Vincenzo Pucci

MK-5172, a Selective Inhibitor of Hepatitis C Virus NS3/4a Protease with Broad Activity across Genotypes and Resistant Variants

ABSTRACT HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.

A novel strategy for reducing phospholipids-based matrix effect in LC–ESI-MS bioanalysis by means of HybridSPE

A novel strategy to minimize phospholipids-based matrix effects in bioanalytical LC-MS/MS assays was evaluated. The phospholipids-based matrix effect was investigated with a commercially available electrospray ionization (ESI) source coupled with a triple quadrupole mass spectrometer. A systematic comparison approach of two sample preparation procedures was performed. In particular, the matrix effect on mass spectrometry response in rat and human plasma samples was studied by comparing sample extracts obtained by means of a conventional plasma protein precipitation with acetonitrile and the novel HybridSPE-Precipitation procedure. The HybridSPE dramatically reduced the levels of residual phospholipids in biological samples, leading to significant reduction in matrix effects. This new procedure which combines the simplicity of precipitation with the selectivity of SPE allows to obtain much cleaner extracts than with conventional procedures. The effective targeted removal of phospholipids and proteins in biological plasma samples achieved with the HybridSPE-Precipitation procedure provides significant improvement in bioanalysis and a practical and fast way to ensure the avoidance of phospholipids-based matrix effects.

Discovery of MK-5172, a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor.

A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.

The Efficacy of the Wee1 Inhibitor MK-1775 Combined with Temozolomide Is Limited by Heterogeneous Distribution across the Blood–Brain Barrier in Glioblastoma

Purpose: Wee1 regulates key DNA damage checkpoints, and in this study, the efficacy of the Wee1 inhibitor MK-1775 was evaluated in glioblastoma multiforme (GBM) xenograft models alone and in combination with radiation and/or temozolomide. Experimental Design: In vitro MK-1775 efficacy alone and in combination with temozolomide, and the impact on DNA damage, was analyzed by Western blotting and γH2AX foci formation. In vivo efficacy was evaluated in orthotopic and heterotopic xenografts. Drug distribution was assessed by conventional mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI)-MS imaging. Results: GBM22 (IC50 = 68 nmol/L) was significantly more sensitive to MK-1775 compared with five other GBM xenograft lines, including GBM6 (IC50 >300 nmol/L), and this was associated with a significant difference in pan-nuclear γH2AX staining between treated GBM22 (81% cells positive) and GBM6 (20% cells positive) cells. However, there was no sensitizing effect of MK-1775 when combined with temozolomide in vitro. In an orthotopic GBM22 model, MK-1775 was ineffective when combined with temozolomide, whereas in a flank model of GBM22, MK-1775 exhibited both single-agent and combinatorial activity with temozolomide. Consistent with limited drug delivery into orthotopic tumors, the normal brain to whole blood ratio following a single MK-1775 dose was 5%, and MALDI-MS imaging demonstrated heterogeneous and markedly lower MK-1775 distribution in orthotopic as compared with heterotopic GBM22 tumors. Conclusions: Limited distribution to brain tumors may limit the efficacy of MK-1775 in GBM. Clin Cancer Res; 21(8); 1916–24. ©2015 AACR.

Synthesis and antiviral properties of novel 7-heterocyclic substituted 7-deaza-adenine nucleoside inhibitors of Hepatitis C NS5B polymerase.

Previous investigations in our laboratories resulted in the discovery of a novel series of potent nucleoside inhibitors of Hepatitis C virus (HCV) NS5B polymerase bearing tetracyclic 7-substituted 7-deaza-adenine nucleobases. The planarity of such modified systems was suggested to play a role in the high inhibitory potency observed. This paper describes how we envisaged to maintain the desired planarity of the modified nucleobase by means of an intra-molecular H-bond, engaging a H-bond donor atom on an appropriately substituted 7-heterocyclic residue with the adjacent amino group of the nucleobase. The success of this strategy is reflected by the identification of several novel potent nucleoside inhibitors of HCV NS5B bearing a 7-heterocyclic substituted 7-deaza-adenine nucleobase. Amongst these, the 1,2,4-oxadiazole analog 11 showed high antiviral potency against HCV replication in replicon cells and efficient conversion to the corresponding NTP in vivo, with high and sustained levels of NTP measured in rat liver following intravenous and oral administration.

MCL1 and BCL-xL Levels in Solid Tumors Are Predictive of Dinaciclib-Induced Apoptosis

Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclibs antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.

The metabolism and disposition of a potent inhibitor of hepatitis C virus NS3/4A protease

Compound A ((1aR,5S,8S,10R,22aR)-5-tert-butyl-N-{(1R,2S)-1-[(cyclopropylsulfonyl)carbamoyl]-2-ethenylcyclopropyl}-14-methoxy-3,6-dioxo-1,1a,3,4,5,6,9,10,18,19,20,21,22,22a-tetradecahydro-8H-7,10-methanocyclopropa[18,19][1,10,3,6]dioxadiazacyclononadecino[12,11-b]quinoline-8-carboxamide) is a prototype of a series of subnanomolar inhibitors of genotypes 1, 2, and 3 hepatitis C virus (HCV) NS3/4A proteases. HCV NS3/4A protease inhibitors have demonstrated high antiviral effects in patients with chronic HCV infection and are likely to form a key component of future HCV therapy. Compound A showed excellent liver exposure in rats, which is essential for compounds intended to treat HCV. The compound was mainly eliminated intact in bile and showed greater than dose proportional systemic exposure in rats. Compound A demonstrated time- and temperature-dependent uptake into rat and human hepatocytes and proved to be a substrate for rat hepatic uptake transporter Oatp1b2 and for human hepatic uptake transporters OATP1B1 and OATP1B3. The liver selectivity observed for this compound is likely to be due to transporter-mediated hepatic uptake together with moderate passive permeability. Metabolism was mainly CYP3A-mediated and generated a reactive epoxide on the vinylcyclopropyl sulfonamide moiety that could be quenched by glutathione. Similar metabolic profiles of Compound A were obtained in liver microsomes of rats and humans. The oral bioavailability at 5 mg/kg was low due to extensive hepatic first-pass effect but clearly the intestinal absorption was enough to deliver a high amount of the compound to the liver. The metabolism and disposition properties of Compound A are particularly attractive to support its evaluation as a drug candidate for the treatment of hepatitis C.

Synthesis and Antiviral Properties of Novel Tetracyclic Nucleoside Inhibitors of Hepatitis C NS5B Polymerase

As part of an ongoing medicinal chemistry effort to identify novel nucleoside inhibitors of HCV NS5B polymerase, we report the discovery of a novel series of 2′-C-Methyl-ribose nucleoside derivatives bearing a 7-aryl and 7-heteroaryl- substituted 7-deaza-adenine nucleobase. A reliable platform for the synthesis and simplified purification of the corresponding nucleoside triphosphates (NTPs) was established, enabling a solid understanding of the SAR relationship within the series. By this approach, we identified the novel analogs 13a and 13b that demonstrated micromolar levels of cellular activity, and the NTPs of which, 16a and 16b, are excellent inhibitors of NS5B with IC50 = 0.1 μM, a level of intrinsic potency similar to that of previous and current clinical candidates.

Enhancement of intestinal absorption of 2-methyl cytidine prodrugs.

The purpose of this study was to investigate the in vivo absorption enhancement of a nucleoside (phosphoramidate prodrug of 2′-methyl-cytidine) anti-viral agent of proven efficacy by means of intestinal permeation enhancers. Natural nucleosides are hydrophilic molecules that do not rapidly penetrate cell membranes by diffusion and their absorption relies on specialized transporters. Therefore, the oral absorption of nucleoside prodrugs and the target organ concentration of the biologically active nucleotide can be limited due to poor permeation across the intestinal epithelium. In the present study, the specificity, concentration dependence, and effect of four classes of absorption promoters, i.e. fatty acids, steroidal detergents, mucoadhesive polymers, and secretory transport inhibitors, were evaluated in a rat in vivo model. Sodium caprate and α-tocopheryl-polyethyleneglycol-1000-succinate (TPGS) showed a significant effect in increasing liver concentration of nucleotide (5-fold). These results suggested that both excipients might be suited in a controlled release matrix for the synchronous release of the drug and absorption promoter directly to the site of absorption and highlights that the effect is strictly dependent on the absorption promoter dose. The feasibility of such a formulation approach in humans was evaluated with the aim of developing a solid dosage form for the peroral delivery of nucleosides and showed that these excipients do provide a potential valuable tool in pre-clinical efficacy studies to drive discovery programs forward.

Abstract C197: Accurins improve the pharmacokinetics, pharmacodynamics, tolerability and anti-tumor activity of the AKT inhibitor MK-2206

Background: BIND-2206 Accurins are novel polymeric nanoparticles encapsulating MK-2206, a Merck AKT inhibitor in phase 2 trials. MK-2206 targets the phosphatidylinositol 3-kinase (PI3K) pathway via AKT inhibition and has demonstrated therapeutic efficacy in the treatment of cancer albeit with dose limiting toxicities. Accurins have shown promise for targeting oncology agents preferentially to tumors, while limiting systemic exposure. To determine if encapsulation of MK-2206 improves therapeutic index, studies were performed to evaluate pharmacokinetics (PK), pharmacodynamics (PD), tolerability and anti-tumor activity compared to parent MK-2206. Materials and Methods: PK of four Accurin formulations with varying in vitro drug release rates was evaluated. Based on these data, two lead Accurins (BIND-2206C and BIND-2206D) were selected for further characterization. Since AKT inhibitors are documented to induce hyperglycemia in mice, blood glucose levels were also evaluated after acute administration of BIND-2206 Accurins or parent MK-2206 as a measure of tolerability. Using a panel of human tumor xenografts (VCaP, SK-OV-3 and BT-474) anti-tumor activity of the Accurins was assessed in mice. When tumors were established mice were dosed orally with parent MK-2206 or intravenously with BIND-2206 Accurins two or three times per week on a three week cycle. Tumor volume was measured post treatment to determine anti-tumor activity. Following final drug administration, tumors were collected for PK and PD evaluation. Extent and duration of tumor target inhibition was evaluated by measuring pAKT and total AKT using a mesoscale discovery method and was correlated to tumor MK-2206 levels. Results: Accurins BIND-2206C and BIND-2206D significantly enhanced the PK profile of parent MK-2206 by increasing Cmax (33 fold) and AUC (222 fold) and decreasing clearance (38 fold) in mice. In addition, both BIND-2206 Accurins improved tolerability as demonstrated by no hyperglycemic response compared to a 300% increase in blood glucose in nude mice treated with parent MK-2206. In the VCaP human prostate cancer tumor model, BIND-2206C inhibited tumor growth by 88% and BIND-2206D induced 16% tumor regression showing significant enhancement of anti-tumor efficacy compared to 55% tumor growth inhibition achieved by parent MK-2206. This correlated with high tumor exposure at 72 hours and prolonged tumor PD at 96 hours post dose for both Accurins compared to parent MK-2206. A similar increase in tumor exposure and prolonged PD response resulted in enhanced anti-tumor efficacy for BIND-2206 Accurins in the SK-OV-3 human ovarian cancer tumor model. In the BT-474 HER2 over-expressing human breast cancer tumor model, BIND-2206 Accurins did not improve efficacy compared to parent drug, although there was significant and prolonged tumor PD correlating with enhanced tumor PK. This suggests that anti-tumor efficacy is model specific and a combination strategy in the BT-474 model may be advantageous. Conclusions: BIND-2206 Accurins showed differentiated PK, increased tumor exposure, prolonged duration of target inhibition, superior efficacy and enhanced tolerability compared to parent MK-2206. These data suggest that nanoparticle formulations of the AKT inhibitor, MK-2206, may provide improved tolerability and therapeutic efficacy in a clinical setting. Citation Format: Louise Cadzow, Michael H. Lam, Young Ho Song, Maria Figueiredo, Hong Wang, David De Witt, Vincenzo Pucci, Jan-Rung Mo, Eric Lewis-Clark, Heidi Ferguson, Marian Gindy, Susan Low, Steve Zale, Jeff Hrkach, Caroline McGregor, Brian J. Long. Accurins improve the pharmacokinetics, pharmacodynamics, tolerability and anti-tumor activity of the AKT inhibitor MK-2206. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C197.