Received: 11 December 2019 Revised: 12 February 2020 Accepted: 2 March 2020

DOI: 10.1111/dth.13293

ORIGINAL PAPER

Efficacy and safety of a new formulation kit (shampoo + lotion)

containing anti-inflammatory and antioxidant agents to treat
hair loss

Alessandra Anzai1 | Amanda F. Pereira2 | Kleber R. Malaquias2 |

Lucas O. Guerra3 | Maurízio Mercuri2

1
Private Practice, S~ao Paulo, SP, Brazil
2
Hypera Pharma, S~ao Paulo, SP, Brazil
Abstract
3
Allergisa—Pesquisa Dermato-cosmética Ltda, Androgenetic alopecia (AGA) and telogen effluvium (TE) are common causes of hair
Campinas, SP, Brazil loss, with limited options for treatment. The aim of this study was to determine the
Correspondence efficacy and safety of a new formulation kit (Pielus Anti-hair loss lotion and shampoo)
Maurízio Mercuri, Hypera Pharma, Ave containing anti-inflammatory and antioxidant agents in patients with TE and/or AGA.
Magalh~aes de Castro, 4800—24 floor—S~ ao
Paulo, SP, Brazil. This was a prospective, single-arm, open-label study with 35 women and 10 men,
Email: maurizio.mercuri@brainfarma.ind.br median age of 44 years, with clinical diagnosis of TE and/or AGA. They used the

Funding information combination of shampoo and lotion daily for 90 days. There was nonoccurrence of
Hypera Pharma adverse events. Phototricogram showed increase in the average number of anagen
hairs at T90 (P < .001). Scalp microphotography showed increase in total follicular
units, total of hairs, and median hair per follicle unit at T90. There was an increase in
diameter (P = .003) and in median maximum load and resistance to traction in T90
(P = .040). Combing and washing test demonstrated a decrease in the number of shed
hairs before washing (P = .005), after washing (P = .01) and in total (P < .001). The
use of shampoo+ lotion daily was safe and effective to attenuate hair loss in men and
women with TE and/or AGA.

KEYWORDS

alopecia, hair disorders, therapy—topical

1 | I N T RO DU CT I O N
Hair loss is a common dermatologic disorder that can be secondary to
many causes. It is a stressful event that can be devastating to a
patient's self-esteem, self-image, and overall quality of life.1,2
Among all causes of alopecia, androgenetic alopecia (AGA) is the
most common: up to 30% of white men will have AGA by age 30, up
to 50% in 50 years and 80% in 70 years.3 The term androgenetic com-
prises the two most important factors of the pathophysiology of the
condition: the genetic predisposition and the role of androgenic hor-
mones. Since the clinical presentation and evolution of alopecia differ
IRB approval status: Approved by Research Ethics Committee “Investiga Research Institute.”
between men and women, the term androgenic has been replaced by
“male pattern hair loss” and “female pattern hair loss.”4
The role of androgens in the development of female AGA is still
debated and other influencing factors are now recognized to contrib-
ute to hair loss. In AGA, scalp biopsies show decreased anagen phase
follicles and increased miniaturized hairs. In addition, there is often
perifollicular lymphocytic inflammatory infiltrate and mild perifollicular
concentric fibrosis.5
Although the role of this inflammation is not completely eluci-
dated the presence of perifollicular microinflammation in AGA biop-
sies was associated with poor response to treatment with minoxidil.6
Telogen effluvium (TE) is a common condition of increased shed-
ding of telogen hairs, characterized as an abnormality of hair cycling.

Dermatologic Therapy. 2020;33:e13293. wileyonlinelibrary.com/journal/dth © 2020 Wiley Periodicals, Inc. 1 of 6

https://doi.org/10.1111/dth.13293
2 of 6
TE is etiologically and pathogenically heterogeneous. Recently, it has
been suggested a new classification of TE that recognizes an “autoim-
mune” TE, related to inflammation.7 Emotional stress has been consid-
ered a typical trigger to TE and the possible mechanisms involve
perifollicular inflammatory events via neuropeptides.8
There is evidence that alopecia can be influenced by environmen-
tal and lifestyle factors in men and women. Among them, smoking is
the most studied factor. Epidemiological studies have associated
smoking with increased hair loss.9,10
The mechanisms are multifactorial: there is alteration of dermal
papilla microvasculature, accumulation of toxic metabolites with dam-
age to hair follicle DNA, imbalance in follicular protease/antiprotease
systems and pro-oxidant effects with release of proinflammatory
cytokines.11 Oxidative stress leads to accumulation of damage to the
organelles of affected cells leading to apoptosis and loss of stem cell
differentiation.
Sun exposure has also been associated with hair loss. Exposure of
the scalp to the sun eventually releases proinflammatory cytokines,
leading to follicular cell damage. Possible mechanisms may involve
photoactivation of porphyrins produced by Propionibacterium sp. in
the pilosebaceous duct, leading to oxidative tissue damage and the
production of radical oxygen species and cytokines by the
12,13
keratinocytes themselves.
Similarly, the use of dyes, which color the shafts through oxida-
tive chemical reactions, may also be implicated in worsening hair
loss.14,15
Excessive proliferation of Malassezia and scalp dermatoses such
as seborrheic dermatitis, psoriasis, and atopic dermatitis can also
affect oxidative balance and treatment aiming a healthy scalp should
be part of the treatment of hair loss.16
All this evidence shows that there may be a therapeutic role in
the use of antioxidants and anti-inflammatories to counteract oxida-
tive stress related to hair loss. According to these data, a new cos-
metic formulation was developed containing anti-inflammatory,
antioxidants, and conditioning agents. The aim of this study was to
assess the safety and efficacy of this new formulation present in
Pielus Anti-hair loss lotion and shampoo through clinical and instru-
mental evaluation and subjective perception of the research partici-
pant under normal use.
2 | P A T I E NT S A N D M E TH O D S
2.1 | Study design and subjects
This was a prospective, single-arm, open-label study developed in a
single private center Allergisa Pesquisa Dermato-Cosmética Ltda,
Campinas—SP—Brazil. The study was carried out in compliance with
the ethical principles of the Declaration of Helsinki and the Good
Clinical Practices International Conference on Harmonization (ICH)
Guidelines. The study received approval from the Research Ethics
Committee (CEP). All subjects were healthy and volunteered for the
study. All subjects provided their informed consent.
ANZAI ET AL.
The study protocol was initiated on 28 November 2017 and
ended on 22 March 2018, total research duration was 90 ± 07 days.
The study was initiated with 45 participants, 35 females and
10 males, aged between 20 and 59 years (median 44 years) who had
a clinical diagnosis of TE and/or AGA. TE diagnosis was made through
the complaint of increased shedding reported by the participant that
presented a positive pull test and did not present criteria for AGA or
other alopecia.
Participants consisted of healthy adults with healthy scalp. Criteria
of noninclusion were: alopecia other than AGA, moderate to intense
dandruff or other skin pathology on scalp; more than 50% of white or
light blond hair; active ocular diseases, insulin-dependent diabetes;
Immune deficiency; systemic corticosteroids or immunosuppressants.
Participants were advised not to modify hair style throughout the
course of the study and not to use hair treatments (ie, hair loss, anti-
dandruff, supplements, etc) throughout the study period.
2.2 | Treatment protocol
Participants were oriented to daily apply the shampoo to wet hair,
gently massage the scalp for 1 minute and rinse off. If desired, they
could use a usual conditioner and rinse. After removal of excessive
water with towel, they should apply the lotion on the scalp and hair
and lightly massage the scalp with fingertips and do not rinse.
2.3 | Phototricogram
Participants had a 1.0 cm2 area of scalp shaved: on vertex of TE
patients and on the most evident alopecic area of AGA patients. The
shaved area was photographed with liquid vaseline using a Nikon D70
camera and an Epiflash Canfield phototricogram system. After 48 hours
of hair shaving, participants returned to the institute and the procedure
for obtaining a photograph from the same location was repeated. The
phototricogram allows the hair count by a trained technician through
the Adobe Photoshop CS4 software and the evaluation of the following
parameters: number of anagen and telogen hairs and total density.
2.4 | Scalp microphotography
An area approximately 10 cm away from the shaved area was photo-
graphed using a Nikon D70 camera at T0 and T90. Captured images
were used for hair counting by trained technician through the
software.
2.5 | Hair extraction procedure
At visits T0 and T90, 20 hairs were extracted from each participant
around the shaved area, to perform tensile strength evaluations and
microscopic analysis of the bulb and shaft. Hair thickness (diameter,
ANZAI ET AL. 3 of 6

determined at the base of the shaft) was measured by a micrometer.
These same hairs were used to evaluate the hair resistance.
2.6 | Tensile strength rating
hypothesis for improvement. Instrumental data were compared by
Student's t test with unilateral hypothesis for improvement. The confi-
dence level considered in the comparative analyzes was 95%. Soft-
ware: XLSTAT 2018 and STATA 10.

For measurements of hair resistance, the Instron Universal Testing 3 | RE SU LT S

Machine Model 5565 was used, equipped with a 10 N load cell, a tensile
speed of 10 mm/min at a temperature of 20 ± 2 C and a relative
humidity of 50% ± 5. The stress and strain tests used at least 10 speci-
mens from each participant (10 hairs, the same ones that were used to
measure thickness), consisting of the part closest to the participant's
scalp, disregarding the opposite end. Hair size has been standardized
wherever possible. Before measuring the diameter, the shaft was moist-
ened with water. The previously moistened wire, evaluated for its diam-
eter with the aid of a micrometer, was fixed through its ends—wrapped
with tape—in the load cell (upper and lower jaws) of the equipment.
The strength evaluation was performed at T0 and T90.
2.7 | Combing and washing method
The study was completed with 41 research participants. Two had
adverse events unrelated to the use of products and two dropped out
of study for unrelated personal reasons.
There was nonoccurrence of adverse events attributed to the use
of products, supervised by dermatologist and ophthalmologist. There-
fore, the products were considered safe under normal conditions
of use.
Phototricogram showed an increase in the average number of
anagen hairs from 121.3 at T0 to 124.60 at T45 (P = .015) and to
131.30 at T90 (P < .001). The analysis of telogen hair count and per-
centage of telogen hairs showed no statistical difference. Scalp micro-
photography showed increased in total follicular units, total of hair
and median hair per follicle unit at T90 (Table 1 and Figure 1).

The participants, after the T0 and T90 visits, remained 48 hours with-
out washing their hair, to perform the combing and washing T A B L E 1 Data obtained through scalp microphotography showed
increase in total follicular units, total of hair, and median hair per
procedures.
follicle unit at T90 compared to baseline
First, in a closed room, a white towel was placed on the floor. The
participant sat on this towel, in a chair, and with a white tray on his Shampoo + lotion
lap, with the help of a comb provided by the institute, combed his hair T0 T90
with 20 movements, from the top of the head to the end of the hair.
Total follicular Median 94.1 102.3
All hairs that came off the participant's head, fell on the tray and/or unit Mean improvement (Δ) in – 8.20
towel, or got caught in the comb were collected. Once this was done, relation toT0
the participants had their hair washed by a trained technician in a % of mean improvement – 8.7
standardized protocol and all hair that has fallen off during the proce- (Δ%) in relation to T0
dure was collected. % of participants with – 85.4
positive effect
P-value – <.001***
2.8 | Clinical efficacy evaluation through Total of hair Median 120.4 137.8
dermatological evaluation, participant's Mean improvement (Δ) in – 17.4
self-assessment, and sensory evaluation relation to T0
% of mean improvement – 14.5
Participants underwent a dermatological clinical efficacy assessment (Δ%) in relation to T0
(5-point questionnaire for hair thickness, density, and new hair growth) % of participants with – 95.1
and were then instructed to complete the perceived efficacy 5-point positive effect

questionnaire for hair loss, strength, breakage, scalp coverage, hair P-value – <.001***
thickness, moisture, greasiness, new hair growth, and volume. Median hairs per Median 1.29 1.36
Participants also answered a 5-point scale questionnaire for ease of follicular unit Mean improvement (Δ) in – 0.07
application, pleasant smell, pleasant foam, freshness/refreshment feeling. relation to T0
% of mean improvement – 5.4
(Δ%) in relation to T0

2.9 | Statistical analyses % of participants with – 61.0

positive effect
P-value – .001**
For clinical efficacy data, the times were compared with baseline using
the nonparametric Wilcoxon Signed Rank test with unilateral ***Significant at a level of .1%; **Significant at a level of 1%.
4 of 6
FIGURE 1 Scalp microphotography of four participants showing increase in hair density and increase in mean total of hairs at T90 compared
to baseline
Regarding thickness and resistance to traction, we observed an
increase in diameter from a median of 56.1 μm in T0 to 58.1 μm in
T90 (P = .003) and increase in the median maximum load from
0.513 N in baseline to 0.542 N in T90 (P = .040). Analysis of change
in extension (mm) and maximum load (N)/diameter (μm) showed no
statistical significance.
In combing and washing test, the number of shed hairs before
washing decreased from a median of 40.10 in T0 to 26.30 in T90
(P = .005), the number of shed hairs after washing decreased dimin-
ished from a median of 31.8 in T0 to 20.60 in T90 (P = .01) and the
total number decreased from a median of 72.00 in T0 to 46.90 in
T90 (P < .001).
In questionnaires assessments, the clinical efficacy by dermatolo-
gist investigators showed a statistically significant improvement in the
thickness and density of hairs in T90 and growth of new hairs in T45
and T90. In the evaluation of participants' subjective efficacy, there
was a significant improvement in T45 and T90 of hair loss, strength,
scalp coverage, hair thickness, new hair growth, and volume. Moisture
showed improvement only in T45 and breakage improved only in T90.
There was no statistical difference in greasiness. All attributes of sen-
sory evaluation had acceptance greater than 85% in both evaluation
times.
4 | DISCUSSION
Multiple factors contribute to hair loss including genetics, hormones,
environmental exposure, medications, nutrition, and intrinsic aging.
Thus, the treatment requires a multimodal approach and the use of
adjuvant treatments may provide additional benefits to the traditional
treatment. Despite recent advances in understanding the biology of
the hair follicle, treatment options for hair disorders are restricted and
ANZAI ET AL.
often do not meet patients' expectations. Response to traditional
treatments is variable and sometimes unsatisfactory because they do
not act on all possibly related mechanisms. Patients are subjected to
prolonged or lifelong use of medicines with possible side effects,
treatments with low cosmetic acceptance or little insertion in the rou-
tine. Therefore, there is a need for adjuvant and newer modalities of
treatment.
Pielus Anti-hair loss shampoo and lotion contain three anti-hair
loss ingredients. NANO R (Redensyl) is a nanoencapsulated compound
derived from green tea extract rich in polyphenols which enhances
stem cell proliferation and differentiation and protects them from the
process of apoptosis, and increases the metabolic activity of dermal
papilla fibroblasts (unpublished data). NANO F (SymHair Force 1631)
is a nanoencapsulated biotechnological blend obtained from Tahiti
blue microalgae, which stimulates the anagen phase of the hair follicle
and decreases the number of apoptotic cells in the hair bulb
(unpublished data). Baicapil is a flavonoid-rich plant complex that pro-
motes prolongation of the anagen phase by increasing ATP produc-
tion of hair follicle cells. It also protects follicle fibroblasts from
oxidative stress, helping to protect against follicular cell
senescence.17-20
Pielus Anti-hair loss shampoo is composed of mild cleansing
agents that provide cleansing without damaging the hair fiber and
Pielus Anti-hair loss lotion has a light, alcohol-free texture and can be
applied to wet or dry scalp.
The combination of shampoo + lotion showed significant
improvement in clinical and subjective efficacy parameters in the
41 participants. The phototricogram revealed anagen count increase
with statistically significant difference in D45 and D90. There was sig-
nificant improvement in the microphotographs in the parameters of
total follicles, sum of hairs and average of hairs per follicle. Thickness
and tensile strength tests showed an increase in diameter and
ANZAI ET AL.
maximum load (N) with statistical difference. Combing and washing
method showed significant improvement in the amount of hair shed
hairs before and after washing and total amount.
The Pielus Anti-hair loss lotion and shampoo also contain condi-
tioning agents that protect the hair shaft promoting hydration, soft-
ness, shine and combability and zinc, pyridoxine and panthenol. In the
sensory evaluation questionnaires, the volunteers attributed the char-
acteristics of ease of application, pleasant smell and freshness/
refreshing sensation with acceptance greater than 85%.
Limitations of this study are lack of placebo group, small sample
size and short time of intervention. A systematic review and meta-
analysis of effectiveness of treatments for AGA showed improvement
also in the placebo group. So we cannot exclude that the results of
this study are not related only to the placebo effect.21 Despite these
limitations, we consider that this pilot study showed that the products
are promising in treating hair loss by addressing the micro-
inflammation associated with hair loss. The follicular micro-
inflammation disturbs dermal papilla fibroblasts and the follicular
epithelium contributing to stem cell apoptosis and perifollicular
fibrosis.22
There is an increasing interest in alternative treatments that are a
simple and cost-effective with no adverse effects for patients
unsatisfied with conventional therapy. Diverse formulations are devel-
oped to address this demand.23-27 Many hair growth promoters topi-
cal formulations are based on herbs and their active constituents.28
Despite similarities in those formulations a comparison between them
are limited by the high heterogeneity in most studies.
5 | C O N CL U S I O N S
Results of the present study show that the formulations under investi-
gation were safe and effective to attenuate hair loss in men and
women with TE and AGA. Further placebo-controlled studies with
larger number of participants are needed to definitively determine the
effectiveness of this formulation to treat hair loss.
CONF LICT OF IN TE RE ST
A.F.P., K.R.M., and M.M. are employees of Hypera Pharma.
ORCID
Alessandra Anzai https://orcid.org/0000-0002-6608-7757
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