Conditions: Vaginal Atrophy
Keywords: vaginal atrophy breast cancer vaginal dryness aromatase inhibitor
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 61
Healthy volunteers: no

Brief summary:
This is a phase II, prospective, randomized, double-blind, placebo-controlled, international (Spain and Sweden) and multicentre study to explore the safety of 0.005% estriol vaginal gel in women with early stage breast cancer in treatment with Non-Steroidal Aromatase Inhibitors (NSAIs) in the adjuvant setting and symptoms of vaginal atrophy.

Detailed description:
This is a phase II, prospective, randomized, double-blind, placebo-controlled, international (Spain and Sweden) and multicentre study.

In the setting of postmenopausal hormone receptor positive breast cancer, treatment with aromatase inhibitors (AIs) is the most effective and well-studied therapy. Vaginal dryness is one of the most frequently reported symptom caused by this adjuvant therapy which may lead to a reduced adherence in breast cancer women.

This study will explore the safety of 0.005% estriol vaginal gel in this oncological context, to demonstrate that this medicinal product is a safe option to treat the vaginal atrophy caused by AIs, without a clinically relevant influence in gonadotropins or systemic estrogen levels.

The main objective is to evaluate the levels of Follicle Stimulating Hormone (FSH) after treatment with 0.005% estriol vaginal gel in hormone receptor-positive postmenopausal women with early stage breast cancer in treatment with Non-Steroidal Aromatase Inhibitors (NSAIs) in the adjuvant setting and symptoms of vaginal atrophy.

Arms:
arm 1: Route: Vaginal. Administration by an applicator inserted deep inside the vagina Dose: 1 g of gel, containing 50 Mcg of estriol Dosage schedule: Weeks 1-3: single daily application Weeks 4-12: twice weekly administration arm 2: Route: Vaginal. Administration by an applicator inserted deep inside the vagina Dose: 1 g of gel. Dosage schedule: Weeks 1-3: single daily application Weeks 4-12: twice weekly administration

Interventions: intervention 1: estriol intervention 2: Placebo
Intervention descriptions: intervention 1: 0.005% estriol vaginal gel intervention 2: placebo vaginal gel

Protocol document:
Prot_000.pdf: 
 
ITFE-2026-C10 (BLISSAFE)                                     
EudraCT number: 2014-004517-84 
Protocol version 1.0: 26-November-2014  
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Protocol ITFE-2026-C10 
A PHASE II PROSPECTIVE, RANDOMIZED, DOUBLE-BLIND, 
PLACEBO-CONTROLLED AND MULTI-CENTRE CLINICAL TRIAL 
TO ASSESS THE SAFETY OF 0.005 % ESTRIOL VAGINAL GEL IN 
HORMONE RECEPTOR-POSITIVE POSTMENOPAUSAL WOMEN 
WITH EARLY STAGE BREAST CANCER  IN TREATMENT WITH 
AROMATASE INHIBITOR IN THE ADJUVANT SETTING. 
“BLISSAFE Study” 
 
Sponsor: ITF RESEARCH PHARMA S.L.U 
Polígono Industrial de Alcobendas 
C/San Rafael, 3 
28108 Alcobendas (Madrid), Spain 
Phone: +34 91 6572323 
 
Fax:  +34 91 6572366 
Email: italfarmaco@itfsp.com  
Protocol-specific Sponsor contact information can be found in the Administrative Binder. 
 
STUDY CHIEF INVESTIGATOR: 
Dr. Pedro Sánchez Rovira, Complejo Hospitalario de Jaén 
Sponsor Study Code: ITFE-2026-C10 
EudraCT Number: 2014-004517-84 
Protocol Approved by ITF RESEARCH PHARMA (ITF): 26-November-2014  
CONFIDENTIAL 
The information and data included in this protocol contain secrets and privileged or confidential information which is the property of 
ITF. No one is authorized to make this information public without the written permission of ITF. These limitations shall also be 
applied to all the information considered to be privileged or confidential and provided in the future. This material can be disclosed 
and used by its equipment and collaborators as necessary for conducting the clinical trial. 


 
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SUMMARY OF THE STUDY PROTOCOL 
Study Title: A PHASE II PROSPECTIVE, RANDOMIZED, DOUBLE-BLIND, PLACEBO-
CONTROLLED AND MULTI-CENTRE CLINICAL TRIAL TO ASSESS THE SAFETY OF 
0.005% 
ESTRIOL 
VAGINAL 
GEL 
IN 
HORMONE 
RECEPTOR-POSITIVE 
POSTMENOPAUSAL WOMEN WITH EARLY STAGE BREAST CANCER IN TREATMENT 
WITH AROMATASE INHIBITOR IN THE ADJUVANT SETTING. “BLISSAFE Study” 
Sponsor Study Code: ITFE-2026-C10 
Sponsor: ITF RESEARCH PHARMA S.L.U  
Countries and number of sites: Spain and Sweden. 5 participating sites approximately 
Study Rationale: 
Breast cancer is the most frequently diagnosed cancer worldwide and the leading cause of cancer 
death among females, accounting for 23% of the total cancer cases and 14% of total cancer deaths 
(1). About 80% of primary breast cancers are hormone sensitive as they contain estrogen receptor 
(ER) and/or progesterone receptor-positive cells (2,3). The presence of hormone receptors makes 
endocrine manipulation one of the most useful therapeutic options for these women. 
Antiestrogenic therapy, either by blocking the estrogen receptor (tamoxifen) or by estrogen 
deprivation (aromatase inhibitor), has become the most effective treatment for endocrine-
responsive breast cancer (4,5). 
In the setting of postmenopausal hormone receptor (HR) positive breast cancer, treatment with 
aromatase inhibitors (AIs) is the most effective and well-studied therapy (6). The recommended 
duration of adjuvant endocrine therapy is 5 years and some patients may benefit from a further 5 
years of treatment. It is well established that lack of adherence to the adjuvant treatment is very 
common due to the side-effects derived of the therapy with AIs, which lead to reduced efficacy 
and can be the most detrimental factor influencing clinical outcome (7). Due to that, with such 
long-term treatment duration, it is critical to address morbidity associated with treatment side-
effects in an effort to optimize quality of life (QoL).  
With regard to AIs, particularly high prevalence of urogenital symptoms such as vaginal dryness 
and sexual dysfunction due to vaginal atrophy, have been observed (5, 8). Taking into account 
that many women diagnosed with breast cancer today will be long-term survivors of their disease, 
the long-term impact of therapy on their well-being and specifically on their sexual adverse 
effects has become a growing topic for research (9). 
In order to overcome symptoms of estrogen deprivation, vaginal moisturizers or, sometimes, low 
dose topical estrogens are used in daily clinical practice, despite the inexistence of adequate 
clinical trials that support their use. Non-hormone vaginal moisturizers relieve urogenital 
symptoms, however their efficacy is significantly lower compared to vaginal estrogens (10). In 
contrast to oral hormone therapy, some consider that local application of estrogens seems to be 
safe in postmenopausal breast cancer patients (11). However, the report of a small prospective 
observation (12) advised caution with the use of vaginal estradiol in breast cancer patients 
receiving aromatase inhibitors, as an increase of serum estradiol levels was found that potentially 


 
ITFE-2026-C10 (BLISSAFE)                                     
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could counteract endocrine breast cancer treatment (13).  
A study using lower doses of estradiol presented in the 2013 ASCO Annual Meeting showed no 
changes in FSH while sporadic elevations of estradiol were seen in 5 out of 26 studied patients. 
The significance of this is difficult to judge as the full results have not been published yet (14).   
According to the publication, “Practical guidelines for managing menopausal symptoms after 
breast cancer”,  the use of vaginal estradiol in patients treated with aromatase inhibitors was 
discouraged, but there was a recommendation for the application of vaginal estriol, a much less 
potent estrogen than estradiol, in case estrogen therapy were deemed necessary (15).  
However, safety data of vaginal estriol in breast cancer patients receiving aromatase inhibitors are 
scarce. Pfeiler et al prospectively investigated the safety of vaginal estriol in 10 postmenopausal 
breast cancer patients receiving AIs by measuring serum hormone levels before and 2 weeks after 
daily application of 0.5 mg vaginal estriol. In this study no elevation of serum estriol or estradiol 
could be detected, but a significant decline in serum follicle stimulating hormone and luteinizing 
hormone was observed. The conclusion of this study was that vaginal estriol did not lead to a 
long term elevation of serum estrogen levels (although the analytical methodology employed 
lacked sufficient sensitivity and the number of patients was very low); nevertheless the significant 
decline in gonadotropins suggested a systemic effect, which had to be kept in mind when offering 
vaginal estriol at the dose of 0.5 mg per application to breast cancer patients on aromatase 
inhibitors (16). 
0.005% Estriol vaginal gel is a new formulation for the local treatment of postmenopausal vaginal 
atrophy, which delivers an ultra-low dose of estriol per application (50 µg), ten times lower than 
the current dose of this hormone used in clinical practice. It is marketed under the tradename of 
Blissel® and Gelistrol® in several European countries since 2012. This new formulation has 
proven to be efficacious in relieving vaginal dryness as well as improving the most typical signs 
of the atrophy in a cohort of postmenopausal women (17). A pharmacokinetic study compared the 
systemic absorption of estriol of the new formulation vs the reference product (Ovestinon vaginal 
cream 0.1%, 0.5 mg of estriol per application). After three weeks daily treatment, it was shown 
that 0.005% Estriol vaginal gel produced negligible plasma estriol levels and significantly lower 
than those produced by Ovestinon (p<0.0001). In addition, 0.005% estriol formulation did not 
change serum FSH or LH while a significant decline of serum FSH was observed in women that 
received Ovestinon (p=0.0425) (18,19). 
These data have suggested that 0.005% estriol vaginal gel could be safe in postmenopausal 
patients treated with aromatase inhibitors; however, they were obtained in a cohort of healthy 
postmenopausal women.  
In order to explore the safety of 0.005% estriol vaginal gel in the oncological context, a new 
safety study is proposed with the hypothesis that 0.005% estriol vaginal gel is a safe therapeutic 
option to treat the vaginal atrophy caused by AIs, without a significant decline in gonadotropin or 
increase in systemic estrogen levels in hormone receptor-positive postmenopausal women with 
early stage breast cancer in treatment with non-steroidal aromatase inhibitors (NSAIs) in the 
adjuvant setting. 
 


 
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Study Design and Treatment: 
This is a phase II, prospective, randomized, double-blind, placebo-controlled, international 
(Spain and Sweden) and multicentre study. 
60 patients will be randomized to receive 0.005% estriol vaginal gel (arm A) and placebo 
moisturizing gel (arm B) in a 4:1 proportion, so 48 patients will enter in arm A and 12 in arm B. 
Arm A: Investigational drug product: 0.005% estriol vaginal gel (Blissel®) 
Route: 
Vaginal. Administration by an applicator inserted deep inside 
the vagina 
Dose: 
1 g of gel, containing 50 g of estriol  
Dosage schedule: 
Weeks 1-3: single daily application  
 
Weeks 4-12: twice weekly administration. 
       Women will be instructed to administer the gel at bedtime.  
Arm B: placebo moisturizing gel administered in the same way. 
Patients will have hormone determinations (estriol, estradiol, estrone, FSH and LH) analyzed in 
a central laboratory at baseline and at weeks 1, 3, and 8 from the beginning of the therapy and at 
week 12 (last day of study therapy). An additional FSH and LH determination will be obtained 
during the screening period in order to assess the intra-individual variation of these hormones in 
baseline conditions. Patients will also undergo gynaecological examination (vaginal smear and 
pH), evaluation of vaginal symptoms and signs, and will answer a female sexual function 
questionnaire at baseline, at week 3 (from the beginning of therapy) and week 12 (last day of 
study therapy). Adverse events will be followed along the study and specifically collected at 
weeks 1, 3 and 8 from the beginning of therapy, at week 12 (last day of study therapy) and at 
safety visit (30 ±5 days after last day of study therapy).  
    
Primary Objective: 
 The primary objective is to evaluate the levels of FSH after treatment with 0.005% estriol 
vaginal gel in hormone receptor-positive postmenopausal women with early stage breast 
cancer in treatment with NSAIs in the adjuvant setting and symptoms of vaginal atrophy. 
Primary End-point: 
 Variation in serum levels of FSH from baseline to 12 weeks of treatment 
Secondary Objectives: 
 To evaluate the levels of estriol, estradiol, estrone, FSH and LH after treatment with 
0.005% estriol vaginal gel in hormone receptor-positive postmenopausal women with 
early stage breast cancer in treatment with NSAIs in the adjuvant setting and symptoms 
of vaginal atrophy. 


 
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 To assess the safety and tolerability of 0.005% estriol vaginal gel in hormone receptor-
positive postmenopausal women with early stage breast cancer in treatment with NSAIs 
in the adjuvant setting and symptoms of vaginal atrophy. 
 To assess the efficacy of 0.005% estriol vaginal gel in the treatment of symptoms and 
signs of vaginal atrophy in hormone receptor-positive postmenopausal women with early 
stage breast cancer in treatment with NSAIs and symptoms of vaginal atrophy.  
 To measure the impact of treatment with 0.005% estriol vaginal gel in sexual function of 
hormone receptor-positive postmenopausal women with early stage breast cancer in 
treatment with NSAIs and symptoms of vaginal atrophy. 
Secondary End-points: 
 Variation in serum levels of FSH at different time points compared to baseline (weeks 1, 3 
and 8). 
 Variation in serum levels of LH and plasma levels of estriol, estradiol and estrone, at 
different time points compared to baseline (weeks 1, 3, 8 and 12). 
 AEs according to the Medical Dictionary for Regulatory Activities (MedDRA).  
 Changes in vaginal dryness and other symptoms and signs of vaginal atrophy; changes in 
vaginal maturation value and changes in vaginal pH at week 3 and week 12 vs baseline. 
 Changes in sexual function measured by the Female Sexual Function Index (FSFI20) scale 
at week 3 and week 12 vs baseline. 
Justification of Sample size determination and interim analysis: 
This is an exploratory study and there are scarce data to allow a precise sample size calculation. 
The most relevant data come from Pfeiler (16) who reported that the levels of FSH in patients 
treated with aromatase inhibitors fell from a mean value of 75.7 to 66.0 mIU/ml after two weeks 
of daily vaginal treatment with 0.5 g of estriol. Although no figures are reported of the variability 
of those measurements, an estimation of the standard deviation can be made applying the 
standard rule of [sd = range/4]. The reported range of FSH values in that report is 45.6 – 134.6, 
thus a reasonable approximation to the sd is 89/4 = 22.3. Under these assumptions, it can be 
calculated that a sample size of 44 would provide 80% power to detect with an alpha=0.05 a 
decrease of FSH levels from 75.7 to 66.0 mIU/ml assuming a standard deviation of 22.3. 
The hormone levels in women treated with IA who are not receiving estrogen therapy are likely 
to suffer physiological temporal oscillations. These variations could make difficult the 
interpretation of any eventual modification of hormone levels observed in the estriol treated 
women.  In the absence of data of naturally occurring variations in hormone levels of women 
treated with IA  that allow us to interpret the results of hormone determinations under study, it is 
considered appropriate to include a placebo group to provide reference data. In this case, the 
placebo is a vaginal moisturizing gel. 
It is estimated that a group of 11 women will allow the assessment of physiological fluctuations 
in hormone levels. Additionally, this sample size for the placebo group will provide 80% power 
and alpha=0.05 to differentiate between active and placebo (ratio 4:1) on the change in the 


 
ITFE-2026-C10 (BLISSAFE)                                     
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Protocol version 1.0: 26-November-2014  
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vaginal maturation value, which is a secondary variable of the study that provides objective 
information about the effectiveness of treatment on vaginal atrophy (calculation based on the 
parameter data from the pivotal Blissel study and considering only the subgroup of women who 
had severe vaginal dryness: change VM active group 24.9 vs 0.6 points in placebo group, SD = 
24.2).  
The differentiation between active and placebo in the subjective parameters of efficacy 
(secondary) would be possible at the expense of a sample size noticeably superior. Since the 
evaluation of effectiveness is not primary but secondary in this study, it is not justified in this 
study to increase the sample for this reason.  
In accordance with the above, the study would include 55 women (44 will receive vaginal estriol 
gel and 11 with the placebo moisturizing gel). Taking into account 10% of dropouts, the sample 
to be considered is of 60 women (48 vaginal estriol gel and 12 placebo moisturizing gel). This is 
an unbalanced study but with an active: placebo ratio of 4:1, which will support an adequate 
patient randomization (blocks of 5). 
10 additional women will be recruited in a first safety phase of the study (8 with vaginal estriol 
gel and 2 placebo moisturizing gel). It is expected that none of them will present with significant 
changes in FSH from postmenopausal to premenopausal levels.    
Statistical: 
 Demographics and Baseline Characteristics 
Standard descriptive statistics, such as the mean, median, range and proportion, will be 
used to summarize the patient sample and to estimate parameters of interest. Ninety-five 
percent confidence intervals will be provided for estimates of interest wherever possible. 
 Blood hormone levels 
The variation of the levels of FSH, estriol, estradiol, estrone and LH after treatment with 
0.005% estriol vaginal gel will be studied in each woman. The variation of levels 
between two arms will be analyzed using a non-parametric test (Mann-Whitney-
Wilcoxon test) or an ANCOVA (if it must be adjusted by initial value). 
 Safety Analyses 
Adverse events data and serious adverse events will be reported in frequency tables 
(overall and by grades). The safety analysis will be performed in the population that has 
received at least one dose of the drug. AEs will be graded according to MedDRA. 
Adverse events will be compared using the chi-square tests (Fisher’s Exact test in the case 
of observing frequencies <5%). 
Symptoms of Vaginal atrophy will be evaluated with a codification from 0 to 3. 
 Efficacy Analyses  
The variations in the intensities for each one of the symptoms and signs of the vaginal 
atrophy, after 3 and 12 weeks, in each treatment arm, will be compared using a non-
parametric test (Mann-Whitney-Wilcoxon). Descriptive statistics will be calculated for 
all variables studied. 


 
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 Other analysis: Sexual function measured by the FSFI scale. It will be used an algorithm 
for determining domain scores and a composite full-scale score. 
Study population and main inclusion and exclusion criteria: 
Patients with hormone receptor positive and HER2 negative early breast cancer who are on 
treatment with NSAI and have vaginal atrophy.   
Inclusion Criteria:  
Patients are eligible to be included in the study only if they meet all of the following criteria: 
1. Written informed consent prior to beginning specific protocol procedures. 
2. Patients must have histological confirmation of breast adenocarcinoma with stage I-IIIA, 
documented at a local pathology department. 
3. The breast tumors must be estrogen-receptor positive and/or progesterone receptor 
positive (≥1% of stained tumor cells by IHC as determined by the local laboratory) with 
any HER2 status. 
4. Postmenopausal status defined as: 12 months of spontaneous amenorrhea or 6 months of 
spontaneous amenorrhea with serum FSH levels > 40 mIU/ml or 6 weeks postsurgical 
bilateral oophorectomy with or without hysterectomy. 
5. Patient must be receiving the non-steroidal aromatase inhibitors anastrozole or letrozole as 
breast cancer treatment in the adjuvant setting for a minimum of 6 months.      
6. Women suffering from moderate to severe vaginal dryness according to the FDA 
guidelines for drug development in postmenopausal women (Center for Drug Evaluation 
and Research, CDER Jan 2003). A moderate symptom will be considered if the symptom 
is present, bothersome and annoying, and a severe symptom will be considered if the 
symptom is present, bothersome and annoying, and interferes with the normal patient 
activity. 
7. Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 or 1. 
8. Adequate bone marrow as defined by the following laboratory values: 
a. Absolute Neutrophil Count (ANC) ≥ 1.5 x 109/L. 
b. Platelets (plt) ≥ 100 x 109/L. 
c. Hemoglobin (Hgb) ≥ 10 g/dl. 
9. Patient has adequate organ function as defined by the following laboratory values: 
d. Serum creatinine ≤ 1.5 x ULN. 
e. Bilirubin ≤ 1.5 × ULN.  
f. Alkaline phosphatase ≤ 2 × ULN. 
g. AST and ALT ≤ 2 × ULN. 
10. Willingness and ability to comply with scheduled visits, treatment plan, laboratory tests 
and other study procedures. 


 
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Exclusion Criteria:   
Patients will be excluded from the study if they meet any of the following criteria: 
1. Stage IIIB-IV breast cancer or bilateral breast cancer. 
2. Treatment with any other current anti-tumoral therapy (chemotherapy, anti-Her2…etc) 
besides the NSAI. Pamidronate or Alendronate are permitted. 
3. Prior history of other malignancy within 5 years of study entry, aside from non-melanoma 
skin cancer or carcinoma-in-situ of the uterine cervix adequately treated. 
4. Postmenopausal uterine bleeding. Vaginal bleeding of unknown etiology. 
5. Patients with endometrial thickness equal to or greater than 4 mm measured by 
transvaginal ultrasound. 
6. Patients who have received any type of vulvovaginal treatment in the 15 days prior to the 
start of the study. 
7. Use of any hormone, natural (phytoestrogens) or herbal products for the treatment of 
menopausal symptoms within the last 3 months. 
8. Current or previous history of thromboembolic disease or coagulopathies. 
9. Severe cardiovascular or respiratory diseases in the previous 6 months. 
10. Renal Impairment. 
11. Hepatitis B and/or hepatitis C carriers (unless with normal hepatic function). 
12. Known human immunodeficiency virus infection. 
13. Known hypersensitivity to NSAI. 
14. Other severe acute or chronic medical or psychiatric condition, or laboratory abnormality 
that would impart, in the judgment of the investigator, excess risk associated with study 
participation or study drug administration, or which, in the judgment of the investigator, 
would make the patient inappropriate for entry into this study. 
15. Previous investigational treatment for any condition or participation in any clinical trial 
within 4 weeks of inclusion date. 


 
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Table of Contents   
Section 
Page 
1. 
Introduction .......................................................................................... 14 
1.1 
Overview of Breast Cancer ............................................................. 14 
1.2 
Adjuvant endocrine treatment of postmenopausal HR+ early 
breast cancer patients ...................................................................... 14 
1.3 
Side effects associated with breast cancer adjuvant endocrine 
therapy ............................................................................................. 14 
1.4 
Management of sexual adverse effects derived from the 
treatment of breast cancer patients .................................................. 15 
1.5 
Role of 0.005% estriol vaginal gel (Blissel®) in the Treatment 
of vaginal atrophy ........................................................................... 16 
1.6 
Study Rationale ............................................................................... 16 
2. 
Objectives ............................................................................................. 17 
2.1 
Primary Objective ........................................................................... 17 
2.2 
Primary End-point ........................................................................... 17 
2.3 
Secondary Objectives ...................................................................... 17 
2.4 
Secondary End-points ..................................................................... 17 
3. 
Investigational Plan .............................................................................. 18 
3.1 
Study Design ................................................................................... 18 
3.1.1 
Early Safety Review ............................................................... 19 
3.2 
Duration of the study ....................................................................... 19 
4. 
Study Population .................................................................................. 20 
4.1 
Inclusion Criteria ............................................................................. 20 
4.2 
Exclusion Criteria ............................................................................ 21 
4.3 
Discontinuations .............................................................................. 21 
4.3.1 
Discontinuation of Study Medication ..................................... 21 
4.3.2 
Discontinuation of Study Sites ............................................... 22 
4.3.3 
Discontinuation of Study ........................................................ 22 
5. 
Treatment ............................................................................................. 23 
5.1 
Treatments Administered ................................................................ 23 
5.1.1 
Blissel® (0.005% estriol vaginal gel) ..................................... 23 
5.1.2 
Vaginal gel placebo ................................................................ 24 
5.2 
Materials and Supplies .................................................................... 24 
5.2.1 
Storage, preparation and administration ................................. 24 
5.2.2 
Accountability ......................................................................... 25 
5.3 
Method of Assigning a Patient to a Treatment ................................ 25 


 
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5.4 
Special Treatment Considerations. Dose Adjustments of Study 
Drug ................................................................................................. 26 
5.4.1 
Dose Interruption .................................................................... 26 
5.4.2 
Special conditions ................................................................... 26 
5.5 
Medication Errors and Overdose .................................................... 27 
5.6 
General Concomitant Medication and Supportive Care 
Guidelines ....................................................................................... 27 
5.6.1 
Prohibited Medications ........................................................... 27 
5.6.2 
Permitted Medications ............................................................ 28 
5.7 
Treatment Compliance .................................................................... 28 
5.8 
Blinding and Unblinding ................................................................. 28 
5.8.1 
Process for urgent unblinding ................................................. 29 
6. 
Efficacy and Safety Evaluations, Sample Collection and Testing 
(Standard Laboratory Testing) and Appropriateness of Assessments . 30 
6.1 
FSH serum levels ............................................................................ 30 
6.2 
Estriol, estradiol, estrone plasma levels and LH serum levels ........ 30 
6.3 
Safety assessments .......................................................................... 31 
6.3.1 
Timing of Assessments ........................................................... 31 
6.3.2 
Safety Definitions ................................................................... 32 
6.3.3 
Management, Timing and Assessment of Adverse Events..... 35 
6.3.4 
Management, Timing and Assessment of SAEs ..................... 36 
6.3.5 
Management, Timing and Assessment of SUSARs ............... 39 
6.4 
Efficacy assesments ........................................................................ 41 
6.5 
Impact on sexual function ............................................................... 41 
7. 
Data Quality Assurance ........................................................................ 43 
7.1 
Data Management and Registries file ............................................. 43 
8. 
Sample Size and Statistical Methods ................................................... 44 
8.1 
Determination of Sample Size ........................................................ 44 
8.2 
Statistical and Analytical Plans ....................................................... 45 
8.2.1 
General Considerations ........................................................... 45 
8.2.1.1. 
Patient Populations ........................................................... 45 
8.2.2. 
Patient Disposition .................................................................. 45 
8.2.3. 
Patient Characteristics............................................................. 46 
8.2.4. 
Concomitant Therapy ............................................................. 46 
8.2.5. 
Treatment Compliance ............................................................ 46 
8.2.6. 
Hormone levels ....................................................................... 46 
8.2.7. 
Efficacy ................................................................................... 47 
8.2.7.1 
Signs and symptoms of vaginal atrophy ............................ 47 
8.2.7.2 
Vaginal Maturation value and pH .................................... 48 


 
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8.2.8. 
Safety Analyses....................................................................... 48 
8.2.9. 
Other Analyses: Patient Reported Outcomes: FSFI ............... 49 
8.2.10. 
Interim Analysis ...................................................................... 49 
8.3 
Data Monitoring Committee ........................................................... 49 
8.4 
Criteria for End of Study ................................................................. 49 
9. 
Informed Consent, Confidentiality, Responsibility Insurance and 
Regulatory Considerations ................................................................... 50 
9.1. 
Informed Consent ............................................................................ 50 
9.2. 
Respect of Confidentiality ............................................................... 50 
9.3. 
Responsibility Insurance ................................................................. 50 
9.4. 
Regulatory Considerations .............................................................. 50 
9.4.1 
Investigator Information ......................................................... 51 
9.4.2 
Protocol Signatures ................................................................. 51 
10. Practical Considerations ....................................................................... 52 
10.1.  Monitoring, Audit and Inspections ................................................ 52 
10.2.  Preservation of Study Documentation ........................................... 52 
10.3.  Protocol Modification .................................................................... 52 
10.4.  Use of the Information and Publication ......................................... 52 
10.5.  Ethics Committees ......................................................................... 53 
11. References ............................................................................................ 54 
Protocol Attachment 1. Study Schedule ....................................................... 55 
Protocol Attachment 2. Eastern Cooperative Oncology Group 
Performance Status ............................................................................... 59 
Protocol Attachment 3. Adverse Events / Serious Adverse Events 
Assessment Guide ................................................................................ 60 
Protocol Attachment 4. FSFI Questionnaire ................................................. 61 


 
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Abbreviations and Definitions  
AE 
Adverse Event 
AI 
Aromatase Inhibitor 
ALT/ALAT (SGPT) 
Alanine Aminotransferase 
ANC 
Absolute Neutrophil Count 
AP 
Alkaline Phosphatase 
AR 
Adverse Reaction 
AST/ASAT (SGOT) 
Aspartate Aminotransferase 
Compliance 
Adherence to all the trial-related requirements, good clinical practice (GCP) 
requirements, and the applicable regulatory requirements. 
CR 
Complete Response 
CTCAE 
Common Terminology Criteria for Adverse Events 
CT Scan 
Computed Tomography Scan 
eCRF 
Electronic Case Report Form (sometimes referred to as Clinical Report Form). An 
electronic form for recording study participants’ data during a clinical study, as 
required by the protocol. 
ECOG 
Eastern Cooperative Oncology Group 
End of Study (Trial) 
The end of study (trial) is the date of the last visit or last scheduled procedure shown 
in the Study Schedule for the last active patient in the study. 
Enroll 
The act of assigning a patient to a treatment.  Patients who are enrolled in the trial 
are those who have been assigned a registration number and treatment. 
Enter 
The act of obtaining informed consent for participation in a clinical trial from 
patients deemed eligible or potentially eligible to participate in the clinical trial.  
Patients entered into a trial are those who sign the informed consent document 
directly or through their legally acceptable representatives. 
ER 
Estrogen Receptor 
ERB/IRB 
Ethical review board/Institutional review board:  A board or committee 
(institutional, regional, or national) composed of medical professional and 
nonmedical members whose responsibility is to verify that the safety, welfare, and 
human rights of the patients participating in a clinical trial are protected. 
ESR 
Expedited Safety Report 
FDA 
Food and Drug Administration 
FSH 
Follicle Stimulating Hormone 


 
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FSFI 
Female Sexual Function Index 
GCP 
Good Clinical Practice 
GEICAM 
Spanish Breast Cancer Research Group 
HER2 
Human Epidermal Growth Factor Receptor 2 
Hgb 
Hemoglobin 
HR 
Hormone Receptor 
ICD 
Informed Consent Document  
IDMC 
Independent Data Monitoring Committee 
IHC 
Immunohistochemistry 
IMP 
Investigational Medicinal Product 
Investigator 
A person responsible for the conduct of the clinical trial at a trial site. If a trial is 
conducted by a team of individuals at a trial site, the investigator is the responsible 
leader of the team and may be called the principal investigator. 
ITF 
ITF Research Pharma S.L.U. 
ITT 
Intent To Treat 
LH 
Luteinizing Hormone 
Legal Representative 
An individual, judicial, or other body authorized under applicable law to consent, on 
behalf of a prospective patient, to the patient’s participation in the clinical trial. 
MedDRA 
Medical Dictionary for Regulatory Activities 
MV 
Maturation Value 
NSAI 
Non-Steroidal Aromatase Inhibitor 
Patient 
A subject with a defined disease. 
PgR 
Progesterone Receptor 
QoL 
Quality of Life 
SAE 
Serious Adverse Event 
SAR 
Serious Adverse Reaction 
SAP 
Statistical Analysis Plan 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
ULN 
Upper Limit of Normal 


 
ITFE-2026-C10 (BLISSAFE)                                     
EudraCT number: 2014-004517-84 
Protocol version 1.0: 26-November-2014  
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Study Title: A PHASE II PROSPECTIVE, RANDOMIZED, DOUBLE-
BLIND, PLACEBO-CONTROLLED AND MULTI-CENTRE CLINICAL 
TRIAL TO ASSESS THE SAFETY OF 0.005% ESTRIOL VAGINAL GEL 
IN HORMONE RECEPTOR-POSITIVE POSTMENOPAUSAL WOMEN 
WITH EARLY STAGE BREAST CANCER IN TREATMENT WITH 
AROMATASE INHIBITOR IN THE ADJUVANT SETTING. 
 
“BLISSAFE Study” 
1. Introduction 
1.1 Overview of Breast Cancer 
Breast cancer is the most frequently diagnosed cancer worldwide and the leading cause of 
cancer death among females, accounting for 23% of the total cancer cases and 14% of total 
cancer deaths (1). About 80% of primary breast cancers are hormone sensitive as they contain 
estrogen receptor (ER) and/or progesterone receptor (PgR) positive cells (2,3). The presence of 
hormone receptors makes endocrine manipulation one of the most useful therapeutic options 
for these women. The role of endocrine therapy in breast cancer is well stablished since more 
than 100 years. Antiestrogenic therapy, either by blocking the estrogen receptor (tamoxifen, 
fulvestrant) or by estrogen deprivation (aromatase inhibitor [AI]), has become the most 
effective treatment for endocrine-responsive breast cancer (4,5). 
1.2 Adjuvant endocrine treatment of postmenopausal HR+ early breast cancer patients   
Since more than 40 years, tamoxifen administered for 5 years has been considered the gold 
standard for postmenopausal early breast cancer patients, however in the last 10 years the AIs 
have been considered the most effective and well-studied therapy (6). The recommended 
duration of adjuvant endocrine therapy is 5 years and some patients may benefit from a further 
5 years of treatment. It is well established that lack of adherence to adjuvant treatment is very 
common due to the side-effects derived of the therapy with AIs, which lead to reduced efficacy 
and can be the most detrimental factor influencing clinical outcome (7). Due to that, with such 
long-term treatment duration, it is critical to address morbidity associated with treatment side-
effects in an effort to optimize quality of life (QoL).  
1.3 Side effects associated with breast cancer adjuvant endocrine therapy 
Across all adjuvant endocrine trials, regardless of the treatment received, vasomotor symptoms 
such as hot flushes are the most common adverse events. Other frequently reported side effects, 
such as vaginal discharge, vaginal dryness, dyspareunia and arthralgia, vary in prevalence 
between tamoxifen and AIs (5). Data from a study specifically addressing menopausal 


 
ITFE-2026-C10 (BLISSAFE)                                     
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symptoms after breast cancer showed that hot flushes (41%), night sweats (36%), loss of 
interest in sex (30%), difficulty sleeping (25%), fatigue (22%) and extreme vaginal dryness 
(19%) were the most common adverse effects, being the urogenital symptoms such as the 
vaginal dryness and sexual dysfunction due to vaginal atrophy of particular high prevalence 
with the treatment with AIs (8).  
1.4 Management of sexual adverse effects derived from the treatment of breast cancer 
patients 
Taking into consideration that many women diagnosed with breast cancer today will be long-
term survivors of their disease, the long-term impact of therapy on their well-being and 
specifically on their sexual adverse effects has become a growing topic for research (9). 
In order to overcome symptoms of estrogen deprivation, vaginal moisturizers or, sometimes, 
low dose topical estrogens are used in daily clinical practice, despite the inexistence of 
adequate clinical trials that support their use. Non-hormone vaginal moisturizers relieve 
urogenital symptoms, however their efficacy is significantly lower compared to vaginal 
estrogens (10). In contrast to oral hormone therapy, some consider that local application of 
estrogens seems to be safe in postmenopausal breast cancer patients (11). However, the report 
of a small prospective observation (12) advised caution with the use of vaginal estradiol in 
breast cancer patients receiving aromatase inhibitors, as an increase of serum estradiol levels 
was found that potentially could counteract endocrine breast cancer treatment (13).  
A study using lower doses of estradiol presented in the 2013 ASCO Annual Meeting showed 
no changes in FSH while sporadic elevations of estradiol were seen in 5 out of 26 studied 
patients. The significance of this is difficult to judge as the full results have not been published 
yet (14).   
According to the publication “Practical guidelines for managing menopausal symptoms after 
breast cancer”, the use of vaginal estradiol in patients treated with aromatase inhibitors was 
discouraged, but there was recommended the application of vaginal estriol, a much less potent 
estrogen than estradiol, in case estrogen therapy were deemed necessary (15).  
However, safety data of vaginal estriol in breast cancer patients receiving aromatase inhibitors 
are scarce. Pfeiler et al prospectively investigated the safety of vaginal estriol in 10 
postmenopausal breast cancer patients receiving AIs by measuring serum hormone levels 
before and 2 weeks after daily application of currently marketed dose of 0.5 mg vaginal estriol 
(Ovestin). In this study no elevation of serum estriol or estradiol could be detected, but a 
significant decline in serum follicle stimulating hormone and luteinizing hormone was 
observed (fell from a mean value of 75.7 to 66.0 mIU/ml after two weeks of daily vaginal 
treatment with 0.5 g of estriol). The conclusion of this study was that vaginal estriol did not 
lead to a long term elevation of serum estrogen levels (although the analytical methodology 
employed lacked sufficient sensitivity and the number of patients was very low); nevertheless 
the significant decline in gonadotropins indicated a systemic effect, which had to be kept in 
mind when offering vaginal estriol at the dose of 0.5 mg per application to breast cancer 
patients on aromatase inhibitors (16). 


 
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1.5 Role of 0.005% estriol vaginal gel (Blissel®) in the Treatment of vaginal atrophy                     
0.005% Estriol vaginal gel is a new formulation for the local treatment of postmenopausal 
vaginal atrophy, which delivers an ultra-low dose of estriol per application (50 µg), ten times 
lower than the current dose of this hormone currently used in clinical practice. It is marketed 
under the trade names of Blissel® or Gelistrol® in several European countries since 2012.  
This new formulation has proven to be efficacious in relieving vaginal dryness as well as 
improving the most typical signs of the atrophy in a cohort of postmenopausal women (17). A 
pharmacokinetic study compared the systemic absorption of estriol of the new formulation vs 
the reference product (Ovestinon vaginal cream 0.1%, 0.5 mg of estriol per application). After 
three weeks daily treatment, it was shown that Blissel produced negligible plasma estriol levels 
and significantly lower than those produced by Ovestinon (p<0.0001). In addition, 0.005% 
estriol formulation did not change serum FSH or LH while a significant decline of serum FSH 
was observed in women that received Ovestinon (p=0.0425) (18,19). 
These data suggested that Blissel could potentially be safe in postmenopausal patients treated 
with aromatase inhibitors; however these results were obtained in a cohort of healthy 
postmenopausal women.  
1.6 Study Rationale 
In order to explore the safety of 0.005% estriol vaginal gel in the oncological context, a new 
safety study is proposed with the hypothesis that 0.005% estriol vaginal gel is a safe therapeutic 
option to treat the vaginal atrophy caused by AIs, without a significant decline in gonadotropin 
or increase in systemic estrogen levels in hormone receptor-positive postmenopausal women 
with early stage breast cancer in treatment with non-steroidal aromatase inhibitors (NSAIs) in 
the adjuvant setting. 
 


 
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2. Objectives 
2.1 Primary Objective 
The primary objective is to evaluate the levels of FSH after treatment with 0.005% estriol 
vaginal gel in hormone receptor-positive postmenopausal women with early stage breast cancer 
in treatment with NSAIs in the adjuvant setting and symptoms of vaginal atrophy. 
2.2 Primary End-point 
Variation in serum levels of FSH from baseline to 12 weeks of treatment. 
2.3 Secondary Objectives 
 To evaluate the levels of estriol, estradiol, estrone, FSH and LH after treatment with 
0.005% estriol vaginal gel in hormone receptor-positive postmenopausal women with 
early stage breast cancer in treatment with NSAIs in the adjuvant setting and symptoms 
of vaginal atrophy. 
 To assess the safety and tolerability of 0.005% estriol vaginal gel in hormone receptor-
positive postmenopausal women with early stage breast cancer in treatment with 
NSAIs in the adjuvant setting and symptoms of vaginal atrophy. 
 To assess the efficacy of 0.005% estriol vaginal gel in the treatment of symptoms and 
signs of vaginal atrophy in hormone receptor-positive postmenopausal women with 
early stage breast cancer in treatment with NSAIs and symptoms of vaginal atrophy.  
 To measure the impact of treatment with 0.005% estriol vaginal gel in sexual function 
of hormone receptor-positive postmenopausal women with early stage breast cancer in 
treatment with NSAIs and symptoms of vaginal atrophy. 
2.4 Secondary End-points 
 Variation in serum levels of FSH at different time points compared to baseline (weeks 
1, 3 and 8). 
 Variation in serum levels of LH and plasma levels of estriol, estradiol and estrone, at 
different time points compared to baseline (weeks 1, 3, 8 and 12). 
 AEs according to the Medical Dictionary for Regulatory Activities (MedDRA). 
 Changes in vaginal dryness and other symptoms and signs of vaginal atrophy; change in 
vaginal maturation value and change in vaginal pH at week 3 and week 12 vs baseline.  
 Changes in sexual function measured by the Female Sexual Function Index (FSFI20) 
scale at week 3 and week 12 vs baseline. 


 
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3. Investigational Plan 
3.1 Study Design 
This is a phase II, prospective, randomized, double-blind, placebo-controlled, international 
(Spain and Sweden) and multicentre study.  
Sixty patients will be randomized (stratified by site) to receive 0.005% estriol vaginal gel (arm 
A) and placebo moisturizing gel (arm B) in a 4:1 proportion, so 48 patients will enter in arm A 
and 12 in arm B. Patients will undergo gynaecological tests, plasma and serum extraction for 
hormone determinations, adverse events collection and will answer a questionnaire according 
to what is outlined in Figure 1. 
A) Patients randomized to Arm A (experimental arm) will receive: 
Investigational drug product: 0.005% estriol vaginal gel (Blissel®) 
Route: 
Vaginal. Administration by an applicator inserted deep 
inside the vagina 
Dose: 
1 g of gel, containing 50 g of estriol  
Dosage schedule: 
Weeks 1-3: single daily application  
 
Weeks 4-12: twice weekly administration. 
Women will be instructed to administer the gel at bedtime.  
B) Patients randomized to Arm B (control arm) will receive: 
Placebo moisturizing gel (vehicle of Blissel®). 
Same route and dose schedule than Blissel®. 
Figure1. Study Design 
Study Drug or Placebo administration
B
w1
w2
w3
w4
w5
w6
w7
w8
w9
w10
w11
w12
Safety visit 
(30 days after 
the end of 
therapy)
Blood samples: Hormonal determinations: FSH, LH, estriol, estradiol and estrone
Gynaecological examination, evaluation of symptoms and signs, pH determination and vaginal smear for maturation value 
FSFI questionnaire
 


 
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3.1.1 
Early Safety Review 
There will be an initial safety phase of the study, in which 10 patients will be included (also 
randomized 4:1) to obtain some preliminary safety data and FSH values prior to the recruitment 
of the rest of the group under study.  
These women will receive the study treatment over 3 weeks (8 will receive estriol vaginal gel 
and 2 placebo moisturizing gel). FSH levels in serum will be analysed at screening, baseline 
and at week 3 (day 22 +/- 3 days). An independent data monitoring committee (IDMC) will 
review the results (together with safety data) which will advise the chief investigators about a 
go /no-go decision with the other 60 patients to complete the study. The study will continue if 
no significant changes in FSH levels from postmenopausal to premenopausal levels are 
observed.   
These 10 patients will be recruited in two sites in Spain, 5 patients in each one. These patients 
will have estrogens, and FSH and LH determined at screening, baseline and at week 3. 
 
Figura 2: Study Design of the Initial Safety Phase 
Study Drug or Placebo administration
B
w1
w2
w3
Safety visit 
(30 days after 
the end of 
therapy)
Blood samples: Hormonal determinations: FSH, LH, estriol, estradiol and estrone
 
3.2 Duration of the study  
It is estimated that the 60 patients will be recruited in approximately 3 months. These patients 
will receive study therapy for 12 weeks.    
The first 10 patients for the early safety review will be recruited in approximately 1 month and 
treated for 3 weeks. The results of the FSH levels will be available in 2-3 additional weeks.  
For safety reasons all patients will have a visit 30 (+/-5) days after finishing treatment with the 
study drugs/medications.  
According to what is outlined above, the length of study will be approximately 11 months.  


 
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4. Study Population 
Patients with hormone receptor positive and HER2 negative early breast cancer who are on 
treatment with NSAI and have vaginal atrophy. 
4.1 Inclusion Criteria 
Patients are eligible to be included in the study only if they meet all of the following criteria: 
1. Written informed consent prior to beginning specific protocol procedures. 
2. Patients must have histological confirmation of breast adenocarcinoma with stage I-
IIIA, documented at a local pathology department. 
3. The breast tumors must be estrogen-receptor positive and/or progesterone receptor 
positive (≥1% of stained tumor cells by IHC as determined by the local laboratory) with 
any HER2 status. 
4. Postmenopausal status defined as: 12 months of spontaneous amenorrhea or 6 months 
of spontaneous amenorrhea with serum FSH levels > 40 mIU/ml or 6 weeks 
postsurgical bilateral oophorectomy with or without hysterectomy. 
5. Patient must be receiving the non-steroidal aromatase inhibitors anastrozole or letrozole 
as breast cancer treatment in the adjuvant setting for a minimum of 6 months.      
6. Women suffering from moderate to severe vaginal dryness according to the FDA 
guidelines for drug development in postmenopausal women (Center for Drug 
Evaluation and Research, CDER Jan 2003). A moderate symptom will be considered if 
the symptom is present, bothersome and annoying, and a severe symptom will be 
considered if the symptom is present, bothersome and annoying, and interferes with the 
normal patient activity. 
7. Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 or 1. 
8. Adequate bone marrow as defined by the following laboratory values: 
a. Absolute Neutrophil Count (ANC) ≥ 1.5 x 109/L. 
b. Platelets (plt) ≥ 100 x 109/L. 
c. Hemoglobin (Hgb) ≥ 10 g/dl. 
9. Patient has adequate organ function as defined by the following laboratory values: 
d. Serum creatinine ≤ 1.5 x ULN. 
e. Bilirubin ≤ 1.5 × ULN.  
f. Alkaline phosphatase ≤ 2 × ULN. 
g. AST and ALT ≤ 2 × ULN. 
10. Willingness and ability to comply with scheduled visits, treatment plan, laboratory tests 
and other study procedures. 


 
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4.2 Exclusion Criteria 
Patients will be excluded from the study if they meet any of the following criteria: 
1. Stage IIIB-IV breast cancer or bilateral breast cancer. 
2. Treatment with any other current anti-tumoral therapy (chemotherapy, anti-Her2…etc) 
besides the NSAI. Pamidronate or Alendronate are permitted. 
3. Prior history of other malignancy within 5 years of study entry, aside from non-
melanoma skin cancer or carcinoma-in-situ of the uterine cervix adequately treated. 
4. Postmenopausal uterine bleeding. Vaginal bleeding of unknown etiology. 
5. Patients with endometrial thickness equal to or greater than 4 mm measured by 
transvaginal ultrasound. 
6. Patients who have received any type of vulvovaginal treatment in the 15 days prior to 
the start of the study. 
7. Use of any hormone, natural (phytoestrogens) or herbal products for the treatment of 
menopausal symptoms within the last 3 months. 
8. Current or previous history of thromboembolic disease or coagulopathies. 
9. Severe cardiovascular or respiratory diseases in the previous 6 months. 
10. Renal Impairment. 
11. Hepatitis B and/or hepatitis C carriers (unless with normal hepatic function). 
12. Known human immunodeficiency virus infection. 
13. Known hypersensitivity to NSAI.  
14. Other severe acute or chronic medical or psychiatric condition, or laboratory 
abnormality that would impart, in the judgment of the investigator, excess risk 
associated with study participation or study drug administration, or which, in the 
judgment of the investigator, would make the patient inappropriate for entry into this 
study. 
15. Previous investigational treatment for any condition or participation in any clinical trial 
within 4 weeks of inclusion date. 
4.3 Discontinuations 
4.3.1 
Discontinuation of Study Medication 
The criteria for enrollment must be followed explicitly. If a patient who does not meet 
enrollment criteria is inadvertently enrolled, that patient should be discontinued from the study 
medication, an exception may be granted if the patient, in the opinion of the investigator, is 
having benefit from the study medication. In these rare cases, the investigator must obtain 
documented approval from the sponsor to allow the patient to continue to receive the study 
drug.      
Patients can be discontinued from the study therapy in the following circumstances: 


 
ITFE-2026-C10 (BLISSAFE)                                     
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 
Patient’s own request. 
 
Unacceptable toxicity.  
 
Tumor recurrence. 
 
Any clinical adverse event (AE), laboratory abnormality or inter-current illness which, in 
the opinion of the investigator, indicates that continued participation in the study is not in 
the best interest of the patient. 
 
Termination of the study by the sponsor. 
 
Physician’s decision, including need of other anti-cancer therapy, not specified in the 
protocol.  
 
If the patient is non-compliant with study procedures.  
 
Loss of ability to freely provide consent through imprisonment or involuntarily 
incarceration for treatment of either a psychiatric or physical (e.g. infectious disease) 
illness. 
 
All patients will be discontinued from the treatment phase and they will perform the safety 
visit in case of a delay of more than 1 week during the daily administration phase or more 
than 2 weeks during the twice weekly administration phase, or permanent discontinuation 
of the study drug unless there is an obvious clinical benefit per the investigator’s medical 
judgment and after discussion with the sponsor.  
If possible, and after the permanent discontinuation of treatment, the patients will be assessed 
using the procedure normally planned for the last dosing day with the study treatment. 
All permanent treatment discontinuation should be recorded by the Investigator in the eCRF 
when considered as confirmed. 
4.3.2 Discontinuation of Study Sites 
Study Site participation may be discontinued if the sponsor, the investigator or the IRB of the 
study site judges it necessary for any reason.  
4.3.3 Discontinuation of Study 
The study may be discontinued by the sponsor if this is medically reasonable and consistent 
with applicable regulations of Good Clinical Practice (GCP). Stopping the study for medical 
reasons may be required if patients experienced adverse reactions under the treatment with the 
study drug or if new information about the safety or effectiveness of the study drug justifies it. 


 
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5. Treatment 
5.1 Treatments Administered 
5.1.1 Blissel® (0.005% estriol vaginal gel) 
Blissel® is a vaginal formulation, estriol-based hydrating gel for the treatment of vaginal 
atrophy.   
The 0.005% estriol gel is provided in 30 g aluminium tubes. The tube is intended for multi-use 
applications, the product dosage being 1 g gel /day (50 µg estriol/g gel), which is applied using 
disposable single-use applicators. 
The list of all components used in the manufacture of the investigational drug, qualitative 
composition (% and per unit dose) and quality of the inactive ingredients are indicated in table 
1. 
Table 1: Qualitative and quantitative composition of the drug 0.005% estriol gel (50 µg/g gel) 
Component 
Function 
% (p/p) 
Quantity/g 
product 
Reference to 
standards 
Estriol 
Drug substance 
0.005 
50 µg 
Ph. Eur. 
Polycarbophil AA-1 
Gelling polymer 
1.5 
15 mg 
Ph. Eur. 
Carbopol 971P 
Gelling polymer 
0.5 
5 mg 
Ph. Eur. 
Glycerine 
Hydrating agent 
10 
100 mg 
Ph. Eur. 
Methylparaben sodium 
Preservative 
0.16 
1.6 mg 
Ph. Eur. 
Propylparaben sodium 
Preservative 
0.02 
0.2 mg 
Ph. Eur. 
10 %NaOH  
pH adjustment 
q.s. pH = 4.5-5.5 
--- 
Ph. Eur. 
37 % HCl  
pH adjustment 
q.s. pH = 2.5-3.5 
--- 
Ph. Eur. 
Purified water 
Carrier 
q.s. 100 % 
q.s. 1 g 
Ph. Eur. 
 
Blissel® will be administered by vaginal route at a dose of 1g/application. Patients will have 1 
application/day during weeks 1 to 3 and 2 applications/week during weeks 4 to 12 (i.e. 
Mondays and Thursdays).  


 
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Route: 
Vaginal. Administration by an applicator inserted deep 
inside the vagina 
Dose: 
1 g of gel, containing 50 g of estriol  
Dosage schedule: 
Weeks 1-3: single daily application  
 
Weeks 4-12: twice weekly administration. 
The treatment will be administered for 12 weeks. 
The Principal Investigator, Sub-Investigator, study nurse or the Gynecologist must explain to 
the patient how to administer the vaginal gel. It is advisable that the first application be 
performed in presence of the gynecologist or the oncologist. Patients will be instructed to 
administer the rest of treatment applications at bedtime.  
5.1.2 Vaginal gel placebo 
The placebo moisturizing gel has the same composition than Blissel® without the active drug 
substance, estriol. 
The placebo moisturizing gel will be administered in the same way. 
5.2 Materials and Supplies 
For this study, the term “study drug” (Investigational Medicinal Product [IMP]) refers to 
0.005% estriol vaginal gel (Blissel®).  
Placebo vaginal gel is the control medicinal product. 
The Sponsor will provide to the study sites with Blissel® and Placebo for the purpose of this 
study.  
Patients will be given three different packages: 
 Baseline: tube of 30 g and 25 administration applicators. For weeks 1-3 of treatment. 
 Week 3: tube of 30 g and 12 administration applicators. For weeks 4-8 of treatment. 
 Week 8: tube of 30 g and 12 administration applicators. For weeks 9-12 of treatment. 
5.2.1 Storage, preparation and administration 
Storage conditions stated in the Study Reference Safety Document may be superseded by the 
label storage. 
Investigators and site staff are reminded to continuously monitor room storage temperatures 
and ensure that thermometers are working correctly as required for proper storage of 
investigational product. Any temperature excursions must be reported immediately to the 
Sponsor and documented. Once a deviation is identified, the investigational product MUST be 
quarantined and not used until the Sponsor provides documentation of permission to use the 
investigational product.  


 
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Blissel® and placebo should be stored at controlled room temperature (≤ 25ºC) in their original 
container.  
Medication should be kept in a secured locked area at the study site in accordance with 
applicable regulatory requirements. Returned medication should be stored separately from 
medication that needs to be dispensed. 
The investigator`s team will hand over the medication to the patient. Patients will only be given 
the medication corresponding to each following period in the visits of day 1 (baseline visit) and 
in the visits of week 3 and week 8. 
The Principal Investigator, Sub-Investigator, study nurse or the Gynecologist must explain to 
the patient how to administer the vaginal gel. It is advisable that the first application be 
performed in presence of the gynecologist or oncologist.  
Women will be instructed to administer the gel themselves, preferably at bedtime, for the rest 
of applications.  
5.2.2 Accountability 
The patient will be indicated to return both the tube and the unused applicators as surplus 
medication. The investigator will collect them and will record the number of unused applicators 
in each period in the eCRF in order to evaluate dosage compliance. 
The surplus medication, the unused applicators in each period, will be stored until the end of 
the study. At that time they will be returned to the sponsor, which shall destroy them. All 
medication supplied for the study will only be used for this protocol and in no case for another 
purpose. 
It is the responsibility of the investigator to ensure that a current record of Blissel® and placebo 
disposition is maintained at each study site where study drug is inventoried and disposed. 
Records or logs must comply with applicable regulations and guidelines. 
GEICAM (on behalf of Sponsor) will provide forms to facilitate inventory control if the staff at 
the investigational site does not have an established system that meets these requirements.  
5.3 Method of Assigning a Patient to a Treatment 
All patients will be screened prior to entry on this study. An explanation of the study and 
discussion of the expected side effects and presentation of the informed consent document will 
take place. Eligible and consenting patients will be enrolled into the study and a screening code 
will be assigned to the patients. 
All patients who meet all inclusion and exclusion criteria will be randomized to receive 0.005% 
estriol vaginal gel (Blissel®) or placebo vaginal gel.  
A randomization list will be generated taking into consideration blocks of 5 (4 actives 1 
placebo). The study medication will be coded according to this randomization list.  Study 
medications will be shipped in blocks of 5 to the sites.  


 
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In each site, all patients that fulfil all inclusion and exclusion criteria and are definitely included 
in the study will be assigned the randomization code. This code will be the one labelled in the 
medications. Thus, the first patient included in a site will receive the first study medication in 
the block, the second patient included will receive the second medication in the block, and this 
procedure will be followed consecutively.  
The eligibility checklist must be completed and signed by the investigator prior to inclusion and 
it must be included into medical history of each patient. 
All eligible patients enrolled in the study will be entered in a patient registration log maintained 
by the Site and in the Trial Master File.  
The study treatment must be administered after definite inclusion of the patient in the study. 
5.4 Special Treatment Considerations. Dose Adjustments of Study Drug 
All dose modifications should be based on the worst preceding toxicity.  
Every effort should be made to administer study treatment on the planned schedule. However, 
in the event of significant treatment-related toxicity, administration of study drug could be 
interrupted or delayed as described in the following sections.  
5.4.1 
Dose Interruption 
In the event of significant treatment-related toxicity, Blissel® or placebo may be interrupted as 
described below. Patients are to be instructed to notify investigators at the first occurrence of 
any adverse sign or symptom. 
Patients experiencing the following adverse events should have their treatment interrupted:  
 Jaundice or deterioration in liver function 
 Significant increase in blood pressure 
 New onset of migraine-type headache 
Treatment may be held as needed until toxicity resolution. Appropriate follow up assessments 
should be done until adequate recovery occurs as assessed by the Investigator. Treatment will 
be resumed once this toxicity completely disappeared; if treatment cannot be resumed within 3 
weeks it will be interrupted permanently. 
All dose interruptions must be clearly documented in the patient's source notes and in the 
eCRF.  
5.4.2 
Special conditions 
The following conditions will require a closer revision of the investigator: 
 Heart disease or circulation problems 
 Hypercholesterolemia 


 
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 Hyperglycemia 
 Breast changes 
 Uterine fibroids or endometriosis 
 Epilepsy 
 Asthma 
 Systemic Lupus Erythematosus 
 Otosclerosis 
5.5 Medication Errors and Overdose 
Medication errors may result in this study from the administration at the wrong time. Such 
medication errors occurring to a study participant are to be captured on the eCRF. In addition 
the Sponsor should be notified immediately.  
Medication errors are reportable irrespective of the presence of an associated AE/SAE, 
including: 
 Medication errors involving patient exposure to the product; 
 Potential medication errors or uses outside of what is foreseen in the protocol that do or 
do not involve the participating patient. 
5.6 General Concomitant Medication and Supportive Care Guidelines 
Patients must be instructed not to take any additional medications (over-the-counter or other 
products) during the study without prior consultation with the investigator. Any medications 
including herbal supplements, vitamins, or treatment taken by the patient from 28 days prior to 
the start of study treatment and up to 30 days (±5 days) following the last dose of 
investigational product and the reason for their administration must be recorded on the eCRF.  
Routine postoperative care, such as dressing changes, suture removal, drain removal, or venous 
access (central or peripheral), does not need to be recorded. Anesthetics used for any surgical 
procedures performed during the patient’s participation in the study can be recorded as 
“unspecified anesthesia” on the concomitant treatment records; it is not necessary to list the 
specific anesthetics.  
5.6.1 Prohibited Medications 
The following treatments are prohibited throughout the duration of the active treatment phase:  
 Anticancer agents: No additional investigational or commercial anticancer agents such 
as chemotherapy, immunotherapy, targeted therapy, biological response modifiers or 
endocrine therapy (different than the NSAI: anastrozole or letrozole) will be permitted 
during the active treatment phase. In general, any drugs containing “for the treatment of 
breast cancer” on the product insert are not permitted on study. 


 
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 Hormone replacement therapy with estrogens or progestins, tibolone, topical 
estrogens (different from the study drug/placebo), phytoestrogen, megestrol acetate 
and selective estrogen-receptor modulators (eg, raloxifene, tibolone) are prohibited 
during the active treatment phase. 
5.6.2 Permitted Medications 
The following treatments are permitted throughout the duration of the active treatment phase:  
 Standard therapies for pre-existing medical conditions, medical and/or surgical 
complications, and palliation. Any medication intended solely for supportive care (eg, 
analgesics, antidiarrheals, antidepressants) may also be used at the investigator’s 
discretion. All medications should be recorded.  
 Bisphosphonates and receptor activator of nuclear factor kappa-B ligand 
(RANKL) inhibitors for the treatment of osteopenia/osteoporosis.  
5.7 Treatment Compliance 
The compliance will be evaluated by the Investigator Team at week 3, 8 and 12 by counting 
and recording the number of unused applicators returned in each period.  
The percentage of pharmacological compliance will be calculated from the count of the 
applicators.  
To evaluate the compliance, the flexibility permitted in the appointments, having to increase or 
reduce applicators from the theoretical calculation of applicators returned, will be taken into 
consideration. Checking the patient completion calendar will facilitate this evaluation. 
5.8 Blinding and Unblinding  
The study drug, 0.005% Estriol vaginal gel, and its vehicle in gel (placebo vaginal gel) will be 
of identical appearance and will have the same aroma and the same texture in order to maintain 
the double blind. 
Throughout the study, the medication with which the patient is being treated will be kept 
strictly confidential and only authorized persons will have access to this information, unless it 
is necessary to break the blinding due to safety reasons. The unblinding must be done only by 
the principal investigator of the site or by an authorized person, only when the knowledge of 
the treatment assignment is deemed essential for the patient’s care. 
The randomization codes will be unblinded and made available for the project statistician to 
perform the data analysis, when the study has been finalized, the database has been verified and 
the protocol violations have been determined. All personnel involved directly in the study shall 
be unaware of the treatment assigned to the patients until the database has been closed. 


 
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5.8.1 
Process for urgent unblinding 
In general, unblinding of participants during the conduct of the clinical trial is not allowed 
unless there are compelling medical or safety reasons to do so. 
Any serious adverse event considered by the investigator to be related to the blinded treatment 
medication (0.005% estriol vaginal gel/placebo vaginal gel) should be reported as such to the 
sponsor via the appropriate SAE reporting form. 
There will be two complete sets of randomization codes. One will be kept by the GEICAM 
Pharmacovigilance department and the other will be distributed among the investigators. The 
emergency codes should be kept in a safe but accessible place in the event they should be 
opened. Each investigator will receive the necessary envelopes closed to randomize a 
determined number of patients. Each envelope will contain the details of the treatment that the 
patient is receiving. In the case of emergency the envelope must be opened to determine the 
treatment being administered. 
The randomization envelopes can only be opened in an emergency. When the investigator 
opens the envelope he/she must record the date, the time and the reason he/she has had to do so 
and will keep this information with the eCRF documentation. He/she should also immediately 
inform the study monitor that a treatment code has been opened As per regulatory reporting 
requirement, it will unblind the identity of the study medication for all unexpected SAES that 
are considered by the investigator to be related to the study drug. Details of subjects who are 
unblended during the study will be included in the Clinical Study Report. 


 
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6. Efficacy and Safety Evaluations, Sample Collection and Testing 
(Standard Laboratory Testing) and Appropriateness of Assessments 
Study procedures and their timing (including tolerance limits for timing) are summarized in the 
Study Schedule, Protocol Attachment 1. 
Patients will receive the treatment for 12 weeks (3 months). 
Patients will be evaluated at baseline (within 2 weeks prior to treatment initiation on day 1), at 
week 1 (day 8 +/-2 days) at week 3 (day 22 +/- 3 days), week 8 (day 57 +/- 3 days) and week 
12 (day 85 +/- 3 days). There will be an additional safety visit 4 weeks (± 5 days) after the last 
study medication administration. 
Before a patient starts her participation in the study, she must have given her written informed 
consent. Through this consent, it is guaranteed that the person voluntarily expresses their wish 
to participate in the clinical trial, after having understood all the information that has been 
provided to her about the study.  
6.1 FSH serum levels 
The primary endpoint is to evaluate the variation of FSH levels in serum from baseline to 12 
weeks of treatment with 0.005% estriol vaginal gel. As a secondary endpoint, we will evaluate 
the variation at different time points along 8 weeks of treatment with 0.005% estriol vaginal 
gel. 
Blood samples to analyze FSH (in serum) will be collected at six time points: 
 Screening: one blood extraction 
 Baseline visit: one blood extraction.  
 Weeks 1-3: one blood extraction in visits at week 1 (day 8 +/- 2 days) and week 3 (day 
22 +/- 3 days). 
 Weeks 4–12: one blood extraction in visits at week 8 (day 57 +/- 3 days) and week 12 
(day 85 +/- 3 days).  
Serum FSH levels will be determined in a Central Laboratory (Laboratorios Echevarne). 
Chemiluminescent assay will be used for their analysis. 
Detailed instructions for the collection, labelling, handling and shipment of samples are 
outlined in the Sample Management Manual. 
6.2 Estriol, estradiol, estrone plasma levels and LH serum levels  
Secondary endpoints are to evaluate the variation of estriol, estradiol and estrone levels in 
plasma and LH levels in serum along 12 weeks of treatment with 0.005% estriol vaginal gel. 
Blood samples to analyze LH (in serum) will be collected at the same six time points: 
 Screening: one blood extraction 


 
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 Baseline visit: one blood extraction 
 Weeks 1-3: blood extraction in visits at week 1 (day 8 +/- 2 days) and week 3 (day 22 
+/- 3 days)  
 Weeks 4–12: blood extraction in visits at week 8 (day 57 +/- 3 days) and week 12 (day 
85 +/- 3 days)  
Blood samples to analyze estriol, estradiol, estrone (in plasma) will be collected at five time 
points: 
 Baseline visit: one blood extraction 
 Weeks 1-3: blood extraction in visits at week 1 (day 8 +/- 2 days) and week 3 (day 22 
+/- 3 days)  
 Weeks 4–12: blood extraction in visits at week 8 (day 57 +/- 3 days) and week 12 (day 
85 +/- 3 days)  
Plasma estradiol, estriol, estrone levels and serum LH levels will be determined in Central 
Laboratories (Laboratorios Echevarne; Pharm-Analyt). Due to the extremely low levels of 
plasma estrogens expected, a newly developed and validated ultrasensitive LC-MS/MS assay 
will be used for the analysis of estradiol, estriol and estrone at Pharm-Analyt. 
Chemiluminescent assay will be used for the analysis of Serum LH at Laboratorios Echevarne. 
Detailed instructions for the collection, labelling, handling and shipment of samples are 
outlined in the different Sample Management Manuals. 
6.3 Safety assessments 
Investigators are responsible for monitoring the safety of patients who have entered this study 
and for alerting GEICAM to any event that seems unusual. 
The investigator is responsible for appropriate medical care of patients during the study. 
The investigator remains responsible for following, through an appropriate health-care option, 
adverse events that are serious or that caused the patient to discontinue before completing the 
study. The patient should be followed until the event is resolved or explained. Frequency of 
follow-up evaluation is left to the discretion of the investigator. 
During the course of the study, all patients entering the trial must be evaluated according to the 
schedule outlined in the flow charts and described below. The results of the evaluation will be 
recorded in the eCRF pages until the patients are not followed anymore. 
6.3.1 
Timing of Assessments 
Physical and gynecological examination including vital signs assessments (blood pressure and 
pulse), will be performed at screening, week 1 (day 8 +/- 2 days), week 3 (day 22 +/- 3 days), 
week 8 (day 57 +/- 3 days), week 12 (day 85 +/- 3 days) and 4 weeks after the last study drug 


 
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administration. Height and weight will be registered at screening. Gynaecological examination 
will comprise breast examination and pelvic examination (examination of uterus and 
appendages), and will be performed at baseline and at weeks 3 and 12. Endometrial ultrasound 
will be performed at screening and at week 12.  
The following safety laboratory assessments will be performed by the local laboratories, at 
screening and at week 12 and 4 weeks (+/- 5 days) after the last study drug administration: 
 Hematology: hemoglobin, WBC, absolute neutrophils, platelet count. 
 Blood Chemistry: fasting glucose, alkaline phosphatase, ALT, AST, total bilirubin, 
serum creatinine and BUN (or urea). 
 Urine test: density, pH, glucose, proteins, cetones, bilirubin, blood, nitrites, 
urobilinogen and leukocites. 
All AEs (and their relatedness to the study drug) occurring during the study will be documented 
in the eCRF at baseline, week 1 (day 8 +/- 2 days), week 3 (day 22 +/- 3 days), week 8 (day 57 
+/- 3 days), week 12 (day 85 +/- 3 days) and 4 weeks after the last study drug administration. 
AEs will be graded according to MedDRA for each type of adverse event will be recorded. 
Additionally, the relationship of the adverse event(s) to the study treatment will be taken into 
consideration. 
6.3.2 
Safety Definitions 
The safety definitions are described in the table 2. 
Table 2: Safety definitions 
Concept 
Definition 
Adverse Event  (AE) 
Any new untoward medical occurrence or worsening of a pre-
existing medical condition in a patient or clinical investigation 
subject administered an investigational (medicinal) product and 
that does not necessarily have a causal relationship with this 
treatment.  
An AE can therefore be any unfavorable and unintended sign, 
symptom or disease temporally associated with the use of 
investigational product, whether or not considered related to the 
investigational product.  
Laboratory abnormalities should be reported as AE only in case 
they lead to an action on study treatment or if they are serious.  
Adverse Reaction (AR) 
All untoward and unintended responses to a medicinal product 
related to any dose administered.  
All expected ARs are listed in the Summary of Product 
Characteristics [SmPC]. If the nature or the severity of an 


 
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adverse reaction is not consistent with the applicable product 
information, the AR is defined as unexpected. The basis for the 
decision is the current version of the corresponding reference 
document that has been submitted and approved by the 
competent authority and the ethics committees. 
Accountability criteria 
The sponsor will classify the adverse event, based in their 
causation relation with the investigational product, following 
the Karch y Lasagna (1977) algorithm, as: 
o Final: there is reasonable temporal sequence between 
the drug administration and the existence of the adverse 
event. This event matches with the adverse reaction 
described for the investigational product, improves with 
the omission and reappears after its re-administration 
and can’t be explained by other causes. 
o Probable: there is reasonable temporal sequence 
between the investigational product administration and 
the appearance of the adverse event. This event matches 
with the adverse reaction described for the drug, 
improves with the omission and can’t be explained by 
other causes. 
o Possible: there is reasonable temporal sequence between 
the investigational product administration and the 
appearance of the adverse event. This event matches 
with the adverse reaction described for the drug but can 
be explained by other causes. 
o Conditional or improbable: there is reasonable temporal 
sequence 
between 
the 
investigational 
product 
administration and the appearance of the adverse event. 
This event does not matches with the adverse reaction 
described for the drug and can be explained by other 
causes. 
o Not related: there is no reasonable temporal sequence 
between the investigational product administration and 
the appearance of the adverse event. This event does not 
matches with the adverse reaction described for the drug 
and can be explained by other causes. 
For expedited reporting purposes it is considered as related the 
categories: final, probable and possible from Karch y Lasagna 
(1977) algorithm and as not related the category conditional or 


 
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improbable of that algorithm.  
The determination of the possible relation with the study 
treatment is responsibility of the principal investigator of the 
site or the person designated by him.  
Serious Adverse Event 
(SAE) and Serious 
Adverse Reaction (SAR) 
Any adverse event or adverse reaction that, at any dose: 
o is fatal (results in death), 
o initial or prolonged inpatient hospitalization, 
o a life-threatening experience (that is, immediate risk of 
dying, defined as an event in which the patient was at 
risk of death at the time of the event; it does not refer to 
an event which hypothetically might have caused death 
if it were more severe), 
o persistent or significant disability/incapacity, 
o congenital anomaly/birth defect or 
o an important medical event, defined as a medical event 
that may not be immediately life-threatening or result in 
death or hospitalization but, based on appropriate 
medical and scientific judgment, may jeopardize the 
patient or may require intervention (eg. medical, 
surgical) to prevent one of the other serious outcomes 
listed above.  
Do not confuse the concept “serious”, described before, with 
“severe” which refers to the intensity of the AE or AR 
(minor/mild/severe). 
Any temporary increase in the severity of a symptom or 
previous sickness that happens after the baseline of the study is 
considered also as an adverse event.  
Suspected Unexpected 
Serious Adverse 
Reaction (SUSAR) 
Any serious adverse reaction whose nature, intensity or 
consequences do not correspond with the reference information 
for the investigational product (Summary of Product 
Characteristics [SmPC]).  
The unexpected nature of an adverse reaction is based in the 
fact of not being observed previously and not in what could be 
advanced based on the pharmacological properties of the drug.  


 
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6.3.3 
Management, Timing and Assessment of Adverse Events 
AE Classification  
 
Adverse events should be classified following MedDRA. The 
investigators team must have access to MedDRA.  
The causal relation between the investigational product and the 
AE will be assessed by the investigator using the Karch y 
Lasagna (1977) algorithm. 
Note that, for the purpose of this protocol, the following 
circumstances 
– 
with 
or 
without 
associated 
adverse 
event/reaction - should be included the data base and will be 
submitted to the sponsor - that is, managed in the same way and 
time as adverse events/reactions: 
 Overdose, abuse, misuse 
 Off-label use 
 Medication error 
Procedure to notify an 
AE to Sponsor 
The site will enter the following events in the eCRF: 
o All adverse events that occur after enrollment.  
o Preexisting conditions that get worse during the study.  
o The evaluation of the possible relationship of each 
adverse event to the study drug or protocol procedure.  
o The circumstances and data that causes the suspension 
of the treatment of a patient due to an adverse event. 
o The events leading to the clinical outcome of death from 
disease progression will be included in the efficacy 
analysis unless and are not recorded as adverse events, 
unless the investigator believes they could  have been 
caused by the study drug.  
Data Management of GEICAM will notify all these events to 
the sponsor as agreed between the parties.  
Timing and assessment 
of AE (see Protocol 
Attachment 3) 
The site staff will report on the eCRF the information of the AE 
in the following  periods: 
 Baseline (after ICD and before study treatment): 
study site personnel will note the occurrence and nature 
of each patient´s medical condition(s) and preexisting 
conditions in the appropriate section of the eCRF. If a 
patient never receives study treatment but experiences 
an adverse event after the ICD is signed, ONLY events 


 
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the investigator believes may have been caused by a 
protocol procedure will be reported to GEICAM via 
eCRF. 
 During treatment with the study treatment: during 
the study, site personnel will record any change in the 
condition(s) and the occurrence and nature of any 
adverse events. A CTCAE grade rating will be assigned 
before each cycle for any adverse event experienced 
during the previous cycle.  
 30-day post-discontinuation follow-up period: each 
patient will have a 30-day post-discontinuation follow-
up evaluation approximately 30 days following the 
discontinuation of study treatment. Patients should be 
closely followed for study treatment adverse events in 
order to detect delayed toxicity. If drug-related toxicity 
is present beyond 30 days post-discontinuation, patients 
must be followed until the toxicity resolves or improved 
to baseline, the relationship is reassessed as unrelated, 
the investigator confirms that no further improvement 
can be expected, another therapy is initiated, or death.  
 Long-Term Follow-up Period (after the 30-day post-
discontinuation): only new and ongoing SAEs thought 
to be related to study treatment or protocol procedures 
should be documented on the eCRF and immediately 
reported to GEICAM via the designated transmission 
method, even if the study has been closed.  
6.3.4 
Management, Timing and Assessment of SAEs 
Timing of SAEs (see 
Protocol Attachment 3) 
All the SAEs (either spontaneously or during the trial visits) 
will be collected since the patient signs the Informed Consent 
Document (ICD). 
All the SAEs must be documented in the medical record of the 
patient and in the eCRF. A follow up of all the SAEs should be 
done until they are solved or until the toxicity is considered 
irreversible.  
SAEs which do not need 
to be notified to the 
Pharmacovigilance 
Department of the 
Sponsor 
The following events are not considered SAEs: 
o A visit to the emergency room or other hospital 
department lasting less than 24 hours that does not 
result in admission (unless considered an “important 


 
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medical event” or a life-threatening event). 
o Elective surgery planned before signing consent. 
o Hospitalization which is due solely to a planned study 
visit and without prolongation. 
o Routine health assessment requiring admission for 
baseline/trending 
of 
health 
status 
(eg. 
routine 
colonoscopy). 
o Medical/surgical admission for purpose other than 
remedying ill health state that was planned before study 
entry. Appropriate documentation is required in these 
cases. 
o Admission encountered for another life circumstance 
that carries no bearing on health status and requires no 
medical/surgical intervention (eg. lack of housing, 
economic 
inadequacy, 
caregiver 
respite, 
family 
circumstances, administrative). 
o Progression of the malignancy during study (including 
signs and symptoms of progression), unless the outcome 
is fatal and death occurred before end of treatment. 
Thereafter death due to disease progression has not to 
be reported as SAE.  
o Hospitalization due to signs and symptoms of disease 
progression. 
o An overnight stay in the hospital that is only due to 
transportation, 
organization 
or 
accommodation 
problems and without medical background.  
They will be reported in the eCRF and in the patient record. 
The rest of SAEs must be notified as described as follows. 
 
 
Procedure to notify a  
SAE to the  
Pharmacovigilance 
Department of GEICAM 
and Sponsor  
 
The SAEs must be notified to the Pharmacovigilance 
Department of GEICAM and to the Sponsor at the same time. 
A member of the investigator team must complete and sign the 
SAE notification form which will be sent by fax/mail, 
immediately and always during the 24 hours following 
knowledge of the SAE: 
 
 


 
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Pharmacovigilance Department of GEICAM 
Fax: +34 916 510 406 
farmacovigilancia@geicam.org  
 
ITF RESEARCH PHARMA S.L.U 
Fax:  +34 91 6572372 
Email: drugsafety@itfsp.com 
Once received, GEICAM and the Sponsor will contact each 
other to make sure that both parties have received the SAE. 
The SAE will be reviewed by the Pharmacovigilance 
Department of the Sponsor and, if necessary, will ask more 
information to the investigator. 
When additional information is obtained about the SAEs, or 
this is solved or is improbable it will change, a follow-up report 
must be also completed and sent by fax/mail, immediately and 
always 
during 
the 
following 
the 
24 
hours 
to 
the 
Pharmacovigilance Department of Sponsor. 
If the Sponsor suspects that the SAE could be a SUSAR, the 
investigator should give the follow up information requested. 
All SAEs from the time the patient have the first dose of the 
study drug through 30 days following the last administration of 
study drug must be reported according to the procedure 
described below. All SAE regardless of timing must be 
reported, if considered related to study drug. 
Likewise, progression of a patient's underlying condition 
leading to one of the above should also not be reported as a 
SAE, but documented as primary study endpoint. 
All SAEs will be followed-up by the investigator until 
satisfactory resolution. Annually all SARs will be reported as 
the DSUR to the competent authorities and the leading ethics 
committee, including all SUSARs.  
Withdrawal from further treatment shall be at the discretion of 
the investigator. 
Death on Study 
Any death occurring during the active treatment part of the 
study and within 30 days following the last treatment must be 
reported to GEICAM and to the sponsor within 24 hours, 
regardless of the relation to study drug(s), and has to be 


 
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reported on the death report form section of the eCRF.  
The cause of death should be documented (cancer-related, 
treatment-related, cancer- and treatment-unrelated). Autopsy 
reports should be collected whenever possible and sent to 
GEICAM.  
Deaths that occur due to tumor progression do not have to be 
reported as a SAE unless they occurred before end of treatment.  
Deaths after the end of study which are considered to be related 
to study treatment have to be reported as SAEs.  
To the extent feasible sufficient information including relevant 
laboratory values, ECG, scan, biopsy or autopsy results must be 
provided by the investigator in the SAE narrative (even if 
investigator determines the SAE is not related) so as to permit 
an independent causality assessment by a Competent Authority. 
6.3.5 
Management, Timing and Assessment of SUSARs 
Expedited Notification of  
SUSAR to the Competent 
Authorities/IRB 
The Pharmacovigilance Department of the GEICAM (on behalf 
of the sponsor) is responsible to notify to each of the competent 
authorities and IRBs of the participating countries, all the 
SUSARs collected in the study, following the procedures shown 
in the current legislation.   
Timing of notification  
The deadline for reporting a SUSAR shall be 15 calendar days 
from when the Sponsor becomes aware of it. When suspected 
SUSAR caused the death of the patient or endangered her life, 
the Sponsor will send the information within 7 calendar days 
from the date on which it becomes aware. This information 
must be completed, if possible, in the next 8 days. 
 
Expedited reporting of 
other relevant safety 
information 
Sponsor will also notify, expeditiously, all the information that 
could modify the balance benefit/risk of the investigational 
product, or determine changes in its administration pattern or in 
the study performance, such as: 
o A qualitative change or an increase in the percentage of 
occurrence of the SAR expected, which are considered 
clinically significant. 
o The SUSAR occurring after completion of the study and 
are reported by the investigator to the sponsor.  


 
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o New events related to the conduct of a trial or the 
development of an IMP likely to affect the safety of 
patients, such as: 
 SAE that could be related with the study 
procedure and could modify the conduct of the 
trial. 
 A significant risk to patients such as lack of 
efficacy in a drug used to treat a life-threatening 
illness. 
 A major safety finding from a newly completed 
animal study (such as carcinogenicity). 
 A temporary halt of a trial for safety reasons if 
the 
trial 
is 
conducted 
with 
the 
same 
investigational medicinal products in another 
country and if this information is known by 
Sponsor. 
 Any recommendation of the IDMC that is 
relevant to the safety of patients (if applicable). 
This relevant information shall be notified as soon as possible 
and no later than 15 days after Sponsor becomes aware of it. 
Additional information will also be notified as quickly as 
possible.  
Annual Safety Reports 
The annual safety reports that include the SAEs and SUSARs 
collected during the study will be sent by the Sponsor to the 
Competent Authorities at the time established by the current 
legislation. 
Notification to  
investigators 
Sponsor will communicate to the investigators any safety 
information that may affect the safety of trial patients, as soon 
as possible.  
Information on SUSAR occurred during the study will be sent 
at the same time that it is notified to competent authorities but 
without submitting any information on the treatment arm.  
They will be informed also, throughout the entire study, of any 
safety aspect that impacts the performance on the clinical trial 
or in the product development, including the interruption or 
modification in the development program of the protocol 
safety-related.  
 


 
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6.4 Efficacy assesments 
The following assessments will be performed at baseline, week 3 (day 22 +/- 3 days) and week 
12 (day 85 +/- 3 days) or early withdrawal visit. 
 Symptoms of vaginal atrophy (vaginal dryness, dyspareunia and pruritus).  
 Signs of vaginal atrophy (mucosa with thinning or flattening of folds, dryness or 
fragility of the vaginal mucosa).  
 Measurement of vaginal pH on the vaginal secretion using a reactive strip (pH 2.0 - 
9.0, provided by the sponsor). The investigator team will insert the strips in the vagina 
and will moisten them as much as possible with the vaginal wall secretion. Then, when 
the strip is coloured, the investigator will check the combination of colours on the strip 
and check the pH value to which it corresponds, recording this value in the eCRF. The 
reading should be made whilst the strip is still damp. The strips will be discarded once 
the pH has been recorded in the eCRF. 
 Vaginal cytology sample to evaluate the vaginal Maturation Value. Two smears (with 
two different spatulas provided by the sponsor one to take the sample and the other to 
take the countersample) will be made per patient (sample and countersample), which 
will be obtained from the bottom of the right and left vaginal sac respectively. Each 
smear will be suitably labelled. The vaginal smears will be fixed whilst they are still 
moist with a water-soluble fixation spray for cytodiagnosis (provided by the sponsor). 
The cytologic samples will be sent to central laboratory (full instructions will be given 
in a laboratory manual). For the cytologic evaluation, the number of parabasal, 
intermediate and superficial cells will be calculated in duplicate on 100 consecutive 
cells of vaginal cytology. The average of the two percentages obtained for each cell type 
will be calculated, which will serve to determine the maturation value (MV) based on 
the following formula:  
0.2 x (% parabasal) + 0.6 x (% intermediate) + 1.0 x (% superficial). 
 
6.5 Impact on sexual function 
Patient reported outcomes of health related with the sexual function will be assessed using the 
FSFI (Female Sexual Function Index) (see attachment 4). It was developed for the specific 
purpose of assessing domains of sexual functioning (e.g. sexual arousal, orgasm, satisfaction, 
pain) in clinical trials.  It is not a measure of sexual experience, knowledge, attitudes, or 
interpersonal functioning in women.  It is not designed for use as a diagnostic instrument and 
should not be used as a substitute for a complete sex history in clinical evaluation. However, 
when initial assessment reveals a sexual concern, using the FSFI (or an equivalent set of 
questions) can help both the physician and the patient measure the nature and severity of the 
problem as well as identify an appropriate course of treatment. 


 
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The FSFI is a brief multidimensional scale with 19-item questionnaire composed of three 
multi-item functional subscales (sexual activity, sexual intercourse and sexual stimulation).  
Patients will complete each questionnaire at baseline, week 3 (day 22 +/- 3 days) and week 12 
(day 85 +/- 3 days) or early withdrawal visit. 
Completed questionnaires are always considered source document and must be filed 
accordingly. 
Patients must preferably complete these instruments in the hospital and prior to having any tests 
and to any discussion of their progress with healthcare personnel at the site. Interviewer 
administration in clinic may be used under special circumstances (eg, patient forgot their 
glasses or feels too ill).   


 
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7. Data Quality Assurance 
To ensure accurate, complete and reliable data, the Sponsor or delegated people on behalf of 
the sponsor will do the following: 
 provide instructional material to the study sites, as appropriate 
 sponsor a start-up training session to instruct the investigators and study 
coordinators. This session will give instructions on the protocol, the completion 
of the eCRFs, and study procedures 
 make periodic visits to the study site to review study progress, investigator and 
patient compliance with the clinical trial protocol requirements and any 
emergent problems. 
 be available for consultation and stay in contact with the study site personnel by 
mail, telephone, and/or fax 
 review and evaluate eCRF data and use standard computer edits to detect errors 
in data collection 
 conduct a quality review of the database     
verify the quality of the data 
To ensure the safety of participants in the study and to ensure accurate, complete, and reliable 
data, the investigator will keep records of laboratory tests, clinical notes, and patient medical 
records in the patient files as original source documents for the study.  If requested, the 
investigator will provide GEICAM (on behalf of the sponsor), applicable regulatory agencies, 
and applicable ethical review boards with direct access to original source documents. 
7.1 Data Management and Registries file 
Data for this study will be recorded via an electronic data capture system using eCRFs. Data will 
be transcribed by the site from the paper or electronic source documents onto the eCRF. In no 
case the eCRF is to be considered as source data for this trial. The eCRFs must be completed in 
an electronic Data Base called ORACLE. That electronic Data Base carries out with the 
regulatory authorities requirements. It is the responsibility of the Investigator to maintain 
adequate and accurate eCRFs (according to the technology used) designed by GEICAM (on 
behalf of the sponsor) to record (according to GEICAM instructions) all observations and other 
data pertinent to the clinical investigation. All eCRFs should be completed in their entirety in a 
neat, to ensure accurate interpretation of data. 
Should a correction be made, the corrected information will be entered in the eCRF overwriting 
the initial information and including a reason for the change. An audit trail allows identifying 
the modification.  
Data are available within the server of GEICAM as soon as they are entered in the eCRF. 
The computerized handling of the data by GEICAM when available in the eCRF may generate 
additional requests to which the Investigator is obliged to respond by confirming or modifying 
the data questioned. The requests with their responses will be managed through the eCRF. 


 
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8. Sample Size and Statistical Methods 
8.1 Determination of Sample Size 
This is an exploratory study and there are scarce data to allow a precise sample size calculation. 
The most relevant data come from Pfeiler16(16) who reported that the levels of FSH in patients 
treated with aromatase inhibitors fell from a mean value of 75.7 to 66.0 mIU/ml after two 
weeks of daily vaginal treatment with 0,5 g of estriol. Although no figures are reported of the 
variability of those measurements, an estimation of the standard deviation can be made 
applying the standard rule of [sd = range/4]. The reported range of FSH values in that report is 
45.6 – 134.6, thus a reasonable approximation to the sd is 89/4 = 22.3. Under these 
assumptions, it can be calculated that a sample size of 44 would provide 80% power to detect 
with an alpha=0.05 a decrease of FSH levels from 75.7 to 66.0 mIU/ml assuming a standard 
deviation of 22.3. 
The hormone levels in women treated with IA who are not receiving estrogen therapy are likely 
to suffer physiological temporal oscillations. These variations could make difficult the 
interpretation of any eventual modification of hormone levels observed in the estriol treated 
women.  In the absence of data of naturally occurring variations in hormone levels of women 
treated with IA  that allow us to interpret the results of hormone determinations under study, it 
is considered appropriate to include a placebo group to provide reference data. In this case, the 
placebo is a vaginal moisturizing gel. 
It is estimated that a group of 11 women will allow the assessment of physiological fluctuations 
in hormone levels. Additionally, this sample size for the placebo group will provide 80% power 
and alpha=0.05 to differentiate between active and placebo (ratio 4:1) on the change in the 
vaginal maturation value, which is a secondary variable of the study that provides objective 
information about the effectiveness of treatment on vaginal atrophy (calculation based on the 
parameter data from the pivotal Blissel study and considering only the subgroup of women who 
had severe vaginal dryness: change VM active group 24.9 vs 0.6 points in placebo group, SD = 
24.2).  
The differentiation between active and placebo in the subjective parameters of efficacy 
(secondary) would be possible at the expense of a sample size noticeably superior. Since the 
evaluation of effectiveness is not primary but secondary in this study, it is not justified in this 
study to increase the sample for this reason.  
In accordance with the above, the study would include 55 women.  44 will receive vaginal 
estriol gel and 11 with the placebo moisturizing gel). Taking into account 10% of dropouts, the 
sample to be considered is of 60 women (48 vaginal estriol gel and 12 placebo moisturizing 
gel). This is an unbalanced study but with an active: placebo ratio of 4:1, which will support an 
adequate patient randomization (blocks of 5). 
10 additional women will be recruited in a first safety phase of the study (8 with vaginal estriol 
gel and 2 placebo moisturizing gel).  


 
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8.2 Statistical and Analytical Plans 
8.2.1 
General Considerations 
Statistical analysis of this study will be the responsibility of GEICAM (on behalf of the 
sponsor). The interpretation of study results will be the responsibility of the chief investigator 
of the study together with the sponsor.   
Detailed methodology for summary and statistical analyses of the data collected in this study 
will be documented in a Statistical Analysis Plan (SAP), which will be dated and maintained by 
GEICAM (on behalf of the sponsor).  This document may modify the plans outlined in the 
protocol; however, any major modifications of the primary endpoint definition and/or its 
analysis will also be reflected in a protocol amendment. 
All the analysis will be performed using the statistical software SAS Enterprise Guide 5.1 
8.2.1.1. 
Patient Populations 
Intent to treat population (ITT): The ITT population will include all patients who are 
randomized, with study treatment (Estriol gel vaginal 0.005% or placebo) assignment 
designated according to initial randomization.   
The ITT population will be the primary population for evaluating patient characteristics, 
hormone levels, efficacy and the FSFI questionnaire. 
Per-protocol population: a subset of the ITT population that received at least one dose of 
study drug and completed the study without any major protocol violations.  
Safety population: will include all patients randomized in the study who received at least one 
dose of treatment, and they will be analyzed according to the actual treatment received. This 
population is for the safety analysis.  
8.2.2. 
Patient Disposition 
A detailed description of patient disposition will be provided. It could include the following: 
 summary of patients entered and by site 
 total number of patients entered 
 total number of patients enrolled 
 summary of reasons for patients entered, but not enrolled 
 total number of patients treated 
 summary of reasons for patients enrolled, but not treated. 
A detailed summary of reasons for patient discontinuation from study treatment will be 
provided. 
A summary of all identified important protocol violations will be provided. 


 
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8.2.3. 
Patient Characteristics 
Patient characteristics could include a summary of the following:  
 patients demographics 
 time of menopause 
 baseline disease characteristics 
 preexisting conditions/secondary conditions 
 prior therapy 
Other patient characteristics will be summarized as deemed appropriate. 
The categorical variables will be summarized by contingency tables. The continuous variables 
will be described by the number of patients with valid values (n), mean, standard deviation, 
median and range in both arms. 
The significance level of all statistical tests is established at 0.05. 
The effect of the treatment on continuous variables will be assessed by paired-t analysis (or 
Mann-Whitney-Wilcoxon in case of lack of normality). Categorical variables will be analyzed 
by chi-cuadrado or Fisher's exact test. 
8.2.4. 
Concomitant Therapy  
A summary of concomitant therapies will be generated in the safety population.  
8.2.5. 
Treatment Compliance  
The percentage of complying patients will be calculated at weeks 3, 8 and 12, defined as those 
with a compliance percentage of 80-110%, in the group of patients treated with 0.005% Estriol 
vaginal gel and in the placebo group. The overall compliance of the study drug will be 
calculated from these partial calculations. It will be summarized the patients and reasons of 
those patients which did not complete the 80% of medication of the study. 
8.2.6. 
Hormone levels 
All these analysis will be based on the ITT population.   
The primary endpoint and one of the secondary endpoints are to evaluate the variation from 
baseline of FSH, LH, estriol, estradiol and estrone blood levels in patients treated with 0.005% 
estriol vaginal gel/placebo.  
The variation of the levels of FSH, estriol, estradiol, estrone and LH after treatment with 
0.005% estriol vaginal gel will be studied in each woman. The variation of levels between two 
arms will be analyzed using a non-parametric test (Mann-Whitney-Wilcoxon test) or an 
ANCOVA (if it must be adjusted by initial value). 
Two determinations of FSH and LH before treatment with 0.005% estriol vaginal gel will be 
studied in each woman in order to study the intra-individual variation. Screening and baseline 
determinations will be used for this purpose.  


 
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Changes in hormone levels will be studied in each woman along the treatment (see sections 6.1 
and 6.2).  
8.2.7. 
Efficacy 
The efficacy analysis will be evaluated in the ITT population. 
The primary endpoint, the variation of FSH between week12 and baseline and comparing by 
arm will be analyzed using a non-parametric test (Mann-Whitney-Wilcoxon test). 
An ANCOVA analysis will be used to corroborate the results obtained for the primary 
endpoint. The ANCOVA analysis will compare the variation of FSH 12 weeks adjusted by 
FSH basal between the two arms. This analysis will be performed if the relation between FSH 
12 and FSH basal is lineal.  
The variations in the intensities for each one of the symptoms and signs of the vaginal atrophy, 
after 3 and 12 weeks, in each treatment arm, will be compared using the non-parametric test 
Mann-Whitney-Wilcoxon.  
8.2.7.1 
Signs and symptoms of vaginal atrophy 
The symptoms evaluated will be the following: 
 Vaginal dryness 
 Pruritus or itching 
 Dyspareunia 
Each symptom will be scored in a numeric scale from 0 to 3, as shown below: 
 0 Absence. The symptom is not present 
 1 The symptom is of mild intensity, without interfering in the patient’s activity 
 2 The symptom is of moderate intensity, causing obvious discomfort to the patient 
 3 The symptom is stated as very irritating and severe in intensity 
A Global Symptoms Score will be evaluated. It will be calculated by summing the intensities of 
all the three symptoms of vaginal atrophy in a certain time point, thus ranging between 0 and 9. 
The variation in the Global Symptom Score vs baseline will be assessed at weeks 3 and 12.  
The signs evaluated will be the following: 
 Vaginal mucosa with flattening of folds or thinning 
 Dryness of the mucosa 
 Fragility of the mucosa 
Each one of these signs will be scored by the investigator on a numerical scale in accordance 
with their presence and degree of severity as follows: 


 
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 0 Absence. The sign is not present. 
 1 The sign is present and is considered a mild alteration 
 2 The sign is present and is considered a moderate alteration 
 3 The sign is present and is considered a severe alteration 
8.2.7.2 
Vaginal Maturation value and pH 
The averages of the differences between the baseline and the final MV and pH of the 0.005% 
Estriol vaginal gel and placebo groups will be shared by a non-parametric test., to determine 
the possible superiority of the 0.005% Estriol vaginal gel treatment compared with placebo 
administration.  
These same tests will be used to analyse the change in the MV and pH after the initial 
observation period of 3 weeks.  
All of the above secondary analyses will be conducted at a two-sided 0.05 level of significance.    
8.2.8. 
Safety Analyses 
The toxicity and tolerability of the study drug will be evaluated in the safety population. Safety 
analyses will include summaries of the incidence of adverse events by MedDRA that occur 
during the study treatment period or within 30 days (+/- 5 days) of the last dose of study 
treatment, regardless of causality and according to the relationship to study drug as assessed by 
the investigator. Additionally, the following safety-related outcomes will be summarized: 
 study treatment discontinuations due to adverse events. 
 deaths 
 SAEs 
 hospitalizations and transfusions  
 use of key concomitant medications or growth factors. 
Analyses for data with discrete dates, for example, deaths, transfusions, and concomitant 
medications, will be done through 30 days after each patient’s last dose of study treatment.  
Adverse events will also be analyzed in this timeframe; that is, if an event starts within 30 days 
of discontinuation from study treatment, but after 30 days after the last dose of study treatment, 
it will not be included. 
Adverse events data and serious adverse events will be presented in frequency tables by grade. 
Hematological and clinical biochemistry toxicities will be assessed from laboratory test 
parameters. The safety analysis will be performed in the safety population.  
Adverse events data and serious adverse events will be reported in frequency tables (overall 
and by grades). Adverse events will be compared using the chi-square tests (Fisher’s Exact test 
in the case of observing frequencies <5%). 


 
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8.2.9. 
Other Analyses: Patient Reported Outcomes: FSFI 
Sexual function measured by the FSFI scale. It will be used an algorithm for determining 
domain scores and a composite full-scale score. The individual domain scores and full scale 
score of the FSFI are derived by a computational formula outlined in the SAP. Individual 
domain scores are obtained by adding the scores of the individual items that comprise the 
domain and multiplying the sum by the domain factor. The full scale score is obtained by 
adding the six domain scores. It should be noted that within the individual domains, a domain 
score of zero indicates that no sexual activity was reported during the previous evaluation. 
8.2.10. 
Interim Analysis 
There will be an interim analysis after the first 10 included patients have completed 3 weeks of 
therapy. These patients will be evaluated after 3 weeks according to the study scheme.  
Only safety data and the analytical values of FSH of these 10 firsts patients will be reviewed. It 
is expected that no significant changes from the postmenopausal to the premenopausal range in 
any patient.  
The 10 patients included in the interim analysis will not be included in the final analysis, as 
they will not complete 4 months of therapy.  For that reason, it will not be necessary to perform 
a correction of the final alpha. 
8.3 Data Monitoring Committee 
The study will use an IDMC. The IDMC membership and governance is outlined in a separate 
charter. 
The IDMC will be responsible to review the FSH and safety data from the first 10 patients 
included in the study and to advise the sponsor and chief investigator about a go /no-go 
decision with the other 60 patients to complete the calculated sample size.   
8.4 Criteria for End of Study 
This study will be considered complete following the data cut-off date and datalock for the 
final analysis. The data cut-off date for the final analysis will occur after all enrolled patients 
have completed the safety visit 30 (+/-5) days after the last study drug administration. 


 
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9. Informed Consent, Confidentiality, Responsibility Insurance and 
Regulatory Considerations 
9.1. 
Informed Consent 
The investigator is responsible for ensuring that the patient understands the risks and benefits of 
participating in the study, including answering any questions the patient may have throughout 
the study and sharing any new information that may be relevant to the patient's willingness to 
continue  her participation in the trial in a timely manner. 
The informed consent document will be used to explain the risks and benefits of study 
participation to the patient in simple terms before the patient is entered into the study, and to 
document that the patient is satisfied with her understanding of the risks and benefits of 
participating in the study and desires to participate in the study. 
The investigator is responsible for ensuring that informed consent is given by each patient or 
legal representative. This includes obtaining the appropriate signatures and dates on the 
informed consent document prior to the performance of any protocol procedures and prior to 
the administration of study treatment.   
As used in this protocol, the term "informed consent" includes all consent and assent given by 
patients or their legal representatives. 
If new safety information results in significant changes in the risk/benefit assessment, the 
consent form should be reviewed and updated if necessary. All patients (including those 
already being treated) should be informed of the new information, given a copy of the revised 
form and give their consent to continue in the study.  
9.2. 
Respect of Confidentiality 
The investigator will be responsible for preserving the suitable information about each patient 
(for example, name, address, telephone number, social security number and study identification) 
so that the competent authorities can have access to said information if necessary. These records 
must be confidentially preserved for the time indicated by the legislation. 
The investigators and the sponsor will maintain the confidentiality of all patients participating 
in the study, according to Good Clinical Practice, GCP and local legislation. 
9.3. 
Responsibility Insurance 
The Sponsor has signed an insurance policy to cover the responsibilities of the investigator and 
those of other parties participating in the study. 
9.4. 
Regulatory Considerations 
This study will be conducted in accordance with the ethical principles that have their origin in 
the Declaration of Helsinki and that are consistent with good clinical practices and the 
applicable laws and regulations.  The investigator, head of the medical institution, or designee 
will promptly submit the protocol to applicable ethical review board(s). 


 
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9.4.1 
Investigator Information 
Physicians with a specialty in medical oncology and gynecology will participate as 
investigators in this clinical trial. 
If investigators are added after the study has been approved by the sponsor, an ERB/IRB, or a 
regulatory agency, these additions will not be considered changes to the protocol. 
9.4.2 
Protocol Signatures 
After reading the protocol, each principal investigator will sign the protocol signature page and 
send a copy of the signed page to Sponsor. 


 
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10. Practical Considerations 
10.1.  Monitoring, Audit and Inspections 
The study will be monitored by means of regular visits of the patients. During the visits to the 
center, the monitor must review the original records of the patients, the records of medication 
stocks and document preservation. The monitor must also evaluate the study procedures and 
discuss the possible problems with the investigator. During the course of the study, audit visits 
can be carried out in the participating centers. The investigator will allow direct access to the 
source documents/data for the tasks of monitoring, audit, reviewed by the ERB and the inspection 
by the Competent Authorities. 
10.2.  Preservation of Study Documentation 
The copies of all the relevant information will be preserved by the investigator for a period of at 
least 5 years after the end of the study, according to current legislation. 
10.3.  Protocol Modification 
Once it has been authorized by the ERB and the competent authority any protocol modification 
must be documented by writing, in the form of an amendment. 
The amendments must be duly identified, by its chronological order number, dated and signed 
by the sponsor and the chief investigator. 
All the protocol amendments must be notified to the ERBs involved in the trial and to the 
competent authority. If the modifications are relevant, the authorization of the involved ERBs 
and/or the competent authority will be necessary before their application. 
After reading the protocol amendment, each principal investigator will sign the protocol 
amendment signature page and send a copy of the signed page to Sponsor. 
10.4.  Use of the Information and Publication 
All the information concerning the study treatment provided by the sponsor in relation to this 
study, and not previously published, is considered to be confidential information with property 
right of the sponsor. This information comprises the basic information about the product, the 
clinical protocol, the work forms where appropriate, the e-CRFs, the assessment methods, the 
technical methodology and the basic scientific data. This confidential information will be the 
property of the sponsor, it must not be disclosed to third parties without the prior written consent 
of the sponsor and it must not be used other than for the purposes of the study. 
The information developed during the practice of this clinical study is also considered to be 
confidential. This information can be disclosed to the extent considered necessary by the sponsor.  
To allow the use of the information derived from this study and to ensure the compliance with the 
current rules, the investigator is obliged to provide GEICAM (on behalf of the sponsor) with all 
the results of examinations and all the data developed in this study. Except in that required by 
law, the information obtained during the study can only be provided to the doctors and to the 


 
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competent authorities by the Sponsor or delegated people on behalf of the sponsor. The sponsor 
commits to publish the results of the study and to present them in scientific meetings either if 
they are positive or negative.  
10.5.  Ethics Committees 
The protocol and the informed consent document will be reviewed by the involved ERBs. The 
single decision of the ERBs referring to the development of the study will be provided in writing 
to GEICAM (on behalf of the sponsor). 
GEICAM (on behalf of the sponsor) will submit the required reports of the progress of the study 
to the ERB and will communicate the possible SAE, the life-threatening adverse events and 
deaths. At the end of the study, GEICAM (on behalf of the sponsor) will inform the ERB of trial 
closure. 
 


 
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16 G. Pfeiler, C. Glatz, R. Königsberg, T. Geisendorfer, A. Fink-Retter, E. Kubista, C. F. Singer and M. Seifert. Vaginal estriol 
to overcome side-effects of aromatase inhibitors in breast cancer patients. Climacteric 2011; 14:339-344. 
17 Antonio Cano, MD, PhD;  José Estévez, MD;  Ramón Usandizaga, MD, PhD; José Luis Gallo, MD,PhD; Misericord Guinot, 
MD, PhD; Juan Luis Delgado, MD;  Elena Castellanos, MD, PhD;  Eloy Moral, MD; Concepción Nieto, MD, PhD;  Jaime 
Moscoso del Prado, PhD; Javier Ferrer, MD, PhD. The therapeutic effect of a new ultra-low concentration estriol gel 
formulation (0.005% Estriol vaginal gel) on symptoms and signs of postmenopausal vaginal atrophy.  Results from a pivotal 
phase III study. Menopause 2012 Oct;19(10):1130-1139. 
18 Delgado JL, de la Calle M, Castellanos E, Usandizaga R, Moscoso P, Nieto C, Estevez J. Safety evaluation of 0.005% 
Estriol vaginal gel: a new ultra-low concentration estriol formulation for the local treatment of postmenopausal vaginal 
atrophy. In 9th Congress of the European Society of Gynecology,  Copenhagen September 2011. P12.03 (pg 215). 
19 Blissel SmPC.  http://www.aemps.gob.es/cima/pdfs/es/ft/72726/FT_72726.pdf 
20 Rosen R, Brown C, Heiman J, et al. The Female Sexual Function Index (FSFI): A Multidimensional Self-Report Instrument 
for the Assessment of Female Sexual Function. Journal of Sex & Marital Therapy, 26:191–208, 2000 
 


 
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Protocol Attachment 1. Study Schedule 
Study Schedule of Events and Timelines. 
ITFE-2026-C10 (BLISSAFE Study) 
During Study Treatment. 
All visits (except week 1) +/- 3 days of scheduled treatment day. 
Post-treatment 
(4 weeks) from 
the last study 
drug dose  
 
Visits 
Screening 
 
 
 
Baseline 
Week 1 
Week 3 
Week 8 
Week 12 or 
early 
withdrawal 
visit 
Post-treatment 
(4 weeks) from 
last study drug 
dose 
Day  
Within 14 days 
prior to the 
registration 
 
1o 
 
8  
(+/- 2 days) 
22 
(+/-3d) 
57 
(+/-3d) 
85 
(+/- 3d) 
+30  
(+/-5d) 
Procedure/Laboratory/  
Diagnostic Test 
 
 
 
 
 
 
ICD for Entry (before any 
study specific tests)a 
X 
 
 
 
 
 
 
Medical and surgical history 
and demographicsb 
X 
 
 
 
 
 
 
Physical examinationc 
X 
 
X 
X 
X 
X 
X 
ECOG PS 
X 
 
X 
X 
X 
X 
X 
Hematologyd 
X 
 
 
 
 
X 
X 
Blood Chemistrye 
X 
 
 
 
 
X 
X 
Urine Testf 
X 
 
 
 
 
X 
X 
Endometrium by transvaginal 
ultrasoundg 
X 
 
 
 
 
X 
 
Moderate or severe vaginal 
dryness 
X 
 
 
 
 
 
 
Review Inclusion/Exclusion 
Criteria 
X 
X 
 
 
 
 
 
Concomitant medications 
X 
X 
X 


 
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Visits 
Screening 
 
 
 
Baseline 
Week 1 
Week 3 
Week 8 
Week 12 or 
early 
withdrawal 
visit 
Post-treatment 
(4 weeks) from 
last study drug 
dose 
Day  
Within 14 days 
prior to the 
registration 
 
1o 
 
8  
(+/- 2 days) 
22 
(+/-3d) 
57 
(+/-3d) 
85 
(+/- 5d) 
+30  
(+/-5d) 
AEs and SAEsh 
X 
X 
X 
Blood (serum) samples for 
FSH and LH determinationi 
X 
Xp 
 
X 
 
 
X 
 
X 
X 
 
REGISTRATION / 
RANDOMIZATION 
 
X 
 
 
 
 
 
Blood (plasma-EDTA) samples 
for estriol, estradiol and estrone 
determinationi 
 
Xp 
 
X 
 
 
X 
 
X 
X 
 
Gynaecological examinationj 
 
Xp 
 
X 
 
X 
 
Symptoms and signs of vaginal 
atrophyk 
 
Xp 
 
X 
 
X 
 
Measurement vaginal pHl 
 
Xp 
 
X 
 
X 
 
Vaginal cytology sample to 
evaluate the vaginal Maturation 
Valuem 
 
Xp 
 
X 
 
X 
 
FSFI questionnairen 
 
Xp 
 
X 
 
X 
 
Study drug/ placebo dosingñ 
Xñ 
Xñ 
 
 


 
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Study Schedule of Events and Timelines. Protocol ITFE-2026-C10 (BLISSAFE Study)  
 
a 
Signed, written informed consent (approved by ERB) obtained prior to any study specific procedure. 
b 
Includes previous treatments, disease characteristics, age, time of menopause.  
c 
Physical examination includes measurements of height and weight (Baseline only), area surface body, blood pressure and pulse rate. 
d 
Hemoglobin, WBC, absolute neutrophils, platelet count.  
e 
Tasting glucose, alkaline phosphatase, ALT, AST, total bilirubin, serum creatinine and BUN (or urea). 
f 
pH, glucose, proteins, cetones, bilirubin, blood, nitrites, urobilinogen and leukocites 
g 
It is advisable that the endometrial ultrasound be performed as the last of  the screening procedures 
h 
After informed consent form signature, but prior to initiation of study medications, only SAEs caused by a protocol-mandated intervention will be collected. 
Adverse events to be monitored continuously during the treatment period. All AEs occurring during the study and until the treatment discontinuation visit 30 
days after the last study drug to be recorded according to MedDRA, thereafter all study drug-related SAEs should continue to be collected.  
i 
Hormonal determinations will be performed by a Central Laboratory.  
j 
It includes breast examination; pelvic examination (examination of uterus and appendages); speculum inspection of vagina and exocervix, vulva and 
rectovaginal component. 
k 
Symptoms of vaginal atrophy (vaginal dryness, dyspareunia and pruritus). 
Signs of vaginal atrophy (mucosa with thinning or flattening of folds, dryness or fragility of the vaginal mucosa). 
l 
Measurement of vaginal pH on the vaginal secretion using a reactive strip (pH 2.0 - 9.0, Merck, provided by the sponsor). The investigator team will insert 
the strips in the vagina and will moisten them as much as possible with the vaginal wall secretion. Then, when the strip is coloured, the investigator will 
check the combination of colours on the strip and check the pH value to which it corresponds, recording this value in the eCRF. The reading should be made 
whilst the strip is still damp. The strips will be discarded once the pH has been recorded in the CRF. 
m 
Two smears (with two different spatulas provided by the sponsor one to take the sample and the other to take the countersample) will be made per patient for 
each sample (sample and countersample), which will be obtained from the bottom of the right and left vaginal sac respectively. Each smear will be suitably 
labelled with the corresponding labels provided to the investigator. The vaginal smears will be fixed whilst they are still moist with a water-soluble fixation 
spray for cytodiagnosis. The cytologic samples will be sent to central laboratory (full instructions will be given in the Laboratory Manual). 
n 
FSFI: Female Sexual Function Index questionnaire; patients must preferably complete these instruments in the hospital and prior to having any tests and to 
any discussion of their progress with healthcare personnel at the site. Interviewer administration in clinic may be used under special circumstances (eg, 
patient forgot their glasses or feels too ill).  
  


 
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ñ 
Dosage schedule: 
Weeks 1-3: single daily application  
 
Weeks 4-12: twice weekly administration. 
The treatment will be administered for 12 weeks. 
The Principal Investigator, Sub-Investigator, study nurse or the Gynecologist must explain to the patient how to administer the vaginal gel. It is advised that 
the first application consider be performed in presence of the gynecologist or oncologist.  
Women will be instructed to administer the gel themselves for the rest of applications. 
The 10 patients included in the safety phase to obtain some preliminary safety data and FSH values prior to the recruitment of the rest of the group under 
study. These women will receive the study treatment over 3 weeks. These 10 patients will only perform the determinations of all hormone (FSH, LH, estriol, 
estradiol and estrone) 
o 
The baseline visit may be performed in two consecutive days (no more), although it will be best performed it in one day. Treatment administration will 
always be the last of the baseline visit procedures.  
p 
These tests should be performed after the definite inclusion of the patient in the study, as part of the baseline visit.  
 


 
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Protocol Attachment 2. Eastern Cooperative Oncology Group 
Performance Status 
ECOG Performance Status 
Grade 
Description 
0 
Fully active, able to carry on all pre-disease performance without restriction 
 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a 
light or sedentary nature, e.g., light housework, office work 
 
2 
Ambulatory and capable of all self-care but unable to carry out any work activities.  Up and 
about more than 50% of waking hours 
 
3 
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours 
 
4 
Completely disabled.  Cannot carry on any self-care.  Totally confined to bed or chair 
 
5 
Dead 
 
Source: Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, Carbone PP. 1982. 
Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol 
5(6):649-65. 
 
                                 
 
 
 
 


 
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Protocol Attachment 3. Adverse Events / Serious Adverse Events 
Assessment Guide 
Time 
After ICD 
Before Drug 
During Therapy 
30-Day  
Post-
discontinuation  
Follow-up Period 
Long-Term  
Follow-up Period 
Events to 
Collect  
AE/SAEs 
 
New/Ongoing AE/SAEs 
 
New/Ongoing SAEs 
 
 
Related to 
Procedures 
Regardless of Relatedness to 
Study Treatment or Procedures 
Related to Study 
Treatment or Procedures 
Abbreviations:  AE = adverse event, ICD = informed consent document, SAE = serious adverse event. 


 
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Protocol Attachment 4. FSFI Questionnaire 
In separate document.  
 

SAP_001.pdf: 
 
 
 
 
Protocol ITFE-2026-C10 
A PHASE II PROSPECTIVE, RANDOMIZED, DOUBLE-BLIND, PLACEBO-
CONTROLLED AND MULTI-CENTRE CLINICAL TRIAL TO ASSESS THE SAFETY OF 
0.005 % ESTRIOL VAGINAL GEL IN HORMONE RECEPTOR-POSITIVE 
POSTMENOPAUSAL WOMEN WITH EARLY STAGE BREAST CANCER  IN 
TREATMENT WITH AROMATASE INHIBITOR IN THE ADJUVANT SETTING. 
“BLISSAFE Study” 
 
 
 
 
 
STATISTICAL ANALYSIS PLAN 
(Version No. 4.0 – 05-Jun-17) 
 
 
 
Sponsor: ITF RESEARCH PHARMA S.L.U 
Polígono Industrial de Alcobendas 
C/San Rafael, 3 
28108 Alcobendas (Madrid), Spain 
Phone: +34 91 6572323 
 
Fax:  +34 91 6572366 
Email: italfarmaco@itfsp.com  
Protocol-specific Sponsor contact information can be found in the Administrative Binder. 
 
STUDY CHIEF INVESTIGATOR: 
Dr. Pedro Sánchez Rovira, Complejo Hospitalario de Jaén 
Sponsor Study Code: ITFE-2026-C10 
EudraCT Number: 2014-004517-84 
Protocol Approved by ITF RESEARCH 
 
 
Protocol No.: ITFE-2026-C10 (BLISSAFE) v1.0 26-Nov-2014 
      
  
 
CONFIDENTIAL 
The information and data included in this Statistical Analysis Plan contain secrets and privileged or confidential 
information which is the property of ITF. No one is authorized to make this information public without the written 
permission of ITF. These limitations shall also be applied to all the information considered to be privileged or confidential 
and provided in the future. This material can be disclosed and used by its equipment and collaborators as necessary for 
conducting the clinical trial. 






 
 
 
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TABLE OF CONTENTS 
1. 
INTRODUCTION ................................................................................................................................................... 8 
2. 
STUDY OBJECTIVES .............................................................................................................................................. 8 
2.1 
MAIN OBJECTIVE .................................................................................................................................................... 8 
2.2 
SECONDARY OBJECTIVE ............................................................................................................................................ 8 
3. 
STUDY DESIGN .................................................................................................................................................... 9 
3.1 
SAMPLE SIZE ........................................................................................................................................................ 10 
3.2 
RANDOMIZATION .................................................................................................................................................. 11 
4. 
STUDY POPULATION ......................................................................................................................................... 11 
4.1 
INCLUSION CRITERIA .............................................................................................................................................. 12 
4.2 
EXCLUSION CRITERIA .............................................................................................................................................. 13 
4.3 
DISCONTINUATIONS ............................................................................................................................................... 14 
4.3.1 
Discontinuation of Study Medication ........................................................................................................... 14 
4.4 
ITT POPULATION .................................................................................................................................................. 15 
4.5 
PER PROTOCOL POPULATION ................................................................................................................................... 15 
4.6 
SAFETY POPULATION .............................................................................................................................................. 15 
5. 
ENDPOINTS AND STUDY VARIABLES .................................................................................................................. 16 
5.1 
PRIMARY ENDPOINT .............................................................................................................................................. 16 
5.2 
SECONDARY ENDPOINTS ......................................................................................................................................... 16 
6. 
DATA SCREENING AND ACCEPTANCE ................................................................................................................ 16 
6.1 
MISSING DATA ...................................................................................................................................................... 16 
6.1.1 
Missing date ................................................................................................................................................. 16 
6.2 
STATISTICAL SOFTWARE .......................................................................................................................................... 17 
6.3 
DATABASE LOCK .................................................................................................................................................... 17 
7. 
INTERIM ANALYSIS ............................................................................................................................................ 17 
7.1 
PURPOSE OF INTERIM ANALYSIS ................................................................................................................................ 17 
7.2 
IDMC (INTERIM DATA MONITORING COMMITTEE) ..................................................................................................... 18 
7.2.1 
IDMC Recommendation ............................................................................................................................... 18 
8. 
CRITERIA FOR END OF STUDY ............................................................................................................................ 19 
9. 
STATISTICAL METHODS AND ANALYSIS ............................................................................................................. 19 
9.1 
STATISTICAL METHODS ........................................................................................................................................... 19 
9.2 
STATISTICAL ANALYSIS ............................................................................................................................................ 26 
9.2.1 
Patient Disposition ....................................................................................................................................... 27 
9.2.2 
Patient Characteristics ................................................................................................................................. 28 
9.2.3 
Concomitant Therapy ................................................................................................................................... 28 
9.2.4 
Treatment Compliance ................................................................................................................................. 28 
9.2.5 
Hormone levels ............................................................................................................................................ 29 
9.2.6 
Main Study Analysis ..................................................................................................................................... 29 
9.2.7 
Efficacy analyses .......................................................................................................................................... 30 
9.2.7.1 
Signs and symptoms of vaginal atrophy .............................................................................................................. 30 


 
 
 
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9.2.7.2 
Vaginal Maturation value and pH ........................................................................................................................ 31 
9.2.8 
Safety Analyses ............................................................................................................................................ 31 
9.2.9 
Other Analyses: Patient Reported Outcomes: FSFI ...................................................................................... 32 
10. 
TABLES AND FIGURES .................................................................................................................................... 34 
11. 
BIBLIOGRAPHY AND REFERENCES ................................................................................................................. 35 
 
 
 


 
 
 
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ABBREVIATIONS AND DEFINITIONS 
 
AE 
Adverse Event 
AI 
Aromatase Inhibitor 
CR 
Complete Response 
CTCAE 
Common Terminology Criteria for Adverse Events 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic Case Report Form (sometimes referred to as Clinical 
Report Form). An electronic form for recording study participants’ data 
during a clinical study, as required by the protocol. 
ER 
Estrogen Receptor 
FDA 
Food and Drug Administration 
FSFI 
Female Sexual Function Index 
FSH 
Follicle Stimulating Hormone 
GCP 
Good Clinical Practice 
GEICAM 
Spanish Breast Cancer Research Group 
HER2 
Human Epidermal Growth Factor Receptor 2 
HR 
Hormone Receptor 
ICD 
Informed Consent Document  
IDMC 
Independent Data Monitoring Committee 
IHC 
Immunohistochemistry 
ITF 
ITF Research Pharma S.L.U. 
ITT 
Intent To Treat 
LH 
Luteinizing Hormone 
MedDRA 
Medical Dictionary for Regulatory Activities 


 
 
 
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MV 
Maturation Value 
NSAI 
Non-Steroidal Aromatase Inhibitor 
Patient 
A subject with a defined disease 
PgR 
Progesterone Receptor 
QoL 
Quality of Life 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
 


 
 
 
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1. 
INTRODUCTION 
The purpose of this Statistical Analysis Plan (SAP) is to give a detailed description of the statistical 
analysis 
to 
be 
performed 
to 
generate 
the 
final 
study 
report 
ITFE-2026-C10  
(BLISSAFE) [1]. This includes, on the one hand, a brief summary of the main features of the study, 
and on the other hand the objective of SAP corresponding to the planning of the statistical analysis 
of the study. 
This Statistical Analysis Plan was created according the ICH Good Clinical Practice [2] [3], 
GEICAM policies and Standard Operating Procedures (SOP); and is consistent with the study 
protocol [1]. 
2. 
STUDY OBJECTIVES 
2.1 
Main Objective 
The primary objective is to evaluate the levels of FSH after treatment with 0.005% estriol vaginal 
gel in hormone receptor-positive postmenopausal women with early stage breast cancer in 
treatment with NSAIs in the adjuvant setting and symptoms of vaginal atrophy. 
2.2 
Secondary Objective 
· 
To evaluate the levels of estriol, estradiol, estrone, FSH and LH after treatment with 
0.005% estriol vaginal gel in hormone receptor-positive postmenopausal women with early 
stage breast cancer in treatment with NSAIs in the adjuvant setting and symptoms of 
vaginal atrophy. 
· 
To assess the safety and tolerability of 0.005% estriol vaginal gel in hormone receptor-
positive postmenopausal women with early stage breast cancer in treatment with NSAIs in 
the adjuvant setting and symptoms of vaginal atrophy. 
· 
To assess the efficacy of 0.005% estriol vaginal gel in the treatment of symptoms and 
signs of vaginal atrophy in hormone receptor-positive postmenopausal women with early 
stage breast cancer in treatment with NSAIs and symptoms of vaginal atrophy.  


 
 
 
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· 
To measure the impact of treatment with 0.005% estriol vaginal gel in sexual function of 
hormone receptor-positive postmenopausal women with early stage breast cancer in 
treatment with NSAIs and symptoms of vaginal atrophy. 
3. 
STUDY DESIGN 
This is a phase II, prospective, randomized, double-blind, placebo-controlled, international (Spain 
and Sweden) and multicentre study. 
60 patients will be randomized to receive 0.005% estriol vaginal gel (arm A) and placebo 
moisturizing gel (arm B) in a 4:1 proportion, so 48 patients will enter in arm A and 12 in arm B. 
Arm A (experimental arm): Investigational drug product: 0.005% estriol vaginal gel (Blissel®) 
Route: 
Vaginal. Administration by an applicator inserted deep inside the 
vagina 
Dose: 
1 g of gel, containing 50 mg of estriol  
Dosage schedule: 
Weeks 1-3: single daily application  
 
Weeks 4-12: twice weekly administration. 
       Women will be instructed to administer the gel at bedtime.  
Arm B (control arm): placebo moisturizing gel (vehicle of Blissel®) administered in the same way. 
Patients will have hormone determinations (estriol, estradiol, estrone, FSH and LH) analyzed in a 
central laboratory at baseline and at weeks 1, 3, and 8 from the beginning of the therapy and at 
week 12 (last day of study therapy). An additional FSH and LH determination will be obtained 
during the screening period in order to assess the intra-individual variation of these hormones in 
baseline conditions. Patients will also undergo gynecological examination (vaginal smear and pH), 
evaluation of vaginal symptoms and signs, and will answer a female sexual function index 
questionnaire at baseline, at week 3 (from the beginning of therapy) and week 12 (last day of study 
therapy). Adverse events will be followed along the study and specifically collected at weeks 1, 3 
and 8 from the beginning of therapy, at week 12 (last day of study therapy) and at safety visit (30 
±5 days after last day of study therapy). 
There will be an initial safety phase of the study (only two sites of Spain will participate), in which 
10 patients will be included (also randomized 4:1) to obtain some preliminary safety data and FSH 


 
 
 
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values prior to the recruitment of the rest of the group under study. These women will receive the 
study treatment over 3 weeks (8 will receive 0.005% estriol vaginal gel and 2 placebo moisturizing 
gel). FSH levels will be analyzed at screening, baseline and after 3 weeks and reviewed (together 
with safety data) by an independent data monitoring committee (IDMC) which will advise the chief 
investigator about a go /no-go decision with the other 60 patients to complete the calculated 
sample size. The study will continue if no significant changes in FSH levels from postmenopausal 
to premenopausal values are demonstrated. 
3.1 
Sample Size 
This is an exploratory study and there are scarce data to allow a precise sample size calculation. 
The most relevant data come from Pfeiler [4] who reported that the levels of FSH in patients treated 
with aromatase inhibitors fell from a mean value of 75.7 to 66.0 mIU/ml after two weeks of daily 
vaginal treatment with 0,5 g of estriol. Although no figures are reported of the variability of those 
measurements, an estimation of the standard deviation can be made applying the standard rule of 
[sd = range/4]. The reported range of FSH values in that report is 45.6 – 134.6, thus a reasonable 
approximation to the sd is 89/4 = 22.3. Under these assumptions, it can be calculated that a 
sample size of 44 would provide 80% power to detect with an alpha=0.05 a decrease of FSH levels 
from 75.7 to 66.0 mIU/ml assuming a standard deviation of 22.3. 
The hormone levels in women treated with AI who are not receiving estrogen therapy are likely to 
suffer physiological temporal oscillations. These variations could make difficult the interpretation of 
any eventual modification of hormone levels observed in the estriol treated women.  In the absence 
of data of naturally occurring variations in hormone levels of women treated with AI  that allow us to 
interpret the results of hormone determinations under study, it is considered appropriate to include 
a placebo group to provide reference data. In this case, the placebo is a vaginal moisturizing gel. 
It is estimated that a group of 11 women will allow the assessment of physiological fluctuations in 
hormone levels. Additionally, this sample size for the placebo group will provide 80% power and 
alpha=0.05 to differentiate between active and placebo (ratio 4:1) on the change in the vaginal 
maturation value, which is a secondary variable of the study that provides objective information 
about the effectiveness of treatment on vaginal atrophy (calculation based on the parameter data 
from the pivotal Blissel study and considering only the subgroup of women who had severe vaginal 
dryness: change MV active group 24.9 vs 0.6 points in placebo group, SD = 24.2). 


 
 
 
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The differentiation between active and placebo in the subjective parameters of efficacy (secondary) 
would be possible at the expense of a sample size noticeably superior. Since the evaluation of 
effectiveness is not primary but secondary in this study, it is not justified in this study to increase 
the sample for this reason. 
In accordance with the above, the study would include 55 women.  44 will receive vaginal estriol 
gel and 11 with the placebo moisturizing gel). Taking into account 10% of dropouts, the sample to 
be considered is of 60 women (48 vaginal estriol gel and 12 placebo moisturizing gel).  
10 additional women will be recruited in a first safety phase of the study (8 with vaginal estriol gel 
and 2 placebo moisturizing gel).  
3.2 
Randomization 
This is an unbalanced study but with an active: placebo ratio of 4:1, which will support an adequate 
patient randomization (blocks of 5). 
This is a double blind study, the study drug, 0.005% Estriol vaginal gel, and its vehicle in gel 
(placebo vaginal gel) will be of identical appearance and will have the same texture in order to 
maintain the double blind. 
The medication will be randomized at ITF Research Pharma according to a randomization list 
performed at ITF Research Pharma. The medication will be sent in blocks of 5 to each site, and the 
first patient included in a specific site, will receive the lowest randomization code indicated in the 
medication received. Codes will be assigned consecutively from the lowest number to the highest. 
ITF provides us and also each site, with opaque and sealed code break envelopes, identified with 
the randomization code, and containing inside a card with the treatment assigned to that patient. At 
the end of the study, and after the clinical Data Base has been closed, ITF Research Pharma will 
send us the complete randomization list.  
For more information about the unblinding process please see the protocol (part 5.8). 
4. 
STUDY POPULATION 
Patients with hormone receptor positive and any HER2 status early breast cancer, who are on 
treatment with NSAI and have vaginal atrophy. 


 
 
 
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4.1 
Inclusion Criteria 
Patients are eligible to be included in the study only if they meet all of the following criteria: 
1. Written informed consent prior to beginning specific protocol procedures. 
2. Patients must have histological confirmation of breast adenocarcinoma with stage I-IIIA, 
documented at a local pathology department. 
3. The breast tumors must be estrogen-receptor positive and/or progesterone receptor positive 
(≥1% of stained tumor cells by IHC as determined by the local laboratory) with any HER2 
status. 
4. Postmenopausal status defined as: 12 months of spontaneous amenorrhea or 6 months of 
spontaneous amenorrhea with serum FSH levels > 40 mIU/ml or 6 weeks postsurgical 
bilateral oophorectomy with or without hysterectomy. 
5. Patient must be receiving the non-steroidal aromatase inhibitors anastrozole or letrozole as 
breast cancer treatment in the adjuvant setting for a minimum of 6 months. 
6. Women suffering from moderate to severe vaginal dryness according to the FDA guidelines 
for drug development in postmenopausal women (Center for Drug Evaluation and 
Research, CDER Jan 2003). A moderate symptom will be considered if the symptom is 
present, bothersome and annoying, and a severe symptom will be considered if the 
symptom is present, bothersome and annoying, and interferes with the normal patient 
activity. 
7. Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 or 1. 
8. Adequate bone marrow as defined by the following laboratory values: 
a. Absolute Neutrophil Count (ANC) ≥ 1.5 x 109/L. 
b. Platelets (plt) ≥ 100 x 109/L. 
c. Hemoglobin (Hgb) ≥ 10 g/dl. 
9. Patient has adequate organ function as defined by the following laboratory values: 
d. Serum creatinine ≤ 1.5 x ULN. 
e. Bilirubin ≤ 1.5 × ULN.  


 
 
 
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f. Alkaline phosphatase ≤ 2 × ULN. 
g. AST and ALT ≤ 2 × ULN. 
10. Willingness and ability to comply with scheduled visits, treatment plan, laboratory tests and 
other study procedures. 
4.2 
Exclusion Criteria 
Patients will be excluded from the study if they meet any of the following criteria: 
1. Stage IIIB-IV breast cancer or bilateral breast cancer. 
2. Treatment with any other current anti-tumoral therapy (chemotherapy, anti-Her2…etc) 
besides the NSAI. Pamidronate or Alendronate are permitted. 
3. Prior history of other malignancy within 5 years of study entry, aside from non-melanoma 
skin cancer or carcinoma-in-situ of the uterine cervix adequately treated. 
4. Postmenopausal uterine bleeding. Vaginal bleeding of unknown etiology. 
5. Patients with endometrial thickness equal to or greater than 4 mm measured by 
transvaginal ultrasound. 
6. Patients who have received any type of vulvovaginal treatment in the 15 days prior to the 
start of the study. 
7. Use of any hormone, natural (phytoestrogens) or herbal products for the treatment of 
menopausal symptoms within the last 3 months. 
8. Current or previous history of thromboembolic disease or coagulopathies. 
9. Severe cardiovascular or respiratory diseases in the previous 6 months. 
10. Renal Impairment. 
11. Hepatitis B and/or hepatitis C carriers (unless with normal hepatic function). 
12. Known human immunodeficiency virus infection. 
13. Known hypersensitivity to NSAI.  


 
 
 
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14. Other severe acute or chronic medical or psychiatric condition, or laboratory abnormality 
that would impart, in the judgment of the investigator, excess risk associated with study 
participation or study drug administration, or which, in the judgment of the investigator, 
would make the patient inappropriate for entry into this study. 
15. Previous investigational treatment for any condition or participation in any clinical trial within 
4 weeks of inclusion date. 
4.3 
Discontinuations 
4.3.1 
Discontinuation of Study Medication 
The criteria for enrollment must be followed explicitly. If a patient who does not meet enrollment 
criteria is inadvertently enrolled, that patient should be discontinued from the study medication, an 
exception may be granted if the patient, in the opinion of the investigator, is having benefit from the 
study medication. In these rare cases, the investigator must obtain documented approval from the 
sponsor to allow the patient to continue to receive the study drug. 
Patients can be discontinued from the study therapy in the following circumstances: 
· 
Patient’s own request. 
· 
Unacceptable toxicity.  
· 
Tumor recurrence. 
· 
Any clinical adverse event (AE), laboratory abnormality or inter-current illness which, in the 
opinion of the investigator, indicates that continued participation in the study is not in the best 
interest of the patient. 
· 
Termination of the study by the sponsor. 
· 
Physician’s decision, including need of other anti-cancer therapy, not specified in the protocol.  
· 
If the patient is non-compliant with study procedures.  
· 
Loss of ability to freely provide consent through imprisonment or involuntarily incarceration for 
treatment of either a psychiatric or physical (e.g. infectious disease) illness. 


 
 
 
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· 
All patients will be discontinued from the treatment phase and they will perform the safety visit 
in case of a delay of more than 1 week during the daily administration phase or more than 2 
weeks during the twice weekly administration phase, or permanent discontinuation of the study 
drug unless there is an obvious clinical benefit per the investigator’s medical judgment and after 
discussion with the sponsor.  
If possible, and after the permanent discontinuation of treatment, the patients will be assessed 
using the procedure normally planned for the last dosing day with the study treatment. 
All permanent treatment discontinuation should be recorded by the Investigator in the eCRF when 
considered as confirmed. 
4.4 
ITT Population 
The intent to treat (ITT) population will include all patients who are randomized, with study 
treatment (Estriol gel vaginal 0.005% or placebo) assignment designated according to initial 
randomization. 
The ITT population will be the primary population for evaluating patient characteristics, hormone 
levels, efficacy and the FSFI questionnaire. 
4.5 
Per protocol population 
Per-protocol population is a subset of the ITT population that received at least one dose of study 
drug and completed the study without protocol violations according to Protocol Deviation Manual. 
4.6 
Safety population 
Safety population will include all patients randomized in the study who received at least one dose 
of treatment, and they will be analyzed according to the actual treatment received. This population 
is for the safety analysis. 
 
 


 
 
 
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5. 
ENDPOINTS AND STUDY VARIABLES 
5.1 
Primary Endpoint 
Variation in serum levels of FSH from baseline to 12 weeks of treatment. 
5.2 
Secondary Endpoints 
· 
Variation in serum levels of FSH at different time points compared to baseline (weeks 1, 3 
and 8). 
· 
Variation in serum levels of LH and plasma levels of estriol, estradiol and estrone, at 
different time points compared to baseline (weeks 1, 3, 8 and 12). 
· 
AEs according to the Medical Dictionary for Regulatory Activities (MedDRA). 
· 
Changes in vaginal dryness and other symptoms and signs of vaginal atrophy; changes in 
vaginal maturation value and changes in vaginal pH at week 3 and week 12 vs baseline. 
· 
Changes in sexual function measured by the Female Sexual Function Index (FSFI [5]) scale 
at week 3 and week 12 vs baseline. 
6. 
DATA SCREENING AND ACCEPTANCE 
6.1 
Missing data 
The frequency of missing data will be examined and reported for each variable in the analysis. 
Missing data for FSH, LH, Estriol, Estradiol and Estrone values will be imputed with the last 
observation carried forward (LOCF) method. Analyses without data imputation will be performed to 
compare the results. For adverse events and rest of study variables, we will not perform data 
imputation for missing data. 
6.1.1 
Missing date 
All non-relevant missing dates that cannot be determined by query should be estimated as follow: 
If the day of the month is missing for any date used in a calculation, the 15th of the month 
will be used to replace the missing date unless the calculation results in a negative time duration 
(e.g., date of onset cannot be prior to day one date). In this case, the date resulting in 1 day 


 
 
 
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duration will be used. If the day of the month and the month are missing for any date used in a 
calculation, the date will be considered missing. 
There will be performed a list with all the date replaced and it will be reviewed by another 
statistician different from the study statistician. 
6.2 
Statistical software 
The statistical analysis will be developed in SAS Enterprise Guide v5.1. 
6.3 
Database lock 
-The data base lock for the interim analysis is planned after the first 10 patients have been included 
and the results of the FSH levels at week 3 will be available and the patient have completed the 
safety visit 30(+/-5) days after the last study drug administration and all the queries for those 10 
patients have been closed. The randomization codes will be unblinded and made available for the 
project statistician to perform the data analysis, when the study for those 10 patients has been 
finalized, the database has been verified and the protocol violations have been determined. The 
data base must be opened again only if there is a mistake affecting a principal variable than can 
affect the integrity of the efficacy or safety results. 
-The database lock for the final analysis will occur approximately 6 months from the inclusion of the 
last patient, if the study meets the plan set. For the database lock all the patients must have been 
performed the safety visit and all the queries must be closed. The randomization codes will be 
unblinded and made available for the project statistician to perform the data analysis, when the 
study has been finalized, the database has been verified and the protocol violations have been 
determined. The data base must be opened again only if there is a mistake affecting a principal 
variable than can affect the integrity of the efficacy or safety results. 
7. 
INTERIM ANALYSIS 
7.1 
Purpose of interim analysis 
There will be an interim analysis after the first 10 included patients have completed 3 weeks of 
therapy. These patients will be evaluated after 3 weeks according to the study scheme. 


 
 
 
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Only safety data and the analytical values of FSH of these 10 first patients will be reviewed. It is 
expected that no significant changes from the postmenopausal to the premenopausal range occurs 
in any patient.  
According to the reference range provided by the central laboratory for the FSH determinations 
(Laboratorios Echevarne), it is shown in the table below, that 95% of postmenopausal women have 
values from 21.7 to 153. A value of 22 mIU/ml will be set as long as baseline value will be greater 
than this value. In any case, all patients will be evaluated by the IDMC. 
 
 
FSH, mIU/ml 
Group 
n 
Median 
Central 95% 
Men  
135 
3,8 
0,7 – 11,1 
Women 
 
 
 
       Postmenopausal 
76 
90,5 
21,7 – 153 
       Postmenopausal (ERT) 
16 
27 
9,7 – 111 
       Oral Contraceptives 
12 
1,7 
ND – 4,9 
*Preliminary 
 
ND: not detectable 
 
The 10 patients included in the interim analysis will not be included in the final analysis, as they will 
not complete 3 months of therapy.  For that reason, it will not be necessary to perform a correction 
of the final alpha. 
7.2 
IDMC (Interim Data Monitoring Committee) 
The study will use an IDMC. The IDMC membership and governance is outlined in a separate 
charter. 
The IDMC will be responsible to review the FSH and safety data from the first 10 patients included 
in the study and to advise the sponsor and chief investigator about a go /no-go decision with the 
other 60 patients to complete the calculated sample size. 
7.2.1 
IDMC Recommendation 
After review of interim analysis by the IDMC, it has been requested to record the time of the last 
administration of Blissel prior to any hormonal extraction to explain possible drastic changes that 
could occur in the hormonal values and if these changes are due to variation between screening 
and basal values or to Blissel administration 
. 


 
 
 
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8. 
 CRITERIA FOR END OF STUDY 
This study will be considered complete following the data cut-off date and datalock for the final 
analysis. The data cut-off date for the final analysis will occur after all enrolled patients have 
completed the safety visit 30 (+/-5) days after the last study drug administration. 
9. 
STATISTICAL METHODS AND ANALYSIS 
9.1 
Statistical Methods 
Descriptive Analysis 
For categorical variables it will be calculated frequencies and percentages. For continuous 
variables it will be calculated standard descriptive statistics, such as total number of observations, 
number of available data, mean, standard deviation, minimum, percentil 25, median, percentil 75 
and maximum. 
Chi-square χ2 Test 
The first type of chi-square test is the goodness of fit test. This is a test which makes a statement 
or claim concerning the nature of the distribution for the whole population. The data in the sample 
is examined in order to see whether this distribution is consistent with the hypothesized distribution 
of the population or not. One way in which the chi-square goodness of fit test can be used is to 
examine how closely a sample matches a population. The chi-square goodness of fit test can be 
used to provide a test for the representativeness of a sample. 
Suppose that a variable has a frequency distribution with k categories into which the data has been 
grouped. The frequencies of occurrence of the variable, for each category, are called the observed 
values. The manner in which the chi-square goodness of fit test works is to determine how many 
cases there would be in each category if the sample data were distributed exactly according to the 
claim. These are termed the expected number of cases for each category. The total of the 
expected number of cases is always made equal to the total of the observed number of cases. The 
null hypothesis is that the observed number of cases in each category is exactly equal to the 
expected number of cases in each category. The alternative hypothesis is that the observed and 
expected number of cases differ sufficiently to reject the null hypothesis. 


 
 
 
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Let !" is the observed number of cases in category i and #" is the expected number of cases in 
each category, for each of the k categories i = 1, 2, 3…., k, into which the data has been grouped. 
The hypotheses are 
$%
$&
:
:
!"
!"
=
'
#"(
#"(
() *
(+,-(.,/0(* 
and the test statistic is 
12 = 3 4!" 5 #"62
#"
"
 
where the summation proceeds across all k categories, and it has k-1 degrees of freedom. 
The second type of chi-square test is the chi-square test for independence of two variables. The 
chi-square test of independence allows researchers to determine whether variables are 
independent of each other or whether there is a pattern of dependence between them. If there is 
dependence, the researcher can claim that the two variables have a statistical relationship with 
each other. 
The only limitation on the use of this test is that the sample sizes must be sufficiently large to 
ensure that the expected number of cases in each category is five or more, if not, the Fisher exact 
test must be used. This rule can be modified somewhat, but as with all approximations, larger 
sample sizes are preferable to smaller sample sizes. There are no other limitations on the use of 
the test, and the chi-square statistic can be used to test any contingency or cross classification 
table for independence of the two variables. 
The chi-square test for independence is conducted by assuming that there is no relationship 
between the two variables being examined. The alternative hypothesis is that there is some 
relationship between the variables. 
H%
H&
:
:
X
X
and
and
Y
Y
are(independent
are(dependent  
Using that we can estimate the expected frequencies in each cell as #"7 = 89"97 where n is the total 
count, 9"(is the proportion in row i and 97 is the proportion in row j. Then the chi-quared statistic is, 
for an r ×c table: 


 
 
 
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12 = 3 4!"7 5 #"762
#"7
";7
~12
4<>&64?>&6 
The observed numbers of cases,!"7, are the numbers of cases in each cell of the cross 
classification table, representing the numbers of respondents for each combination of the variables. 
The expected numbers of cases #" for each of the cell can be obtained from the multiplication rule 
of probability for independent events. 
Fisher Exact Test 
When the assumptions for the Chi-squared test are not met, i.e. one of the expected values in a 
2x2 table is less than 5, and especially when it is less than 1, then Fisher’s Exact test must be 
applied. The null hypothesis for the test is that there is no association between the rows and 
columns of the 2x2 table, such that the probability of a subject being in a particular row is not 
influenced by being in a particular column. If the columns represent the study group and the rows 
the outcome, then the null hypothesis could be interpreted as the probability of having a particular 
outcome not being influenced by the study group, and the test evaluate whether the two study 
groups differ in the proportions with each outcome. An important assumption for Fisher’s Exact 
test, is that the binary data are independent. If the proportions are correlated then more advanced 
techniques should be applied. 
The test is based upon calculating directly the probability of obtaining the results that we have 
shown (or results more extreme) if the null hypothesis is actually true, using all possible 2×2 tables 
that could have been observed, for the same row and column totals as the observed data. These 
row and column totals are also known as marginal totals. What we are trying to establish is how 
extreme our particular table (combination of cell frequencies) is in relation to all the possible ones 
that could have occurred given the marginal totals. 
For tables rxk, with r or/and k major than 2, it could be used the extension of Freeman-Halton to the 
Fisher Exact Test. Other alternative is to obtain the p-value using Monte Carlo simulation [6]. 
 
 
 
 
 


 
 
 
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Mann-Whitney-Wilcoxon Test 
Mann Whitney U o Wilcoxon rank-sum test 
The Mann-Whitney U test is a nonparametric method for comparing two independent samples. This 
test is also called the Wilcoxon rank-sum test. The Mann-Whitney U test assumes only that we 
have independent random samples from the two groups. It does not assume anything about a 
normal distribution. 
The null hypothesis for the Mann-Whitney U test is that the population distribution of the response 
variable—whatever that distribution might be— is the same for both groups. The alternative 
hypothesis is that the response variable tends to be larger for one group than for the other group. 
To calculate the test statistic for the Mann-Whitney U test, we start by summing the ranks in each 
group. Define 
R1 = sum of ranks for Group 1; R2 = sum of ranks for Group 2: 
Then just pick one group or the other—it really doesn’t matter which. Let’s say we pick Group 1. 
Then the Mann-Whitney U test statistic1 is 
@ = A& 5 8&48& B C6
D
 
where n1 is the number of subjects in Group 1. 
If the null hypothesis is true, then every possible arrangement of the ranks among the two groups is 
equally likely. We can use this fact to calculate the probability of getting various values of the test 
statistic U under the null hypothesis, which in turn lets us calculate the p-value of the test. 
If the p-value is less than or equal to α, then we reject the null hypothesis and conclude that one 
group tends to have larger response variable values than the other (one-sided alternative) or 
merely that there is a difference one way or the other (two-sided alternative). 
If the p-value is greater than α, then we fail to reject the null hypothesis, which means we think the 
null hypothesis is reasonable, which means we think it’s reasonable that there’s no difference 
between the response variable values for the two groups. 
                                               
1 There are a lot of other ways to calculate U, all of which give the same answer. 


 
 
 
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Wilcoxon signed-ranks test 
Wilcoxon signed-ranks test is a non-parametric test for correlated samples. The rationale of the test 
is similar to that of the Mann-Whitney test for independent samples. This test assumes that it can 
rank differences between paired observations. The data for the test will consist of a number of pairs 
of scores, each derived from a single subject, or from a pair of matched subjects. 
The Wilcoxon signed ranks test requires that the differences are approximately symmetric and that 
the data are measured on an ordinal, interval, or ratio scale. When the assumptions for the 
Wilcoxon signed ranks test are met but the assumptions of the t test are violated, the Wilcoxon 
signed ranks test is usually more powerful in detecting a difference between the two populations.  
The Wilcoxon signed ranks test uses the test statistic W computing as follow 
1. For each item in a sample of n items, compute a difference score, E" between the two 
paired values. 
2. Neglect the + and - signs and list the set of n absolute differences, |FG| 
3. Omit any absolute difference score of zero from further analysis, thereby yielding a set of n’ 
nonzero absolute difference scores, where n’< n. After you remove values with absolute 
difference scores of zero, n’ becomes the actual sample size. 
4. Assign ranks RG from 1 to n’ to each of the(|FG| such that the smallest absolute difference 
score gets rank 1 and the largest gets rank n’. If two or more|FG| are equal, assign each of 
them the mean of the ranks they would have been assigned individually if ties had not 
occurred in the data.  
5. Reassign the symbol + or - to each of the n’ ranks,RG, depending on whether FG  was 
originally positive or negative. 
6. Compute the Wilcoxon test statistic, W, as the sum of the positive ranks 
W = 3 RG
4I6
JK
GL&
 
The Wilcoxon signed rank test rejects the hypothesis that there are no systematic differences 
within pairs when the rank sum W+ is far from its mean. 
 


 
 
 
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ANCOVA 
The analysis of covariance (ANCOVA) is typically used to adjust or control for differences between 
the groups based on another, typically interval level, a variable called the covariate. The ANCOVA 
is an extension of ANOVA that typically provides a way of statistically controlling the effects of 
continuous or scale variables that you are concerned about, but that are not the focal point or 
independent variable(s) in the study. The ANCOVA F test evaluates whether the population means 
on the dependent variable, adjusted for differences on the covariate, differ across levels of a factor. 
If a factor has more than two levels and the F is significant, follow-up tests should be conducted to 
determine where there are differences on the adjusted means between groups. 
One-way analysis of covariance model 
M"7 = N B O" B (P(4Q"7 5(QST T 6 B(U"7  i = 1,…,k ; j = 1,…, ni 
N!!grand mean 
VG((effect of group i. 
Z(  lineal regression coefficient. 
xG[ covariate value corresponding to observation yG[. 
x\TT mean of covariate. 
]G[ random error. 
ni: number of subjects in treatment i.  
The null hypothesis of ANCOVA is no difference among the adjusted population means. 
$% ^
$_ ^(O" (=
O" ('(O`
O`
(+,-(.,/0(*; b 
But it is necessary to test the following hypothesis previously. 
c$% ^
$_ ^(P( =
P( '(f
f 
If the covariate has not predictive power for dependent variable (null hypothesis) we will use 
ANOVA instead of ANCOVA because it would be exactly the same statistical test. 


 
 
 
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ASSUMPTIONS FOR ANCOVA 
There are two major assumptions that underlie the use of ANCOVA; both concern the nature of the 
relationship between the dependent variable and the covariate. 
The first is that the relationship is linear. If the relationship is nonlinear, the adjustments made in 
the ANCOVA will be biased; the magnitude of this bias depends on the degree of departure from 
linearity, especially when there are substantial differences between the groups on the covariate. 
Thus it is important for the researcher, in preliminary analyses, to investigate the nature of the 
relationship between the dependent variable and the covariate (by looking at a scatter plot of the 
data points), in addition to conducting an ANOVA on the covariate. 
The second assumption is about the regression lines within each of the groups. We assume that 
the relationship must be linear. Additionally, however, the regression lines for these individual 
groups are assumed to be parallel; in other words, they have the same slope. This assumption is 
often called homogeneity of regression slopes or parallelism and is necessary in order to use the 
pooled within-groups regression coefficient for adjusting the sample means; this is one of the most 
important assumptions for the ANCOVA. Failure to meet this assumption implies that there is an 
interaction between the covariate and the treatment. This assumption can be checked with an F 
test on the interaction of the independent variable(s) with the covariate(s). If the F test is significant 
(i.e., significant interaction) then this assumption has been violated and the covariate should not be 
used as it is. A possible solution is converting the continuous scale of the covariate to a categorical 
(discrete) variable and making it a subsequent independent variable, and then use a factorial 
ANOVA to analyze the data. 
The assumptions underlying the ANCOVA had a slight modification from those for the ANOVA, 
however, conceptually, they are the same. 
Assumption 1: The cases represent a random sample from the population, and the scores on the 
dependent variable are independent of each other, known as the assumption of independence. The 
test will yield inaccurate results if the independence assumption is violated. This is a design issue 
that should be addressed prior to data collection. Using random sampling is the best way of 
ensuring that the observations are independent; however, this is not always possible. The most 
important thing to avoid is having known relationships among participants in the study. 


 
 
 
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Assumption 2: The dependent variable is normally distributed in the population for any specific 
value of the covariate and for any one level of a factor (independent variable), known as the 
assumption of normality. This assumption describes multiple conditional distributions of the 
dependent variable, one for every combination of values of the covariate and levels of the factor, 
and requires them all to be normally distributed. To the extent that population distributions are not 
normal and sample sizes are small, p values may be invalid. In addition, the power of ANCOVA 
tests may be reduced considerably if the population distributions are non-normal and, more 
specifically, thick-tailed or heavily skewed. The assumption of normality can be checked with 
skewness values (e.g., within +3.29 standard deviations). 
Assumption 3: The variances of the dependent variable for the conditional distributions are equal, 
known as the assumption of homogeneity of variance. To the extent that this assumption is violated 
and the group sample sizes differ, the validity of the results of the one-way ANCOVA should be 
questioned. Even with equal sample sizes, the results of the standard post hoc tests should be 
mistrusted if the population variances differ. The assumption of homogeneity of variance can be 
checked with the Levene’s F test. 
If the assumptions are not met then it will be applied nonparametric ANCOVA [7]. For example, 
Akritas et al [8] proposed a completely nonparametric model that avoids restrictive modeling 
assumptions. 
9.2 
Statistical Analysis 
Standard descriptive statistics, such as the mean, median, range and proportion, will be used to 
summarize the patient sample and to estimate parameters of interest. Ninety-five percent 
confidence intervals will be provided for estimates of interest wherever possible. 
The variation of the levels of FSH, estriol, estradiol, estrone and LH after treatment with 0.005% 
estriol vaginal gel will be studied in each woman using Wilcoxon-signed rank test. The variation of 
levels between two arms will be analyzed using a non-parametric test (Mann-Whitney-Wilcoxon 
test) or an ANCOVA (if it must be adjusted by initial value). 
Adverse events data and serious adverse events will be reported in frequency tables. The safety 
analysis will be performed in the population that has received at least one dose of the drug. AEs 


 
 
 
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will be graded according to MedDRA. Adverse events between groups will be compared using the 
chi-square tests of independence (Fisher’s Exact test in the case of observing frequencies <5%). 
Symptoms of Vaginal atrophy will be evaluated with a codification from 0 to 3. 
The variations in the intensities for each one of the symptoms and signs of the vaginal atrophy, 
after 3 and 12 weeks, in each treatment arm, will be compared using a non-parametric test (Mann-
Whitney-Wilcoxon). Descriptive statistics will be calculated for all variables studied. A global 
symptoms score will be evaluated; it will be calculated by summing the intensities of all the three 
symptoms of vaginal atrophy in a certain time point.  
Sexual function measured by the FSFI [5] scale. It will be used an algorithm for determining domain 
scores and a composite full-scale score. 
Time between previous study medication administration dose time and blood sample (serum and 
plasma) collection time will be summarized (mean, median, range,..) for each of visits and will be 
compared using a non-parametric test (Wilcoxon-signed rank test).  
Primary and secondary objectives will be analyzed in the same way, without those patients who 
may cause baseline differences, in a sensitivity analysis to evaluate robustness of the conclusions. 
The significance level of all statistical tests is established at 0.05. 
9.2.1 
Patient Disposition 
A detailed description of patient disposition will be provided. It could include the following: 
· 
summary of patients entered and by site 
· 
total number of patients entered 
· 
total number of patients enrolled 
· 
summary of reasons for patients entered, but not enrolled 
· 
total number of patients treated 
· 
summary of reasons for patients enrolled, but not treated. 
A detailed summary of reasons for patient discontinuation from study treatment will be provided. 
A summary of all identified important protocol violations will be provided. 


 
 
 
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9.2.2 
Patient Characteristics 
Patient characteristics could include a summary of the following:  
· 
patients demographics 
· 
time of menopause 
· 
baseline disease characteristics 
· 
preexisting conditions/secondary conditions 
· 
prior therapy 
Other patient characteristics will be summarized as deemed appropriate. 
The categorical variables will be summarized by contingency tables. The continuous variables will 
be described by the number of patients with valid values (n), mean, standard deviation, median and 
range in both arms. 
The effect of the treatment on continuous variables will be assessed by Mann-Whitney-Wilcoxon 
test. Categorical variables will be analyzed by chi-square or Fisher's exact test. 
9.2.3 
Concomitant Therapy 
A summary of concomitant therapies will be generated in the safety population. 
9.2.4 
Treatment Compliance 
The percentage of complying patients will be calculated at weeks 3, 8 and 12, defined as those 
with a compliance percentage of 80-110%, in the group of patients treated with 0.005% Estriol 
vaginal gel and in the placebo group. The overall compliance of the study drug will be calculated 
from these partial calculations and in the population that has received at least one dose of the drug. 
It will be summarized the patients and reasons of those patients which did not complete the 80% of 
medication of the study.  
The estimate of percentage compliance will be given by: 
g0-h08j(k,/9l*m8h0( =
ohjqml(s,.0(ms/*8*.j0-0s(90-(u00b
E,.0(0Q90hj0s(j,(v0(ms/*8*.j0-0s(90-(u00b (Q(Cff 


 
 
 
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According to the protocol:  
Weeks 1-3 single daily application 1 g of gel (7g/week). 
Weeks 4-12 twice weekly administration 1 g of gel (2g/week). 
Dose expected to be administered: (7gx3w) + (2gx9w) = 39g in 12 weeks 
9.2.5 
Hormone levels 
All these analysis will be based on the ITT population. 
The primary endpoint and one of the secondary endpoints are to evaluate the variation from 
baseline of FSH, LH, estriol, estradiol and estrone blood levels in patients treated with 0.005% 
estriol vaginal gel.  
Two determinations of FSH and LH before treatment with 0.005% estriol vaginal gel will be studied 
in each woman in order to study the intra-individual variation. Screening and baseline 
determinations will be used for this purpose. This intra-individual variation will be compared to the 
variation between baseline and the values obtained at every study visits to study the FSH and LH 
variation caused by treatment.  
Changes in hormone levels will be studied in each woman along the treatment. Also, changes in 
hormone levels will be compared by arm.  
9.2.6 
Main Study Analysis 
The primary endpoint, the variation of FSH between week 12 and baseline and also comparing by 
arm will be analyzed using a non-parametric test (Mann-Whitney-Wilcoxon test). 
An ANCOVA analysis will be used to corroborate the results obtained for the primary endpoint. The 
ANCOVA analysis will compare the variation of FSH 12 weeks adjusted by FSH basal between the 
two arms. This analysis will be performed if the relation between FSH 12 and FSH basal is lineal.  
These analyses will be evaluated in the ITT population, and also in Per-Protocol population to 
confirm the results. 
 


 
 
 
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9.2.7 
Efficacy analyses 
9.2.7.1 Signs and symptoms of vaginal atrophy 
 
The symptoms evaluated will be the following: 
· 
Vaginal dryness 
· 
Pruritus or itching 
· 
Dyspareunia 
Each symptom will be scored in a numeric scale from 0 to 3, as shown below: 
· 
0 Absence. The symptom is not present 
· 
1 The symptom is of mild intensity, without interfering in the patient’s activity 
· 
2 The symptom is of moderate intensity, causing obvious discomfort to the patient 
· 
3 The symptom is stated as very irritating and severe in intensity 
A Global Symptoms Score will be evaluated. It will be calculated by summing the intensities of all 
the three symptoms of vaginal atrophy in a certain time point, thus ranging between 0 and 9. The 
variation (difference between the sum of the intensity in week 3 or 12 minus the sum of the 
intensity in baseline) in the Global Symptom Score vs baseline will be assessed at weeks 3 and 12. 
Two different versions of this analysis will be done taking into consideration if the patients have had 
previous sexual intercourse: 
- 
Version 1: dyspareunia will not be score for patients that did not have previous sexual 
intercourse, and therefore it will not be taken into consideration for the global score and the 
statistical analysis.  
- 
Version 2: dyspareunia will be score as 0 for patients that did not have previous sexual 
intercourse. 
Version 1 will be the one used for the evaluation and interpretation of the study results.  
The signs evaluated will be the following: 
· 
Vaginal mucosa with flattening of folds or thinning 


 
 
 
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· 
Dryness of the mucosa 
· 
Fragility of the mucosa 
Each one of these signs will be scored by the investigator on a numerical scale in accordance with 
their presence and degree of severity as follows: 
· 
0 Absence. The sign is not present. 
· 
1 The sign is present and is considered a mild alteration 
· 
2 The sign is present and is considered a moderate alteration 
· 
3 The sign is present and is considered a severe alteration 
The variations in the intensities for each one of the symptoms and signs of the vaginal atrophy, 
after 3 and 12 weeks, in each treatment arm, will be compared using the non-parametric test 
Mann-Whitney-Wilcoxon.  
9.2.7.2  Vaginal Maturation value and pH 
The averages of the differences between the baseline and the final MV and pH of the 0.005% 
Estriol vaginal gel and placebo groups will be shared by a non-parametric test, to determine the 
possible superiority of the 0.005% Estriol vaginal gel treatment compared with placebo 
administration. 
These same tests will be used to analyse the change in the MV and pH after the initial observation 
period of 3 weeks.  
All of the above secondary analyses will be conducted at a two-sided 0.05 level of significance. 
9.2.8 
Safety Analyses 
The safety analysis will be evaluated in the safety population. 
The toxicity and tolerability of the study drug will be evaluated in the safety population. Safety 
analyses will include summaries of the incidence of adverse events by MedDRA that occur during 
the study treatment period or within 30 days (+/- 5 days) of the last dose of study treatment, 
regardless of causality and according to the relationship to study drug as assessed by the 
investigator. Additionally, the following safety-related outcomes will be summarized: 


 
 
 
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· 
study treatment discontinuations due to adverse events. 
· 
deaths 
· 
SAEs 
· 
hospitalizations and transfusions 
· 
use of key concomitant medications or growth factors. 
Analyses for data with discrete dates, for example, deaths, transfusions, and concomitant 
medications, will be done through 30 days after each patient’s last dose of study treatment.  
Adverse events will also be analyzed in this timeframe; that is, if an event starts within 30 days of 
discontinuation from study treatment, but after 30 days after the last dose of study treatment, it will 
not be included. 
Adverse events data and serious adverse events will be presented in frequency tables by grade. 
Hematological and clinical biochemistry toxicities will be assessed from laboratory test parameters. 
The safety analysis will be performed in the safety population. 
Adverse events data and serious adverse events will be reported in frequency tables (overall and 
by grades). Adverse events will be compared using the chi-square tests (Fisher’s Exact test in the 
case of observing frequencies <5%). 
9.2.9 
Other Analyses: Patient Reported Outcomes: FSFI 
Sexual function measured by the Female Sexual Function Index (FSFI) scale, a 19-item 
questionnaire, has been developed as a brief, multidimensional self-report instrument for assessing 
the key dimensions of sexual function in women. It will be used an algorithm for determining 
domain scores and a composite full-scale score. The individual domain scores and full scale 
(overall) score of the FSFI can be derived from the computational formula outlined in the table 
below. Individual domain scores are obtained by adding the scores of the individual items that 
comprise the domain and multiplying the sum by the domain factor. The full scale score is obtained 
by adding the six domain scores. It should be noted that within the individual domains, a domain 
score of zero indicates that no sexual activity was reported during the previous evaluation. 
 


 
 
 
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Domain 
Questions 
Score 
Range 
Factor 
Minimum 
Score 
Maximum 
Score 
Score 
Desire 
1, 2 
1 – 5 
0.6 
1.2 
6.0 
 
Arousal 
3, 4, 5, 6 
0 – 5 
0.3 
0 
6.0 
 
Lubrication 
7, 8, 9, 10 
0 – 5 
0.3 
0 
6.0 
 
Orgasm 
11, 12, 13 
0 – 5 
0.4 
0 
6.0 
 
Satisfaction 
14, 15, 16 
0 (or 1) – 5* 
0.4 
0.8 
6.0 
 
Pain 
17, 18, 19 
0 – 5 
0.4 
0 
6.0 
 
 
 
Full Scale Score Range 
2.0 
36.0 
 
 
 
 
 
 
Total 
 
*Range for item 14 = 0–5; range for items 15 and 16 = 1–5 
 
A score w 26.55 is classified as FSD (Female Sexual Dysfunction) 


 
 
 
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10. 
TABLES AND FIGURES 
Table/Figure No. 
Title of Table/Figure 
1. 
 
Recruitment of Site 
2. 
 
Consort Flowchart 
 
2.1 
Protocol deviations 
 
2.2 
Analysis Populations 
3. 
 
Characteristics of patients 
4. 
 
Treatment  
5. 
 
Safety analysis  
6. 
 
End of treatment 
7. 
 
Efficacy Analysis 
8. 
 
Signs and symptoms of vaginal atrophy analysis 
9. 
 
Vaginal Maturation value and pH analysis 
10. 
 
Patient Reported Outcomes: FSFI analysis 
The numbers of tables and figures do not have to match exactly with those of the statistical report. 


 
 
 
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11. 
BIBLIOGRAPHY AND REFERENCES 
 
[1] GEICAM, "Protocol ITFE-2026-C10". 
[2] "ICH E9, Statistical Principles for Clinical Trials.". 
[3] "ICH E3, Structure and Content of Clinical Study Reports". 
[4] G. Pfeiler, C. Glatz, R. Königsberg, T. Geisendorfer, A. Fink-Retter, E. Kubista, C. F. Singer and 
M. Seifert, "Vaginal estriol to overcome side-effects of aromatase inhibitors in breast cancer 
patients," Climateric, pp. 339-344, 2011.  
[5] R. Rosen, C. Brown and J. Heiman, "The Female Sexual Function Index (FSFI): A 
Multidimensional Self-Report Instrument for the Assessment of Female Sexual Function," 
Journal of Sex & Marital Therapy, vol. 26, no. 191-208, pp. 191-208, 2000.  
[6] A. C. Hope, «A Simplified Monte Carlo Significance Test Procedure,» Journal of the Royal 
Statistical Society, vol. 30, nº 3, pp. 582-598, 1968.  
[7] H. &. N. N. Dette, «Nonparametric analysis of covariance,» The Annals of Statistics, vol. 29, nº 
5, p. 1361–1400, 2001.  
[8] S. A. Y. D. M.G. Akritas, «Nonparametric models and methods for nonlinear analysis of 
covariance,» Biometrika, vol. 87, nº 3, p. 507–526, 2000.