Dulbecco’s modified Eagle’s medium (DMEM), keratinocyte serum-free medium, fetal bovine serum (FBS), penicillin-streptomycin, and phosphate-buffered saline (PBS) were obtained from Gibco BRL (Grand Island, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, Mo., USA). Trichoxidil^TM, Trichosol^TM, and minoxidil solution were supplied by Fagron^TM (Brazil). The purity and quality of the raw materials used, as well as the formulation of Trichoxidil^TM and Trichosol^TM were monitored by the Fagron^TM Brazil quality control department.

The CCD1072Sk cell line was obtained from Rio de Janeiro Cell Bank (CCD1072Sk - ATCC CRL2088). The cells were cultured in a monolayer using IMDM (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 UI/mL penicillin/streptomycin, and 0.25 μg/mL Fungizone (Gibco) in a humidified atmosphere at 37˚C in 5% CO₂. These cells were trypsinized three times per week using 0.25% trypsin/EDTA (Cultilab, Brazil). For the 24-hour cell viability assessment, the control and treated cells were centrifuged and resuspended in equal parts medium and trypan blue (0.05% solution) and counted using a hemocytometer. To evaluate the cytotoxicity of Trichoxidil^TM, the essential oil was dissolved in the culture medium in appropriate concentrations. The cell viability of control and Trichoxidil^TM (0.05% - 5.0%)-treated fibroblasts cells were measured using a standard MTT assay. Briefly, 5 × 10⁴ viable cells were seeded into clear 96-well flat-bottom plates (Corning) in IMDM medium supplemented with 10% fetal bovine serum (FBS) and incubated with different concentrations of the extract for 24 h. Then, 10 μL/well of MTT (5 mg/mL) was added and the cells were incubated for 4 h. Following incubation, 100 μL of 10% sodium dodecyl sulfate (SDS) solution in deionized water was added to each well and left overnight. The absorbance was measured at 595 nm using a FlexStation 3 Multi-Mode Benchtop Reader (Molecular Devices, Sunnyvale, CA, USA).

The effect of Trichoxidil^TM on the expression of mRNA of KGF; IGF-1; and VEGF in fibroblasts was evaluated by RT-qPCR. Cells were treated with Trichoxidil^TM (2.5% and 5.0%) for 24 hours. Total RNA extracted from cells samples was converted to cDNA using a SuperScript® III RT kit (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. The concentration of RNA was detected using a NanoDrop 2000 (Thermo Fisher Scientific, Inc.). GAPDH was used as the internal control. The thermocycling conditions were as follows: 95˚C for 10 min followed by 36 cycles of 94˚C for 15 sec and 57˚C for 40 sec. The 2-ΔΔCq method was used to quantify the relative gene expression levels of the target genes. Relative standard curves were generated by serial dilutions and all samples were run in triplicates. Following, the sequence of primers used in the qRT-PCR analysis: primer forward (5'-AGAGACCCTTTGCGGGGCTGA-3') and reverse primer (5'-CTTCTGAGTCTTGGGCATGT-3') for IGF-1; primer forward (5'-ATCAGGACAGTGGCAGTTGGA-3') and reverse primer (5'-AAC ATTTCCCCTCCGTTGTGT-3') for KGF, primer forward (5'-CTTTAGAGATC AGCCCAACC-3') and reverse primer (5'-CTACCCAGAGGGAAGAAATAAC-3') for VEGF; and primer forward (5'-GGAAGGTGAAGGTCGGAGTC-3') and reverse primer (5'-CTCAGCCTTGACGGTGCCATG-3') for GAPDH.

This study was carried out after approval by the local Ethics Committee and written consent was obtained from each subject (3.150.582 - approbation number). After obtaining informed written consent, all the patients were subjected to a detailed medical history followed by thorough general physical, dermatological and systemic examinations. Both the image acquisition by the Trichoscan equipment and the cylindrical fragments, 33 volunteers, diagnosed with androgenetic alopecia, men and women, aged between 25 and 50 years, were evaluated. Exclusion criteria were: 1) volunteers who were not diagnosed with AGA; and 2) volunteers who were not in the age range (25 and 50 years). The subjects were divided into four groups and assigned the drugs in a randomized manner. Control group, without treatment (n = 5); Trichosol^TM vehicle group, without active (n = 5); Hydroalcoholic vehicle group, without active (n = 4); Trichosol^TM vehicle group, with 2.5% minoxidil (n = 5); Hydroalcoholic vehicle group, with 2.5% minoxidil (n = 5); Trichosol^TM group with 2.5% Trichoxidil^TM (n = 5) and Hydroalcoholic vehicle group with 2.5% Trichoxidil^TM (n = 4). They were instructed to apply the solution to the balding area with a calibrated dropper twice daily at 12-hour intervals. The volunteers were evaluated in 2 phases throughout the study. Time zero (T = 0), the first evaluation and time ninety (T = 90), the second evaluation, after 90 days of treatment with the formulations developed for the study. In the evaluation, T = 0, data and images were obtained using the Trichoscan, followed by the collection of the cylindrical fragment for extraction of the total mRNA and histological analysis. The same procedure was repeated after 90 days of treatment (T = 90).

The FotoFinder Trichoscale softwear was used to evaluate the parameters of the hair growth phases, as the anagen and telogen phases. All patients were assessed and subjected to photographic records with a 10x magnification dermatoscope and a digital camera with 20x and 40x magnification on the small area of the shaved headscalp. The dermoscopy findings, for example, the numerical data report, as well as the photos generated by the equipment, were stored for statistical analysis and compared between the groups evaluated.

Cylindrical scalp fragments were obtained on the vertex region. After trimming 1.0 cm² of hair, 4-mm punch biopsies were taken at T = 0 and T = 90. The punches were obtained parallel to the direction of hair growth. For follicular peribulbar evaluation and molecular biology analysis, scalp fragments were fixed for four h to 48 h, using 10% buffered formalin, and embedded in pure paraffin. To histologic analysis, the scalp fragments were sectioned and stained with hematoxylin and eosin. The following parameters were evaluated: total follicles, vellus follicles, terminal follicle and fibrous tract. The presence of peribulbar changes (i.e. inflammation) were assessed by a dermatologist. Part of the scalp fragments were used to extract total RNA followed by the quantification of mRNA of KGF; IGF-1; and VEGF, as described above in the in vitro tests. All tissues were stored at −80˚C until the RNA extraction procedure.

The obtained results were expressed as the mean ± standard error of mean (SEM) from at least three independent experiments, unless stated otherwise. Paired data was evaluated by Student’s t-test. One-way analysis of variance (ANOVA) was used for multiple comparisons. A p value of <0.05 was considered significant.

Figure 1 shows the different concentrations of Trichoxidil^TM, which were tested for cell proliferation on fibroblast cell line. The highest concentrations tested induce cell proliferation (*p < 0.01).

To investigate whether Trichoxidil^TM regulates KGF, IGF-1, and VEGF expression at transcriptional level, RT-PCR was performed. As shown in Figure 2, Trichoxidil^TM significantly increased the expression of KGF, IGF-1, and VEGF mRNA levels in fibroblasts cells. Trichoxidil^TM was superior to the normalized control (arbitrary unit 1.0). It was also shown to be superior to minoxidil, considering the levels of KGF and IGF-1 mRNA (a p < 0.05) and (#p < 0.05), respectively. In addition, Figure 2 shows the dose-response effect of both Trichoxidil^TM (C) and minoxidil (D) on the mRNA levels of the growth factors evaluated.

The results obtained at times T = 0 and T = 90 days are shown in Table 1. Anagen and telogen follicles, follicular units and capillary density were evaluated. Both Trichoxidil^TM and minoxidil increased all parameters evaluated. Analysis of the capillary density showed that Trichoxidil^TM associated with Trichosol^TM vehicle, was the most effective association (Table 1). Figure 3(A) shows that the formulations containing Trichoxidil^TM and minoxidil, which used Trichosol^TM as vehicle, significantly increased the percentage of follicular units (*p < 0.05). Figure 3(B) shows that Trichoxidil^TM formulated with Trichosol^TM increased more effectively the percentage of anagen phase and reduction of the telogen when compared to other formulations.

Total follicles, vellus follicles, terminal follicles and fibrous tract were evaluated, shown in Table 2. Figure 4(A) shows the percentage of total follicles in volunteers who used Trichoxidil^TM or minoxidil formulations. Total follicles percentage was higher in volunteers treated with Trichoxidil^TM associated with Trichosol^TM,

when compared to other formulations (*p < 0.05). Trichoxidil^TM also significantly reduced (*p < 0.05) the percentage of vellus follicles when compared to minoxidil, regardless of the vehicles used in the formulations (Figure 4(B)). The percentage increase in terminal follicles was significant (*p < 0.05) in the volunteers who used the formulation of Trichoxidil^TM in Trichosol^TM (Figure 4(C)). Figures 5(A)-(G) shows histological sections of anagen terminal follicles (capillary channel diameter greater than the inner root sheath), without signs of apoptosis in the outer root sheath. Finally, the results described in Table 2 showed that the volunteers who received formulations with Trichoxidil^TM, both in Trichosol^TM vehicle and hydroalcoholic vehicle, showed a significant reduction (*p < 0.05) in the number of fibrous tracts.

We also investigated the effects of Trichoxidil^TM on the mRNA levels of growth factors KGF, IGF-1, and VEGF in scalp fragments. The expression of growth factors at mRNAs levels increased after treatment with Trichoxidil^TM and minoxidil formulations. Regarding IGF-1, the results shown in Figure 6(A), indicate that both Trichoxidil^TM and minoxidil increased the mRNA levels. Although

formulations with Trichoxidil^TM showed an increase, there was no statistical significance. Figure 6(B) indicates that Trichoxidil^TM independent of the vehicle, significantly increased the expression of KGF when compared to other formulations, including minoxidil formulations (*p < 0.05). Finally, Figure 6(C) shows that formulations containing minoxidil significantly increased the expression of VEGF mRNA levels compared to the other groups (*p < 0.05).