Efficacy of the association of topical minoxidil and topical finasteride compared to their use in monotherapy in men with androgenetic alopecia: A prospective, randomized, controlled, assessor blinded, 3‐arm, pilot trial

Topical minoxidil (MNX) 2%–5% and oral finasteride (F) 1 mg/day are the only two pharmacological treatments authorized for androgenetic alopecia (AGA). Recently, a 2.2 mg/mL topical formulation of F was developed to minimize the systemic adverse effects associated with the oral formula. MNX and F act through different mechanisms; therefore, their association could improve clinical efficacy. To evaluate the efficacy of the association of 5% MNX and 0.25% topical F compared to their use in monotherapy, a 6‐month, prospective, randomized, assessor‐blinded trial was conducted.


| INTRODUC TI ON
Androgenetic alopecia (AGA) is a hair disorder with a multifactorial pathogenesis.Dihydrotestosterone (DHT) has a crucial role. 1 Men predisposed to AGA show an increased conversion of testosterone into DHT that induces miniaturization of hair follicles, determining the transformation of terminal hair into vellus hair. 2 The incidence of AGA increases with age.Up to 30% of 30 year-old men and up to 50% of 50 year-old men could have AGA. 3 Minoxidil (MNX) and finasteride (F) are the only two drugs authorized for AGA treatment.
Products containing 2%-5% minoxidil are available in the market.
Minoxidil solutions generally contain propylene glycol (PG), useful for the solubilization and penetration of MNX.However, PG frequently induces local irritation 4 : For this reason, a PG-free MNX formulation was developed and is commercially available. 5MNX acts as an arteriolar vasodilatation that opens potassium channels, playing a key role in cell proliferation. 4In addition, it is able to prolong the anagen phase in the dermal papilla by inducing β-catenin activity. 6MNX affects follicular cells by enhancing hair growth and reducing hair loss. 70][11][12][13][14] Although F is generally well-tolerated, it can determine some sexual adverse effects related to the inhibition of 5α-reductase and increase the risk of depression. 9To minimize these potential systemic effects, a topical finasteride formulation was recently developed and registered.A phase III study confirmed the efficacy and safety of 0.25% topical finasteride, which showed a similar effect to 1 mg /day oral finasteride. 9MNX and F act through two different mechanisms; therefore, their association could improve the efficacy of treatment compared to their respective use in monotherapy.Considering the potential systemic effects of oral finasteride, the efficacy of the combination of topical minoxidil and finasteride was evaluated.Previous studies showed that the combined use of oral F and topical MNX showed better therapeutic results compared to the application of single treatments alone during 1-year follow-up. 15,16e combination of 0.1% topical finasteride with 3%-5% minoxidil showed greater efficacy compared to MNX alone. 17,18However, the difference in hair count did not reach statistical significance. 17other study observed that the combined topical treatment of 0.25% finasteride and 3% minoxidil in an ethanol/propylene glycol/ water base promoted significantly greater hair growth in males with AGA as compared to 3% MNX alone. 19

| S TUDY AIM
Although several studies have evaluated the efficacy of different concentrations of combined topical MNX and F, this study aims to evaluate and compare the efficacy and safety of the combination of 5% PG-free MNX and 0.25% topical F (concentration of 2.275 mg/ mL), applied in different times of the day (MNX in the morning and topical F in the evening), compared to their use in monotherapy in subjects with AGA.

| Population and Study design
Between February 2021 and December 2022, 75 AGA subjects were assessed and screened for inclusion in the trial.Thirty-three subjects were excluded for different reasons (not meeting the inclusion/exclusion criteria or declining to participate).The study was designed as a 6-month, prospective, randomized, assessorblinded trial in men with AGA.The trial took place in a Dermatology clinic in Italy between February 2021 and December 2022.
The study protocol was approved by the Investigational Review Board.The trial was conducted according to Good Clinical Practice Guidelines and Helsinki Declaration.All subjects provided signed informed consent.Forty-two males (mean age 24 ± 3 years), with AGA (I-VII Hamilton-Norwood grading scale), were enrolled.The main inclusion criteria were sex (male), age >18 years, AGA (I-VII), compared to group B (p < 0.001) and group C (p < 0.05) both at 3 and 6 months (2.0 ± 0.7 vs. 0.6 ± 0.8 and 1.3 ± 0.6; respectively).A significantly greater percentage of subjects in Group A achieved a GPAS score of ≥2 in comparison with Groups B and C both after 3 and 6 months (79% vs. 8% and 41%, respectively).No significant differences were observed in mean hair diameter and hormonal levels between the three groups.Good tolerability was observed in all treated groups.

Conclusion:
The association of 5% MNX lotion and 0.25% F in spray formulation in patients with AGA showed a significantly higher clinical and instrumental efficacy compared to the monotherapies, with comparable tolerability and safety profile.

| Study outcomes
The efficacy of treatments was assessed at each visit.The primary efficacy endpoint was evaluated by a trichoscopic evaluation (Medicam 1000) with Trichoscale (hair count, hair density, vellus rate count, terminal rate count, hair thickness, analysis area: 0.9 cm 2 ).The secondary efficacy endpoint was the global photographic assessment score (GPAS), a 7-point score from −3 (severe worsening) to +3 (excellent improvement), that evaluates the AGA severity by serial digital camera photography.Both trichoscopic and photographic evaluations were performed by an investigator unaware of treatment allocation.A secondary efficacy outcome was the percentage distribution of subjects achieving a GPAS of ≥2 at months 3 and 6.In addition, at baseline and after 3 months of treatment the serum level of follicle-stimulating hormone (FSH), luteinizing hormone (LH), DHEA-S, and testosterone were evaluated.
The tolerability and cosmetic acceptability of products were evaluated by patients after 6 months through a self-administered questionnaire based on different criteria (fragrance, ease of application, satisfaction of applicability, product absorption, hair volume, sensation of greasiness, hair manageability (combing, hairstyle), using a score from −2 (low acceptability) to +2 (greater acceptability).

| Statistical analysis and sample size calculation
A total of 42 participants were enrolled and randomized in 2:1:1 allocation ratio.Sample size calculation was performed (G*Power software, version 3.1.9.4,Heinrich Heine University) starting from the data of Gupta et al. 20 (+15 hair density/cm 2 after 5% MNX, +10 hair density/cm 2 after F, compared to placebo), and a hypothesis to find an additional effect of +25 hair density/cm 2 after MNX + F. Considering an effect size (Choen's d value) of 1, an alpha value of 0.05, and a power of 80%, a total of at least 42 subjects should be enrolled.A one-way ANOVA with post hoc Tukey analysis was used for comparing treatments, and a p-value <0.05 was considered significant (GraphPad Software, version 5.0, Inc.).

| RE SULTS
Between February 2021 and December 2022, 75 subjects were assessed and screened for inclusion in the trial.Thirty-three subjects were excluded for different reasons (not meeting the inclusion/exclusion criteria or declining to participate).Therefore, in this study 42 men with AGA were enrolled, and all subjects completed the study (Figure 1).

Demographics and baseline characteristics are reported in
Table 1 and were similar between groups with the exception of hair density, which was statistically different (p < 0.001) in Group A (MNX + F) compared to Group C (MNX).No differences in terms of hair density were observed between other groups.Only in group A were there subjects with IV-V alopecia grading.
Global photographs and a macro-photograph of some patients in group A (MNX + F) taken at baseline and after 6 months are shown in Figures 6 and 7.

| Tolerability and cosmetic acceptability
The tolerability and cosmetic acceptability of products were evaluated by patients after 6 months through a self-administered questionnaire using a score from −2 (low acceptability) to +2 (greater acceptability).The results are illustrated in Table 2.
Good tolerability was observed in all treated groups.The association of 5% MNX and F (Group A) was significantly more appreciated in terms of fragrance compared to F alone and in terms of hair greasiness compared to MNX alone.Overall speaking, the tolerability and cosmetic acceptability were significant higher in Group A (MNX + F) compared to Group C (MNX).No significant differences were observed in hormonal levels between the three groups (data not shown).

| DISCUSS ION
European evidence-based guidelines recommend topical minoxidil (MNX) and oral finasteride (F) for the treatment of AGA in men. 9,21However, oral finasteride can be associated with adverse reactions or systemic side effects, such as sexual dysfunction and depression, that could be reduced with the use of the topical formulation 22 Different studies have reported the efficacy of topical finasteride in the range of 0.005%-1% 19,23,24 ; however, a recent phase III, randomized, 28-week, controlled clinical trial, that enrolled 458 adult males suggested that 0.25% topical finasteride could be considered an optimal concentration in terms of efficacy and safety. 9,19MNX and F act through two different mechanisms.
Although the mechanism of action of MNX is still unclear, it probably exerts a vasodilator effect improving microcirculation at the scalp level. 25F inhibits type II 5α-reductase, reducing the conversion of testosterone into DHT. 8,9Considering the different action mechanisms of MNX and F, their association could improve the efficacy of treatment compared to their use in monotherapy.Different studies present in the literature have underlined the superior efficacy of a combined solution of 0.1% or 0.25% finasteride mixed with 3% or 5% MNX in the galenic formulation; however, further investigations are warranted to determine the most effective formulation. 22The aim of the present work was to evaluate the efficacy of the combined use of two registered separate solutions of 0.25% F and propylene glycol (PG)-free 5% Minoxidil lotion (selected to limit potential adverse reactions associated with PG). 4,5,26e present study showed that the combined administration of 5% MNX and 0.25% topical finasteride provided a fast and good clinical response in the treatment of male AGA over 6 months.All the treatments evaluated in this study resulted in an increase in hair density over time.The greatest increase in hair density was observed after 6 months in the MNX + F group compared to the other two monotherapy groups (+81 hairs/cm  Ease of application 1.3 ± 0.9 1.6 ± 0.5 0.9 ± 0.7 ns ns Satisfaction-applicability 1.2 ± 0.9 1.6 ± 0.5 0.9 ± 1.  after the treatment with 0.25% topical or oral finasteride. 9The global photographic assessment score (GPAS) was significantly higher in Group A as compared to Group B (p < 0.001) and Group C (p < 0.05), both at 3 and 6 months.Good tolerability was observed in all treated groups.The tolerability and cosmetic acceptability were significantly higher in Group A (MNX + F) as compared to Group C (MNX).Some limitations should be taken into account in interpreting the results of this pilot trial.This study was an open trial; therefore, to improve the internal validity of the study, we adopted an assessorblinded evaluation study design.Another limit of our study could be the relatively small sample size considered.However, based on the primary outcome (hair density) and literature review, we conducted a sample size calculation and decided to enroll 42 subjects.
Therefore, in relation to the primary outcome, the sample size calculation could be considered adequately powered.Finally, another limitation of the present study is the relatively short treatment period.However, the combined used of MNX + F showed significant efficacy starting from the third month, providing a useful approach to motivate patients, facilitating compliance to the therapy.In fact, a recent trial 26 demonstrated that in subjects treated with topical MNX the lack of improvement experienced by patients was the main reason for discontinuing.Therefore, the use of combination therapy with topical MNX and topical F could increase adherence to the AGA treatment thanks to its faster efficacy in comparison to monotherapy.

| CON CLUS IONS
Our study suggests that the association of 0.25% topical finasteride and 5% PG-free topical minoxidil could be a good option for the treatment of AGA in men, showing superior efficacy compared to their use in monotherapy.This association was well-tolerated and could be recommended in different clinical situations such as men with severe (≥II Norwood-Hamilton grade) AGA or in the absence of response with other topical therapies.

K
E Y W O R D S androgenetic alopecia, association therapy, finasteride, Minoxidil, topical combination and treatment naive or free from any therapy for at least 6 months.The main exclusion criteria were the presence of other hair loss conditions or allergies to the ingredients used in the study.The study was conducted over 6 months, and patients were evaluated at baseline and after 3 and 6 months.Patients were randomly divided into three arms (2:1:1): group A (n = 19), 5% MNX (Carexidil, 5% MNX PG-free, Cantabria Labs Difa Cooper) and topical 0.25% F (Caretopic, Cantabria Labs Difa Cooper); group B (n = 12), topical finasteride alone; and group C (n = 11), 5% MNX alone.The randomization list was generated by a dedicated computer program.In group A, subjects applied 1 mL of 5% MNX in the morning and 4 puffs of F (0.2 mL) spray in the evening; in group B, subjects applied 4 puffs of F spray in the evening; in group C, subjects applied 1 mL of 5% MNX both in the morning and in the evening.
time (158.2± 12.5 and 183.5 ± 15.3 hair/cm 2 at T3 and T6 for Group A, 149.5 ± 14.3 and 154.6 ± 18.6 hair/cm 2 at T3 and T6 for Group B, and 212.3 ± 18.4 and 223.7 ± 19.8 hair/cm 2 at T3 and T6 for Group C) although it was statistically significant only in group A (Figure2).The mean change in hair density from baseline was statistically different between group A (MNX + F) and group B (F) (p < 0.01) but no statistical differences were observed between group A (MNX + F) and group C (MNX), both after 3 and 6 months.At baseline, mean ± SE hair terminal density was 63.6 ± 6.4, 66.2 ± 7.7 and 108.4 ± 8.1 terminal hair/cm 2 , for group A (MNX + F), group B (F), and group C (MNX), respectively.All groups showed increasing terminal hair counts over time, statistically significant however only in group A (Figure3A).The mean change in hair terminal density from baseline was statistically different between group A (MNX + F) and group B (F) (p < 0.05) but no statistical differences were observed between group A (MNX + F) and group C (MNX), both after 3 and 6 months.On the contrary, although it was possible to observe a general increasing trend in vellus hair density, no statistical differences were observed in this parameter during the treatment for all the groups considered (Figure3B).At baseline, mean ± SE hair diameter was 52.3 ± 3.1, 47.3 ± 2.1, and 42.0 ± 3.0 μm, for group A (MNX + F), group B (F), and group C

F I G U R E 2 1
Mean change from baseline in hair density in MNX + F, F and MNX groups.Data are expressed as mean ± standard error (SE).*p < 0.05; ***p < 0.001.Demographics and baseline characteristics.

F I G U R E 3 F I G U R E 4 F I G U R E 5 6
Mean terminal hair density (A) and vellus hair density (B) in MNX + F, F and MNX groups.Data are expressed as mean ± SE. *p < 0.05, ***p < 0.001.Mean hair diameter in MNX + F, F and MNX groups.Data are expressed as mean ± SE.Global photographic assessment score (GPAS).*p < 0.05; ***p < 0.001.The statistical analyses were conducted between T3 data and between T6 data.Data are expressed as mean±SD.Global photographs of a patient in group A (MNX + F), taken at baseline (A) and after 6 months (B).

F I G U R E 7
Global photographs and macro-photographs of a patient in group A (MNX + F), taken at baseline (A, B), after 3 months (C, D), and after 6 months (E, F).TA B L E 2Tolerability and cosmetic acceptability of tested products.
Only the association of MNX and F caused a statistically significant increase in hair density after both 3 (p < 0.05) and 6 (p < 0.001) months treatment as compared to baseline.Previous studies evaluating the combination of MNX and F reported an increase in hair density in a range from 4.8 to 61.8 hairs/cm 2 ,17,19,22 suggesting that 5% MNX and 0.25% (+81 hairs/cm 2 ) could represent the most effective combination therapy.Although vellus hair density remained essentially unchanged over time, all groups showed an increasing terminal hair density during the treatment period, although it was statistically significant only in group A (MNX + F).This could be explained by the recruitment of less active follicular units in the first 3 months, followed by the transformation of vellus hairs into terminal hairs in the subsequent months.No significant differences in mean hair diameter were observed between the three groups.Similar results, in terms of hair width, were observed byPiraccini et al., 2MNX + F, +18 hairs/cm 2 F,