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ABI-H0731, a first-generation hepatitis B virus (HBV) core protein inhibitor, has demonstrated effective antiviral activity in chronic hepatitis B (CHB) patients in a phase 1b clinical trial and is currently being further evaluated in phase 2 clinical trials. Here, we report the preclinical profile of ABI-H0731. In in vitro cell culture systems (HepG2-derived cell lines HepAD38 and HepG2-NTCP and primary human hepatocytes [PHHs]), ABI-H0731 exhibited selective inhibition of HBV DNA replication (50% effective concentration [EC50] from 173 nM to 307 nM)., ABI-H0731, a first-generation hepatitis B virus (HBV) core protein inhibitor, has demonstrated effective antiviral activity in chronic hepatitis B (CHB) patients in a phase 1b clinical trial and is currently being further evaluated in phase 2 clinical trials. Here, we report the preclinical profile of ABI-H0731. In in vitro cell culture systems (HepG2-derived cell lines HepAD38 and HepG2-NTCP and primary human hepatocytes [PHHs]), ABI-H0731 exhibited selective inhibition of HBV DNA replication (50% effective concentration [EC50] from 173 nM to 307 nM). Most importantly, ABI-H0731 suppressed covalently closed circular DNA (cccDNA) formation in two de novo infection models with EC50s from 1.84 μM to 7.3 μM. Mechanism-of-action studies indicated that ABI-H0731 is a direct-acting antiviral that targets HBV core protein, preventing HBV pregenomic RNA (pgRNA) encapsidation and subsequent DNA replication. The combination of ABI-H0731 with entecavir appears to decrease viral DNA faster and deeper than nucleoside/nucleotide analogue (NrtI) therapy alone. In addition, ABI-H0731 disrupts incoming nucleocapsids, causing the premature release of relaxed circular DNA (rcDNA) before delivery to the nucleus, and thus prevents new cccDNA formation. ABI-H0731 exhibits pangenotypic activity and is additive to moderately synergistic when combined with an NrtI. In addition to its potency and novel mechanism of action, ABI-H0731 possesses drug-like properties and a preclinical pharmacokinetic profile supportive of once-daily dosing in patients with CHB. Taken together, these data support the ongoing clinical development of ABI-H0731 as a treatment for HBV.

The development and optimization of a series of quinolinylpurines as potent and selective PI3Kδ kinase inhibitors with excellent physicochemical properties are described. This medicinal chemistry effort led to the identification of 1 (AMG319), a compound with an IC50 of 16 nM in a human whole blood assay (HWB), excellent selectivity over a large panel of protein kinases, and a high level of in vivo efficacy as measured by two rodent disease models of inflammation.

Structure-based rational design led to the synthesis of a novel series of potent PI3K inhibitors. The optimized pyrrolopyridine analogue 63 was a potent and selective PI3Kβ/δ dual inhibitor that displayed suitable physicochemical properties and pharmacokinetic profile for animal studies. Analogue 63 was found to be efficacious in animal models of inflammation including a keyhole limpet hemocyanin (KLH) study and a collagen-induced arthritis (CIA) disease model of rheumatoid arthritis. These studies highlight the potential therapeutic value of inhibiting both the PI3Kβ and δ isoforms in the treatment of a number of inflammatory diseases.

The optimization of a series of 8-aza-quinazolinone analogs for antagonist activity against the CXCR3 receptor is reported. Compounds were optimized to avoid the formation of active metabolites and time-dependent-inhibitors of CYP3A4. In addition, antagonists showed potent against CXCR3 activity in whole blood and optimized to avoid activity in the chromosomal aberration assay. Compound 25 was identified as having the optimal balance of CXCR3 activity and pharmacokinetic properties across multiple pre-clinical species, which are reported herein.

The 2-methyl substituted indole, 2MI [2-(4-(4-(2,4-dichlorophenylsulfonamido)-2-methyl-1 H -indol-5-yloxy)-3-methoxyphenyl)acetic acid] is a potent dual inhibitor of 1) chemoattractant receptor-homologous molecule expressed on T-helper type-2 cells and 2) d-prostanoid receptor. During evaluation as a potential treatment for asthma and allergic rhinitis, 2MI was identified as a mechanism-based inactivator of CYP3A4 in vitro. The inactivation was shown to be irreversible by dialysis and accompanied by an NADPH-dependent increase in 2MI covalent binding to a 55- to 60-kDa microsomal protein, consistent with irreversible binding to CYP3A4. Two glutathione (GSH) adducts, G1 and G2, were identified in vitro, and the more abundant adduct (G1) was unambiguously determined via NMR to be GSH adducted to the 3-position of the 2-methylindole moiety. The potential for a clinical drug-drug interaction arising from mechanism-based inactivation of CYP3A4 by 2MI was predicted using a steady-state model, and a 4.3- to 7.5-fold increase in the exposure of midazolam was predicted at anticipated therapeutic concentrations. To better assess the potential for in vivo drug-drug interactions, the Sprague-Dawley rat was used as an in vivo model. An excellent in vitro-in vivo correlation was observed for the reduction in enzyme steady-state concentration ( E′ ss/ E ss) as well as the change in the exposure of a prototypical CYP3A substrate, indinavir (area under the curve (AUC) for indinavir/AUC). In summary, 2MI was identified as a potent mechanism-based inactivator of CYP3A and was predicted to elicit a clinically relevant drug-drug interaction in humans at an anticipated therapeutic concentration.

A general way of improving the potency of CXCR3 antagonists with fused hetero-bicyclic cores was identified. Optimization efforts led to the discovery of a series of imidazo-pyrazine derivatives with improved pharmacokinetic properties in addition to increased potency. The efficacy of the lead compound 21 is evaluated in a mouse lung inflammation model.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as an attractive therapeutic target for cardiovascular disease. Monoclonal antibodies (mAbs) that bind PCSK9 and prevent PCSK9:low-density lipoprotein receptor complex formation reduce serum low-density lipoprotein-cholesterol (LDL-C) in vivo. PCSK9-mediated lysosomal degradation of bound mAb, however, dramatically reduces mAb exposure and limits duration of effect. Administration of high-affinity mAb1:PCSK9 complex (1:2) to mice resulted in significantly lower mAb1 exposure compared with mAb1 dosed alone in normal mice or in PCSK9 knockout mice lacking antigen. To identify mAb-binding characteristics that minimize lysosomal disposition, the pharmacokinetic behavior of four mAbs representing a diverse range of PCSK9-binding affinities at neutral (serum) and acidic (endosomal) pH was evaluated in cynomolgus monkeys. Results revealed an inverse correlation between affinity and both mAb exposure and duration of LDL-C lowering. High-affinity mAb1 exhibited the lowest exposure and shortest duration of action (6 days), whereas mAb2 displayed prolonged exposure and LDL-C reduction (51 days) as a consequence of lower affinity and pH-sensitive PCSK9 binding. mAbs with shorter endosomal PCSK9:mAb complex dissociation half-lives (

CYP3A4-mediated biotransformation of (R)-N-(1-(3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)ethyl)-N-(pyridin-3-ylmethyl)-2-(4-(trifluoromethoxy)phenyl)acetamide (AMG 487) was previously shown to generate an inhibitory metabolite linked to dose- and time-dependent pharmacokinetics in humans. Although in vitro activity loss assays failed to demonstrate CYP3A4 time-dependent inhibition (TDI) with AMG 487, its M2 phenol metabolite readily produced TDI when remaining activity was assessed using either midazolam or testosterone (K(I) = 0.73-0.74 μM, k(inact) = 0.088-0.099 min(-1)). TDI investigations using an IC(50) shift method successfully produced inhibition attributable to AMG 487, but only when preincubations were extended from 30 to 90 min. The shift magnitude was ∼3× for midazolam activity, but no shift was observed for testosterone activity. Subsequent partition ratio determinations conducted for M2 using recombinant CYP3A4 showed that inactivation was a relatively inefficient process (r = 36). CYP3A4-mediated biotransformation of [(3)H]M2 in the presence of GSH led to identification of two new metabolites, M4 and M5, which shifted focus away from M2 being directly responsible for TDI. M4 (hydroxylated M2) was further metabolized to form reactive intermediates that, upon reaction with GSH, produced isomeric adducts, collectively designated M5. Incubations conducted in the presence of [(18)O]H(2)O confirmed incorporation of oxygen from O(2) for the majority of M4 and M5 formed (>75%). Further evidence of a primary role for M4 in CYP3A4 TDI was generated by protein labeling and proteolysis experiments, in which M4 was found to be covalently bound to Cys239 of CYP3A4. These investigations confirmed a primarily role for M4 in CYP3A4 inactivation, suggesting that a more complex metabolic pathway was responsible for generation of inhibitory metabolites affecting AMG 487 human pharmacokinetics.

Background: The use of dried blood spot (DBS) sampling technique is of particular interest for drug discovery pharmacokinetic studies due to the small blood volume requirement. In addition, automated blood sampling is an attractive approach for rat pharmacokinetic studies as animal handling work is minimized. The goal of this study was to use an automated DBS sampler for automated blood collection and spotting onto DBS paper for pharmacokinetic studies in rats. AMG 517, a potent and selective vanilloid receptor antagonist, was dosed to rats (n = 3) intravenously and blood samples were collected at nine time points over a 24 h period using the automated DBS sampler. After drying, storage and shipment, the DBS samples were extracted and analyzed by LC–MS/MS. Results: The developed bioanalytical method for the analysis of DBS samples had good accuracy and precision within the context of a discovery, non-GLP analysis. The concentration–time data and pharmacokinetic parameters generated from automated spotted samples were very similar to those derived from manually spotted DBS samples. The manual DBS data were also comparable to plasma data after correction for blood-to-plasma ratio. Conclusion: The automated DBS sampling is a promising technique for rodent pharmacokinetic studies and will improve the efficiency and quality of DBS sampling.

Abstract 4964 Immune receptors such as the B cell receptor (BCR) require key signaling intermediate phosphatidylinositol-3 kinase delta (PI3Kδ) for normal immune cell survival, development and function. PI3Kδ is a class IA lipid kinase, is expressed primarily within the hematopoietic system and is composed of a catalytic subunit p110δ and a regulatory subunit p85. Recently, deregulated BCR-PI3Kδ signaling has been reported to play a role in B-cell malignancies such as chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL) by mediating abnormal B-cell growth and survival. Indeed, the constitutive phosphorylation of downstream signaling intermediate AKT is associated with poor prognosis in several B cell malignancies. Here, we have investigated the potential of a novel small molecule inhibitor of PI3Kδ, AMG 319, to suppress PI3K signaling in human B cell lines and assessed the subsequent effects on viability as a single agent and in combination with chemotherapeutic drugs in preclinical models. Small molecule AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent preclinical pharmacokinetic (PK) properties. AMG 319 was found to potently inhibit PI3Kδ in enzyme assays (IC50 5000 fold). Furthermore, AMG 319 was considered inactive at 10 μM on non-PI3K class I kinases in a broader kinome screen of 402 kinases. In preclinical PK studies, AMG 319 had low systemic clearance, T1/2 range of 2–4 hr, oral bioavailability of >45% and unbound fractions in plasma of 5–19%. Here, we have investigated the potential for AMG 319 to inhibit constitutive PI3K mediated signaling and effects on human B cell line viability. In a broad screen of >20 cell lines derived from B cell malignancies, the majority of lines were found to express PI3Kδ protein, all cells lines expressed the PI3Kα and β isoforms and variable levels of constitutive pAKTS473 were detected. AMG 319 was found to potently suppress constitutive pAKTS473 in the cell lines with IC50 in the low single to double digit nM range. Cellular viability was inhibited by AMG 319 though lines were variably sensitive to drug (range low double digit nM to μM IC50). As cell lines were variably sensitive to AMG 319 as a single agent, we examined if AMG 319 could enhance the efficacy of chemotherapeutic agents in vitro and in vivo. These studies focused on a DLBCL cell line HT which was relatively insensitive to AMG 319 as a single agent (IC50 ∼10 μM) in viability assays even though pAKTS473 was potently suppressed (IC50 ∼ 0.030 μM). Treatment with AMG 319 was found to synergize with the effects of vincristine to reduce cell viability in vitro using a 72 hr viability assay. Next we examined whether the enhanced cytotoxicity using these drugs in combination could be observed in vivo. Using the human B-cell lymphoma HT xenograft model, we found that AMG 319 in combination with vincristine enhanced tumor growth inhibition above that observed with either agent alone. Taken together, these findings suggest that the inhibition of PI3Kδ with AMG 319 may enhance the effects of chemotherapeutic agents in B cell malignancies. In conclusion, AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent PK properties. AMG 319 inhibited constitutive pAKTS473, reduced the viability of B cell lines and synergized with vincristine in vitro and in vivo. The safety, PK and preliminary efficacy of AMG 319 are currently being investigated in a Phase I trial in patients with relapsed or refractory lymphoid malignancies. Disclosures: Sinclair: Amgen: Employment, Stock and Options. Metz:Amgen, Inc: Employment, Stock and Options. Cushing:Amgen, Inc: Employment, Stock and Options. Liu:Amgen, Inc: Employment, Stock and Options. Brake:Amgen, Inc: Employment, Stock and Options. Starnes:Amgen, Inc: Employment, Stock and Options. Means:Amgen, Inc: Employment, Stock and Options. Henne:Amgen, Inc: Employment, Stock and Options. Archibeque:Amgen: Employment, Stock and Options. Mattson:Amgen, Inc: Employment, Stock and Options. Drew:Amgen, Inc: Employment, Stock and Options. Busse:Amgen, Inc: Employment, Stock and Options. Wang:Amgen, Inc: Employment, Stock and Options. Al-Assaad:Amgen, Inc: Employment, Stock and Options. Molineux:Amgen: Employment, Stock and Options.