Subject(s)
Patents as Topic/legislation & jurisprudence , Precision Medicine , DNA , Humans , United StatesABSTRACT
We previously reported that interferon (IFN)-free direct-acting antiviral combination treatment succeeded in eradicating genotype 1b hepatitis C virus (HCV) in human hepatocyte chimeric mice. In this study, we examined the effect of vaniprevir (MK7009, NS3/4A protease inhibitor) and BMS-788329 (NS5A inhibitor) combination treatment on HCV genotype 1b and the expression of IFN-stimulated genes (ISGs) using a subgenomic replicon system and the same animal model. Combination treatment with vaniprevir and BMS-788329 significantly reduced HCV replication compared to vaniprevir monotherapy in HCV replicon cells (Huh7/Rep-Feo cells). HCV genotype 1b-infected human hepatocyte chimeric mice were treated with vaniprevir alone or in combination with BMS-788329 for four weeks. Vaniprevir monotherapy reduced serum HCV RNA titers in mice, but viral breakthrough was observed in mice with high HCV titers. Ultra-deep sequence analysis revealed a predominant replacement by drug-resistant substitutions at 168 in HCV NS3 region in these mice. Conversely, in mice with low HCV titers, HCV was eradicated by vaniprevir monotherapy without viral breakthrough. In contrast to monotherapy, combination treatment with vaniprevir and BMS-788329 succeeded in completely eradicating HCV regardless of serum viral titer. IFN-alpha treatment significantly increased ISG expression; however, vaniprevir and BMS-788329 combination treatment caused no increase in ISG expression both in cultured cells and in mouse livers. Therefore, combination treatment with vaniprevir and BMS-788329 eliminated HCV via a non-ISG-mediated mechanism. This oral treatment might offer an alternative DAA combination therapy for patients with chronic hepatitis C.
Subject(s)
Antiviral Agents/administration & dosage , Hepatitis C/drug therapy , Indoles/administration & dosage , Animals , Cell Line , Cyclopropanes , Disease Models, Animal , Drug Resistance, Viral , Drug Therapy, Combination/methods , Gene Expression Profiling , Hepacivirus/drug effects , Hepacivirus/physiology , Hepatocytes/virology , High-Throughput Nucleotide Sequencing , Humans , Isoindoles , Lactams, Macrocyclic , Leucine/analogs & derivatives , Mice , Mice, Transgenic , Mutation, Missense , Proline/analogs & derivatives , RNA, Viral/genetics , Sulfonamides , Treatment Outcome , Virus Replication/drug effectsABSTRACT
Neonatal candidiasis is an increasingly common occurrence causing significant morbidity and mortality and a higher risk of dissemination to the central nervous system (CNS) than that seen with older patients. The current understanding of optimal antifungal therapy in this setting is limited. We have developed a model of disseminated candidiasis with CNS involvement in juvenile mice to assess the efficacy of the echinocandin caspofungin relative to amphotericin B (AmB). Juvenile mice were inoculated intravenously with 5.64 × 10(4) CFU of Candida albicans MY1055. Treatment with caspofungin at 1, 2, 4, and 8 mg/kg of body weight/day, AmB at 1 mg/kg/day, or a vehicle control (VC) was initiated 30 h after infection and continued for 7 days. Pharmacokinetic parameters for caspofungin were also determined. Culture and histology showed evidence of disseminated candidiasis with multifocal encephalitis at the start of antifungal therapy. Survival was 100% in all treated groups, while mortality was 100% in the VC by day 11 after infection. By day 5, all mice in the caspofungin treatment (four doses) groups showed reductions in kidney and brain burden relative to the VC, while AmB treatment reduced kidney burden but gave no reduction of brain fungal burden. Systemic levels of caspofungin were similar in infected and uninfected mice, while brain levels were higher in infected animals. In this juvenile mouse model, caspofungin demonstrated dose-dependent activity, equivalent to or better than that of AmB at 1 mg/kg, against disseminated candidiasis with CNS involvement.
Subject(s)
Antifungal Agents/therapeutic use , Candidiasis/drug therapy , Central Nervous System Fungal Infections/drug therapy , Echinocandins/therapeutic use , Animals , Antifungal Agents/pharmacokinetics , Brain/drug effects , Brain/microbiology , Caspofungin , Echinocandins/pharmacokinetics , Kidney/drug effects , Kidney/microbiology , Lipopeptides , MiceABSTRACT
Brain penetration of drugs which are subject to P-glycoprotein (Pgp)-mediated efflux is attenuated, as manifested by the fact that the cerebrospinal fluid concentration (C(CSF)), a good surrogate of the unbound brain concentration (C(ub)), is lower than the unbound plasma concentration (C(up)) for Pgp substrates. In rodents, the attenuation magnitude of brain penetration by Pgp-mediated efflux has been estimated by correlating the ratio of CSF to plasma exposures (C(CSF)/C(p)) with the unbound fraction in plasma (f(u)) upon the incorporation of the in vivo or in vitro Pgp-mediated efflux ratios (ERs). In the present work, we investigated the impact of Pgp-mediated efflux on C(CSF) in monkeys. Following intravenous administration to cisterna magna ported rhesus monkeys, the CSF and plasma concentrations were determined for 25 compounds from three discovery programs. We also evaluated their f(u) in rhesus plasma and ER in human and African green monkey MDR-transfected LLC-PK1 cells. These compounds varied significantly in the f(u) (0.025-0.73), and 24 out of 25 are considered Pgp substrates based on their appreciable directional transport (ER>2). The C(CSF)/C(p) was significantly lower than the corresponding f(u) (>or=3-fold) for 16 compounds regardless of a significant correlation (R(2)=0.59, p=4 x 10(-5)) when the C(CSF)/C(p) was plotted against the f(u). When the f(u) was normalized to the ER (f(u)/ER) the correlation was improved (R(2)=0.75, p=8 x 10(-8)). More importantly, only one compound showed the C(CSF)/C(p) that exceeded 3-fold of the normalized f(u). The results suggest that the impact of Pgp-mediated efflux in monkeys, similar to the case in rodents, is reasonably reflected by the gradient between the free concentrations in plasma and in CSF. Therefore, f(u) and Pgp ER may serve as useful measurements in estimating in vivo C(CSF)/C(p) ratios in monkeys, and potentially in humans.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Biological Transport/drug effects , Blood-Brain Barrier/physiology , Brain/drug effects , Plasma/drug effects , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Biological Transport/physiology , Blood Proteins/metabolism , Brain/metabolism , Cells, Cultured , Humans , Macaca mulatta , Male , Molecular Weight , Organic Chemicals/chemical synthesis , Organic Chemicals/metabolism , Plasma/chemistry , TransfectionABSTRACT
beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Protein Precursor/cerebrospinal fluid , Amyloid beta-Protein Precursor/antagonists & inhibitors , Animals , Aspartic Acid Endopeptidases/antagonists & inhibitors , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacology , Humans , Infusions, Intravenous , Macaca mulatta , Mice , Mice, Transgenic , TransfectionABSTRACT
HIV-1 integrase catalyzes the insertion of viral DNA into the genome of the host cell. Integrase inhibitor N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide selectively inhibits the strand transfer process of integration. 4-Substituted pyrrolidinones possessing various groups on the pyrrolidinone nitrogen were introduced at the 5-position of the naphthyridine scaffold. These analogs exhibit excellent activity against viral replication in a cell-based assay. The preparation of these compounds was enabled by a three-step, two-pot reaction sequence from a common butenolide intermediate.
Subject(s)
Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , HIV Integrase Inhibitors/chemical synthesis , HIV Integrase Inhibitors/pharmacology , HIV Integrase/metabolism , HIV-1/drug effects , Naphthyridines/chemical synthesis , Naphthyridines/pharmacology , Administration, Oral , Animals , Anti-HIV Agents/chemistry , HIV Integrase Inhibitors/chemistry , Molecular Structure , Naphthyridines/chemistry , Rats , Structure-Activity RelationshipABSTRACT
beta-Secretase (BACE) cleavage of amyloid precursor protein (APP) is one of the first steps in the production of amyloid beta peptide Abeta42, the putative neurotoxic species in Alzheimer's disease. Recent studies have shown that BACE1 knockdown leads to hypomyelination, putatively caused by a decline in neuregulin (NRG)-1 processing. In this study, we have tested a potent cell-permeable BACE1 inhibitor (IC(50) approximately 30 nM) by administering it directly into the lateral ventricles of mice, expressing human wild-type (WT)-APP, to determine the consequences of BACE1 inhibition on brain APP and NRG-1 processing. BACE1 inhibition, in vivo, led to a significant dose- and time-dependent lowering of brain Abeta40 and Abeta42. BACE1 inhibition also led to a robust brain secreted (s)APPbeta lowering that was accompanied by an increase in brain sAPPalpha levels. Although an increase in full-length NRG-1 levels was evident in 15-day-old BACE1 homozygous knockout (KO) (-/-) mice, in agreement with previous studies, this effect was also observed in 15-day-old heterozygous (+/-) mice, but it was not evident in 30-day-old and 2-year-old BACE1 KO (-/-) mice. Thus, BACE1 knockdown led to a transient decrease in NRG-1 processing in mice. Pharmacological inhibition of BACE1 in adult mice, which led to significant Abeta lowering, was without any significant effect on brain NRG-1 processing. Taken together, these results suggest that BACE1 is the major beta-site cleavage enzyme for APP and that its inhibition can lower brain Abeta and redirect APP processing via the potentially nonamyloidogenic alpha-secretase pathway, without significantly altering NRG-1 processing.
Subject(s)
Amyloid Precursor Protein Secretases/biosynthesis , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Protein Precursor/biosynthesis , Aspartic Acid Endopeptidases/antagonists & inhibitors , Brain/metabolism , Neuregulin-1/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Amyloid Precursor Protein Secretases/physiology , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Brain/drug effects , Down-Regulation/drug effects , Down-Regulation/physiology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neuregulin-1/genetics , Protease Inhibitors/pharmacology , Up-Regulation/drug effects , Up-Regulation/physiologyABSTRACT
A series of potent novel dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 6 is active against replication of HIV with a CIC(95) of 0.31 microM and exhibits no shift in potency in the presence of 50% normal human serum. It displays a good pharmacokinetic profile when dosed in rats and no covalent binding with microsomal proteins in both in vitro and in vivo models.
Subject(s)
HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacology , Pyrazines/chemistry , Pyrazines/pharmacology , Animals , Benzene/chemistry , Cell Line , HIV/drug effects , HIV/enzymology , HIV/physiology , HIV Integrase Inhibitors/chemical synthesis , HIV Integrase Inhibitors/pharmacokinetics , Humans , Microsomes, Liver/drug effects , Models, Molecular , Molecular Structure , Pyrazines/chemical synthesis , Pyrazines/pharmacokinetics , Rats , Structure-Activity Relationship , Virus Replication/drug effectsABSTRACT
beta-Secretase inhibition offers an exciting opportunity for therapeutic intervention in the progression of Alzheimer's disease. A series of isonicotinamides derived from traditional aspartyl protease transition state isostere inhibitors has been optimized to yield low nanomolar inhibitors with sufficient penetration across the blood-brain barrier to demonstrate beta-amyloid lowering in a murine model.
Subject(s)
Amides/chemical synthesis , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Isonicotinic Acids/chemical synthesis , Peptide Fragments/metabolism , Amides/chemistry , Amides/pharmacology , Animals , Biological Availability , Brain/metabolism , Dose-Response Relationship, Drug , Isonicotinic Acids/pharmacokinetics , Isonicotinic Acids/pharmacology , Mice , Rats , Structure-Activity RelationshipABSTRACT
A 1,6-naphthyridine inhibitor of HIV-1 integrase has been discovered with excellent inhibitory activity in cells, good pharmacokinetics, and an excellent ability to inhibit virus with mutant enzyme.
Subject(s)
HIV Integrase Inhibitors/chemical synthesis , HIV Integrase Inhibitors/pharmacokinetics , Naphthyridines/chemical synthesis , Administration, Oral , Animals , Cells, Cultured , HIV Integrase/drug effects , HIV Integrase/genetics , HIV Integrase Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Mutation , Naphthyridines/pharmacokinetics , Naphthyridines/pharmacology , Rats , Structure-Activity RelationshipABSTRACT
A macrocyclic inhibitor of beta-secretase was designed by covalently cross-linking the P1 and P3 side chains of an isophthalamide-based inhibitor. Macrocyclization resulted in significantly improved potency and physical properties when compared to the initial lead structures. More importantly, these macrocyclic inhibitors also displayed in vivo amyloid lowering when dosed in a murine model.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Macrocyclic Compounds/chemical synthesis , Protease Inhibitors/chemical synthesis , Amides/chemistry , Amyloid beta-Peptides/metabolism , Animals , Blood-Brain Barrier , Brain/metabolism , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacokinetics , Mice , Molecular Conformation , Phthalic Acids/chemistry , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacokinetics , Stereoisomerism , Structure-Activity Relationship , Tissue DistributionABSTRACT
Introduction of a 5,6-dihydrouracil functionality in the 5-position of N-(4-fluorobenzyl)-8-hydroxy-[1,6]naphthyridine-7-carboxamide 1 led to a series of highly active HIV-1 integrase inhibitors. These compounds displayed low nanomolar activity in inhibiting both the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 11 is a 150-fold more potent antiviral agent than 1, with a CIC(95) of 40 nM in the presence of human serum. It displays good pharmacokinetics when dosed in rats and dogs.
Subject(s)
Benzyl Compounds/pharmacology , HIV Integrase Inhibitors/pharmacology , HIV-1/drug effects , Naphthyridines/pharmacology , Uracil/analogs & derivatives , Virus Replication/drug effects , Animals , Benzyl Compounds/chemistry , Benzyl Compounds/pharmacokinetics , Biological Availability , Crystallography, X-Ray , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacokinetics , HIV-1/physiology , Naphthyridines/chemistry , Naphthyridines/pharmacokinetics , Rats , Uracil/chemistryABSTRACT
The increasing incidence of resistance to current HIV-1 therapy underscores the need to develop antiretroviral agents with new mechanisms of action. Integrase, one of three viral enzymes essential for HIV-1 replication, presents an important yet unexploited opportunity for drug development. We describe here the identification and characterization of L-870,810, a small-molecule inhibitor of HIV-1 integrase with potent antiviral activity in cell culture and good pharmacokinetic properties. L-870,810 is an inhibitor with an 8-hydroxy-(1,6)-naphthyridine-7-carboxamide pharmacophore. The compound inhibits HIV-1 integrase-mediated strand transfer, and its antiviral activity in vitro is a direct consequence of this ascribed effect on integration. L-870,810 is mechanistically identical to previously described inhibitors from the diketo acid series; however, viruses selected for resistance to L-870,810 contain mutations (integrase residues 72, 121, and 125) that uniquely confer resistance to the naphthyridine. Conversely, mutations associated with resistance to the diketo acid do not engender naphthyridine resistance. Importantly, the mutations associated with resistance to each of these inhibitors map to distinct regions within the integrase active site. Therefore, we propose a model of the two inhibitors that is consistent with this observation and suggests specific interactions with discrete binding sites for each ligand. These studies provide a structural basis and rationale for developing integrase inhibitors with the potential for unique and nonoverlapping resistance profiles.
Subject(s)
HIV Infections/drug therapy , HIV Integrase Inhibitors/pharmacology , HIV-1/drug effects , Naphthyridines/pharmacology , Animals , Cells, Cultured , Dogs , Drug Resistance, Multiple , Drug Resistance, Viral , HIV Integrase/genetics , HIV Integrase/metabolism , HIV Integrase Inhibitors/chemistry , HIV-1/enzymology , HIV-1/genetics , HIV-2/drug effects , Humans , Macaca mulatta , Male , Mutagenesis, Site-Directed , Naphthyridines/chemistry , Rats , Simian Immunodeficiency Virus/drug effects , T-Lymphocytes/cytology , T-Lymphocytes/virology , Virus Integration/drug effectsABSTRACT
The burgeoning problem of overweight and obesity in US children, adolescents, and adults has reached epidemic proportions. Data from the CRRIC I study conducted in 2000 in Jackson, Mississippi among elementary African-American children indicated that 39% of the boys and 49% of the girls had BMIs at or above the 85th percentile. The current study replicated CRRIC I in a sample of 113 African American Children in the third and fourth grades in the rural town of Canton, Mississippi. Results revealed that 46% of the boys and 46% of the girls had BMI at or above the 85th percentile. Fitness profile results revealed that only 11% of the children were classified as fit, 84% were classified as fair, and 5% were deemed unfit. As in CRRIC I, health histories of grandparents were positive for the comorbities of overweight and obesity: diabetes, stroke, and heart disease. These data indicate a critical need to aggressively intervene to impact the lifestyle choices of this generation.
Subject(s)
Black or African American/statistics & numerical data , Cardiovascular Diseases/etiology , Child Welfare , Obesity/ethnology , Adolescent , Anthropometry , Body Mass Index , Child , Exercise , Female , Health Surveys , Humans , Life Style , Male , Mississippi/epidemiology , Needs Assessment , Obesity/classification , Obesity/complications , Obesity/prevention & control , Physical Fitness , Population Surveillance , Risk Factors , Risk Reduction BehaviorABSTRACT
Antagonism of the bradykinin B(1) receptor was demonstrated to be a potential treatment for chronic pain and inflammation. Novel benzodiazepines were designed that display subnanomolar affinity for the bradykinin B(1) receptor (K(i) = 0.59 nM) and high selectivity against the bradykinin B(2) receptor (K(i) > 10 microM). In vivo efficacy, comparable to morphine, was demonstrated for lead compounds in a rodent hyperalgesia model.