Ligand recognition and allosteric regulation of DRD1-Gs signaling complexes.

Dopamine receptors, including D1- and D2-like receptors, are important therapeutic targets in a variety of neurological syndromes, as well as cardiovascular and kidney diseases. Here, we present five cryoelectron microscopy (cryo-EM) structures of the dopamine D1 receptor (DRD1) coupled to Gs heterotrimer in complex with three catechol-based agonists, a non-catechol agonist, and a positive allosteric modulator for endogenous dopamine. These structures revealed that a polar interaction network is essential for catecholamine-like agonist recognition, whereas specific motifs in the extended binding pocket were responsible for discriminating D1- from D2-like receptors. Moreover, allosteric binding at a distinct inner surface pocket improved the activity of DRD1 by stabilizing endogenous dopamine interaction at the orthosteric site. DRD1-Gs interface revealed key features that serve as determinants for G protein coupling. Together, our study provides a structural understanding of the ligand recognition, allosteric regulation, and G protein coupling mechanisms of DRD1.

Ciliotherapy: Remote Control of Primary Cilia Movement and Function by Magnetic Nanoparticles.

Patients with polycystic kidney disease (PKD) are characterized with uncontrolled hypertension. Hypertension in PKD is a ciliopathy, an abnormal function and/or structure of primary cilia. Primary cilia are cellular organelles with chemo and mechanosensory roles. In the present studies, we designed a cilia-targeted (CT) delivery system to deliver fenoldopam specifically to the primary cilia. We devised the iron oxide nanoparticle (NP)-based technology for ciliotherapy. Live imaging confirmed that the CT-Fe2O3-NPs specifically targeted primary cilia in cultured cells in vitro and vascular endothelia in vivo. Importantly, the CT-Fe2O3-NPs enabled the remote control of the movement and function of a cilium with an external magnetic field, making the nonmotile cilium exhibit passive movement. The ciliopathic hearts displayed hypertrophy with compromised functions in left ventricle pressure, stroke volume, ejection fraction, and overall cardiac output because of prolonged hypertension. The CT-Fe2O3-NPs significantly improved cardiac function in the ciliopathic hypertensive models, in which the hearts also exhibited arrhythmia, which was corrected with the CT-Fe2O3-NPs. Intraciliary and cytosolic Ca2+ were increased when cilia were induced with fluid flow or magnetic field, and this served as a cilia-dependent mechanism of the CT-Fe2O3-NPs. Fenoldopam-alone caused an immediate decrease in blood pressure, followed by reflex tachycardia. Pharmacological delivery profiles confirmed that the CT-Fe2O3-NPs were a superior delivery system for targeting cilia more specifically, efficiently, and effectively than fenoldopam-alone. The CT-Fe2O3-NPs altered the mechanical properties of nonmotile cilia, and these nano-biomaterials had enormous clinical potential for ciliotherapy. Our studies further indicated that ciliotherapy provides a possibility toward personalized medicine in ciliopathy patients.

The Synergistic Roles of Cholecystokinin B and Dopamine D5 Receptors on the Regulation of Renal Sodium Excretion.

Renal dopamine D1-like receptors (D1R and D5R) and the gastrin receptor (CCKBR) are involved in the maintenance of sodium homeostasis. The D1R has been found to interact synergistically with CCKBR in renal proximal tubule (RPT) cells to promote natriuresis and diuresis. D5R, which has a higher affinity for dopamine than D1R, has some constitutive activity. Hence, we sought to investigate the interaction between D5R and CCKBR in the regulation of renal sodium excretion. In present study, we found D5R and CCKBR increase each other's expression in a concentration- and time-dependent manner in the HK-2 cell, the specificity of which was verified in HEK293 cells heterologously expressing both human D5R and CCKBR and in RPT cells from a male normotensive human. The specificity of D5R in the D5R and CCKBR interaction was verified further using a selective D5R antagonist, LE-PM436. Also, D5R and CCKBR colocalize and co-immunoprecipitate in BALB/c mouse RPTs and human RPT cells. CCKBR protein expression in plasma membrane-enriched fractions of renal cortex (PMFs) is greater in D5R-/- mice than D5R+/+ littermates and D5R protein expression in PMFs is also greater in CCKBR-/- mice than CCKBR+/+ littermates. High salt diet, relative to normal salt diet, increased the expression of CCKBR and D5R proteins in PMFs. Disruption of CCKBR in mice caused hypertension and decreased sodium excretion. The natriuresis in salt-loaded BALB/c mice was decreased by YF476, a CCKBR antagonist and Sch23390, a D1R/D5R antagonist. Furthermore, the natriuresis caused by gastrin was blocked by Sch23390 while the natriuresis caused by fenoldopam, a D1R/D5R agonist, was blocked by YF476. Taken together, our findings indicate that CCKBR and D5R synergistically interact in the kidney, which may contribute to the maintenance of normal sodium balance following an increase in sodium intake.

Rapid determination of fenoldopam in human plasma by UPLC-MS/MS for pharmacokinetic analysis in patients.

We developed and validated a rapid, selective, and sensitive ultra-performance liquid-chromatography mass-spectrometry (UPLC-MS/MS) method for quantifying fenoldopam in human plasma for pharmacokinetic studies. Fenoldopam and the internal-standard (IS), oxazepam, were isolated from human plasma by liquid-liquid extraction using ethyl acetate after alkalization, and were separated on a 2.1×100 mm Acquity UPLC HSS T3 C18 column (inside diameter, 1.8 µm) using a mobile phase of water (0.05% formic acid) and acetonitrile gradient elution. The fenoldopam and IS were eluted at 1.07 and 2.32 min, respectively. Quantification was performed using positive-ion electrospray-ionization (ESI), and the fenoldopam and IS responses were optimized at the m/z 306.16→107.10 and m/z 287.1→241.01 transitions, respectively. The assay was validated over the linear range of 0.1-40 ng/mL fenoldopam with intra- and interassay precision <13.21%. The matrix effect of normal and hemolyzed plasma was 94.9-101.6%. Fenoldopam was stable for ≥34 days at -70 °C in normal and hemolyzed plasma containing ascorbic acid as a stabilizer. This method can be successfully applied in pharmacokinetic studies of fenoldopam in hypertensive patients.

Stability of fenoldopam mesylate in two infusion solutions.

The physical and chemical stability of fenoldopam as the mesylate salt were studied. Test samples of fenoldopam mesylate, with fenoldopam concentrations ranging from 4 to 300 micrograms/mL, were prepared in polyvinyl chloride (PVC) bags of 5% dextrose injection and 0.9% sodium chloride injection and were stored at 4 degrees C in the dark and 23 degrees C in ambient fluorescent light. Physical and chemical stability were evaluated at 0, 24, 48, and 72 hours. Physical stability was assessed by visual observation in fluorescent light and using a high-intensity monodirectional light beam. Turbidity and particle content were measured electronically. Chemical stability of fenoldopam mesylate was evaluated by using a stability-indicating high-performance liquid chromatographic analytical technique. All samples were physically stable throughout the study. Little or no change in particulate burden and haze level was found. Little or no loss of fenoldopam occurred in concentrations of 40 micrograms/mL or greater at either temperature throughout the study period. At the lowest concentrations, minor losses of 3-5% were found. Fenoldopam mesylate, prepared in fenoldopam concentrations ranging from 4 to 300 micrograms/mL, was physically and chemically stable in PVC bags of 5% dextrose and 0.9% sodium chloride injections for 72 hours when stored at 4 degrees C in the dark and 23 degrees C when exposed to ambient fluorescent light.

Development of controlled-release SK&F 82526-J buffer bead formulations with tartaric acid as the buffer.

The purpose of this research was to design and develop a novel controlled-release bead formulation for oral administration with buffer crystals as a carrier for loading of fenoldopam mesylate, an intravenous antihypertensive agent, which provides an in vitro release rate of 30-50 mg/hr for 6-8 hr. Buffer crystals were coated in a fluid-bed granulator with a blend of gastrointestinal (GI) insoluble/enteric Eudragit polymers (such as RSPM/S100 polymer blend), drug was layered on these polymer-subcoated buffer beads by a slurry coating process, and the drug-layered beads were subsequently overcoated using a blend of GI insoluble/enteric polymers. The release of fenoldopam and tartaric acid was monitored by a two-stage dissolution procedure using USP Apparatus 2 (paddles at 50 rpm) and HPLC methodologies. The overcoating of drug-layered tartaric acid crystals with Eudragit polymers with different permeabilities significantly affected the release of fenoldopam. However, even the least permeable polymer, Eudragit RS, could not sustain the release of tartaric acid beyond 2 hr, suggesting the need to subcoat freely water-soluble tartaric acid crystals prior to drug layering. By varying the type/ratios of the GI insoluble/enteric polymers for subcoat and overcoat, capsule formulations were developed, which released fenoldopam and tartaric acid at different rates. The use of Eudragit polymer blends at the optimized ratios for both subcoat and overcoat resulted in a significant retardation in the release of tartaric acid; still, the tartaric acid release was faster than that of fenoldopam, suggesting the need to use a less water-soluble fumaric or succinic acid crystals as the buffer. However, the technology described using tartaric acid crystals as the buffer will be highly useful for weakly basic drug substances with less stringent pH-dependent solubility profiles.

The pH-independent release of fenoldopam from pellets with insoluble film coats.

Various ratios of succinic acid to fenoldopam mesylate, ranging from 0:1 to 18:1 were incorporated in pellets and coated with 1.5-12% w/w Surelease. Even though the coating level did influence the rate and amount of fenoldopam release, the influence of the succinic acid to drug ratio was much more important and evident at all coating levels. Being a weakly basic drug, fenoldopam release ceased when testing in SIF for succinic acid to drug ratios of 0:1-4:1, with the end of release being more abrupt for the 0:1 than for the 4:1 ratio. Only for a succinic acid to drug ratio of > or =5 was fenoldopam release constant for 6-8 h and independent of the pH-value of dissolution media. For a thin coat of about 2.5% w/w Surelease, those pellets showed an ideal controlled release behaviour with release rates of about 5-10%/h and a total release of almost 80% in 8 h. The dissolution profiles of Surelease coated pellets with high succinic acid to drug ratios (> or =5) and different coating levels, were evaluated for best fits to commonly used kinetic models. Sustained release mechanisms are discussed according to best fit models. The quantification of the pH-adjuster succinic acid, released from pellets with an acid to drug ratio of < or =1 showed, that despite their failure as a controlled release system for fenoldopam, the investigated coats could control the release of succinic acid effectively at optimized coating levels. For increasing succinic acid to drug ratios (< or =4) succinic acid was released at an ever more constant rate and release rates, though still faster than the release rates of fenoldopam, decreased steadily for increasing ratios. At a 5:1 ratio finally release rates of succinic acid and fenoldopam were almost identical. Therefore those pellet cores were almost completely emptied during dissolution testing, with both fenoldopam and succinic acid leaving at a constant rate and a total release of about 80% each for a 2.5% Surelease coat, while lower succinic acid to drug ratios had failed to show any sustained release for such thin Surelease coats. A similar formulation with fumaric acid instead of succinic acid failed to show the desired release pattern, indicating that it is the presence of a sufficiently high amount of succinic acid rather than the presence of an acidic compound in general, that ensures fenoldopam solubility at higher pH-values.