Preclinical and clinical antiviral characterization of AB-836, a potent capsid assembly modulator against hepatitis B virus.

HBV capsid assembly modulators (CAMs) target the core protein and inhibit pregenomic RNA encapsidation and viral replication. HBV CAMs also interfere with cccDNA formation during de novo infection, which in turn suppresses transcription and production of HBV antigens. In this report, we describe the antiviral activities of AB-836, a potent and highly selective HBV CAM.

Conformationally Constrained Isoquinolinones as Orally Efficacious Hepatitis B Capsid Assembly Modulators.

Isoquinolinone-based HBV capsid assembly modulators that bind at the dimer:dimer interface of HBV core protein have been shown to suppress viral replication in chronic hepatitis B patients. Analysis of their binding mode by protein X-ray crystallography has identified a region of the small molecule where the application of a constraint can lock the preferred binding conformation and has allowed for further optimization of this class of compounds. Key analogues demonstrated single digit nM EC values in reducing HBV DNA in a HepDE19 cellular assay in addition to favorable ADME and pharmacokinetic properties, leading to a high degree of oral efficacy in a relevant hydrodynamic injection mouse model of HBV infection, with effecting a 3 log decline in serum HBV DNA levels at a once daily dose of 1 mg/kg.

Rational Design, Synthesis, and Structure-Activity Relationship of a Novel Isoquinolinone-Based Series of HBV Capsid Assembly Modulators Leading to the Identification of Clinical Candidate AB-836.

Inhibition of Hepatitis B Virus (HBV) replication by small molecules that modulate capsid assembly and the encapsidation of pgRNA and viral polymerase by HBV core protein is a clinically validated approach toward the development of new antivirals. Through definition of a minimal pharmacophore, a series of isoquinolinone-based capsid assembly modulators (CAMs) was identified. Structural biology analysis revealed that lead molecules possess a unique binding mode, exploiting electrostatic interactions with accessible phenylalanine and tyrosine residues.

Discovery of -Linked Nucleoside Analogues with Antiviral Activity against SARS-CoV-2.

The recent COVID-19 pandemic underscored the limitations of currently available direct-acting antiviral treatments against acute respiratory RNA-viral infections and stimulated major research initiatives targeting anticoronavirus agents. Two novel nsp5 protease (MPro) inhibitors have been approved, nirmatrelvir and ensitrelvir, along with two existing nucleos(t)ide analogues repurposed as nsp12 polymerase inhibitors, remdesivir and molnupiravir, but a need still exists for therapies with improved potency and systemic exposure with oral dosing, better metabolic stability, and reduced resistance and toxicity risks. Herein, we summarize our research toward identifying nsp12 inhibitors that led to nucleoside analogues and , which showed favorable pan-coronavirus activity in cell-infection screens, were metabolized to active triphosphate nucleotides in cell-incubation studies, and demonstrated target (nsp12) engagement in biochemical assays.

Preclinical Antiviral and Safety Profiling of the HBV RNA Destabilizer AB-161.

HBV RNA destabilizers are a class of small-molecule compounds that target the noncanonical poly(A) RNA polymerases PAPD5 and PAPD7, resulting in HBV RNA degradation and the suppression of viral proteins including the hepatitis B surface antigen (HBsAg). AB-161 is a next-generation HBV RNA destabilizer with potent antiviral activity, inhibiting HBsAg expressed from cccDNA and integrated HBV DNA in HBV cell-based models. AB-161 exhibits broad HBV genotype coverage, maintains activity against variants resistant to nucleoside analogs, and shows additive effects on HBV replication when combined with other classes of HBV inhibitors.

Design, synthesis, and structure-activity relationship of a bicyclic HBV capsid assembly modulator chemotype leading to the identification of clinical candidate AB-506.

Disruption of the HBV capsid assembly process through small-molecule interaction with HBV core protein is a validated target for the suppression of hepatitis B viral replication and the development of new antivirals. Through combination of key structural features associated with two distinct series of capsid assembly modulators, a novel aminochroman-based chemotype was identified. Optimization of anti-HBV potency through generation of SAR in addition to further core modifications provided a series of related functionalized aminoindanes.

Preclinical characterization of AB-506, an inhibitor of HBV replication targeting the viral core protein.

AB-506, a small-molecule inhibitor targeting the HBV core protein, inhibits viral replication in vitro (HepAD38 cells: EC of 0.077 μM, CC > 25 μM) and in vivo (HBV mouse model: ∼3.0 log reductions in serum HBV DNA compared to the vehicle control).

Preclinical Profile of AB-423, an Inhibitor of Hepatitis B Virus Pregenomic RNA Encapsidation.

AB-423 is a member of the sulfamoylbenzamide (SBA) class of hepatitis B virus (HBV) capsid inhibitors in phase 1 clinical trials. In cell culture models, AB-423 showed potent inhibition of HBV replication (50% effective concentration [EC] = 0.08 to 0.

Efficacy of CPX-351, (cytarabine:daunorubicin) liposome injection, against acute lymphoblastic leukemia (ALL) xenograft models of the Pediatric Preclinical Testing Program.

Background: CPX-351, a liposomal formulation of cytarabine and daunorubicin co-encapsulated at an optimized synergistic 5:1 molar ratio, has demonstrated improved clinical outcomes over conventional cytarabine/daunorubicin treatment in a randomized phase 2 trial in patients with AML as well as superior efficacy against preclinical leukemia models when compared to the free drugs in combination.

Procedures: Given the promising phase 2 data, limited toxicities observed, and the known clinical activities of cytarabine/daunorubicin, we assessed the efficacy of CPX-351 against a panel of childhood ALL xenograft models. Plasma pharmacokinetics of cytarabine and daunorubicin following CPX-351 treatment were determined by HPLC in order to correlate efficacy with drug exposure.

Liposomal encapsulation of a synergistic molar ratio of cytarabine and daunorubicin enhances selective toxicity for acute myeloid leukemia progenitors as compared to analogous normal hematopoietic cells.

Objective: To evaluate the possibility of improved selective killing of acute myeloid leukemia (AML) cells with CPX-351 (a liposomal formulation of cytarabine and daunorubicin). CPX-351 and the same molar ratio of free drugs were compared for cytotoxicity against colony-forming cells (CFCs) and subpopulations of cells enriched for primitive progenitors from AML patients and normal granulocyte colony-stimulating factor-mobilized peripheral blood (PB) and bone marrow (BM) donors.

Materials And Methods: AML blasts (n = 13) and normal PB and BM cells (n = 7) were incubated for 24 hours in various concentrations of CPX-351 or free drugs before plating in CFC assay or staining with anti-CD34 and anti-CD38 antibodies, Annexin-V, and propidium iodide followed by fluorescence-activated cell sorting analysis.

Schedule- and dose-dependency of CPX-351, a synergistic fixed ratio cytarabine:daunorubicin formulation, in consolidation treatment against human leukemia xenografts.

CPX-351, a liposomal formulation co-encapsulating cytarabine (Cyt) and daunorubicin (Daun), has been developed, which delivers synergistic Cyt:Daun molar ratios to bone marrow. CPX-351 has demonstrated markedly superior anti-leukemic activity over free Cyt:Daun drug cocktails in preclinical models. Given the prolonged plasma lifetime of CPX-351, we examined the relationship between therapeutic efficacy and the frequency of treatment in the consolidation setting using a bone marrow-engrafting human leukemia xenograft model.

Leukemia-selective uptake and cytotoxicity of CPX-351, a synergistic fixed-ratio cytarabine:daunorubicin formulation, in bone marrow xenografts.

The objective of this study was to examine the pharmacodynamic basis for the potent preclinical and clinical anti-leukemic activity of CPX-351, a nano-scale liposome formulation of cytarabine and daunorubicin co-encapsulated at a synergistic 5:1 molar ratio. A bone marrow-engrafting CCRF-CEM leukemia model in Rag2-M mice was utilized to correlate the therapeutic and myelosuppressive properties of CPX-351 with bone marrow delivery and drug uptake in leukemia cells relative to normal bone marrow cell populations. When administered to mice bearing CCRF-CEM human leukemia xenografts, CPX-351 ablated bone marrow (BM) leukemic cells to below detectable levels for multiple weeks, whereas the free-drug cocktail only transiently suppressed leukemia growth.

Drug ratio-dependent antagonism: a new category of multidrug resistance and strategies for its circumvention.

A newly identified form of multidrug resistance (MDR) in tumor cells is presented, pertaining to the commonly encountered resistance of cancer cells to anticancer drug combinations at discrete drug:drug ratios. In vitro studies have revealed that whether anticancer drug combinations interact synergistically or antagonistically can depend on the ratio of the combined agents. Failure to control drug ratios in vivo due to uncoordinated pharmacokinetics could therefore lead to drug resistance if tumor cells are exposed to antagonistic drug ratios.

Drug ratio-dependent antitumor activity of irinotecan and cisplatin combinations in vitro and in vivo.

Irinotecan and cisplatin are two established anticancer drugs, which together constitute an effective combination for treating small-cell lung cancer. We investigated whether the efficacy of this combination could be improved by controlling drug ratios following in vivo administration. Irinotecan and cisplatin combinations were evaluated systematically for drug ratio-dependent synergy in vitro using a panel of 20 tumor cell lines.

Modulating the therapeutic activity of nanoparticle delivered paclitaxel by manipulating the hydrophobicity of prodrug conjugates.

A series of paclitaxel prodrugs designed for formulation in lipophilic nanoparticles are described. The hydrophobicity of paclitaxel was increased by conjugating a succession of increasingly hydrophobic lipid anchors to the drug using succinate or diglycolate cross-linkers. The prodrugs were formulated in well defined block copolymer-stabilized nanoparticles.

Increased preclinical efficacy of irinotecan and floxuridine coencapsulated inside liposomes is associated with tumor delivery of synergistic drug ratios.

Whether anticancer drug combinations act synergistically or antagonistically often depends on the ratio of the agents being combined. We show here that combinations of irinotecan and floxuridine exhibit drug ratio-dependent cytotoxicity in a broad panel of tumor cell lines in vitro where a 1:1 molar ratio consistently provided synergy and avoided antagonism. In vivo delivery of irinotecan and floxuridine coencapsulated inside liposomes at the synergistic 1:1 molar ratio (referred to as CPX-1) lead to greatly enhanced efficacy compared to the two drugs administered as a saline-based cocktail in a number of human xenograft and murine tumor models.

Ratiometric dosing of anticancer drug combinations: controlling drug ratios after systemic administration regulates therapeutic activity in tumor-bearing mice.

Anticancer drug combinations can act synergistically or antagonistically against tumor cells in vitro depending on the ratios of the individual agents comprising the combination. The importance of drug ratios in vivo, however, has heretofore not been investigated, and combination chemotherapy treatment regimens continue to be developed based on the maximum tolerated dose of the individual agents. We systematically examined three different drug combinations representing a range of anticancer drug classes with distinct molecular mechanisms (irinotecan/floxuridine, cytarabine/daunorubicin, and cisplatin/daunorubicin) for drug ratio-dependent synergy.

Immunogenicity and rapid blood clearance of liposomes containing polyethylene glycol-lipid conjugates and nucleic Acid.

Polyethylene glycol (PEG) is used widely in the pharmaceutical industry to improve the pharmacokinetics and reduce the immunogenicity of therapeutic and diagnostic agents. The incorporation of lipid-conjugated PEG into liposomal drug delivery systems greatly enhances the circulation times of liposomes by providing a protective, steric barrier against interactions with plasma proteins and cells. Here we report that liposome compositions containing PEG-lipid derivatives and encapsulated antisense oligodeoxynucleotide (ODN) or plasmid DNA elicit a strong immune response that results in the rapid blood clearance of subsequent doses in mice.

In vitro and in vivo characterization of doxorubicin and vincristine coencapsulated within liposomes through use of transition metal ion complexation and pH gradient loading.

Purpose: There is an opportunity to augment the therapeutic potential of drug combinations through use of drug delivery technology. This report summarizes data obtained using a novel liposomal formulation with coencapsulated doxorubicin and vincristine. The rationale for selecting these drugs is due in part to the fact that liposomal formulations of doxorubicin and vincristine are being separately evaluated as components of drug combinations.

Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures.

Typical methods used for encapsulating antisense oligodeoxynucleotides (ODN) and plasmid DNA in lipid vesicles result in very low encapsulation efficiencies or employ cationic lipids that exhibit unfavorable pharmacokinetic and toxicity characteristics when administered intravenously. In this study, we describe and characterize a novel formulation process that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities (0.15--0.

Clearance properties of liposomes involving conjugated proteins for targeting.

This review examines methods of protein conjugation onto liposomes and the effects of surface bound protein on the liposomes' biological behavior. It is evident that the presence of a conjugated protein significantly alters the attributes of targeted liposomes. Specifically, protein conjugation can result in dramatic increases in liposome size, enhanced immunogenicity, and increased plasma elimination.

Antibody conjugation methods for active targeting of liposomes.

Liposomes are useful drug delivery vehicles since they may protect encapsulated drugs from enzymatic degradation and rapid clearance in vivo, or alter biodistribution, potentially leading to reduced toxicities (1,2). A major limitation to the development of many specialized applications is the problem of directing liposomes to tissues where they would not normally accumulate. Consequently, a great deal of effort has been made over the years to develop liposomes that have targeting vectors attached to the bilayer surface.

Application of oligo-(14-amino-3,6,9,12-tetraoxatetradecanoic acid) lipid conjugates as steric barrier molecules in liposomal formulations.

Lipid conjugates of oligo-(14-amino-3,6,9,12-tetraoxatetradecanoic acid) (ATTAn) were synthesized as monodisperse analogues of poly(ethylene glycol) (PEG) derivatives used in liposomal drug delivery systems. The new lipids were shown to be at least equivalent to MePEGA-2000-DSPE in assays designed to evaluate the effectiveness of polymers as steric barrier molecules in liposomes. Liposomes containing 1-5% of ATTA8-DSPE (octamer) showed comparable long circulation behavior relative to PEG-2000-DSPE analogues.

An immune response to ovalbumin covalently coupled to liposomes is prevented when the liposomes used contain doxorubicin.

It is now well established that liposomes with surface associated proteins are immunogenic. Repeated administration of protein coated liposomes elicits the generation of antibodies and the elimination of proteoliposome increases markedly in animals 'immunized' with such liposomes. This immune response compromises the therapeutic potential of liposomal formulations that rely on the use of protein- or peptide-based targeting ligands to enhance cell specificity.