kg or twice a day at showed antitumor activity against HCT116 tumor xenograft for 500 mg/kg to sustain therapeutic exposures. Concentrations the duration of the 5-day treatment with T/C values in the range measured after 500 mg/kg orally twice daily showed that the of 79% to 65%. A comparable T/C value of 47% was reported simulation was quite predictive for days 1 to 4 of dosing. for the MESS-DX5 human uterine carcinoma xenograft using a However, the decrease in plasma and tumor levels after 5 days similar regimen of 500 mg/kg orally thrice a day for 4 days (13) suggests metabolic induction or decreased absorption. Impor- and the same schedule has been used in a Phase I clinical trial tantly, tumor concentrations were well above the growth of CYC202 (51, 52). More prolonged drug administration at inhibitory concentrations before day 5. the dose level used was not well tolerated. Progressive reduction

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of tumor RB phosphorylation and cyclin D1 expression We have shown that a detailed understanding of the in vitro occurred over the 5 days of treatment. As mentioned, a CDK inhibitory potency and cellular activity of this group of concentration of 18 Amol/L CYC202 is required to inhibit RB trisubstituted aminopurines, coupled to a similarly detailed phosphorylation by 50% in cell culture after a 24-hour analysis of pharmacokinetic and pharmacodynamic properties exposure. Tumor concentrations measured 4 hours after the in vivo, led to the rational selection of CYC202 for tumor second dose exceeded 18 Amol/L on days 1 and 3. Given that xenograft studies. Based on the knowledge of the drug exposures the exposure to CYC202 is not continuous in the mouse but required for antiproliferative activity and of the PK properties of rather is intermittent due to repeated dosing and subsequent CYC202, computer simulation of drug concentrations was clearance of the compound, it is probably not surprising that helpful in developing an oral dosing schedule that allowed >24 hours is needed to achieve a measurable effect on RB therapeutic activity to be shown at exposures causing inhibition phosphorylation in the HCT116 xenograft. The sustained PD of RB phosphorylation and cyclin D1 depletion in tumor tissue. effects at 5 days, which occur despite the lower CYC202 tumor The data presented constitute a ‘‘pharmacologic audit trail,’’ concentrations at this time, remain unexplained. Differences in particularly in relation to pharmacokinetic-pharmacodynamic tumor microenvironment for in vivo tumor xenografts com- relationships (31, 32). The construction of such an audit trail can pared with in vitro cell culture may play a role. improve the quality of decision-making in preclinical drug It is important to stress that although the inhibition of RB development. Together with the antitumor activity in other phosphorylation in the human tumor xenograft is consistent xenograft models reported elsewhere using similar schedules with CYC202 acting as a CDK inhibitor in vivo, whether this is (13), these results support the ongoing development of CYC202, direct via inhibition of CDK catalytic activity or indirect via which is now undergoing Phase II clinical trial (51). depletion of cyclins and CDK4 is unclear. Regardless of the precise mechanism, our results indicate that RB phosphoryla- tion may be a useful pharmacodynamic marker that generally Acknowledgments correlates with the antitumor activity of CYC202, and suggest that cyclin D1 may be an additional biomarker. Cyclin D1 may We thank the members of the Signal Transduction and Molecular Pharmacology be especially useful in situations where RB phosphorylation is Team and the Cell Cycle ControlTeam in the Cancer Research UKCentre for Cancer Therapeutics, together with colleagues at Cyclacel Ltd., for valuable discussions; not detectable, as in RB-negative tumors. With respect to JennyTitley of the Cell Cycle Control Team for flow cytometry; and the protein bio- clinical application, these measurements require suitable tumor chemistry and the assay development/screening teams at Cyclacel for kinase or surrogate tissue to be available before and after treatment. assays.

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