The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregulation, by activation or transforming mutations of the p110α subunit, is associated with the development of many cancers. 2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) is a novel small molecule inhibitor of PI3K currently being evaluated in the clinic as an anticancer agent. The objectives of these studies were to characterize the relationships between GDC-0941 plasma concentrations and tumor reduction in MCF7.1 breast cancer xenografts and to evaluate the association between the tumor pharmacodynamic biomarker [phosphorylated (p) Akt and phosphorylated proline-rich Akt substrate of 40 kDa (pPRAS40)] responses and antitumor efficacy. MCF7.1 tumor-bearing mice were treated for up to 3 weeks with GDC-0941 at various doses (12.5–200 mg/kg) and dosing schedules (daily to weekly). An indirect response model fitted to tumor growth data indicated that the GDC-0941 plasma concentration required for tumor stasis was approximately 0.3 μM. The relationship between GDC-0941 plasma concentrations and inhibition of pAkt and pPRAS40 in tumor was also investigated after a single oral dose of 12.5, 50, or 150 mg/kg. An indirect response model was fitted to the inhibition of Akt and PRAS40 phosphorylation data and provided IC50 estimates of 0.36 and 0.29 μM for pAkt and pPRAS40, respectively. The relationship between pAkt inhibition and tumor volume was further explored using an integrated pharmacokinetic biomarker tumor growth model, which showed that a pAkt inhibition of at least 30% was required to achieve stasis after GDC-0941 treatment of the MCF7.1 xenograft.
The phosphatidylinositol 3-kinase (PI3K) pathway plays a major role in cell survival, motility, differentiation, and metabolism (Engelman et al., 2006). As part of this pathway, the PI3K family of lipid kinases catalyzes the phosphorylation of the 3′-hydroxyl group of phosphatidylinositols, leading to the activation of the serine/threonine protein kinase Akt and further downstream effectors, such as PRAS40, part of the mTOR complex 1, and S6 kinases (Engelman, 2009). The PI3Ks are divided into three classes based on their substrate specificity and sequence homology; in class I, four isoforms of the catalytic subunit p110 have been identified. The α and β isoforms, belonging to class IA, are ubiquitously expressed, whereas the δ and γ isoforms, belonging to class IA and IB, respectively, are mainly present in leukocytes (Ghigo and Hirsch, 2008). Over the past few years, numerous mutations, leading to the deregulation of the pathway, have been associated with the development of cancers. Activating mutations of the p110α subunit have been noted in breast, colon, prostate, and ovarian cancers (Engelman, 2009; Wong et al., 2010). In addition, mutations or loss of the phosphatase and tensin homolog have also been identified in a large variety of cancers (Chalhoub and Baker, 2009). Thus, the PI3K pathway has emerged as a major target in the development of small molecule anticancer drugs, including the PI3K inhibitors N-(3-(benzo[c][1,2,5]thiadiazol-5-ylamino)quinoxalin-2-yl)-4-methylbenzenesulfonamide (XL147), 5-(2,6-dimorpholinopyrimidin-4-yl)-4-(trifluoromethyl)pyridin-2-amine (BKM120), and (Z)-5-((4-(pyridin-4-yl)quinolin-6-yl)methylene)thiazolidine-2,4-dione (GSK1059615) and dual PI3K/mTor inhibitors (Liu et al., 2009).
GDC-0941 (Fig. 1) is a novel small molecule inhibitor of PI3K currently being evaluated in phase I studies as an anticancer agent. This compound was shown to be selective for the class I PI3K against a panel of 228 kinases (Folkes et al., 2008). Of the kinases tested, only two, the human tyrosine kinase Flt3 and the human kinase TrkA, were inhibited by more than 50% with 1 μM GDC-0941 (59 and 61%, respectively). GDC-0941 can be considered equipotent against the four class I PI3K isoforms with IC50 values of 0.003, 0.033, 0.003, and 0.075 μM against p110α, β, δ, and γ, respectively, and potently inhibits the phosphorylation of Akt in PC3-NCI (prostate) and MCF7.1 cells (breast), with IC50 values ranging from 0.028 to 0.037 μM. It is also able to inhibit the proliferation of MCF7.1 and PC3-NCI cells with IC50 values of 0.72 and 0.28 μM, respectively (Folkes et al., 2008), and was shown to be efficacious against the U87MG glioblastoma and IGROV-1 human ovarian cancer xenograft models in athymic mice (Raynaud et al., 2009).
Pharmacokinetic (PK)-pharmacodynamic (PD) modeling in preclinical settings can be a valuable tool to explore and understand the relationships between the pharmacokinetic properties of a compound and the PD marker modulation and efficacy data. Once established in preclinical models, these relationships may be useful in assessing the viability of a compound for further development and can be extrapolated to determine potential efficacious levels in humans (Chien et al., 2005) or guide dose escalation in clinical studies.
The objectives of the present studies were to characterize the in vivo efficacy of GDC-0941 and to investigate the PK-PD relationship of the GDC-0941 plasma concentration to MCF7.1 tumor reduction and PD marker (Akt and PRAS40) phosphorylation.
Materials and Methods
GDC-0941 was synthesized by Genentech, Inc. (South San Francisco, CA). All solvents used in analytical assays were from Thermo Fisher Scientific (Waltham. MA) and were of analytical or high-performance liquid chromatography grade. All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless specified.
Female athymic nude nu/nu mice (Charles River Laboratories, Hollister, CA) weighing 20 to 27 g received oral doses (100 μl) of 5, 50, 75, 100, and 200 mg/kg GDC-0941 in 0.5% methylcellulose-0.2% Tween 80 (MCT). Blood samples (0.2 ml) were collected from each mouse by terminal cardiac puncture at 0.083, 0.25, 0.5, 1, 3, 6, 9, and 24 h postdose into tubes containing K2EDTA as an anticoagulant. Samples were taken from three different animals at each time point. After the blood was mixed with K2EDTA, the samples were stored on ice and within 1 h of collection were centrifuged for 5 min at 2000g and 2 to 8°C. Plasma was collected and stored at −80°C until analysis. Total concentrations of GDC-0941 were determined by liquid chromatography-tandem mass spectrometry, after plasma protein precipitation with acetonitrile and injection of the supernatant onto the column. A CTC HTS PAL autosampler (LEAP Technologies, Carrboro, NC) linked to a Shimadzu SCL-10A controller with LC-10AD pumps (Shimadzu, Columbia, MD), coupled with a Sciex API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA), was used for the liquid chromatography-tandem mass spectrometry assay. The aqueous mobile phase was water with 0.1% formic acid and the organic mobile phase was acetonitrile with 0.1% formic acid. The total run time was 4 min, and the ionization was conducted in the positive ion mode using the transition m/z 514.3 → 338.2. The internal standard was the deuterated (D8) analog of GDC-0941. The lower and upper limits of quantitation of the assay were 0.005 and 10 μM, respectively.
MCF7.1 Breast Cancer Xenografts Studies.
MCF7.1 is an in vivo selected cell line developed at Genentech, Inc. and derived from the parental MCF7 human breast cancer cell line (American Type Tissue Collection, Manassas, VA). Because the tumorigenicity of the MCF7.1 cells in mice is estrogen-dependent, estrogen pellets (17β-estradiol, 0.36 mg/pellet, 60-day release) obtained from Innovative Research of America (Sarasota, FL) were implanted into the dorsal shoulder blade area of female athymic nude nu/nu mice. After 3 to 7 days, 20 million human breast cancer MCF7.1 cells, resuspended in a 1:1 mixture of Hanks' buffered salt solution and Matrigel basement membrane matrix (BD Biosciences, San Jose, CA), were subcutaneously implanted into the right flank of each mouse. After implantation, tumors were monitored until they reached a mean tumor volume of 200 to 300 mm3. Tumor size and body weight were recorded twice per week during the study. Animal body weights were measured using an Adventurer Pro AV812 scale (Ohaus Corporation, Pine Brook, NJ). Tumor lengths and widths were measured using Ultra Cal-IV calipers (model 54-10-111; Fred V. Fowler Company, Inc., Newton, MA). Mice were euthanized if body weight loss was greater than 20% from their initial body weight or if the tumors exceeded 2000 mm3. The mean tumor volume (TV) across all groups was 269 mm3 at the initiation of dosing.
Tumor volume was calculated with Excel version 11.2 (Microsoft, Redmond, WA) using the following equation: GDC-0941 was administered orally in MCT for 16 to 21 days. Animals in the control groups received the vehicle, MCT (100 μl). The animals treated with GDC-0941 received the following doses in 100 μl of MCT. Ten mice were assigned to each dose group: experiment 1: vehicle, 12.5, 25, 50, 75, 100, and 200 mg/kg q.d.; experiment 2: vehicle, 12.5, 25, 37.5, 50, and 100 mg/kg b.i.d.; and experiment 3: vehicle, 200 mg/kg q.d., 200 mg/kg every other day, and 200 mg/kg every 3rd day. The tumor volumes at the end of the study for each group were compared with the tumor volume of the corresponding vehicle group.
Mean tumor volumes and S.E.M. were calculated using JMP software (version 5.1.2; SAS Institute, Cary, NC). Statistical analyses of tumor volumes were performed using Dunnett's t test with JMP software.
Modulation of pAKT and pPRAS40 in MCF7.1 Xenografts.
Tumor cells were implanted as described previously for the efficacy studies and tumors were monitored until they reached a mean volume of 250 to 350 mm3. Afterward, the animals were divided into four dose groups and received a single oral dose of vehicle (100 μl of MCT; n = 7), 12.5, (n = 4), 50 (n = 4), or 150 mg/kg (n = 4) GDC-0941 in 100 μl of MCT. Tumors were collected at 0.5, 4, 8, and 24 h postdose from the 12.5, 50, and 150 mg/kg treatment groups and at 0.5 (n = 3) and 24 h (n = 4) postdose from the vehicle group. Excised tumors were flash-frozen and stored at −80°C until analysis.
Frozen tumors were weighed and lysed with a pestle PP (Scienceware, Pequannock, NJ) in cell extract buffer (BioSource, Carlsbad, CA) supplemented with protease inhibitors (F. Hoffman-La Roche, Ltd., Mannheim, Germany), 1 mM phenylmethylsulfonyl fluoride, and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich). Protein concentrations were determined using the BCA Protein Assay Kit (Pierce Chemical, Rockford, IL). The BioSource and Luminex Bio-Plex systems (Bio-Rad Laboratories, Hercules, CA) were used to determine the levels of Akt, phosphorylated at serine 473 (pAkt); PRAS40, phosphorylated at threonine 246 (pPRAS40); and total Akt. These assays quantify protein levels based on measurements of fluorescence intensity. The data were normalized by dividing mean fluorescent values for the pPRAS40 and pAkt Luminex data by the mean fluorescent values of the total Akt Luminex assay.
PK and PK-PD modeling was performed using SAAMII (Saam Institute, University of Washington, Seattle, WA).
A one-compartment model with first-order oral absorption was used to fit the mean plasma concentration-time data from female nu/nu mice. The pharmacokinetics of GDC-0941 appeared linear over the range of doses tested, and the five single doses (5, 50, 75, 100, and 200 mg/kg) were fitted simultaneously. The 40-fold change in dose was associated with an equivalent change in area under the curve between 5 and 200 mg/kg (2.4 μM · h at 5 mg/kg and 115 μM · h at 200 mg/kg). The mean estimates of the absorption rate constant (ka), the elimination rate constant (ke), and the apparent volume of distribution (V/F) were used to simulate the GDC-0941 plasma concentrations for the modeling of the xenograft efficacy studies.
An indirect response model, described by differential eq. 1, was fitted to the xenograft efficacy data: where t is time (hours), TV is the tumor volume (millimeters cubed), kng is the net growth rate constant (hour−1), and K is the rate constant (hour−1) associated with the reduction of tumor by GDC-0941. K is further described as where Kmax is the maximum value of K (hour−1), C is the plasma concentration of GDC-0941 (micromolar), n is the Hill coefficient, and KC50 is the concentration of GDC-0941 where K is 50% of Kmax.
Blood was collected at a single time point, 1 h postdose at the end (day 21) of the efficacy studies in the q.d. arm (experiment 1). The plasma concentrations determined in these samples (data not shown) were not used further for any analyses or modeling. However, they were similar to the plasma concentrations obtained in the single-dose PK study, indicating the absence of time dependence for the pharmacokinetics of GDC-0941 in mice. Consequently, GDC-0941 plasma concentrations in mice were simulated based on the parameters determined in the pharmacokinetic studies, and mean tumor volumes from each dose group were used in the modeling. The ED50 was determined by fixing PD parameter estimates and simulating the daily dose required for 50% reduction of the tumor volume relative to that of the vehicle control group at the end of the study. The plasma concentration needed to achieve tumor stasis was determined when K was equal to kng.
Modulation of phosphorylation of Akt and PRAS40.
The relationship between Akt and PRAS40 phosphorylation in tumors and GDC-0941 plasma concentrations was characterized using an indirect response model, in which the inhibition of the formation of the PD markers pAkt or pPRAS40 by GDC-0941 could be described by eq. 2: where PD corresponds to the level of PD marker (pAkt or pPRAS40) measured (fluorescence intensity), t is time (hours), kin is the rate of formation of the PD marker (fluorescence intensity per hour), C is the plasma concentration of GDC-0941 (micromolar), IC50 is the plasma concentration of GDC-0941 producing 50% inhibition of the PD marker (micromolar), and kout (hour−1) is the rate constant defining the loss of the PD marker. Under homeostatic conditions, kin = kout (PD) and kout can be substituted with kin/PDb, where PDb represents the level of PD marker at baseline (predose or vehicle control).
GDC-0941 plasma concentrations in mice were simulated based on the parameters determined in the pharmacokinetic studies. Because, as stated above, the plasma concentrations achieved did not seem to markedly change throughout the efficacy study compared with the single-dose PK study, it was assumed that the PD response in the efficacy study could be represented well by the single-dose PD study.
Integrated tumor growth model.
To further define the relationship between pAkt inhibition and tumor growth inhibition, the PK-PD model used to characterize the relationship between GDC-0941 plasma concentrations and tumor volumes in the efficacy studies was combined with the model describing the modulation of pAkt. This integrated model assumes that the inhibition of tumor growth is mediated by the decrease in pAkt (i.e., inhibition of phosphorylation of Akt). The changes in pAkt levels were simulated for each dose used in the efficacy study. Thus, the levels of pAkt were related to the tumor volumes using eqs. 3 and 4: where %I describes the percentage of inhibition of pAkt and K(%I)50 corresponds to the percentage of inhibition of pAkt where K is 50% of Kmax. The percentage of inhibition of pAkt needed for tumor stasis was determined when K was equal to kng.
GDC-0941 plasma concentrations in mice were simulated based on the parameters determined in the pharmacokinetics studies. Mean tumor volumes from all dose groups (experiments 1–3) were fitted simultaneously.
The pharmacokinetic profiles at the five doses used are presented in Fig. 2A. The parameters ka, ke, and V/F were estimated for GDC-0941 after single oral administrations to mice of 5, 50, 75, 100, and 200 mg/kg GDC-0941 in MCT and were 10.4 h−1, 0.23 h−1, and 19.9 l/kg, respectively (Table 1). These estimated parameters were used to simulate plasma concentration-time profiles when the xenograft efficacy and PD marker modulation data were modeled, because serial blood samples adequate for modeling were not collected from tumor-bearing mice during the studies. The comparison between the observed plasma concentrations and model predictions is presented in Fig. 2B and indicates that the PK model used described these data appropriately.
Xenograft efficacy studies.
GDC-0941 was administered orally to MCF7.1 tumor-bearing mice at various doses and dosing schedules. Tumor growth inhibition results are presented in Fig. 3, A to C. The inhibition of the MCF7.1 tumor growth appeared to be dose-dependent. Tumor regression was observed at daily doses higher than 50 mg/kg (Fig. 3A) and twice-daily doses higher than 25 mg/kg (Fig. 3B). All doses and regimen showed statistically significant (p < 0.05) tumor growth inhibition at the end of the study compared with their respective vehicle group, except the 200 mg/kg every third day (Fig. 3C) treatment group.
An indirect response model was fitted to the tumor data from all studies (eq. 1) and appeared to describe them adequately. The comparison between the observed tumor volumes and model predictions is presented in Fig. 3D. The estimates of the pharmacodynamic parameters describing MCF7.1 tumor growth and reduction effect by GDC-0941 are presented in Table 2.
Modulation of phosphorylation of Akt and PRAS40.
The levels of the PD markers pAkt and pPRAS40 were measured in tumors after a single oral administration of GDC-0941 at 12.5, 50, or 150 mg/kg in MCF7.1 tumor-bearing mice. The results are presented in Fig. 4. A sharp decrease in pAkt (Fig. 4A) and pPRAS40 (Fig. 4B) was observed at the three doses, indicative of a potent inhibition of the PI3K pathway. The suppression of the PD markers was pronounced at 30 min postdose and sustained for 8 h at the 50 and 150 mg/kg doses. In contrast, the signal was mostly recovered 4 h postdose at the 12.5 mg/kg dose level. In general, higher GDC-0941 plasma concentrations (simulated) resulted in greater suppression of the PD markers (Fig. 4, A and B).
The comparisons between the observed and predicted pAkt and pPRAS40 levels after fitting with an indirect response model (eq. 2) are presented in Fig. 4, C and D, respectively. The PD parameters are shown in Table 3.
Integrated tumor growth model.
The relationship between inhibition of the PI3K pathway and efficacy was further investigated using a PK-PD model integrating GDC-0941 plasma concentrations, pAkt modulation, and tumor growth inhibition (Fig. 5A). The indirect response model describing the relationship between GDC-0941 plasma concentrations and pAkt levels was used to simulate pAkt inhibition for all dose groups included in the efficacy studies. An indirect response model relating the pAkt inhibition to tumor growth inhibition (eqs. 3 and 4) was fitted to the tumor volumes from experiments 1 to 3 simultaneously. The comparison between the observed tumor volumes and model predictions is presented in Fig. 5B, and the estimated parameters are in Table 4. Based on these parameters and the relationship between pAkt inhibition and the rate constant K, representing a tumor reduction effect by GDC-0941 (Fig. 5C), it appeared that a constant inhibition of pAkt of at least 30% would be necessary to achieve tumor stasis. In addition, the simulation of the time course of the pAkt suppression at the daily ED50, based on the parameters listed in Table 3, is presented in Fig. 6. This plot suggests that ∼30% inhibition of pAkt is achieved for approximately 10 h at the ED50.
The PI3K-Akt pathway is one of the most commonly altered signaling pathways in cancer (Liu et al., 2009). After activation by receptor tyrosine kinases, class I PI3K is recruited to the plasma membrane and converts phosphatidylinositol biphosphate to phosphatidylinositol triphosphate. This initial reaction leads to the activation of the serine/threonine kinase Akt, a major effector of PI3K, which in turn triggers downstream signaling events, including PRAS40 phosphorylation. Mutations of the p110α catalytic subunit of the PI3K have been associated with 27% of breast cancers, 24% of endometrial cancers, and more than 10% of colon and upper digestive tract cancers (Liu et al., 2009). GDC-0941, a selective inhibitor of class I PI3K, is currently being evaluated in clinical trials as an anticancer agent (Sarker et al., 2009).
Indirect response models are relevant when the response measured is the product of an indirect mechanism (Dayneka et al., 1993; Mager et al., 2003), such as the inhibition or stimulation of the formation (kin) or loss (kout) of the mediator controlling the physiological effect. In the present studies, the levels of pAkt and pPRAS40 in tumor were monitored after upstream inhibition of PI3K by GDC-0941 administered as a single dose to MCF7.1 tumor-bearing mice. The inhibition of the production of these two markers (Fig. 3C) was characterized using indirect response models and the in vivo IC50 estimates were 0.36 and 0.29 μM for inhibition of pAkt and pPRAS40, respectively (Table 2). For pAkt, this value was approximately 10-fold higher than the IC50 measured in vitro (0.028 μM) (Folkes et al., 2008). This difference may be partly explained by the binding to plasma protein in vivo, because GDC-0941 is 97% bound in mouse plasma (E. G. Plise, unpublished data), and/or by limited distribution to the subcutaneous tumor. The IC50 for pPRAS40 inhibition was not determined in vitro.
The relationship between GDC-0941 plasma concentrations and MCF7.1 tumor growth inhibition was also adequately described by an indirect response model (Fig. 2). This PK-PD model suggested that GDC-0941 plasma concentrations of approximately 0.3 μM would be necessary to achieve tumor stasis (Table 1), which was comparable to the IC50 estimated for both pAkt and pPRAS40 inhibition (Table 2). The similar IC50 values established in separate models between GDC-0941 concentrations and inhibition of PD marker phosphorylation or tumor growth imply that approximately 50% inhibition of Akt and PRAS40 phosphorylation would be associated with tumor stasis. This approach, comparing parameters in independent models, was used by Yamazaki et al. (2008) in their investigations of the cMet inhibitor (R)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamine (PF02341066), in which the EC90 for the inhibition of cMet phosphorylation was similar to the EC50 for tumor growth inhibition, leading the authors to conclude that more than 90% inhibition of cMet phosphorylation would be needed to inhibit tumor growth by more than 50%. We investigated further the relationship between pAkt modulation and tumor growth by using an integrated tumor growth model, as proposed by Wong et al. (2009) and Bueno et al. (2008). This model assumes that tumor growth inhibition is mediated by the decrease in pAkt (Fig. 5A). The curve describing the relationship between inhibition of Akt phosphorylation and tumor growth inhibition, represented by the rate constant K (Fig. 5C), indicated that the increase in effect would be less than linear beyond 28.6% inhibition [K(%I)50] and that little improvement in effect would be achieved with pAkt inhibition greater than 50%. The modeling also suggested that 30% continuous inhibition of pAkt signal would be required to achieve tumor stasis. This level of pAkt inhibition was achieved for approximately 10 h at the daily ED50 of 16 mg/kg (Fig. 6). This is, to our knowledge, the first report describing a PK-PD relationship between modulation of the PI3K pathway and tumor growth inhibition. A similar PK-PD model integrating pPRAS40 modulation and tumor growth inhibition may be built; however, although a decrease in PRAS40 phosphorylation can be considered a marker of the inhibition of the pathway, it remains to be established as a direct driver of tumor growth inhibition (Manning and Cantley, 2007). The integrated model described here may be helpful in trying to predict the likelihood of tumor response in humans from the extent of suppression of the pathway measured by pAkt inhibition. Such an extrapolation assumes that the relationship between PD marker response and antitumor activity is similar in human and xenograft models. The MCF7.1 cells used in these studies harbor a mutation (E545K) of the p110α catalytic subunit, resulting in constitutive PI3K pathway activation (Edgar et al., 2010). Thus, they are considered highly dependent on the pathway, making them sensitive to its inhibition. It is likely that a stronger inhibition of pAkt would be required to achieve stasis of tumor cells less “addicted” to this pathway. However, despite limitations in the translation to human efficacy, the PK-PD modeling performed in the current studies may nevertheless be useful to make “no-go” decisions in compound development, select the optimal times for PD marker data collection, or guide clinical trial design. Such an application was described by Tanaka et al. (2008), who extrapolated the relationship established in rats between the plasma concentrations of everolimus (an mTor inhibitor) and S6 kinase 1 inhibition to simulate the effects of various dosing regimen in human using scaled pharmacokinetics.
It is also worth noting that the IC50 for pAkt estimated preclinically in our model appears consistent with data collected in humans. Dose-dependent inhibition of pAkt measured in platelet-rich plasma was reported in patients, with an IC50 that could be estimated to be approximately 0.3 μM (Sarker et al., 2009). Although pAkt was measured in tumor in our studies, this agreement with preliminary clinical data from platelet-rich plasma suggests that the preclinical PK-PD model may be useful in designing future clinical trials.
We thank the Drug Metabolism and Pharmacokinetics and Translational Oncology Departments and the In Vivo Studies Group for their contributions to the results presented.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
- phophatidylinositol 3-kinase
- proline-rich Akt substrate of 40kDa
- mammalian target of rapamycin
- 0.5% methylycellulose/0.2% Tween 80
- tumor growth inhibition
- tumor volume
- Received February 23, 2010.
- Accepted June 10, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics