Purpose
Tepotinib is a highly selective, potent, mesenchymal–epithelial transition factor (MET) inhibitor, approved for the treatment of non-small cell lung cancer (NSCLC) harboring
MET
exon 14 ...skipping. Objectives of this population pharmacokinetic (PK) analysis were to evaluate the dose–exposure relationship of tepotinib and its major circulating metabolite, MSC2571109A, and to identify the intrinsic/extrinsic factors that are predictive of PK variability.
Methods
Data were included from 12 studies in patients with cancer and in healthy participants. A sequential modeling approach was used to analyze the parent and metabolite data, including covariate analyses. Potential associations between observed covariates and PK parameters were illustrated using bootstrap analysis-based forest plots.
Results
A two-compartment model with sequential zero- and first-order absorption, and a first-order elimination from the central compartment, best described the plasma PK of tepotinib in humans across the dose range of 30–1400 mg. The bioavailability of tepotinib was shown to be dose dependent, although bioavailability decreased primarily at doses above the therapeutic dose of 500 mg. The intrinsic factors of race, age, sex, body weight, mild/moderate hepatic impairment and mild/moderate renal impairment, along with the extrinsic factors of opioid analgesic and gefitinib intake, had no relevant effect on tepotinib PK. Tepotinib has a long effective half-life of ~ 32 h.
Conclusions
Tepotinib shows dose proportionality up to at least the therapeutic dose, and time-independent clearance with a profile appropriate for once-daily dosing. None of the covariates identified had a clinically meaningful effect on tepotinib exposure or required dose adjustments.
Interaction and cross-talk of G-protein-coupled receptors (GPCRs) are of considerable interest because an increasing number of examples implicate a profound functional and physiological relevance of ...homo- or hetero-oligomeric GPCRs. The ghrelin (growth hormone secretagogue receptor (GHSR)) and melanocortin-3 (MC3R) receptors are both known to have orexigenic effects on the hypothalamic control of body weight. Because in vitro studies indicate heterodimerization of GHSR and MC3R, we investigated their functional interplay. Combined in situ hybridization and immunohistochemistry indicated that the vast majority of GHSR-expressing neurons in the arcuate nucleus also express MC3R. In vitro coexpression of MC3R and GHSR promoted enhanced melanocortin-induced intracellular cAMP accumulation compared with activation of MC3R in the absence of GHSR. In contrast, agonist-independent basal signaling activity and ghrelin-induced signaling of GHSR were impaired, most likely due to interaction with MC3R. By taking advantage of naturally occurring GHSR mutations and an inverse agonist for GHSR, we demonstrate that the observed enhanced MC3R signaling capability depends directly on the basal activity of GHSR. In conclusion, we demonstrate a paradigm-shifting example of GPCR heterodimerization allowing for mutually opposite functional influence of two hypothalamic receptors controlling body weight. We found that the agonist-independent active conformation of one GPCR can determine the signaling modalities of another receptor in a heterodimer. Our discovery also implies that mutations within one of two interacting receptors might affect both receptors and different pathways simultaneously. These findings uncover mechanisms of important relevance for pharmacological targeting of GPCR in general and hypothalamic body weight regulation in particular.
Background: The melanocortin-3 (MC3R) and ghrelin (GHSR) receptors are important key components in hypothalamic weight regulation.
Results: MC3R and GHSR di/oligomerize and have an opposite impact on each other's function.
Conclusion: The high basal activity of GHSR is a determinant of heterodimer function, and MC3R may constrain GHSR function.
Significance: Receptor di/oligomerization and its functional relevance contribute to the complex network of hypothalamic weight regulation.
Most Ca2+-permeable ion channels are inhibited by increases in the intracellular Ca2+ concentration (Ca2+i), thus preventing potentially deleterious rises in Ca2+i. In this study, we demonstrate that ...currents through the osmo-, heat- and phorbol ester-sensitive, Ca2+-permeable nonselective cation channel TRPV4 are potentiated by intracellular Ca2+. Spontaneous TRPV4 currents and currents stimulated by hypotonic solutions or phorbol esters were reduced strongly at all potentials in the absence of extracellular Ca2+. The other permeant divalent cations Ba2+ and Sr2+ were less effective than Ca2+ in supporting channel activity. An intracellular site of Ca2+ action was supported by the parallel decrease in spontaneous currents and Ca2+i on removal of extracellular Ca2+ and the ability of Ca2+ release from intracellular stores to restore TRPV4 activity in the absence of extracellular Ca2+. During TRPV4 activation by hypotonic solutions or phorbol esters, Ca2+ entry through the channel increased the rate and extent of channel activation. Currents were also potentiated by ionomycin in the presence of extracellular Ca2+. Ca2+-dependent potentiation of TRPV4 was often followed by inhibition. By mutagenesis, we localized the structural determinant of Ca2+-dependent potentiation to an intracellular, C-terminal calmodulin binding domain. This domain binds calmodulin in a Ca2+-dependent manner. TRPV4 mutants that did not bind calmodulin lacked Ca2+-dependent potentiation. We conclude that TRPV4 activity is tightly controlled by intracellular Ca2+. Ca2+ entry increases both the rate and extent of channel activation by a calmodulin-dependent mechanism. Excessive increases in Ca2+i via TRPV4 are prevented by a Ca2+-dependent negative feedback mechanism.
Mammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1–7) are Ca2+-permeable cation channels involved in receptor-mediated increases in intracellular Ca2+. ...Unlike most other TRP-related channels, which are inhibited by La3+ and Gd3+, currents through TRPC4 and TRPC5 are potentiated by La3+. Because these differential effects of lanthanides on TRPC subtypes may be useful for clarifying the role of different TRPCs in native tissues, we characterized the potentiating effect in detail and localized the molecular determinants of potentiation by mutagenesis. Whole cell currents through TRPC5 were reversibly potentiated by micromolar concentrations of La3+or Gd3+, whereas millimolar concentrations were inhibitory. By comparison, TRPC6 was blocked to a similar extent by La3+ or Gd3+ at micromolar concentrations and showed no potentiation. Dual effects of lanthanides on TRPC5 were also observed in outside-out patches. Even at micromolar concentrations, the single channel conductance was reduced by La3+, but reduction in conductance was accompanied by a dramatic increase in channel open probability, leading to larger integral currents. Neutralization of the negatively charged amino acids Glu543and Glu595/Glu598, situated close to the extracellular mouth of the channel pore, resulted in a loss of potentiation, and, for Glu595/Glu598 in a modification of channel inhibition. We conclude that in the micromolar range, the lanthanide ions La3+ and Gd3+ have opposite effects on whole cell currents through TRPC5 and TRPC6 channels. The potentiation of TRPC4 and TRPC5 by micromolar La3+ at extracellular sites close to the pore mouth is a promising tool for identifying the involvement of these isoforms in receptor-operated cation conductances of native cells.
Institut für Pharmakologie, Freie Universität Berlin,
14195 Berlin, Germany
To investigate the
possible role of members of the mammalian transient receptor potential
(TRP) channel family (TRPC1-7) in ...vasoconstrictor-induced
Ca 2+ entry in vascular smooth muscle cells, we studied
Arg 8 -vasopressin (AVP)-activated channels in A7r5
aortic smooth muscle cells. AVP induced an increase in free cytosolic
Ca 2+ concentration (Ca 2+ i )
consisting of Ca 2+ release and Ca 2+ influx.
Whole cell recordings revealed the activation of a nonselective cation
current with a doubly rectifying current-voltage relation strikingly
similar to those described for some heterologously expressed TRPC
isoforms. The current was also stimulated by direct activation of G
proteins as well as by activation of the phospholipase C -coupled
platelet-derived growth factor receptor. Currents were not activated by
store depletion or increased Ca 2+ i .
Application of 1-oleoyl-2-acetyl- sn -glycerol stimulated the current independently of protein kinase C, a characteristic property of
the TRPC3/6/7 subfamily. Like TRPC6-mediated currents, cation currents
in A7r5 cells were increased by flufenamate. Northern hybridization
revealed mRNA coding for TRPC1 and TRPC6. We therefore suggest that
TRPC6 is a molecular component of receptor-stimulated Ca 2+ -permeable cation channels in A7r5 smooth muscle cells.
transient receptor potential channel; calcium ion influx; receptor-operated channel
Several Ca(2+)-permeable channels, including the non-selective cation channel TRPV4, are subject to Ca(2+)-dependent facilitation. Although it has been clearly demonstrated in functional experiments ...that calmodulin (CaM) binding to intracellular domains of TRP channels is involved in this process, the molecular mechanism remains elusive. In this study, we provide experimental evidence for a comprehensive molecular model that explains Ca(2+)-dependent facilitation of TRPV4. In the resting state, an intracellular domain from the channel N terminus forms an autoinhibitory complex with a C-terminal domain that includes a high-affinity CaM binding site. CaM binding, secondary to rises in intracellular Ca(2+), displaces the N-terminal domain which may then form a homologous interaction with an identical domain from a second subunit. This represents a novel potentiation mechanism that may also be relevant in other Ca(2+)-permeable channels.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The range of actions of the second messenger Ca 2+ is a key determinant of neuronal excitability and plasticity. For dendritic spines, there is on-going debate regarding how
diffusional efflux of Ca ...2+ affects spine signalling. However, the consequences of spino-dendritic coupling for dendritic Ca 2+ homeostasis and downstream signalling cascades have not been explored to date. We addressed this question by four-dimensional
computer simulations, which were based on Ca 2+ -imaging data from mice that either express or lack distinct endogenous Ca 2+ -binding proteins. Our simulations revealed that single active spines do not affect dendritic Ca 2+ signalling. Neighbouring, coactive spines, however, induce sizeable increases in dendritic Ca 2+ i when they process slow synaptic Ca 2+ signals, such as those implicated in the induction of long-term plasticity. This spino-dendritic coupling is mediated by
buffered diffusion, specifically by diffusing calbindin-bound Ca 2+ . This represents a central mechanism for activating calmodulin in dendritic shafts and therefore a novel form of signal integration
in spiny dendrites.
RGS9-deficient mice show drug-induced dyskinesia but normal locomotor activity under unchallenged conditions.
Genes related to Ca2+ signaling and their functions were regulated in RGS9-deficient ...mice.
Changes in Ca2+ signaling that compensate for RGS9 loss-of-function can explain the normal locomotor activity in RGS9-deficient mice under unchallenged conditions.
Identified signaling components may represent novel targets in antidyskinetic therapy. The long splice variant of the regulator of G-protein signaling 9 (RGS9-2) is enriched in striatal medium spiny neurons and dampens dopamine D2 receptor signaling. Lack of RGS9-2 can promote while its overexpression prevents drug-induced dyskinesia. Other animal models of drug-induced dyskinesia rather pointed towards overactivity of dopamine receptor-mediated signaling. To evaluate changes in signaling pathways mRNA expression levels were determined and compared in wild-type and RGS9-deficient mice. Unexpectedly, expression levels of dopamine receptors were unchanged in RGS9-deficient mice, while several genes related to Ca2+ signaling and long-term depression were differentially expressed when compared to wild type animals. Detailed investigations at the protein level revealed hyperphosphorylation of DARPP32 at Thr34 and of ERK1/2 in striata of RGS9-deficient mice. Whole cell patch clamp recordings showed that spontaneous synaptic events are increased (frequency and size) in RGS9-deficient mice while long-term depression is reduced in acute brain slices. These changes are compatible with a Ca2+-induced potentiation of dopamine receptor signaling which may contribute to the drug-induced dyskinesia in RGS9-deficient mice.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We identified and examined a candidate gene for local directional selection in Europeans, TRPV6, and conclude that selection has acted on standing genetic variation at this locus, creating parallel ...soft sweep events in humans. A novel modification of the extended haplotype homozygosity (EHH) test was utilized, which compares EHH for a single allele across populations, to investigate the signature of selection at TRPV6 and neighboring linked loci in published data sets for Europeans, Asians and African-Americans, as well as in newly-obtained sequence data for additional populations. We find that all non-African populations carry a signature of selection on the same haplotype at the TRPV6 locus. The selective footprints, however, are significantly differentiated between non-African populations and estimated to be younger than an ancestral population of non-Africans. The possibility of a single selection event occurring in an ancestral population of non-Africans was tested by simulations and rejected. The putatively-selected TRPV6 haplotype contains three candidate sites for functional differences, namely derived non-synonymous substitutions C157R, M378V and M681T. Potential functional differences between the ancestral and derived TRPV6 proteins were investigated by cloning the ancestral and derived forms, transfecting cell lines, and carrying out electrophysiology experiments via patch clamp analysis. No statistically-significant differences in biophysical channel function were found, although one property of the protein, namely Ca(2+) dependent inactivation, may show functionally relevant differences between the ancestral and derived forms. Although the reason for selection on this locus remains elusive, this is the first demonstration of a widespread parallel selection event acting on standing genetic variation in humans, and highlights the utility of between population EHH statistics.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Polyuria, hypernatremia, and hypovolemia are the major clinical signs of inherited nephrogenic diabetes insipidus (NDI). Hypernatremia is commonly considered a secondary sign caused by the net loss ...of water due to insufficient insertion of aquaporin-2 water channels into the apical membrane of the collecting duct cells. In the present study, we employed transcriptome-wide expression analysis to study gene expression in V2 vasopressin receptor (Avpr2)-deficient mice, an animal model for X-linked NDI. Gene expression changes in NDI mice indicate increased proximal tubular sodium reabsorption. Expression of several key genes including Na+-K+-ATPase and carbonic anhydrases was increased at the mRNA levels and accompanied by enhanced enzyme activities. In addition, altered expression was also observed for components of the eicosanoid and thyroid hormone pathways, including cyclooxygenases and deiodinases, in both kidney and hypothalamus. These effects are likely to contribute to the clinical NDI phenotype. Finally, our data highlight the involvement of the renin-angiotensin-aldosterone system in NDI pathophysiology and provide clues to explain the effectiveness of diuretics and indomethacin in the treatment of NDI.
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