Simultaneous pharmacokinetic and pharmacodynamic analysis of 5α-reductase inhibitors and androgens by liquid chromatography tandem mass spectrometry.

Benign prostatic hyperplasia and prostate cancer can be treated with the 5α-reductase inhibitors, finasteride and dutasteride, when pharmacodynamic biomarkers are useful in assessing response. A novel method was developed to measure the substrates and products of 5α-reductases (testosterone, 5α-dihydrotestosterone (DHT), androstenedione) and finasteride and dutasteride simultaneously by liquid chromatography tandem mass spectrometry, using an ABSciex QTRAP(®) 5500, with a Waters Acquity™ UPLC. Analytes were extracted from serum (500 µL) via solid-phase extraction (Oasis(®) HLB), with (13)C3-labelled androgens and d9-finasteride included as internal standards. Analytes were separated on a Kinetex C18 column (150 × 3 mm, 2.6 µm), using a gradient run of 19 min. Temporal resolution of analytes from naturally occurring isomers and mass +2 isotopomers was ensured. Protonated molecular ions were detected in atmospheric pressure chemical ionisation mode and source conditions optimised for DHT, the least abundant analyte. Multiple reaction monitoring was performed as follows: testosterone (m/z 289 → 97), DHT (m/z 291 → 255), androstenedione (m/z 287 → 97), dutasteride (m/z 529 → 461), finasteride (m/z 373 → 317). Validation parameters (intra- and inter-assay precision and accuracy, linearity, limits of quantitation) were within acceptable ranges and biological extracts were stable for 28 days. Finally the method was employed in men treated with finasteride or dutasteride; levels of DHT were lowered by both drugs and furthermore the substrate concentrations increased.

Is there seasonal variation in symptom severity, uroflowmetry and frequency-volume chart parameters in men with lower urinary tract symptoms?

PURPOSE: There is a widely held perception that lower urinary tract symptoms may be exacerbated by cold weather. In this study, we examine the effect of seasonal variation in ambient temperatures on frequency-volume chart derivatives, symptom severity scores and uroflowmetry parameters in men with lower urinary tract symptom. METHODS: Between January 2000 and April 2004, men presenting with lower urinary tract symptom were prospectively recruited and assessed in Edinburgh, UK (55°52'N) with maritime temperate climates (Köppen classification Cfb). Local monthly temperatures were extracted from national meteorological records. Patients completed the International Prostate Symptom Score and 3-day frequency volume chart before undergoing free uroflowmetry with post-micturition volume measurement. Exclusion criteria were previous bladder outflow surgery and anti-cholinergic medication. RESULTS: Data on 296 patients were suitable for analysis. Mean age was 62.3 years (range, 26-90). Over the period of study, the coldest month was January (mean = 4.7℃) and the warmest month was August (mean = 15.8℃). There was no significant variation in either International Prostate Symptom Score symptom scores by season (p > 0.05) or any frequency-volume chart parameters, with the exception on an increase in median actual nightly voids over the summer months (p = 0.021). There was no significant correlation between maximal flow rate and post-micturition residual volumes and mean monthly temperatures (p > 0.05). CONCLUSIONS: Seasonal variation in nocturia, but not other frequency-volume parameters, symptom severity or uroflowmetry parameters, is significant in men with lower urinary tract symptom. Future work should consider the impact of seasonal variation in lower urinary tract symptoms in both sexes across a wider range of climates.

5α-reductase type 1 modulates insulin sensitivity in men.

CONTEXT: 5α-Reductase (5αR) types 1 and 2 catalyze the A-ring reduction of steroids, including androgens and glucocorticoids. 5α-R inhibitors lower dihydrotestosterone in benign prostatic hyperplasia; finasteride inhibits 5αR2, and dutasteride inhibits both 5αR2 and 5αR1. In rodents, loss of 5αR1 promotes fatty liver. OBJECTIVE: Our objective was to test the hypothesis that inhibition of 5αR1 causes metabolic dysfunction in humans. DESIGN, SETTING, AND PARTICIPANTS: This double-blind randomized controlled parallel group study at a clinical research facility included 46 men (20-85 years) studied before and after intervention. INTERVENTION: Oral dutasteride (0.5 mg daily; n = 16), finasteride (5 mg daily; n = 16), or control (tamsulosin; 0.4 mg daily; n = 14) was administered for 3 months. MAIN OUTCOME MEASURE: Glucose disposal was measured during a stepwise hyperinsulinemic-euglycemic clamp. Data are mean (SEM). RESULTS: Dutasteride and finasteride had similar effects on steroid profiles, with reduced urinary androgen and glucocorticoid metabolites and reduced circulating DHT but no change in plasma or salivary cortisol. Dutasteride, but not finasteride, reduced stimulation of glucose disposal by high-dose insulin (dutasteride by -5.7 [3.2] µmol/kg fat-free mass/min, versus finasteride +7.2 [3.0], and tamsulosin +7.0 [2.0]). Dutasteride also reduced suppression of nonesterified fatty acids by insulin and increased body fat (by 1.6% [0.6%]). Glucose production and glycerol turnover were unchanged. Consistent with metabolic effects of dutasteride being mediated in peripheral tissues, mRNA for 5αR1 but not 5αR2 was detected in human adipose tissue. CONCLUSION: Dual inhibition of 5αRs, but not inhibition of 5αR2 alone, modulates insulin sensitivity in human peripheral tissues rather than liver. This may have important implications for patients prescribed dutasteride for prostatic disease.

Measurement of tamsulosin in human serum by liquid chromatography-tandem mass spectrometry.

A simple, sensitive and robust method to extract tamsulosin from human serum, and quantify by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated and is applicable as a measure of compliance in clinical research. Tamsulosin was extracted from human serum (100µL) via liquid-liquid extraction with methyl tert-butyl ether (2mL) following dilution with 0.1M ammonium hydroxide (100µL), achieving 99.9% analyte recovery. Internal standard, d9-finasteride, was synthesised in-house. Analyte and internal standard were separated on an Ascentis(®) Express C18 (100mm×3mm, 2.7µm) column using a gradient elution with mobile phases methanol and 2mM aqueous ammonium acetate (5:95, v/v). Total run-time was 6min. Tamsulosin was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring (MRM) mode using positive electrospray ionisation. Mass transitions monitored for quantitation were: tamsulosin m/z 409→228 and d9-finasteride m/z 382→318, with the structural formulae of ions confirmed by Fourier transform ion cyclotron resonance mass spectrometry (within 10ppm). The limit of quantitation was 0.2ng/mL, and the method was validated in the linear range 0.2-50ng/mL with acceptable inter- and intra-assay precision and accuracy and stability suitable for routine laboratory practice. The method was successfully applied to samples taken from research volunteers in a clinical study of benign prostatic hyperplasia.

Volume-induced responses in the isolated bladder: evidence for excitatory and inhibitory elements.

OBJECTIVE: To quantify changes in autonomous activity in response to alterations in intravesical volume, to explore the possible underlying regulatory mechanisms. MATERIALS AND METHODS: Experiments were conducted using whole isolated bladders from six female guinea pigs (280-400 g). A cannula was inserted into the urethra to monitor intravesical pressure and the bladder was suspended in a heated chamber containing carboxygenated physiological solution at 33-36 degrees C. All drugs were added to the solution on the ablumenal bladder surface. RESULTS: An increase in intravesical volume followed by a rapid reduction lead to a complex series of activity comprising of several distinct phases. After a volume increase there was an initial 'burst' of frequency which gradually declined to a 'steady state'. After a volume reduction there was a period of quiescence with spontaneous activity gradually returning to levels seen before the increase, termed the 'inhibitory phase'. The frequency of transient contractions, both immediately after a volume increase and at steady state, increased both with increasing intravesical volume and dose of arecaidine. The length of the inhibitory phase increased both with the duration and magnitude of volume increase. However, the inhibitory phase was not entirely dependent n the magnitude of volume change, as the inhibitory phase was shorter when the volume was not returned to baseline levels. Increasing doses of arecaidine shortened the inhibitory phase. CONCLUSIONS: These observations suggest that the regulation of volume-induced spontaneous activity relies on complex excitatory and inhibitory inputs. The rapid burst of activity resulting from a rise in volume suggests the presence of a rapidly adapting mechanism. Rapid reduction in intravesical volume leads to a quiescent period, i.e. the inhibitory phase. This is related to both the duration of intravesical volume increase and its magnitude. However, similar volume changes are more effective when the volume is reduced back to baseline, as opposed to the bladder being incompletely emptied. Furthermore, the frequency of transient contractions remained constant once a steady state was reached, with no evidence of inhibition before volume reduction. This suggests that mechanisms involved in the generation of the inhibitory phase initiated during bladder filling require >30 s to have a significant effect, but depend on a reduction in volume to be triggered, with the response dependent on the volume reduced. The mechanisms involved in generating and modulating the inhibitory phase seem to be regulated by a strong cholinergic input but the exact nature of these mechanisms is unknown. The potential importance of these results in terms of the general physiology and pharmacology of the bladder is discussed.

Cholinergic activation of phasic activity in the isolated bladder: possible evidence for M3- and M2-dependent components of a motor/sensory system.

OBJECTIVES: To analyse pressure changes induced by muscarinic agonists on the isolated bladder in order to examine whether there are different responses representing different components of a motor/sensory system within the bladder wall. MATERIALS AND METHODS: Whole isolated bladders from 19 female guinea-pigs (280-400 g) were used. A cannula was inserted into the urethra to monitor intravesical pressure and the bladder was suspended in a heated chamber containing carboxygenated physiological solution at 33-36 degrees C. Initially, the responses to the cholinergic agonists, arecaidine but-2-ynyl ester tosylate and carbachol were assessed. Then, in an attempt to identify the muscarinic receptor subtypes involved, the effects of selective muscarinic antagonists on the arecaidine-induced bladder responses were assessed. The antagonists used were the relatively M(3)-selective 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) and darifenicin, and relatively M(2)-selective AFDX-116. All drugs were added to the solution bathing the ablumenal surface of the bladder. RESULTS: The whole bladders exposed to cholinergic agonists respond with complex changes in intravesical pressure. Immediately after application of the agonist there was a burst of high frequency transient contractions. During continued application of agonist the frequency of the transients decreased and their amplitude increased. Thus, there appear to be two components to the response: an initial fast phase and a later slow component. The maximum frequency of the initial burst increased with increasing concentrations of agonist. By contrast, the frequency of the transients in the steady state showed little dependence on agonist concentration. There were quantitative differences between the responses to arecaidine and carbachol. Arecaidine was less effective in generating the initial burst of high-frequency activity and the transients were significantly larger. At low dose, arecaidine was more effective in producing the large transients in the steady state. Pre-exposure of the bladder to 4-DAMP (0.1-10 nM) or darifenicin (0.1-10 nM) significantly reduced the frequency of the initial burst of activity; 0.3 nM 4-DAMP reduced the frequency by half. In this concentration range, 4-DAMP reduced the amplitude of the initial transients but did not affect the frequency of the transients in the steady state. There were similar results with darifenicin. However, darifenicin was less effective in reducing the amplitude of the initial transients. By contrast, ADFX-116 had little effect on the frequency of the initial transients but did reduce amplitude; 300 nM AFDX-116 was needed to reduce the frequency of the initial burst by half. CONCLUSIONS: This analysis suggests that there are different but interrelated mechanisms in the isolated bladder contributing to complex contractile activity. Three components can be identified: a mechanism operating during voiding to produce a global contraction of the whole bladder and two mechanisms, pacemaker and conductive, involved in generating and propagating local contractions in the bladder wall. The pacemaker component is more sensitive to darifenicin and 4-DAMP than to AFDX-116 suggesting that the underlying processes rely predominantly on M(3) receptors and less so on M(2) (M(3) > M(2)). The phasic activity in the later stages is less affected by M(3) antagonists and might therefore involve predominantly M(2) receptors (M(2) > M(3)). The potential importance of these results in terms of the general physiology and pharmacology of the bladder is discussed.

Nocturia, nocturia indices and variables from frequency-volume charts are significantly different in Asian and Caucasian men with lower urinary tract symptoms: a prospective comparison study.

OBJECTIVE: To describe and compare the patterns of nocturia in Asian and Caucasian men presenting with lower urinary tract symptoms (LUTS), and to identify associations or correlations between LUTS and variables from a frequency-volume chart (FVC), as nocturia is common among men with LUTS, and analysis of FVCs shows nocturnal polyuria and reduced nocturnal bladder capacity (NBC) as the predominant causes in Western patients, but there are few comparisons with other ethnic groups. PATIENTS AND METHODS: Consecutive men aged > or = 40 years, presenting with LUTS and nocturia to an Asian and a Caucasian tertiary centre, were recruited prospectively. The men completed the International Prostate Symptom Score and a 3-day FVC. Men having had bladder outlet surgery and/or receiving anticholinergics were excluded. We computed the nocturia ratio, i.e. the nocturnal urine volume/ 24-h urine volume, nocturia index, predicted nocturnal voids and NBC index (NBCI), and analysed comparisons and correlations. RESULTS: In all, 93 Asian and 200 Caucasian men were recruited prospectively, with a similar age and overall severity of LUTS. The nocturia ratio was larger in the Caucasian men, whereas the NBCI was larger in the Asians (P < 0.001). The prevalence of nocturnal polyuria in men aged > or =60 years (nocturia ratio > or =0.3) was significantly higher in the Caucasian population. Conversely, the prevalence of reduced NBC appeared to be higher in the Asians (based on a NBCI of >2; P < 0.001). CONCLUSIONS: The patterns of nocturia and FVC variables differed significantly in age-matched Asian and Caucasian groups. There are also possible ethnic differences in the causes of nocturia, with nocturnal polyuria being more prevalent in Caucasians.

Antimuscarinic drugs in detrusor overactivity and the overactive bladder syndrome: motor or sensory actions?

Antimuscarinic drugs are generally thought to exert their therapeutic action on detrusor overactivity by reducing the ability of the detrusor muscle to contract. We review currently available published data to establish whether there is any evidence to support this contention. Using a PubMed data search, only 14 original articles (including two abstracts) were found that contained cystometric data for both filling and voiding phases and where the actions of antimuscarinic drugs have been reported in detail. These articles were separated into three groups dealing with neuropathic patients (three papers), patients with idiopathic overactive bladder (four papers) and a group whose aetiology was unclear (seven papers). Variables relating to bladder function during the filling phase (time of first desire to void, time to first unstable contraction, and bladder capacity) were identified. Similarly, variables relating to voiding were identified and compared (e.g. maximum detrusor pressure and detrusor pressure at maximum flow rate). The antimuscarinic drugs have a clearly significant effect on sensations of urge, time to first sensation to void, maximum bladder capacity, decrease in voiding frequency and reduction in incontinence episodes. However, only one article (studying neuropaths) reported a significant reduction of the variables associated with detrusor contraction. The remaining four studies (idiopaths/not stated), reported no change in bladder contractility with antimuscarinic drugs. Thus the available data do not support the conclusion that antimuscarinic drugs at doses used in current clinical practice exert their therapeutic action by inhibiting detrusor contractility, but they suggest effects on variables associated with sensation.

How do you stretch a bladder? A survey of UK practice, a literature review, and a recommendation of a standard approach.

AIMS: To assess how and why hydrodistension of the bladder is performed by UK urologists and to compare this practise with the published literature on distension. To suggest a standardised technique for hydrodistension to allow comparison of diagnostic and therapeutic studies. METHODS: A questionnaire was sent to all UK consultant urologists. Questions addressed the indications for short bladder distension (SBD), details of technique, evaluation of outcome, and awareness of evidence base. The literature on bladder distension was reviewed. RESULTS: The majority of respondents perform SBD, principally in the diagnosis and therapy of interstitial cystitis (IC). There was considerable variation in the duration of distension, repetition of distension, the pressure used for distension, and the measurement of bladder capacity. The literature on the technique of hydrodistension is imprecise and no respondent was able to cite literature to support his or her practice. We suggest a simple, more objective technique for performing hydrodistension. CONCLUSIONS: SBD is widely used. There is marked variability in technique and little more than anecdotal evidence to support any particular approach. Research into the evaluation and treatment of painful bladder syndrome in general and IC in particular would be facilitated by the adoption of a standardised technique.

Oral complications after buccal mucosal graft harvest for urethroplasty.

OBJECTIVE: To report long-term oral complications after buccal mucosal graft (BMG) harvesting for urethroplasty. PATIENTS AND METHODS: In a retrospective study of all patients who had BMG harvesting for urethroplasty from April 1996 to September 2002, telephone interviews were conducted using a standard proforma. RESULTS: Thirty-five patients were identified but only 30 (mean age 48.3 years, range 24-86) could be contacted; they had had 31 operations. Soon after surgery (the first 48 h), 22 (73%) of the patients had little or no oral pain; 70% and 90% of the patients were able to eat and drink, respectively; 59% complained of numbness and 75% complained of tightness of the mouth. At discharge 6 days after surgery 90% of patients had little or no oral pain and all were able to eat and drink, but 10% had moderate-to-severe oral pain, 39% had oral numbness, and 52% had tightness of the mouth. At the time of interview, 16% of patients had oral numbness (mean duration 13.6 months) and 32% had tightness of the mouth (mean duration 20.9 months). In answer to the question of whether they would have their cheek mucosa harvested again if required, 74% responded 'yes', 3% 'no', and 23% had mixed feelings. CONCLUSIONS: BMG harvesting is a good operation, as most patients were satisfied, but it is not without long-term complications and patients should be adequately informed.

Magnetic resonance imaging for urinary incontinence.

Magnetic resonance imaging provides the most accurate, versatile and safe imaging of the pelvic floor. Images can be produced to show sections in any plane and even in three dimensions. The resolution is such that detail as good as that seen in histological sections is possible. Once standardization of data acquisition and patient positioning is agreed we look forward to a new era of increasingly accurate diagnoses of incontinence, allowing tailored management, both surgical and nonsurgical.