WO2000006168A1

WO2000006168A1 - USE OF ALFUZOSIN FOR THE MANUFACTURE OF DRUGS INTENDED FOR THE TREATMENT OF DISORDERS INDUCED BY SMOOTH MUSCLE CONTRACTION IN THE URINARY TRACT, EXCLUDING CONTRACTION OF α-ADRENERGIC ORIGIN

Info

Publication number: WO2000006168A1
Application number: PCT/EP1999/005278
Authority: WO
Inventors: Ralph Eckert
Original assignee: Sanofi Synthelabo SA
Current assignee: Sanofi Aventis France
Priority date: 1998-07-28
Filing date: 1999-07-23
Publication date: 2000-02-10
Anticipated expiration: 2001-01-28
Also published as: AU5163799A

Abstract

The present invention relates to the use of alfuzosin and its pharmaceutically acceptable salts for manufacturing drugs intended for the treatment of disorders induced by smooth muscle contraction in the urinary tract, excluding contraction of α-adrenergic origin.

Description

USE OF ALFUZOSIN FOR THE MANUFACTURE OF DRUGS INTENDED FOR THE TREATMENT OF DISORDERS INDUCED BY SMOOTH MUSCLE CONTRACTION IN THE URINARY TRACT, EXCLUDING CONTRACTION OF α-ADRENERGIC ORIGIN

Alfuzosin and its salts, particularly the hydrochloride are known drugs used in the symptomatic treatment of disorders of the lower urinary tract, in which hyperactivity of the α-adrenergic receptors of the vesicosphincter system disturbs the continence/micturition cycle. The increased symptomatic neurotransmission in these diseases results in a αi-adrenoceptor mediated increase in prostatic smooth muscle tone which seems to be responsible for the dynamic infravesical obstruction. Alfuzosin is acknowledged to be a powerful and uroselective αi-adrenoceptor blocker. Alfuzosin is used to treat dysuria, being induced by a range of disease of α-adrenergic origin, which was disclosed in EP 204 597. Among these diseases bladder neck disease, benign prostate hyperplasia (BPH) , neurological disorders like paraplegia may be mentioned.

Up to now the general approach adopted to the treatment of smooth muscle contraction symptoms, for example in the urinary tract, is closely bound to α_la-adrenoceptor blockade (Walden P.D. et al, "localization of mRNA and receptor binding sites for the α_la-adrenoceptor subtype in the rat, monkey and human urinary bladder and prostate", The Journal of Urology (1997), 157, 1032-1033).

The applicant showed that alfuzosin induces a surprisingly smooth muscle relaxation, this relaxation being completely independent from competitive α_x-adrenoceptor blockade. This observation has never been made on any α-_L-adrenoceptor blocker .

Accordingly, the present invention relates to the use of alfuzosin and its pharmaceutically acceptable salts for manufacturing drugs intended for the treatment of disorders induced by smooth muscle contraction in the urinary tract, excluding contraction of α-adrenergic origin.

The elucidation of this direct relaxation of smooth muscle enables to enlarge the scope of the indications for alfuzosin to the following diseases of the urinary tract, where contraction of smooth muscle is not induced by α-adrenergic transmission, like, for example: functional urethral strictures; detrusor-sphincter dyssynergia; enuresis diurna and/or nocturna (children with bladder outlet obstruction, urethral stricture etc) if dependant on subvesical obstruction; vesico-renal reflux due to subvesical obstruction; neurogenic bladder dysfunction (facilitating emptying and thereby reducing recurrent urinary tract infection) .

Indeed, the smooth muscle contraction observed in these previous diseases of the urinary tract may also be mediated by some neurotransmitters or mediators like: prostaglandins, leukotriens, thromboxanes, endothelin... , constituting the infection mediators.

Pharmacological tests were carried out in order to point out the effect of alfuzosin on cellular contractility regulation.

To simulate the smooth muscle contraction phenylephrine (PE) can be used. PE induces prostatic smooth muscle contraction dose-dependently and fully reversibly. It increases intracellular Ca²⁺ concentration via activation of voltage-dependent Ca²⁺ - channels by intracellular Ca²⁺ release.

The free Ca²⁺ concentration [Ca^2"1"^ plays a key role in the regulation of smooth muscle contractility. Elevation of [Ca²⁺]_i increase smooth muscle tone by Ca²⁺ - calmodulin dependent protein kinase induced phosphorylation of the myosin light chaine kinase (MLCK) initiating myofilamental crossbridging whereby decrease in [Ca²⁺]_i results in reduced activation of this pathway leading to smooth muscle relaxation. [Ca²⁺]_i is determined by the concerted interactions of voltage-dependent Ca ⁺ - channels, intracellular Ca²⁺ - pools and Ca²⁺ - extrusion systems. In the smooth muscle tissue, voltage-dependant Ca²⁺ - channels of L-type is a long-lasting and slowly inactivating Ca²⁺ - channel and the more important one for the regulation of smooth muscle tone. The threshold potential of the L-type Ca²⁺ - channel starts opening at potentials above -30 mV and has its maximal open probability at around 0 mV.

GENERAL EXPERIMENTAL METHODS

Human prostatic tissue

Human prostatic tissue, obtained from patients undergoing TURP because of obstructive and irritative symptoms, is used. Tissue strips are obtained from various regions of the prostate included the 6 o'clock position. The electrocaustically altered tissue borders are dissected before contractility studies are started. In case of radical suprapubic prostatectomy because of localized prostate cancer, tissue is obtained from the entire prostate including the prostate capsule.

Cell isolation

To isolate human prostatic smooth muscle myocytes a similar protocol is used as described by Eckert et al. , "regulation of the prostatic smooth muscle contractility by intracellular second messengers. Implications for the conservative treatment of benign prostatic hyperplasia", Urol . Int . ,

54(1995) , 6-21. The mean cell length is 115 ± 20 μm, width 20 ± 5 μm (n, number of experiments = 24) . Only cells with a homogenous cytoplasm and without intracellular granulation as a character for protoelytic cell damages are used for further recordings .

Organ bath experiment

Contractile properties of human prostatic tissue strips are studied in the SCHULER organ bath FMI IOA-5301. Contractions are evoked pharmacologically by application of the α-_L-adrenoceptor selective agonists phenylephrine and norepinephrine (10^~8 to 10^~2 M) or electrically by transmural field stimulation (TMS) to establish the contractile reponse of the tissue strips. TMS is performed by FMI UBA-3165 stimulator delivering single square wave pulses. The polarity of the electodes is reversed automatically after each pulse by a polarity-changing unit. The train duration is 5 s and the intervall 120 s. The pulse duration is kept at 0,4 ms and the output voltage adjusted to supra maximal levels. Isometric contractions of the tissue is registered on a FMI GM-2 force displacement transducer and stored digitally on a 486 IBM-compatible PC. Dose-response curves are obtained non-cumulatively, e.g. after 20 min before increasing the drug concentration.

Patch clamp and FURA II fluorescence technique

The patch clamp technique consists in the following steps : Isolated myocytes are transferred to the experimental chamber and perfused with extracellular solution (35 ± 1°C) composed of 115 rruM NaCl, 5,4 mM KC1, 1,8 mM CaCl₂, 1 mM NaH₂P0₄, 1 mM MgCl₂, 10 mM glucose, 5 mM HEPES (adjusted with 2 mM NaOH to pH 7,4). During the experiments all solutions are bubbled with 95% 0₂ plus 5% C0₂. Pipettes with tip diameters of 1-3 μm and resistances of 1-3 MΩ filed with 80 mM potassium aspartate, 50 mM KC1, 10 mM KH₂P0₄, 3 mM MgS0₄, 5 mM Na₂ATP, 5 mM HEPES, 0-10 mM EGTA. After giga-seal formation and patch breakthrough, voltage clamp is aplied with a conventional patch-clamp amplifier (L/M-EPC 7, List Medical Electronic, Darmastadt, FRG) using the single-pipette whole-cell configuration of the patch-clamp technique. (Hamill et al. , "improved patch-clamp techniques for high resolution current recordings from cells and cell-free membrane patches", Pfl ϋgers Arch . , 391(1981), 85)

Experimental setup is as described as in Eckerct et al., "regulation of the prostatic smooth muscle contractility by intracellular second messengers. Implications for the conservative treatment of benign prostatic hyperplasia", Urol . In t . , 54 (1995) , 6-21. In order to activate the calcic current, I_Ca cells are depolarized from a holding potential of -80 mV to a test potential of 0 V where I_Ca is about maximum. To isolate I_Ca fast sodium current is inactivated by applying a 100 ms lasting prepulse from -80 to -40 mV.

According to FURA II technique, for the whole-cell recordings (ionic currents from the complete cell surface) the myocytes are loaded with the free dye (Fura II pentapotassium salt, 100-400 μM) included in the pipette solution. The dye diffuses into the cell within 20-60 s depending on the cell size. The apparent concentration of free calcium [Ca²⁺]_i is calculated from the fluorescence ratio according to Grynkiowicz et al . , "A new generation of Ca²⁺ indicators with greatly improved fluorescence properties", J. Biol . Chem, 260 (1985), 3440-3450. The time resolution is between 1 and 50 ms . At the beginning of the experiment a cell is placed into the center of the field of view within the area of light measurement, then the cell-attached configuration (single-channel current recording) is established. Autofluorescence of the cell and fluorescence of the pipette-entrapped dye is cancelled by means of background substraction. The whole-cell configuration is then established by a pulse of suction and the free dye starts to diffuse into the cytosol.

Intracellular dialysis

The pipette perfusion device for intracellular application of various chemicals consists in brief of a fine fused silica tube coated with polyamide (outer diameter : 170 μm, inner diameter : 130 μm, Scientific Glass Engeneering, Weiterstadt, Germany) as the inlet tube inside of the patch pipette. The tip of tubing is tapered (about 50 μm at the end) by pulling the heated capillary and positioned 100 μm from the tip opening of the patch electrodes. The distal end of the inlet tubing is connected to reservoirs of different internal solutions. The pipette tip is perfused by applying negative pressure to the back end of the patch pipette. The dead space between reservoirs and the tip of the inlet tubing causes a delay of 1-2 minutes before effects of a new solution become apparent. The pipette is continuously perfused throughout the experiment except for the period of switching between the reservoirs. On the top of an inverted microscope a cell bath is mounted where the isolated myocytes are placed. UV-light of 340 nm and 380 nm is generated by two monochromators which are linked to the microscope via glasfiber wires. The signal from the patch-pipette is delivered to the patch-clamp amplifier EPC 7. The converted signal from the photomultiplier tube (PMT) is connected to the computer for calculation of intracellular Ca²⁺ concentration from two consecutive light pulses of 340 and 380 nm.

Statistical analysis

Standard statistical tests such as Students t-test, Mann Whitney and Chi Square are used for statistical analysis, correlations among different variables are calculated, p values < 0,05 are considered significant. Results are expressed as mean values ± S.E.M..

RESULTS AND DISCUSSION

Experiment No.l: Spasmolytic potency of alfuzosin - organ bath experiment

A contraction of smooth muscle tissue is evoked by extracellular application of potassium chloride (KCl) in a concentration of 30 mM that leads to a comparable contraction as PE by opening of voltage-dependant L-type channels via depolarization of the cellular resting potential. The contraction by KCl is based on a direct depolarization of the cellular membrane by changing the potassium gradient which determines the membrane potential. Therefore the KCl contraction occurs independant from an norepinephrine release with consecutive activation of α₁-adrenoceptors .

As a result, alfuzosin inhibits dose-dependently and fully reversibly the KCl [30 mM] contraction up to 80% (see table I) .

Experiment No.2: Spasmolytic potency of alfuzosin in isolated myocytes - patch clamp and FURA II

The cellular resting potential of studied smooth muscle cells is -72,45 ± 4,8 mV (n = 21). Na⁺, K⁺ and Ca²⁺ currents are detectable. Extracellular administration of PE augments I_Ca from 8 μA/cm² to 18,5 μA/cm² in a dose-dependent and fully reversible manner. Plotting the peak amplitude versus the respective test potential leads to a current-voltage relation, showing that beside the stimulation of I_Ca other membrane currents are not affected by PE . The EC₅₀ is around 0,1 μM.

To investigate how PE alters the membrane conductance for Ca²⁺, L-type Ca²⁺ - channel currents (I_Ca) are evoked by depolarizing the myocyte from a holding potential of -70 mV to a prepotential of -40 mV to inactivate Na⁺ - channel current (I_Na) and finally to the test potential of 0 mV where I_Ca is about maximum. By plotting the peak of I_Ca versus time, a so called time course is obtained which is the ideal protocol for illustration of hormone effects.

As a result, EC₅₀ concentration of alfuzosin (by intracellular application) is in the range of 300 nM for exerting an inhibition of the basal Ca²⁺ - current (see table

ID • TABLE I

Table 1 : Spasmolytic potency of alfuzosin. contractility [%] represents the percentage of contraction based on the control contraction by 30 mM KCl.

TABLE II

Table 2: Spasmolytic potency of alfuzosin in isolated myocytes. Intracellular application of alfuzosin occurs at t=3 min (alfuzosin concentration reaches = 100 nM) , t=7 min (300 nM) , t=14 min (600 nM) , t=21 min (900 nM) , t=26 min ( 1200 nM) .

These results clearly show that alfuzosin can be used for the treatment of disorders induced by smooth muscle contraction of the urinary tract, excluding contraction of α-adrenergic origin. Indeed the prostate, the inner bladder sphincter as well as the bladder neck (men and women) develop embryologically from the same origin and behave pharmacologically similarly.

Functional urethral strictures; detrusor-sphincter dyssynergia; enuresis diurna and/or nocturna if dependent on subvesical obstruction; vesico-renal reflux due to subvesical obstruction; neurogenic bladder dysfunction can for example be cited.

For these purposes, alfuzosin or pharmaceutically salts thereof can be administrated orally, parenterally or transdermally. The corresponding pharmaceutical composition can be presented in any suitable form such as gelatine capsules, tablets, solutions, tablets and the like. Alfuzosin can be combined with any suitable excipient.

The daily dosage can range from 0,5 to 50 mg for adult humans .

Claims

claims

1. Use of alfuzosin and its pharmaceutically acceptable salts for manufacturing drugs intended for the treatment of disorders induced by smooth muscle contraction in the urinary tract, excluding contraction of α-adrenergic origin.

2. Use of alfuzosin and its pharmaceutically acceptable salts for manufacturing drugs, according to claim 1, intended for the treatment of: functional urethral strictures; detrusor-sphincter dyssynergia; enuresis diurna and/or nocturna; vesico-renal reflux due to subvesical obstruction; neurogenic bladder dysfunction.