Unremarkable impact of Oatp inhibition on the liver concentration of fluvastatin, lovastatin and pitavastatin in wild-type and Oatp1a/1b knockout mouse

<p>1. Oatp inhibitors have been shown to significantly increase the plasma exposure of statins. However, understanding alterations of liver concentration is also important. While modeling has simulated liver concentration changes, availability of experimental data is limited, especially when concerning drug–drug interactions (DDI). The objective of this work was to determine blood and liver concentrations of fluvastatin, lovastatin and pitavastatin, when blocking uptake transporters.</p> <p>2. In wild-type mouse, rifampin pre-treatment decreased the unbound liver-to-plasma ratio (<i>K</i>_p,uu) of fluvastatin by 4.2-fold to 2.2, lovastatin by 4.9-fold to 0.81 and pitavastatin by 10-fold to 0.21. Changes in <i>K</i>_p,uu were driven by increases in systemic exposures as liver concentrations were not greatly altered.</p> <p>3. In Oatp1a/1b knockout mouse (KO), rifampin exerted no additional effect on fluvastatin and lovastatin. Contrarily, rifampin further decreased pitavastatin <i>K</i>_p,uu by 3.4-fold, suggesting that the KO is inadequate to completely block liver uptake of pitavastatin as there are additional rifampin-sensitive uptake mechanism(s) not captured in the KO model.</p> <p>4. This work provides experimental data showing that the plasma compartment is more sensitive to Oatp modulation than the liver compartment, even for rifampin-mediated DDI. Consistent with previous simulations, inhibiting or targeting Oatps may change <i>K</i>_p,uu, but exhibit only a minimal effect on absolute liver concentrations.</p

Role of P‑Glycoprotein on the Brain Penetration and Brain Pharmacodynamic Activity of the MEK Inhibitor Cobimetinib

Cobimetinib is a MEK inhibitor currently in clinical trials as an anticancer agent. The objectives of this study were to determine in vitro and in vivo if cobimetinib is a substrate of P-glycoprotein (P-gp) and/or breast cancer resistance protein (Bcrp1) and to assess the implications of efflux on cobimetinib pharmacokinetics (PK), brain penetration, and target modulation. Cell lines transfected with P-gp or Bcrp1 established that cobimetinib was a substrate of P-gp but not a substrate of Bcrp1. In vivo, after intravenous and oral administration of cobimetinib to FVB (wild-type; WT), <i>Mdr1a/b­(−/−)</i>,<i> Bcrp1 (−/−)</i>, and <i>Mdr1a/b­(−/−)/Bcrp­(−/−)</i> knockout (KO) mice, clearance was similar in WT (35.5 ± 16.7 mL/min/kg) and KO animals (22.0 ± 3.6 to 27.6 ± 5.2 mL/min/kg); oral exposure was also similar between WT and KO animals. After an oral 10 mg/kg dose of cobimetinib, the mean total brain to plasma ratio (Kp) at 6 h postdose was 0.3 and 0.2 in WT and <i>Bcrp1­(−/−)</i> mice, respectively. In <i>Mdr1a/b­(−/−)</i> and <i>Mdr1<i>a</i>/1b/Bcrp1­(−/−)</i> KO mice and WT mice treated with elacridar (a P-gp and BCRP inhibitor), Kp increased to 11, 6, and 7, respectively. Increased brain exposure in <i>Mdr1a/b­(−/−)</i> and <i>Mdr1<i>a</i>/1b/Bcrp1­(−/−)</i> KO and elacridar treated mice was accompanied by up to ∼65% suppression of the target (pErk) in brain tissue, compared to WT mice. By MALDI imaging, the cobimetinib signal intensity was relatively high and was dispersed throughout the brain of <i>Mdr1<i>a</i>/1b/Bcrp1­(−/−)</i> KO mice compared to low/undetectable signal intensity in WT mice. The efflux of cobimetinib by P-gp may have implications for the treatment of patients with brain tumors/metastases

Characterizing the <i>in vitro</i> species differences in N-glucuronidation of a potent pan-PIM inhibitor GNE-924 containing a 3,5-substituted 6-azaindazole

<p>1.Glucuronidation of amines has been shown to exhibit large species differences, where the activity is typically more pronounced in human than in many preclinical species such as rat, mouse, dog and monkey. The purpose of this work was to characterize the <i>in vitro</i> glucuronidation of GNE-924, a potent pan-PIM inhibitor, to form M1 using liver microsomes (LM) and intestinal microsomes (IM).</p> <p>2.M1 formation kinetics varied highly across species and between liver and intestinal microsomes. In LM incubations, rat exhibited the highest rate of M1 formation (CL_int,app) at 140 ± 10 µL/min/mg protein, which was approximately 30-fold higher than human. In IM incubations, mouse exhibited the highest CL_int,app at 484 ± 40 µL/min/mg protein, which was >1000-fold higher than human. In addition, CL_int,app in LM was markedly higher than IM in human and monkey. In contrast, CL_int,app in IM was markedly higher than LM in dog and mouse.</p> <p>3.Reaction phenotyping indicated that UGT1A1, UGT1A3, UGT1A9, UGT2B4 and the intestine-specific UGT1A10 contributed to the formation of M1.</p> <p>4.This is one of the first reports showing that N-glucuronidation activity is significantly greater in multiple preclinical species than in humans, and suggests that extensive intestinal N-glucuronidation may limit the oral exposure of GNE-924.</p

Discovery of 5‑Azaindazole (GNE-955) as a Potent Pan-Pim Inhibitor with Optimized Bioavailability

Pim kinases have been identified as promising therapeutic targets for hematologic–oncology indications, including multiple myeloma and certain leukemia. Here, we describe our continued efforts in optimizing a lead series by improving bioavailability while maintaining high inhibitory potency against all three Pim kinase isoforms. The discovery of extensive intestinal metabolism and major metabolites helped refine our design strategy, and we observed that optimizing the pharmacokinetic properties first and potency second was a more successful approach than the reverse. In the resulting work, novel analogs such as <b>20</b> (GNE-955) were discovered bearing 5-azaindazole core with noncanonical hydrogen bonding to the hinge

A Unique Approach to Design Potent and Selective Cyclic Adenosine Monophosphate Response Element Binding Protein, Binding Protein (CBP) Inhibitors

The epigenetic regulator CBP/P300 presents a novel therapeutic target for oncology. Previously, we disclosed the development of potent and selective CBP bromodomain inhibitors by first identifying pharmacophores that bind the KAc region and then building into the LPF shelf. Herein, we report the “hybridization” of a variety of KAc-binding fragments with a tetrahydroquinoline scaffold that makes optimal interactions with the LPF shelf, imparting enhanced potency and selectivity to the hybridized ligand. To demonstrate the utility of our hybridization approach, two analogues containing unique Asn binders and the optimized tetrahydroquinoline moiety were rapidly optimized to yield single-digit nanomolar inhibitors of CBP with exquisite selectivity over BRD4(1) and the broader bromodomain family

Structure-Based Discovery of Novel Amide-Containing Nicotinamide Phosphoribosyltransferase (Nampt) Inhibitors

Crystal structures of several urea- and thiourea-derived compounds in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein were utilized to design a potent amide-containing inhibitor bearing an aza-indole moiety (<b>7</b>, Nampt BC IC₅₀ = 9.0 nM, A2780 cell proliferation IC₅₀ = 10 nM). The Nampt–<b>7</b> cocrystal structure was subsequently obtained and enabled the design of additional amide-containing inhibitors which incorporated various other fused 6,5-heterocyclic moieties and biaryl sulfone or sulfonamide motifs. Additional modifications of these molecules afforded many potent biaryl sulfone-containing Nampt inhibitors which also exhibited favorable in vitro ADME properties (microsomal and hepatocyte stability, MDCK permeability, plasma protein binding). An optimized compound (<b>58</b>) was a potent inhibitor of multiple cancer cell lines (IC₅₀ <10 nM vs U251, HT1080, PC3, MiaPaCa2, and HCT116 lines), displayed acceptable mouse PK properties (F = 41%, CL = 52.4 mL/min/kg), and exhibited robust efficacy in a U251 mouse xenograft model

GNE-781, A Highly Advanced Potent and Selective Bromodomain Inhibitor of Cyclic Adenosine Monophosphate Response Element Binding Protein, Binding Protein (CBP)

Inhibition of the bromodomain of the transcriptional regulator CBP/P300 is an especially interesting new therapeutic approach in oncology. We recently disclosed in vivo chemical tool <b>1</b> (GNE-272) for the bromodomain of CBP that was moderately potent and selective over BRD4(1). In pursuit of a more potent and selective CBP inhibitor, we used structure-based design. Constraining the aniline of <b>1</b> into a tetrahydroquinoline motif maintained potency and increased selectivity 2-fold. Structure–activity relationship studies coupled with further structure-based design targeting the LPF shelf, BC loop, and KAc regions allowed us to significantly increase potency and selectivity, resulting in the identification of non-CNS penetrant <b>19</b> (GNE-781, TR-FRET IC₅₀ = 0.94 nM, BRET IC₅₀ = 6.2 nM; BRD4(1) IC₅₀ = 5100 nΜ) that maintained good in vivo PK properties in multiple species. Compound <b>19</b> displays antitumor activity in an AML tumor model and was also shown to decrease Foxp3 transcript levels in a dose dependent manner

Discovery of a Potent and Selective in Vivo Probe (GNE-272) for the Bromodomains of CBP/EP300

The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure–activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded <b>59</b> (GNE-272, TR-FRET IC₅₀ = 0.02 μM, BRET IC₅₀ = 0.41 μM, BRD4(1) IC₅₀ = 13 μM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound <b>59</b> showed a marked antiproliferative effect in hematologic cancer cell lines and modulates <i>MYC</i> expression in vivo that corresponds with antitumor activity in an AML tumor model