Identification of the Hypertension Drug Guanfacine as an Antivirulence Agent in Pseudomonas aeruginosa.

An alternative solution to the cyclical development of new antibiotics is the concept of disarming pathogens without affecting their growth, thereby eliminating the selective pressures that lead to resistant phenotypes. Here, we have employed our previously developed HiTES methodology to identify one such compound against the ESKAPE pathogen Pseudomonas aeruginosa. Rather than induce silent biosynthetic gene clusters, we used HiTES to suppress actively expressed virulence genes. By screening a library of 770 FDA-approved drugs, we identified guanfacine, a clinical hypertension drug, as an antivirulence agent in P. aeruginosa. Follow-up studies showed that guanfacine reduces biofilm formation and pyocycanin production without altering growth. Moreover, we identified a homologue of QseC, a sensor His kinase used by multiple pathogens to turn on virulence, as a target of guanfacine. Our studies suggest that guanfacine might be an attractive antivirulence lead in P. aeruginosa and provide a template for uncovering such molecules by screening for downregulators of actively expressed biosynthetic genes.

Cytokine IL-10, activators of PI3-kinase, agonists of α-2 adrenoreceptor and antioxidants prevent ischemia-induced cell death in rat hippocampal cultures.

In the present work we compared the protective effect of anti-inflammatory cytokine IL-10 with the action of a PI3-kinase selective activator 740 Y-P, selective agonists of alpha-2 adrenoreceptor, guanfacine and UK-14,304, and compounds having antioxidant effect: recombinant human peroxiredoxin 6 and B27, in hippocampal cell culture during OGD (ischemia-like conditions). It has been shown that the response of cells to OGD in the control includes two phases. The first phase was accompanied by an increase in the frequency of spontaneous synchronous Ca2+-oscillations (SSCO) in neurons and Ca2+-pulse in astrocytes. Spontaneous Ca2+ events in astrocytes during ischemia in control experiments disappeared. The second phase started after a few minutes of OGD and looked like a sharp/avalanche, global synchronic (within 20 s) increase in [Ca2+]i in many cells. Within 1 h after OGD, a mass death of cells, primarily astrocytes, was observed. To study the protective action of the compounds, cells were incubated in the presence of the neuroprotective agents for 10-40 min or 24 h before ischemia. All the neuroprotective agents delayed a global [Ca2+]i increase during OGD or completely inhibited this process and increased cell survival.

Alpha-2A Adrenoceptor Agonist Guanfacine Restores Diuretic Efficiency in Experimental Cirrhotic Ascites: Comparison with Clonidine.

BACKGROUND: In human cirrhosis, adrenergic hyperfunction causes proximal tubular fluid retention and contributes to diuretic-resistant ascites, and clonidine, a sympatholytic drug, improves natriuresis in difficult-to-treat ascites. AIM: To compare clonidine (aspecific α2-adrenoceptor agonist) to SSP-002021R (prodrug of guanfacine, specific α2A-receptor agonist), both associated with diuretics, in experimental cirrhotic ascites. METHODS AND RESULTS: Six groups of 12 rats were studied: controls (G1); controls receiving furosemide and potassium canrenoate (G2); rats with ascitic cirrhosis due to 14-week CCl4 treatment (G3); cirrhotic rats treated (over the 11th-14th CCl4 weeks) with furosemide and canrenoate (G4), furosemide, canrenoate and clonidine (G5), or diuretics and SSP002021R (G6). Three rats of each group had their hormonal status and renal function assessed at the end of 11th, 12th, 13th, and 14th weeks of respective treatments.Cirrhotic rats in G3 and G4 gained weight over the 12th-14th CCl4 weeks. In G4, brief increase in sodium excretion over the 11th-12th weeks preceded worsening of inulin clearance and natriuresis (diuretic resistance). In comparison with G4, the addition of clonidine (G5) or guanfacine (G6) to diuretics improved, respectively, sodium excretion over the 11th-12th CCl4 weeks, or GFR and electrolytes excretion over the 13th-14th CCl4 weeks. Natriuretic responses in G5 and G6 were accompanied by reduced catecholamine serum levels. CONCLUSIONS: α2A-receptor agonists restore glomerular filtration rate and natriuresis, and delay diuretic-resistant ascites in experimental advanced cirrhosis. Clonidine ameliorates diuretic-dependent natriuresis just for a short time.

Guanfacine for the treatment of attention deficit hyperactivity disorder in children and adolescents.

Guanfacine is an α2A-adrenoreceptor agonist currently indicated for the treatment of attention deficit hyperactivity disorder (ADHD). This article reviews the chemistry, pharmacodynamics and pharmacokinetics of guanfacine, as well as the clinical trial literature on guanfacine for the treatment of ADHD in children and adolescents, mainly focusing on the use of guanfacine extended-release (GXR). Six already published prospective randomized controlled trials (RCTs) and one unpublished RCT study were identified for GXR in the treatment of ADHD. All RCTs trials showed superiority over placebo on the primary outcome measure. Guanfacine, especially XR, seems to be an effective and safe treatment option for ADHD in children and adolescents.

Pharmacokinetics and pharmacodynamics of guanfacine extended release in adolescents aged 13-17 years with attention-deficit/hyperactivity disorder.

The safety and efficacy of guanfacine extended release (up to 4 mg/day) for attention-deficit/hyperactivity disorder (ADHD) in children and adolescents aged 6-17 years is well documented. Data suggest that weight-adjusted doses of guanfacine extended release >0.08 mg/kg but ≤0.12 mg/kg, if tolerated, may provide additional clinical benefits. For many adolescents, such dosing would exceed 4 mg/day, the highest approved dose. This open-label multicenter study evaluated the safety, tolerability, and steady-state pharmacokinetics of guanfacine extended release at escalated forced doses ≤9 mg/day in adolescents (N = 31) aged 13-17 years with ADHD. Following doses of approximately 0.12 mg/kg, the highest weight group (>70-90 kg) exhibited lower mean clearance at steady-state than the lowest weight group (≥30-50 kg). Consistent with its known antihypertensive effects, guanfacine extended release was associated with dose-dependent decreases in blood pressure (BP) and heart rate (HR). The physiologic response of increased BP upon standing was blunted in a dose-related manner while the physiologic response of increased HR upon standing was not substantively affected. The most common treatment-emergent adverse events were somnolence, dizziness, and sinus bradycardia. These results, and those from prior studies, support further examination of the efficacy and safety of higher weight-adjusted doses of guanfacine extended release for ADHD.

Development and validation of a simple, sensitive and accurate LC-MS/MS method for the determination of guanfacine, a selective α2A -adrenergicreceptor agonist, in plasma and its application to a pharmacokinetic study.

A simple, practical, accurate and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for the quantitation of guanfacine in beagle dog plasma. After protein precipitation by acetonitrile, the analytes were separated on a C18 chromatographic column by methanol and water containing 0.1% (v/v) formic acid with a gradient elution. The subsequent detection utilized a mass spectrometry under positive ion mode with multiple reaction monitoring of guanfacine and enalaprilat (internal standard) at m/z 246.2 → 159.0 and m/z 349.2 → 205.9, respectively. Good linearity was obtained over the concentration range of 0.1-20 ng/mL for guanfacine in dog plasma and the lower limit of quantification of this method was 0.1 ng/mL. The intra- and inter-day precisions were <10.8% relative standard deviation with an accuracy of 92.9-108.4%. The matrix effects ranged from 89.4 to 100.7% and extraction recoveries were >90%. Stability studies showed that both analytes were stable during sample preparation and analysis. The established method was successfully applied to an in vivo pharmacokinetic study in beagle dogs after a single oral dose of 4 mg guanfacine extended-release tablets.

Adjuvants to prolong the local anesthetic effects of coated microneedle products.

The objective of this study was to identify an adjuvant for anesthetics coated on microneedles to provide rapid onset and prolonged analgesic action with minimal skin tissue reaction. Aqueous lidocaine or prilocaine formulations with or without clonidine or the related analogs, guanfacine and apraclonidine, were dip-coated onto polymeric microneedles. The amount of lidocaine or prilocaine coated onto the microneedles was assessed by high performance liquid chromatography (HPLC). Delivery efficiency and dermal pharmacokinetics associated with lidocaine or prilocaine delivered via the microneedles were characterized in vivo using domestic swine. Skin punch biopsies were collected and analyzed to determine the anesthetic concentrations in the skin using HPLC-mass spectrometry (LC-MS). Addition of clonidine to the formulations decreased the systemic absorption rate of the anesthetics from the patch application site without impacting the coating performance or the rapid onset of anesthesia. Formulations with 0.3 wt.% clonidine, identified as the optimal dose for lidocaine-delivery via microneedles, maintained the lidocaine skin concentration above the estimated therapeutic level (100 ng/mg) for 1 h without causing any skin irritation or color change. The other two clonidine analogs, guanfacine and apraclonidine, also led to delayed systemic absorption of lidocaine from the skin, indicating utility in providing prolonged analgesia.

A novel method for the determination of guanfacine in urine by gas chromatography-mass spectrometry.

Guanfacine (Tenex), an antihypertensive available since 1975, has recently been indicated for the treatment of attention deficit hyperactivity disorder in children (Intuniv). Because of this new usage, a gas chromatography-mass spectrometry method was developed and validated for the determination of guanfacine in urine. Guanfacine and 100 ng of protriptyline (internal standard) were extracted from 1.0 mL urine with 0.5 mL of saturated carbonate/bicarbonate buffer and 2 mL of ethyl acetate. The solvent extract was evaporated and derivatized with heptaflurobutyric anhydride in n-butyl chloride. Chromatographic separation was achieved using a DB-5 capillary column (30 m x 0.32 mm, 0.25 microm). Ions monitored for guanfacine were m/z 86.1, 272.1, and 274.1, and ions monitored for protriptyline were m/z 191.1 and 189.1. Concentrations were determined using calibrators over the range of 0.1-2.0 mg/L. The linear regression for all calibration curves had r2 values > or = 0.99. The limit of detection was 0.05 mg/L; limit of quantitation was 0.1 mg/L; and upper limit of linearity was 10.0 mg/L. Percent recovery of guanfacine at 0.1 and 2.0 mg/L was 93% and 71%, respectively. The method was found acceptable for routine quantitative analysis of guanfacine in urine.

Heme coordination states of unfolded ferrous cytochrome C.

The structural changes of ferrous Cyt-c that are induced by binding to SDS micelles, phospholipid vesicles, DeTAB, and GuHCl as well as by high temperatures and changes in the pH have been studied by RR and UV-Vis absorption spectroscopies. Four species have been identified in which the native methionine-80 ligand is removed from the heme iron. This coordination site is either occupied by a histidine (His-33 or His-26) to form a 6cLS configuration, which is the prevailing species in GuHCl at pH 7.0 and ambient temperature, or remains vacant to yield a 5cHS configuration. The three identified 5cHS species differ with respect to the hydrogen-bond interactions of the proximal histidine ligand (His-18) and include a nonhydrogen-bonded, a hydrogen-bonded, and a deprotonated imidazole ring. These structural motifs have been found irrespective of the unfolding conditions used. An unambiguous spectroscopic distinction of these 5cHS species is possible on the basis of the Fe-N(imidazole) stretching vibrations, the RR bands in the region between 1300 and 1650 cm(-1), and the electronic transitions in the Soret- and Q-band regions. In acid and neutral solutions, the species with a hydrogen-bonded and a nonhydrogen-bonded His-18 prevail, whereas in alkaline solutions a configuration with a deprotonated His-18 ligand is also observed. Upon lowering the pH or increasing the temperature in GuHCl solutions, the structure on the proximal side of the heme is perturbed, resulting in a loss of the hydrogen-bond interactions of the His-18 ligand. Conversely, the hydrogen-bonded His-18 of ferrous Cyt-c is stabilized by electrostatic interactions which increase in strength from phospholipid vesicles to SDS micelles. The results here suggest that unfolding of Cyt-c is initiated by the rupture of the Fe-Met-80 bond and structural reorganizations on the distal side of the heme pocket, whereas the proximal part is only affected in a later stage of the denaturation process.