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ORIGINAL ARTICLE
Comparative phase I randomized open-label pilot clinical trial
of Gynophilus
®
(Lcr regenerans
®
) immediate release capsules
versus slow release muco-adhesive tablets
Caroline Dausset
1
&Stéphane Patrier
1
&Pawel Gajer
2
&Claudia Thoral
1
&Yann Lenglet
3
&Jean-Michel Cardot
4
&
Philippe Judlin
5
&Jacques Ravel
2
&Adrien Nivoliez
1
Received: 2 March 2018 /Accepted: 2 July 2018 /Pu blished online: 21 July 2018
#The Author(s) 2018
Abstract
Gynophilus
®
(Lcr regenerans
®
) is a live biotherapeutic product (LBP) that contains the live biotherapeutic microorganism
Lactobacillus rhamnosus Lcr35
®
, which is indicated to restore vaginal health. The aim of the study was to compare the safety,
ease of use, and compliance of two formulations (immediate release: IR capsule and slow release: SR muco-adhesive tablets) as
well as the colonization of Lcr35
®
in healthy women. This phase I study (Comprigel) is a parallel, randomized, 4-arm, and open-
label clinical trial evaluating an IR daily capsule formulation vs. a SR tablet administered every 3, 4, or 5 days for 21 days. Self-
collected vaginal swabs were used to quantify Lcr35
®
and characterize the composition and structure of the vaginal microbiota.
Both LBPs were well-tolerated, and no severe adverse effects were reported. All groups had Lcr35
®
vaginal concentrations over
10
7
colony forming unit per milliliter of vaginal secretion on each day in the study. The new Gynophilus
®
slow release tablets
administered either every 3, 4, or 5 days provided vaginal concentrations that were not significantly different from those of classic
Gynophilus
®
(capsule) once-a-day regimen. The LBPs and the different regimens did not adversely influence the abundance of
native Lactobacillus spp. and indeed tended to favor their growth and reduce colonization by non-Lactobacillus spp. This study
illustrates that the SR muco-adhesive LBP tablet (Gynophilus
®
SR) administered every 3 or 4 days as a safe, well-tolerated, and
efficacious alternative to a more demanding IR daily capsule and could protect women’s healthy vaginal microbiome by
promoting endogenous Lactobacillus spp.
Keywords Live biotherapeutic product .Lcr35 .Vaginal infections .Va gina l mic rob i ota .Slow release .Muco-adhesive tablet .
Safety
Introduction
The healthy vaginal microbiota is dominated by Lactobacillus
spp. which play important roles in protecting women, partic-
ularly against vaginal infections [1,2]. Bacterial vaginosis
(BV) and vulvovaginal candidiasis (VVC) are the most prev-
alent vaginal infections in women of reproductive age [3,4].
These infections have a negative impact on a woman’squality
of life and represent a significant cost to the healthcaresystem,
especially due to medical consultations and antimicrobial
treatments. In addition, recurrence is common and can lead
to multiple relapses over time [5,6]. The use of probiotic
products or live biotherapeutic products (LBPs) represent an
alternative or complement to traditional treatment regimens to
help control and/or re-establish a beneficial vaginal microbio-
ta [7,8]. Indeed, LBPs are thought to stimulate the
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10096-018-3321-8) contains supplementary
material, which is available to authorized users.
*Caroline Dausset
c.dausset@biose.com
1
Research and Development Department, BIOSE, Aurillac, France
2
Institute for Genome Sciences, University of Maryland School of
Medicine, Baltimore, MD, USA
3
Gynecology and Obstetrics Department, Jacques Lacarin Hospital
Center, Vichy, France
4
Biopharmaceutical Department, UMR MEDIS, Faculty of Pharmacy,
University of Clermont Auvergne, Clermont-Ferrand, France
5
Gynecology and Obstetrics Department, Nancy University Hospital,
Nancy, France
European Journal of Clinical Microbiology & Infectious Diseases (2018) 37:1869–1880
https://doi.org/10.1007/s10096-018-3321-8
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
recolonization of endogenous Lactobacillus spp., which can
inhibit the growth of pathogens such as Gardnerella vaginalis
and Candida albicans [7,8]. LBPs have been used success-
fully as an adjunct to traditional antibiotics for BV and as an
antifungal for VVC to improve the cure rates and prevent
recurrence by driving the restoration of beneficial commensal
microbiota [3,9,10].
LBP Gynophilus immediate release (IR) capsule, contain-
ing Lactobacillus rhamnosus Lcr35
®
, is commonly recom-
mended for preventive therapies for gynecological indica-
tions. Indeed, several clinical trials validated its efficiency
for preventing recurrent VVC and BV [11–13].
LBPs were first delivered in the form of vaginal dosage
capsules because vaginal administration affects vaginal health
more quickly than does oral LBP and enhances the viability of
the live biotherapeutic microorganism (LBM) [14]. To be ef-
ficacious, a sufficient amount of the probiotic bacteria must be
delivered locally. Previous studies have shown that in healthy
women, the concentration of Lactobacillus spp. is greater than
or equal to 10
7
CFU/ml of vaginal secretion [15,16]. Capsule
administration results in an immediate release of all the LBMs
that are linked to the natural vaginal secretions induced by the
daily elimination of approximately 10
7
to 10
8
CFU per day
and therefore requires daily administrations [17]. To limit this
rapid loss, Gynophilus
®
SR tablet was developed to ensure the
continuous release and presence of the LBM in sufficient
quantities for many days while conserving its in vitro intrinsic
characteristics (viability, stability, growth, and pathogen inhi-
bition) [18,19]. For the first time, a LBM was formulated as
slow release tablet for local vaginal administration to enhance
women’s quality of life by spacing LBP intake [14,20].
The objective of this pilot phase I clinical trial was to de-
termine the optimal posology for the new tablet form (SR)
compared to the daily capsule (IR). The following criteria
were analyzed (i) safety, (ii) ease of use of two galenic forms,
(iii) vaginal concentration of the LBM L. rhamnosus Lcr35
®
in healthy women, and (iv) impact of the products on the
vaginal microbiota.
Materials and methods
Live biotherapeutic products
Two industrial LBPs, provided by BIOSE (Aurillac, France),
were used for this study: Gynophilus
®
(immediate release
capsule), and Gynophilus
®
SR (slow release tablet), both of
which were administered vaginally. Gynophilus
®
capsule
contains 350 mg administered daily and the new muco-
adhesive Gynophilus
®
SR vaginal tablet contains 1000 mg.
LBPs batch release correspond to L. rhamnosus Lcr35
®
con-
centration greater than 10
9
CFU per dosage forms (capsule
and tablet). The LBPs and their corresponding characteristics
are shown in Table 1. In this article, the LPBs are called by
their respective commercial names: Gynophilus
®
and
Gynophilus
®
SR.
Study design
The study was designed to include at least 32 female volun-
teers. Following the management of the recruitment centers,
the cohort comprised of 35 healthy reproductive age women
(> 18 years old). All women were not pregnant (negative urine
pregnancy test) and used adapted contraception throughout
the trial. Exclusion criteria included, in addition to the classi-
cal criteria, gynecological bacterial, fungal, or viral infection
in the month prior to or at time of enrollment; use of vaginal or
oral probiotics in the last 30 days; or allergy to one of the
active ingredients or one of the excipients in the products.
Further, additional specific exclusion criteria included an in-
ability to comply with the constraints of the protocol, currently
breastfeeding, being post-menopausal (no menstruation in the
last 6 months), menstrual bleeding lasting more than 8 days,
participated in a clinical study in the last 3 months, severe
acute or chronic conditions deemed incompatible with partic-
ipation in the trial by the clinician, and immunosuppression.
At enrollment, 1 day after the last day of menstruation, each
participant completed a medical history questionnaire and was
examined by a clinician who collected cervico-vaginal sam-
ples to test for Trichomonas vaginalis, Chlamydia
trachomatis, Neisseria gonorrhoeae,andCandida spp. A
clinical evaluation was performed at Alfred Fournier
Institute according to the Amsel criteria [21]. A second vagi-
nal swab was also collected using the Copan ESwab system
(Cat# 480C) bythe clinician and stored at BIOSE at −80 °C in
1 ml of Amies transport medium for L. rhamnosus Lcr35
®
quantification and vaginal microbiota analysis by 16S rRNA
gene amplicon sequencing.
Among the 35 volunteer women, one of them had taken a
prohibited treatment before the inclusion, and one other wom-
an did not take the test drug after inclusion because of men-
struation. Therefore, after obtaining written consent and en-
rollment, 33 women were randomized to one of four arms:
Gynophilus
®
capsules administered daily which served as
the reference arm (REF, n= 9) or Gynophilus
®
SR tablets
administered either every 3 (TRT1, n= 8), 4 (TRT2, n=9),
or 5 (TRT3, n= 7) days. Randomization was stratified within
enrollment centers (Centre Hospitalier of Aurillac or Vichy,
Table 1 Industrial LBPs and their characteristics
LBP commercial name Dosage form Dosage LBM Application
Gynophilus
®
Capsule 350 mg Lcr35
®
Vaginal
Gynophilus
®
SR Tablet 1000 mg Lcr35
®
Vaginal
1870 Eur J Clin Microbiol Infect Dis (2018) 37:1869–1880
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
France). The randomization list was prepared using the SAS
®
software version 9.3.
Women self-administered the first LBPs according the
product package insert on the day of enrollment and continued
for 21 days according to their assigned regimen. Each day,
women self-collected a vaginal swab (Copan ESwab system)
and stored the sample at 4 °C in their home fridge. Volunteers
delivered all self-collected vaginal swabs to the clinic at the
second visit. This second clinical visit occurred within 4 days
of the last administration of the assigned LBP. A clinical eval-
uation similar to that performed at enrollment was performed
and a vaginal swab was collected by the clinician using a
Copan ESwab. All sample collected were stored at −80 °C
at BIOSE until additional analysis.
DNA extraction from vaginal samples
An optimized and standardized DNA extraction protocol ded-
icated to bacterial DNA extraction from swab samples has
been used (Genoscreen, Lille, France). ESwabs were thawed
on ice and a total of 500 μl of cell suspension in Amies trans-
port medium was processed using the Nucleospin 96 tissue kit
(Macherey Nagel, Germany) including a bead-beating step, as
recommended by the manufacturer. DNA quantification was
performed using SYBR green (Life Technologie, Paisley,
UK). A 16S rRNA gene PCR amplification was performed
using 2 μl of extracted DNA to confirm the presence of bac-
terial DNA using standard 16S rRNA gene PCR. DNA ex-
traction and quantification steps were performed by
GenoScreen Lille, France.
Quantification of L. rhamnosus Lcr35
®
by qPCR
Quantification of L. rhamnosus Lcr35
®
was estimated by a
specific qPCR run in triplicate as previously described
(Darbaky et al. 2016). L. rhamnosus Lcr35
®
quantities were
expressed in CFU/ml using a standard curve correlating Ct
values to colony forming unit (CFU) from in vitro culture in
De Man, Rogosa, and Sharpe (MRS) medium.
Quantification of total 16S rRNA copies
The number of 16S rRNA gene copies was measured using the
BactQuant qPCR assay as previously reported [22]. Estimates
of taxa absolute abundance were obtained by multiplying the
relative abundance of a specific taxa with the 16S rRNA gene
copy number in a sample (Online Resources 1and 2).
Vaginal microbiota characterization
The V3-V4 regions of the 16S rRNA gene were amplified
from 50 ng of DNA from each sample using the methods of
Fadrosh et al. [23]andsequencedonanIlluminaMiSeq
(Illumina, San Diego, CA, USA) using the 300-bp paired-
end protocol. Raw sequence reads were processed using in
house scripts and QIIME version 1.6.0 [24] to remove the
amplification primer sequences and quality screen was per-
formed according the following criteria: sequence reads with
average quality score of less than 20 over a 30-bp sliding
window were truncated at the first base pair of the window
and evaluated for length. If the trimmed read was < 75% of its
original length, it was discarded. Paired sequences were as-
sembled according to Fadrosh et al. [23] and each sequence
was assigned to a bacterial taxonomy using the PECAN soft-
ware [25]. Community state types (CST) according to the
previously reported method [26], taxa relative abundance
and CST assignments are shown in Online Resources 1and 2.
Statistical analysis
Data are presented as the mean and standard deviation for
continuous variables and as numbers or proportions (%) for
categorical variables. Comparisons between groups were per-
formed by using the Kruskal-Wallis test or Fisher exact test for
categorical variables. To compare the mean concentration of
Lcr35
®
over time between treatment and reference arms, the
Wilcoxon test was used. All clinical statistical analyses were
performed using the SAS software (SAS
®
institute Cary, NC,
USA, www.sas.com); pvalues < 0.05 were considered
statistically significant.
Inter-group differences in numbers of 16S rRNA copies
and microbiota were assessed using a Kruskal-Wallis test.
Theses statistical analyses were carried out using the
GraphPad Prism 5 software (www.graphpad.com/prism); p
values < 0.05 were considered statistically significant.
As a classic pilot study, small arm population induces a low
power calculation so the statistical analysis was focused on
midpoint and variability.
Results
Volunteer population
A total of 35 volunteer women were enrolled, and 34 were
randomized into 4 treatment arms; 33 women completed the
study (Fig. 1). Two women were excluded because of use of
treatment before product initiation or menstruation during the
treatment.
The population characteristics and statistic results are
shown in Table 2. The median participant age was 32 years
(32.3 ± 7.7 years). A variability was observed in the median
participant’s age among the 4 treatment arms (TRT1, TRT2,
TRT3, and REF) (p= 0.02). However, all women were be-
tween 21 and 52 years of age. Body mass index (BMI) was
not significantly different (p= 0.60) between each arm, with a
Eur J Clin Microbiol Infect Dis (2018) 37:1869–1880 1871
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
mean of 23 ± 5 (kg/m
2
). Overall, 54.5% of the participants had
at least one previous live birth. The mean number of children
was 2 for the entire cohort, varying from 1 in TRT2 to 3 in
TRT1 (Table 2).
Safety of Lcr regenerans
®
products
No severe adverse events were observed (Table 3). Ten
adverse events (AE) were reported, but no differences
(p= 0.40) in the frequency of AEs were detected among
the 4 arms (Table 3). All AEs were considered not serious.
One of the AEs in TRT2 was deemed to be of severe in-
tensity with vulvar itching. Other reported AEs included
three that were linked to product use, and the women re-
ported vulvar itching, brown discharges, and vulvovaginal
discomfort after administration of the first tablet. However,
none of the AEs led to permanent or temporary withdrawal
from the study (Table 3).
Tolerance/acceptance
Clinical tolerance was evaluated at the last visit by the study
clinician through questioning and examination of the partici-
pants. Factors evaluated were onset of clinical signs and/or
urogenital symptoms (Table 4). The products were well-
tolerated (score: excellent) by half the subjects and were tol-
erated in the other half (score: good). Specific criteria evalua-
tion (tingling, dryness, burning, itching, and pelvic pain) was
rarely reported, and no difference in these reported factors
between arms was observed (Table 5).
Table 2 Demographic characteristics of the population—FAS population (N=33
Δ
)
Parameter Statistics TRT1 (1 tab/3 days)
(N = 8)
TRT2 (1 tab/4 days)
(N = 9)
TRT3 (1 tab/5 days)
(N = 7)
REF (1 caps/ day)
(N = 9)
p(< 0.05)
Age at inclusion (years) Mean ± SD 39.4 ± 6.5 28.9 ± 4.8 32.4 ± 9.7 29.4 ± 5.7 0.0220**
Body mass index (kg/m
2
)Mean ± SD 24.53 ± 4.95 22.67 ± 6.19 22.47± 3.21 22.44 ± 4.29 0.5988**
Children
No n(%) 2 (25.0%) 5 (55.6%) 3 (42.9%) 5 (55.6%) 0.6115*
Yes n(%) 6 (75.0%) 4 (44.4%) 4 (57.1%) 4 (44.4%)
If yes, number of children Mean ± SD 2.7 ± 0.5 1.0 ± 0.0 2.0 ± 0.8 1.5 ± 0.6 0.0652**
*Fisher’s exact test
**Kruskal-Wallis test
Δ
N= 33 because only 33 received the treatment
Fig. 1 Study design of
CompriGel pilot clinical trial
1872 Eur J Clin Microbiol Infect Dis (2018) 37:1869–1880
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Administration and dosage regimen demonstrated strong
satisfaction (Table 6). The tablet regimen showed significantly
higher acceptance after the 3-week administration, especially
in the less restrictive arms, in which the tablets were taken
every 5 days compared to the daily capsule (Table 6).
Lcr35
®
colonization
Women self-collected a daily vaginal swab that was used to
measure the concentration of Lactobacillus rhamnosus
Lcr35
®
in each arm (Fig. 2). The average vaginal Lcr35
®
concentration showed no major difference among the four
arms (p= 0.77). A decrease in the daily Lcr35
®
concentration
was observed post-tablet administration as the frequency of
administration increased from every 3 to every 5 days (Fig. 2).
Although the daily Lcr35
®
concentration decreased post-ad-
ministration, it remained greater than 10
7
CFU/ml in TRT1
and TRT2, with the exception of 1 day in TRT2. In TRT3,
5 days post-administration, the Lcr35
®
concentration was ap-
proximately 10
7
CFU/ml.
Effect of Gynophilus
®
formulations on the vaginal
microbiota
The daily composition and absolute abundance of the
vaginal microbiota were evaluated using 16S rRNA
gene amplicon sequencing and quantitative PCR. No
significant difference (p= 0.12) in the concentration of
non-Lcr35
®
Lactobacillus spp. was observed before
treatment (D0), during treatment and at the end of treat-
ment (D21) (Fig. 3a). The overall composition in
Lactobacillus spp. remained consistent during and after
treatment.
The vaginal microbiota was classified into six distinct
community state types (CSTs) based on the microbiota
bacterial composition and abundance. The before and af-
ter treatment CST frequencies are shown in Table 7,
where CST-I is dominated by L. crispatus, CST-II is dom-
inated by L. gasseri, CST-III is dominated by L. iners,
CST-V is dominated by L. jensenii, CST-IV is character-
ized by a paucity of Lactobacillus spp. and a diverse set
of strict and facultative anaerobes, and CST-VI is domi-
natedbytheLBMLcr35
®
. The frequency of CSTs was
not different between visit 1 and visit 2, indicating that
Gynophilus
®
treatments did not disturb the indigenous
vaginal microbiota. Only one on 35 women is classified
as CST-VI (dominance Lcr35
®
) because the last tablet
was administrated just before the V2. So, Lcr35
®
did
not colonize and replace the endogenous microbiota.
Moreover, the CST proportions were not drastically mod-
ified which suggest that Lcr35
®
did not promote only one
CST type.
Table 3 Safety analysis—adverse events during the treatment period (N=33
Δ
)
Volunteer with at least one TRT1 (1 tab/3 days)
(N = 8)
TRT2 (1 tab/4 days)
(N = 9)
TRT3 (1 tab/5 days)
(N = 7)
REF (1 caps/ day)
(N = 9)
Total
(N = 33)
p*(< 0.05)
SAE (n) 00000NA
AE (n) 4 1 2 3 10 0.4017
AE of severe intensity (n)01000NA
AE linked to administration of the trial
treatment (n)
111030.7773
AE that led to permanent or temporary
discontinuation of study treatment (n)
00000NA
*Fisher’s exact test
Δ
N= 33 because only 33 received the treatment
Table 4 Overall tolerance (N=33
Δ
)
Volunteer evaluation TRT1 (1 tab/3 days)
(N = 8)
TRT2 (1 tab/4 days)
(N = 9)
TRT3 (1 tab/5 days)
(N = 7)
REF (1 caps/day)
(N = 9)
Tot al
(N = 33)
p*(<0.05)
Very good tolerance 4 6 2 4 16 0.5446
Good tolerance 4 3 5 4 16
Average tolerance 0 0 0 0 0
Poor tolerance 0 0 0 0 0
*Fisher’s exact test
Δ
N= 33 because only 33 received the treatment
Eur J Clin Microbiol Infect Dis (2018) 37:1869–1880 1873
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Representative vaginal microbiota profiles for each
study arm and the absolute abundance of Lcr35
®
are
showninFig.4and highlight the absence of a rapid
change in the composition and structure of the vaginal
microbiota during treatment. Of note, it was observed that
the Escherichia coli present at visit 1 was rapidly elimi-
nated after the initiation of treatment and that the indige-
nous Lactobacillus spp. re-colonized the vagina (Fig. 4a).
Moreover, E. coli species was detected at least once dur-
ing the study in vaginal samples of 28 women. For 21 of
them, E. coli is eliminated quickly after treatment (Online
Resource 3). Overall, after the initiation of Gynophilus
®
treatments, Lactobacillus spp. abundance remained stable
or promoted (Fig. 4).
Discussion
LBPs aim to re-establish a Lactobacillus-dominated vag-
inal microbiota by promoting the recolonization of the
indigenous Lactobacillus spp., thereby restoring the pro-
tection against infections [7,8,27,28]. The LBP
Gynophilus
®
is vaginally delivered and is indicated to
prevent recurrent VVC and BV by restoring vaginal
health [11–13]. This LBP contains L. rhamnosus
Lcr35
®
, which was previously reported to have potent
antimicrobial properties [18,19,29–32]. Gynophilus
®
(Lcr regenerans
®
) LBP formulated in vaginal capsules
was shown to be well-tolerated and significantly enhanced
the quality of life of women after treatment [13]. A muco-
adhesive slow release tablet formulation (SR) was
developed to limit the rapid loss of the LBP associated
with natural vaginal secretions and ultimately enhance the
ease of use by minimizing the frequency of administration
[14,18–20] resulting in an increased compliance.
CompriGel is a pilot phase I clinical trial, in which 35
volunteers were randomized to four arms to compare the
LBP vaginal concentrations between daily IR capsule ad-
ministration and SR tablets taken every 3, 4, or 5 days for
21 days. As most of the pilot clinical studies, the power
calculation is limited due to the small size population but
minimize the risks in healthy subjects while allowing se-
lection of an interesting posology. So, this pilot study was
designed to determine the more adapted posology of the
new SR tablet.
Inthisstudy,wehaveshownnodifferenceinLcr35
®
colonization between SR tablets administered every 3 or
4 days and daily capsules. Importantly, the mean con-
centration of L. rhamnosus Lcr35
®
in all treatment arms
remained greater than 10
7
CFU/ml, which is the neces-
sary minimal concentration for activity and, ultimately,
to prevent vaginal infections [17]. The Lcr35
®
coloniza-
tion was quantified by molecular biology. This method
was not used to differentiate viable and dead microor-
ganisms and we know that the delivered LBM concen-
tration is at least 1.10
9
CFU/posology and we observe a
quickly remove of the LBM probably induced by the
vaginal secretions [17]. So, administration of tablets ev-
ery 3 or 4 days maintained a mean Lcr35
®
concentra-
tion greater than 10
9
CFU/ml, similar to that obtained
with the daily capsules. This finding supports the pos-
sibility of a clinical regimen in which Gynophilus
®
SR
Table 5 Satisfaction of female volunteers with regard to treatment tolerance
Parameter Statistics TRT1
(1 tab/3 days)
(N = 8)
TRT2
(1 tab/4 days)
(N = 9)
TRT3
(1 tab/5 days)
(N = 7)
REF
(1 caps/day)
(N = 9)
Tot al
(N = 33)
Global p value**
(< 0.05)
Tingling Mean ± SD 0.43 ± 0.69 1.13 ± 3.18 0.67 ± 1.43 0.28 ± 0.22 0.63 ± 1.77 0.5856
0 = no tingling
10 = severe tingling
Dryness Mean ± SD 0.30 ± 0.38 0.11 ± 0.15 0.50 ± 0.98 0.29 ± 0.19 0.29 ± 0.50 0.3568
0 = no dryness
10 = severe dryness
Burning Mean ± SD 0.29 ± 0.38 0.14 ± 0.25 0.14 ± 0.18 0.23 ± 0.19 0.20 ± 0.26 0.5970
0 = no burning
10 = severe burning
Itching Mean ± SD 0.66 ± 0.86 1.22 ± 3.26 0.97 ± 2.35 1.37 ± 3.24 1.07 ± 2.56 0.5747
0 = no itching
10 = severe itching
Pelvic pain Mean ± SD 0.29 ± 0.34 1.18 ± 2.41 0.11 ± 0.15 0.48 ± 0.82 0.55 ± 1.35 0.6258
0 = no pelvic pain
10 =severe pelvic pain
**Kruskal-Wallis test
1874 Eur J Clin Microbiol Infect Dis (2018) 37:1869–1880
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
tablets are administered every 3 or 4 days while main-
taining maximum efficacy.
The formulation of the muco-adhesive Gynophilus
®
SR tablet was developed to provide a three-fold increase
in the quantity of LBM brought in one dosage form to
reduce the rapid loss of LBM Lcr35
®
delivered by a cap-
sule. Moreover, this new dosage form ultimately im-
proved the comfort and ease of use, as the controlled
muco-adhesive vaginal formulation affords a prolonged
residence time of the LBM at efficacious concentrations
with less frequent administration. Slow release delivery
strategies have been successfully developed for antifungal
and antibacterial drugs and for other vaginal LBPs using
different muco-adhesive tablets or vaginal rings [33–37].
The main goal of slow release strategies is to promote
compliance when the recommended treatment duration is
longer than 2–3 days and, ultimately, to improve the
comfort and ease of use by reducing the burden on wom-
en through spacing administrations. The results presented
have confirmed that over a 3-week treatment period,
Gynophilus
®
SR tablets administered every 3 or 4 days
were reported to be convenient and less burdensome than
were daily Gynophilus
®
capsules (Table 6). Daily treat-
ment schedules generally induce poor compliance over
long administration periods. Gynophilus
®
SR promoted
compliance and improved treatment adherence. This find-
ing supports a benefit of the LBP for treating recurrent
vulvovaginal infections, which have been shown to re-
quire a 3-week treatment [13]. While compliance and ease
of use are important factors, the safety of the product is of
the utmost importance. In this phase I clinical trial, no
severe adverse events (AE) and only a few AEs were
reported; these AEs were reported equally in all arms of
the study. None of the AEs led to temporary
Table 6 Satisfaction of female volunteers with regard to administration and dosage regimen
Parameter Statistics TRT1
(1 tab/3 days)
(N = 8)
TRT2
(1 tab/4 days)
(N = 9)
TRT3
(1 tab/5 days)
(N = 7)
REF
(1 caps/day)
(N = 9)
Tot al
(N = 33)
Global
p value**
(< 0.05)
Administration of the treatment Mean ± SD 0.84 ± 0.61 1.04 ± 1.11 1.00 ± 1.20 1.00 ± 1.20 0.92 ± 0.86 0.9740
0 = easy administration
10 = difficult administration
Size of the administered capsules/tablets Mean ± SD 1.33 ± 2.45 1.08 ± 0.97 1.61 ± 2.50 0.58 ± 0.36 1.12 ± 1.70 0.5983
0 = an appropriate size
10 = an inappropriate size
Use of intravaginal administration
of gynecological-related
treatments
Mean ± SD 0.91 ± 0.93 2.01 ± 1.59 1.43 ± 1.41 1.99 ± 1.82 1.62 ± 1.50 0.2635
0 = is practical
10 = is restrictive
Administration of capsules/tablets Mean ± SD 0.59 ± 0.72 1.52 ± 1.66 0.79 ± 0.54 1.12 ± 1.12 1.03 ± 1.14 0.5565
0 = an appropriate shape
10 = not practical
Use of the capsule/tablet Mean ± SD 0.84 ± 0.79 2.50 ± 1.66 1.47 ± 1.18 3.09 ± 2.82 2.04 ± 1.97 0.1381
0 = is ideal
10 = is restrictive
Administration of a capsule every
day/every 3, 4, and 5 days
Mean ± SD 2.33 ± 3.48 2.61 ± 2.38 0.96 ± 1.46 5.38 ± 3.35 2.95 ± 3.15 0.0270
0 = is not restrictive
10 = is restrictive
The 3-week period of administration Mean ± SD 2.94 ± 3.32 2.39 ± 1.41 1.27 ± 1.53 5.61 ± 2.95 3.16 ± 2.86 0.0364
0 = is not restrictive
10 = is restrictive
Release of the capsule/tablet Mean ± SD 3.20 ± 2.83 4.16 ± 3.63 3.49 ± 3.06 2.88 ± 2.72 3.43 ± 2.98 0.8958
0 = complete Bdisintegration^of the
capsule/tablet
10 = no Bdisintegration^of the
administered capsule/tablet which
was found completely expelled
from the vagina
**Kruskal-Wallis test
Eur J Clin Microbiol Infect Dis (2018) 37:1869–1880 1875
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
discontinuation or withdrawal from the study. Overall, the
tolerance of the LBPs was reported to be excellent in half
of the participants and good in the other half and was
associated with an overall strong satisfaction with the
use of the products. These results are in line with previous
clinical trials for the prevention of recurrent VVC and BV
with various Gynophilus
®
formulations [11,13].
Gynophilus
®
SR appears to be a well-accepted, safe, and
easy-to-use LBP with high compliance that delivers an
adequate concentration of LBM Lcr35
®
forupto5days
post-administration. The efficacy of Gynophilus
®
SR
must be confirmed in a large-scale study with the final
selected dose.
The impact of Gynophilus
®
LBP formulation and treat-
ment frequency on the composition and structure of the
vaginal microbiota was evaluated. The results showed that
at study entry, 54.6% of the volunteer’s vaginal microbi-
ota harbored microorganisms that were phylogenetically
related to Lactobacillus spp. as the numerically dominant
members (Table 7). The frequency of vaginal CSTs in
these otherwise healthy French women is similar to that
observed by Ravel et al. in North American women [26,
38]. In this phase I clinical study, Gynophilus
®
capsules
and SR tablet administration did not disturb the endoge-
nous Lactobacillus spp. and trends towards an increase in
the abundance of indigenous Lactobacillus spp. without
Lcr35
®
colonization (Fig. 3and Fig. 4). This finding sup-
ports a beneficial effect of treatment by restoring a
healthy Lactobacillus-dominated vaginal microbiota.
However, because all participants are healthy and disease
free, a clinical trial designed to evaluate treatment efficacy
in women with VVC or BV is warranted to confirm that
Gynophilus
®
SR administered every 3 or 4 days will also
promote the restoration of a resilient Lactobacillus spp.-
dominated microbiota while efficiently treating the vagi-
nal infections.
Even if statistical results showed no differences be-
tween both galenic forms, it has to be remind that low
number of subject does not allow any extrapolation to
the global population. This pilot clinical trial will afford
1876 Eur J Clin Microbiol Infect Dis (2018) 37:1869–1880
Fig. 2 Mean vaginal concentrations of Lactobacillus rhamnosus Lcr35
®
over time in each arm measured by the targeted qPCR method. Delivery of 1
tablet every 3 days (a), 1 tablet every 4 days (b), and 1 tablet every 5 days (c) were compared to the reference treatment of 1 capsule per day
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
statistical power calculation to design a larger and more
robust clinical trial to confirm the Gynophilus
®
SR effi-
cacy on vaginal infections.
In conclusion, this study showed that the SR formula-
tion of Gynophilus
®
administered every 3, 4, or 5 days is
well-tolerated, easy-to-use, and less burdensome than the
capsule form. More importantly, the findings support the
conclusion that the muco-adhesive Gynophilus
®
SR ad-
ministered every 3, 4, or 5 days is a safe, well-tolerated,
well-accepted, and efficient alternative to provide a daily
Fig. 3 Composition of the
vaginal microbiota in all
treatment arms. aEffect of all
treatments combined on the
estimates of Lactobacillus spp.
absolute abundance at visit 1,
during treatment and at visit 2. b
Community state type distribution
over all samples collected in the
study based on the species
composition and abundance, as
determined by the heatmap of
percentage abundance of
microbial taxa found in the
vaginal microbial communities of
all samples
Table 7 CST frequency at visit 1
andvisit2(N=33) CST-I CST-II CST-III CST-IV CST-V CST-VI
Visit 1 (D 0) n(%) 10 (30.3%) 2 (6.1%) 6 (18.2%) 15 (45.4%) 0 (0.0%) 0 (0.0%)
Visit 2 (D21) n(%) 10 (30.3%) 2 (6.1%) 4 (12.1%) 16 (48.5%) 0 (0.0%) 1 (3.0%)
Eur J Clin Microbiol Infect Dis (2018) 37:1869–1880 1877
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Fig. 4 Representative longitudinal vaginal microbiota profiles of women
enrolled in atreatment 1; btreatment 2. Lactobacillus casei (pink)
indicates the live biotherapeutic microorganism Lcr35
®
. Vertically,
each column represents one sampling day. Horizontally, community
state type (CST) distribution for all samples collected by women was
determined by the abundance of phylotype composition represented in
the taxa relative abundance histogram. CST reflect the dominant
microorganisms, CST-I is dominated by L. crispatus,CST-IIbyL.
gasseri,CST-IIIbyL. iners, CST-V by L. jensenii, CST-VI by L. casei
(Lcr35
®
), and CST-IV is characterized by a diverse set of strict and
facultative anaerobes. The vaginal microbiota study of each volunteer
women is presented in Online Resource 3
1878 Eur J Clin Microbiol Infect Dis (2018) 37:1869–1880
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
vaginal concentration of LBM greater than 10
7
CFU/mL
while maintaining and even favoring a woman’sindige-
nous Lactobacillus spp. Thus, Gynophilus
®
SR adminis-
tered every 3 or 4 days is an efficacious alternative for
patients with poor compliance profiles.
Acknowledgements We wish to thank all gynecologists and volunteers
who have participated to the CompriGel clinical trial. Thanks to all those
who contributed in the clinical trial, in particular Richard Boissiere for the
monitoring.
Funding This pilot CompriGel study was supported by BIOSE.
Compliance with ethical standards
Conflict of interest Adrien Nivoliez, Caroline Dausset, Claudia Thoral,
and Stéphane Patrier have an institutional affiliation with BIOSE. Jean-
Michel Cardot, Philippe Judlin, and Yann Lenglet have a consultancy
contract with BIOSE, the financial and logistic support of CompriGel
study.
Ethical approval The protocol for CompriGel clinical trial was designed
according to the recommendations of the ICH Steering Committee rec-
ommendations (ICH E6), Good Clinical Practice guidelines, the declara-
tion of Helsinki and the French legislation. Then this study was evaluated
favorably by the Ethics Committee South-East IV and approved by the
Competent Authority (ANSM: Agence Nationale de Sécurité du
Médicament [French National Agency for Medicines and Health
Products Safety]) which granted the authorization. This clinical trial
was registered under 2014-001746-21 EudraCT Number.
Informed consent In accordance with the law of 6 January 1978 mod-
ified, the woman volunteer was informed in the information leaflet and
gave her written consent for the processing of her personal data imple-
mented as part of this study and to enable analysis of the research results.
Before inclusion in the study, the investigator provided the woman vol-
unteer with all the information about the study. When the woman volun-
teer gives her consent, she and the investigator signed and dated the
consent form.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a link
to the Creative Commons license, and indicate if changes were made.
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