Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

Discovery of potent and selective inhibitors of calmodulin-dependent kinase II (CaMKII).

We hereby disclose the discovery of inhibitors of CaMKII (7h and 7i) that are highly potent in rat ventricular myocytes, selective against hERG and other off-target kinases, while possessing good CaMKII tissue isoform selectivity (cardiac γ/δ vs. neuronal α/ß). In vitro and in vivo ADME/PK studies demonstrated the suitability of these CaMKII inhibitors for PO (7h rat F = 73%) and IV pharmacological studies.

Preclinical disposition of GDC-0973 and prospective and retrospective analysis of human dose and efficacy predictions.

[3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-((S)-3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-methanone (GDC-0973) is a potent and highly selective inhibitor of mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase (ERK) 1/2 (MEK1/2), a MAPK kinase that activates ERK1/2. The objectives of these studies were to characterize the disposition of GDC-0973 in preclinical species and to determine the relationship of GDC-0973 plasma concentrations to efficacy in Colo205 mouse xenograft models. The clearance (CL) of GDC-0973 was moderate in mouse (33.5 ml · min(-1) · kg(-1)), rat (37.9 ± 7.2 ml · min(-1) · kg(-1)), and monkey (29.6 ± 8.5 ml · min(-1) · kg(-1)). CL in dog was low (5.5 ± 0.3 ml · min(-1) · kg(-1)). The volume of distribution across species was large, 6-fold to 15-fold body water; half-lives ranged from 4 to 13 h. Protein binding in mouse, rat, dog, monkey, and human was high, with percentage unbound, 1 to 6%. GDC-0973-related radioactivity was rapidly and extensively distributed to tissues; however, low concentrations were observed in the brain. In rats and dogs, [(14)C]GDC-0973 was well absorbed (fraction absorbed, 70-80%). The majority of [(14)C]GDC-0973-related radioactivity was recovered in the bile of rat (74-81%) and dog (65%). The CL and volume of distribution of GDC-0973 in human, predicted by allometry, was 2.9 ml · min(-1) · kg(-1) and 9.9 l/kg, respectively. The predicted half-life was 39 h. To characterize the relationship between plasma concentration of GDC-0973 and tumor growth inhibition, pharmacokinetic-pharmacodynamic modeling was applied using an indirect response model. The KC(50) value for tumor growth inhibition in Colo205 xenografts was estimated to be 0.389 µM, and the predicted clinical efficacious dose was ∼10 mg. Taken together, these data are useful in assessing the disposition of GDC-0973, and where available, comparisons with human data were made.

SAR and in vivo evaluation of 4-aryl-2-aminoalkylpyrimidines as potent and selective Janus kinase 2 (JAK2) inhibitors.

We report the discovery of a series of 4-aryl-2-aminoalkylpyrimidine derivatives as potent and selective JAK2 inhibitors. High throughput screening of our in-house compound library led to the identification of hit 1, from which optimization resulted in the discovery of highly potent and selective JAK2 inhibitors. Advanced lead 10d demonstrated a significant dose-dependent pharmacodynamic and antitumor effect in a mouse xenograft model. Based upon the desirable profile of 10d (XL019) it was advanced into clinical trials.

Discovery of a novel class of highly potent, selective, ATP-competitive, and orally bioavailable inhibitors of the mammalian target of rapamycin (mTOR).

A series of novel, highly potent, selective, and ATP-competitive mammalian target of rapamycin (mTOR) inhibitors based on a benzoxazepine scaffold have been identified. Lead optimization resulted in the discovery of inhibitors with low nanomolar activity and greater than 1000-fold selectivity over the closely related PI3K kinases. Compound 28 (XL388) inhibited cellular phosphorylation of mTOR complex 1 (p-p70S6K, pS6, and p-4E-BP1) and mTOR complex 2 (pAKT (S473)) substrates. Furthermore, this compound displayed good pharmacokinetics and oral exposure in multiple species with moderate bioavailability. Oral administration of compound 28 to athymic nude mice implanted with human tumor xenografts afforded significant and dose-dependent antitumor activity.

Bridging the gap between preclinical and clinical studies using pharmacokinetic-pharmacodynamic modeling: an analysis of GDC-0973, a MEK inhibitor.

PURPOSE: GDC-0973 is a potent and selective mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor. Pharmacokinetic-pharmacodynamic (PK-PD) modeling was used to relate GDC-0973 plasma and tumor concentrations, tumor pharmacodynamics and antitumor efficacy to establish pharmacokinetic endpoints and predict active doses in the clinic. EXPERIMENTAL DESIGN: A PK-PD model was used to characterize GDC-0973 tumor disposition and in vivo potency in WM-266-4 xenograft mice. Simulations were conducted using the PK-PD model along with human pharmacokinetics to identify a target plasma concentration and predict active doses. In vivo potency and antitumor efficacy were characterized in A375 melanoma xenograft mice, and a population-based integrated PK-PD-efficacy model was used to relate tumor pharmacodynamics (%pERK decrease) to antitumor activity. RESULTS: GDC-0973 showed a sustained tumor pharmacodynamic response due to longer residence in tumor than in plasma. Following single doses of GDC-0973, estimated in vivo IC(50) values of %pERK decrease based on tumor concentrations in xenograft mice were 0.78 (WM-266-4) and 0.52 µmol/L (A375). Following multiple doses of GDC-0973, the estimated in vivo IC(50) value in WM-266-4 increased (3.89 µmol/L). Human simulations predicted a minimum target plasma concentration of 83 nmol/L and an active dose range of 28 to 112 mg. The steep relationship between tumor pharmacodynamics (%pERK decrease) and antitumor efficacy suggests a pathway modulation threshold beyond which antitumor efficacy switches on. CONCLUSIONS: Clinical observations of %pERK decrease and antitumor activity were consistent with model predictions. This article illustrates how PK-PD modeling can improve the translation of preclinical data to humans by providing a means to integrate preclinical and early clinical data.

Discovery and characterization of an inhibitor of glucosylceramide synthase.

Targeting glycosphingolipid synthesis has emerged as a novel approach for treating metabolic diseases. 32 (EXEL-0346) represents a new class of glucosylceramide synthase (GCS) inhibitors. This report details the elaboration of hit 8 with the goal of achieving and maintaining maximum GCS inhibition in vivo. 32 inhibited GCS with an IC(50) of 2 nM and achieved maximum hepatic GCS inhibition after four or five daily doses in rodents. Robust improvements in glucose tolerance in DIO mice and ZDF rats were observed after 2 weeks of q.d. dosing. Four weeks of dosing resulted in decreased plasma triglycerides and reduced hepatic fat deposition. Thus, 32 provides insight into the amount of metabolic regulation that can be restored following achievement of maximal target knockdown.