Effects Of Receptor Occupancy Research Articles

Differential Binding of Sarilumab and Tocilizumab to IL-6Rα and Effects of Receptor Occupancy on Clinical Parameters.

We evaluated interleukin-6 (IL-6) receptor-α subunit (IL-6Rα) signaling inhibition with sarilumab and tocilizumab, the association between IL-6Rα receptor occupancy (RO) and C-reactive protein (CRP), and the potential clinical relevance of any differences. For this, we measured IL-6Rα binding and signaling inhibition with sarilumab and tocilizumab invitro, simulated soluble IL-6Rα RO over time for approved sarilumab subcutaneous (SC) and tocilizumab intravenous (IV) and SC doses, and assessed associations between calculated RO and CRP reduction, 28-joint Disease Activity Score based on CRP, and 20%/50%/70% improvement in American College of Rheumatology responses from clinical data. Sarilumab binds IL-6Rα in vitro with 15- to 22-fold higher affinity than tocilizumab, and inhibits IL-6-mediated classical and trans signaling via membrane-bound and soluble IL-6Rα. Sarilumab 200 and 150 mg SC every 2 weeks achieved >90% RO after first and second doses, respectively, maintained throughout the treatment period. At steady-state trough, RO was greater with sarilumab 200mg (98%) and 150 mg SC every 2 weeks (94%), and tocilizumab 162 mg SC weekly (>99%) and 8mg/kg IV every 4 weeks (99%), vs tocilizumab 162 mg SC every 2 weeks (84%) and 4 mg/kg IV every 4 weeks (60%). Higher RO was associated with greater CRP reduction and 28-joint Disease Activity Score based on CRP reduction, and more sarilumab patients achieving 20%/50%/70% improvement in American College of Rheumatology responses. The greatest reduction in CRP levels was observed with sarilumab (both doses) and tocilizumab 8mg/kg IV every 4 weeks (reductions proportionally smaller with 4mg/kg IV every 4 weeks). Higher IL-6Rα binding affinity translated into higher RO with sarilumab vs tocilizumab 4mg/kg every 4 weeks or 162 mg every 2 weeks; tocilizumab required the higher dose or increased frequency to maintain the same degree of RO and CRP reduction. Higher RO was associated with clinical parameter improvements.

Effect of Receptor Occupancy on Folate Receptor Internalization

The folate receptor (FR) is a GPI anchored cell surface glycoprotein that functions to facilitate folic acid uptake and mediate signal transduction. With the introduction of multiple folate-targeted drugs into the clinic, the question has arisen regarding how frequently a patient can be dosed with a FR-targeted drug or antibody and whether dosing frequency exerts any impact on the availability of FR for subsequent rounds of FR-mediated drug uptake. Although the rate of FR recycling has been examined in murine tumor models, little or no information exists on the impact of FR occupancy on its rate of endocytosis. The present study quantitates the number of cell surface FR-α and FR-β following exposure to saturating concentrations of a variety of folate-linked molecules and anti-FR antibodies, including the unmodified vitamin, folate-linked drug mimetics, multifolate derivatized nanoparticles, and monoclonal antibodies to FR. We report here that FR occupancy has no impact on the rate of FR internalization. We also demonstrate that multivalent conjugates that bind and cross-link FRs at the cell surface internalize at the same rate as monovalent folate conjugates that have no impact on FR clustering, even though the multivalent conjugates traffic through a different endocytic pathway.

The relationship between dopamine D2 receptor occupancy and the vacuous chewing movement syndrome in rats.

A dose-response relationship between dopamine D(2) occupancy and acute extrapyramidal symptoms (EPS) has been well established. However, the link with the induction of tardive dyskinesia (TD) is less clear. To ascertain the nature and extent of D(2) receptor occupancy effects on haloperidol-induced vacuous chewing movements (VCMs) in a rat model of TD. Groups of eight rats received haloperidol decanoate injections corresponding to daily doses of 0, 0.08, 0.17, 0.33, or 1 mg/kg for 10-12 weeks. VCMs were measured on a weekly basis and D(2) occupancy levels were measured in vivo using [(3)H]-raclopride at the end of the experiment. Final VCM scores were significantly different between haloperidol doses ( P=0.001). Moderate but significant correlations were found between dose and average VCM scores (r=0.69, P<0.001) and between D(2) occupancy and average VCM scores (r=0.65, P<0.001). The rats that developed the VCM syndrome (>/=8 VCMs) had higher occupancies than rats that did not. Of the rats with an occupancy above 70%, 63% developed VCMs, compared with 37% of the rats with D(2) occupancy below that. These results indicate that chronic haloperidol induces VCMs in a dose-dependent manner, with doses leading to high D(2) occupancy increasing the likelihood of emergence of the VCM syndrome. While a certain level of D(2) occupancy may be necessary for inducing VCMs, it is not sufficient in and of itself to induce the VCM syndrome.

Internalization of the Hyaluronan Receptor CD44 by Chondrocytes

Chondrocytes express CD44 as a primary receptor for the matrix macromolecule hyaluronan. Hyaluronan is responsible for the retention and organization of proteoglycan within cartilage, and hyaluronan–chondrocyte interactions are important for the assembly and maintenance of the cartilage matrix. Bovine articular chondrocytes were used to study the endocytosis and turnover of CD44 and the effects of receptor occupancy on this turnover. Matrix-intact chondrocytes exhibit approximately a 6% internalization of cell surface CD44 by 4 h. Treatment with Streptomyces hyaluronidase to remove endogenous pericellular matrix increased internalization to approximately 20% of cell surface CD44 at 4 h. This turnover could be partially inhibited by the addition of exogenous hyaluronan to these matrix-depleted chondrocytes. Cell surface biotin-labeled CD44 was internalized by chondrocytes and this internalization was decreased in the presence of hyaluronan. Colocalization of internalized CD44 and fluorescein-labeled hyaluronan in intracellular vesicles correlates with the previous results of receptor-mediated endocytosis pathway for the degradation of hyaluronan by acid hydrolases. Taken together, our results indicate that CD44 is internalized by chondrocytes and that CD44 turnover is modulated by occupancy with hyaluronan.

Pharmacokinetics and Pharmacodynamics of a Second Dose of Atracurium† in Anaesthetised Patients

To determine whether the rapid onset of a second dose of a muscle relaxant was explained by pharmacokinetic (PK) or pharmacodynamic (PD) factors, atracurium PK-PD modelling was performed after 2 bolus doses of 0.5 mg/kg given approximately 1 hour apart to 6 anaesthetised patients. Neuromuscular function was monitored using single twitch stimulation during onset and train-of-four during recovery. Arterial concentrations were measured every 10 seconds for the first 2 minutes following each atracurium injection, then at frequent intervals thereafter. After the second dose, in spite of mean residual atracurium plasma concentrations of 227 µg/L, kinetics were unchanged. In contrast, onset times were markedly more rapid after the second dose (1.6 ± 0.1 vs 9.0 ± 1.8 minutes after the first dose) and clinical recovery prolonged (55.8 ± 2.2 vs 42.5 ± 2.0 minutes after the first dose). Taking into account residual plasma concentrations and neuromuscular block, the equilibration rate constant between plasma and effect site concentrations, Keo, was not significantly altered (0.035 ± 0.001 vs 0.043 ± 0.004 min−1). Therefore, the Keo seems to represent the rate of transfer to the neuromuscular junction without the confounding effect of receptor occupancy. Conversely, onset times would be extremely dependent on receptor events. Hence, the more rapid onset time following the second atracurium dose is most likely caused by residual concentrations at the effect site.

Human insulin-like growth factor I receptor internalization. Role of the juxtamembrane domain.

Cytoplasmic submembrane domains of the insulin-like growth factor I (IGF-1) receptor ranging from glycine 940 to proline 959 were investigated for their role in endocytosis of the IGF-1 ligand in rat pituitary GC cells stably expressing mutant human IGF-1 receptors. Replacement of each of three tyrosine residues within the juxtamembrane domain reduced the internalization rate (Ke) by 33% (943Y-->A), 47% (950Y-->A), and 41% (957Y-->A), respectively. Other substitutions within the submembrane region variably retarded receptor-mediated IGF-1 ligand endocytosis. Thus, each amino acid appears to independently contribute to the overall ligand-stimulated IGF-1 receptor internalization rate. The effect of receptor occupancy on IGF-1 receptor endocytosis was also tested. The rate of 125I-labeled IGF-1 internalization by transfectants overexpressing the wild type IGF-1 receptor was rapid but decreased markedly in the presence of increasing unlabeled IGF-1 (ED50 = 44,000 receptors/cell). Substitution by alanine for any of the three submembrane tyrosines increased the ED50 to > 56,000 receptors/cell, decreased the slope (Kic), and had a variable effect on Vmax in the presence of increasing unlabeled IGF-1. In contrast, 125I-labeled IGF-1 endocytosis by the mutant kinase-deficient 952STOP cells was slow and not further retarded by unlabeled IGF-1. These results suggest that ligand-mediated internalization of the human IGF-I is consistent with saturable interactions between the IGF-I receptor juxtamembrane region (glycine 940-tyrosine 957) and components of the endocytic apparatus.

Peptide-major histocompatibility complex class II complexes with mixed agonist/antagonist properties provide evidence for ligand-related differences in T cell receptor-dependent intracellular signaling.

Clonal activation of CD4+ and CD8+ T lymphocytes depends on binding of peptide-major histocompatibility complex (MHC) molecule complexes by their alpha/beta receptors, eventually resulting in sufficient aggregation to initiate second messenger generation. The nature of intracellular signals resulting from such T cell receptor (TCR) occupancy is believed to be independent of the specific structure of the ligand being bound, and to vary quantitatively, not qualitatively, with the concentration of ligand offered and the affinity of the receptor for the peptide-MHC molecule complex. In contrast to the expectations of this model, the analysis of the response of a T helper type 1 clone to mutant E alpha E beta k molecules in the absence or presence of a peptide antigen revealed that peptide inhibited the interleukin 2 (IL-2) response to an otherwise allostimulatory mutant form of this MHC class II molecule. The inhibition was not due to competition for formation of alloantigen, it required TCR recognition of peptide-mutant MHC molecule complexes, and it decreased IL-2 production without affecting receptor-dependent IL-3, IL-2 receptor alpha, or size enlargement responses. This preferential reduction in IL-2 secretion could be correlated with the costimulatory signal dependence of this cytokine response, but could not be overcome by crosslinking the CD28 molecule on the T cell. These results define a new class of TCR ligands with mixed agonist/antagonist properties, and point to a ligand-related variation in the quality of clonotypic receptor signaling events or their integration with other signaling processes. It was also found that a single TCR ligand showed greatly different dose thresholds for the elicitation of distinct effector responses from a cloned T cell population. The observations that changes in ligand structure can result in qualitative alterations in the effects of receptor occupancy and that quantitative variations in ligand density can be translated into qualitative differences in T cell responses have important implications for models of intrathymic selection and control of the results of active immunization.

Insulin receptors internalize by a rapid, saturable pathway requiring receptor autophosphorylation and an intact juxtamembrane region.

The effect of receptor occupancy on insulin receptor endocytosis was examined in CHO cells expressing normal human insulin receptors (CHO/IR), autophosphorylation- and internalization-deficient receptors (CHO/IRA1018), and receptors which undergo autophosphorylation but lack a sequence required for internalization (CHO/IR delta 960). The rate of [125I]insulin internalization in CHO/IR cells at 37 degrees C was rapid at physiological concentrations, but decreased markedly in the presence of increasing unlabeled insulin (ED50 = 1-3 nM insulin, or 75,000 occupied receptors/cell). In contrast, [125I]insulin internalization by CHO/IRA1018 and CHO/IR delta 960 cells was slow and was not inhibited by unlabeled insulin. At saturating insulin concentrations, the rate of internalization by wild-type and mutant receptors was similar. Moreover, depletion of intracellular potassium, which has been shown to disrupt coated pit formation, inhibited the rapid internalization of [125I]insulin at physiological insulin concentrations by CHO/IR cells, but had little or no effect on [125I]insulin uptake by CHO/IR delta 960 and CHO/IRA1018 cells or wild-type cells at high insulin concentrations. These data suggest that the insulin-stimulated entry of the insulin receptor into a rapid, coated pit-mediated internalization pathway is saturable and requires receptor autophosphorylation and an intact juxtamembrane region. Furthermore, CHO cells also contain a constitutive nonsaturable pathway which does not require receptor autophosphorylation or an intact juxtamembrane region; this second pathway is unaffected by depletion of intracellular potassium, and therefore may be independent of coated pits. Our data suggest that the ligand-stimulated internalization of the insulin receptor may require specific saturable interactions between the receptor and components of the endocytic system.

Rate constants for binding, dissociation, and internalization of EGF: effect of receptor occupancy and ligand concentration

We measured the kinetic parameters for interaction of epidermal growth factor (EGF) with fetal rat lung (FRL) cells under two sets of experimental conditions and applied sensitivity analysis to see which parameters were well-defined. In the first set of experiments (method 1), the kinetics of internalization and dissociation of radiolabeled EGF were measured with a temperature-shift protocol in medium initially devoid of free ligand. The initial concentration of radiolabeled EGF bound to the cell surface corresponded to levels of receptor occupancy ranging from approximately 200 receptors per cell to approximately 18,000 receptors per cell, a level at which EGF binding approaches saturation. In the second set of experiments (method 2), carried out at a constant temperature, we began with no surface-bound or internalized ligand. The initial free ligand concentration was varied from 0.2 to 50 ng/mL. In both sets of experiments, we measured surface-bound, internalized, and free 125I-EGF as functions of time and evaluated the parameters of a mathematical model of endocytosis. Sensitivity analysis showed that three rate constants were well-defined in this combination of two experimental approaches: ke, the endocytic rate constant; ka, the association rate constant; and kd, the dissociation rate constant. The endocytic parameter ke was found to be independent of initial surface receptor occupancy (method 1); there was some indication that it increased with initial free ligand concentration in method 2. Neither kd nor ka was found to change with extent of initial surface receptor occupancy or initial free ligand concentration, respectively, a finding of significance, since diffusion theory predicts these parameters will vary with surface receptor occupancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced sensitivity of heart cells to adenosine and up-regulation of receptor number after treatment of guinea pigs with theophylline.

Experiments were carried out in hearts from guinea pigs that were fed either the adenosine receptor antagonist theophylline (0.6 mg/ml) or no drug. The A1 adenosine receptor radioligand [125I]aminobenzyladenosine bound to a single affinity class of receptors in heart cell membranes from control animals with Bmax and KD of 18.3 +/- 1.0 fmol/mg protein and 3.7 +/- 0.6 nM, respectively (n = 8). Heart cell membranes from animals fed theophylline for 2, 7, and 14 days bound the radioligand with about the same affinity, but the number of binding sites was significantly increased (p less than 0.01) to 30.6 +/- 1.7 (n = 3), 30.0 +/- 0.8 (n = 3), and 27.3 +/- 2.9 (n = 4), respectively. Nearly identical results were obtained with membranes prepared from enzymatically dispersed ventricular myocytes. Fourteen days of theophylline treatment also produced a small increase (12%, p less than 0.01) in the number of binding sites in membranes derived from cerebral cortexes. Isolated ventricular myocytes prepared from animals fed no drug or theophylline for 7 days were used to determine the effect of adenosine on 20 nM isoproterenol-stimulated calcium current (ICa) measured by the whole-cell patch-clamp technique. Adenosine reduced isoproterenol-stimulated ICa without affecting the activation or inactivation kinetics of the current; ICa density was reduced less by 5 microM adenosine in cells from control (25 +/- 3 to 21 +/- 3 microA/microF) than in cells from theophylline-fed animals (26 +/- 5 to 17 +/- 2 microA/microF). Although a high concentration (0.5 mM) of adenosine abolished isoproterenol-stimulated ICa in cells from control or theophylline-fed animals, the IC50 for adenosine was sixfold less in cells derived from theophylline-fed animals than in cells from control animals (4.6 +/- 0.6 microM and 28.3 +/- 1.4 microM, respectively, p less than 0.01). In contrast, the increase in ICa in response to isoproterenol alone and the potency of acetylcholine to antagonize this effect of isoproterenol were the same in both groups of cells. A maximally effective concentration of R-phenylisopropyladenosine (0.1 mM) inhibited isoproterenol-stimulated cyclic AMP accumulation less in cardiomyocytes from control than from theophylline-fed animals (28.7 +/- 1.8% vs. 42.0 +/- 4.2%, p less than 0.05). In summary, exposure of the myocardium to theophylline increases the number of adenosine receptors and the effects of receptor occupancy by agonists. These findings imply that the endogenous concentration of adenosine is high enough in the normoxic guinea pig heart to chronically maintain adenosine receptors in a partially down-regulated state.

The Human Platelet Vasopressin Receptor and Its Intracellular Messengers

The effects of divalent cations on human platelet vasopressin receptor binding characteristics and effects of receptor occupancy on endogenous protein phosphorylation were investigated. Binding of vasopressin to its receptor is modulated by both the nature and the concentration of ions. Whatever the cation present, guanosine 5'-triphosphate or 5' guanylylimidodiphosphate do not alter the receptor binding characteristics. In the presence of extracellular calcium, vasopressin stimulates the phosphorylation of a 45,000-dalton protein and to a lesser degree of a 20,000-dalton protein following a pattern observed with thrombin and 12-O-tetradecanoylphorbol-13-acetate, a phorbol ester. Phosphorylation is also stimulated by a V1 vascular agonist, but not V2 renal agonists, and is more potently blocked by a V1 vascular antagonist than by a V2 renal antagonist. These results suggest that human platelets bear typical V1 vascular vasopressin receptors which stimulate the phosphorylation of specific substrates of protein kinase C and myosin light-chain kinase.

Studies on the Glucocorticoid Receptor in Human Peripheral Lymphocytes

There have been several reports about the glucocorticoid receptor (GC-R) in human peripheral lymphocytes (HPL). However, little has been investigated about the validity of the assay method of GC-R in HPL. such as the effect of receptor occupancy by intrinsic cortisol or administered GC. In the present study, it was clarified that receptor-bound prednisolone or cortisol was almost completely dissociated from the receptor by washing lymphocytes three times with 30 min equilibration at 37 degrees C between each centrifugation. The GC receptor was measured by whole cell binding assay using [3H] dexamethasone as a ligand. The receptor number and dissociation constant (Kd) of lymphocytes, which were preincubated with prednisolone (10(-7) M) or cortisol (10(-6) M) at 37 degrees C for 1 h, were equal to those lymphocytes preincubated without GC. These data indicated that the receptor number and Kd of HPL can be determined without the effects of receptor occupancy by this assay procedure. The GC and GC-R complex in the nucleus are quite important for the action of GC. However, there have been few reports about the assay of nuclear GC-R in HPL. In this study, it was elucidated that nuclear receptor-bound [3H] dexamethasone could be measured by centrifugation (2000 g, 10 min) of the nuclei of lymphocytes after being lysed in 1.5 mM MgCl2 (0 degree C). By these assay procedures, the receptor number in whole lymphocytes of normal HPL (n = 22) was determined to be 10.48 +/- 2.40 (M +/- SD) fmoles/10(6) cells, and Kd was 5.8 +/- 1.24 nM, while the receptor number and Kd in the nuclei were 5.68 +/- 1.53 and 6.29 +/- 1.49, respectively.

Inositol trisphosphate and diacylglycerol as intracellular second messengers in liver.

Receptor occupation by a variety of Ca2+-mobilizing hormones, such as alpha 1-adrenergic agents, vasopressin and angiotensin II, causes a rapid phosphodiesterase-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate in the plasma membrane with the production of the water soluble compound myo-inositol-1,4,5-trisphosphate (IP3) and the lipophilic molecule 1,2-diacylglycerol (DG). This review summarizes the recent evidence obtained in the liver that defines the roles of these products as intracellular messengers of hormone action. Intracellular Ca2+ mobilization is mediated by IP3, which releases Ca2+ from a subpopulation of the endoplasmic reticulum, resulting in a rapid increase of the cytosolic free Ca2+ concentration ( [Ca2+]i). Further effects of receptor occupancy are inhibition of the plasma membrane Ca2+-ATPase, despite net Ca2+ efflux, and an increased permeability of the plasma membrane to extracellular Ca2+. The activation of the phospholipid-dependent protein kinase C by DG does not alter Ca2+ fluxes across the plasma membrane. In contrast to some secretory cells, a synergism between protein kinase C activation and increased [Ca2+]i is not observed in liver. Activation of protein kinase C profoundly inhibits the response to alpha 1-adrenergic agonists, with only minimal effects on the vasopressin response. It is concluded that in liver the two inositol-lipid messenger systems, IP3 and DG, exert their effects by essentially separate pathways.

Effect of receptor occupancy on [3H]dexamethasone binding to circulating leukocytes.

To determine the effect of steroid occupancy of the glucocorticoid receptor on the measurement of total receptor concentration using Scatchard plot of [3H]dexamethasone (Dex) binding to circulating leukocytes, leukocytes were incubated with 10(-7)M cortisol or 10(-9)M Dex prior to [3H]Dex binding study. Total binding capacity calculated from Scatchard plot analysis of [3H]Dex binding was not significantly reduced after pre-incubation with cortisol. However, total binding capacity was significantly lower after pre-incubation with Dex. These data suggest that total glucocorticoid receptor concentration can be measured from a 3 h incubation of leukocytes obtained from untreated patients.