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Voiding Dysfunction

Combination of Alfuzosin and Tadalafil Exerts an Additive

Relaxant Effect on Human Detrusor and Prostatic Tissues In Vitro

Stephanie Oger a, Delphine Behr-Roussel a, Diane Gorny a, Thierry Lebret b,Yves Denoux b, Laurent Alexandre a, Francois Giuliano c,*

a Pelvipharm, Orsay Parc, Batiment Cedre, 86 rue de Paris, 91400 Orsay, Franceb Foch Hospital, Department of Urology, Suresnes, Francec AP-HP, Neuro-Uro-Andrology, Department of Physical Medicine and Rehabilitation Raymond Poincare Hospital, Garches, France

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Article history:

Accepted April 21, 2009Published online ahead ofprint on May 3, 2009

Keywords:

PDE5 inhibitor

a1-adrenoceptor antagonist

Lower urinary tract symptoms

Bladder

Prostate

Smooth muscle

Abstract

Background: Lower urinary tract symptoms (LUTS) suggestive of benign prostatic

hyperplasia (BPH) and erectile dysfunction (ED) are highly prevalent in aging menand are strongly linked. Alpha1-blockers such as alfuzosin are effective monotherapiesfor LUTS. Phosphodiesterase type 5 (PDE5) inhibitors such as tadalafil are the first-line

treatment for ED. Both drugs act by two different mechanisms of action on commonurogenital target organs and, thus, may have additive effects.Objectives: We evaluated in vitro the effects of alfuzosin, tadalafil, and the combinationof both on human detrusor and prostatic smooth muscle.

Design, setting, and participants: Prostatic and bladder tissue were obtained frompatients (n = 20 and n = 17, respectively) undergoing cystoprostatectomy for bladdercancer.Measurements: In organ baths, isolated prostatic strips and isolated bladder strips were

incubated with vehicle, tadalafil (10�6 M and 10�5 M), alfuzosin (3 � 10�8 M or 10�6 Mand 10�5 M) or a combination. Concentration-response curves (CRCs) to norepinephrinewere generated on prostatic strips and detrusor strips precontracted with carbachol.

Strips were also submitted to electrical field stimulation (EFS).Results and limitations: When alfuzosin and tadalafil were combined, the maximalrelaxation to norepinephrine on carbachol-precontracted detrusor strips was signifi-cantly increased compared with tadalafil alone, and EFS-induced detrusor contractions

were better inhibited compared with each compound alone. Tadalafil significantlyinhibited norepinephrine-induced prostatic strip contractions by reducing the maximaleffect, whereas alfuzosin shifted the CRC of norepinephrine to the right. Combining bothtadalafil and alfuzosin resulted in a greater relaxant effect. Likewise, the combination

was more effective at reducing EFS-induced contractions compared with each com-pound alone.Conclusions: The combination of alfuzosin and tadalafil exerts an additive effect of

inhibiting adrenergic smooth muscle tone of prostatic tissue and EFS-induced detrusorcontractions and conversely, of enhancing adrenergic relaxation of detrusor precontractedwith carbachol. These experiments provide experimental support for the clinical inves-tigation of the combination of a1-blockers and PDE5 inhibitors in the treatment of LUTS.

# 2009 European Association of Urology. Published by Elsevier B.V. All rights reserved.

* Corresponding author. AP-HP, Neuro-Uro-Andrology, Department of Physical Medicine and Reha-bilitation, Raymond Poincare Hospital, 104 bd Raymond Poincare, 92380 Garches, France.Tel. +33147107748; Fax: +33147104443.E-mail address: giuliano@cyber-sante.org (F. Giuliano).

0302-2838/$ – see back matter # 2009 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.eururo.2009.04.039

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1. Introduction

Lower urinary tract symptoms (LUTS) increase with

advancing age [1]. LUTS comprise storage symptoms, often

related to detrusor overactivity, and voiding symptoms. The

latter could result either from bladder outlet obstruction as

a result of benign prostatic enlargement resulting from the

histologic condition of benign prostatic hyperplasia (BPH)

or from impaired detrusor contractility.

Alpha-adrenoceptor antagonists are considered to be the

most effective monotherapy for LUTS suggestive of BPH [2].

They improve both voiding and storage symptoms of BPH

and are characterized by a rapid onset of action [3]. All

a1-adrenoceptor antagonists are considered to have a

comparable efficacy in LUTS. Nevertheless, alfuzosin offers

the advantages of good cardiovascular tolerability, even in

elderly and hypertensive patients, and less deleterious effect

on sexual function [4].

Phosphodiesterase type 5 (PDE5) inhibitors are the first-

line treatment of erectile dysfunction (ED) [5]. Moreover,

randomized placebo-controlled trials have shown that the

three available PDE5 inhibitors (ie, sildenafil, vardenafil, or

tadalafil), also improve voiding and storage LUTS in patients

with BPH [6–9]. A recent pilot study has also shown that a

single vardenafil administration (20 mg) can improve

urodynamic parameters in men with spinal cord injuries

[10].

LUTS and ED are strongly linked independent of age and

cardiovascular comorbidities, as shown in several epide-

miologic studies [11,12]. Thus, coprescription of a-adreno-

ceptor antagonists and PDE5 inhibitors is likely to increase.

The concept is currently emerging that treatment with a

combination of an a1-adrenoceptor antagonist and a PDE5

inhibitor is the most effective therapy to treat LUTS related

to BPH. Specific precautions, however, should be taken

regarding the concomitant use of these two therapeutic

classes due to their potential additive hypotensive effects.

Interestingly, alfuzosin (10 mg once daily [OD]) shows no

relevant hemodynamic interaction with tadalafil at the

highest prescribed dose (20 mg OD) [13]. Moreover, very

recent reports of combination of a-adrenoceptor antagonists

and PDE5 inhibitors have each reported no symptomatic

hemodynamic effects [14,15]. Finally, a pilot clinical study

has shown that combining sildenafil 25 mg OD with alfuzosin

10 mg OD was superior to each therapy alone (same dosages)

in relieving LUTS and ED in BPH patients [16].

The mechanism of action responsible for the greater

benefit of the combination in LUTS is not yet fully

understood and should be explored. Increasing recent data

indicate that the benefit observed from the combination in

clinical practice on LUTS could be due to an effect of both

drugs, not only on the prostate but also on the smooth

muscle of the bladder [17–19]. Thus, our goal was to

evaluate in vitro whether the combination of alfuzosin and

tadalafil was more effective than each compound alone in

inhibiting pharmacologically induced or electrical field

stimulation (EFS)–induced contractions of human prostatic

smooth muscle and/or in enhancing pharmacologically

induced relaxation of human bladder smooth muscle.

2. Materials and methods

2.1. Preparation of human prostatic strips and detrusor

strips

Prostatic samples (from the transition zone) and bladder samples (from

the dome) were obtained from 20 patients (age: 69 � 3 yr) and from 17

patients (age: 67 � 2.5 yr), respectively, undergoing cystoprostatectomy for

infiltrating bladder cancer. The subjects had no history of bladder

dysfunction according to their medical charts and had not received

radiotherapy. All patients provided their informed consent.

Tissue samples were stored at 4 8C in Krebs-HEPES buffer (118 mM

sodium chloride, 4.7 mM potassium chloride (KCl), 1.2 mM magnesium

sulfate, 1.2 mM potassium dihydrogen phosphate, 2.5 mM calcium

chloride, 4.2 mM sodium bicarbonate, 11.1 mM glucose, and 20.8 mM

HEPES; pH: 7.4) containing penicillin (100 IU/ml) and streptomycin

(0.1 mg/ml) for optimal conservation until use (within 24 h maximum).

Samples were cleaned of adherent tissue and blood, and sections

(4 � 2 � 2 mm) were excised from each donor sample for each

experiment.

Strips were suspended in 5-ml organ chambers filled with Krebs-

HEPES buffer (37 8C, pH: 7.4) that was continuously bubbled with 95% O2

and 5% CO2. They were connected to force transducers for isometric

tension recording (Pioden Controls Ltd, UK) and an initial tension was

applied (0.5–1 g). Following amplification, the tension changes were

computerized with Mac Lab/8 using Chart 5 software (ADInstruments

Ltd, Oxfordshire, UK). The tissue preparations were allowed to

equilibrate for 60 min while being washed periodically with fresh

Krebs-HEPES buffer, and they were primed by KCl (100 mM, 10 min).

2.2. In vitro contractile experiments with prostatic strips

Concentration-response curves (CRCs) to norepinephrine (10�8 M to

10�4 M) were constructed on each strip. Then, after washings, and

following a 20-min incubation period with either vehicle (corresponding

to the high dose combination), tadalafil (10�6 M or 10�5 M), alfuzosin

(3 � 10�8 M), or two combinations of both compounds (10�6 M tadalafil

plus 3 � 10�8 M alfuzosin or 10�5 M tadalafil plus 3 � 10�8 M alfuzosin),

CRCs to norepinephrine were repeated.

In another set of experiments, frequency response curves (FRCs) (at

5 Hz, 10 Hz, 15 Hz, 20 Hz, 30 Hz, and 40 Hz; 5-ms pulse duration, 5-s

train duration; 300 mA) were constructed. After washings, and following

a 20-min incubation period with the same conditions as for norepi-

nephrine CRCs, FRCs were repeated.

The concentrations used for alfuzosin were in the same range as its

pKi determined for the a1-adrenoceptor in human prostate [20].

The concentrations used for tadalafil were previously reported to be

efficient concentrations in prostatic tissue [21] and were used to

evaluate whether the combination could improve the efficacy of each

compound.

2.3. In vitro contractile experiments with detrusor strips

Strips were incubated for a 20-min period with alfuzosin alone (at 10�6

and 10�5 M), tadalafil alone (at 10�6 M and 10�5 M), or both compounds

combined at two different concentrations (10�6 M tadalafil plus 10�6 M

alfuzosin or 10�5 M tadalafil plus 10�5 M alfuzosin) and vehicle

(corresponding to the dosage combination). Strips were then precon-

tracted with carbachol (10�6 M) and allowed to reequilibrate until a

stable response was obtained (20 min), and then CRCs for norepinephr-

ine (10�9 M to 3 � 10�5 M) were performed.

In a second set of experiments, the detrusor strips were submitted to

EFS (20 Hz; 5-ms pulse duration, 5-s train duration; 300 mA) applied by

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Table 1 – Maximal effect (Emax) and pD2� values of norepinephrine-induced contractions of human prostatic strips in presence of tadalafil,

alfuzosin, or a combination of both

Pretreatment pD2 Emax, %

Vehicle (n = 9) 5.6 � 0.1 65.4 � 3.0

Tadalafil 10�6 M (n = 7) 5.5 � 0.1 64.6 � 3.3

Tadalafil 10�5 M (n = 8) 5.7 � 0.2 48.4 � 4.6y

Alfuzosin 3.10�8 M (n = 7) 5.1 � 0.1yy 79.2 � 5.1y

Tadalafil 10�6 M plus alfuzosin 3.10�8 M (n = 5) 5.2 � 0.1yy,§ 68.9 � 4.4

Tadalafil 10�5 M plus alfuzosin 3.10�8 M (n = 7) 5.0 � 0.1yy,**,## 68.3 � 3.1*

� pD2 value is the negative log concentration of compound that produces 50% reduction of the maximal response. Data are the mean plus or minus standard

error of the mean of (n) experiments using prostate samples from (n) patients.y p < 0.05.yy p < 0.001 versus vehicle.§ p < 0.05 versus tadalafil 10�6.* p < 0.05.** p < 0.01 versus tadalafil 10�5M.## p < 0.01 versus tadalafil 10�6M plus alfuzosin 10�6 M.

Fig. 1 – Effect of combinations of tadalafil and alfuzosin on norepinephrine-induced contractions of human prostatic tissue. Concentration-responsecurves (CRCs) to norepinephrine were performed on human prostatic strips in presence of vehicle (n = 9) or of (A) tadalafil at 10S6 M (n = 7) or alfuzosin at3 T 10S8 M (n = 7) or a combination of both (n = 5), (B) tadalafil at 10S5 M (n = 8) or alfuzosin at 3 T 10S8 M (n = 7) or a combination of both (n = 7), or (C)combination of tadalafil 10S6 M plus alfuzosin 3 T 10S8 M (n = 5) or tadalafil 10S5 M plus alfuzosin 3 T 10S8 M (n = 7). Data are the mean plus or minusstandard error of the mean of (n) experiments using prostate samples from (n) patients.* p < 0.05.** p < 0.01, two-way analysis of variance (ANOVA) followed by Bonferroni post tests, d p < 0.05.### p < 0.001, two-way ANOVA followed by a modified student t test with the Bonferroni adjustment for multiple comparisons because of interaction inthe two-way ANOVA analysis, §p < 0.05.

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groups of three stimulations at 1-min intervals and followed by a 3-min

period of rest. After 20 min of equilibration to obtain stable twitch

contractions, strips were incubated with vehicle or alfuzosin (20 min at

10�6 M and at 10�5 M). CRCs for tadalafil (10�7 M to 10�5 M) or vehicle in

log increments were then performed every 18 min.

The concentrations used for alfuzosin were higher than the one used

in experiments with prostate tissue because a-adrenoceptors play a

lesser role in human bladder contraction [20].

2.4. Data analysis

For experiments on detrusor strips using norepinephrine, results were

expressed as a percentage of inhibition of the contractile response to

carbachol. For experiments using EFS on detrusor tissue, results were

expressed in percentage of the initial contractile response before

addition of tadalafil or vehicle.

For experiments on prostatic strips, results were expressed as a

percentage of maximal contraction obtained during the first CRCs to

norepinephrine or the first FRCs. For each CRC for norepinephrine, a pD2

value (negative log concentration of compound that produces 50%

reduction of the maximal response) and a maximal effect were

determined in each experimental condition using the four-parameter

logistic model. Statistical comparisons were performed according to the

extra sum of squares F-test principle with GraphPad Prism 4.03 software

(GraphPad Software, La Jolla, CA, USA). The data were expressed as mean

plus or minus standard error of the mean of the number (n) of

experiments performed with different bladder or prostatic human

samples. Statistical comparisons of norepinephrine CRCs and EFS

experiments were performed with a two-way analysis of variance

(ANOVA) followed by Bonferroni posttest. In case of interaction between

the two factors (concentration and drug) in the two-way ANOVA, a

modified student t test with the Bonferroni adjustment for multiple

comparisons was performed.

2.5. Drugs and chemicals

All drugs and chemicals were purchased from Sigma (St. Louis, MO, USA)

except tadalafil and alfuzosin, which were provided by Sanofi-Aventis

Fig. 2 – Effect of combinations of tadalafil and alfuzosin on electrical field stimulation –induced contractions on human prostatic tissue. Frequency-response curves (FRCs) (at 5 Hz, 10 Hz, 15 Hz, 20 Hz, 30 Hz, and 40 Hz) were performed on human prostatic strips in presence of vehicle (n = 9) or of (A)tadalafil at 10S6 M (n = 7) or alfuzosin at 3 T 10S8 M (n = 7) or a combination of both (n = 8), (B) tadalafil at 10S5 M (n = 8) or alfuzosin at 3�10S8 M (n = 7) ora combination of both (n = 7), or (C) combination of tadalafil 10S6 M plus alfuzosin 3 T 10S8 M (n = 8) or tadalafil 10S5 M plus alfuzosin 3 T 10S8 M (n = 7).Data are the mean plus or minus standard error of the mean of (n) experiments using prostate samples from (n) patients.**p < 0.01.***p < 0.001, two-way analysis of variance.

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(Paris, France). Tadalafil was prepared in dimethyl sulfoxide (DMSO).

Final concentrations of DMSO in the organ bath were 0.033 and 0.1% for

concentrations of 10�6 M and 10�5 M, respectively. Norepinephrine was

prepared in 0.1% ascorbic acid to avoid oxidation. Other drugs were

prepared in distilled water.

3. Results

3.1. Effect of tadalafil, alfuzosin, or a combination of both on

norepinephrine-induced contractions of human prostatic strips

In the presence of tadalafil 10�5 M, the maximal response to

norepinephrine was significantly decreased, whereas the

presence of tadalafil 10�6 M had no effect (Table 1).

Alfuzosin (at 3 � 10�8 M) significantly antagonized

norepinephrine-induced contractions of prostatic strips by

shifting the curve to the right by 3.2-fold (Table 1). The

combination of tadalafil (10�6 M) and alfuzosin (3� 10�8 M)

seemed to exert a greater inhibitory effect on norepine-

phrine-induced contractions compared with tadalafil 10�6 M

alone, although this comparison was not significant (Fig. 1A).

This combination did not further enhance the inhibitory

effect exerted by alfuzosin 3 � 10�8 M alone. The in-

hibitory effect on norepinephrine-induced contractions of

both compounds combined at high concentrations was

significantly greater than alfuzosin 3 � 10�8 M alone and

was also close to significantly greater than tadalafil alone

(Fig. 1B). The high-dose combination significantly dis-

placed further to the right, by 5-fold, the CRC for

norepinephrine obtained with tadalafil 10�5 M alone

(Table 1). Moreover, the maximal effect of norepinephrine

was lower with the high-dose combination compared with

alfuzosin 3 � 10�8 M alone, although the difference was

not significant (Table 1).

Combination of tadalafil and alfuzosin at high doses on

norepinephrine-induced contraction had greater relaxant

effects than the combination at the lower doses (Fig. 1C).

3.2. Effect of tadalafil, alfuzosin, or a combination of both on

electrical field stimulation–induced contractions of human

prostatic strips

Tadalafil (10�5 M) and alfuzosin (3� 10�8 M) significantly

inhibit EFS-induced prostatic-strip contractions (Fig. 2).

The combination of tadalafil (10�6 M) and alfuzosin

(3� 10�8 M) exerted a greater inhibitory effect on

EFS-induced contractions compared with tadalafil alone

but not with alfuzosin alone. The combination with tadalafil

at 10�5 M and alfuzosin at 3 � 10�8 M tended to exert a

greater inhibitory effect than each compound alone, although

these effects were not significantly different.

3.3. Effect of tadalafil, alfuzosin, or a combination of both

drugs on the relaxation induced by norepinephrine on

carbachol-precontracted human detrusor strips

In presence of vehicle, norepinephrine induced a concen-

tration-dependent relaxation of carbachol-induced con-

traction of bladder strips when compared to the cumulative

addition of vehicle ( p < 0.0001; data not shown).

The presence of tadalafil (at both 10�6 M and 10�5 M) or

alfuzosin (10�6 M) in the organ bath did not modify the

relaxant effect of norepinephrine. Conversely, alfuzosin

(10�5 M) significantly enhanced the maximal relaxant

response of norepinephrine (Table 2).

In presence of the combination of tadalafil 10�6 M and

alfuzosin 10�6 M, norepinephrine exerted a greater

significant relaxant effect versus alfuzosin 10�6 M and

close to significantly greater effect versus tadalafil 10�6 M

alone (Fig. 3A). The maximal relaxant response of norepi-

nephrine when both drugs were combined was significantly

increased compared with alfuzosin alone (Table 2).

At higher concentrations (10�5 M), the combination of

tadalafil and alfuzosin further enhanced the relaxant effect

of norepinephrine when compared to tadalafil and alfuzosin

Table 2 – Maximal effect (Emax) and pD2� values of norepinephrine-induced relaxation of human detrusor strips precontracted with

carbachol in presence of tadalafil, alfuzosin, or a combination of both

Pretreatment pD2 Emax, %

Vehicle (n = 7) 6.3 � 0.1 �63.3 � 2.5

Tadalafil 10�6 M (n = 6) 6.4 � 0.1 �63.8 � 3.4

Tadalafil 10�5 M (n = 6) 6.5 � 0.1 �65.1 � 2.4

Alfuzosin 10�6 M (n = 7) 6.3 � 0.1 �58.2 � 2.7

Alfuzosin 10�5 M (n = 6) 6.5 � 0.1 �70.6 � 2.0y, *

Tadalafil 10�6 M plus alfuzosin 10�6 M (n = 7) 6.5 � 0.1 �66.8 � 2.8§

Tadalafil 10�5 M plus alfuzosin 10�5 M (n = 7) 6.5 � 0.1 �75.9 � 2.2yy,**,#

� pD2 value is the negative log concentration of compound that produces 50% reduction of the maximal response. Data are the mean plus or minus standard

error of the mean of (n) experiments using prostate samples from (n) patients.y p < 0.05.yy p < 0.001 versus vehicle.§ p < 0.05 versus alfuzosin 10�6 M.* p < 0.05.** p < 0.01 versus tadalafil 10�5 M.# p < 0.05 versus tadalafil 10�6 M plus alfuzosin 10�6 M.

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alone (Fig. 3B). The maximal relaxant response to norepi-

nephrine in presence of the combination was significantly

increased when compared with the maximal response

reached with tadalafil alone (Table 2).

The combination of tadalafil and alfuzosin exerted a

dose-dependent relaxant effect on human detrusor strips;

indeed, the combination at the higher doses was signifi-

cantly greater than the effect at the lower doses (Fig. 3C).

3.4. Influence of alfuzosin on the effect of tadalafil on electrical

field stimulation–induced human detrusor strips contractions

The preincubation of the human detrusor strips with

alfuzosin without further addition of tadalafil induced an

inhibitory effect on EFS-induced contractions that was

significantly different at 10�5 M when compared to vehicle

(Fig. 4A and B). The successive addition of tadalafil alone to

the strips preincubated with vehicle had no significant

effect on EFS-induced contractions. The preincubation of

alfuzosin followed by the successive addition of three

increasing concentrations of tadalafil (10�7 M,10�6 M, and

10�5 M) enhanced the effect of tadalafil’s dose response

alone or alfuzosin preincubation alone. This effect was

significant for alfuzosin at 10�5 M.

4. Discussion

The present study provides experimental support to the

reported clinical benefit on male LUTS of using a combina-

tion of a PDE5 inhibitor with an a1-adrenoceptor antagonist

Fig. 3 – Effect of tadalafil or alfuzosin or a combination of both on norepinephrine-induced relaxations of human detrusor strips precontracted withcarbachol. Concentration-response curves (CRCs) to norepinephrine were performed on human detrusor strips in the presence of vehicle (n = 7) or of (A)tadalafil at 10S6 M (n = 6) or alfuzosin at 10S6 M (n = 7) or a combination of both (n = 7), (B) tadalafil at 10S5 M (n = 6) or alfuzosin at 10S5 M (n = 6) or acombination of both (n = 7), or (C) combination of tadalafil 10S6 M plus alfuzosin 10S6 M (n = 7) or tadalafil 10S5 M plus alfuzosin 10S5 M (n = 7). Data arethe mean plus or minus standard error of the mean of (n) experiments using prostate samples from (n) patients.* p < 0.05.** p < 0.01.*** p < 0.001 two-way analysis of variance followed by Bonferroni post tests, dp < 0.05.

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[16]. We demonstrated that the combination of the a1-

adrenoceptor antagonist alfuzosin and the PDE5 inhibitor

tadalafil had an additive effect of inhibiting adrenergic or

EFS-elicited smooth muscle tone of human prostatic tissue,

inhibiting detrusor contractions induced by EFS, and

enhancing adrenergic relaxation of human detrusor tissue

precontracted with carbachol.

Alpha-1 adrenoceptors have a crucial role in conveying

human prostate smooth muscle contraction in response to

norepinephrine [20]. By reducing the adrenergic tone of the

prostate, a1-adrenoceptor antagonists relieve LUTS/BPH

[22]. PDE5 isoenzymes might also be involved in controlling

relaxant and/or contractile processes of the human

prostatic tissue. They are expressed in the transition zone

of human prostate [17], and we showed in this experi-

mental study that inhibition of PDE5 isoenzymes by

tadalafil reduced prostatic tissue contractions induced by

endogenous norepinephrine released by sympathetic

nerves during EFS [23] and by exogenous norepinephrine.

This effect is likely to be due to a subsequent increase in

cyclic guanosine monophosphate (cGMP) levels, although

an effect on the cyclic adenosine 30,50-monophosphate

(cAMP) cascade cannot be excluded [21]. The combination

of tadalafil with alfuzosin exerted a greater inhibition

of endogenous or exogenous norepinephrine-induced

contractions of human prostatic strips compared with each

compound alone. We postulate that this reflects an additive

effect of both drugs through their different mechanisms of

action. Thus, combining an a1-adrenoceptor antagonist

such as alfuzosin with a PDE5 inhibitor such as tadalafil

could improve the obstructive component of LUTS as

previously reported in a pilot single-center study where

Fig. 4 – Influence of alfuzosin preincubation on tadalafil effect on electrical field stimulation (EFS)–induced human detrusor strips contractions.Concentration-response curves (CRCs) to tadalafil or vehicle were performed on EFS-induced detrusor contractions in presence of alfuzosin at 10S6 M or10S5 M or vehicle. The data are mean plus or minus standard error of the mean of six experiments using bladder samples from six different patients.* p < 0.05.** p < 0.001, two-way analysis of variance (ANOVA).# p < 0.05, two-way ANOVA followed by a modified student t test with the Bonferroni adjustment for multiple comparisons because of interaction in thetwo-way ANOVA analysis, §p < 0.05.

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the combination of alfuzosin 10 mg OD and sildenafil 25 mg

OD was superior to each compound taken in monotherapy

[16].

Although tadalafil was found to exert a relaxant effect in

vitro on human prostate strips, there was no effect of

tadalafil (5 mg, then 20 mg daily) on maximum flow rate in

patients with LUTS and ED [7,9]. It should be noted that in

the population studied, maximum flow rate was initially

elevated and, consequently, was difficult to further

improve, which may explain the lack of improvement

with tadalafil. Alternatively, the sites of action of

tadalafil in patients with LUTS could be extraprostatic.

Detrusor represents a potential site of action for tadalafil on

LUTS because PDE5 isoenzymes are expressed in human

detrusor [24] and a functional role of PDE5 isoenzymes in

rat bladder tissue has been described in vitro [18]. In our

experiments, however, tadalafil alone failed either to

enhance norepinephrine-induced relaxation of precon-

tracted human detrusor or to inhibit EFS-induced detrusor

contractions.

The concentrations of tadalafil used on both detrusor and

prostatic tissues were rather high; consequently, this raises

questions about whether the in vitro effects observed in the

present study are clinically relevant. Tadalafil was also

required at high concentrations to achieve relaxation of

human cavernosal strips [25], although its clinical benefit

on ED is clearly demonstrated. This highlights the difficulty

in determining the clinical relevance of the concentrations

used in in vitro experiments.

The combination of alfuzosin with tadalafil enhanced

norepinephrine-induced relaxation when compared with

each compound alone, suggesting that alfuzosin amplified

the relaxant effect of tadalafil which could not be identified

when it was used alone. This amplifying effect could be a

consequence of several modifications in signaling pathways.

First, in human detrusor, b-adrenoceptors predominate over

a-adrenoceptors; thus, the response to norepinephrine,

which does not discriminate between a-adrenoceptors and

b-adrenoceptors, is relaxation. Although a-adrenoceptors

are only weakly involved in human detrusor contraction,

high concentrations of a1-adrenoceptor agonists never-

theless evoke detrusor contractions [26,27]. By blocking the

signaling pathway mediating contraction induced by

norepinephrine, alfuzosin might even favor the cAMP-

dependent signaling pathway involved in the relaxation

mediated via b-adrenoceptors [28]. Consequently, alfuzosin

alone might enhance norepinephrine-induced relaxation of

human detrusor as shown in the present study. If the cAMP-

dependent signaling pathway is favored in the presence of

alfuzosin during norepinephrine-induced detrusor relaxa-

tion, this could increase cGMP levels initially stimulated by

tadalafil but that had remained, until then, functionally

silent. Such positive cross-talk mechanisms between cGMP

and cAMP signaling, previously described in other systems

[29], could be the origin of the enhanced relaxant effect of the

combination.

In parallel, the combination of alfuzosin and tadalafil

exerted a greater inhibitory effect on EFS-induced contrac-

tions compared with each compound alone. This benefit is

probably due to additive effects of both drugs. Indeed,

a1-adrenoceptors are expressed on cholinergic nerve term-

inals present in the bladder wall and facilitate acetylcholine

release to yield detrusor contraction [30]. Alfuzosin could

thus reduce acetylcholine release induced by EFS and

responsible for detrusor contractions, thereby indirectly

decreasing detrusor contractile tone and facilitating

tadalafil’s inhibitory effect.

5. Conclusions

The combination of alfuzosin and tadalafil has previously

been shown to exert an additive relaxant effect of human

corpus cavernosum [25], supporting the potential benefit of

such combination in ED patients not responding to PDE5

inhibitors as reported in pilot clinical studies [16,31,32].

These new experimental data suggest that the combination

of alfuzosin and tadalafil also shows an additive relaxant

effect on human prostate and detrusor tissue. This finding

suggests that the combination of alfuzosin and tadalafil

could be more effective than monotherapy in relieving LUTS

associated with BPH. Because LUTS and ED are strongly

linked, these results strengthen the concept that a

combination of a PDE5 inhibitor with an a1-adrenoceptor

antagonist might represent a promising pharmacologic

strategy to treat LUTS associated with BPH and ED. The

value of such a combination deserves further investigation

in placebo-controlled trials.

Author contributions: Francois Giuliano had full access to all the data in the

study and takes responsibility for the integrity of the data and the accuracy

of the data analysis.

Study concept and design: Oger, Behr-Roussel, Giuliano.

Acquisition of data: Gorny, Lebret, Denoux.

Analysis and interpretation of data: Gorny, Oger, Behr-Roussel.

Drafting of the manuscript: Oger, Behr-Roussel.

Critical revision of the manuscript for important intellectual content: Behr-

Roussel, Giuliano.

Statistical analysis: Gorny, Oger.

Obtaining funding: Alexandre, Giuliano.

Administrative, technical, or material support: Alexandre, Giuliano.

Supervision: Behr-Roussel, Giuliano.

Other (specify): None.

Financial disclosures: I certify that all conflicts of interest, including specific

financial interests and relationships and affiliations relevant to the subject

matter or materials discussed in the manuscript (eg, employment/affilia-

tion, grants or funding, consultancies, honoraria, stock ownership or

options, expert testimony, royalties, or patents filed, received, or pending),

are the following: None.

Funding/Support and role of the sponsor: This work was supported by

Sanofi-Aventis in the form of a restricted grant. Sanofi-Aventis approved

of the manuscript.

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