CHARACTERIZATION OF CYTOCHROME P450 2C8 ACTIVITY IN VIVO: PHARMACOGENETIC AND PHARMACOKINETIC STUDIES OF ROSIGLITAZONE METABOLISM

The Cytochrome P450 (CYP) superfamily of drug metabolizing enzymes is responsible for the metabolism of a variety of drugs and endogenous compounds. The CYP2C enzyme subfamily (CYP2C8, CYP2C9, and CYP2C19) mediates the metabolism of approximately twenty percent of these compounds. While CYP2C9 and CYP2C19 have been well characterized in vivo, relatively little is known about the in vivo contribution of CYP2C8. However, as the number of substrates and interest in polymorphic expression has grown, so too has the importance of CYP2C8. The in vivo relevance of CYP2C8 can be estimated with a drug predominately metabolized by this enzyme as a probe substrate. Thus, the overall purpose of this research was to investigate the utility of rosiglitazone as an in vivo probe of CYP2C8 activity. To accomplish this goal, we characterized the pharmacokinetics of rosiglitazone in the presence of the CYP2C8 inhibitor, trimethoprim, the CYP inducer, St. John's wort, and in subjects genotyped for variant CYP2C8 alleles. Novel liquid chromatographic methods were developed for the determination of rosiglitazone and trimethoprim plasma concentrations with fluorescence and ultraviolet wavelength detection, respectively. CYP2C8 genotyping was accomplished with a newly developed method based on Pyrosequencing technology, which facilitates high-throughput analysis in a cost-effective manner. Trimethoprim was an effective inhibitor of rosiglitazone metabolism in vitro and it increased rosiglitazone concentrations in vivo by 31%. In addition, there was a strong relationship (r2=0.97, p=0.0021) between trimethoprim plasma concentration and fold inhibition in subjects who did not carry the CYP2C8*3 allele, suggesting genotype influences the extent of CYP2C8 inhibition. Administration of St. John's wort increased rosiglitazone clearance by 35%, but CYP2C8 genotype did not affect the magnitude of induction. Finally, genotype did not affect basal rosiglitazone metabolism. Since changes have been observed with other CYP2C8 metabolized drugs, polymorphic effects of CYP2C8 may be substrate dependent. In conclusion, these results support the use of rosiglitazone as an in vivo probe of CYP2C8 activity, as it is affected by CYP2C8 inhibitors and inducers. The clinical benefits of CYP2C8 substrates may be influenced by these and other CYP2C8 modulators and therefore rosiglitazone could serve as a probe to detect these interactions

Synaptic nanomodules underlie the organization and plasticity of spine synapses.

Experience results in long-lasting changes in dendritic spine size, yet how the molecular architecture of the synapse responds to plasticity remains poorly understood. Here a combined approach of multicolor stimulated emission depletion microscopy (STED) and confocal imaging in rat and mouse demonstrates that structural plasticity is linked to the addition of unitary synaptic nanomodules to spines. Spine synapses in vivo and in vitro contain discrete and aligned subdiffraction modules of pre- and postsynaptic proteins whose number scales linearly with spine size. Live-cell time-lapse super-resolution imaging reveals that NMDA receptor-dependent increases in spine size are accompanied both by enhanced mobility of pre- and postsynaptic modules that remain aligned with each other and by a coordinated increase in the number of nanomodules. These findings suggest a simplified model for experience-dependent structural plasticity relying on an unexpectedly modular nanomolecular architecture of synaptic proteins

Updates in the chronic kidney disease-mineral bone disorder show the role of osteocytic proteins, a potential mechanism of the bone-vascular paradox, a therapeutic target, and a biomarker

The chronic kidney disease-mineral bone disorder (CKD-MBD) is a complex multi-component syndrome occurring during kidney disease and its progression. Here, we update progress in the components of the syndrome, and synthesize recent investigations, which suggest a potential mechanism of the bone-vascular paradox. The discovery that calcified arteries in chronic kidney disease inhibit bone remodeling lead to the identification of factors produced by the vasculature that inhibit the skeleton, thus providing a potential explanation for the bone-vascular paradox. Among the factors produced by calcifying arteries, sclerostin secretion is especially enlightening. Sclerostin is a potent inhibitor of bone remodeling and an osteocyte specific protein. Its production by the vasculature in chronic kidney disease identifies the key role of vascular cell osteoblastic/osteocytic transdifferentiation in vascular calcification and renal osteodystrophy. Subsequent studies showing that inhibition of sclerostin activity by a monoclonal antibody improved bone remodeling as expected, but stimulated vascular calcification, demonstrate that vascular sclerostin functions to brake the Wnt stimulation of the calcification milieu. Thus, the target of therapy in the chronic kidney disease-mineral bone disorder is not inhibition of sclerostin function, which would intensify vascular calcification. Rather, decreasing sclerostin production by decreasing the vascular osteoblastic/osteocytic transdifferentiation is the goal. This might decrease vascular calcification, decrease vascular stiffness, decrease cardiac hypertrophy, decrease sclerostin production, reduce serum sclerostin and improve skeletal remodeling. Thus, the therapeutic target of the chronic kidney disease-mineral bone disorder may be vascular osteoblastic transdifferentiation, and sclerostin levels may be a useful biomarker for the diagnosis of the chronic kidney disease-mineral bone disorder and the progress of its therapy

Sotatercept safety and effects on hemoglobin, bone, and vascular calcification

Introduction: Patients with end-stage kidney disease (ESKD) exhibit anemia, chronic kidney disease‒mineral bone disorder (CKD-MBD), and cardiovascular disease. The REN-001 and REN-002 phase II, multicenter, randomized studies examined safety, tolerability, and effects of sotatercept, an ActRIIA-IgG1 fusion protein trap, on hemoglobin concentration; REN-001 also explored effects on bone mineral density (BMD) and abdominal aortic vascular calcification. Methods: Forty-three patients were treated in REN-001 (dose range: sotatercept 0.3‒0.7 mg/kg or placebo subcutaneously [s.c.] for 200 days) and 50 in REN-002 (dose range: 0.1‒0.4 mg/kg i.v. and 0.13‒0.5 mg/kg s.c. for 99 days). Results: In REN-001, frequency of achieving target hemoglobin response (\u3e10 g/dl [6.21 mmol/l]) with sotatercept was dose-related and greater than placebo (0.3 mg/kg: 33.3%; 0.5 mg/kg: 62.5%; 0.7 mg/kg: 77.8%; 0.7 mg/kg [doses 1 and 2]/0.4 mg/kg [doses 3‒15]: 33.3%; placebo: 27.3%). REN-002 hemoglobin findings were similar (i.v.: 16.7%-57.1%; s.c.: 11.1%‒42.9%). Dose-related achievement of ≥2% increase in femoral neck cortical BMD was seen among only REN-001 patients receiving sotatercept (0.3‒0.7 mg/kg: 20.0%‒57.1%; placebo: 0.0%). Abdominal aortic vascular calcification was slowed in a dose-related manner, with a ≤15% increase in Agatston score achieved by more REN-001 sotatercept versus placebo patients (60%‒100% vs. 16.7%). The most common adverse events during treatment were hypertension, muscle spasm, headache, arteriovenous fistula site complication, and influenza observed in both treatment and placebo groups. Conclusion: In patients with ESKD, sotatercept exhibited a favorable safety profile and was associated with trends in dose-related slowing of vascular calcification. Less-consistent trends in improved hemoglobin concentration and BMD were observed

Nanoscale rules governing the organization of glutamate receptors in spine synapses are subunit specific

Heterotetrameric glutamate receptors are essential for the development, function, and plasticity of spine synapses but how they are organized to achieve this is not known. Here we show that the nanoscale organization of glutamate receptors containing specific subunits define distinct subsynaptic features. Glutamate receptors containing GluA2 or GluN1 subunits establish nanomodular elements precisely positioned relative to Synaptotagmin-1 positive presynaptic release sites that scale with spine size. Glutamate receptors containing GluA1 or GluN2B specify features that exhibit flexibility: GluA1-subunit containing AMPARs are found in larger spines, while GluN2B-subunit containing NMDARs are enriched in the smallest spines with neither following a strict modular organization. Given that the precise positioning of distinct classes of glutamate receptors is linked to diverse events including cell death and synaptic plasticity, this unexpectedly robust synaptic nanoarchitecture provides a resilient system, where nanopositioned glutamate receptor heterotetramers define specific subsynaptic regions of individual spine synapses

Ephrin-B3 controls excitatory synapse density through cell-cell competition for EphBs

Cortical networks are characterized by sparse connectivity, with synapses found at only a subset of axo-dendritic contacts. Yet within these networks, neurons can exhibit high connection probabilities, suggesting that cell-intrinsic factors, not proximity, determine connectivity. Here, we identify ephrin-B3 (eB3) as a factor that determines synapse density by mediating a cell-cell competition that requires ephrin-B-EphB signaling. In a microisland culture system designed to isolate cell-cell competition, we find that eB3 determines winning and losing neurons in a contest for synapses. In a Mosaic Analysis with Double Markers (MADM) genetic mouse model system in vivo the relative levels of eB3 control spine density in layer 5 and 6 neurons. MADM cortical neurons in vitro reveal that eB3 controls synapse density independently of action potential-driven activity. Our findings illustrate a new class of competitive mechanism mediated by trans-synaptic organizing proteins which control the number of synapses neurons receive relative to neighboring neurons

Neuron Glia-Related Cell Adhesion Molecule (NrCAM) Promotes Topographic Retinocollicular Mapping

NrCAM (Neuron-glial related cell adhesion molecule), a member of the L1 family of cell adhesion molecules, reversibly binds ankyrin and regulates axon growth, but it has not been studied for a role in retinotopic mapping. During development of retino-collicular topography, NrCAM was expressed uniformly in retinal ganglion cells (RGCs) along both mediolateral and anteroposterior retinal axes, and was localized on RGC axons within the optic tract and superior colliculus (SC). Anterograde tracing of RGC axons in NrCAM null mutant mice at P10, when the map resembles its mature form, revealed laterally displaced ectopic termination zones (eTZs) of axons from the temporal retina, indicating defective mediolateral topography, which is governed by ephrinB/EphBs. Axon tracing at P2 revealed that interstitial branch orientation of ventral-temporal RGC axons in NrCAM null mice was compromised in the medial direction, likely accounting for displacement of eTZs. A similar retinocollicular targeting defect in EphB mutant mice suggested that NrCAM and EphB interact to regulate mediolateral retino-collicular targeting. We found that EphB2 tyrosine kinase but not an EphB2 kinase dead mutant, phosphorylated NrCAM at a conserved tyrosine residue in the FIGQY ankyrin binding motif, perturbing ankyrin recruitment in NrCAM transfected HEK293 cells. Furthermore, the phosphorylation of NrCAM at FIGQY in SC was decreased in EphB1/3 and EphB1/2/3 null mice compared to WT, while phospho-FIGQY of NrCAM in SC was increased in EphB2 constitutively active (F620D/F620D) mice. These results demonstrate that NrCAM contributes to mediolateral retinocollicular axon targeting by regulating RGC branch orientation through a likely mechanism in which ephrinB/EphB phosphorylates NrCAM to modulate linkage to the actin cytoskeleton