This first-in-human dose-escalation trial evaluated the safety, tolerability, maximal tolerated dose (MTD), dose limiting toxicities (DLTs), pharmacokinetics, pharmacodynamics and preliminary clinical activity of pictilisib (GDC-0941), an oral, potent and selective inhibitor of the Class I phosphatidylinositol-3-kinases (PI3K).
Patients and Methods
Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450mg once-daily, initially on days 1-21 every 28 days and later, utilizing continuous dosing for selected dose levels. Pharmacodynamic studies incorporated 18F-FDG-PET, and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma and tumor tissue.
Pictilisib was well-tolerated. The most common toxicities were grade 1-2 nausea, rash and fatigue while the DLT was grade 3 maculopapular rash (450mg, 2 of 3 patients; 330mg, 1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed >90% in platelet rich plasma at 3 hours post-dose at the MTD and in tumor at pictilisib doses associated with AUC >20uM.hr. Significant increase in plasma insulin and glucose levels, and >25% decrease in 18F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria, respectively.
Pictilisib was safely administered with a dose-proportional pharmacokinetic profile, on-target pharmacodynamic activity at dose levels ≥100mg and signs of antitumor activity. The recommended Phase II dose was continuous dosing at 330mg once-daily.
The bromodomain containing proteins
BAZ2A/B play essential roles
in chromatin remodeling and regulation of noncoding RNAs. We present
the structure based discovery of a potent, selective, and cell active
inhibitor 13 (BAZ2-ICR) of the BAZ2A/B bromodomains through
rapid optimization of a weakly potent starting point. A key feature
of the presented inhibitors is an intramolecular aromatic stacking
interaction that efficiently occupies the shallow bromodomain pockets. 13 represents an excellent chemical probe for functional studies
of the BAZ2 bromodomains in vitro and in vivo.
The Aurora kinases regulate key stages of mitosis including centrosome maturation, spindle assembly, chromosome segregation and cytokinesis. Aurora A and B overexpression has also been associated with various human cancers and as such, they have been extensively studied as novel anti-mitotic drug targets. Here we characterise the Aurora kinase inhibitor CCT137690, a highly selective, orally bioavailable imidazo[4,5-b]pyridine derivative that inhibits Aurora A and B kinases with low nanomolar IC50 values in both biochemical and cellular assays and exhibits anti-proliferative activity against a wide range of human solid tumour cell lines. CCT137690 efficiently inhibits histone H3 and TACC3 phosphorylation (Aurora B and Aurora A substrates, respectively) in HCT116 and HeLa cells. Continuous exposure of tumour cells to the inhibitor causes multipolar spindle formation, chromosome misalignment, polyploidy and apoptosis. This is accompanied by p53/p21/BAX induction, thymidine kinase 1 (TK1) downregulation and PARP cleavage. Furthermore, CCT137690 treatment of MYCN-amplified neuroblastoma cell lines inhibits cell proliferation and decreases MYCN protein expression. Importantly, in a transgenic mouse model of neuroblastoma (TH-MYCN) that overexpresses MYCN protein and is predisposed to spontaneous neuroblastoma formation, this compound significantly inhibits tumour growth. The potent preclinical activity of CCT137690 suggests that this inhibitor may benefit patients with MYCN amplified neuroblastoma.
Aurora; Neuroblastoma; Inhibitor; MYCN
We undertook a comprehensive clinical and biological investigation of serial medulloblastoma biopsies obtained at diagnosis and relapse. Combined MYC family amplifications and P53 pathway defects commonly emerged at relapse, and all patients in this group died of rapidly progressive disease postrelapse. To study this interaction, we investigated a transgenic model of MYCN-driven medulloblastoma and found spontaneous development of Trp53 inactivating mutations. Abrogation of p53 function in this model produced aggressive tumors that mimicked characteristics of relapsed human tumors with combined P53-MYC dysfunction. Restoration of p53 activity and genetic and therapeutic suppression of MYCN all reduced tumor growth and prolonged survival. Our findings identify P53-MYC interactions at medulloblastoma relapse as biomarkers of clinically aggressive disease that may be targeted therapeutically.
•Combined P53 and MYC family defects emerge at medulloblastoma relapse•P53-MYC defects are a biomarker for rapidly progressive relapsed disease•Trp53 and MYCN interact to drive aggressive medulloblastoma development in mice•Targeting MYCN or P53 pathway reactivation reduces tumor growth and prolongs survival
Hill et al. find that coincident MYC amplifications and p53 pathway defects are common in relapsed medulloblastoma (MB) and correlate with poor postrelapse prognosis. The authors go on to explore this MYC-p53 interaction in a mouse MB model and show that these tumors are dependent on both aberrations.
To provide rationale for using PI3K and/or MAPK pathway inhibitors to treat rhabdomyosarcomas (RMS), a major cause of pediatric/adolescent cancer deaths.
The prevalence of PI3K/MAPK pathway activation in RMS clinical samples was assessed using immunohistochemistry. Compensatory signaling and crosstalk between PI3K/MAPK pathways was determined in RMS cell lines following p110α shRNA-mediated depletion. Pharmacological inhibition of reprogrammed signaling in stable p110α knockdown lines was used to determine the target-inhibition profile inducing maximal growth inhibition. The in vitro and in vivo efficacy of inhibitors of TORC1/2(AZD8055), MEK(AZD6244) and P13K/mTOR(NVP-BEZ235) were evaluated alone and in pair-wise combinations.
PI3K pathway activation was seen in 82.5% rhabdomyosarcomas with co-activated MAPK in 36% and 46% of alveolar and embryonal sub-types respectively. p110α knockdown in cell lines over the short and long term was associated with compensatory expression of other p110 isoforms, activation of the MAPK pathway and cross-talk to reactivate the PI3K pathway. Combinations of PI3K pathway and MEK inhibitors synergistically inhibited cell growth in vitro. Treatment of RD cells with AZD8055 plus AZD6244 blocked reciprocal pathway activation, as evidenced by reduced AKT/ERK/S6 phosphorylation. In vivo, the synergistic effect on growth and changes in pharmacodynamic biomarkers was recapitulated using the AZD8055/AZD6244 combination but not NVP-BEZ235/AZD6244. Pharmacokinetic analysis provided evidence of drug-drug interaction with both combinations.
Dual PI3K/MAPK pathway activation and compensatory signaling in both rhabdomyosarcoma subtypes predicts a lack of clinical efficacy for single agents targeting either pathway, supporting a therapeutic strategy combining a TORC1/2 with a MEK inhibitor.
Rhabdomyosarcoma; PI3K pathway inhibitors; MAPK pathway inhibitor; synergistic inhibition; pharmacodynamics/pharmacokinetics
protein kinase MPS1 is a crucial component of the spindle assembly
checkpoint signal and is aberrantly overexpressed in many human cancers.
MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal
instability and aneuploidy. PTEN-deficient breast tumor cells are
particularly dependent upon MPS1 for their survival, making it a target
of significant interest in oncology. We report the discovery and optimization
of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based
design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes
an inactive conformation of MPS1 with the activation loop ordered
in a manner incompatible with ATP and substrate-peptide binding; it
displays a favorable oral pharmacokinetic profile, shows dose-dependent
inhibition of MPS1 in an HCT116 human tumor xenograft model, and is
an attractive tool compound to elucidate further the therapeutic potential
of MPS1 inhibition.
Aurora-A differs from Aurora-B/C
at three positions in the ATP-binding
pocket (L215, T217, and R220). Exploiting these differences, crystal
structures of ligand–Aurora protein interactions formed the
basis of a design principle for imidazo[4,5-b]pyridine-derived
Aurora-A-selective inhibitors. Guided by a computational modeling
approach, appropriate C7-imidazo[4,5-b]pyridine derivatization
led to the discovery of highly selective inhibitors, such as compound 28c, of Aurora-A over Aurora-B. In HCT116 human colon carcinoma
cells, 28c and 40f inhibited the Aurora-A
L215R and R220K mutants with IC50 values similar to those
seen for the Aurora-A wild type. However, the Aurora-A T217E mutant
was significantly less sensitive to inhibition by 28c and 40f compared to the Aurora-A wild type, suggesting
that the T217 residue plays a critical role in governing the observed
isoform selectivity for Aurora-A inhibition. These compounds are useful
small-molecule chemical tools to further explore the function of Aurora-A
The ribosomal P70 S6 kinases play a crucial role in PI3K/mTOR regulated signalling pathways and are therefore potential targets for the treatment of a variety of diseases including diabetes and cancer. In this study we describe the identification of three series of chemically distinct S6K1 inhibitors. In addition, we report a novel PKA-S6K1 chimeric protein with five mutations in or near its ATP-binding site, which was used to determine the binding mode of two of the three inhibitor series, and provided a robust system to aid the optimisation of the oxadiazole-substituted benzimidazole inhibitor series. We show that the resulting oxadiazole-substituted aza-benzimidazole is a potent and ligand efficient S6 kinase inhibitor, which blocks the phosphorylation of RPS6 at Ser235/236 in TSC negative HCV29 human bladder cancer cells by inhibiting S6 kinase activity and thus provides a useful tool compound to investigate the function of S6 kinases.
S6 kinase; P70S6K; cancer; inhibitor; structure-based drug design
The ALKF1174L mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALKF1174L in the neural crest. Compared to ALKF1174L and MYCN alone, coexpression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance and enhanced lethality. ALKF1174L/MYCN tumors exhibited increased MYCN dosage due to ALKF1174L-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2, overcame the resistance of ALKF1174L/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALKF1174L in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.
Many tumors exhibit defective cell cycle checkpoint control and increased replicative stress. CHK1 is critically involved in the DNA damage response and maintenance of replication fork stability. We have therefore discovered a novel, potent, highly selective, orally active, ATP competitive CHK1 inhibitor, CCT244747, and present its preclinical pharmacology and therapeutic activity.
Cellular CHK1 activity was assessed using an ELISA assay and cytotoxicity a SRB assay. Biomarker modulation was measured using immunoblotting and cell cycle effects by flow cytometry. Single agent, oral CCT244747 antitumor activity was evaluated in a MYCN-driven transgenic mouse model of neuroblastoma by MRI and in genotoxic combinations in human tumor xenografts by growth delay.
CCT244747 inhibited cellular CHK1 activity (IC50 29-170nM), significantly enhanced the cytotoxicity of several anticancer drugs and abrogated drug-induced S and G2 arrest in multiple tumor cell lines. Biomarkers of CHK1 (pS296 CHK1) activity and cell cycle inactivity (pY15 CDK1) were induced by genotoxics and inhibited by CCT244747 both in vitro and in vivo, producing enhanced DNA damage and apoptosis. Active tumor concentrations of CCT244747 were obtained following oral administration. The antitumor activity of both gemcitabine and irinotecan were significantly enhanced by CCT244747 in several human tumor xenografts, giving concomitant biomarker modulation indicative of CHK1 inhibition. CCT244747 also showed marked antitumor activity as a single agent in a MYCN-driven neuroblastoma.
CCT244747 represents the first structural disclosure of a highly selective, orally active CHK1 inhibitor and warrants further evaluation alone or combined with genotoxic anticancer therapies.
CHK1; CCT244747; pharmacology; biomarkers; neuroblastoma
Acquired resistance to selective FLT3 inhibitors, is an emerging clinical problem in the treatment of FLT3-ITD+ acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has limited investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD+ human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD+ allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD+ patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13- RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild type allele and duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.
FLT3; Aurora; Kinase; AML; Resistance; Inhibitor
Although daily rhythms regulate multiple aspects of human physiology, rhythmic control of the metabolome remains poorly understood. The primary objective of this proof-of-concept study was identification of metabolites in human plasma that exhibit significant 24-h variation. This was assessed via an untargeted metabolomic approach using liquid chromatography–mass spectrometry (LC-MS). Eight lean, healthy, and unmedicated men, mean age 53.6 (SD ± 6.0) yrs, maintained a fixed sleep/wake schedule and dietary regime for 1 wk at home prior to an adaptation night and followed by a 25-h experimental session in the laboratory where the light/dark cycle, sleep/wake, posture, and calorific intake were strictly controlled. Plasma samples from each individual at selected time points were prepared using liquid-phase extraction followed by reverse-phase LC coupled to quadrupole time-of-flight MS analysis in positive ionization mode. Time-of-day variation in the metabolites was screened for using orthogonal partial least square discrimination between selected time points of 10:00 vs. 22:00 h, 16:00 vs. 04:00 h, and 07:00 (d 1) vs. 16:00 h, as well as repeated-measures analysis of variance with time as an independent variable. Subsequently, cosinor analysis was performed on all the sampled time points across the 24-h day to assess for significant daily variation. In this study, analytical variability, assessed using known internal standards, was low with coefficients of variation <10%. A total of 1069 metabolite features were detected and 203 (19%) showed significant time-of-day variation. Of these, 34 metabolites were identified using a combination of accurate mass, tandem MS, and online database searches. These metabolites include corticosteroids, bilirubin, amino acids, acylcarnitines, and phospholipids; of note, the magnitude of the 24-h variation of these identified metabolites was large, with the mean ratio of oscillation range over MESOR (24-h time series mean) of 65% (95% confidence interval [CI]: 49–81%). Importantly, several of these human plasma metabolites, including specific acylcarnitines and phospholipids, were hitherto not known to be 24-h variant. These findings represent an important baseline and will be useful in guiding the design and interpretation of future metabolite-based studies. (Author correspondence: Jooern.Ang@icr.ac.uk or Florence.Raynaud@icr.ac.uk)
Acylcarnitines; Daily variation; Human; Liquid chromatography–mass spectrometry; Metabolomics; Plasma metabolites
Paediatric glioblastoma (pGBM), although rare, is one of the leading causes of cancer-related deaths in children, with tumours essentially refractory to existing treatments. We have identified IGF1R to be a potential therapeutic target in pGBM due to gene amplification and high levels of IGF2 expression in some tumour samples, as well as constitutive receptor activation in pGBM cell lines. In order to evaluate the therapeutic potential of strategies targeting the receptor, we have carried out in vitro and in vivo preclinical studies using the specific IGF1R inhibitor NVP-AEW541. A modest inhibitory effect was seen in vitro, with GI50 values of 5-6μM, and concurrent inhibition of receptor phosphorylation. Specific targeting of IGF1R with siRNA decreased cell viability, diminished downstream signalling through PI3-kinase and induced G1 arrest, effects mimicked by NVP-AEW541, both in the absence and presence of IGF2. Hallmarks of PI3-kinase inhibition were observed after treatment with NVP-AEW541 by expression profiling and Western blot analysis. Phospho-RTK arrays demonstrated phosphorylation of PDGFRα/β in pGBM cells suggesting co-activation of an alternative RTK pathway. Treatment of KNS42 with the PDGFR inhibitor imatinib showed additional effects targeting the MAP-kinase pathway, and co-treatment of the PDGFR inhibitor imatinib with NVP-AEW541 resulted in a highly synergistic interaction in vitro, and increased efficacy after 14 days therapy in vivo compared with either agent alone. These data provide evidence that inhibition of IGF1R, in combination with other targeted agents, may be a useful and novel therapeutic strategy in pGBM.
IGF1R; PDGFR; PI3-kinase; MAP-kinase; paediatric glioblastoma
The phosphatidylinositide 3-kinase (PI3K) pathway is very commonly activated in a wide range of human cancers and is a major driving force in oncogenesis. One of the class I lipid kinase members of the PI3K family, p110α, is probably the most commonly mutated kinase in the human genome. Alongside genetic, molecular biological and biochemical studies, chemical inhibitors have been extremely helpful tools in understanding the role of PI3K enzymes in signal transduction and downstream physiological and pathological processes, and also in validating PI3Ks as therapeutic targets. Although they have been valuable in the past, the early and still frequently employed inhibitors, wortmannin and LY294002, have significant limitations as chemical tools. Here, we discuss the case history of the discovery and properties of an increasingly used chemical probe, the pan-class I PI3K and mTOR inhibitor PI-103 (a pyridofuropyrimidine) and its very recent evolution into the thienopyrimidine drug GDC-0941 that exhibits excellent oral anticancer activity in preclinical models and is now undergoing Phase I clinical trials in cancer patients. We also illustrate the impact of structural biology on the design of PI3K inhibitors and on the interpretation of their effects. The challenges and outlook for drugging the PI3 kinome are discussed in the more general context of the role of structural biology and chemical biology in innovative drug discovery.
A Phase I study to define toxicity and recommend a Phase II dose of the HSP90 inhibitor alvespimycin (17-DMAG; 17-dimethylaminoethylamino-17-demethoxygeldanamycin). Secondary endpoints included evaluation of pharmacokinetic profile, tumor response and definition of a biologically effective dose (BED).
Patients and Methods
Patients with advanced solid cancers were treated with weekly, intravenous (IV) 17-DMAG. An accelerated titration dose escalation design was used. The maximum tolerated dose (MTD) was the highest dose at which ≤ 1/6 patients experienced dose limiting toxicity (DLT). Dose de-escalation from the MTD was planned with mandatory, sequential tumor biopsies to determine a BED. Pharmacokinetic and pharmacodynamic assays were validated prior to patient accrual.
Twenty five patients received 17-DMAG (range 2.5 to 106 mg/m2). At 106mg/m2 of 17-DMAG 2/4 patients experienced DLT, including one treatment related death. No DLT occurred at 80mg/m2. Common adverse events were gastrointestinal, liver function changes and ocular. AUC and Cmax increased proportionally with 17-DMAG doses ≤ 80mg/m2. In peripheral blood mononuclear cells significant (p <0.05) HSP72 induction was detected (≥ 20mg/m2) and sustained for 96 hours (≥ 40mg/m2). Plasma HSP72 levels were greatest in the two patients who experienced DLT. At 80mg/m2 client protein (CDK4, LCK) depletion was detected and tumor samples from 3/5 patients confirmed HSP90 inhibition. Clinical activity included complete response (castration refractory prostate cancer, CRPC 124 weeks), partial response (melanoma, 159 weeks) and stable disease (chondrosarcoma, CRPC and renal cancer for 28, 59 and 76 weeks respectively).
The recommended Phase II dose of 17-DMAG is 80mg/m2 weekly, IV.
Abiraterone acetate is a prodrug of abiraterone, a selective inhibitor of CYP17, the enzyme catalyst for two essential steps in androgen biosynthesis. In castration-resistant prostate cancers (CRPCs), extragonadal androgen sources may sustain tumor growth despite a castrate environment. This phase I dose-escalation study of abiraterone acetate evaluated safety, pharmacokinetics, and effects on steroidogenesis and prostate-specific antigen (PSA) levels in men with CPRC with or without prior ketoconazole therapy.
Patients and Methods
Thirty-three men with chemotherapy-naïve progressive CRPC were enrolled. Nineteen patients (58%) had previously received ketoconazole for CRPC. Bone metastases were present in 70% of patients, and visceral involvement was present in 18%. Three patients (9%) had locally advanced disease without distant metastases. Fasted or fed cohorts received abiraterone acetate doses of 250, 500, 750, or 1,000 mg daily. Single-dose pharmacokinetic analyses were performed before continuous daily dosing.
Adverse events were predominantly grade 1 or 2. No dose-limiting toxicities were observed. Hypertension (grade 3, 12%) and hypokalemia (grade 3, 6%; grade 4, 3%) were the most frequent serious toxicities and responded to medical management. Confirmed ≥ 50% PSA declines at week 12 were seen in 18 (55%) of 33 patients, including nine (47%) of 19 patients with prior ketoconazole therapy and nine (64%) of 14 patients without prior ketoconazole therapy. Substantial declines in circulating androgens and increases in mineralocorticoids were seen with all doses.
Abiraterone acetate was well tolerated and demonstrated activity in CRPC, including in patients previously treated with ketoconazole. Continued clinical study is warranted.
The Wnt signaling pathway is frequently deregulated in cancer due to mutations in the genes encoding APC, β-catenin and axin. To identify small molecule inhibitors of Wnt signaling as potential therapeutics, a diverse chemical library was screened using a TCF-reporter cell line in which the activity of the pathway was induced at the level of the Disheveled protein. A series of deconvolution studies was used to focus on 3 compound series that selectively killed cancer cell lines with constitutive Wnt signaling. Activities of the compounds included the ability to induce degradation of β-catenin that had been stabilized by a GSK-3 inhibitor. This screen illustrates a practical approach to identify small molecule inhibitors of Wnt signaling that can seed the development of agents suitable to treat patients with Wnt-dependent tumors.
The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus (31P)- and proton (1H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103–induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase α (ChoKα), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r2 = 0.9, P = 0.009) was found only between PC concentrations and ChoKα expression but not with FAS or pACL. This study identified inhibition of ChoKα as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of 1H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with 31P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors.
PI3K; Choline Kinase; MRS; phosphocholine; biomarkers
The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110α with IC50 ≤ 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% anti-proliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. GDC-0941 has recently entered phase I clinical trials.
Purpose. The study was performed to assess the antitumour activity and toxicity of a 72-h continuous infusion of
single-agent etoposide as second-line treatment for patients with locally advanced or metastatic soft tissue sarcoma (STS),
following reports of substantial activity using this schedule of etoposide administration as first-line treatment in
combination with ifosfamide.
Patients/method. This was an open phase I/II trial performed at a single institution in
patients with metastatic or locally
advanced STS who had failed first-line treatment with doxorubicin + ifosfamide combination chemotherapy or, less
commonly, single-agent treatment with doxorubicin or ifosfamide. Etoposide was given as a continuous intravenous
infusion over 72 h. The starting dose level was
200 mg m-2
day-1 × 3 escalating in 10% steps in cohorts of three patients
until dose-limiting toxicity was encountered.
Results. Seventeen patients were treated, median age 47 years (range 26–71 years). No responses were seen in 16
assessable patients despite etoposide levels in the cotoxic range. The steady-state plasma concentration exceeded
8 μg ml−1 in all patients and in patients treated at ≥ 600 mg m −2
the mean steady-state level was 14.4 μg ml −1. The
median event-free survival was 6 weeks (95% confidence interval (CI) 3.31–8.69) and the overall survival 16 weeks (95%
CI 9.28–22.72). The maximum tolerated dose in this pretreated patient group was 200 mg mm-2
day-1 × 3. The dose-limiting toxicity was myelosuppression.
Discussion. Etoposide given by 72-h infusion is inactive as second-line chemotherapy in STS.
It is associated with significant toxicity when given in these doses, in this patient group.
Protein kinase B (PKB or Akt) is an important component of intracellular signaling pathways regulating growth and survival. Signaling through PKB is frequently deregulated in cancer, and inhibitors of PKB therefore have potential as antitumor agents. The optimization of lipophilic substitution within a series of 4-benzyl-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amines provided ATP-competitive, nanomolar inhibitors with up to 150-fold selectivity for inhibition of PKB over the closely related kinase PKA. Although active in cellular assays, compounds containing 4-amino-4-benzylpiperidines underwent metabolism in vivo, leading to rapid clearance and low oral bioavailability. Variation of the linker group between the piperidine and the lipophilic substituent identified 4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamides as potent and orally bioavailable inhibitors of PKB. Representative compounds modulated biomarkers of signaling through PKB in vivo and strongly inhibited the growth of human tumor xenografts in nude mice at well-tolerated doses.
Pyrazolopyridine inhibitors with low micromolar potency
and good selectivity against CHK2 were previously identified by fragment-based
screening. The optimization of the pyrazolopyridines to a series of
potent and CHK1-selective isoquinolines demonstrates how fragment-growing
and scaffold morphing strategies arising from a structure-based understanding
of CHK1 inhibitor binding can be combined to successfully progress
fragment-derived hit matter to compounds with activity in vivo. The
challenges of improving CHK1 potency and selectivity, addressing synthetic
tractability, and achieving novelty in the crowded kinase inhibitor
chemical space were tackled by multiple scaffold morphing steps, which
progressed through tricyclic pyrimido[2,3-b]azaindoles
to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately
to imidazo[4,5-c]pyridines and isoquinolines. A potent
and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was
identified, which potentiated the efficacies of irinotecan and gemcitabine
in SW620 human colon carcinoma xenografts in nude mice.
Optimization of the imidazo[4,5-b]pyridine-based
series of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurora
kinases (Aurora-A Kd = 7.5 nM, Aurora-B Kd = 48 nM), FLT3 kinase (Kd = 6.2 nM), and FLT3 mutants including FLT3-ITD (Kd = 38 nM) and FLT3(D835Y) (Kd = 14 nM). FLT3-ITD causes constitutive FLT3 kinase
activation and is detected in 20–35% of adults and 15% of children
with acute myeloid leukemia (AML), conferring a poor prognosis in
both age groups. In an in vivo setting, 27e strongly
inhibited the growth of a FLT3-ITD-positive AML human
tumor xenograft (MV4–11) following oral administration, with
in vivo biomarker modulation and plasma free drug exposures consistent
with dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was
selected as a preclinical development candidate for the treatment
of human malignancies, in particular AML, in adults and children.
Inhibitors of checkpoint kinase 1 (CHK1) are of current
as potential antitumor agents, but the most advanced inhibitor series
reported to date are not orally bioavailable. A novel series of potent
and orally bioavailable 3-alkoxyamino-5-(pyridin-2-ylamino)pyrazine-2-carbonitrile
CHK1 inhibitors was generated by hybridization of two lead scaffolds
derived from fragment-based drug design and optimized for CHK1 potency
and high selectivity using a cell-based assay cascade. Efficient in
vivo pharmacokinetic assessment was used to identify compounds with
prolonged exposure following oral dosing. The optimized compound (CCT244747)
was a potent and highly selective CHK1 inhibitor, which modulated
the DNA damage response pathway in human tumor xenografts and showed
antitumor activity in combination with genotoxic chemotherapies and
as a single agent.