Design of a silicone reservoir intravaginal ring for the delivery of oxybutynin.

Oxybutynin, a drug of choice in the treatment of urinary incontinence, has low oral bioavailability due to extensive first-pass metabolism. A toxic metabolite, N-desethyloxybutynin, has been linked to adverse reactions to oral oxybutynin. This study, therefore, reports on the design of an oxybutynin intravaginal ring (IVR) of reservoir design, comprising an oxybutynin silicone elastomer core encased in a non-medicated silicone sheath, manufactured by reaction injection moulding at 50oC. An unusually high initial burst release of oxybutynin (42.7 mg in 24 h) was observed in vitro with a full length core (100 mg drug loading), with subsequent non-zero order drug release. Use of fractional segment cores substantially reduced the burst effect, yielding linear cumulative drug release versus time plots from days 2 to 14. Thus, a 1/8 fractional segment core gave a 24 h burst of 11.28 mg oxybutynin and, thereafter, zero order release at the target dose of 5 mg/day over 14 days. Two oxybutynin cores, each 1/16 of full length, gave a greater release than a single 1/8 core, due to core segment end effects resulting in an increased surface area for release. The burst release was investigated by determining drug solubilities in the propan-1-ol product of elastomer condensation cure (390 mg/ml) and in the elastomer itself (13.9-20.21 mg/ml, by direct extraction and indirect thermal methods). These high oxybutynin solubilities were considered the major contributors to the burst effect. It was concluded that use of a fractional segment core would allow development of a suitable oxybutynin reservoir IVR.

Examination of surface properties and in vitro biological performance of amorphous diamond-like carbon-coated polyurethane

Despite the emerging use of diamond-like carbon (DLC) as a coating for medical devices, few studies have examined the resistance of DLC coatings onto medical polymers to both microbial adherence and encrustation. In this study, amorphous DLC of a range of refractive indexes (1.7-1.9) and thicknesses (100-600 nm) was deposited onto polyurethane, a model polymer, and the resistance to microbial adherence (Escherichia coli; clinical isolate) and encrustation examined using in vitro models. In comparison to the native polymer, the advancing and receding contact angles of DLC-coated polyurethane were lower, indicating greater hydrophilic properties. No relationship was observed between refractive index, thickness, and advancing contact angle, as determined using multiple correlation analysis. The resistances of the various DLC-coated polyurethane films to encrustation and microbial adherence were significantly greater than that to polyurethane; however, there were individual differences between the resistances of the various DLC coatings. In general, increasing the refractive index of the coatings (100 nm thickness) decreased the resistance of the films to both hydroxyapatite and struvite encrustation and to microbial adherence. Films of lower thicknesses (100 and 200 nm; of defined refractive index, 1.8), exhibited the greatest resistance to encrustation and to microbial adherence. In conclusion, this study has uniquely illustrated both the microbial antiadherence properties and resistance to urinary encrustation of DLC-coated polyurethane. The resistances to encrustation and microbial adherence were substantial, and in light of this, it is suggested that DLC coatings of low thickness and refractive index show particular promise as coatings of polymeric medical devices. (c) 2006 Wiley Periodicals, Inc.

Evaluation of a poly(vinyl pyrollidone)-coated biomaterial for urological use

The associated problems of bacterial biofilm formation and encrustation that may cause obstruction or blockage of urethral catheters and ureteral stents often hinders the effective use of biomaterials within the urinary tract. In this in vitro study, we have investigated the surface properties of a hydrophilic polyvinyl pyrollidone) (PVP)-coating applied to polyurethane and determined its suitability for use as a urinary tract biomaterial by comparing its lubricity and ability to resist bacterial adherence and encrustation with that of uncoated polyurethane and silicone. The PVP-coated polyurethane was significantly more hydrophilic and more lubricious than either uncoated polyurethane or silicone. Adherence of a hydrophilic Escherichia coli isolate to PVP-coated polyurethane and uncoated polyurethane was similar but significantly less than adherence to silicone. Adherence of a hydrophobic Enterococcus faecalis isolate to PVP-coated polyurethane and silicone was similar but was significantly less than adherence to uncoated polyurethane. Struvite encrustation was similar on the PVP-coated polyurethane and silicone but significantly less than on uncoated polyurethane. Furthermore, hydroxyapatite encrustation was significantly less on the PVP-coated polyurethane than on either uncoated polyurethane or silicone. The results suggest that the PVP-coating could be useful in preventing complications caused by bacterial biofilm formation and the deposition of encrustation on biomaterials implanted in the urinary tract and, therefore, warrants further evaluation. (C) 2002 Elsevier Science Ltd. All rights reserved.

Oxaliplatin induces hyperexcitability at motor and autonomic neuromuscular junctions through effects on voltage-gated sodium channels

Oxaliplatin, an effective cytotoxic treatment in combination with 5-fluorouracil for colorectal cancer, is associated with sensory, motor and autonomic neurotoxicity. Motor symptoms include hyperexcitability while autonomic effects include urinary retention, but the cause of these side-effects is unknown. We examined the effects on motor nerve function in the mouse hemidiaphragm and on the autonomic system in the vas deferens. In the mouse diaphragm, oxaliplatin (0.5 mM) induced multiple endplate potentials (EPPs) following a single stimulus, and was associated with an increase in spontaneous miniature EPP frequency. In the vas deferens, spontaneous excitatory junction potential frequency was increased after 30 min exposure to oxaliplatin; no changes in resting Ca(2+) concentration in nerve terminal varicosities were observed, and recovery after stimuli trains was unaffected.In both tissues, an oxaliplatin-induced increase in spontaneous activity was prevented by the voltage-gated Na(+) channel blocker tetrodotoxin (TTX). Carbamazepine (0.3 mM) also prevented multiple EPPs and the increase in spontaneous activity in both tissues. In diaphragm, beta-pompilidotoxin (100 microM), which slows Na(+) channel inactivation, induced multiple EPPs similar to oxaliplatin's effect. By contrast, blockers of K(+) channels (4-aminopyridine and apamin) did not replicate oxaliplatin-induced hyperexcitability in the diaphragm. The prevention of hyperexcitability by TTX blockade implies that oxaliplatin acts on nerve conduction rather than by effecting repolarisation. The similarity between beta-pompilidotoxin and oxaliplatin suggests that alteration of voltage-gated Na(+) channel kinetics is likely to underlie the acute neurotoxic actions of oxaliplatin.

Effect of purinergic blockers on outward current in isolated smooth muscle cells of the sheep bladder

Freshly dispersed cells from sheep urinary bladder were voltage clamped using the whole cell and inside-out patch-clamp technique. Cibacron and Basilen blue increased outward current in a dose-dependent manner with a half-maximal response at 10(-5) M. Suramin, in concentrations to 10(-3) M, had no such effect. The Cibacron blue response was abolished in Ca2+-free physiological salt solution, suggesting that it was acting on a Ca2+-dependent current. Similarly, the Cibacron blue-sensitive current was significantly attenuated by charybdotoxin. Cibacron blue did not modulate inward current nor were its effects modified by caffeine or heparin, suggesting that its effect on outward current was not secondary to an increase in intracellular Ca2+. Application of 10(-4) M Cibacron blue to the inside membrane of excised patches caused a rapid increase in open probability of a large-conductance (300 pS) K+ channel. These results suggest that Cibacron blue is a potent activator of a Ca2+-dependent outward current in bladder smooth muscle cells in addition to its action as a purinergic blocker.

The relationship of clinical and inflammatory markers to outcome in stable patients with cystic fibrosis

Decreased survival in patients with cystic fibrosis has been related to FEV1, BMI, and infection with Burkholderia cepacia complex (BCC). We have assessed the relationship of blood, sputum, and urine inflammatory markers to lung function, BMI, colonization with B cenocepacia (Bc), and patient survival. Thirty-nine stable cystic fibrosis (CF) patients (10 with Bc) were enrolled in a study to determine the effect of alpha-1-antitrypsin on airways inflammation. Pre-treatment measurements were used in this study. Demographics, sputum microbiology, heart rate, oxygen saturation, lung function were recorded. Blood samples were obtained for white blood count (WBC), C-Reactive Protein (CRP), and plasma neutrophil elastase/AAT complexes (pNEC). Neutrophil elastase (NE), neutrophil elastase/AAT complexes (sNEC), interleukin-8 (IL-8), TNF-receptor 1 (sTNFr), and myeloperoxidase (MPO) were measured in sputum and urinary desmosine concentration determined. Patients with Bc had significantly higher levels of pNEC, 332?±?91.4 ng/ml (mean?±?SEM) versus 106?±?18.2 ng/ml (P?=?0.0005) and sNEC, 369?±?76.6 ng/ml versus 197?±?36.0 ng/ml compared to those who were not. Five deaths were reported at the end of 1 year, (four with Bc) (P?=?0.011). Patients who subsequently died had significantly lower lung function FEV1, 1.2?±?0.2 L versus 2.0?±?0.1 L (P?=?0.03) and FVC, 2?±?0.3 L versus 3.1?±?0.2 L (P?=?0.01), compared to those that survived. There was significantly higher NE activity, 3.6?±?1.6 U/ml versus 1.5?±?0.6 U/ml (P?=?0.03), pNEC, 274?±?99 ng/ml versus 142?±?30 ng/ml (P?=?0.05), MPO, 163?±?62 mcg/ml versus 54?±?6.9 mcg/ml (P?=?0.03), and urinary desmosines 108?±?19.9 pM/mg creatinine versus 51.1?±?3.3 pM/mg creatinine (P?=?0.001), in those patients who subsequently died compared to those that survived. These data suggest there is increased neutrophil degranulation in patients infected with Bc and these patients have a poor outcome.

Transient receptor potential melastatin 8 (TRPM8) channel involvement in the regulation of vascular tone

The transient receptor potential melastatin 8 (TRPM8) channel has been characterized as a cold and menthol receptor expressed in a subpopulation of sensory neurons but was recently identified in other tissues, including the respiratory tract, urinary system, and vasculature. Thus TRPM8 may play multiple functional roles, likely to be in a tissue- and activation state-dependent manner. We examined the TRPM8 channel presence in large arteries from rats and the functional consequences of their activation. We also aimed to examine whether these channels contribute to control of conscious human skin blood flow. TRPM8 mRNA and protein were detected in rat tail, femoral and mesenteric arteries, and thoracic aorta. This was confirmed in single isolated vascular myocytes by immunocytochemistry. Isometric contraction studies on endothelium-denuded relaxed rat vessels found small contractions on application of the TRPM8-specific agonist menthol (300 microM). However, both menthol and another agonist icilin (50 microM) caused relaxation of vessels precontracted with KCl (60 mM) or the alpha-adrenoceptor agonist phenylephrine (2 microM) and a reduction in sympathetic nerve-mediated contraction. These effects were antagonized by bromoenol lactone treatment, suggesting the involvement of Ca(2+)-independent phospholipase A(2) activation in TRPM8-mediated vasodilatation. In thoracic aorta with intact endothelium, menthol-induced inhibition of KCl-induced contraction was enhanced. This was unaltered by preincubation with either N(omega)-nitro-l-arginine methyl ester (l-NAME; 100 nM), a nitric oxide synthase inhibitor, or the ACh receptor antagonist atropine (1 microM). Application of menthol (3% solution, topical application) to skin caused increased blood flow in conscious humans, as measured by laser Doppler fluximetry. Vasodilatation was markedly reduced or abolished by prior application of l-NAME (passive application, 10 mM) or atropine (iontophoretic application, 100 nM, 30 s at 70 microA). We conclude that TRPM8 channels are present in rat artery vascular smooth muscle and on activation cause vasoconstriction or vasodilatation, dependent on previous vasomotor tone. TRPM8 channels may also contribute to human cutaneous vasculature control, likely with the involvement of additional neuronal mechanisms.

PdT-2: a novel myotropic Type-2 tryptophyllin from the skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor.

Amphibian skin secretions represent a unique resource for the discovery of new bioactive peptides. Here we report the isolation, structural and functional characterization of a novel heptapeptide amide, DMSPPWHamide, from the defensive skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor. This peptide is of unique primary structure and has been classified as a member of the rather heterogenous tryptophyllin-2 (T-2) family of amphibian skin peptides and named P. dacnicolor Tryptophyllin-2 (PdT-2) in accordance. PdT-2 is the first Type 2-tryptophyllin to possess discrete bioactivity. Both natural and synthetic replicates of the peptide were found to contract the smooth muscle of rat urinary bladder, the latter displaying an EC50 of 4 nM.

Sauvatide - a novel amidated myotropic decapeptide from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei.

Here we describe the structural and functional characterization of a novel myotropic peptide, sauvatide, from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei. Sauvatide is a C-terminally amidated decapeptide with the following primary structure – LRPAILVRTKamide – monoisotopic mass 1164.77 Da, which was found to contract the smooth muscle of rat urinary bladder with an EC50 of 2.2 nM. The sauvatide precursor, deduced from cloned skin cDNA, consists of 62 amino acid residues with a single copy of sauvatide located near the C-terminus. The mature peptide is generated from the precursor by cleavage at a classical –KR-cleavage site located proximal to the N-terminus and by removal of a –GKGK sequence at the C-terminus, the first glycyl residue acting as amide donor. Amphibian skin secretions thus continue to be a source of novel and potent biologically active peptides acting through functional targets in mammalian tissues.

The Effects of Cilostazol on Exercise-induced Ischaemia-reperfusion Injury in Patients with Peripheral Arterial Disease

Objectives: Cilostazol improves walking distance in peripheral arterial disease (PAD) patients. The study objectives were to assess the effects of cilostazol on walking distance, followed by the additional assessment of cilostazol on exercise-induced ischaemiaereperfusion injury in such patients.

Methods: PAD patients were prospectively recruited to a double-blinded, placebo-controlled trial. Patients were randomised to receive either cilostazol 100 mg or placebo twice a day. The primary end-point was an improvement in walking distance. Secondary end-points included the assessment of oxygen-derived free-radical generation, antioxidant consumption and other markers of the in?ammatory cascade. Initial and absolute claudication distances (ICDs and ACDs, respectively) were measured on a treadmill. In?ammatory response was assessed before and 30 min post-exercise by measuring lipid hydroperoxide, ascorbate, atocopherol, b-carotene, P-selectin, intracellular and vascular cell-adhesion molecules (I-CAM and V-CAM), thromboxane B2 (TXB2), interleukin-6, interleukin-10, high-sensitive C-reactive protein (hsCRP), albuminecreatinine ratio (ACR) and urinary levels of p75TNF receptor. All tests were performed at baseline and 6 and 24 weeks.

Results: One hundred and six PAD patients (of whom 73 were males) were recruited and successfully randomised from December 2004 to January 2006. Patients who received cilostazol demonstrated a more signi?cant improvement in the mean percentage change from baseline in ACD (77.2% vs. 26.6% at 6 weeks, pZ0.026 and 161.7% vs. 79.0% at 24 weeks, pZ0.048) as compared to the placebo. Cilostazol reduced lipid hydroperoxide levels compared to a placebo-related increase before and after exercise (6 weeks: pre-exercise: 11.8% vs.

GK-22 amide: a novel myotropic peptide from the skin secretion of the bamboo leaf odorous frog, Odorrana versabilis

The skin secretions produced by many amphibians are formidable chemical/biological weapons deployed as a defence against predators. Bioactive peptides are often the predominant class of biochemical within these secretions and the inventory of such remains incomplete with each individual taxon producing unique cocktails contained within which are some signature peptides, such as bradykinins and tachykinins. These secretions have been the source of many peptides subsequently found to have structural homologues in vertebrate neuroendocrine systems (bombesin/GRP; sauvagine/CRF; caerulein/CCK) and vice versa (bradykinin, CGRP, NMU). They are thus unequivocally intriguing resources for novel bioactive peptide discovery. Here we describe a novel 22-mer amidated peptide, named GK-22 amide (N-terminal Gly (G) and C-terminal Lys (K) amide) with an internal disulphide bridge between Cys (C) 11 and 20 from the skin secretion of Odorrana versabilis. Molecular cloning indicated that it is encoded as a single copy on a biosynthetic precursor of 59 amino acid residues consisting of a signal peptide, an acidic amino acid residue-rich spacer domain and a mature peptide encoding domain flanked N-terminally by a classical -Lys-Arg- (KR) propeptide convertase processing site and C-terminally by a Gly (G) residue amide donor. A synthetic replicate of this peptide produced potent and dose-dependent contraction of the smooth muscle of rat urinary bladder. GK-22 amide thus represents the prototype of a novel class of myotropic peptide from amphibian skin and its discovery illustrates the continuing potential of this resource to this end.

The use of lytic bacteriophages in the prevention and eradication of biofilms of Proteus mirabilis and Escherichia coli.

Antibiotics have been the cornerstone of the clinical management of bacterial infections since their discovery in the early part of the last century. Eight decades later, their widespread, often indiscriminate use, has resulted in an overall reduction in their effectiveness, with reports of multidrug-resistant bacteria now commonplace. Increasing reliance on indwelling medical devices, which are inherently susceptible to biofilm-mediated infections, has contributed to unacceptably high rates of nosocomial infections, placing a strain on healthcare budgets. This study investigates the use of lytic bacteriophages in the treatment and prevention of biofilms of bacterial species commonly associated with infections of indwelling urological devices and catheter-associated urinary tract infections. The use of lytic bacteriophages against established biofilms of Proteus mirabilis and Escherichia coli is described, whereby biofilm populations have been reduced successfully by three to four log cycles (99.9-99.99% removal). The prevention of biofilm formation on Foley catheter biomaterials following impregnation of hydrogel-coated catheter sections with a lytic bacteriophage has also been investigated. This has revealed an approximate 90% reduction in both P. mirabilis and E. coli biofilm formation on bacteriophage-treated catheters when compared with untreated controls.

Kassorins: Novel innate immune system peptides from skin secretions of the African hyperoliid frogs, Kassina maculata and Kassina senegalensis

From defensive skin secretions acquired from two species of African hyperoliid frogs, Kassina maculata and Kassina senegalensis, we have isolated two structurally related, C-terminally amidated tridecapeptides of novel primary structure that exhibit a broad spectrum of biological activity. In reflection of their structural novelty and species of origin, we named the peptides kassorin M (FLEGLLNTVTGLLamide; 1387.8 Da) and kassorin S (FLGGILNTITGLLamide; 1329.8 Da), respectively. The primary structure and organisation of the biosynthetic precursors of kassorins M and S were deduced from cloned skin secretion-derived cDNA. Both open-reading frames encoded a single copy of kassorin M and S, respectively, located at the C-terminus. Kassorins display limited structural similarities to vespid chemotactic peptides (7/13 residues), temporin A (5/13 residues), the N-terminus of Lv-ranaspumin, a foam nest surfactant protein of the frog, Leptodactylus vastus, and an N-terminal domain of the equine sweat surfactant protein, latherin. Both peptides elicit histamine release from rat peritoneal mast cells. However, while kassorin S was found to possess antibacterial activity against Staphylococcus aureus, kassorin M was devoid of such activity. In contrast, kassorin M was found to contract the smooth muscle of guinea pig urinary bladder (EC50 = 4.66 nM) and kassorin S was devoid of this activity. Kassorins thus represent the prototypes of a novel family of peptides from the amphibian innate immune system as occurring in defensive skin secretions.

Genetic Polymorphisms in Nitric Oxide Synthase 3 Gene and Implications for Kidney Disease: A Meta-Analysis

Background/Aims: The NOS3 gene is a biological and positional candidate for diabetic nephropathy. However, the relationship between NOS3 polymorphisms and renal disease is inconclusive. This study aimed to clarify the association of NOS3 variants with nephropathy in individuals with type 1 diabetes. Methods: We conducted a case-control study examining all common SNPs in the NOS3 gene by a tag SNP approach. Individuals with type 1 diabetes and persistent proteinuria (cases, n = 718) were compared with individuals with type 1 diabetes but no evidence of renal disease (controls, n = 749). Our replication collection comprised 1,105 individuals with type 1 diabetes recruited to a nephropathy case group and 862 control individuals with normal urinary albumin excretion rates. Meta-analysis was conducted for SNPs where more than three genotype datasets were available. Results: A novel association was identified in the discovery collection (rs1800783, p(genotype) = 0.006, p(allele) = 0.002, OR = 1.26, 95% CI: 1.08-1.47) and supported by independent replication using a tag SNP (rs4496877, pairwise r(2) = 0.96 with rs1800783) in the replication collection (p(genotype) = 0.002, p(allele) = 0.0006, OR = 1.27, 95% CI: 1.10-1.45). Conclusion: The A allele of rs1800783 is a significant risk factor for nephropathy in individuals with type 1 diabetes, and further comprehensive studies are warranted to confirm the definitive functional variant in the NOS3 gene. Copyright (C) 2010 S. Karger AG, Basel