Preparation and comparison of chitosan nanoparticles with different degrees of glutathione thiolation

Background

Chitosan has gained considerable attentions as a biocompatible carrier to improve delivery of active agents. Application of this vehicle in the form of nanoparticle could profit advantages of nanotechnology to increase efficacy of active agents.The purpose of this study was to provide detailed information about chitosan–glutathione (Cht-GSH)nanoparticles which are gaining popularity because of their high mucoadhesive and extended drug release properties.

Methods

Depolymerization of chitosan was carried out using sodium nitrite method.Glutathione was covalently attached to chitosan and the solubility of the resulting conjugates was evaluated. Nanoparticles were prepared by ionic gelation method and then the effect of glutathione immobilization on properties of nanoparticles was investigated.

Results

Thiolation efficiency was higher in lower molecular weight chitosan polymers compared to unmodified chitosan nanoparticles. Cht-GSH conjugates of the same molecular weight but with different degrees of thiolation had the same hydrodynamic diameter (995± nm) and surface charge (102± mV) as unmodified chitosan, but comprised of a denser network structure and lower concentration. Cht-GSH nanoparticles also exhibited greater mucoadhesive strength which was less affected by ionic strength and pH of the environment.

Conclusion

Thiolation improves the solubility of chitosan without any significant changes in size and charge of nanoparticles, but affects the nanogel structure.     

Drug packaging and delivery using perfluorocarbon nanoparticles for targeted inhibition of vascular smooth muscle cells

Aim:

To investigate the in vitro release profile of drugs encapsulated within perfluorocarbon (PFC) nanoparticles (NPs) and their ability to inhibit the activity of vascular smooth muscle cells (SMCs).

Methods:

Dexamethasone phosphate (DxP) or dexamethasone acetate (DxA) was encapsulated into PFC nanoparticles using a high-pressure homogenous method. The morphology and size of the NPs were examined using scanning electron microscopy (SEM) and a laser particle size analyzer. Drug loading and in vitro release were assessed by high-performance liquid chromatography (HPLC). The impact of NP capsules on SMC proliferation, migration and apoptosis in vitro was assessed using cell counting kit-8, transwell cell migration and flow cytometry assays.

Results:

The sizes of DxP-NPs and DxA-NPs were 224±6 nm and 236±9 nm, respectively. The encapsulation efficiency (EE) of DxP-NPs was 66.4%±1.0%, with an initial release rate of 77.2%, whereas the EE of DxA-NPs was 95.3%±1.3%, with an initial release rate of 23.6%. Both of the NP-coated drugs could be released over 7 d. Human umbilical artery SMCs were harvested and cultured for four to six passages. Compared to free DxP, SMCs treated with tissue factor (TF)-directed DxP-NPs showed significant differences in the inhibition of proliferation, migration and apoptosis (P<0.05).

Conclusion:

The results collectively suggest that PFC nanoparticles will be beneficial for targeted drug delivery because of the sustained drug release and effective inhibition of SMC proliferation and migration.     

Novel gastroretentive sustained-release tablet of tacrolimus based on self-microemulsifying mixture: in vitro evaluation and in vivo bioavailability test

Aim:

To develop a novel gastroretentive drug delivery system based on a self-microemulsifying (SME) lipid mixture for improving the oral absorption of the immunosuppressant tacrolimus.

Methods:

Liquid SME mixture, composed of Cremophor RH40 and monocaprylin glycerate, was blended with polyethylene oxide, chitosan, polyvinylpyrrolidone and mannitol, and then transformed into tablets via granulation, with ethanol as the wetting agent. The tablets were characterized in respect of swelling, bioadhesive and SME properties. In vitro dissolution was conducted using an HCl buffer at pH 1.2. Oral bioavailability of the tablets was examined in fasted beagle dogs.

Results:

The tablet could expand to 13.5 mm in diameter and 15 mm in thickness during the initial 20 min of contact with the HCl buffer at pH 1.2. The bioadhesive strength was as high as 0.98±0.06 N/cm². The SME gastroretentive sustained-release tablets preserved the SME capability of the liquid SME formations under transmission electron microscope. The drug-release curve was fit to the zero-order release model, which was helpful in reducing fluctuations in blood concentration. Compared with the commercially available capsules of tacrolimus, the relative bioavailability of the SME gastroretentive sustained-release tablets was 553.4%±353.8%.

Conclusion:

SME gastroretentive sustained-release tablets can enhance the oral bioavailability of tacrolimus with poor solubility and a narrow absorption window.     

Degarelix, a novel GnRH antagonist, causes minimal histamine release compared with cetrorelix, abarelix and ganirelix in an ex vivo model of human skin samples

AIMS

Early studies on gonadotrophin-releasing hormone (GnRH) antagonists pointed out histamine-mediated anaphylactic reactions as a potential adverse effect of these drug candidates. In this study we have compared the histamine-releasing potential of four approved and marketed antagonists, degarelix, cetrorelix, abarelix and ganirelix in an ex vivo model of human skin samples.

METHODS

Human skin samples were obtained during cosmetic plastic surgery and kept in oxygenated saline solution. The samples were incubated either without or at different concentrations of the antagonists (3, 30 or 300 µg ml⁻¹ for all, except for ganirelix 1, 10 or 100 µg ml⁻¹). The drug-induced effect was expressed as the increase relative to basal release. The histamine-releasing capacity of the skin was verified by a universal histamine releaser, compound 40/80.

RESULTS

Degarelix had no significant effect on basal histamine release in the 3 to 300 µg ml⁻¹ concentration range. The effect of ganirelix was moderate causing a nonsignificant increase of 81 ± 27% at the 100 µg ml⁻¹ concentration. At 30 and 300 µg ml⁻¹ concentrations abarelix (143 ± 29% and 362 ± 58%, respectively, P < 0.05) and cetrorelix (228 ± 111% and 279 ± 46%, respectively, P < 0.05) caused significantly increased histamine release.

CONCLUSIONS

In this ex vivo human skin model, degarelix displayed the lowest capacity to release histamine followed by ganirelix, abarelix and cetrorelix. These findings may provide indirect hints as to the relative likelihood of systemic anaphylactic reactions in clinical settings.     

Cannabinoid CB(2) receptor attenuates morphine-induced inflammatory responses in activated microglial cells

BACKGROUND AND PURPOSE

Among several pharmacological properties, analgesia is the most common feature shared by either opioid or cannabinoid systems. Cannabinoids and opioids are distinct drug classes that have been historically used separately or in combination to treat different pain states. In the present study, we characterized the signal transduction pathways mediated by cannabinoid CB₂ and µ-opioid receptors in quiescent and LPS-stimulated murine microglial cells.

EXPERIMENTAL APPROACH

We examined the effects of µ-opioid and CB₂ receptor stimulation on phosphorylation of MAPKs and Akt and on IL-1β, TNF-α, IL-6 and NO production in primary mouse microglial cells.

KEY RESULTS

Morphine enhanced release of the proinflammatory cytokines, IL-1β, TNF-α, IL-6, and of NO via µ-opioid receptor in activated microglial cells. In contrast, CB₂ receptor stimulation attenuated morphine-induced microglial proinflammatory mediator increases, interfering with morphine action by acting on the Akt-ERK1/2 signalling pathway.

CONCLUSIONS AND IMPLICATIONS

Because glial activation opposes opioid analgesia and enhances opioid tolerance and dependence, we suggest that CB₂ receptors, by inhibiting microglial activity, may be potential targets to increase clinical efficacy of opioids.     

Enhancement of oral bioavailability of the poorly water-soluble drug silybin by sodium cholate/ phospholipid-mixed micelles

Aim:

To evaluate a mixed micellar drug delivery system composed of sodium cholate and phospholipid for oral administration of silybin, a promising hepatoprotectants.

Methods:

The optimum formulation of sodium cholate/phospholipid-mixed micelles containing silybin was obtained based on the study of pseudo-ternary phase diagram. The dissolution of silybin-mixed micelles was investigated. The pharmacokinetic characteristics and bioavailability after oral administration of silybin-mixed micelles and silybin-N-methylglucamine were compared in dogs.

Results:

The mean particle size of prepared mixed micelles was 75.9±4.2 nm. The largest solubility of silybin was found to be 10.0±1.1 mg/mL in the optimum formulation of mixed micelles. The silybin-sodium cholate/phospholipid-mixed micelles showed a very slow release of silybin 17.5% (w/w) within 72 h in phosphate buffer (pH 7.4) and 15.6% (w/w) in HCl solution (pH 1.2). After oral administration to dogs, the relative bioavailability of mixed micelles versus silybin-N-methylglucamine in dogs was 252.0%.

Conclusion:

Sodium cholate/phospholipid-mixed micelles are promising carriers in orally delivery of silybin, considering their capability of enhancing bioavailability and large-scale production.     

Role of 5-HT(1) receptor subtypes in the modulation of pain and synaptic transmission in rat spinal superficial dorsal horn

BACKGROUND AND PURPOSE

5-HT receptor agonists have variable nociceptive effects within the spinal cord. While there is some evidence for 5-HT_1A spinally-mediated analgesia, the role of other 5-HT₁ receptor subtypes remains unclear. In the present study, we examined the spinal actions of a range of 5-HT₁ agonists, including sumatriptan, on acute pain, plus their effect on afferent-evoked synaptic transmission onto superficial dorsal horn neurons.

EXPERIMENTAL APPROACH

For in vivo experiments, 5-HT agonists were injected via chronically implanted spinal catheters to examine their effects in acute mechanical and thermal pain assays using a paw pressure analgesymeter and a Hargreave''s device. For in vitro experiments, whole-cell patch-clamp recordings of primary afferent-evoked glutamatergic EPSC were made from lamina II neurons in rat lumbar spinal slices.

KEY RESULTS

Intrathecal (i.t.) delivery of the 5-HT_1A agonist R ± 8-OH-DPAT (30–300 nmol) produced a dose-dependent thermal, but not mechanical, analgesia. Sumatriptan and the 5-HT_1B, 5-HT_1D, 5-HT_1F agonists CP93129, PNU109291 and {"type":"entrez-nucleotide","attrs":{"text":"LY344864","term_id":"1257802930","term_text":"LY344864"}}LY344864 (100 nmol) had no effect on either acute pain assay. R ± 8-OH-DPAT (1 µM) and sumatriptan (3 µM) both reduced the amplitude of the evoked EPSC. In contrast, CP93129, PNU109291 and {"type":"entrez-nucleotide","attrs":{"text":"LY344864","term_id":"1257802930","term_text":"LY344864"}}LY344864 (0.3–3 µM) had no effect on the evoked EPSC. The actions of both R ± 8-OH-DPAT and sumatriptan were abolished by the 5-HT_1A antagonist WAY100635 (3 µM).

CONCLUSIONS AND IMPLICATIONS

These findings indicate that the 5-HT_1A receptor subtype predominantly mediates the acute antinociceptive and cellular actions of 5-HT₁ ligands within the rat superficial dorsal horn.     

Stability of celecoxib oral suspension

Background:

Celecoxib is a selective cyclo-oxygenase 2 inhibitor that relieves pain without affecting platelet function, causing gastrointestinal toxic effects, or increasing the risk of bleeding.

Objectives:

To develop a suspension formulation for oral celecoxib and to determine its physical and chemical stability when packaged in amber polyvinyl chloride (PVC) bottles and stored with refrigeration (5°C) and at room temperature (23°C).

Methods:

The contents of celecoxib capsules were used to prepare a single suspension, with Ora-Blend used as the suspending and flavouring agent. The suspension (10 mg/mL) was then packaged in amber PVC bottles and stored at either 5°C or 23°C. Samples were collected on days 0, 7, 14, 21, 27, 56, and 93. Chemical stability was determined using a validated stability-indicating high-performance liquid chromatography method. At each sampling time, the suspensions were checked visually for changes in appearance (i.e., colour, layering, caking, and ease of resuspension), odour, and pH.

Results:

All of the suspensions were stable for at least 93 days, regardless of storage conditions. There were no apparent changes in physical appearance, nor were there any substantial changes in odour or pH.

Conclusions:

Suspensions of celecoxib (10 mg/mL in Ora-Blend) packaged in amber PVC bottles were stable for up to 93 days when stored at 5°C or 23°C. A 3-month expiry date has been established for this oral suspension on the basis of physical compatibility and chemical stability.     

Stability of aseptically prepared tazocin solutions in polyvinyl chloride bags

Background:

Tazocin, a mixture of piperacillin and tazobactam, has recently been reformulated to include edetate disodium (EDTA) and citric acid. Since the introduction of this new formulation, there have been no studies of stability in polyvinylchloride (PVC) bags.

Objective:

To complete a physical compatibility and chemical stability study of the new formulation of Tazocin, prepared at 2 concentrations in each of 2 diluents and stored in PVC bags.

Methods:

Tazocin, at 22.5 or 90 mg/mL, was compounded in dextrose 5% in water (D5W) or 0.9% sodium chloride (normal saline [NS]) in PVC bags. The bags were stored at 5°C with protection from light for 14, 21, or 28 days, followed in each case by storage at 23°C with exposure to light for 72 h. Triplicate samples collected at each of the 7 time points were analyzed in duplicate using a stability-indicating high-performance liquid chromatography method. Physical compatibility was determined by monitoring the solutions for changes in colour, clarity, and pH.

Results:

The amount of each drug remaining for each concentration in each diluent was above 95% of the initial concentration after storage at 5°C with protection from light and above 94% of the initial concentration after an additional 72 h at 23°C with exposure to light. The pH of the solutions changed only slightly over the course of the study, and all solutions remained clear and colourless.

Conclusions:

Tazocin solutions at 22.5 and 90 mg/mL, prepared in PVC bags of either D5W or NS, were chemically stable after storage for up to 28 days at 5°C with protection from light followed by 72 h at 23°C with exposure to light.     

Mixed micelles loaded with silybin-polyene phosphatidylcholine complex improve drug solubility

Aim:

To prepare a novel formulation of phosphatidylcholine (PC)-bile salts (BS)-mixed micelles (MMs) loaded with silybin (SLB)-PC complex for parenteral applications.

Methods:

SLB-PC-BS-MMs were prepared using the co-precipitation method. Differential scanning calorimetry (DSC) analysis was used to confirm the formation of the complex and several parameters were optimized to obtain a high quality formulation. The water-solubility, drug loading, particle size, zeta potential, morphology and in vivo properties of the SLB-PC-BS-MMs were determined.

Results:

The solubility of SLB in water was increased from 40.83±1.18 μg/mL to 10.14±0.36 mg/mL with a high drug loading (DL) of 14.43%±0.44% under optimized conditions. The SLB-PC-BS-MMs were observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and showed spherical shapes. The particle size and zeta potential, as measured by photon correlation spectroscopy (PCS), were about 30±4.8 nm and −39±5.0 mV, respectively. In vivo studies showed that incorporation of the SLB-PC complex into PC-BS-MMs led to a prolonged circulation time of the drug.

Conclusion:

This novel formulation appears to be a good candidate for drug substances that exhibit poor solubility for parenteral administration.     

Development of diclofenac sodium-loaded alginate-PVP K 30 microbeads using central composite design

Background and the purpose of the study

Diclofenac sodium is a non-steroidal anti-inflammatory agent with a short biological half-life (1–2 hr) and requires multiple dosing. This research was carried out to develop and optimize diclofenac sodium loaded alginate-PVP K 30 microbeads to eliminate the need for multiple dosing and adverse effects.

Methods

Diclofenac sodium loaded alginate-PVP K 30 microbeads were prepared by ionotropic gelation. Particle size, drug release, swelling, FTIR and SEM analyses were performed.

Results

Optimized microbeads showed particle size of 0.589±0.054 to 0.620±0.067 mm, and drug entrapment efficiency of 97.88±2.86 to 98.60±3.55%. The in vitro drug release from microbeads was sustained over 10 hrs and followed controlled-release pattern. FTIR analysis indicated the possibility of intermolecular hydrogen bonding interactions, i.e., –OH…O=C in microbeads.

Conclusion

Microbeads for oral controlled delivery of diclofenac sodium were successfully developed by ionotropic gelation.     

Safety, tolerability and pharmacokinetics of an intravenous bolus of sildenafil in patients with pulmonary arterial hypertension

AIMS

To assess pharmacokinetics and pharmacodynamics of a 10 mg intravenous sildenafil bolus in pulmonary arterial hypertension (PAH) patients stabilized on 20 mg sildenafil orally three times daily.

METHODS

Pharmacokinetic parameters were calculated using noncompartmental analysis.

RESULTS

After an acute increase, plasma concentrations stabilized within the range reported previously for a 20 mg oral tablet. At 0.5 h, mean ± SD changes from baseline were −8.4 ± 11.7 mmHg (systolic pressure), −2.6 ± 7.3 mmHg (diastolic pressure) and −3.5 ± 10.4 beats min⁻¹ (heart rate). There was no symptomatic hypotension.

CONCLUSIONS

Although further research is warranted, a 10 mg sildenafil intravenous bolus appears to provide similar exposure, tolerability and safety to the 20 mg tablet.     

Carvedilol inhibits the cardiostimulant and thermogenic effects of MDMA in humans

BACKGROUND AND PURPOSE

The use of ±3,4-methylenedioxymethamphetamine (MDMA, ‘ecstasy’) is associated with cardiovascular complications and hyperthermia.

EXPERIMENTAL APPROACH

We assessed the effects of the α₁- and β-adrenoceptor antagonist carvedilol on the cardiostimulant, thermogenic and subjective responses to MDMA in 16 healthy subjects. Carvedilol (50 mg) or placebo was administered 1 h before MDMA (125 mg) or placebo using a randomized, double-blind, placebo-controlled, four-period crossover design.

KEY RESULTS

Carvedilol reduced MDMA-induced elevations in blood pressure, heart rate and body temperature. Carvedilol did not affect the subjective effects of MDMA including MDMA-induced good drug effects, drug high, drug liking, stimulation or adverse effects. Carvedilol did not alter the plasma exposure to MDMA.

CONCLUSIONS AND IMPLICATIONS

α₁- and β-Adrenoceptors contribute to the cardiostimulant and thermogenic effects of MDMA in humans but not to its psychotropic effects. Carvedilol could be useful in the treatment of cardiovascular and hyperthermic complications associated with ecstasy use.     

Enhanced antibacterial activity of roxithromycin loaded pegylated poly lactide-co-glycolide nanoparticles

Background and the purpose of the study

The purpose of this study was to prepare pegylated poly lactide-co-glycolide (PEG-PLGA) nanoparticles (NPs) loaded with roxithromycin (RXN) with appropriate physicochemical properties and antibacterial activity. Roxithromycin, a semi-synthetic derivative of erythromycin, is more stable than erythromycin under acidic conditions and exhibits improved clinical effects.

Methods

RXN was loaded in pegylated PLGA NPs in different drug;polymer ratios by solvent evaporation technique and characterized for their size and size distribution, surface charge, surface morphology, drug loading, in vitro drug release profile, and in vitro antibacterial effects on S. aureus, B. subtilis, and S. epidermidis.

Results and conclusion

NPs were spherical with a relatively mono-dispersed size distribution. The particle size of nanoparticles ranged from 150 to 200 nm. NPs with entrapment efficiency of up to 80.0±6.5% and drug loading of up to 13.0±1.0% were prepared. In vitro release study showed an early burst release of about 50.03±0.99% at 6.5 h and then a slow and steady release of RXN was observed after the burst release. In vitro antibacterial effects determined that the minimal inhibitory concentration (MIC) of RXN loaded PEG-PLGA NPs were 9 times lower on S. aureus, 4.5 times lower on B. subtilis, and 4.5 times lower on S. epidermidis compared to RXN solution. In conclusion it was shown that polymeric NPs enhanced the antibacterial efficacy of RXN substantially.     

Double-transfected MDCK cells expressing human OCT1/MATE1 or OCT2/MATE1: determinants of uptake and transcellular translocation of organic cations

BACKGROUND AND PURPOSE

The organic cation transporters 1 (OCT1) and 2 (OCT2) mediate drug uptake into hepatocytes and renal proximal tubular cells, respectively. Multidrug and toxin extrusion protein 1 (MATE1) is a major component of subsequent export into bile and urine. However, the functional interaction of OCTs and MATE1 for uptake and transcellular transport of the oral antidiabetic drug metformin or of the cation 1-methyl-4-phenylpyridinium (MPP⁺) has not fully been characterized.

EXPERIMENTAL APPROACH

Single-transfected Madin-Darby canine kidney (MDCK) cells as well as double-transfected MDCK-OCT1-MATE1 and -OCT2-MATE1 cells were used to study metformin and MPP⁺ uptake into and transcellular transport across cell monolayers, along with their concentration and pH dependence.

KEY RESULTS

Cellular accumulation of MPP⁺ and metformin was significantly reduced by 31% and 46% in MDCK-MATE1 single-transfected cells compared with MDCK control cells (10 µM; P < 0.01). Over a wide concentration range (10–2500 µM) metformin transcellular transport from the basal into the apical compartment was significantly higher in the double-transfected cells compared with the MDCK control and MDCK-MATE1 monolayers. This process was not saturated up to metformin concentrations of 2500 µM. In MDCK-OCT2-MATE1 cells basal to apical MPP⁺ and metformin transcellular translocation decreased with increasing pH from 6.0 to 7.5.

CONCLUSIONS AND IMPLICATIONS

Our data demonstrate functional interplay between OCT1/OCT2-mediated uptake and efflux by MATE1. Moreover, MATE1 function in human kidney might be modified by changes in luminal pH values.     

Modulation of drug block of the cardiac potassium channel KCNA5 by the drug transporters OCTN1 and MDR1

BACKGROUND AND PURPOSE

A common site for drug binding on voltage-gated ion channels is at the interior face of the channel pore. In this study, we tested the hypothesis that the extent of drug block of the human cardiac KCNA5 (K_v1.5) channel underlying the atrial-specific, ultra-rapidly activating, delayed K⁺ current (I_Kur) is modulated by the drug uptake and efflux transporters encoded by organic cation transporter 1 (OCTN1) and multiple drug-resistant gene 1 (MDR1) and expressed in human heart.

EXPERIMENTAL APPROACH

Drug block of KCNA5 was assessed in Chinese hamster ovary cells transiently transfected with KCNA5 alone or in combination with the OCTN1 or MDR1 transporter construct, as well as in an MDR1 stably expressed cell line.

KEY RESUTLS

Co-expression of OCTN1 significantly facilitated block by quinidine (10 µM), verapamil (20 µM), propafenone (5 µM) and clofilium (30 µM). Further evidence of drug transport modulating drug block was the finding that with OCTN1, block developed faster and only partially washed-out, and that block potentiation was prevented by cimetidine, an inhibitor of OCTN1. MDR1 expression attenuated KCNA5 block by erythromycin (an MDR1 substrate). Block was restored by reversin-205 (10 µM, an MDR1 inhibitor). MDR1 did not affect KCNA5 inhibition by KN-93 (1 µM), a blocker acting on the outer mouth of the channel pore.

CONCLUSIONS AND IMPLICATIONS

The extent of drug block of KCNA5 can be modulated by drug uptake and efflux transporters. These data provide further support for the idea that modifying intracellular drug concentrations could modulate the effects of blocking ion channels in patients.     

Formulation and in vitro characterization of cefpodoxime proxetil gastroretentive microballoons

Background and the purpose of the study

The objective of the present work was to improve bioavailability of cepodoxime proxetil through gastroretentive microballoon formulation.

Methods

Microballoons of cefpodoxime proxetil were formulated by solvent evaporation and diffusion method employing hydroxypropylmethyl cellulose (HPMC) and ethyl cellulose (EC) polymers and characterized for particle size, surface morphology, incorporation efficiency, floating behavior, in vitro drug release study and differential scanning calorimetry (DSC).

Results

The average particle size of formulated microballoons was in the range of 54.23±2.78–95.66±2.19µm. Incorporation efficiencies of over 83.77±0.85% were achieved for the optimized formulations. Most of formulations remained buoyant (having buoyancy percentage maximum of 81.36±1.96%) for more than 12 hrs indicating good floating behavior of microballoons. Higher values of correlation coefficients were obtained with Higuchi''s square root of time kinetic treatment heralding diffusion as predominant mechanism of drug release.

Conclusion

Inferences drawn from in vitro studies suggest that microballoons may be potential delivery system for cefpodoxime proxetil with improvement in bioavailability in comparison to conventional dosage forms.     

Spontaneous release of acetylcholine from autonomic nerves in the bladder

Background and purpose:

Bladder contractility is regulated by intrinsic myogenic mechanisms interacting with autonomic nerves. In this study, we have investigated the physiological role of spontaneous release of acetylcholine in guinea pig and rat bladders.

Experimental approach:

Conventional isotonic or pressure transducers were used to record contractile activity of guinea pig and rat bladders.

Key results:

Hyoscine (3 µmol·L⁻¹), but not tetrodotoxin (TTX, 1 µmol·L⁻¹), reduced basal tension, distension-evoked contractile activity and physostigmine (1 µmol·L⁻¹)-evoked contractions of the whole guinea pig bladder and muscle strips in vitro. ω-Conotoxin GVIA (0.3 µmol·L⁻¹) did not affect physostigmine-induced contractions when given either alone or in combination with ω-agatoxin IVA (0.1 µmol·L⁻¹) and SNX 482 (0.3 µmol·L⁻¹). After 5 days in organotypic culture, when extrinsic nerves had significantly degenerated, the ability of physostigmine to induce contractions was reduced in the dorso-medial strips, but not in lateral strips (which have around 15 times more intramural neurones). Most muscle strips from adult rats lacked intramural neurones. After 5 days in culture, physostigmine-induced or electrical field stimulation-induced contractions of the rat bladder strips were greatly reduced. In anaesthetized rats, topical application of physostigmine (5–500 nmol) on the bladder produced a TTX-resistant tonic contraction that was abolished by atropine (4.4 µmol·kg⁻¹ i.v.).

Conclusions and implications:

The data indicate that there is spontaneous TTX-resistant release of acetylcholine from autonomic cholinergic extrinsic and intrinsic nerves, which significantly affects bladder contractility. This release is resistant to blockade of N, P/Q and R type Ca²⁺ channels.British Journal of Pharmacology (2009) 157, 607–619; doi:10.1111/j.1476-5381.2009.00166.x; published online 3 April 2009     

Stability of ciprofloxacin in polyvinylchloride minibags

Background:

Ciprofloxacin is a fluoroquinolone antibiotic used to treat infections caused by both gram-positive and gram-negative organisms.

Objective:

To determine the physical and chemical stability of ciprofloxacin diluted in 5% dextrose in water (D5W) or 0.9% sodium chloride (normal saline [NS]) and stored in polyvinylchloride (PVC) minibags at various temperatures.

Methods:

Solutions of ciprofloxacin (1 and 2 mg/mL) were prepared by diluting a commercially available concentrate (10 mg/mL) with either D5W or NS. The prepared solutions were then packaged in PVC mini-bags. Three minibags of each concentration–diluent combination were stored at 2°C to 8°C with protection from light, at 21°C to 24°C with exposure to light, and at 29°C to 31°C with protection from light. Samples were collected from each minibag on days 0, 7, 14, and 30 and then analyzed. Colour, clarity, and pH were monitored when the samples were collected. On each day of analysis, the samples were accurately diluted before duplicate analysis with a stability-indicating high-performance liquid chromatography assay. A solution was considered stable if the concentration remained above 90% of the initial values.

Results:

There were no changes in the physical characteristics of any of the solutions. At both concentrations (1 and 2 mg/mL), the ciprofloxacin solutions prepared in D5W remained above 93.9% of the initial concentration over the 30-day study period under all 3 storage conditions. Similarly, at both concentrations, solutions diluted in NS remained above 95.9% of the initial concentration over the 30-day study period under all 3 storage conditions.

Conclusions:

Ciprofloxacin prepared in either D5W or NS and stored in PVC minibags was stable for 30 days under 3 separate storage conditions: 2°C to 8°C with protection from light, 21°C to 24°C with exposure to light, and 29°C to 31°C with protection from light.     

Inositol trisphosphate-dependent Ca2+ stores and mitochondria modulate slow wave activity arising from the smooth muscle cells of the guinea pig prostate gland

Background and purpose

Changes in smooth muscle tone of the prostate gland are involved in aetiology of symptomatic prostatic hyperplasia, however the control mechanisms of prostatic smooth muscle are not well understood. Here, we have examined the role of internal Ca²⁺ compartments in regulating slow wave activity in the guinea pig prostate.

Experimental approach

Standard intracellular membrane potential recording techniques were used.

Key results

The majority (89%) of impaled cells displayed ‘slow wave’ activity. Cyclopiazonic acid (10 µmol·L⁻¹) transiently depolarized (3–9 mV) the membrane potential of the prostatic stroma and transiently increased slow wave frequency. Thereafter, slow wave frequency slowly decreased over 20–30 min. Ryanodine transiently increased slow wave frequency, although after 30 min exposure slow wave frequency and time course returned to near control values. Caffeine (1 mmol·L⁻¹) reduced slow wave frequency, accompanied by membrane depolarization of about 8 mV. Blockade of inositol trisphosphate receptor (IP₃R)-mediated Ca²⁺ release with 2-aminoethoxy-diphenylborate (60 µmol·L⁻¹) or Xestospongin C (3 µmol·L⁻¹) or inhibiting phospholipase C and IP₃ formation using {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (5 µmol·L⁻¹) or neomycin (1 and 4 mmol·L⁻¹) reduced slow wave frequency, amplitude and duration. The mitochondrial uncouplers, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (1–10 µmol·L⁻¹), carbonyl cyanide m-chlorophenylhydrazone (1–3 µmol·L⁻¹) or rotenone (10 µmol·L⁻¹), depolarized the membrane (8–10 mV) before abolishing electrical activity.

Conclusion and implications

These results suggest that slow wave activity was dependent on the cyclical release of Ca²⁺ from IP₃-controlled internal stores and mitochondria. This implies that intracellular compartments were essential in the initiation and/or maintenance of the regenerative contractile activity in the guinea pig prostate gland.