Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition -- Shangary et al. 105 (10): 3933 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Shangary et al. 10.1073/pnas.0708917105.

Supporting Information

Files in this Data Supplement:

SI Methods
SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Figure 11
SI Figure 12
SI Figure 13
SI Table 1
SI Figure 14
SI Figure 15
SI Figure 16
SI Figure 17
SI Figure 18
SI Figure 19
SI Figure 20
SI Table 2
SI Figure 21
SI Figure 22
SI Table 3

SI Figure 6

Fig. 6. Predicted binding models of MI-219 and MI-63 to MDM2. Predicted binding models of MDM2 inhibitors, in ball and stick representation, with MDM2 using the GOLD program. Carbons are in cyan, nitrogen in blue, oxygen in red, fluorine in light blue and chlorine in green. Surface representation of MDM2 is shown with carbons in gray, nitrogen in blue, oxygen in red, and sulfur in yellow. p53 peptide is represented in violet with the key residues Phe-19, Leu-22, Trp-23, and Leu-26 shown as sticks. Hydrogen bonds are depicted with dashed yellow lines. Hydrogen atoms are not included for clarity. Figures were generated by using the program Pymol. (A) Binding model of p53 peptide with MDM2 (from crystal structure PDB code:1YCR), indicating the residues providing key interactions with MDM2 protein. (B) Binding model of MI219 to MDM2. (C) Binding model of MI-219, superpositioned to the bound conformation of p53 peptide in MDM2-p53 crystal structure (MDM2 is not shown). MI-219 is seen to interact in the regions occupied by the four key interacting residues of p53: Phe-19, Leu-22, Trp-23, and Leu-26.

SI Figure 7

Fig. 7. Binding affinity of MI-219 to Bcl-2 and Bcl-xL, and of p53 peptide to MDM2. (A) Binding to human Bcl-2 and Bcl-xL proteins was determined by using competitive FP-based binding assays. (B) Saturation curve of fluorescently labeled p53-based peptide (PMDM6-F) to MDMX. (C) Competitive binding curve of unlabeled PMDM6 peptide to MDM2 protein.

SI Figure 8

Fig. 8. MI-219 blocks the MDM2-p53 interaction and activates the p53 pathway. (A) The indicated cancer cell lines, differing in p53 status, were treated for 24 h in the absence or presence of MI-219, Nutlin-3 or MI-61. Whole cell lysates were analyzed for p53 and its target gene products by Western blot analysis. (B) SJSA-1 cell line was treated for 15 h with or without MDM2 inhibitors or proteasome inhibitor MG132. The levels of p53 and MDM2 proteins in whole cell lysates (WCL) were determined by Western blot analysis. Coimmunoprecipitation assay was performed by immunoprecipitating p53 and detecting MDM2 and p53 proteins in the immunoprecipitates.

SI Figure 9

Fig. 9. MI-219 activates p53 pathway, induces cell cycle arrest, but not cell death in normal cells. A panel of normal human cells was used to test the activation of the p53 pathway, and induction of cell cycle arrest and cell death/apoptosis by MI-219. Human mammary epithelial cells (HMEC), human renal epithelial cells (HREC), WI-38 lung fibrobalsts, CCD-18Co colon fibroblasts, foreskin fibroblasts, and MCF-12F normal-like cell line were used. (A) p53 activation was tested upon treatment with MI-219 or MI-61 for 24 h (except HREC, which were treated for 48 h). Cell cycle progression was analyzed at 24 h time point by PI staining (B), loss of cell viability at 4 days of treatment (except HREC, which were treated for 48 h) (C), and apoptosis by Annexin V/PI double staining at 2 day treatment (D). Adherent and suspension cells were pooled for all cell death and apoptosis analyses. In general, we observed that MI-219 induces p53 activation and cell cycle arrest in all normal cells, but nor or minimal cells death and apoptosis.

SI Figure 10

Fig. 10. MI-219 and Nutlin-3 do not induce cell cycle arrest in cancer cells with deleted or mutated p53, and p53 is essential for cell cycle arrest. (A) Cell cycle arrest in SaOS-2 and PC-3 with deleted p53 and DU-145 with mutant p53 was examined by flow cytometric analysis of DNA content. Cells were incubated in the absence or presence of the inhibitors for 24 h, fixed in 70% ethanol, stained with propidium iodide and analyzed by flow cytometry. Percentage of cells in G₁, S and G₂/M phase were computed by ModFit analysis, and values from a representative experiment are shown as mean and the error bars represent SEM. (B) LNCaP cells were transfected for 48 h with p53 siRNA or control GFP siRNA. Posttransfection, cells were treated with inhibitors for 24 h as indicated, and whole cell lysates were analyzed by Western blot analysis (Upper). Transfected cells were treated for 24 h with inhibitors and cell cycle analysis was performed by staining with propidium iodide (Lower). Percentage of cells in G₁, S, and G₂/M phase were computed by ModFit analysis, and data from a representative experiment are shown as mean and the error bars represent SEM (Lower).

SI Figure 11

Fig. 11. MI-219 and Nutlin-3 induce selective and strictly p53-dependent cell death in cancer cells. (A) Cells were treated in the absence or presence of the inhibitors at indicated concentrations. Adherent and suspension cells were pooled and cell viability was determined by trypan blue dye exclusion. Blue or morphologically unhealthy cells are scored dead. Shown are the mean values from a representative experiment and the error bars show SEM. (B) LNCaP cancer cells, transfected for 48 h with p53 siRNA or control GFP siRNA (from experiment described in SI Fig. 10B), were incubated for 2 days with or without MI-219, Nutlin-3, or MI-61. Cells were stained with Annexin V/PI and flow cytometric analysis of apoptosis assay was performed. Data are expressed as mean ± SD of the duplicates from a representative experiment. Values at top of each panel represent Annexin V/PI-double positive (early and late apoptosis) cells and those at bottom are Annexin V-positive cells (early apoptosis).

SI Figure 12

Fig. 12. Induction of apoptosis by MI-219 is caspase-dependent. (A) SJSA-1 cancer cell line was treated for 16, 24, and 48h after in the absence or presence of MI-219 or MI-61. Whole cell lysates were analyzed for cleavage of PARP and caspase 3. p53 and its target gene products by Western blot analysis. (B) Cells were treated with 100 mM of pan caspase inhibitor ZVAD, followed by analysis of apoptosis after 48 h treatment with MI-219 or Nutlin-3.

SI Figure 13

Fig. 13. Down-regulation of MDMX increases PARP cleavage by MI-219. LNCaP cancer cells, transfected for 48h with MDMXsiRNA (Fig. 3D), were treated for 24h in the absence or presence of MI-219. Whole cell lysates were analyzed for MDMX, p53 and cleaved PARP (p85).

SI Figure 14

Fig. 14. MI-219 induces p53 activation, inhibition of cell proliferation and induction of apoptosis in tumor xenografts. (A) Western blot analysis of the p53 activation in SJSA-1 and LNCaP xenograft tumor tissue lysates was performed. (B) SJSA-1 xenograft tumor tissues, harvested from mice treated for 1 and 3 h with a single dose of MI-219, were examined for cell proliferation by IHC staining of BrdU and apoptosis by TUNEL staining. (C) Cleaved PARP was detected as a marker of apoptosis induced by MI-219. Irinotecan was used a control.

SI Figure 15

Fig. 15. Oral administration of MI-219 achieves robust in vivo anti-tumor activity in SJSA-1 xenograft model. Nude mice, bearing established s.c. SJSA-1 xenograft tumors, were administered with MI-219, irinotecan or vehicle control. Each group of mice (eight mice per group) received MI-219 p.o., irinotecan i.p, or vehicle p.o. for 2 weeks at the indicated doses. Irinotecan was given once a week. Treatment began at day 13 and ended at day 26 postinoculation for the SJSA-1 xenograft model. Shown are the pictures taken 1 day after the treatment was stopped. Because one mouse in vehicle control group had to be euthanized due to excessive tumor size (>10% of body weight, as defined by UCUCA guidelines) before photography, only seven mice are shown.

SI Figure 16

Fig. 16. Oral administration of MI-219 does not have a significant effect on animal body weight. Percentage of change in mean animal body weight body weights was analyzed. Data represent mean tumor volumes and percentage of change in mean animal body weight, and error bars represent SEM.

SI Figure 17

Fig. 17. Oral administration of MI-219 does not achieve in vivo anti-tumor activity in xenograft tumor model with mutant p53. (A) Cells were treated for 24 h with MI-219, and the levels of p53, MDM2, and p21 in whole cell lysates, obtained from MDA-MB-231 (2LMP clone) breast cancer cell line, were analyzed by Western blot analysis. (B) Cell growth inhibition by MI-219 in 2LMP was examined in a WST-based assay. (C) Nude athymic mice, bearing established s.c. 2LMP breast tumors with mutant p53, were administered with MI-219, taxotere i.v. or vehicle control. Each group of mice (eight mice per group) received drugs for 2 weeks at the indicated doses. Taxotere was given once a week and MI-219 was given once a day with MI 219. Tumor volumes were measured three times each week. Data shown are mean tumor volumes and error bars represent SEM. Taxotere (P < 0.0001 ANOVA), but not MI-219 (each dose P > 0.05 ANOVA), caused significant reduction in tumor volume. The body weights of the mice treated with vehicle control or MI-219 did not differ significantly throughout the experimental protocol in each xenograft model.

SI Figure 18

Fig. 18. MI-219 does not induce apoptosis in mouse small intestinal crypts. (A) Apoptosis in normal small-intestine crypts from BALB/c mice (three mice per group) treated with a single oral dose of MI-219 or IR was examined by TUNEL and H&E staining. Note damage to the crypts in IR-exposed animals. (B) p53 accumulation by IR was analyzed by IHC analysis. Note robust p53 accumulation by IR in the crypts of the IR-exposed animals.

SI Figure 19

Fig. 19. IR, but not MI-219, induces toxicity in thymus and small-intestine crypts. (A) Histopathological examination of thymus (A) and small intestine crypts (B) harvested from C57BL/6 mice (three mice per group) treated with a single oral dose of vehicle, MI-219, or IR was examined by H&E staining. Because p53 accumulation is known to cause severe damage to thymus, leading to loss of thymus weight, we additionally measured thymus weight in vehicle, MI-219 or IR-exposed animals. Note a marked loss of thymus weight by IR, but not by MI-219. Also note damage to the crypts in IR-exposed animals.

SI Figure 20

Fig. 20. MI-219 does not induce toxicity in radio-resistant and -sensitive tissues. Nude mice (two per group) were treated with MI-219 (300 mg/kg BID) for 14 days (A) and BALB/c mice (four mice per group) for 7 days (B), and tissues were stained with H&E. MI-219 did not induce any damage to these tissues.

SI Figure 21

Fig. 21. MI-219 induces minimal accumulation in mouse cells in vitro and in vivo, but activates p53. (A) Nude mice were treated with a single oral dose of MI-219 (300 mg/kg) and the levels of p53 and p21 in the indicated tissues were examined by Western blot analysis. (B) NIH 3T3 and B16 melanoma mouse cell lines were treated for 24 h with MI-219, MI-61 or Nutlin-3. The levels of p53 and p21 in whole cell lysates were detected by Western blot analysis.

SI Figure 22

Fig. 22. Computational modeling of mouse MDM2, its interaction with MI-219 and compared with human MDM2. (A) Modeled mouse MDM2 structure in superposition with the crystal structure of human MDM2. For clarity, only the key residues in the p53-binding site are shown. Carbon atoms of mouse MDM2 are shown in green and carbon atoms of human MDM2 are shown in yellow. Side chains of MDM2 residues in the binding site are shown in stick, except residues 54 and 57, which are shown in ball-stick. (B) Predicted binding models of MI219 in complex with human MDM2 (Left) and mouse MDM2 (Right). MI-219 was shown in ball-and-stick model.

Table 1. Pharmacokinetics of MI-219 in mice

Parameter	Mean ± SD
C _max, ng/mL	9,561 ± 728
T _max, h	1.58 ± 0.7
AUC _0→9 h, h×ng/mL	19,206 ± 6,655
t 1/2, h	1.61 ± 0.5
F , %	54.9 ± 19.0

MI-219 was administered p.o. at a dose of 50 mg/kg.

Data represent means ± SD of values obtained in three animals. C_max, measured maximal plasma concentration; T_max, time to reach the measured maximal plasma concentration; AUC_0→9h, area under plasma level-time curve from 0 to 9 h; t_1/2, elimination half life; F, oral bioavailability.

Table 2. Distribution of MI-219 in tissues and blood plasma of tumor bearing nude mice 1 h after administration

Tissue	Concentration mean ± SD, µM	Relative distribution*
Plasma	145.8 ± 20.4	1.00
Tumor	165.3 ± 10.8	1.13
Lung	686.8 ± 120.6	4.71
Liver	686.8 ± 120.6	4.71
Duodenum	645.2 ± 519.4	4.43
Kidney	405.1 ± 43.3	2.78
Heart	283.0 ± 48.4	1.94
Stomach	267.2 ± 8.72	1.83
Spleen	157.4 ± 10.4	1.08
Ovary	92.8 ± 17.5	0.64
Fat	73.5 ± 9.7	0.50
Muscle	69.1 ± 3.7	0.47
Skin	44.4 ± 13.8	0.30
Brain	2.8 ± 0.4	0.02

MI-219 was administered as a single oral dose of 200 mg/kg in nude mice bearing SJSA-1 tumor xenografts.

*Relative distribution, concentration of MI-219 in tissue/concentration of MI-219 in plasma.

Table 3. Scores obtained for docked pose of MI-219 to human and mouse MDM2

using different scoring functions

MDM2	GoldChemScore	ChemScore	XScore (-Log(K_d)	Drugscore
Human	24.86	-31.63	6.9	-336447
Mouse	22.96	-29.14	6.9	-337665

SI Methods

Cell Lines.
SJSA-1 and SaOS-2 osteosarcoma cell lines, LNCaP, 22Rv1, DU-145, and PC-3 prostate cancer cell lines; MCF-12F normal-like cell line; WI-38 human lung fibroblasts; human mammary epithelial cells; CCD-18Co primary normal human colon fibroblasts (ATCC); PrEC primary normal human prostate epithelial cells (Clonetics, Cambrex); and human renal epithelial cells (ScienCell Research Laboratories) were obtained commercially. Isogenic HCT-116 colon cancer cell lines were a kind gift from B. Vogelstein (Johns Hopkins University, Baltimore). Foreskin fibroblasts were a kind gift from M. Soengas (University of Michigan). Design, Chemical Synthesis, and Binding Models Prediction of MDM2 Inhibitors.
MI-219, MI-63 and MI-61 were synthesized by using our published method (1, 2). The binding models for MDM2 inhibitors were predicted by using the GOLD program (3, 4) (version 2.1) with the ChemScore fitness function. The structures of MDM2 inhibitors were constructed by using the SYBYL molecular modeling software (SYBYL, 7.3; Tripos Associates: St. Louis, MO) and were energy-minimized with the Tripos force field. The MDM2 structural coordinates were extracted from the crystal structure (5) of MDM2 complexed with a p53 transactivation domain peptide available from the Protein Data Bank (PDB ID code 1YCR). Hydrogen atoms were added to the protein using SYBYL. The binding site was defined to encompass all atoms within a 12Å radius sphere, whose origin was located at the center of the Trp-23 of the p53 peptide ligand. The standard Genetic Algorithm protocol was used for docking.

Binding models for each MDM2 inhibitor were obtained by 20 individual docking runs, yielding 20 solutions that were ranked according to their scores calculated by the ChemScore fitness function in the GOLD program.

MI-63 was extensively modified to derive MDM2 inhibitor with desirable PK properties. It was found that the N-2-morpholinoethyl group in MI-63 played a major role for its low oral bioavailability (Fig. 1A). The 2-morpholinoethyl group is partially exposed to solvent in our predicted binding model (2) and mimicked Leu-22 residue in p53. Assisted by our predicted binding model, extensive modifications have been performed on this group. Replacement of the N-2-morpholinoethyl group in MI-63 with an N-(S)-3,4-dihydroxybutyl group led to a dramatic improvement in oral bioavailability of the resulting compound (data not shown) and had no detrimental effect on binding affinity to MDM2 (Fig. 1B). Installation of a fluorine substituent on the 5-position in the oxindole ring enhanced the metabolic stability and the overall oral bioavailability. The 2-F fluorine in the phenyl ring in MI-63 was found to have no significant effect on PK and in vivo stability, and was therefore removed. These optimization efforts have led to the design of MI-219 (Fig. 1 A and B). Computational modeling predicted that MI-219 mimics the four key binding residues in p53 (Phe-19, Leu-22, Trp-23 and Leu-26) (Fig. 1C and SI Fig. 6 A-C) and achieves optimal hydrogen bonding and hydrophobic interactions with MDM2. In PK studies MI-219 achieved good oral bioavailability (SI Table 1).

Based on the predicted binding models for this class of MDM2 inhibitors, the 6-Cl in the oxindole ring in MI-63 and MI-219 inserts into a small and narrow hydrophobic cavity in MDM2. MI-61 was thus designed as an inactive analogue by replacing the 6-Cl atom in the oxindole with a larger and more hydrophilic methoxyl group to disrupt its interaction with MDM2. MI-61 was determined to have a K_i value of >10 mM binding to MDM2 and is >2,000 times weaker than MI-219 (Fig. 1 A and B), serving as an excellent inactive control inhibitor in this study.

Modeling of the Mouse MDM2 and Prediction of the Binding Model of MI-219 in Complex with Mouse MDM2.
The mouse MDM2 protein was homology-modeled by using the crystal structure of human MDM2 (PDB ID code 1YCR)(5) as the template. The sequence alignment used was based on first 110 residues of MDM2. Multiple 3D models of mouse MDM2 were generated by using the program Modeller (6), and the structure with the lowest violation and energy scores was chosen as the candidate. In case of mouse and human MDM2 proteins, the binding pocket for the p53 peptide has only 2 variant residues viz. Leu-54 and Leu-57 in human MDM2 are Ile-54 and Ile-57 respectively in mouse MDM2. Given the high degree of conservation between the proteins, the residues in the binding pocket were examined and as part of refinement side chain orientations of residues in the binding site were adjusted to the template structure. For the two variant residues, a side chain scan was performed initially with SYBYL followed by energy minimization of the residues in the presence of p53 peptide. The resulting model was then used for docking of MI219 for evaluating the relative binding orientations and affinities of the ligand for these proteins. The final structure was checked and validated by Procheck and Verify-3D (7, 8).

The binding poses of MI-219 with mouse MDM2 and human MDM2 (PDB ID code 1YCR) were generated with the GOLD program (version 3.1) (3, 4). The centers of the binding sites for mouse MDM2 and human MDM2 were set at centre of the Trp-23 in the p53 peptide with a radius of 12 Å, large enough to cover the binding pockets. For each genetic algorithm (GA) run, a maximum number of 200,000 operations were performed on a population of five islands of 100 individuals. Operator weights for cross-over, mutation, and migration were set to 95, 95, and 10, respectively. The docking simulations were terminated after 20 runs for each ligand. ChemScore implemented in Gold 3.1 was used as the fitness function to evaluate the docked conformations. The 20 conformations ranked highest by each fitness function were saved for analysis of the predicted docking modes. For the docking poses reported, these were the highest ranked conformations from the docking simulations.

Analysis of the binding models of MI-219 to human and mouse MDM2 showed that this compound occupies the same region in the binding site of both proteins. Due to the conserved residues in the binding pocket similar formation of hydrogen bonds by MI-219 with backbone atoms were also seen. Further evaluation of the predicted binding models, involved rescoring of the obtained poses using different scoring functions. The scores obtained for the predicted binding mode of MI-219 to human and mouse MDM2 utilizing the ChemScore function in Gold is shown with that obtained from Chemscore(9), Xscore(10) and Drugscore(11) in SI Table 3. It was seen that the predicted binding pose of MI-219 to both human and mouse MDM2 score similarly, further indicating that MI-219 should have identical affinity to both human and mouse MDM2 proteins.

MDMX, Bcl-2 and Bcl-xL Binding Assays.
For testing the selectivity of MDM2 inhibitors and natural p53 peptide (¹³PLSQETFSDLWKLLPEN²⁹-NH2) against MDMX, an MDM2 homologue, we have optimized and established a sensitive and quantitative fluorescence polarization-based (FP-based) binding assay using a recombinant human MDMX protein (residues 1-153). As a fluorescent labeled probe, we used PMDM6-F (5-FAM-bAla-bAla-Phe-Met-Aib-pTyr-(6-Cl-L-Trp)-Glu-Ac3c-Leu-Asn-NH2), a high affinity p53-based peptide (12), in our MDM2 binding assay. The K_d value of PMDM6-F with the MDMX protein was determined to be 51.56 nM (SI Fig. 7B). The specificity of the assay was confirmed by competitive displacement of PMDM6-F from MDM2 protein by its corresponding unlabeled peptide (termed PMDM6) without the fluorescence tag 5-FAM (SI Fig. 7C).

The dose-dependent binding experiments were carried out with serial dilutions of the tested compounds in DMSO. A 5-ml sample of the tested samples and preincubated MDMX protein (300 nM) and PMDM6-F peptide (10 nM) in the assay buffer (100 mM potassium phosphate, pH 7.5; 100 mg/ml bovine gamma globulin; 0.02% sodium azide), were added in Dynex 96-well, black, round-bottom plates to produce a final volume of 125 ml. For each assay, the controls included the MDMX protein and PMDM6-F (equivalent to 0% inhibition), only PMDM6-F peptide (equivalent to 100% inhibition). The polarization values were measured after 3 h of incubation using an ULTRA READER (Tecan). The IC₅₀ values, i.e., the inhibitor concentration at which 50% of bound peptide is displaced, were determined from a plot using nonlinear least-squares analysis. Curve fitting was performed by using GRAPHPAD PRISM software (GraphPad Software, Inc.). K_i values were calculated as described in ref. 2.

The binding selectivity of the MDM2 inhibitors against Bcl-2 and Bcl-xL was determined according to our already established FP-based binding assays (2).

Real-Time PCR.
Total RNA was extracted by TRIzol extraction (Invitrogen) from cells treated with or without MDM2 inhibitor for 15 h. cDNA was synthesized from 1 mg of total RNA using random hexamers and AMV reverse transcriptase (Promega). Next, TaqMan gene expression assays were performed by using TP53 (Hs00153349_m1), MDM2 (Hs00242813_m1) and p21 (CDKN1A) (Hs00355782_m1) gene-specific primer/probe sets (Applied Biosystems) for real-time PCR amplification in a BioRad iCycler PCR machine. GAPDH (Hs99999905_m1) was used for normalization and relative quantification of mRNA was calculated by comparative cycle threshold (Ct) method. Immunohistochemistry.
To determine p53 activation in tumor xenografts and normal tissues, nude mice bearing established tumor were treated with a single oral dose of MI-219 or vehicle (20% PEG 400, 3% Cremophor, 77% PBS). At each time point, tissues were harvested and immunohistochemical examination of p53 expression was performed on formaldehyde-fixed, paraffin-embedded tissues. Paraffin-embedded tissue sections, mounted on slides, were deparaffinized and rehydrated by washing in a descending series of alcohol solutions (100%, 95%, 70%, 50%, 30%) and PBS. Antigen retrieval was performed by microwave oven method using a commercially available antigen retrieval buffer (Covance Research Products, Inc.). After cooling down the slides for 30 min at room temperature, the slides were washed with PBS and sections were permeabilized for 10 min at room temperature with 0.3% Triton-X-100 in PBS. Slides were washed with PBS and immersed in 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. The slides were rehydrated, washed in PBS (pH 7.4) for 15 min and tissue sections were blocked with 2% normal goat serum in PBS for 30 min to block unspecific binding sites. After decanting the blocking serum, sections were incubated overnight in a cold room with anti-p53 rabbit polyclonal antibody (FL-393 clone; Santa Cruz Biotechnology), which cross-reacts with human and mouse p53. Dilutions of anti-p53 antibody were prepared in 2% normal goat serum in PBS. After washing, specimens were incubated with biotin-labeled goat-anti-rabbit secondary antibody for 1 h, followed by washing with PBS for 15 min. Next, the specimens were incubated for 30 min with a mixture of avidin:biotinylated horseradish peroxidase complex, which has been prepared 30 min before use by mixing avidin and biotinylated horseradish peroxidase (VECTASTAIN Elite ABC Kit). p53 was detected by diaminobenzidine tetrahydrochloride (DAB) substrate using DAB/buffer system (Sigma). Incubations with substrate were carefully monitored to achieve the desired stain intensity. Sections were washed with water, counterstained with hematoxylin stain for 5-10 seconds and washed under running water. The slides were dehydrated by washing through an ascending series of alcohol, and finally in xylene, and permanently mounted and visualized under the microscope.

Cell proliferation was examined by immunohistochemical staining of BrdU using the 5-Bromo-2'-deoxy-uridine labeling and detection kit II (Roche Applied Science). Apoptosis was examined by TUNEL staining using the In situ Cell Death Detection Kit (Roche Applied Science).

Histopathological analyses of normal tissues
. To determine the effect of MDM2 inhibitors on normal tissues, nude mice (three per group) were orally dosed twice daily at 300 mg/kg of MI-219 or vehicle (20% PEG 400, 3% Cremophor, 77% PBS) for a total period of 2 weeks. One group of mice was euthanized 2 weeks after starting the treatment and the second group was euthanized 4 weeks after starting the treatment. From each group, after euthanizing, nine different organ tissues were excised, fixed in formaldehyde, paraffin embedded and tissue sections were stained with hematoxylin and eosin (H&E). To examine tissue damage, H&E stained tissue samples from mice receiving vehicle control were compared with tissues from mice receiving 300 mg/kg BID of MI-219. Histopathology analyses were performed by an experienced pathologist. Pharmacokinetics Study.
To determine its oral bioavailability, pharmacokinetic (PK) evaluations of MI-219 were performed in mice. The PK studies involved administration of a single oral (p.o.) dose and a single bolus i.v. dose of MI-219. PK parameters were generated from the plasma concentration-time data by a noncompartment method.

For p.o. administration, MI-219 was dissolved in DMA, followed by addition of the Cremophor EL. The resulting solution was then diluted in 0.9% saline to the volume of 8.5 ml, the pH value of which was pH 7.0, adjusted by 0.1 M sodium hydroxide solution in saline. For i.v administration, MI-219 was dissolved in the PEG400/Cremophor mixture and then diluted in 0.9% saline to the volume of 4 ml. The solution was then adjusted to pH 7 with 0.1 mM sodium hydroxide solution in saline. The MI-219 was administered p.o. in mice and rats. The samples for p.o. and i.v. dosing were prepared freshly and were used within 30 min after the preparation.

After treatment, blood was withdrawn at selected time points and plasma samples were prepared. Plasma samples (50 ml) were extracted with 1 ml of ethyl acetate by vortex-shaking for 5 min; the sample was then centrifuged at 13,000 rpm for 5 min, and the upper ethyl acetate phase (800 ml) was removed and reduced to dryness under a stream of nitrogen at 35°C. The residue was reconstituted in 50 ml of 50% CH3CN. After centrifugation at 13,000 rpm for 5 min, 10 ml of the supernatant were introduced into the LC/MS/MS system.

LC conditions: Column: Phenomenex Synergi (particle size 5 mm, 50 mm ´ 2.00 mm i.d.; Torrance) with a precolumn 0.2-mm filter (Upchurch Scientific). The LC isocratic mobile phase used was CH3CN-H₂O (40:60, vol/vol) delivered at the flow rate 0.25 ml/min. The sample size for LC/MS/MS analysis was 10 ml.

The LC/MS/MS system consisted of a Thermo Finnigan TSQ Quantum triple[sbond]quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) probe with Surveyor Modular LC system consisting of an LC pump, an autosampler, and a PDA-UV detector controlled by the Finnigan Xcalibur software package.

Calibration Curves were prepared by spiking drug-free mouse plasma with MI-219 and then serially diluting with blank mouse plasma to generate a range of nominal concentrations of MI 219.

To determine the plasma PK parameters of MI-219, the concentration-time data were analyzed by noncompartmental methods using the KineticaTM2000 software package, Version 3.0 (Innaphase). The area under the plasma concentration-time curve up to the last measured time point (9 h) after p.o and i.v dosing (AUC0®9 h) and the maximum concentration (C_max) were used as parameters for assessing exposure in mouse PK study. The AUC0® t was calculated by the trapezoidal rule. The C_max and the time taken to achieve peak concentration (T_max) were obtained directly from the data without interpolation. The terminal elimination half-life (t_1/2) was calculated by using the relationship 0.693/k, where k is the elimination rate constant. The absolute oral bioavailability (F) was estimated from the ratio of the AUC0®t values after p.o. administration over AUC0®t values after i.v. administration. Results were expressed as arithmetic mean ± standard deviation (SD).

1. Ding K, Lu Y, Nikolovska-Coleska Z, Qiu S, Ding Y, Gao W, Stuckey J, Krajewski K, Roller PP, Tomita Y, Parrish DA, Deschamps JR, Wang S (2005) J Am Chem Soc 127:10130-1.

2. Ding K, Lu Y, Nikolovska-Coleska Z, Wang G, Qiu S, Shangary S, Gao W, Qin D, Stuckey J, Krajewski K, Roller PP, Wang S (2006) J Med Chem 49:3432-3435.

3. Jones G, Willett P, Glen RC, Leach AR, Taylor R (1997) J Mol Biol 267:727-748.

4. Verdonk ML, Cole JC, Hartshorn MJ, Murray CW, Taylor RD (2003) Proteins 52:609-623.

5. Kussie PH, Gorina S, Marechal V, Elenbaas B, Moreau J, Levine AJ, Pavletich NP (1996) Science 274:948-953.

6. Sali A, Blundell TL (1993) J Mol Biol 234:779-815.

7. Bowie JU, Luthy R, Eisenberg D (1991) Science 253:164-170.

8. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) J Appl Cryst 26:283-291.

9. Eldridge MD, Murray CW, Auton TR, Paolini GV, Mee RP (1997) J Comput-Aided Mol Des 11:425-445.

10. Wang R, Lai L, Wang W (2002) J Comput-Aided Mol. Des 16:11-26.

11. Gohlke H, Hendlich M, Klebe G (2000) J Mol Biol 295:337-356.

12. Garcia-Echeverria C, Chene P, Blommers MJ, Furet P (2000) J Med Chem 43:3205-3208.