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Drug Design, Development and Therapy 2013:7 413–423
Drug Design, Development and erapy
Preparation and evaluation of a multimodal
minoxidil microemulsion versus minoxidil alone
in the treatment of androgenic alopecia of mixed
etiology: a pilot study
Farouk M Sakr
1
Ali MI Gado
2
Haseebur R Mohammed
3
Abdel Nasser Ismail Adam
4
1
Department of Pharmaceutics, College of
Pharmacy, Riyadh Colleges of Dentistry
and Pharmacy, Riyadh, Kingdom of Saudi
Arabia;
2
Department of Pharmacology and
Toxicology, College of Pharmacy, Riyadh
Colleges of Dentistry and Pharmacy, Riyadh,
Kingdom of Saudi Arabia;
3
Department
of Pharmaceutical Chemistry, College of
Pharmacy, Riyadh Colleges of Dentistry
and Pharmacy, Riyadh, Kingdom of Saudi
Arabia;
4
Department of Human Biology and
Physiology, College of Pharmacy, Riyadh
Colleges of Dentistry and Pharmacy, Riyadh,
Kingdom of Saudi Arabia
Correspondence: Farouk M Sakr
Pharmacy and Applied Medical Sciences,
Riyadh Colleges of Dentistry and
Pharmacy, PO Box 84891, Riyadh 11681,
Kingdom of Saudi Arabia
Tel +96 61 9200 0018
Email farouksakr@riyadh.edu.sa
Background: The variable success of topical minoxidil in the treatment of androgenic
alopecia has led to the hypothesis that other pathways could mediate this form of hair loss,
including infection and/or microinflammation of the hair follicles. In this study, we prepared
a multimodal microemulsion comprising minoxidil (a dihydrotestosterone antagonist),
diclofenac (a nonsteroidal anti-inflammatory agent), and tea tree oil (an anti-infective agent).
We investigated the stability and physicochemical properties of this formulation, and its
therapeutic efficacy compared with a formulation containing minoxidil alone in the treatment
of androgenic alopecia.
Methods: We developed a multimodal oil/water (o/w) microemulsion, a formulation
containing minoxidil alone, and another containing vehicle. A three-phase diagram was
constructed to obtain the optimal concentrations of the selected oil, surfactant, and cosur-
factant. Thirty-two men aged 18–30 years were randomized to apply 1 mL of microemulsion
containing the multimodal formulation (formulation A, n = 11), minoxidil alone (formulation
B, n = 11) or placebo (formulation C, n = 10) twice daily to the affected area for 32 weeks.
Efficacy was evaluated by mean hair count, thickness, and weight on the targeted area of the
scalp. Global photographs were taken, changes in the area of scalp coverage were assessed
by patients and external investigators, and the benefits and safety of the study medications
were evaluated. The physical stability of formula A was examined after a shelf storage
period of 24 months.
Results: Formulation A achieved a significantly superior response than formulations B
and C in terms of mean hair count (P , 0.001), mean hair weight (P , 0.001), and mean
hair thickness (P , 0.05). A patient self-assessment questionnaire demonstrated that the
multimodal minoxidil formulation significantly (P , 0.001) slowed hair loss, increased
hair growth, and improved appearance, and showed no appreciable side effects, such as
itching and/or inflammation of the scalp compared with the minoxidil alone and placebo
formulations. These improvements were in agreement with the photographic assessments
made by the investigators. Formula A was shown to be an o/w formulation with consistent
pH, viscosity, specific gravity, and homogeneity, and was physically stable after 24 months
of normal storage.
Conclusion: A multimodal microemulsion comprising minoxidil, diclofenac, and tea tree
oil was significantly superior to minoxidil alone and placebo in terms of stability, safety, and
efficacy, and achieved an earlier response in the treatment of androgenic alopecia compared
with minoxidil alone in this 32-week pilot study.
Keywords: androgenic alopecia, diclofenac, microemulsion, minoxidil, nonsteroidal anti-
inflammatory agents, tea tree oil
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ORIGINAL RESEARCH
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http://dx.doi.org/10.2147/DDDT.S43481
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Introduction
The success rate of treatment for androgenic alopecia barely
exceeds 30% using antihypertensive agents or modulators of
androgen metabolism, implying that other pathophysiologic
pathways may be involved in this condition.
1–4
Androgenic
alopecia is considered to be an alteration of hair growth
and/or premature aging of the pilosebaceous unit, with a
multifactorial and even polygenic etiology.
1
Various hypotheses have been put forward to explain the
causes of androgenic alopecia, of which none can be said to
account fully for the condition. Infection and microinflam-
mation of the hair follicles has been considered to contribute
to the pathogenesis of androgenic alopecia.
5–9
However, only
55% of patients with male-pattern androgenic alopecia and
microinflammation were shown to have hair regrowth in
response to treatment with minoxidil, which was less than
the 77% recorded for patients with no signs of inflamma-
tion.
3
This suggests that perifollicular inflammation may be a
contributing factor in some cases of androgenic alopecia that
do not respond to minoxidil. Inflammation may be effective
in initiating androgenic alopecia by altering local androgenic
hormone metabolism.
2
Several studies of hair follicles taken
from subjects with androgenic alopecia have reported moder-
ate lymphohistiocytic inflammation in 30% of subjects with
androgenic alopecia versus 15% of controls.
9
Localization of the inflammatory infiltrate at the level
of the upper follicle suggests that the primary causal event
triggering inflammation might occur in the vicinity of the fol-
licular infundibulum. One could speculate that colonization
of the infundibulum by microbes and/or antigens could be
involved in generation of an inflammatory response due to oxi-
dative tissue injury.
9,10
In line with this hypothesis, Piérard et al
11
found that use of topical antimicrobials may be beneficial for
the treatment of androgenic alopecia. Combinations contain-
ing minoxidil-pyrithione zinc,
12
minoxidil-ketoconazole,
13
and
minoxidil-hydrocortisone
14
have been shown to improve hair
growth compared with use of minoxidil alone.
The aim of the present investigation was to prepare and
evaluate a pharmaceutically stable and therapeutically effective
multimodal topical treatment that can address the multifactorial
etiology of androgenic alopecia, including excessive sensitiv-
ity of the hair follicle to androgen, microbial infection, and/or
microinflammation. The formula tested was a microemulsion
containing minoxidil (an antiandrogenic agent), tea tree oil (a
widely used natural antifungal/antibacterial agent),
15,16
and
diclofenac (a topical nonsteroidal anti-inflammatory agent
marked in the form of Voltaren
®
Emulgel™ 1% and Voltaren
Ophthalmic 0.1% eyedrops by Novartis, Basel, Switzerland).
Diclofenac was chosen to diminish hair follicle microinflam-
mation because it has less pronounced side effects in compari-
son with cortisone derivatives. A microemulsion formulation
was used because of its many advantages, including ease of
preparation, improved drug solubility, ability to protect drugs
with poor stability from environmental conditions, better
transdermal drug delivery, and long shelf-life.
19,20
The physical
properties of a freshly prepared multimodal microemulsion,
including pH, viscosity, refractive index, dilution test, dye
staining test, optical transparency, specific gravity, and cen-
trifugal stress, were compared with those after a shelf storage
period of 24 months. A pilot study was also performed in men
with androgenic alopecia to compare the efficacy and safety of
microemulsions containing multimodal minoxidil, minoxidil
alone, and placebo.
Materials and methods
Materials
Minoxidil USP was sourced from Alan Pharmaceuticals
(London, UK), pharmaceutical grade diclofenac sodium
from Ningbo Samreal Chemical Co, Ltd (Zhejiang, People’s
Republic of China), premium grade tea tree oil (Malaleuca
alternifolia) from Malaleuca Plantation of Bungawalbyn Pty
Limited (Coraki, NSW, Australia), and high-performance liq-
uid chromatography grade acetone and pharmaceutical grade
propylene glycol and absolute alcohol from Sigma-Aldrich
(St Louis, MO, USA). Labrasol
®
(containing caprylocaproyl
polyoxyl-8 glycerides EP) was obtained from Gattefossé
(Saint-Priest, Cedex, France).
Preparation of microemulsions
Construction of ternary phase diagram
The ternary phase diagram shown in Figure 1 was constructed
to investigate the range of concentrations needed for the
components of the microemulsions using the water titration
method and different oil/surfactant/cosurfactant ratios. The
optimum composition was 0.5% diclofenac, 5% tea tree oil,
5% minoxidil, 5% lauryl alcohol, and 35% water in combi-
nation with 55% Labrasol
®
[(surfactant)/(propylene glycol-
ethanol mixture at 1:1 as cosurfactant) at (1:1)].
The compositions of the microemulsions containing mul-
timodal minoxidil, minoxidil alone, and placebo are shown in
Table 1. The microemulsion containing multimodal minoxidil was
prepared by homogenously mixing 5% w/v of tea tree oil with 5%
w/v of lauryl alcohol, chosen for its good drug solubilizing capac-
ity. Specified amounts of minoxidil and diclofenac were dissolved
in the Labrasol
®
surfactant/propylene glycol-ethanol mixture as
a cosurfactant. Next, the two mixtures were combined by gentle
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Sakr et al
Drug Design, Development and Therapy 2013:7
stirring. A designated amount of double-distilled water was added
to the mixtures dropwise, and the multimodal microemulsion was
obtained at ambient temperature. The procedure was repeated
to prepare the microemulsions containing minoxidil alone and
placebo. The preparations obtained were individually packed in
50 mL amber brown glass bottles each equipped with a calibrated
dropper. All bottles were labeled in a blinded fashion as A (multi-
modal minoxidil), B (minoxidil alone), or C (placebo).
Characterization and physical stability of multimodal
microemulsion
Characterization of microemulsions is difficult because
of the complex structures and components involved in
these systems, as well as the limitations of the assessment
techniques that can be used, but such knowledge is essential
for their successful commercial exploitation. Therefore,
studies using a combination of complementary techniques
are usually required to obtain a comprehensive under-
standing of the physicochemical properties and structure
of microemulsions. Because this was a pilot study, the
multimodal microemulsion was characterized on the basis
of its physical properties, which can not only explain the
performance of the system but also help in predicting its
physical stability over time, prior to and after 24 months
of storage.
The multimodal microemulsions (formulation A) were
examined for basic physical stability after 24 months of
normal storage conditions and compared with the results
obtained for the freshly prepared multimodal samples.
The tests were carried out on five specimens from each
preparation and observed for phase separation, flocculation,
or precipitation. The microemulsions were centrifuged for
30 minutes at 25°C and inspected for any change in their
homogeneity after storage for 24 months under ambient
conditions and away from direct light. Chemical assays
and testing of the active ingredients are planned in further
broader investigations of the study formulation.
Staining/dye solubility tests
A few drops of the microemulsion were spread on a slide after
mixing with a diluted drop of aqueous solution containing
methylene blue as a water-soluble dye or a drop of Sudan III
solution as an oil-soluble dye, and observed under an optical
microscope. The blue color of the water-soluble dye appeared
as a continuous phase within which microscopic oil droplets
were scattered. The red color of the oil-soluble dye appeared
as dispersible red microscopic droplets in a noncolored
continuous phase, indicating that the multimodal formula-
tion was an oil/water (o/w) microemulsion. These tests were
repeated on the stored samples, and the results were similar,
indicating no phase conversion.
Transparency/translucency
The droplets of the microemulsions being smaller than
1/4th the wavelength of visible light, permit white light
to pass through the dispersed system. The microemul-
sion systems were inspected for optical transparency and
homogeneity prior to and after storage by routine obser-
vation under strong light. The formulations were also
checked for the presence of undissolved drug or other solid
ingredients.
100
90
80
70
100
% oil
% water
908070
60
50
40
30
20
10
0
Labrasol
®
+ Propylene glycol-ethanol (1:1)
% surfactant + cosurfactant
100
90
80
70
60
50
40
30
20
10
0
6050403020100
Figure 1 Ternary phase diagram of the oil, surfactant/cosurfactant mixture-water
system at a 1:1 weight ratio of Labrasol
®
+ propylene glycol-ethanol at ambient
temperature.
Note: Shaded area shows microemulsion zone.
Table 1 Composition of formulations A, B, and C
Ingredients Formulation A Formulation B Formulation C
Minoxidil 5% w/v
(50 mg/mL)
5% w/v
(50 mg/mL)
–
Diclofenac
sodium
0.5% w/v
(5 mg/mL)
– –
Tea tree oil 5% w/v
(50 mg/mL)
– –
Lauryl
alcohol
5% (50 mg/mL) 5% (50 mg/mL) 5% (50 mg/mL)
Propylene
glycol
13.75% w/v
(0.1375 mg/mL)
13.75% w/v
(0.1375 mg/mL)
13.75% w/v
(0.1375 mg/mL)
Ethanol 30% w/v
(0.30 mg/mL)
30% w/v
(0.30 mg/mL)
30% w/v
(0.3 mg/mL)
Labrasol
®
27.5% w/v 27.5% w/v 27.5% w/v
Puried
water
35–100 mL 35–100 mL 35–100 mL
Notes: w/v represents weight/volume. Formulation A, minoxidil, diclofenac and tea
tree oil; formulation B, minoxidil only; formulation C, placebo.
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Comparison of two topical minoxidil formulations
Drug Design, Development and Therapy 2013:7
Dilution testing
O/w microemulsions are homogeneous when diluted with
water, whereas a w/o microemulsion is not and undergoes
phase inversion into an o/w microemulsion. Dilution tests
were used to compare the freshly prepared A, B, and C
microemulsions with corresponding formulations that had
been stored for 24 months to determine if the microemulsion
underwent phase inversion from o/w to w/o during storage. To
do this, a few drops of the freshly prepared and stored micro-
emulsions were placed on clean glass slides. A couple of drops
of water were mixed in using a glass rod, and the transparency
or turbidity of the microemulsions were examined.
19
Centrifugation stress testing
Next, 2.5 mL samples of the freshly prepared and stored
microemulsions were aliquoted into centrifuge tubes and
centrifuged at 3000 rpm and 5000 rpm for 30 minutes at
ambient temperature to assess their physical instability,
including phase separation, phase inversion, aggregation,
creaming, and cracking.
Measurements of pH
pH changes could affect product stability and drug bio-
availability from a microemulsion at the site of permeation.
Accordingly, the pH of the freshly prepared and stored
microemulsions was determined using a microprocessor pH
meter (pH 211, Hanna Instruments, Cluj-Napoca, Romania)
calibrated using pH buffer solution. The pH of each micro-
emulsion was determined in triplicate.
20
Assessment of specic gravity
The specific gravity of the freshly prepared and stored multi-
modal samples was determined using a specific gravity bottle.
The bottle was washed, dried completely, and weighed empty
at room temperature (about 25°C). The bottle was then filled
with the microemulsion and weighed. Specific gravity (g/mL)
was calculated using the following formula:
Specific
=
(Weight of filled bottle-weight of empty bottl
ee)
Volume of bottle)
gravity
(
(1)
Measurement of viscosity
Viscosity measurements were performed using a viscometer
(Brookhaven Instruments Corporation, Holtsville, NY, USA)
operating in single mode (Spindle C50).
21
All measurements
were done in triplicate for 60 seconds at a temperature of
about 25°C.
Selection of subjects
Thirty-two men aged 25–30 years with early signs of
stage II–IV frontal or vertex pattern androgenic alopecia
based on the Norwood-Hamilton scale of baldness in men
22
were enrolled into the study. Men with stage V–VII androgenic
alopecia were excluded. All subjects were healthy Saudis or
Egyptians with fair skin and black hair who also complained
of a noticeably oily and slightly itchy scalp. Subjects who
were using hair restorers or systemically active drugs, includ-
ing steroids, nonsteroidal anti-inflammatories, cytotoxic
agents, topical antiseptic and/or antifungals, vasodilators,
antibiotics, antihypertensive agents, diuretics, or specifically
contraindicated agents (such as spironolactone, cimetidine,
diazoxide, cyclosporine, finasteride, or ketoconazole) during
the previous six months were also excluded. All patients
were informed of the likely pharmacological and therapeutic
effects of the study formulations as well as their poten-
tial adverse effects, based on available drug information.
Institutional review board approval and written informed
consent were obtained before the participants were enrolled
in the study.
Study design
This was a randomized, 32-week, placebo-controlled,
double-blind investigation of the safety and efficacy of a
multimodal emulsion containing minoxidil, diclofenac,
and tea tree oil (n = 11) versus minoxidil formulation alone
(n = 11) and a placebo control formulation (n = 10) for the
treatment of early-stage frontal and vertex male-pattern
androgenic alopecia in men.
22
The patients applied 1 mL of
each test solution at approximately 12-hour intervals to the
frontoparietal and vertex areas of the scalp for 32 weeks. All
cases were followed up and evaluated weekly by an external
investigator and by the patients themselves.
Hair sampling
Sites for hair sampling were selected from the frontal and
vertex scalp area most affected by alopecia.
23
Hair in the
designated area was hand-clipped during the initial baseline
screening procedure and at eight-week intervals thereafter,
for a total period of 32 weeks. Treatment was started immedi-
ately after the first clipping and continued for four successive
clippings at eight-week intervals.
Hair clipping technique
A 2 cm
2
rigid transparent plastic template was placed over
the chosen scalp site. The hair within the square area was
carefully pulled through, then grasped and hand-clipped
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close to the scalp with small straight surgical scissors. After
removing the template, two diagonal corners of the outlined
square were marked permanently with sterile Indian ink from
a 21-gauge needle.
23
Hair clipping was performed at eight-
week intervals, and the cut hairs were carefully placed on
collection paper. Before removing the template, the clipped
area was inspected with a magnifying lens for any missed or
loose hairs that should be included.
Application of treatment
Following hair clipping, the subjects were provided with the
test solutions in a randomized manner. The assigned medi-
cations were provided in identically appearing prelabeled
bottles. The subjects were instructed to use a calibrated
dropper for application of 1 mL of each test microemulsion
twice daily to the frontoparietal or vertex area of the scalp,
starting at the clipped site. The test products were applied
to a dry scalp and spread with a finger tip, and the hair was
allowed to dry naturally without a hair dryer.
Assessment
Numerous methods are available for assessment of hair
variables, ie, density, growth rate, and mean hair weight,
count, and thickness. These techniques can be classified as
invasive (eg, biopsy), semi-invasive (trichogram, unit area
trichogram), or noninvasive (eg, global hair count, global
photographic assessment, and phototrichogram).
The phototrichogram
24
is a simple, reproducible, and
sensitive noninvasive technique that is used to determine
the rate of hair growth, size of hair fibers, and propor-
tion of hair follicles in telogen phase, and to quantify the
amount of hair shed. All hairs in a 2 cm
2
area are trimmed
1 mm from the skin surface and a baseline photograph is
taken. After a week, the same region is photographed and
the hairs are trimmed again. This process is repeated until
an adequate number of photographs are available for com-
parison. Comparison with baseline photographs identifies
hair fibers that have grown (follicles in anagen) and those
that have not (follicles in telogen), rate of hair growth over
seven days, hair density (number of hairs counted in the
photograph), and which hair fibers are missing five days
later (hair shedding).
In 2001, Hoffmann developed a commercial fully
computerized phototrichogram technique using automated
software
25
whereby a 1.8 cm
2
area of hair loss showing a
transition between normal hair growth and a balding area
for men with androgenic alopecia and an area in the mid
vertex for women with diffuse hair loss is chosen for clip-
ping. The area on the scalp is marked with a central black
tattoo. Clipped hairs within the target area are dyed and
photographs are taken immediately afterwards and then
2–3 days after shaving using a digital closeup camera
with epiluminescence microscopy. Two photographs are
then compared using a software system that can recognize
Check appropriate box below
Strongly agree
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2
3
4
5
5
6
7
5
5
5
5
6
7
4
4
4
4
4
4
3
3
3
3
3
3
2
2
2
2
2
2
1
1
1
1
1
1
Disagree
Strongly disagree
Agree
No opinion either way
A lot better
A lot worse
Somewhat worse
Somewhat better
A little better
A little worse
Same
Greatly increased
Moderately increased
Slightly increased
Slightly decreased
Moderately decreased
Greatly decreased
No change
Very effective
Not very effective
Not effective at all
Somewhat effective
I am very satisfied
I am satisfied
I am neutral
I am dissatisfied
I am very dissatisfied
a) the hair line at the front of your head ?
b) the hair on top of your head ?
c) your hair overall ?
1. Since the start of the study, I can see my bald spot getting smaller
2. Because of the treatment I have received since the start of the study, the appearance of my hair is
3. Since the start of the study, how would you describe the growth of your hair?
4. Since the start of the study, how effective do you think the treatment has been in slowing
down your hair loss?
5. Compared to the start of the study, which statement best describes your satisfaction
with the appearance of:
Figure 2 Patient self-assessment questionnaire concerning changes in their scalp hair after treatment.
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Comparison of two topical minoxidil formulations
Drug Design, Development and Therapy 2013:7
images of individual hair fibers. By comparing the two
photographs, the computer can determine which hairs
are growing and which are not. Because the results are
reproducible, this software can be used to monitor the
success of treatment. However, in this pilot study, we used
previously reported noninvasive techniques,
4,23
including
determination of mean hair weight, hair count, and hair
thickness, as well as a global photographic method.
Determination of mean hair weight, count,
and thickness
The hair samples were degreased and then weighed in high-
performance liquid chromatography grade acetone for at least
one hour. When completely dry, the hair was transferred to
a light aluminum weighing pan with a 5 digits readability
balance. The relative humidity of the balance chamber was
measured using a digital hygrometer calibrated at 20% and
90% relative humidity using saturated solutions of potassium
acetate and zinc sulfate, respectively. The relative humidity
of the balance chamber was recorded at 65% after condition-
ing for two hours, and the hair samples were then weighed.
The entire hair samples were then individually laid out for
counting in groups of ten on a marked grid using a wide-field
zone projection microscope.
23
After counting, subsamples
containing 25 hairs each were measured for thickness. For
convenience and reproducibility, only two subsamples were
taken from each subject at baseline and after 32 weeks of
treatment. The hair subsamples were mounted on large slides
and covered with a piece of thin plastic wrap film to enable
simple visual observation. Hair thickness was measured at
the midpoint of each individual hair by tracking the projected
image with a 10 × reticle, and dividing by a lens magnifica-
tion of 71.2.
23
Patient self-assessment
Patients assessed their scalp hair using a validated, self-
administered hair growth questionnaire
26
containing four
questions on treatment efficacy and three questions on
satisfaction with appearance (Figure 2). Mean ranking of
responses by patients treated with the different microemul-
sions and their significance were assessed using a computer-
ized Kruskal–Wallis test accounting for different weighting
of answers and covariance between the questions.
Investigator assessment
Investigators assessed the participants at all time points
using a standardized seven-point rating scale of hair growth
compared with baseline (–3, greatly decreased; –2, moder-
ately decreased; –1, slightly decreased; 0, no change; +1,
slightly increased; +2, moderately increased; +3, greatly
increased). Baseline global photographs for each patient
were given to the investigator for reference.
24
Global photographic assessment
Standardized global color photographs of the frontal and
vertex scalp were taken. Paired baseline and post-treatment
slides were examined independently by two investigators
using the standardized seven-point rating scale.
26
This tech-
nique has previously been demonstrated to have excellent
test-retest reproducibility.
27
Statistical analysis
Statistical analysis of the results for hair count, responses to
the individual patient self-assessment questionnaire, and the
investigator and global photographic assessments were done
using analysis of variance.
Results
The ternary phase diagram shown in Figure 1 indicates that
the composition and ratios of the surfactant, cosurfactant,
and water used for preparation of the microemulsions
existing in the designated region of the microemulsion zone.
The compositions chosen for microemulsions A, B, and C
(Table 1) were identical, except for the number of active
ingredients in each formulation.
Physical properties and stability
The results in Table 2 shows the physical properties, includ-
ing appearance, color, odor, viscosity, water dilution, optical
transparency, dye staining tests, specific gravity, pH and
centrifugal stress tests, for the freshly prepared multimodal
60.00
Mean thickness of the hair (µm)
40.00
20.00
0.00
Control Multimodal formula Minoxidil
Before
After
Figure 3 Comparison of mean hair thickness (µm) at baseline and after 32 weeks
of treatment.
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Sakr et al
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period increased from baseline to 32 weeks, the hair count
increased progressively in the group treated with the mul-
timodal emulsion and to a greater extent than in the group
treated with the microemulsion containing minoxidil alone,
while the placebo group showed a marked decrease in hair
count. The last column in Table 3 shows that the effect of
the multimodal emulsion on hair count is highly significant
compared with that of the minoxidil and placebo formulations
(P = 0.009 and P , 0.001, respectively).
Table 4 shows the effects of emulsions A, B, and C on
mean hair weight, and are in agreement with those seen in
Table 3, ie, during the 32-week treatment period, there was a
progressive increase in hair weight, which was significantly
better for the multimodal minoxidil microemulsion than
for the formulations containing minoxidil alone or placebo
(P , 0.001).
Table 5 shows the statistical analysis of results for the
patient evaluation questionnaire. The multimodal micro-
emulsion containing minoxidil showed the best rank values,
followed by the minoxidil only and placebo microemulsions,
with highly statistically significant treatment differences
(P = 0.000). Table 6 shows the percentages of patients with
improvement in hair growth on the scalp as assessed by
the investigators and global photographic evaluation. The
multimodal treatment microemulsion showed a 75%–79%
increase in hair growth versus 37%–41% for minoxidil alone
and 13%–16% for placebo.
Figure 4 shows representative photographs for
subjects with vertex alopecia treated using the multimodal
minoxidil microemulsion at baseline (Figure 4A),
indicating a moderate increase in hair growth at
week 8 (Figure 4B) and greatly increased hair growth at
week 16 (Figure 4C). Figure 5 shows similarly representative
photographs for subjects with frontal alopecia treated using
the multimodal minoxidil microemulsion at baseline
(Figure 5A), indicating a moderate increase in hair growth
at week 8 (Figure 5B) and markedly increased hair growth
at week 16 (Figure 5C). Figure 6 shows representative
Table 2 Results of stability testing for formulation A containing
minoxidil, diclofenac, and tea tree oil at baseline and after
24 months of storage
Test Initial product After 24 months of
storage
Appearance Clear and transparent Clear and transparent
Color Conforms
Odor Characteristically
aromatic
Conforms
Specic gravity
1.21 ± 0.015 g/mL 1.25 ± 0.01 g/mL
pH
5.7 ± 0.01 5.9 ± 0.025
Water dilution test Clear/transparent o/w Clear/transparent o/w
Staining tests Conrm o/w emulsion Conrm o/w emulsion
with no phase inversion
Optical
transparency
Transparent/
translucent/clear
Transparent/
translucent/clear
Mean viscosity (PaS)
0.79 ± 0.012 0.81 ± 0.071
Centrifugation
at 3000 rpm
No phase separation,
turbidity, cracking,
or creaming (stable)
No phase separation,
turbidity, cracking, or
creaming (stable)
Centrifugation
at 5000 rpm
No phase separation,
turbidity, cracking,
or creaming (stable)
No phase separation,
turbidity, cracking, or
creaming (stable)
microemulsion and the corresponding values after 24 months
of storage. It is clear that the multimodal formulation is an
o/w emulsion and that both the freshly prepared and stored
formulations did not show any appreciable differences in
physical properties, indicating good stability.
Hair growth
A summary of the results for hair growth measured on four
occasions at eight-week intervals is provided in Figures 3–7
and Tables 3–6. Figure 3 compares the mean ± standard
deviation thickness (µm) of hair at baseline and after 32
weeks of treatment with formulation A, B, or C. The dif-
ferences in column height before and after treatment were
in the order of multimodal microemulsion . minoxidil .
placebo.
The effect of microemulsions A, B, and C on mean hair
count is shown in Table 3, indicating that, as the treatment
Figure 4 Representative photographs of a subject with vertex alopecia treated with the multimodal minoxidil microemulsion. (A) Baseline, (B) at week 8 (moderately
increased hair growth), and (C) week 16 (markedly increased hair growth).
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Comparison of two topical minoxidil formulations
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Figure 5 Representative photographs of a subject with frontal alopecia treated with the multimodal minoxidil microemulsion. (A) Baseline, (B) at week 8 (moderately
increased hair growth), and (C) week 16 (markedly increased hair growth).
Figure 6 Representative photographs of a subject with frontal alopecia treated with minoxidil alone. (A) Baseline, (B) at week 8 (slightly increased hair growth), and
(C) week 16 (moderately increased hair growth).
Figure 7 Representative photographs of a subject with frontal alopecia treated with placebo. (A) Baseline, (B) at week 8 (slight decrease in hair growth), and (C) week 16
(moderate decrease in hair growth).
Table 4 Statistical evaluation of effects of formulations A, B, and C on mean hair weight
Hair weight (mg, mean ± standard deviation)
F P-value
Formulation A Formulation B Formulation C
Baseline
8.06 ± 1.09 7.80 ± 0.53 7.37 ± 0.76
1.765 0.190
8 weeks
9.24 ± 1.46
a
8.40 ± 0.71
a
6.95 ± 0.75
a
12.554 0.000***
16 weeks
10.62 ± 1.59
b
9.13 ± 0.95
b
6.48 ± 0.60
a
34.918 0.000***
24 weeks
11.83 ± 1.74
b
9.77 ± 0.94
c
5.99 ± 0.52
a
62.755 0.000***
32 weeks
12.30 ± 1.76
b
10.32 ± 1.02
c
5.34 ± 0.41
a
89.650 0.000***
Notes: F and P-value calculated using one-way analysis of variance; ***denotes difference signicant at P , 0.001.
a,b,c
denote signicant difference at P , 0.05 calculated using
the Scheffe’s post hoc test.
Table 3 Statistical evaluation of effects of formulations A, B, and C on mean hair count
Hair count (mean ± standard deviation)
F P-value
Formulation A Formulation B Formulation C
Baseline
133.6 ± 26
a
122.3 ± 14.9
a
139.8 ± 23.9
a
1.597 0.221
8 weeks
157.2 ± 29.9
b
136.1 ± 11.4
a,b
124.2 ± 23.1
a
5.599 0.009**
16 weeks
157.8 ± 29.9
b
136.1 ± 11.3
c
111.1 ± 21.5
a
23.333 0.000***
24 weeks
201.4 ± 28.3
b
159.4 ± 12.4
c
100.0 ± 16.7
a
63.146 0.000***
32 weeks
217.3 ± 27.9
b
170.5 ± 11.9
c
82.9 ± 13.5
a
126.165 0.000***
Notes: F and P-value calculated using one-way analysis of variance; **difference signicant at P , 0.01; ***difference signicant at P , 0.001.
a,b,c
denote signicance of
difference at P , 0.05 calculated by Scheffe’s post hoc test.
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Table 6 Percentages of patients with improvement in scalp hair as per the investigator’s evaluation and global photographic
assessment
Multimodal Minoxidil Placebo Difference Difference
Formulation A Formulation B Formulation C A versus C B versus C
Investigator assessment
Increased hair growth 75% 37% 16% 59 (43, 76) 21 (17, 30)
Photographic assessment
Increased hair growth 79% 41% 13% 66 (63, 84) 28 (21, 48)
Table 5 Statistical evaluation
a,b
of the mean ranks between the three treatment groups based on patient evaluation questionnaire
Questions Placebo (mean rank) Minoxidil (mean rank) Multimodal (mean rank) DF Chi square P-value
Patient self-assessment
Q1 27.05 14.00 9.41 2 22.237 0.000***
Q2 27.10 16.23 7.14 2 25.065 0.000***
Q3 26.90 17.09 6.45 2 26.027 0.000***
Q4 27.30 16.27 6.91 2 26.899 0.000***
Q5 27.50 16.09 6.91 2 26.513 0.000***
Q6 28.54 17.45 7.06 2 27.55 0.000***
Q7 28.65 17.99 7.46 2 27.85 0.000***
Notes:
a
Kruskal–Wallis test;
b
grouping variable; ***denotes difference signicant at P , 0.001; Q1, size of bald spots; Q2, appearance of hair; Q3, growth of hair; Q4, slowing
hair loss; Q5, satisfaction with hair line at front of head; Q6, satisfaction with hair on top of head; Q7, hair overall.
Abbreviation: DF, degrees of freedom.
photographs for subjects with frontal alopecia treated
with the microemulsion containing minoxidil alone at
baseline (Figure 6A), indicating a slight increase in hair
growth at week 8 (Figure 6B) and a moderate increase
in hair growth at week 16 (Figure 6C). Figure 7 shows
representative photographs of placebo-treated subjects
with frontal alopecia at baseline (Figure 7A), indicating
a slight decrease in hair growth at week 8 (Figure 7B)
and a moderate decrease in hair growth at week 32
(Figure 7C).
Discussion
Physical and stability tests
for microemulsion A
The results of physical testing of the freshly prepared and
stored multimodal minoxidil microemulsions were similar,
with no marked changes seen. The microemulsions remained
translucent and visually clear. Dilatability with aqueous
phase and dye staining tests showed that the products were
o/w microemulsions with no sign of phase inversion. Physical
stability tests were done 10 and 30 days after preparation and
again after 24 months of storage to detect any appreciable
changes in consistency, color, odor, pH, viscosity, specific
gravity, and centrifugal stress. The stored samples are found
to be physically stable, with no signs of creaming, cracking,
or phase separation.
Hair growth
Mean hair count
Table 3 shows a steady increase in mean hair count in
the group treated with the multimodal minoxidil micro-
emulsion which was more pronounced from baseline
onwards throughout the study than in the group treated
with the microemulsion containing minoxidil only, which
in turn was more pronounced than in the placebo group.
One-way analysis of variance showed no significant
difference in mean hair count between the three treat-
ment groups at baseline (P . 0.2). Improvement in hair
count with the multimodal minoxidil microemulsion was
significantly greater than with minoxidil alone or placebo
throughout the treatment period (P , 0.01 and P , 0.001,
respectively).
Mean hair weight
The mean hair weight results shown in Table 4 are consistent
with those for mean hair count in Table 3. Mean hair weight
for the multimodal minoxidil microemulsion was greater
from baseline onwards compared with that for the minoxidil
only and placebo formulations. One-way analysis of vari-
ance showed no statistically significant difference between
the three treatment formulations at baseline (P . 0.19).
However, improvement in mean hair weight for the mul-
timodal minoxidil formulation was significantly greater
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Comparison of two topical minoxidil formulations
Drug Design, Development and Therapy 2013:7
(P , 0.001) than that from the minoxidil only and placebo
formulations throughout the study.
Mean hair thickness
Figure 3 shows mean hair thickness in the three treatment
groups at baseline and at the end of the 32-week treatment
period. Variation in hair thickness measurements taken at
eight-week intervals was found to be erratic, irreproducible,
and too time-consuming a procedure for comparison between
the different treatment groups. There was no appreciable
change in column height in the placebo group before and
after treatment. However, statistically significant differences
in mean hair thickness were found between baseline and the
end of treatment using the multimodal minoxidil microemul-
sion (P , 0.05, paired t-test). These findings are in agreement
with the abovementioned evaluation results and are indicative
of the superiority of the multimodal minoxidil formulation
versus the minoxidil only and placebo formulations.
Patient self-assessment
Statistical evaluations of the mean ranks between the treat-
ment groups for the patient self-evaluation questionnaire
are presented in Table 5, and the results for responses to the
patient self-assessment questionnaire are shown in Table 6.
The results indicate that the multimodal minoxidil formula-
tion was significantly superior (P , 0.001) according to the
patients, and had an earlier onset of action in comparison
with the minoxidil only and placebo formulations. Patients
treated with the multimodal minoxidil formulation reported
alleviation of scalp itching, reduced sebum production, and
a pronounced reduction in amount of hair shedding during
the first week of treatment compared with the subjects treated
with minoxidil only, who reported a delayed effect, starting
after 4–5 weeks of treatment. Treatment with the placebo
formulation resulted in significant hair shedding.
Investigator and photographic assessments
Figures 4 and 5 show representative photographs of subjects
with vertex and frontal alopecia, respectively, treated with
the multimodal minoxidil microemulsion at baseline and
after 8 and 16 weeks of treatment. These were rated as mod-
erately improved at week 16 and greatly improved at week
32. Figure 5 shows representative photographs for subjects
treated with the minoxidil only formulation, which were rated
as slightly improved at week 16 and moderately improved
at week 32. Figure 6 shows a representative example from
the placebo-treated group, which was rated as showing a
slight and moderate decrease in hair growth at weeks 16
and 32, respectively. The results for patient self-assessment
shown in Table 5 are further confirmed by the results of
investigator and photographic assessment shown in Table 6,
indicating a 75%, 37%, and 16% improvement in scalp
hair growth according to investigator assessment in the
multimodal minoxidil, minoxidil only, and placebo groups,
respectively, and a 79%, 41%, and 13% improvement accord-
ing to photographic assessment, all favoring the multimodal
minoxidil microemulsion.
Both the investigator assessment and patient reports
indicated that signs and symptoms of contact dermatitis
(ie, stinging, burning, itching, dryness, scaling) were more
prevalent in the group treated with the 5% minoxidil solution
(seven of 11 patients, about 64%) than in the placebo group
(four of 10 patients, about 40%). In contrast, symptoms in
the group treated with the multimodal minoxidil formula-
tion were less prevalent (one of 11 patients, about 9%).
These results are indicative of better tolerability and safety
using the multimodal minoxidil formulation in comparison
with the minoxidil only and placebo options. The superior
tolerability of the multimodal minoxidil formulation could
be attributed to the inclusion of ibuprofen, a nonsteroidal
anti-inflammatory agent, that ameliorates inflammation of
both the scalp and hair follicles. Reduced inflammation
could minimize sebum production, which provides a good
nutrition medium for bacterial and fungal infections. Inclu-
sion of tea tree oil has the additional advantage of helping to
eliminate bacterial and fungal organisms that could modify
androgen metabolism and lead to hair fall. It appears that
the multimodal formulation further potentiates the effect of
minoxidil in increasing hair density, improving hair growth,
and reducing inflammation and risk of bacterial infection.
Conclusion
All evaluations showed highly significant improvements using
the multimodal minoxidil microemulsion in comparison with
the minoxidil only formulation which, in turn, was signifi-
cantly more effective than placebo. The pronounced effects
of the multimodal minoxidil formulation compared with
minoxidil alone could be attributed to the anti-inflammatory
effect of diclofenac in reducing scalp and follicular micro-
inflammation and the tea tree oil in resolving any existing
microbial or fungal colonization of the hair follicles. These
results indicate that androgenic alopecia is multifactorial and
perhaps polygenic in nature. Hence, an effective multimodal
microemulsion comprising minoxidil, an anti-inflammatory
agent, and an anti-infective agent is recommended as being
more promising than minoxidil alone. Diclofenac was used
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Drug Design, Development and Therapy 2013:7
in the multimodal formulation instead of a cortisone deriva-
tive to reduce the risk of side effects, and tea tree oil was
used on the basis that it is a highly effective natural anti-
infective agent.
15,16
Further study is planned in a larger patient
population for an extended period to monitor the long-term
effects in individuals with androgenic alopecia accompanied
by hair follicle infection and/or microinflammation.
Acknowledgments
The authors thank Abdullah Al Shammery from the Riyadh
Colleges of Dentistry and Pharmacy for providing the
encouragement and support that made this work possible. They
are also grateful to H Mosadomi from the Research Center,
and to Sharat Pani and other colleagues for their constructive
observations and suggestions throughout this investigation.
Disclosure
The authors report no conflicts of interest in this work.
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