ABSTRACT
BACKGROUND AND OBJECTIVES: In the rare disease primary hyperoxaluria type 1, overproduction of oxalate by the liver causes kidney stones, nephrocalcinosis, kidney failure, and systemic oxalosis. Lumasiran, an RNA interference therapeutic, suppresses glycolate oxidase, reducing hepatic oxalate production. The objective of this first-in-human, randomized, placebo-controlled trial was to evaluate the safety, pharmacokinetic, and pharmacodynamic profiles of lumasiran in healthy participants and patients with primary hyperoxaluria type 1. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This phase 1/2 study was conducted in two parts. In part A, healthy adults randomized 3:1 received a single subcutaneous dose of lumasiran or placebo in ascending dose groups (0.3-6 mg/kg). In part B, patients with primary hyperoxaluria type 1 randomized 3:1 received up to three doses of lumasiran or placebo in cohorts of 1 or 3 mg/kg monthly or 3 mg/kg quarterly. Patients initially assigned to placebo crossed over to lumasiran on day 85. The primary outcome was incidence of adverse events. Secondary outcomes included pharmacokinetic and pharmacodynamic parameters, including measures of oxalate in patients with primary hyperoxaluria type 1. Data were analyzed using descriptive statistics. RESULTS: Thirty-two healthy participants and 20 adult and pediatric patients with primary hyperoxaluria type 1 were enrolled. Lumasiran had an acceptable safety profile, with no serious adverse events or study discontinuations attributed to treatment. In part A, increases in mean plasma glycolate concentration, a measure of target engagement, were observed in healthy participants. In part B, patients with primary hyperoxaluria type 1 had a mean maximal reduction from baseline of 75% across dosing cohorts in 24-hour urinary oxalate excretion. All patients achieved urinary oxalate levels ≤1.5 times the upper limit of normal. CONCLUSIONS: Lumasiran had an acceptable safety profile and reduced urinary oxalate excretion in all patients with primary hyperoxaluria type 1 to near-normal levels. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Study of Lumasiran in Healthy Adults and Patients with Primary Hyperoxaluria Type 1, NCT02706886.
Subject(s)
Hyperoxaluria, Primary/drug therapy , Oxalates/urine , RNA, Small Interfering/pharmacology , RNA, Small Interfering/pharmacokinetics , Renal Agents/pharmacology , Renal Agents/pharmacokinetics , Adolescent , Adult , Child , Female , Glycolates/blood , Humans , Hyperoxaluria, Primary/blood , Hyperoxaluria, Primary/urine , Male , RNA, Small Interfering/adverse effects , Renal Agents/adverse effects , Single-Blind Method , Young AdultABSTRACT
By sequencing autozygous human populations, we identified a healthy adult woman with lifelong complete knockout of HAO1 (expected ~1 in 30 million outbred people). HAO1 (glycolate oxidase) silencing is the mechanism of lumasiran, an investigational RNA interference therapeutic for primary hyperoxaluria type 1. Her plasma glycolate levels were 12 times, and urinary glycolate 6 times, the upper limit of normal observed in healthy reference individuals (n = 67). Plasma metabolomics and lipidomics (1871 biochemicals) revealed 18 markedly elevated biochemicals (>5 sd outliers versus n = 25 controls) suggesting additional HAO1 effects. Comparison with lumasiran preclinical and clinical trial data suggested she has <2% residual glycolate oxidase activity. Cell line p.Leu333SerfsTer4 expression showed markedly reduced HAO1 protein levels and cellular protein mis-localisation. In this woman, lifelong HAO1 knockout is safe and without clinical phenotype, de-risking a therapeutic approach and informing therapeutic mechanisms. Unlocking evidence from the diversity of human genetic variation can facilitate drug development.
Subject(s)
Alcohol Oxidoreductases/genetics , Hyperoxaluria, Primary/therapy , RNAi Therapeutics , Adult , Alcohol Oxidoreductases/antagonists & inhibitors , Animals , CHO Cells , Cricetulus , Female , Glycolates/metabolism , Humans , Hyperoxaluria, Primary/metabolismABSTRACT
Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin and leptin receptor pathways and thus an attractive therapeutic target for diabetes and obesity. Starting with a high micromolar lead compound, structure-based optimization of novel PTP1B inhibitors by extension of the molecule from the enzyme active site into the second phosphotyrosine binding site is described. Medicinal chemistry, guided by X-ray complex structure and molecular modeling, has yielded low nanomolar PTP1B inhibitors in an efficient manner. Compounds from this chemical series were found to be actively transported into hepatocytes. This active uptake into target tissues could be one of the possible avenues to overcome the poor membrane permeability of PTP1B inhibitors.
Subject(s)
Models, Molecular , Phosphotyrosine/metabolism , Protein Tyrosine Phosphatases/antagonists & inhibitors , Thiophenes/chemical synthesis , Animals , Binding Sites , Caco-2 Cells , Catalytic Domain , Cell Membrane Permeability , Crystallography, X-Ray , Half-Life , Hepatocytes , Humans , In Vitro Techniques , Male , Mice , Mice, Inbred C57BL , Microsomes, Liver/metabolism , Molecular Structure , Phosphotyrosine/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/chemistry , Rats , Rats, Sprague-Dawley , Solubility , Structure-Activity Relationship , Thiophenes/pharmacokinetics , Thiophenes/pharmacology , Tissue DistributionABSTRACT
OBJECTIVE AND DESIGN: Elevated blood pressure and insulin resistance are strongly associated in patients. We explored the potential for the anti-hypertensive angiotensin II type 1-receptor (ATR(1)) antagonists to improve insulin sensitivity through modulation of the nuclear receptor PPARgamma, in vitro and in vivo compared to the potent insulin sensitizer, rosiglitazone. METHODS: PPARgamma modulation by ATR(1) antagonists was measured first by direct recruitment of PGC-1, followed by trans-activation reporter assays in cells, and promotion of adipogenesis in fibroblast and pre-adipocyte cell lines. Improvement of insulin sensitivity was measured as changes in levels of glucose, insulin, and adiponectin in ob/ob mice. RESULTS: Telmisartan, candesartan, irbesartan, and losartan (but not valsartan or olmesartan) each served as bona fide PPARgamma ligands in vitro, with EC(50) values between 3 and 5 micro mol/l. However, only telmisartan, and to a lesser extent candesartan, resulted in significant PPARgamma agonism in cells. In vivo, although rosiglitazone significantly lowered both glucose (33%, p<0.01) and insulin (61%, p<0.01) levels and increased expression of adiponectin (74%, p<0.001), sartan treatment had no effect. CONCLUSIONS: Many members of the sartan family of ATR(1) antagonists are PPARgamma ligands in cell-free assays but their modulation of PPARgamma in cells is relatively weak. Furthermore, none appear to improve insulin sensitivity in a rodent model under conditions where other insulin sensitizers, including rosiglitazone, do. These results question whether reported effects of sartans on insulin sensitivity may be through other means, and should guide further efforts to develop dual agents to treat hypertension and insulin resistance.
Subject(s)
Angiotensin II Type 1 Receptor Blockers/pharmacology , Hypoglycemic Agents/pharmacology , Insulin Resistance , PPAR gamma/agonists , 3T3-L1 Cells , Adipogenesis/drug effects , Adiponectin/blood , Angiotensin II Type 1 Receptor Blockers/chemistry , Animals , Blood Glucose/drug effects , Chlorocebus aethiops , Dose-Response Relationship, Drug , Hypoglycemic Agents/chemistry , Insulin/blood , Male , Mice , Mice, Obese , Obesity/blood , Obesity/metabolism , PPAR gamma/genetics , PPAR gamma/metabolism , Promoter Regions, Genetic/drug effects , Recombinant Proteins/agonists , Rosiglitazone , Structure-Activity Relationship , Thiazolidinediones/pharmacology , Transcription Factors/metabolism , Transcription, Genetic/drug effects , TransfectionABSTRACT
Antagonists of the B7 family of co-stimulatory molecules have the potential for altering immune responses therapeutically. To better define the requirements for such inhibitors, we have mapped the binding of an entire panel of blocking antibodies specific for human B7.1. By mutagenesis, each of the residues critical for blocking antibody binding appeared to fall entirely within the N-terminal V-set domain of B7.1. Thus, although antibody-antigen interacting surfaces can be quite large, these results indicate that a relatively small portion of the GFCC'C" face of this domain is crucial for further antagonist development.
Subject(s)
B7-1 Antigen/immunology , Amino Acid Sequence , Animals , Antibodies, Blocking/genetics , Antibodies, Blocking/immunology , B7-1 Antigen/genetics , COS Cells , Epitope Mapping , Epitopes, B-Lymphocyte , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/immunology , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunologyABSTRACT
The long circulating half-life of serum albumin, the most abundant protein in mammalian plasma, derives from pH-dependent endosomal salvage from degradation, mediated by the neonatal Fc receptor (FcRn). Using yeast display, we identified human serum albumin (HSA) variants with increased affinity for human FcRn at endosomal pH, enabling us to solve the crystal structure of a variant HSA/FcRn complex. We find an extensive, primarily hydrophobic interface stabilized by hydrogen-bonding networks involving protonated histidines internal to each protein. The interface features two key FcRn tryptophan side chains inserting into deep hydrophobic pockets on HSA that overlap albumin ligand binding sites. We find that fatty acids (FAs) compete with FcRn, revealing a clash between ligand binding and recycling, and that our high-affinity HSA variants have significantly increased circulating half-lives in mice and monkeys. These observations open the way for the creation of biotherapeutics with significantly improved pharmacokinetics.
Subject(s)
Histocompatibility Antigens Class I/chemistry , Receptors, Fc/chemistry , Serum Albumin/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding, Competitive , Female , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Kinetics , Ligands , Macaca fascicularis , Male , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Mimicry , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Rats , Sequence Homology , Serum Albumin/genetics , beta 2-Microglobulin/chemistryABSTRACT
Effective therapies are needed to control excessive bleeding in a range of clinical conditions. We improve hemostasis in vivo using a conformationally pliant variant of coagulation factor Xa (FXa(I16L)) rendered partially inactive by a defect in the transition from zymogen to active protease. Using mouse models of hemophilia, we show that FXa(I16L) has a longer half-life than wild-type FXa and does not cause excessive activation of coagulation. Once clotting mechanisms are activated to produce its cofactor FVa, FXa(I16L) is driven to the protease state and restores hemostasis in hemophilic animals upon vascular injury. Moreover, using human or murine analogs, we show that FXa(I16L) is more efficacious than FVIIa, which is used to treat bleeding in hemophilia inhibitor patients. FXa(I16L) may provide an effective strategy to enhance blood clot formation and act as a rapid pan-hemostatic agent for the treatment of bleeding conditions.
Subject(s)
Enzyme Precursors/therapeutic use , Factor Xa/therapeutic use , Hemophilia A/drug therapy , Hemostatics/therapeutic use , Animals , Blood Coagulation/genetics , Disease Models, Animal , Enzyme Precursors/pharmacokinetics , Factor VIIa/genetics , Factor VIIa/metabolism , Factor Xa/pharmacokinetics , Gene Expression , HEK293 Cells , Hemorrhage/drug therapy , Hemostasis/genetics , Hemostatics/pharmacokinetics , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Recombinant Proteins/genetics , Recombinant Proteins/therapeutic use , Thrombelastography , Thrombin/metabolismABSTRACT
The prevalence of obesity in the USA and worldwide has reached epidemic proportions during the last two decades. Drugs currently available for the treatment of obesity provide no more than 5% placebo-adjusted weight loss and are associated with undesirable side effects. Peroxisome proliferator-activated receptor (PPAR) modulators offer potential benefits for the treatment of obesity and its associated complications but their development has been complicated by biological, technical, and regulatory challenges. Despite significant challenges, PPAR modulators are attractive targets for the treatment of obesity and could offer a viable alternative to the millions of patients who fail to lose weight following rigorous dieting and exercise protocols. In addition, PPAR modulators have the potential-added benefit of ameliorating the associated comorbidities.
ABSTRACT
Considerable effort exists within drug discovery to develop novel compounds to improve the underlying metabolic defects in type 2 diabetes. One approach is focused on inhibition of the tyrosine phosphatase, PTP1B, an important negative regulator of both insulin and leptin signaling. Historically, tyrosine phosphatase assays have used either small organic phosphates or, alternatively, phosphorylated peptides from the target proteins themselves. In characterizing inhibitors of PTP1B, measuring turnover of small organic phosphates is limited to evaluation of compounds that bind the active site itself. Peptide substrates allow identification of additional subsets of inhibitors (e.g., those that bind the second aryl-phosphate site), but assays of peptide turnover often involve detection steps that then limit full kinetic evaluation of inhibitors. Here we use a polyclonal antibody specific for the phosphorylated insulin receptor to allow much more sensitive detection of peptide phosphorylation. This kinetically robust enzyme-linked immunosorbent assay (ELISA) gives k(cat) and K(m) values for a phosphorylated insulin receptor peptide consistent with values determined by a continuous fluorescence-based assay. Furthermore, IC50 values determined for well-behaved active site inhibitors agree well with values determined for p-nitrophenyl phosphate cleavage. This assay permits full characterization of a larger subset of inhibitors as drug candidates for this promising target.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Protein Tyrosine Phosphatases/metabolism , Receptor, Insulin/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Inhibitory Concentration 50 , Molecular Structure , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/antagonists & inhibitors , Sensitivity and Specificity , Time FactorsABSTRACT
A series of monocyclic thiophenes was designed and synthesized as PTP1B inhibitors. Guided by X-ray co-crystal structural information and computational modeling, rational design led to key interactions with Asp48 and improved inhibitory potency against PTP1B.
Subject(s)
Aspartic Acid/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/metabolism , Thiophenes/chemistry , Thiophenes/pharmacology , Aspartic Acid/metabolism , Crystallography, X-Ray , Cyclization , Enzyme Inhibitors/chemistry , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Structure , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/chemistry , Structure-Activity Relationship , Thiophenes/chemical synthesisABSTRACT
Ertiprotafib belongs to a novel class of insulin sensitizers developed for treatment of type 2 diabetes. In insulin-resistant rodent models, ertiprotafib and a close analog lowered both fasting blood glucose and insulin levels and improved glycemic excursion during an oral glucose tolerance test. In addition, treatment of rodents improved lipid profiles, with significantly lowered triglyceride and free fatty acid levels. These results suggested that this therapeutic activity might involve mechanisms in addition to PTP1b inhibition. In this study, we demonstrate that ertiprotafib activates peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma at concentrations comparable with those of known agonists of these regulators. Furthermore, it is able to drive adipocyte differentiation of C3H10T(1/2) cells, a hallmark of PPARgamma activation. Livers from ertiprotafib-treated animals showed significant induction of acyl-CoA oxidase activity, probably caused by PPARalpha engagement in these animals. We also show that ertiprotafib inhibits PTP1b in vitro with nonclassic kinetics at concentrations above its EC(50) for PPAR agonism. Thus, the complete mechanism of action for ertiprotafib and related compounds in vivo may involve multiple independent mechanisms, including (but not necessarily limited to) PTP1b inhibition and dual PPARalpha/PPARgamma agonism. Ertiprotafib pharmacology and interpretation of clinical results must be seen in light of this complexity.
Subject(s)
Adipocytes/cytology , Diabetes Mellitus, Type 2/drug therapy , Hypoglycemic Agents/pharmacology , Hypolipidemic Agents/pharmacology , Phenylpropionates/pharmacology , Thiophenes/pharmacology , Adipocytes/drug effects , Animals , Blood Glucose/drug effects , Blood Glucose/metabolism , Cell Differentiation/drug effects , Humans , Insulin/blood , Kinetics , Lipids/blood , Male , Mice , Mice, Obese , PPAR alpha/genetics , PPAR gamma/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/antagonists & inhibitors , Recombinant Proteins/metabolism , Triglycerides/bloodABSTRACT
The interaction of co-stimulatory molecules on T cells with B7 molecules on antigen presenting cells plays an important role in the activation of naive T cells. Consequently, agents that disrupt these interactions should have applications in treatment of transplant rejection as well as autoimmune diseases. To this end, specific small molecule inhibitors of human B7.1 were identified and characterized. These compounds inhibit the binding of B7.1 to both CD28 and CTLA4. Both classes of compounds appear to bind the same site, a relatively small portion of the GFCC'C" face of the N-terminal V-set domain of human B7.1, not present in the homologous B7.2 or even mouse B7.1. This site may represent a rare hot spot for small molecule antagonist design of inhibitors of cell-cell interactions, whose ligands may yield leads for the development of novel immunomodulatory medicines.