Platinum Priority – Pelvic PainEditorial by George A. Barbalias on pp. 366–368 of this issue

Trigonal Injection of Botulinum Toxin A in Patients with

Refractory Bladder Pain Syndrome/Interstitial Cystitis

Rui Pinto a,b, Tiago Lopes a, Barbara Frias b,c, Andre Silva a,b, Joao Alturas Silva a,b,Carlos Martins Silva a,b,c, Celia Cruz b,c, Francisco Cruz a,b,c,*, Paulo Dinis a,b,c

a Department of Urology, Hospital de Sao Joao, Portugalb Faculty of Medicine, University of Porto, Portugalc Institute of Histology and Embryology (Faculty of Medicine of Porto) and Instituto de Biologia Molecular e Celular, University of Porto, Portugal

E U R O P E A N U R O L O G Y 5 8 ( 2 0 1 0 ) 3 6 0 – 3 6 5

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Article info

Article history:

Accepted February 23, 2010Published online ahead ofprint on March 6, 2010

Keywords:

Bladder pain syndrome

Interstitial cystitis

Botulinum toxin

NGF

BDNF

Bladder trigone

Pain

Abstract

Background: Bladder pain syndrome/interstitial cystitis (BPS/IC) is a chronic dis-

ease without an effective treatment, characterized by pain during bladder filling.

Most nociceptive bladder afferents course in the trigone.

Objective: To evaluate efficacy and tolerability of trigonal injection of botulinum

toxin A (BoNTA) in patients with BPS/IC. Urine concentration of nerve growth factor

(NGF) and brain-derived neurotrophic factor (BDNF) were also evaluated.

Design, setting, and participants: Women with refractory BPS/IC were included in

an open, exploratory study.

Intervention: Under sedation, 100 U of BoNTA (Botox) were injected in 10 trigonal

sites (10 U per 1 ml saline). Retreatment was allowed 3 mo after injection.

Measurements: Pain, urinary frequency, O’Leary-Sant score (OSS), quality of life,

(QoL), and urodynamic testing at 1 and 3 mo and every 3 mo thereafter. Urine NGF

and BDNF were assessed at the same points. Patients who were retreated were

evaluated every 3 mo.

Results and limitations: All patients reported subjective improvement at 1- and

3-mo follow-up. Pain, daytime and nighttime voiding frequency, OSS, and QoL

improved significantly. Bladder volume to first pain and maximal cystometric

capacity more than doubled. Treatment remained effective in >50% of the patients

for 9 mo. Retreatment was also effective in all cases, with similar duration. A

significant, transient reduction in urinary NGF and BDNF was observed. No cases of

voiding dysfunction occurred.

The low number of patients and the lack of a placebo arm are obvious limitations

of this study.

Conclusions: Trigonal injection of BoNTA is a safe and effective treatment for

refractory BPS/IC.

soc

. H513r@m

# 2010 European As

* Corresponding authorPortugal. Fax: +351 225E-mail address: cruzfjm

0302-2838/$ – see back matter # 2010 European Association of Urology. Publis

iation of Urology. Published by Elsevier B.V. All rights reserved.

ospital S. Joao, Faculty of Medicine of Porto and IBMC of Porto, Porto,655.ed.up.pt (F. Cruz).

hed by Elsevier B.V. All rights reserved. doi:10.1016/j.eururo.2010.02.031

Fig. 1 – Location of trigonal injection sites. Each site was injected withbotulinum toxin A, 10 U per 1 ml saline.

E U R O P E A N U R O L O G Y 5 8 ( 2 0 1 0 ) 3 6 0 – 3 6 5 361

1. Introduction

The International Continence Society (ICS) has defined

painful bladder syndrome as the complaint of suprapubic

pain related to bladder filling accompanied by other

symptoms such as daytime and nighttime voiding fre-

quency in the absence of a proven urinary infection or other

obvious pathology [1]. The European Society for the Study of

Interstitial Cystitis (ESSIC), in an attempt to use a consistent

pain syndrome terminology, has preferred the term bladder

pain syndrome (BPS). BPS is defined as pelvic pain, pressure,

or discomfort perceived to be related to the urinary bladder,

accompanied by at least one other urinary symptom such as

a persistent urge to void or increased daytime and

nighttime voiding frequency [2]. The definition of interstitial

cystitis (IC) should probably be restricted to cases that

include typical cystoscopic and histologic features [1,2]. In

this paper, the designation BPS/IC will be used.

The etiology of BPS/IC is unknown. Therefore, BPS/IC

management is directed to pain relief, as bladder pain is

believed to drive both voiding frequency and nocturia.

Botulinum toxin A (BoNTA) has been shown to decrease

noxious input [3,4]. The analgesic effect of BoNTA pre-

sumably results from decreased neuropeptide release at

peripheral extremities [5], and glutamate, substance P, and

calcitonin gene-related peptides from the central endings of

bladder sensory nerves [5]. In the first case, neurogenic

inflammation is prevented, and in the second, nociceptive

transmission becomes inhibited at the spinal cord.

Most nociceptive bladder afferents are concentrated in

the trigone [6]. This may suggest that previous studies that

injected the entire bladder of BPS/IC patients with BoNTA

[7,8] placed most of the neurotoxin far from the nociceptive

fibers and increased the risk of decreasing detrusor

contractility. The hazard of vesicoureteral reflux after

trigonal BoNTA is nonexistent [9]. Therefore, we undertook

a pilot study evaluating the effectiveness, safety, and

duration of treatment/retreatment of BPS/IC patients with

BoNTA applied exclusively to the trigone. Additionally, the

urine concentration of two neurotrophic factors known to

influence the activity of nociceptive afferent fibers—nerve

growth factor (NGF) and brain-derived neurotrophic factor

(BDNF)—was assessed. The urinary levels of NGF were

previously shown to be increased in BPS/IC patients [10].

However, BDNF, an equally ubiquitous neurotrophin, has

never been investigated in this disease.

2. Material and methods

Twenty-six women with BPS/IC gave written informed consent to

receive BoNTA, a practice that has been approved by the ethics

committee of our institution in refractory cases. Patients were informed

about the possible complications of BoNTA administration, including the

necessity of occasional self-catheterization.

All patients had experienced symptoms for >6 mo and had

undergone cystoscopy with hydrodistension and bladder biopsy under

general anesthesia. Previous unsuccessful treatments included oral

pentosan polysulphate, amitriptyline, intravesical heparinoids, and

intravesical dimethyl sulfoxide. Two patients had had intravesical

resiniferatoxin 2 yr previously. Pregnancy, neurologic diseases, urinary

tract infections (UTIs), aminoglycoside use, bladder outlet obstruction,

detrusor overactivity, urinary incontinence, bladder stones, and previous

pelvic radiotherapy were exclusion criteria.

All patients were evaluated by history, physical examination, serum

biochemistry, urinalysis, urine culture and cytology, and imaging

assessing the upper and lower urinary tracts. Pain intensity was scored

using a 10-point visual analog scale (VAS). Patients provided a 3-d

voiding frequency volume chart [11], and the O’Leary-Sant score (OSS)

was used to assess symptoms and problems. Quality of life (QoL) was

evaluated using question 8 of the International Prostate Symptoms

Score. Pressure-flow studies (Dantec Dynamics Ltd., Bristol, England),

followed ICS directives. A 6-French dual-channel catheter was used,

infusing saline at room temperature (25 ml/min). Bladder volumes for

first sensation/pain of bladder filling, maximum cystometric capacity,

maximum urinary flow rate during voiding phase (Qmax), bladder

contractility index, and postvoid residual (PVR) volume were assessed.

BoNTA (Allergan, Irvine, CA, USA) was injected under light sedation

through a 23-gauge needle (Coloplast A/S, Humlebaek, Denmark)

inserted 3 mm into the trigonal wall underneath cystoscopic control.

A total of 100 U was distributed throughout 10 sites (10 U per 1 ml saline,

Fig. 1). Patients received prophylactic ciprofloxacin; specifically, 500 mg

twice a day for 3 d.

After 2 wk, PVR volume and muscle weakness were assessed. Urine

was collected for culture. Patients were further evaluated at 1 and 3

mo and every 3 mo thereafter using VAS for pain, a 3-d voiding chart,

OSS, QoL, and urodynamic investigations, as performed at baseline.

Patients could ask for retreatment after the 3-mo visit if they reported

symptom reappearance and OSS and VAS confirmed a return to

baseline values.

Urinary NGF and BDNF were also measured in the last 10 patients, at

baseline and at 1, 3, and 6 mo after BoNTA injection. Urine samples were

collected at a full bladder sensation, immediately put on ice, and

centrifuged (3000 rpm; 10 min at 4 8C). The supernatant was collected in

1.5-ml tubes and stored at �80 8C. NGF and BDNF concentrations were

determined by ELISA (Emax Immunoassay System, Promega, Madison,

WI, USA), with a minimum sensitivity of 3.9 pg/ml and 7.8 pg/ml,

respectively, following the manufacturer’s instructions. Briefly, 96-well

plates were coated with anti-NGF/BDNF polyclonal antibodies. Urine or

NGF/BDNF standards were added to each well, and plates were

incubated. After several washes, anti-NGF/BDNF monoclonal antibodies

were added, and plates were again incubated. After thorough washes,

the amount of bound monoclonal antibody was detected using

immunoglobulin G–horseradish peroxidase-conjugated antibody. The

unbound conjugate was washed out, followed by a 10-min incubation

with a 100 ml substrate solution at room temperature. Hydrochloric acid

was added to terminate the reactions. Color change was measured with a

Synergy HT Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA)

Table 1 – Classification of patients according to European Society for the Study of Interstitial Cystitis criteria [2]

Bladder pain syndrome classification Biopsy

X (not done) A (normal) B (inconclusive) C (positive)

Cystoscospy with hydrodistension X (not done) 0 0 0 0

1 (normal) 0 0 0 0

2 (glomerulations) 0 1 1 6

3 (Hunner’s lesion) 0 1 2 15

Table 2 – Data at baseline and after first and second treatments

First treatmentbaseline

First treatment,first month

First treatment,third month

Secondtreatmentbaseline

Secondtreatment,

third month

Pain visual analog scale (0–10) 5.8 � 1.8 2.1 � 0.3* 1.7 � 0.8* 4.3 � 0.6 1.6 � 0.6*

Daytime frequency 11.4 � 3.6 5.5 � 1.2* 4.7 � 0.9* 8 � 2 5 � 2*

Nighttime frequency 5.5 � 1.9 2.7 � 1.1* 2.1 � 0.7* 4.3 � 0.6 3 � 1*

O’Leary-Sant score for symptoms (0–20) 15.6 � 3 8.4 � 1.1* 6.8 � 1.2* 11.3 � 1.2 7.5 � 1.5*

O’Leary-Sant score for problems (0–16) 13.4 � 3.4 4.9 � 2.1* 4.6 � 1.3* 7 � 3 5 � 2.6*

Quality of life (0–6) 5.1 � 0.9 1.8 � 0.4* 0.7 � 0.5* 4 � 0.6 1.7 � 0.6*

Bladder volume for first sensation/pain (ml) 44 � 20 104 � 23* 107.5 � 20.8* 44 � 14 90 � 23*

Maximum cystometric capacity (ml) 106 � 42 244 � 56* 279 � 82* 139 � 33 214 � 61*

Bladder contractility index

(PdetQmax + 5 � Qmax)

70.2 � 42 78 � 15.2 80.8 � 14 70.5 � 13.5 82 � 13.5

Maximum urinary flow rate (ml/s) 13.3 � 1.8 14.6 � 2.8 15.3 � 2.5 13.1 � 2.4 15.4 � 2.8

Postvoid residual volume (ml) 3.1 � 4.3 10.9 � 4.9 13.8 � 6.3 12 � 4.5 12.4 � 6.7

* p < 0.05.

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at 450 nm. All samples were run in duplicate, and values were averaged.

NGF and BDNF levels were normalized to the urinary concentration of

creatinine.

Results are presented as mean values plus or minus standard

deviations. A paired t test was used for intragroup comparisons. A

Wilcoxon test for nonparametric data was used for multiple compar-

isons. Kaplan-Meyer plots were applied to the duration of treatment. A p

value of <0.05 was considered statistically significant. Analysis of data

was performed using IBM SPSS v.18.0 for the Macintosh (SPSS Inc., an

IBM Company, Chicago, IL, USA).

3. Results

Patients had a mean age of 48.1 � 13 yr. All patients had

typical cystoscopic findings, and the majority had positive

findings in bladder samples. The ESSIC classification for all 26

patients is presented in Table 1.

Fig. 2 – O’Leary-Sant score for symptoms (0–20) and problems (0–16) at baseli* = p < 0.05.

Symptoms were present for 5 � 2 yr. Table 2 shows VAS

value, daytime and nighttime voiding frequency, OSS and QoL

scores, and urodynamic parameters at baseline. No patient

had detrusor overactivity.

After 2 wk of treatment, no patients reported voiding

difficulties, high PVR, or UTI. Urine cultures were all

negative.

Sixteen patients were followed for 2 yr. The last 10 cases

had a follow-up of 6 mo. At 1-mo and 3-mo follow-up,

voiding difficulties—namely, hesitancy, a weak or inter-

mittent stream, or high PVR (Table 2)—were not observed.

UTIs did not occur. A marked decrease in OSS symptoms and

problems at 1 and 3 mo was observed (Fig. 2). In addition,

pain intensity, daytime and nighttime voiding frequency,

and QoL decreased significantly, whereas bladder volume to

first pain sensation and maximum cystometric capacity

increased significantly (Table 2). Bladder contractility

ne and after first treatmen.

Fig. 3 – Kaplan-Meyer plot for duration of first and second treatment. Thetwo curves overlap in most of their extension (in the first treatments, 10patients currently have only 6-mo follow-up).

Fig. 5 – Urine concentration (pg/mg) of nerve growth factor (NGF) andbrain-derived neurotrophic factor (BDNF) at baseline and aftertreatment.* = p < 0.05.

E U R O P E A N U R O L O G Y 5 8 ( 2 0 1 0 ) 3 6 0 – 3 6 5 363

index, Qmax, and PVR were not changed (Table 2). At 6-mo

follow-up, 3 of 26 patients requested retreatment. At 9 mo, 4

of 13 patients made the same request. At 12-mo follow-up,

nine patients that reached this visit were retreated.

Considering only the patients followed-up to retreatment

(16 for the first treatment and 16 for the second treatment),

the average duration of clinical improvement was 10.1� 2.4

mo after the first treatment and 10.3 � 2.2 after retreatment.

Sixteen patients were retreated. Before reinjection, OSS

symptom and problem score, VAS, daytime and nighttime

voiding frequency, and QoL were slightly inferior to baseline

values at presentation. Although not accompanied by

hydrodistension, the appearance of the mucosa was similar

to baseline at reinjection cystoscopy. A significant improve-

ment was nevertheless detected in all these parameters at

the 3-mo visit (Table 2). No cases of voiding dysfunction

were reported. Five UTIs were detected. All 16 patients were

observed at the 6-mo visit, and 2 asked for a third BoNTA

injection. At the 9-mo visit, 5 of the 14 remaining patients

Fig. 4 – O’Leary-Sant symptom and problem combined score of all patientsdistributed by tertiles after first and second treatment. A score <14 isfound in normal subjects [11].

also asked for retreatment. At 12 mo, all the remaining nine

patients asked for a third injection (Fig. 3).

OSS applied to normal individuals shows combined

scores below 14 [12]. Using this criteria, all patients had a

combined OSS score below 14 after the first treatment, and

only one had a score above that cut off after the second

treatment (Fig. 4, Table 2).

NGF changed from 16.4 � 11.2 pg/mg at baseline to

2.2 � 2.1, 6.1 � 3.5, and 16.2 � 11.7 pg/mg after 1, 3, and 6 mo,

respectively ( p < 0.05 at first and third months). BDNF

changed from 39.9 � 35.3 to 13 � 9.5, 25.9 � 9.5, and

28.3 � 12.7, respectively, at the same time points ( p < 0.05

at the first month) (Fig. 5). At 1 mo, a strong correlation was

found between the absolute decrease in NGF and BDNF

concentration and the absolute decrease of pain intensity in

the VAS (Fig. 6).

4. Discussion

This work showed that trigonal injection of BoNTA

improved lower urinary tract symptoms, including pain

and frequency, in the entire cohort. More than half the

patients reported symptom improvement during at least

the first 9 mo of treatment. At 1-yr follow-up, all patients

requested retreatment, which was as effective and safe

during a similar period of time. At reinjection, the

Fig. 6 – Correlation between the absolute decrease of urinaryconcentration of nerve growth factor (D NGF) and brain-derivedneurotrophic factor (D BDNF) and absolute decrease of pain intensity (D

VAS; VAS = visual analog scale) at 1-mo follow-up.R2 = multivariate coefficient of determination.

E U R O P E A N U R O L O G Y 5 8 ( 2 0 1 0 ) 3 6 0 – 3 6 5364

cystoscopic appearance of the mucosa was similar to that at

baseline. As interim cystoscopies were not planned, we do

not know whether BoNTA treatment improved mucosa

appearance in parallel with symptom regression.

The trigone contains a dense suburothelial network of

nociceptive fibers [6] that participate in pain generation and

neurogenic inflammation [4,5,13]. This network is mark-

edly increased in BPS/IC patients [14]. BoNTA blocks the

release of neurotransmitters from those fibers [5]. In

addition, BoNTA reduces TRPV1 trafficking from the

cytoplasm to the cell membrane of sensory neurons [15].

As a consequence, a marked analgesic effect of BoNTA can

be expected. BoNTA also decreases adenosine triphosphate

(ATP) release from the bladder [16], providing an additional

explanation for the analgesic effect. ATP is released from

urothelial cells [17,18] and causes pain after binding

purinergic receptors in suburothelial sensory fibers [19].

Urothelium from BPS/IC patients [20] releases more ATP

than control urothelium. The mechanism leading to ATP

release impairment is, however, unclear. Urothelium, in

contrast to sensory nerves, does not express SV2 or SNAP-25

[21], precluding the direct action of BoNTA. Future studies

will elucidate this phenomenon.

BoNTA reduces the urine concentration of NGF [10], a

neurotrophic agent essential for the growth and main-

tenance of a large subset of nociceptors. As NGF has noxious

effects [22], its reduction in the urine can also contribute to

a decrease in bladder pain. The present study confirmed

NGF reduction but added the novel information that BDNF,

another ubiquitous neurothrophin with nociceptive activity

[23], is also decreased upon BoNTA treatment. NGF is

released from urothelial [24] and smooth muscle cells [25],

whereas BDNF immunoreactivity was observed in urothelial

cells [26] and in sensory fibers [23]. The mechanism for

such a decrease is unknown, and any explanation at this

time can be considered only speculative. It is, however,

interesting to observe that whole bladder [10] or trigonal

injections (as done in this study) might cause a similar

neurothrophin decrease. However, this decrease might be

the consequence of sensory fiber concentration in the

trigone.

Previous studies with BoNTA in BPS/IC patients have

reported conflicting results. Giannantoni [7] injected 200 U

of BoNTA in 20 sites in 15 patients. However, only 60 U were

applied in the trigone, helping to explain the short

antinociceptive effect observed. Pain recurred in 73% of

the patients at 5 mo. Kuo et al. [8] injected 100 or 200 U in

40 sites in the posterior and lateral walls, combined with

hydrodistension. Curiously, 71% of the patients still showed

improvement at 6 mo, 55% at 12 mo, and 30% at 24 mo. This

surprisingly long-lasting effect might be attributable to

hydrodistension. However, the treated population might

also differ between the Kuo et al study [8] and this one. Pain

was less intense and occurred at bladder volumes of 150 ml

in the Kuo et al study [8]. In ours, pain appeared at volumes

of <50 ml. Furthermore, no cases of ulcerative disease were

included by Kuo et al. [8]. Thus, patients enrolled in our

study might have had a more severe form of BPS/IC and

therefore responded to BoNTA for shorter periods. The use

of a common classification system such as the ESSIC model

might help in future comparisons [2,27].

Restraining BoNTA to the trigone might have prevented

voiding dysfunction, a result often reported by other studies

that injected the whole bladder [7,8]. As a fixed part of the

bladder, predominantly innervated by sympathetic nerves,

the human trigone does not contract during voiding [28].

Although some BoNTA could have diffused from the trigone

to the lateral walls, in fact, there was no clinical or

urodynamic evidence of poor emptying. Stress urinary

incontinence eventually caused by BoNTA diffusion to

adjacent sphincteric structures did not occur. Although UTIs

were not observed after the first treatment, five patients

developed UTIs after reinjection. This is a concern, as UTIs

might worsen BPS/IC symptoms [29].

The duration of clinical improvement was comparable to

that reported in detrusor overactivity [3,30]. Nevertheless,

BPS/IC being a chronic disease, retreatments are foresee-

able. In patients retreated through the duration of this

study, BoNTA has remained effective. This is an important

consideration for BPS/IC patients, because BoNTA injections

have no curative effect. Although reinjections every 8–10

mo might constitute a major burden to patients and health

systems, detrusor overactivity is being managed cost

effectively with repeated injections for >8 yr [3,30].

5. Conclusions

Trigonal injection of 100 U BoNTA improves BPS/IC

symptoms without significant complications. Reinjections

remain effective. The low number of patients and the lack of

E U R O P E A N U R O L O G Y 5 8 ( 2 0 1 0 ) 3 6 0 – 3 6 5 365

a placebo arm are obvious limitations of this study.

Therefore, placebo-controlled dose-escalating studies are

warranted.

Author contributions: Francisco Cruz 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: Dinis, F. Cruz, Pinto.

Acquisition of data: Pinto, Lopes, Frias, J.A. Silva, C.M. Silva.

Analysis and interpretation of data: Pinto.

Drafting of the manuscript: Pinto.

Critical revision of the manuscript for important intellectual content: F. Cruz,

Dinis, C. Cruz.

Statistical analysis: Pinto.

Obtaining funding: Dinis, F. Cruz.

Administrative, technical, or material support: None.

Supervision: Dinis, F. Cruz.

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/affiliation, grants or funding, consultancies, honoraria,

stock ownership or options, expert testimony, royalties, or patents filed,

received, or pending), are the following: F. Cruz is a consultant with

Allergan, Astellas, and Recordati.

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

INComb FP7 HEALTH project no. 223234 and the Portuguese Association

of Urology.

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