Proof of Principle for Local Delivery of a c-Met Inhibitor.

The reported proof of principle study demonstrated the feasibility of local delivery of a c-Met inhibitor (VXc-140) in a subcutaneous xenograft tumor model. VXc-140 was formulated in a wafer delivery system for direct implantation into the tumor. Systemic and local tumor exposure of VXc-140 was analyzed. High tumor exposures coupled with fast release of compound were associated with significant tumor regression and reduction in tumor levels of phosphorylated c-Met. High VXc-140 tumor-to-plasma ratios (∼42 at the tumor periphery) were achieved. The tumor response achieved (7/11 partial response) with VXc-140 with the local delivery in the wafer (4 mg over 15 days) was comparable to the regression observed (11/15 partial response) for VXc-140 in the oral delivery (∼8 mg total administered once a day for 2 weeks). Notably, the plasma levels in animals implanted with VXc-140 wafers ranged from 2 to 4 µM, which, although higher than trough levels achieved with oral administration, were well below oral Cmax levels (∼42 µM) suggesting that toxicities associated with Cmax exposure may be reduced or eliminated by local delivery. The high tumor to plasma exposure of VXc-140 and the efficacy observed with local wafer delivery warrants further exploration into the utility of local delivery.

Discovery of Novel Allosteric HCV NS5B Inhibitors. 2. Lactam-Containing Thiophene Carboxylates.

Lomibuvir (1) is a non-nucleoside, allosteric inhibitor of the hepatitis C virus NS5B polymerase with demonstrated clinical efficacy. Further development efforts within this class of inhibitor focused on improving the antiviral activity and physicochemical and pharmacokinetic properties. Recently, we reported the development of this series, leading to compound 2, a molecule with comparable potency and an improved physicochemical profile relative to 1. Further exploration of the amino amide-derived side chain led to a series of lactam derivatives, inspired by the X-ray crystal structure of related thiophene carboxylate inhibitors. This series, exemplified by 12f, provided 3-5-fold improvement in potency against HCV replication, as measured by replicon assays. The synthesis, structure-activity relationships, in vitro ADME characterization, and in vivo evaluation of this novel series are discussed.

Discovery of Novel Thiophene-Based, Thumb Pocket 2 Allosteric Inhibitors of the Hepatitis C NS5B Polymerase with Improved Potency and Physicochemical Profiles.

The hepatitis C viral proteins NS3/4A protease, NS5B polymerase, and NS5A are clinically validated targets for direct-acting antiviral therapies. The NS5B polymerase may be inhibited directly through the action of nucleosides or nucleotide analogues or allosterically at a number of well-defined sites. Herein we describe the further development of a series of thiophene carboxylate allosteric inhibitors of NS5B polymerase that act at the thumb pocket 2 site. Lomibuvir (1) is an allosteric HCV NS5B inhibitor that has demonstrated excellent antiviral activity and potential clinical utility in combination with other direct acting antiviral agents. Efforts to further explore and develop this series led to compound 23, a compound with comparable potency and improved physicochemical properties.

Synthesis and evaluation of NS5A inhibitors containing diverse heteroaromatic cores.

Inhibitors of the HCV NS5A nonstructural protein are showing promising clinical potential in the treatment of hepatitis C when used in combination with other direct-acting antiviral agents. Current NS5A clinical candidates such as daclatasvir, ledipasvir, and ombitasvir share a common pharmacophore that features a pair of (S)-methoxycarbonylvaline capped pyrrolidines linked to various cores by amides, imidazoles and/or benzimidazoles. In this Letter, we describe the evaluation of NS5A inhibitors which contain alternative heteroaromatic replacements for these amide mimetics. The SAR knowledge gleaned in the optimization of scaffolds containing benzoxazoles was parlayed toward the identification of potent NS5A inhibitors containing other heteroaromatic replacements such as indoles and imidazopyridines.

Benzimidazole-containing HCV NS5A inhibitors: effect of 4-substituted pyrrolidines in balancing genotype 1a and 1b potency.

The treatment of HCV with highly efficacious, well-tolerated, interferon-free regimens is a compelling clinical goal. Trials employing combinations of direct-acting antivirals that include NS5A inhibitors have shown significant promise in meeting this challenge. Herein, we describe our efforts to identify inhibitors of NS5A and report on the discovery of benzimidazole-containing analogs with subnanomolar potency against genotype 1a and 1b replicons. Our SAR exploration of 4-substituted pyrrolidines revealed that the subtle inclusion of a 4-methyl group could profoundly increase genotype 1a potency in multiple scaffold classes.

Discovery of thienoimidazole-based HCV NS5A inhibitors. Part 1: C2-symmetric inhibitors with diyne and biphenyl linkers.

The discovery of C2-symmetric bis-thienoimidazoles HCV NS5A inhibitors is herein reported. Two straightforward approaches to access the requisite diyne and biphenyl linker moieties are described. This study revealed the paramount importance of the aromatic character of the linker to achieve high genotype 1a potency.

Discovery of thienoimidazole-based HCV NS5A inhibitors. Part 2: non-symmetric inhibitors with potent activity against genotype 1a and 1b.

The discovery of non-symmetric thienoimidazole-containing HCV NS5A inhibitors is described. The inhibitors herein reported display high potencies against both genotype 1a and 1b. In this follow-up manuscript, we discuss the importance of the linker aromaticity to achieve high potency, particularly against genotype 1a.

Discovery of Thienoimidazole-Based HCV NS5A Genotype 1a and 1b Inhibitors.

The discovery of potent thienoimidazole-based HCV NS5A inhibitors is herein reported. A novel method to access the thienoimidazole [5,5]-bicyclic system is disclosed. This method gave access to a common key intermediate (6) that was engaged in Suzuki or Sonogashira reactions with coupling partners bearing different linkers. A detailed study of the structure-activity relationship (SAR) of the linkers revealed that aromatic linkers with linear topologies are required to achieve high potency for both 1a and 1b HCV genotypes. Compound 20, with a para-phenyl linker, was identified as a potential lead displaying potencies of 17 and 8 pM against genotype 1a and 1b replicons, respectively.

In vitro and in vivo isotope effects with hepatitis C protease inhibitors: enhanced plasma exposure of deuterated telaprevir versus telaprevir in rats.

Telaprevir 2 (VX-950), an inhibitor of the hepatitis C virus (HCV(a)) NS3-4A protease, is in phase 3 clinical trials. One of the major metabolites of 2 is its P1-(R)-diastereoisomer, 3 (VRT-394), containing an inversion at the chiral center next to the alpha-ketoamide on exchange of a proton with solvent. Compound 3 is approximately 30-fold less active against HCV protease. In an attempt to suppress the epimerization of 2 without losing activity against the HCV protease, the proton at that chiral site was replaced with deuterium (d). The compound 1 (d-telaprevir) is as efficacious as 2 in in vitro inhibition of protease activity and viral replication (replicon) assays. The kinetics of in vitro stability of 1 and 2 in buffered pH solutions and plasma samples, including human plasma, suggest that 1 is significantly more stable than 2. Oral administration (10 mg/kg) in rats resulted in a approximately 13% increase of AUC for 1.

In vivo release from a drug delivery MEMS device.

A drug delivery microelectromechanical systems (MEMS) device was designed to release complex profiles of multiple substances in order to maximize the effectiveness of drug therapies. The device is based on micro-reservoirs etched into a silicon substrate that contain individual doses of drug. Each dose is released by the electrochemical dissolution of the gold membrane that covers the reservoir. The first in vivo operation of this device was reported in this study. Subcutaneous release was demonstrated in rats using two tracer molecules, fluorescein dye and radiolabeled mannitol, and one radiolabeled chemotherapeutic agent, carmustine (BCNU). BCNU was chosen because of the need to improve the direct delivery of chemotherapy to malignant tumors. The spatial profile of fluorescein dye release from the drug delivery device was evaluated by fluorimetry, the temporal profile of 14C labeled mannitol release was evaluated by liquid scintillation counting, and the temporal profile of 14C labeled BCNU release was evaluated by accelerator mass spectrometry (AMS). Release profiles obtained from injected controls were compared with those from activated devices. The in vivo dye release results showed high concentration of fluorescein in the flank tissue surrounding the devices 1 h after activation. The 14C labeled mannitol released from the drug delivery devices was rapidly cleared (1 day) from the rat urine. In vivo release of 14C labeled BCNU from activated devices showed slightly slower kinetics than the injected and in vitro controls, and the time to reach the steady-state plasma 14C concentration was on the order of 1 h. All these results demonstrated the capability of this drug delivery device to achieve localized delivery of various compounds with well-defined temporal profiles.

Repeated in vivo electrochemical activation and the biological effects of microelectromechanical systems drug delivery device.

The repeated activation of a microelectromechanical systems (MEMS) drug delivery device was studied in vivo in rats to examine the effect of implantation on the device operation and the effect of electrochemical activation on the inflammatory and wound-healing response. The MEMS devices were fabricated from a silicon wafer into which reservoirs were etched and covered with gold membranes. The membranes were electrochemically removed when an anodic voltage was applied. Devices were implanted subcutaneously both with and without stainless steel mesh cages for 4, 7, 14, 21, or 28 days before activation. Devices were activated every other day for five activations. Leukocyte concentrations indicated that both the application of voltage and the gold corrosion products elevated the inflammatory response which was resolved within 48 h after each activation. The efficiency of gold membrane removal was not impaired throughout the implantation, although a bimodal distribution of background current densities was observed after long implantation times. The thickness of the fibrous capsule surrounding the MEMS devices was similar between activated and control devices explanted at each time point. It was concluded that the repeated activation of MEMS drug delivery devices was successful and the activation produced an acceptable biological response that resolved promptly.

In vivo inflammatory and wound healing effects of gold electrode voltammetry for MEMS micro-reservoir drug delivery device.

The in vivo biocompatibility and biofouling of gold electrodes for a microelectromechanical systems drug delivery device were investigated in a rodent model. The role of the applied voltage and gold electrolysis products in modulating the inflammatory response (biocompatibility), and the temporal adhesion of cellular populations onto macroscopic gold film electrodes (biofouling) were analyzed in reference to two controls, devices to which voltage was not applied (uncorroded) or voltage was applied to inert platinum electrodes (electrical controls). Voltammetry was applied to the gold surfaces once (day 4, 7, 14, 21, 28, 35, 42, or 49), while voltage of identical magnitude was applied to the electrical controls. An inflammatory response characterized by a rapid decrease of leukocyte concentration to control levels was observed 48 h following voltage application with no significant cell concentration difference (p > 0.05) between the corroded devices and electrical controls. The histological evaluation of the direct implant fibrous capsule showed comparable thickness of voltage applied and control specimens. The gold corrosion peak current showed no significant difference (p > 0.05) among peak values at all time points. It was concluded that gold electrode corrosion was biocompatible and its electrochemical performance was not hindered by fibrous capsule formation.

Electronic MEMS for triggered delivery.

Implantable electronic devices such as pacemakers and neural implants are often used for electrical stimulation. The usage of microfabrication techniques to produce microelectromechanical systems (MEMS) has allowed engineers to address a wider range of clinical indications. A new direction in the area of MEMS technology is the goal of achieving pulsatile drug delivery. The digital capabilities of MEMS may allow greater temporal control over drug release compared to traditional polymer-based systems, while the batch-processing techniques used in the microelectronics industry can lead to greater device uniformity and reproducibility than is currently available to the pharmaceutical industry. A repertoire of structures, including microreservoirs, micropumps, valves, and sensors, is being developed that will provide a strong foundation for the design of integrated, responsive MEMS for drug delivery.

Biocompatibility and biofouling of MEMS drug delivery devices.

The biocompatibility and biofouling of the microfabrication materials for a MEMS drug delivery device have been evaluated. The in vivo inflammatory and wound healing response of MEMS drug delivery component materials, metallic gold, silicon nitride, silicon dioxide, silicon, and SU-8(TM) photoresist, were evaluated using the cage implant system. Materials, placed into stainless-steel cages, were implanted subcutaneously in a rodent model. Exudates within the cage were sampled at 4, 7, 14, and 21 days, representative of the stages of the inflammatory response, and leukocyte concentrations (leukocytes/microl) were measured. Overall, the inflammatory responses elicited by these materials were not significantly different than those for the empty cage controls over the duration of the study. The material surface cell density (macrophages or foreign body giant cells, FBGCs), an indicator of in vivo biofouling, was determined by scanning electron microscopy of materials explanted at 4, 7, 14, and 21 days. The adherent cellular density of gold, silicon nitride, silicon dioxide, and SU-8(TM) were comparable and statistically less (p<0.05) than silicon. These analyses identified the MEMS component materials, gold, silicon nitride, silicon dioxide, SU-8(TM), and silicon as biocompatible, with gold, silicon nitride, silicon dioxide, and SU-8(TM) showing reduced biofouling.