In silico trial for the assessment of givinostat dose adjustment rules based on the management of key hematological parameters in polycythemia vera patients.

Polycythemia vera (PV) is a chronic myeloproliferative neoplasm characterized by excessive levels of platelets (PLT), white blood cells (WBC), and hematocrit (HCT). Givinostat (ITF2357) is a potent histone-deacetylase inhibitor that showed a good safety/efficacy profile in PV patients during phase I/II studies. A phase III clinical trial had been planned and an adaptive dosing protocol had been proposed where givinostat dose is iteratively adjusted every 28 days (one cycle) based on PLT, WBC, and HCT. As support, a simulation platform to evaluate and refine the proposed givinostat dose adjustment rules was developed. A population pharmacokinetic/pharmacodynamic model predicting the givinostat effects on PLT, WBC, and HCT in PV patients was developed and integrated with a control algorithm implementing the adaptive dosing protocol. Ten in silico trials in ten virtual PV patient populations were simulated 500 times. Considering an eight-treatment cycle horizon, reducing/increasing the givinostat daily dose by 25 mg/day step resulted in a higher percentage of patients with a complete hematological response (CHR), that is, PLT ≤400 × 109 /L, WBC ≤10 × 109 /L, and HCT < 45% without phlebotomies in the last three cycles, and a lower percentage of patients with grade II toxicity events compared with 50 mg/day adjustment steps. After the eighth cycle, 85% of patients were predicted to receive a dose ≥100 mg/day and 40.90% (95% prediction interval = [34, 48.05]) to show a CHR. These results were confirmed at the end of 12th, 18th, and 24th cycles, showing a stability of the response between the eighth and 24th cycles.

Novel Thienoduocarmycin-Trastuzumab ADC Demonstrates Strong Antitumor Efficacy with Favorable Safety Profile in Preclinical Studies.

New antibodies-drug conjugate (ADC) payloads overcoming chemoresistance and killing also poorly proliferating tumors at well-tolerated doses are much desired. Duocarmycins are a well-known class of highly potent cytotoxic agents, with DNA minor groove-binding and alkylation properties, active also in chemoresistant tumors. Although different duocarmycin derivatives have been used during the years as payloads for ADC production, unfavorable physicochemical properties impaired the production of ADCs with optimal features. Optimization of the toxin to balance reactivity and stability features and best linker selection allowed us to develop the novel duocarmycin-like payload-linker NMS-P945 suitable for conjugation to mAbs with reproducible drug-antibody ratio (DAR) >3.5. When conjugated to trastuzumab, it generated an ADC with good internalization properties, ability to induce bystander effect and immunogenic cell death. Moreover, it showed strong target-driven activity in cells and cytotoxic activity superior to trastuzumab deruxtecan tested, in parallel, in cell lines with HER2 expression. High in vivo efficacy with cured mice at well-tolerated doses in HER2-driven models was also observed. A developed pharmacokinetic/pharmacodynamic (PK/PD) model based on efficacy in mice and cynomolgus monkey PK data, predicted tumor regression in patients upon administration of 2 doses of trastuzumab-NMS-P945-ADC at 0.5 mg/kg. Thus, considering the superior physicochemical features for ADC production and preclinical results obtained with the model trastuzumab ADC, including bystander effect, immunogenic cell death and activity in chemoresistant tumors, NMS-P945 represents a highly effective, innovative payload for the creation of novel, next-generation ADCs.

Population pharmacokinetic-pharmacodynamic analysis of givinostat.

BACKGROUND: Givinostat (ITF2357), an oral, synthetic histone deacetylase inhibitor, significantly improved all histological muscle biopsy parameters in a Phase II study in boys with Duchenne muscular dystrophy (DMD). RESEARCH DESIGN AND METHODS: A population pharmacokinetic (PK) model, including seven clinical studies, was developed to explore the effect of covariates on givinostat PK. The final model was qualified to simulate pediatric dosing recommendations. A PK/pharmacodynamic (PD) model was developed to simulate the link between givinostat plasma concentration and platelet time course in 10-70-kg children following 6 months of givinostat 20-70 mg twice daily. RESULTS: A two-compartment model, with first-order input with lag and first-order elimination from the central compartment, described givinostat PK, demonstrating increasing apparent clearance with increasing body weight. The PK/PD model well-described platelet count time course. Weight-based dosing (arithmetic mean systemic exposure of 554-641 ng·h/mL) produced an average platelet count decrease from baseline of 45% with maximum decrease within 28 days. After 1 week and 6 months, ~1% and ~14-15% of patients, respectively, had a platelet count <75 × 109/L. CONCLUSIONS: Based on these data, givinostat dosing will be body weight adjusted and include monitoring of platelet counts to support efficacy and safety in a Phase III DMD study.

PK/PD analysis of trazodone and gabapentin in neuropathic pain rodent models: Translational PK-PD modeling from nonclinical to clinical development.

A pharmacokinetic/pharmacodynamic (PK/PD) model was developed to describe the time course of writhings after intraperitoneal injection of acetic acid in mice. The model was applied to investigate the antinociceptive effect of trazodone and gabapentin alone and in combination. Writhings time course was described by a transit compartment model with the delay due to the transit of the acetic acid being represented by a chain of intermediate compartments. In the drug-treated animals, the number of writhings decreases according to a k2 factor linking drug concentration and antinociceptive effect. Compounds' potency parameters were 10.9 and 0.0459 L/µmoles/min for trazodone and gabapentin, respectively, indicating a much higher in vivo potency of trazodone in the PD writhing test. The PK/PD parameters were used to simulate the expected writhing counts in mice at combined doses without efficacy alone, assuming pharmacological additivity. Simulation results indicated that, at low dose combinations, experimental data were mostly below the simulated writhings median, suggesting possible synergic effect. Such hypothesis was tested by adding the γ parameter in the PK/PD model to represent the deviation from the assumption of no-interaction, leading to a reduction of the objective function compared to the additive model. On this basis, several simulations were performed to identify possible starting dose combinations of trazodone and gabapentin in humans, by selecting doses yielding systemic exposures close to those being synergic in the mouse. Simulations indicated that doses of 50-100 mg trazodone could enhance gabapentin antinociceptive effect in humans, supporting the development of a low dose combination for optimal analgesia treatment.

Phase I dose escalation study of NMS-1286937, an orally available Polo-Like Kinase 1 inhibitor, in patients with advanced or metastatic solid tumors.

Background Pharmacological inhibition of polo-like kinase 1 (PLK1) represents a new approach for the treatment of solid tumors. This study was aimed at determining the first cycle dose-limiting toxicities (DLTs) and related maximum tolerated dose (MTD) of NMS-1286937, a selective ATP-competitive PLK1-specific inhibitor. Secondary objectives included evaluation of its safety and pharmacokinetic (PK) profile in plasma, its antitumor activity, and its ability to modulate intracellular targets in biopsied tissue. Methods This was a Phase I, open-label, dose-escalation trial in patients with advanced/metastatic solid tumors. A treatment cycle comprised 5 days of oral administration followed by 16 days of rest, for a total of 21 days (3-week cycle). Results Nineteen of 21 enrolled patients with confirmed metastatic disease received study medication. No DLTs occurred at the first 3 dose levels (6, 12, and 24 mg/m2/day). At the subsequent dose level (48 mg/m2/day), 2 of 3 patients developed DLTs. An intermediate level of 36 mg/m2/day was therefore investigated. Four patients were treated and two DLTs were observed. After further cohort expansion, the MTD and recommended phase II dose (RP2D) were determined to be 24 mg/m2/day. Disease stabilization, observed in several patients, was the best treatment response observed. Hematological toxicity (mostly thrombocytopenia and neutropenia) was the major DLT. Systemic exposure to NMS-1286937 increased with dose and was comparable between two cycles of treatment following oral administration of the drug. Conclusions This study successfully identified the MTD and DLTs for NMS-1286937 and characterized its safety profile.

Discovery of Entrectinib: A New 3-Aminoindazole As a Potent Anaplastic Lymphoma Kinase (ALK), c-ros Oncogene 1 Kinase (ROS1), and Pan-Tropomyosin Receptor Kinases (Pan-TRKs) inhibitor.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for the development of different tumor types. Despite the remarkable clinical activity of crizotinib (Xalkori), the first ALK inhibitor approved in 2011, the emergence of resistance mutations and of brain metastases frequently causes relapse in patients. Within our ALK drug discovery program, we identified compound 1, a novel 3-aminoindazole active on ALK in biochemical and in cellular assays. Its optimization led to compound 2 (entrectinib), a potent orally available ALK inhibitor active on ALK-dependent cell lines, efficiently penetrant the blood-brain barrier (BBB) in different animal species and highly efficacious in in vivo xenograft models. Moreover, entrectinib resulted to be strictly potent on the closely related tyrosine kinases ROS1 and TRKs recently found constitutively activated in several tumor types. Entrectinib is currently undergoing phase I/II clinical trial for the treatment of patients affected by ALK-, ROS1-, and TRK-positive tumors.

PA401, a novel CXCL8-based biologic therapeutic with increased glycosaminoglycan binding, reduces bronchoalveolar lavage neutrophils and systemic inflammatory markers in a murine model of LPS-induced lung inflammation.

RATIONALE: Neutrophils play a fundamental role in a number of chronic lung diseases. Among the mediators of their recruitment to the lung, CXCL8 (IL-8) is considered to be one of the major players. CXCL8 exerts its chemotactic activity by binding to its GPCR receptors (CXCR1/R2) located on neutrophils, as well as through interactions with glycosaminoglycans (GAGs) on cell surfaces including those of the microvascular endothelium. Binding to GAG co-receptors is required to generate a solid-phase haptotactic gradient and to present IL-8/CXCL8 in a proper conformation to its receptors on circulating neutrophils. METHODS: We have engineered increased GAG-binding affinity into human CXCL8, thereby obtaining a competitive inhibitor that displaces wild-type IL-8/CXCL8 from GAGs. By additionally knocking-out the GPCR binding domain of the chemokine, we generated a dominant negative protein (dnCXCL8; PA401) with potent anti-inflammatory characteristics proven in vivo in a murine model of LPS-induced lung inflammation (Adage et al., 2015). Here we have further investigated PA401 activity in this pulmonary model by evaluating plasma changes induced by LPS on white blood cells (WBC) and a broad range of inflammatory markers, especially chemokines, by addressing immediate effects of PA401 on these parameters in healthy and LPS exposed mice. RESULTS: Aerosolized LPS induced a significant increase in bronchoalveolar lavage (BAL) neutrophils after 3 and 7h, as well as an increase in total WBC and changes in 21 of the 59 measured plasma markers, mostly belonging to the chemokine family. PA401 treatment in saline exposed mice didn't induce major changes in any of the measured parameters. When administered to LPS aerosolized mice, PA401 caused a significant normalization of KC/mCXCL1 and other inflammatory markers, as well as of blood WBC count. In addition, BAL neutrophils were significantly reduced, confirming the previously observed lung anti-inflammatory activity of PA401 in this experiment. CONCLUSIONS: PA401 is a new promising biologic therapeutic with a novel and unique mechanism of action for interfering with neutrophilic lung inflammation, that also normalizes plasma inflammatory markers.

CHF5074 reduces biomarkers of neuroinflammation in patients with mild cognitive impairment: a 12-week, double-blind, placebo-controlled study.

As neuroinflammation is an early event in the pathogenesis of Alzheimer' s disease, new selective antiinflammatory drugs could lead to promising preventive strategies. We evaluated the safety, tolerability, pharmacokinetics and pharmacodynamics of CHF5074, a new microglial modulator, in a 12-week, double-blind, placebo-controlled, parallel groups, ascending dose study involving 96 MCI patients. Subjects were allocated into three successive study cohorts to receive ascending, titrated doses of CHF5074 (200, 400 or 600 mg/day) or placebo. Vital signs, cardiac safety, neuropsychological performance and safety clinical laboratory parameters were assessed on all subjects. Plasma samples were collected throughout the study for measuring drug concentrations, soluble CD40 ligand (sCD40L) and TNF-α. At the end of treatment, cerebrospinal fluid (CSF) samples were optionally collected after the last dose to measure drug levels, ß- amyloid1-42 (Aß42), tau, phospho-tau181, sCD40L and TNF-α. Ten patients did not complete the study: one in the placebo group (consent withdrawn), two in the 200-mg/day treatment group (consent withdrawn and unable to comply) and seven in the 400-mg/day treatment group (five AEs, one consent withdrawn and one unable to comply). The most frequent treatment-emergent adverse events were diarrhea, dizziness and back pain. There were no clinically significant treatmentrelated clinical laboratory, vital sign or ECG abnormalities. CHF5074 total body clearance depended by gender, age and glomerular filtration rate. CHF5074 CSF concentrations increased in a dose-dependent manner. At the end of treatment, mean sCD40L and TNF-α levels in CSF were found to be inversely related to the CHF5074 dose (p=0.037 and p=0.001, respectively). Plasma levels of sCD40L in the 600-mg/day group were significantly lower than those measured in the placebo group (p=0.010). No significant differences between treatment groups were found in neuropsychological tests but a positive dose-response trend was found on executive function in APOE4 carriers. This study shows that CHF5074 is well tolerated in MCI patients after a 12-week titrated treatment up to 600 mg/day and dose-dependently affects central nervous system biomarkers of neuroinflammation.

Preclinical studies in the mdx mouse model of duchenne muscular dystrophy with the histone deacetylase inhibitor givinostat.

Previous work has established the existence of dystrophin-nitric oxide (NO) signaling to histone deacetylases (HDACs) that is deregulated in dystrophic muscles. As such, pharmacological interventions that target HDACs (that is, HDAC inhibitors) are of potential therapeutic interest for the treatment of muscular dystrophies. In this study, we explored the effectiveness of long-term treatment with different doses of the HDAC inhibitor givinostat in mdx mice--the mouse model of Duchenne muscular dystrophy (DMD). This study identified an efficacy for recovering functional and histological parameters within a window between 5 and 10 mg/kg/d of givinostat, with evident reduction of the beneficial effects with 1 mg/kg/d dosage. The long-term (3.5 months) exposure of 1.5-month-old mdx mice to optimal concentrations of givinostat promoted the formation of muscles with increased cross-sectional area and reduced fibrotic scars and fatty infiltration, leading to an overall improvement of endurance performance in treadmill tests and increased membrane stability. Interestingly, a reduced inflammatory infiltrate was observed in muscles of mdx mice exposed to 5 and 10 mg/kg/d of givinostat. A parallel pharmacokinetic/pharmacodynamic analysis confirmed the relationship between the effective doses of givinostat and the drug distribution in muscles and blood of treated mice. These findings provide the preclinical basis for an immediate translation of givinostat into clinical studies with DMD patients.

Pharmacokinetics and pharmacodynamics of CHF5074 after short-term administration in healthy subjects.

CHF5074 has been shown to inhibit brain ß-amyloid deposition and attenuate memory deficits in different transgenic mice models of Alzheimer disease. We evaluated the safety, pharmacokinetics, and pharmacodynamics of 3 ascending dose regimens of CHF5074 (200, 400, and 600 mg/d for 14 d) in a double-blind, placebo-controlled, parallel group study involving 48 healthy subjects. Plasma, urine, and cerebrospinal fluid (CSF) samples were collected for measuring drug and main metabolite concentrations and potential biomarkers of pharmacodynamic activity (ß-amyloid1-40, ß-amyloid1-42, soluble CD40 ligand, and tumor necrosis factor-α). All subjects completed the study, and no serious or severe adverse events were reported. The maximum tolerated dose was close to 600 mg/d with mild diarrhea being the most frequent adverse event at this dose. CHF5074 reached peak plasma levels 2 to 3 hours after drug administration and then was slowly eliminated (t(1/2z)=30 h) in the urine as glucoronide. Systemic exposure to the drug appeared to be dose-proportional with a 2-fold accumulation ratio at steady state. Metabolite plasma levels peaked at 4 to 5 hours and accounted for about 25% of the parent compound. Drug levels in the CSF were dose-proportional. The drug dose-dependently lowered the levels of the soluble CD40 ligand, a marker of microglia activation, in both plasma and CSF samples.

NMS-P937, an orally available, specific small-molecule polo-like kinase 1 inhibitor with antitumor activity in solid and hematologic malignancies.

Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase considered to be the master player of cell-cycle regulation during mitosis. It is indeed involved in centrosome maturation, bipolar spindle formation, chromosome separation, and cytokinesis. PLK1 is overexpressed in a variety of human tumors and its overexpression often correlates with poor prognosis. Although five different PLKs are described in humans, depletion or inhibition of kinase activity of PLK1 is sufficient to induce cell-cycle arrest and apoptosis in cancer cell lines and in xenograft tumor models. NMS-P937 is a novel, orally available PLK1-specific inhibitor. The compound shows high potency in proliferation assays having low nanomolar activity on a large number of cell lines, both from solid and hematologic tumors. NMS-P937 potently causes a mitotic cell-cycle arrest followed by apoptosis in cancer cell lines and inhibits xenograft tumor growth with clear PLK1-related mechanism of action at well-tolerated doses in mice after oral administration. In addition, NMS-P937 shows potential for combination in clinical settings with approved cytotoxic drugs, causing tumor regression in HT29 human colon adenocarcinoma xenografts upon combination with irinotecan and prolonged survival of animals in a disseminated model of acute myelogenous leukemia in combination with cytarabine. NMS-P937, with its favorable pharmacologic parameters, good oral bioavailability in rodent and nonrodent species, and proven antitumor activity in different preclinical models using a variety of dosing regimens, potentially provides a high degree of flexibility in dosing schedules and warrants investigation in clinical settings.

5-(2-amino-pyrimidin-4-yl)-1H-pyrrole and 2-(2-amino-pyrimidin-4-yl)-1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one derivatives as new classes of selective and orally available Polo-like kinase 1 inhibitors.

The discovery and characterization of two new chemical classes of potent and selective Polo-like kinase 1 (PLK1) inhibitors is reported. For the most interesting compounds, we discuss the biological activities, crystal structures and preliminary pharmacokinetic parameters. The more advanced compounds inhibit PLK1 in the enzymatic assay at the nM level and exhibit good activity in cell proliferation on A2780 cells. Furthermore, these compounds showed high levels of selectivity on a panel of unrelated kinases, as well as against PLK2 and PLK3 isoforms. Additionally, the compounds show acceptable oral bioavailability in mice making these inhibitors suitable candidates for further in vivo activity studies.

Phase I study of the safety, tolerability and pharmacokinetics of PHA-848125AC, a dual tropomyosin receptor kinase A and cyclin-dependent kinase inhibitor, in patients with advanced solid malignancies.

PURPOSE: This phase I trial assessed the safety, maximally tolerated dose (MTD) and pharmacokinetics of TRKA/CDK inhibitor PHA-848125AC in adult patients with advanced/metastatic solid tumors. PATIENTS AND METHODS: Patients with relapsed or refractory solid tumors, for which no standard therapy existed, were eligible. PHA-848125AC was administered orally in two schedules: daily for 7 consecutive days in 2-week cycles (i.e. 7 days on/7 days off q2wks; S1) or daily for 4 consecutive days a week for 3 weeks in 4-week cycles (i.e. 4 days on/3 days off x 3wks q4wks; S2). RESULTS: Thirty-seven patients were treated in this study, 22 in S1 and 15 in S2. The recommended phase II dose (RP2D) was 150 mg/day for either schedule. The dose-limiting toxicities (DLTs) in S1 included ataxia (Grade 2-4) and tremors (Grade 2-3). In S2, DLTs included tremors (Grade 2-3), elevated lipase (Grade 3), increased creatinine (Grade 2), and nausea and vomiting (Grade 3). These events were all reversible. In S2, out of 14 patients evaluable for efficacy, 2 patients with thymic carcinoma, showed partial response and stable disease was observed in 3 patients. Stable disease was observed in 6 out 14 patients evaluable for efficacy on S1. Drug pharmacokinetics demonstrated a half-life of approximately 33 h, and dose-proportionality with accumulation by a factor of 3 after repeated administrations. CONCLUSION: The RP2D of PHA-848125AC was 150 mg/day on both schedules. Based on the responses noted in thymic carcinoma, a phase II study for patients with that disease is currently enrolling.

NMS-P937, a 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivative as potent and selective Polo-like kinase 1 inhibitor.

As part of our drug discovery effort, we identified and developed 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as PLK1 inhibitors. We now report the optimization of this class that led to the identification of NMS-P937, a potent, selective and orally available PLK1 inhibitor. Also, in order to understand the source of PLK1 selectivity, we determined the crystal structure of PLK1 with NMS-P937. The compound was active in vivo in HCT116 xenograft model after oral administration and is presently in Phase I clinical trials evaluation.

4,5-Dihydro-1H-pyrazolo[4,3-h]quinazolines as potent and selective Polo-like kinase 1 (PLK1) inhibitors.

A series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives was optimized as Polo-like kinase 1 inhibitors. Extensive SAR afforded a highly potent and selective PLK1 compound. The compound showed good antiproliferative activity when tested in a panel of tumor cell lines with PLK1 related mechanism of action and with good in vivo antitumor efficacy in two xenograft models after i.v. administration.

Cdc7 kinase inhibitors: 5-heteroaryl-3-carboxamido-2-aryl pyrroles as potential antitumor agents. 1. Lead finding.

Cdc7 serine/threonine kinase is a key regulator of DNA synthesis in eukaryotic organisms. Cdc7 inhibition through siRNA or prototype small molecules causes p53 independent apoptosis in tumor cells while reversibly arresting cell cycle progression in primary fibroblasts. This implies that Cdc7 kinase could be considered a potential target for anticancer therapy. We previously reported that pyrrolopyridinones (e.g., 1) are potent and selective inhibitors of Cdc7 kinase, with good cellular potency and in vitro ADME properties but with suboptimal pharmacokinetic profiles. Here we report on a new chemical class of 5-heteroaryl-3-carboxamido-2-substituted pyrroles (1A) that offers advantages of chemistry diversification and synthetic simplification. This work led to the identification of compound 18, with biochemical data and ADME profile similar to those of compound 1 but characterized by superior efficacy in an in vivo model. Derivative 18 represents a new lead compound worthy of further investigation toward the ultimate goal of identifying a clinical candidate.

Dual targeting of CDK and tropomyosin receptor kinase families by the oral inhibitor PHA-848125, an agent with broad-spectrum antitumor efficacy.

Altered expression and activity of cyclin-dependent kinase (CDK) and tropomyosin receptor kinase (TRK) families are observed in a wide variety of tumors. In those malignancies with aberrant CDK activation, the retinoblastoma protein (pRb) pathway is deregulated, leading to uncontrolled cell proliferation. Constitutive activation of TRKs is instead linked to cancer cell survival and dissemination. Here, we show that the novel small-molecule PHA-848125, a potent dual inhibitor of CDKs and TRKs, possesses significant antitumor activity. The compound inhibits cell proliferation of a wide panel of tumoral cell lines with submicromolar IC(50). PHA-848125-treated cells show cell cycle arrest in G(1) and reduced DNA synthesis, accompanied by inhibition of pRb phosphorylation and modulation of other CDK-dependent markers. The compound additionally inhibits phosphorylation of TRKA and its substrates in cells, which functionally express this receptor. Following oral administration, PHA-848125 has significant antitumor activity in various human xenografts and carcinogen-induced tumors as well as in disseminated primary leukemia models, with plasma concentrations in rodents in the same range as those found active in inhibiting cancer cell proliferation. Mechanism of action was also confirmed in vivo as assessed in tumor biopsies from treated mice. These results show that the dual CDK-TRK inhibitor PHA-848125 has the potential for being a novel and efficacious targeted drug for cancer treatment.

Transcriptional analysis of an E2F gene signature as a biomarker of activity of the cyclin-dependent kinase inhibitor PHA-793887 in tumor and skin biopsies from a phase I clinical study.

A transcriptional signature of the pan-cyclin-dependent kinase (Cdk) inhibitor PHA-793887 was evaluated as a potential pharmacodynamic and/or response biomarker in tumor and skin biopsies from patients treated in a phase I clinical study. We first analyzed the expression of a number of known E2F-dependent genes that were predicted to be modulated after Cdk2 and Cdk4 inhibition in xenograft tumor and skin samples of mice treated with the compound. This panel of 58 selected genes was then analyzed in biopsies from seven patients treated with PHA-793887 in a phase I dose escalation clinical trial in solid tumors. Quantitative real-time PCR or microarray analyses were done in paired skin and tumor biopsies obtained at baseline and at cycle 1. Analysis by quantitative real-time PCR of the signature in skin biopsies of patients treated at three different doses showed significant transcriptional downregulation with a dose-response correlation. These data show that PHA-793887 modulates genes involved in cell cycle regulation and proliferation in a clinical setting. The observed changes are consistent with its mechanism of action and correlate with target modulation in skin and with clinical benefit in tumors.

Identification of potent pyrazolo[4,3-h]quinazoline-3-carboxamides as multi-cyclin-dependent kinase inhibitors.

Abnormal proliferation mediated by disruption of the mechanisms that keep the cell cycle under control is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDKs) and cyclins (Cy) and inhibiting their activity are regarded as promising antitumor agents to complement the existing therapies. An expansion of pyrazolo[4,3-h]quinazoline chemical class oriented to the development of three points of variability was undertaken leading to a series of compounds able to inhibit CDKs both in vitro and in vivo. Starting from the CDK selective but poorly soluble hit compound 1, we succeeded in obtaining several compounds showing enhanced inhibitory activity both on CDKs and on tumor cells and displaying improved physical properties and pharmacokinetic behavior. Our study led to the identification of compound 59 as a highly potent, orally bioavailable CDK inhibitor that exhibited significant in vivo efficacy on the A2780 ovarian carcinoma xenograft model. The demonstrated mechanisms of action of compound 59 on cancer cell lines and its ability to inhibit tumor growth in vivo render this compound very interesting as potential antineoplastic agent.