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1.
Toxicol Appl Pharmacol ; 409: 115285, 2020 12 15.
Article in English | MEDLINE | ID: mdl-33069749

ABSTRACT

The OX40 receptor plays a crucial co-stimulatory role in T effector cell survival, expansion, cytokine production, and cytotoxicity to tumor cells; therefore, OX40 agonists are being evaluated as anti-cancer immunotherapies, especially in combination with checkpoint inhibitors. To support clinical development of BMS-986178 (an OX40 agonist antibody), two repeat-dose toxicity studies were conducted in cynomolgus monkeys. In the first study, BMS-986178 was administered intravenously (IV) once weekly for one month at doses from 30 to 120 mg/kg. BMS-986178 was well tolerated; surprisingly, immune function was suppressed rather than increased based on pharmacodynamic (PD) and flow cytometry readouts (e.g. T-cell dependent antibody response [TDAR]). To determine whether immune suppression was due to a bi-phasic response, a follow-up study was conducted at lower doses (1 and 10 mg/kg). Although receptor engagement was confirmed, immune function was still suppressed at both doses. In addition, treatment-emergent anti-drug antibodies (ADAs) at 1 mg/kg resulted in hypersensitivity reactions and reduced BMS-986178 exposure after repeated dosing, which precluded a full PD assessment at this dose. In conclusion, BMS-986178 was clinically well-tolerated by monkeys at weekly IV doses from 10 to 120 mg/kg (AUC[0-168] ≤ 712,000 µg●h/mL). However, despite target engagement, PD assays and other immune endpoints demonstrated immune suppression, not stimulation. Due to the inverted immune response at higher doses and the onset of ADAs, additional repeat-dose toxicity studies of BMS-986178 in monkeys (that would typically be required to support Phase 3 clinical trials and registration) would not add value for human safety assessment.


Subject(s)
Antibodies, Monoclonal/immunology , Immunity/immunology , Receptors, OX40/immunology , T-Lymphocytes/immunology , Animals , Female , Follow-Up Studies , Humans , Immunotherapy/methods , Macaca fascicularis , Male
2.
Antimicrob Agents Chemother ; 57(7): 3250-61, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23629699

ABSTRACT

While new direct-acting antiviral agents for the treatment of chronic hepatitis C virus (HCV) infection have been approved, there is a continued need for novel antiviral agents that act on new targets and can be used in combination with current therapies to enhance efficacy and to restrict the emergence of drug-resistant viral variants. To this end, we have identified a novel class of small molecules, exemplified by PTC725, that target the nonstructural protein 4B (NS4B). PTC725 inhibited HCV 1b (Con1) replicons with a 50% effective concentration (EC50) of 1.7 nM and an EC90 of 9.6 nM and demonstrated a >1,000-fold selectivity window with respect to cytotoxicity. The compounds were fully active against HCV replicon mutants that are resistant to inhibitors of NS3 protease and NS5B polymerase. Replicons selected for resistance to PTC725 harbored amino acid substitutions F98L/C and V105M in NS4B. Anti-replicon activity of PTC725 was additive to synergistic in combination with alpha interferon or with inhibitors of HCV protease and polymerase. Immunofluorescence microscopy demonstrated that neither the HCV inhibitors nor the F98C substitution altered the subcellular localization of NS4B or NS5A in replicon cells. Oral dosing of PTC725 showed a favorable pharmacokinetic profile with high liver and plasma exposure in mice and rats. Modeling of dosing regimens in humans indicates that a once-per-day or twice-per-day oral dosing regimen is feasible. Overall, the preclinical data support the development of PTC725 for use in the treatment of chronic HCV infection.


Subject(s)
Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Hepacivirus/drug effects , Hepatitis C/drug therapy , Indoles/pharmacology , Sulfonamides/pharmacology , Viral Nonstructural Proteins/metabolism , Amino Acid Substitution , Animals , Antiviral Agents/pharmacokinetics , Cell Line, Tumor , Drug Resistance, Viral/genetics , Drug Synergism , Humans , Indoles/metabolism , Indoles/pharmacokinetics , Interferon-alpha/pharmacology , Male , Mice , Microbial Sensitivity Tests , Rats , Rats, Sprague-Dawley , Sulfonamides/metabolism , Sulfonamides/pharmacokinetics , Viral Nonstructural Proteins/genetics , Virus Replication/drug effects
3.
Bioorg Med Chem Lett ; 17(20): 5543-7, 2007 Oct 15.
Article in English | MEDLINE | ID: mdl-17804230

ABSTRACT

Bioassay-guided fractionation of an active fraction from an extract of a marine starfish, Novodinia antillensis, led to the isolation and identification of two new saponins, Sch 725737 (1) and Sch 725739 (2). Compound 1 was identified as the NaV1.8 inhibitor with IC(50) of approximately 9 microM. The purification and the structure elucidation of these two saponins are described.


Subject(s)
Saponins/chemistry , Starfish/chemistry , Steroids/chemistry , Animals , Carbohydrate Sequence , Magnetic Resonance Spectroscopy , Membrane Potentials/drug effects , Saponins/isolation & purification , Saponins/pharmacology , Sodium Channel Blockers/chemistry , Sodium Channel Blockers/isolation & purification , Sodium Channel Blockers/pharmacology , Sodium Channels/metabolism , Steroids/isolation & purification , Steroids/pharmacology
4.
Mol Pharmacol ; 71(1): 19-29, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17005902

ABSTRACT

Ezetimibe is the first in class 2-azetidinone that decreases plasma cholesterol by blocking intestinal cholesterol absorption. Ezetimibe effectively reduces plasma cholesterol in several species including human, monkey, dog, hamster, rat, and mouse, but the potency ranges widely. One potential factor responsible for this variation in responsiveness is diversity in ezetimibe metabolism. After oral administration, ezetimibe is glucuronidated. Both ezetimibe and the glucuronide lower plasma cholesterol; however, the glucuronide exhibits greater potency. Recent identification of Niemann-Pick C1 Like-1 (NPC1L1) as the molecular target of ezetimibe enables direct binding studies to be performed. Here, we report the cloning of NPC1L1 derived from multiple species and assess amino acid sequence homology among human, monkey, dog, hamster, rat, and mouse. The rank order of affinity of glucuronidated ezetimibe for NPC1L1 in each species correlates with the rank order of in vivo activity with monkey > dog > hamster and rat >> mouse. Ezetimibe analogs that bind to NPC1L1 exhibit in vivo cholesterol-lowering activity, whereas compounds that do not bind NPC1L1 are inactive. Specific structural components of ezetimibe are identified as critical for binding to NPC1L1. The results demonstrate that small variations in ezetimibe structure or in NPC1L1 amino acid sequence can profoundly influence ezetimibe/NPC1L1 interaction and consequently in vivo activity. The results demonstrate that the ability of compounds to bind to NPC1L1 is the major determinant of in vivo responsiveness.


Subject(s)
Azetidines/pharmacology , Azetidines/pharmacokinetics , Membrane Proteins/physiology , Amino Acid Sequence , Animals , Anticholesteremic Agents/pharmacology , Binding Sites , Cells, Cultured , Cholesterol/metabolism , Cloning, Molecular , DNA, Complementary/genetics , Ezetimibe , Humans , Intestinal Absorption , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Transport Proteins , Models, Molecular , Molecular Sequence Data , Niemann-Pick Diseases , Protein Conformation , Rats
5.
Proc Natl Acad Sci U S A ; 102(23): 8132-7, 2005 Jun 07.
Article in English | MEDLINE | ID: mdl-15928087

ABSTRACT

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.


Subject(s)
Azetidines/pharmacology , Membrane Proteins/metabolism , Membrane Transport Proteins/metabolism , Proteins/metabolism , Animals , Azetidines/chemistry , Binding Sites , Cell Line , Cell Membrane/metabolism , Enterocytes/cytology , Enterocytes/metabolism , Ezetimibe , Humans , Intestinal Mucosa/metabolism , Intestines/cytology , Macaca mulatta , Membrane Proteins/genetics , Membrane Transport Proteins/deficiency , Membrane Transport Proteins/genetics , Mice , Mice, Inbred C57BL , Microvilli/metabolism , Niemann-Pick Diseases , Protein Binding , Proteins/genetics , Rats , Rats, Sprague-Dawley , Species Specificity
6.
Biochim Biophys Acta ; 1722(3): 282-92, 2005 Apr 15.
Article in English | MEDLINE | ID: mdl-15777641

ABSTRACT

The exact mechanistic pathway of cholesterol absorption in the jejunum of the small intestines is a poorly understood process. Recently, a relatively novel gene, Niemann-Pick C1 Like 1 (NPC1L1), was identified as being critical for intestinal sterol absorption in a pathway which is sensitive to sterol absorption inhibitors such as ezetimibe. NPC1L1 is a multi-transmembrane protein, with a putative sterol sensing domain. Very little else is known about the NPC1L1 protein. In this report, we characterize the native and recombinant rat NPC1L1 protein. We show that NPC1L1 is a 145 kDa membrane protein, enriched in the brush border membrane of the intestinal enterocyte and is highly glycosylated. In addition, sequential detergent extraction of enterocytes result in highly enriched preparations of NPC1L1. An engineered Flag epitope tagged rat NPC1L1 cDNA was expressed as recombinant protein in CHO cells and demonstrated cell surface expression, similar to the native rat protein. These biochemical data indicate that NPC1L1 exists as a predominantly cell surface membrane expressed protein, consistent with its proposed role as the putative intestinal sterol transporter.


Subject(s)
Membrane Transport Proteins/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Base Sequence , DNA Primers , Membrane Transport Proteins/immunology , Molecular Sequence Data , Rats , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Subcellular Fractions/metabolism
7.
J Pharmacol Exp Ther ; 306(2): 498-504, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12730276

ABSTRACT

Vinpocetine is a clinically used synthetic vincamine derivative with a diverse pharmacological profile that includes action at several ion channels, principally "generic" populations of sodium channels that give rise to tetrodotoxin-sensitive conductances. A number of cell types are known to express tetrodotoxin-resistant (TTXr) sodium conductances, the molecular bases of which have remained elusive until recently. One such TTXr channel, termed NaV1.8, is of particular interest because of its prominent and selective expression in peripheral afferent nerves. The effects of vinpocetine on TTXr channels specifically, are unknown. We have assessed the effects of the drug on cloned rat NaV1.8 channels expressed in a dorsal root ganglion-derived cell line, ND7/23. Vinpocetine produced a concentration- and state-dependent inhibition of NaV1.8 sodium channel activity. Voltage-clamp experiments revealed an approximately 3-fold increase in vinpocetine potency when whole-cell NaV1.8 conductances were elicited from relatively depolarized potentials (-35 mV; IC50 = 3.5 microM) compared with hyperpolarized holding potentials (-90 mV; IC50 = 10.4 microM). Vinpocetine also produced an approximately 22 mV leftward shift in the voltage dependence of NaV1.8 channel inactivation but did not affect the voltage range of channel activation. These properties are reminiscent of several other known sodium channel blockers and suggested that vinpocetine may exhibit frequency-dependent block. Accordingly, tonic block of NaV1.8 channels by vinpocetine (3 microM) increased proportionally with increasing depolarizing commands over the frequency range 0.1 to 1Hz. In summary, the present data demonstrate that vinpocetine is capable of blocking NaV1.8 sodium channel activity and suggest a potential additional utility in various sensory abnormalities arising from abnormal peripheral nerve activity.


Subject(s)
Sodium Channel Blockers/pharmacology , Sodium Channels/metabolism , Vinca Alkaloids/pharmacology , Animals , Cells, Cultured , Drug Resistance , Electrophysiology , Patch-Clamp Techniques , Rats , Sodium Channels/drug effects , Sodium Channels/physiology , Tetrodotoxin/pharmacology
8.
Biochim Biophys Acta ; 1580(1): 77-93, 2002 Jan 30.
Article in English | MEDLINE | ID: mdl-11923102

ABSTRACT

The molecular mechanisms of cholesterol absorption in the intestine are poorly understood. With the goal of defining candidate genes involved in these processes a fluorescence-activated cell sorter-based, retroviral-mediated expression cloning strategy has been devised. SCH354909, a fluorescent derivative of ezetimibe, a compound which blocks intestinal cholesterol absorption but whose mechanism of action is unknown, was synthesized and shown to block intestinal cholesterol absorption in rats. Pools of cDNAs prepared from rat intestinal cells enriched in enterocytes were introduced into BW5147 cells and screened for SCH354909 binding. Several independent clones were isolated and all found to encode the scavenger receptor class B, type I (SR-BI), a protein suggested by others to play a role in cholesterol absorption. SCH354909 bound to Chinese hamster ovary (CHO) cells expressing SR-BI in specific and saturable fashion and with high affinity (K(d) approximately 18 nM). Overexpression of SR-BI in CHO cells resulted in increased cholesterol uptake that was blocked by micromolar concentrations of ezetimibe. Analysis of rat intestinal sections by in situ hybridization demonstrated that SR-BI expression was restricted to enterocytes. Cholesterol absorption was determined in SR-B1 knockout mice using both an acute, 2-h, assay and a more chronic fecal dual isotope ratio method. The level of intestinal cholesterol uptake and absorption was similar to that seen in wild-type mice. When assayed in the SR-B1 knockout mice, the dose of ezetimibe required to inhibit hepatic cholesterol accumulation induced by a cholesterol-containing 'western' diet was similar to wild-type mice. Thus, the binding of ezetimibe to cells expressing SR-B1 and the functional blockade of SR-B1-mediated cholesterol absorption in vitro suggest that SR-B1 plays a role in intestinal cholesterol metabolism and the inhibitory activity of ezetimibe. In contrast studies with SR-B1 knockout mice suggest that SR-B1 is not essential for intestinal cholesterol absorption or the activity of ezetimibe.


Subject(s)
CD36 Antigens/metabolism , Cholesterol/metabolism , Intestinal Mucosa/metabolism , Membrane Proteins , Receptors, Immunologic , Receptors, Lipoprotein , Animals , Anticholesteremic Agents/pharmacology , Azetidines/pharmacology , CD36 Antigens/biosynthesis , CD36 Antigens/genetics , CHO Cells , Cholesterol/blood , Cloning, Molecular , Cricetinae , Dose-Response Relationship, Drug , Ezetimibe , Flow Cytometry , Gene Library , In Situ Hybridization , Intestinal Absorption/drug effects , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Knockout , Microscopy, Fluorescence , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Scavenger , Scavenger Receptors, Class B
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