Hypothesis / aims of study
Parkinson’s disease (PD) is known to exhibit not only motor symptoms but also various non-motor symptoms. Of the non-motor symptoms, autonomic neuropathy is reported to highly impair patients’ quality of life, especially overactive bladder is noted from the early stage of PD1). The prefrontal cortex-basal ganglion dopamine D1 receptor direct pathway is reduced with lesion of the substantial nigra, and dopamine D2 receptor indirect pathway is enhanced, suggesting that micturition reflex is elevated in PD condition. However, it remains difficult to explain urinary symptoms induced by depletion of dopamine based on this mechanism alone. Rotigotine is a dopamine agonist with affinity for all dopamine D1 to D5 receptors (especially D3) delivered through transdermal patch. It has been reported that Rotigotine could reduce frequency of urination at night in patients with PD, but its mechanism of action has not been fully clarified2). Indeed, the effects of dopamine D1 and D2 receptor agonists on urinary symptoms vary with individual severity and staging, and complicated to clarify in clinical practice. Micturition reflex has been mainly targeted using small animals for investigating dysuria. In this study, we investigated the micturition reflex in PD model rats using the method established by our group3). Despite the differences between rodent and human species, useful findings have been often obtained from the stage of drug discovery. We therefore investigated the direct effect of rotigotine on the micturition reflex using PD model rats.
Study design, materials and methods
Female Sprague-Dawley (SD) rats with 12-14 weeks weighting 250-350 g were used in all experiments. 6-OHDA (10 μg) was injected into the lateral striatum of those rats 2 weeks before measuring the intravesical pressure to prepare unilateral PD model rats. They had been confirmed to have motor dysfunction in order to prove successful PD models. Intravenous administration was selected as an acute reaction, and subcutaneous administration was selected as a chronic reaction. This study was conducted after being approved by our institution ethics committee.
Measurement of intercontraction interval (ICI) and voiding pressure (VP)
Rats were anesthetized with isoflurane. The lower abdomen was dissected, and a polyethylene tube (PE-50) was inserted/placed at the top of bladder and ligated/fixed with thread. After confirming that no liquid was leaking from the ligated part, the tube passed through the subcutaneous tissue of back in each rat and managed until the time of use. When measuring the intravesical pressure, the catheter which was put outside from the neck of each rat was connected to a three-way stopcock, one end was connected to a syringe pump, and saline at room temperature was continuously injected into the bladder at a constant rate (3 ml/hour) to measure the intravesical pressure. The other end was connected to a pressure transducer and the intravesical pressure was monitored/amplified by a pressure amplifier, incorporated into a personal computer via PowerLab system, and recorded by Chart Program. Voiding parameters were measured overtime as follows: ICI, VP, non-voiding pressure, and residual urine volume (Figure 1). ICI is defined as the time to contraction under constant filling conditions. VP is defined as maximum intravesical pressure with detrusor contractility.
In intravenous administration, Rotigotine was prepared with saline with 0.125, 0.25, and 0.5 mg/kg. In subcutaneous administration, Rotigotine was suspended in corn oil with 0.125, 0.25, and 0.5 mg/mg. Rotigotine (0.125, 0.25 and 0.5 mg/kg) were administrated intravenously or subcutaneously to PD model rats. 0.9% w/v Saline (0.1ml/kg) was intravenously administrated, or cone oil (0.1ml/kg) was subcutaneously administrated as control vehicle.
Administration of test drug or control
Rats were allowed to stabilize for approximately 2 hours before the experimental protocol was initiated. The voiding parameters were continuously measured for 4 hours in total. In subcutaneous administration, control vehicle was injected at beginning of protocol. Rotigotine, which was prepared by the designated method, was then administered intravenously 1 hour and 2 hours after initiation of protocol. In intravenous administration, rats were performed according to the time course as described above as for the subcutaneous administration.
The results of intravesical pressure were expressed as mean ± standard error and transition diagram, and data in the PD model were statistically analyzed with one-way ANOVA. Changes in other urinary parameters were expressed as mean ± standard error and compared with paired-t test before and after drug administration. The level of significance was designated to be 5%.
Interpretation of results
This is the first evidence that Rotigotine inhibited bladder overactivity in PD model rats. Regarding administration of Rotigotine, ICI significantly decreased by intravenous administration, but ICI increased by subcutaneous administration. Possible underlying mechanisms to explain contrary effect of Rotigotine are the features of dopamine receptor family in micturition reflex and the affinity state of dopamine receptors. Firstly, it is known that dopamine D2 like (D2, 3 and 4) receptors can facilitate micturition reflex whereas dopamine D1 like (D1 and 5) receptors can inhibit micturition reflex. Dopamine D1 and D2 like receptors are considered to be different affinity status. D1 receptor was in a low affinity agonist state, whereas the D2 receptor was primarily in the high affinity agonist state. Secondly, Rotigotine is a unique dopamine agonist with affinity for all dopamine D1 through D5 receptors subtypes (especially highest affinity for D3), mainly acting as dopamine D2 like receptor agonist rather than D1 like receptor agonist. Thus, the effects of dopamine receptors on micturition reflex are suggested to depend on dopamine subtypes. Intravenous administration of Rotigotine excited micturition reflex in acute phase due to activated D2 like receptors, contrarily subcutaneous administration of Rotigotine inhibited micturition reflex in chronic phase due to activated D1 like receptors.
The clinical study, investigating the effect of Rotigotine on bladder function in patients with PD, reported that bladder overactivity was suppressed probably due to stimulation of D1 receptors. Our findings showed that subcutaneous administration of Rotigotine significantly improved the urinary symptoms in PD model rats, which might support previous clinical studies2). Rotigotine transdermal patch has been commonly used in clinical situation after being approved by European Medicines Agency for use in 2006, and by the Food and Drug Administration in 2007. To date, the meta-analysis stated that Rotigotine can reduce the motor symptoms of PD. Therefore, Rotigotine would improve not only motor symptoms but also micturition symptoms by transdermal administration, and the overall impact may be effective for improving pollakiuria in patients with PD.