Although Erbitux (cetuximab) has proven therapeutic benefit in the clinical setting, the molecular determinants predicting responsiveness to this agent are still not very well understood. Here, we assessed the relationship between basal total and activated (pY1068) epidermal growth factor receptor (EGFR) levels in a tumor and the responsiveness to cetuximab monotherapy or combination-based treatment using human xenograft models. Cetuximab treatment alone (0.25-1 mg/mouse/injection, q3d, i.p.) effectively delayed the growth of GEO and L2987 tumors by a minimum of 10 days corresponding to log cell kill values of >or=1.0. Borderline activity was seen in the A549 and WiDr xenografts. However, cetuximab failed to show any significant antitumor activity in the HT29, HCT116, LOVO, Colo205, LX-1, HCC70, and N87 models. All of the studied tumors had detectable yet variable levels of EGFR. For combination regimens, cetuximab (1 mg/mouse/injection, q3dx5, i.p.) and cisplatin (4.5 mg/kg/injection, q3dx5, i.v.) proved to be significantly more efficacious than individual monotherapies in the cisplatin-refractory yet cetuximab-responsive GEO tumor model (P < 0.001). However, no therapeutic enhancement was observed in the cisplatin and cetuximab weakly responsive A549 xenograft. Similarly, combinations of CPT-11 (48 mg/kg/injection, q3dx5, i.v.) with cetuximab (1 mg/mouse/injection, q3dx5, i.p.) failed to show any improvements over individual monotherapies in the cetuximab resistant/weakly responsive HT29, A549, and WiDr models. We conclude that preclinical activity associated with cetuximab monotherapy does not correlate directly with relative basal levels of total or activated (pY1068) EGFR in a tumor. Moreover, robust single-agent activity by cetuximab may be the best predictor for this agent to potentiate chemotherapy-mediated antitumor activities.

12017 Background: Ixabepilone belongs to a class of structurally novel, microtubule-stabilizing agents that exert their antimitotic action by binding to tubulin with a binding mode that is distinct from the taxanes. Preclinical findings that ixabepilone has antitumor activity in a broad spectrum of tumor types, including taxane-resistant tumors, is borne out by Phase II clinical trials where ixabepilone has demonstrated activities in multiple tumor types including breast, renal, pancreatic, prostate and lymphoma. The aim of this series of studies was to further characterize the therapeutic potential of ixabepilone in combination with currently approved chemotherapy agents. Methods: Antitumor activity was evaluated in the GEO human colon and L2987 human lung carcinoma xenografts. Therapeutic synergism of the combination was defined as the attainment of efficacy that was significantly better than the best response of the individual single agents administered at their maximum-tolerated dose (MTD) or optimal dose (OD). Results: In the GEO tumors, single-agent ixabepilone produced 1.1 log cell kill (LCK) at its MTD. Cetuximab at its OD yielded 0.8 LCK. The combination of ixabepilone and cetuximab produced 1.7 LCK which was significantly superior to ixabepilone alone (P=0.0173) and cetuximab alone (P=0.0002). Similar synergistic efficacy was observed in the L2987 tumors. The combined efficacy of capecitabine plus ixabepilone was evaluated in the GEO tumors. In this tumor, single-agent ixabepilone was modestly active (LCK = 0.8) at its MTD. Single-agent capecitabine was not effective (LCK = 0.4) at its MTD. However, the combination of the two agents produced therapeutic synergism, yielding antitumor efficacy (1.9 LCK) that was superior to either of the agents alone at their MTDs (P=0.035 and 0.0004, respectively). Conclusions: Ixabepilone demonstrates robust synergistic antitumor efficacy when used in combination with cetuximab or capecitabine in human xenografts providing a biologic rationale for these combinations in the treatment of cancer. [Table: see text]

Purpose: The epidermal growth factor receptor (EGFR), a protein tyrosine kinase expressed in many types of human cancers including colon and breast, has been strongly associated with tumor progression. Cetuximab, an IgG1 anti-EGFR chimeric mouse/human monoclonal antibody, has been proven to be effective in the treatment of advanced colon cancer. To date, there has not been a study to systematically evaluate the pharmacokinetics (PK) of Cetuximab in a preclinical model and to further explore any correlation of drug exposure between animal models and cancer patients. In the present study, we characterized the PK of Cetuximab in nude mice at efficacious dose levels and further compared the preclinical optimal dose and active plasma drug concentration with those determined in clinical studies. Experimental design: The antitumor activity of Cetuximab was evaluated using the GEO human colon carcinoma xenografts implanted subcutaneously in nude mice. The drug was administered ip every 3 days for five total injections (inj) (q3dx5) at dose levels ranging from 1 mg/inj to 0.04 mg/inj. The plasma PK of Cetuximab was determined at dose levels of 1.0, 0.25, and 0.04 mg/inj with a single bolus iv or ip administration in nude mice. The tumoral PK of Cetuximab was determined at dose levels of 0.25, and 0.04 mg/inj with a single bolus ip administration in nude mice bearing GEO tumor xenografts. The plasma and tumoral levels of Cetuximab were quantitated by an ELISA assay. Results: Cetuximab demonstrated a dose-dependent antitumor activity at dose levels of 0.25, 0.1, and 0.04 mg/inj, with a statistically significant tumor growth delay (in reaching a tumor target size of 1 gm) of 18 days (P<0.001), 12.3 days (P<0.01), and 10 days (P<0.01) for 0.25, 0.1, and 0.04 mg/inj, respectively. A separate study employing the same treatment schedule showed that Cetuximab was equally active at dose levels ranging from 0.25 mg/inj to 1 mg/inj. Therefore, dose levels of Cetuximab from 1 mg/inj to 0.04 mg/inj can be considered to be within the efficacious range, while dose levels of 0.25 mg/inj or higher appeared to be optimal for the antitumor activity of Cetuximab in the GEO tumor model. When Cetuximab was given iv to mice, the elimination half life (t1/2) was 39.6, 37.8, and 42.2 h for doses of 1.0, 0.25, and 0.04 mg/inj, respectively, suggesting a similar disposition kinetics of Cetuximab within this dose range. The volume of distribution (Vd) ranged from 0.062 l/kg to 0.070 l/kg, suggesting that Cetuximab is primarily confined to the plasma compartment with limited peripheral tissue distribution. Clearance (CL) was similar and no apparent PK saturation was observed across the dose ranging from 0.04 mg/inj to 1.0 mg/inj. When mice were administered with a single bolus ip administration at doses of 1, 0.25, and 0.04 mg/inj, the maximum plasma concentration (Cmax) was 407.6, 66.4, and 16.5 μg/ml. The area under the curve of plasma drug concentration (AUC) was 19212.4, 3182.4, and 534.5 μg/ml h, for 1.0, 0.25, and 0.04 mg/inj, respectively. The average steady state plasma concentration (Css avg) for the multiple dosing schedule was estimated to be 73.1 μg/ml at 0.25 mg/inj and was considered as an active plasma drug concentration. The maximum tumoral concentration of Cetuximab was 2.6 and 0.53 ng/mg-tumor while the tumoral drug exposure was 112.6 and 18.3 ng/mg h for 0.25 and 0.04 mg/inj, respectively. The EGFR was estimated to be nearly completely occupied by Cetuximab at the optimal dose of 0.25 mg/inj. Conclusion: In the present study, we compared the preclinical optimal dose and the corresponding active plasma concentration determined in mice with those being observed in cancer patients, i.e. 65–100 μg/ml. The preclinical optimal dose of 0.25 mg/inj was significantly lower than the current clinical dose. However, the active plasma concentration at 0.25 mg/inj is within the range of the active drug concentrations in cancer patients treated with Cetuximab under the current optimal dosing regimen. It appears that the active plasma drug concentration determined in preclinical model predicts better than the optimal preclinical dose for the clinical development of antibody drugs.

Resistance to imatinib is a growing concern in CML, particularly in advanced disease. The most common cause of resistance is mutations in BCR-ABL, but other mechanisms have also been identified, including over-expression of BCR-ABL, activation of SRC family kinases and the P-glycoprotein (PGP) efflux pump (via MDR1 over-expression). Dasatinib (BMS-354825) is a novel, oral, multi-targeted tyrosine kinase inhibitor that targets BCR-ABL and SRC kinases. Dasatinib has 325-fold greater potency versus imatinib in cell lines transduced with wild-type BCR-ABL and is active against 18 out of 19 BCR-ABL mutations tested that confer imatinib resistance (Shah et al, Science305:399, 2004; O’Hare et al, Cancer Res65:4500–5, 2005), and preliminary results from a Phase I study show that it is well tolerated and has significant activity in imatinib-resistant patients in all phases of CML (Sawyers et al, J Clin Oncol23:565s, 2005; Talpaz et al, J Clin Oncol23:564s, 2005). We assessed the ability of dasatinib to overcome a variety of mechanisms of imatinib resistance. First, the leukemic-cell killing activity of dasatinib was tested in vitro in three human imatinib-resistant CML cell lines (K562/IM, MEG-01/IM and SUP-B15/IM). Based on IC50 values, dasatinib had >1000-fold more potent leukemic-cell killing activity compared with imatinib versus all three cell lines. Furthermore, in mice bearing K562/IM xenografts, dasatinib was curative at doses >5 mg/kg, while imatinib had little or no impact at doses as high as 150 mg/kg, its maximum tolerated dose. We determined that the MEG-01/IM and SUP-B15/IM cell lines carried BCR-ABL mutations known to confer imatinib resistance to imatinib clinically (Q252H and F359V, respectively). In K562/IM cells, BCR-ABL mutations or BCR-ABL over-expression were not detected, but the SRC family member FYN was over-expressed. PP2, a known inhibitor of SRC family kinases but not BCR-ABL, could reverse the imatinib resistance in these cells. Together, these data suggest that activation of FYN may be a cause of imatinib resistance in K562/IM. Based on cell proliferation IC50, we found that the anti-leukemic activity of dasatinib in K562/IM cells was 29-fold more potent compared with AMN107 (a tyrosine kinase inhibitor that inhibits BCR-ABL but not SRC family kinases). Given that the human serum protein binding of dasatinib, imatinib and AMN107 were 93, 92 and >99% respectively, the difference in potency between dasatinib and AMN107 in vivo may be far greater than the simple fold-difference in the in vitro IC50 values. Finally, in K562 cells over-expressing PGP (K562/ADM), we found that dasatinib was only 6-fold less active than in parental K562 cells. Because of the extreme potency of dasatinib in K562 cells, this reduced potency still afforded an IC50 of 3 nM, which is readily achievable in vivo. Indeed, in mice bearing K562/ADM xenografts, dasatinib was curative at 30 mg/kg, with significant anti-leukemic activity at 15 mg/kg. In conclusion, the rational design of dasatinib as a multi-targeted kinase inhibitor allows this agent to overcome a variety of mechanisms of resistance to imatinib in CML, including mechanisms that are not overcome by agents with a narrower spectrum of inhibition, such as AMN107. Dasatinib is currently in Phase II evaluation in imatinib-resistant/-intolerant patients in the ‘START’ program, and in Phase I evaluation in solid tumors.

The major concern in the treatment of CML is resistance to the approved agent imatinib mesylate at all stages of disease, most commonly due to mutations in BCR-ABL (but other mechanisms have also been identified). Experimental agents such as dasatinib (BMS-354825), a novel, oral kinase inhibitor that targets BCR-ABL and SRC kinases, or AMN107, which targets BCR-ABL but not SRC, were designed to address all or parts of these mechanisms and are currently under clinical testing. A second concern in CML is persistence of BCR-ABL-positive cells or ‘residual disease' in the majority of patients on imatinib therapy, including those with complete cytogenetic responses. Bone marrow studies reveal that the residual disease resides at least in part in the primitive CD34+ progenitor compartment, suggesting that imatinib may not be effective against these cell populations (Bhatia et al, Blood 101:4701, 2003). Moreover, several imatinib-resistant ABL kinase domain mutations have been detected in CD34+/BCR-ABL+ progenitors (Chu et al, Blood 105:2093, 2005), a scenario for eventual disease relapse. A hallmark of CD34+ primitive CML progenitors is quiescence (Elrick et al, Blood 105:1862, 2005). We hypothesized that BCR-ABL inhibitors like imatinib may not be effective in killing CML cells in this non-proliferative state. This was tested by comparing cytotoxicity of imatinib or dasatinib in proliferating K562 cells and in cells forced into quiescence by nutrient depletion. Proliferating K562 cells were effectively killed by imatinib (IC50 250–500 nM) and dasatinib (IC50 <1.00 nM). However, cells in quiescent cultures were far more resistant (imatinib IC50 >5000 nM; dasatinib IC50 >12 nM), suggesting that these inhibitors may be less effective in eradicating quiescent CD34+ progenitors. BMS-214662 is a FTI in Phase I clinical development. Unlike many other FTI, BMS-214662 exhibits potent cytotoxic activity against a variety of human tumor cells, and uniquely, its cytotoxicity is highly selective against non-proliferating cancer cells of epithelial origin (Lee et al, Proceedings of the AACR 42:260s, 2001). We now demonstrate similar selectivity in K562 CML cells. BMS-214662 was 68-fold more potent in killing quiescent (IC50 = 0.7 uM) than proliferating K562 cells (IC50 = 47.5 uM). Because BCR-ABL inhibitors and BMS-214662 target distinct cell populations (proliferating vs quiescent), there may be a positive therapeutic interaction when these agents are used in combination. In vitro studies in quiescent K562 cultures demonstrated that the combination of BMS-214662 and dasatinib, at concentrations readily achievable in the clinic, produced supra-additive cytotoxicity (% cell kill: dasatinib alone = 0%, BMS-214662 alone = 21%, combination = 71%). In vivo studies in K562 xenografts implanted SC in mice also showed that the combination of BMS-214662 and dasatinib produced a superior anti-leukemic activity than either dasatinib alone (P=0.0157) or BMS-214662 alone (P=0.0002). These results highlight the potential utility of BMS-214662 for targeting the quiescent progenitor compartment which, in combination with targeted agents such as dasatinib, address both BCR-ABL-dependent and -independent mechanisms of resistance, and may produce more durable responses and suppress the emergence of resistance.

Purpose: Epidermal growth factor receptor (EGFR), a protein tyrosine kinase expressed in many types of human cancers, has been strongly associated with tumor progression. Cetuximab is an IgG(1) anti-EGFR chimeric mouse/human monoclonal antibody that has been approved for the treatment of advanced colon cancer. Using human tumor xenografts grown in nude mice, we have determined the in vivo pharmacodynamic response of cetuximab at efficacious doses. Three pharmacodynamic end points were evaluated: tumoral phospho-EGFR, tumoral mitogen-activated protein kinase (MAPK) phosphorylation, and Ki67 expression.Experimental Design: The pharmacodynamic study was conducted in nude mice bearing Geo tumors following a single i.p. administration of 0.25 and 0.04 mg. The tumors were analyzed by immunohistochemistry. The levels of phospho-EGFR were quantitated by an ELISA assay.Results: At 0.25 mg, phospho-EGFR was maximally inhibited by 91% at 24 hours, whereas the level of inhibition decreased to 72% by 72 hours. At 0.04 mg, the maximum inhibition of phospho-EGFR was 53% at 24 hours, whereas the level of inhibition decreased to 37% by 72 hours. The time course of phospho-EGFR inhibition and recovery seemed to correlate with the pharmacokinetics of cetuximab. Immunohistochemical analysis showed that phospho-MAPK and Ki67 expression were inhibited between 24 and 72 hours at 0.25 and 0.04 mg. A pharmacokinetic/ pharmacodynamic model was established and predicted that the plasma concentration of cetuximab required to inhibit 90% of phospho-EGFR was 67.5 mu g/mL.Conclusions: Phospho-EGFR/phospho-MAPK could be useful clinical biomarkers to assess EGFR inhibition by cetuximab.

Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) originates from multipotential stem cells and is caused by a reciprocal translocation between chromosomes 9 and 22, resulting in the formation of the fusion protein BCR-ABL, a constitutively activated tyrosine kinase. The discovery of imatinib, which selectively targets BCR-ABL, represents a breakthrough treatment for this disorder. However, emerging evidence indicates that a significant proportion of patients in the early chronic phase of the disease fail to achieve optimal response to imatinib due to innate or acquired drug resistance. Moreover, patients in the accelerated or blast crisis phases of the disease in general respond far less favorably to imatinib therapy. There is an urgent need for improved treatment options for these patient populations. BMS-354825, a dual-selective inhibitor of SRC and ABL kinases has demonstrated promising antileukemic activity in vitro and in vivo against preclinical models of human CML, including several that were resistant to imatinib through a variety of mechanisms (Lee et al., Proceedings of the AACR, 2004; Donato et al., Proceedings of the AACR, 2004; Shah et al., Science, 16:399–401, 2004). BMS-354825 is currently being evaluated in a Phase I clinical trial. We performed studies to gain better understanding of the relationship between the pharmacokinetics (PK) and pharmacodynamics (PD) of BMS-354825 and its antileukemic activity. Efficacy was determined in a human CML model (K562) grown subcutaneously in mice. Mouse plasma PK was determined by liquid chromatography/mass spectrometry. Inhibitions of the phosphorylation of tumoral BCR-ABL and its down-stream substrate CrkL, were used as PD markers and were measured by Western blot analysis. BMS-354825 administered orally was efficacious (curative) over a wide range of doses (1.25–50 mg/kg/dose) and exhibited predictable and dose-dependent pharmacokinetics. The time-course of tumoral BCR-ABL and CrkL inhibition and recovery directly correlated with plasma level of BMS-354825. At the minimum effective dose of 1.25 mg/kg/dose, maximum inhibition of BCR-ABL was observed at ~3 hr post-dose, and the inhibition was partially reversed at 7 hr and completely recovered between 7–17 hr post-dose. Based on these data, a PK/PD model was established which predicted that the plasma concentration of BMS-354825 required to effectively inhibit BCR-ABL in K562 cells was ~20 nM. In addition, the model predicted that at a given total dose, twice-a-day split-dose regimens should be more efficacious than once-a-day regimens. This was supported by in vivo efficacy studies in K562 xenografts where BMS-354825 was curative at 1.25 mg/kg/dose when administered twice-a-day, whereas once-a-day dosing required 5 mg/kg/dose to achieve the same response. In summary, BCR-ABL and/or CrkL phosphorylation in CML cells can serve as in vivo PD markers for BMS-354825. The safety/efficacy of BMS-354825, administered either once daily or twice-a-day, is currently being evaluated in Phase I clinical trials in CML patients.

Chronic myeloid leukemia (CML) is a stem cell disorder caused by a constitutively activated tyrosine kinase, the BCR-ABL oncoprotein. Imatinib (STI571, Gleevec) is a small-molecule inhibitor of this kinase that produces clinical remissions in CML patients and is now frontline therapy for this disease. While this agent has a high rate of clinical success in early phases of CML, development of resistance to this drug is increasingly becoming problematic, particularly in later stages of the disease. Moreover, growing evidence suggests that imatinib has very poor penetration of the blood brain barrier, likely due at least partly to its being a substrate of P-glycoprotin (Pgp), resulting in subtherapeutic levels in the CNS. As a result, several clinical cases have been reported where CNS relapses occurred in imatinib treated CML patients despite peripheral blood and bone marrow complete responses (Leis et al., Leuk Lymphoma. 2004 Apr;45(4):695–8). This phenomenon has also been recapitulated in at least one preclinical model, where the limited ability of imatinib to cross the blood-brain barrier allowed the CNS to become a sanctuary for BCR-ABL-induced leukemia (Wolff et al., Blood. 2003 Jun 15;101(12):5010–3). BMS-354825, a small-molecule dual-function SRC/ABL tyrosine kinase inhibitor, was designed to overcome many of the limitations associated with imatinib therapy. BMS-354825 has more than 500-fold increased potency relative to imatinib versus BCR-ABL and more importantly retains activity against 14 of 15 imatinib-resistant BCR-ABL mutants (Shah et al., Science, 2004 Jul 16;305(5682):399–401). In addition, BMS-354825 proved to be equally effective against several preclinically- and clinically-derived tumor models of imatinib resistance (Lee et al., Proceedings of the AACR, Volume 45, March 2004). In the current study, we assessed the efficacy of BMS-354825, which is not a Pgp substrate, in a model of established intracranial CML tumors. SCID-beige mice bearing K562 CML tumors implanted intracranially (2x106 cells per animal) were treated with BMS-354825 orally b.i.d. for a period of up to 40 days. BMS-354825 proved to be exceptionally efficacious resulting in increased lifespan of animals by 450% and 268% for the 15 mg/kg and 5 mg/kg dose levels, respectively. In order to more directly assess the anti-tumor activities of BMS-354825 in this intracranial CML model, we implanted K562 cells stably transfected with the firefly luciferase gene intracranially into SCID-beige animals. Bioluminescent imaging (BLI) then allowed the non-invasive monitoring of in vivo growth of these tumors. BMS-354825 at 15 mg/kg (2qdx14;6 po) achieved tumor regressions and subsequent complete stasis of intracranial K562 growth while animals were on therapy. In summary, these results suggest that BMS-354825 may have therapeutic advantages over imatinib in the management of intracranial CML disease and warrants further clinical investigation.

PURPOSE:Inhibition of the enzyme cyclooxygenase with celecoxib is cytotoxic in a variety of solid tumor cell lines. Previous work has shown that by charting circadian rhythms, it has been possible to find optimal times to deliver a dose of drug, such that it is most efficacious in killing cancer cells and least harmful to normal tissues. Therefore, we examined the time dependence of toxicity (chronotoxicity) and of antitumor effects (chronotherapy) of celecoxib to determine optimal time of day for dosing with respect to light-dark cycles.EXPERIMENTAL DESIGN:Celecoxib was administered i.p. for 10 days (5 days on, 2 days off, 5 days on) to nude mice bearing s.c. breast xenografts. Body weight, peripheral blood cells, clinical chemistry, and tumor growth were monitored.RESULTS:The highest tolerance (100% survival) was found at 7 HALO and the least occurred at 17 h after light onset (HALO; 10% survival). Chronotherapy at a 20-mg/kg dose varied between the seven HALO evaluated and between the three breast tumors (MCF-7, ZR-75-30, and MDA-MB-468) studied. When the maximum tolerated dose (MTD) of celecoxib was optimized for each HALO, we found that at 7-10 HALO, the MTD was 25 mg/kg, whereas at 17-20 HALO; the MTD was only 10 mg/kg. Tumor regression was observed when dosing was done at 23 HALO to 7 HALO (5 a.m. to 1 p.m.), whereas no therapeutic response was observed when dosing was done at 10-13 HALO (4 p.m. to 7 p.m.), and rapid tumor growth was noted when dosing was done at 17 HALO (11 p.m.).CONCLUSIONS:Tumor growth response to the MTD at each HALO revealed that there was no clear relationship between dose administered and therapeutic response. COX-2 expression was not able to explain either the chronotherapy or the chronotoxicity results obtained.