78 ItalIan Journal of Dermatology anD Venereology february 2022 O R I G I N A L A R T I C L E a new combination of molecules for the treatment of androgenetic alopecia and telogen effluvium: a double-blind randomized, monocentric, placebo-controlled study alfredo roSSI 1 *, francesca magrI 1 , marco DI fraIa 1 , gemma Caro 1 , maria C. fortuna 1 , marco PIaCentInI 2 , leonardo Celleno 3 1 Unit of Dermatology, Sapienza University, Rome, Italy; 2 Eurofins Cosmetics & Personal Care, Rome, Italy; 3 Sacred Heart Catholic University, Rome, Italy *Corresponding author: Alfredo Rossi, Unit of Dermatology, Sapienza University, Viale del Policlinico 155, 00161 Rome, Italy. E-mail: alfredo.rossi@uniroma1.it a B S t r a C t BACKGROUND: Androgenetic alopecia (AGA) is the most frequent form of alopecia. Telogen effluvium (TE) is a common form of diffuse hair loss mainly observed in women. The aim of our study was to evaluate the efficacy of a topical trichological treatment containing a new combination of molecules for the treatment of aga and te. METHODS: In-vitro tests were performed analyzing different combinations and concentrations of arginine, zinc and a third enzymatically neutral substance called AA on human follicles dermal papillae cells. These tests evaluated the capability of inhibiting the 5α-reductase ( 5-AR ) enzyme and the 5-AR gene expression. We also performed an in-vivo study. Forty individuals affected by AGA and TE were divided into two groups. One group was administered a combination of zinc and arginine (lotion A), whilst the other placebo (lotion B). Therapy duration was 23 consecutive weeks. Follow-up examinations and pull tests occurred at baseline, after 6 weeks and at the end of the therapy. On 20 randomly selected patients we also performed noninvasive phototrichograms. RESULTS: In-vitro tests showed that the combination had a strong statistically significant inhibitory activity on 5-AR of dermal papillae cells. Number of hairs removed by pull-test significantly decreased at T0, T1 and T2 in patient treated with lotion A. We also observed an increase in the percentage of anagen hair and a decrease in telogen hairs. Concerning phototrichograms, all objective parameters evaluated showed better results in the lotion a group when compared with the placebo group. CONCLUSIONS: Based on our results, the combination of arginine and zinc tested in our study could represent a good therapeutic option for the treatment of AGA and TE and it might represent a valid alternative to finasteride. ( Cite this article as: rossi a, magri f, Di fraia m, Caro g, fortuna mC, Piacentini m, et al. a new combination of molecules for the treatment of an- drogenetic alopecia and telogen effluvium: a double-blind randomized, monocentric, placebo-controlled study. Ital J Dermatol Venereol 2022;157:78- 83. DOI: 10.23736/S2784-8671.21.06915-7) K ey words : Alopecia; Therapeutics; Arginine; Zinc. Italian Journal of Dermatology and Venereology 2022 February;157(1):78-83 DOI: 10.23736/S2784-8671.21.06915-7 © 2021 EDIZIONI MINERVA MEDICA Online version at http://www.minervamedica.it a ndrogenetic alopecia (AGA) is the most frequent form of non-scarring alopecia affecting up to 80% of male and 55% of female individuals during their life. 1 It is characterized by a progressive miniaturization of hair follicles, especially involving the frontal and temporal regions of the scalp and the vertex. In male AGA, hair thinning is caused by the activity of testosterone metabo - lite dihydrotestosterone (DHT) on androgen-sensitive hair follicles of genetically predisposed subjects. 1 on the oth- er hand, the pathophysiology of female aga is still not completely understood. Even though genetic and hormon - al factors are considered involved as in the more common male form, its link to androgen hormones is less direct than in females affected by complete androgen insensitiv - ity syndrome. 2, 3 the main treatment options for male aga are systemic finasteride (1 mg per day) and topical (2% and 5%) mi - noxidil, which represent the only two products currently CoPyrIgHt © 2022 eDIZIonI mInerVa meDICa This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies (either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo, or other proprietary information of the Publisher. neW ComBInatIon of moleCuleS for aga anD te roSSI Vol. 157 - no. 1 ItalIan Journal of Dermatology anD Venereology 79 molecules of inhibiting the 5-AR enzyme, and the 5-AR gene expression. Eight *103 cells are seeded in 96-well plates and treated with the tested compounds plus 100 nm testosterone for 24 hours 20 ng/mL of BSA-DHT are coated ON at 4 °C in 100 uL of 50 mM sodium carbonate pH 9.0. After washing in PBS, the plate containing BSa-DHt is incubated with 50 ul of cells supernatant plus 50 ul of primary antibody anti-DHT conjugated with Biotin dissolved in PBS con - taining 1% BSa. two hours later, the plate is washed in 1X PBS 3 times, and incubated with 100 ul of streptavidin- HRP 5 ug/mL in 1X PBS containing 1% BSA. The amount of DHt is measured by a colorimetric reaction using a 0.5 mg/mL solution of OPD, 0.012%. The plate is incubated at room temperature up to color development and the ab - sorbance at 490 nm is measured by a plate reader Victor3 (PerkinElmer; Waltham, MA, USA). We also performed an in-vivo double-blind, randomized, monocentric, placebo-controlled study on 26 individuals affected by hair loss due to female and male AGA and 14 affected by TE. All 26 patients with female and male AGA enrolled in our study presented a mild-to-moderate form of the disorder (II-IV stage of the Norwood-Hamilton Scale and I-II stage of the Ludwig Scale). All 14 patients with TE had a mild-to-moderate form (I-III stage of the Savin Scale). The individuals of our sample were randomly and equally divided into two groups. One group was administered the active substance (lo - tion A), whilst the other placebo (lotion B). Lotion A contained a combination of ZNGL and ARG in associa - tion with AA (INCI: aqua, ascorbic acid, glycol, arginine, gluconolactone, zinc gluconate, trans-anethole, ethylhex- approved by the American Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Currently, minoxidil solution is the only FDA approved therapeutic option for female aga. In addition, many other topical and systemic not-ap- proved therapeutic options are currently available for the management of aga, including systemic dutasteride, top- ical finasteride, topical high and medium potent steroids, oral minoxidil, topical cetirizine, platelet-rich plasma (PRP) therapy, mesotherapy, laser therapy. finasteride inhibits the type-II 5α-reductase , which catalyzes the conversion of testosterone (T) to the more active form dihydrotestosterone (DHT). 3 However, this molecule may be associated with several adverse events, mostly sexually related (0.7-5%), 4, 5 such as decreased li- bido, erectile disfunction, reduced spermatic volume; oth - er possible alterations are gynecomastia and depression. Telogen effluvium (TE) is a common form of diffuse hair loss mainly observed in women. It occurs approxi - mately three months after the exposition to several triggers, such as major surgery, infections, trauma, post-partum hormonal alterations, dysthyroidism and iron deficiency. 6 all these conditions alter the hair follicle cycle, inducing a large number of hairs in anagen phase to abruptly enter the telogen phase. 7 As a consequence, TE is characterized by shortening of the anagen phase and by the extension of the telogen phase, with consequent reduction of the total hair volume. 8 The spontaneous remission of TE is frequent. Obvi - ously, therapeutic strategies are influenced by the eventual presence of underlying causes, which must be carefully investigated. Topical steroids, topical minoxidil, oral iron, dietary and antioxidant supplementation are the most fre- quently prescribed therapies for TE. the aim of our double-blind randomized, monocentric, placebo-controlled study was to evaluate the efficacy of a topical trichological treatment containing a new combina- tion of molecules for the treatment of aga and te. Materials and methods In this article we described the results of two studies: one in vitro and one in vivo In-vitro tests analyzing different combinations and concentrations of arginine gluconate/ascorbate (ARG) and zinc gluconate (ZNGL) and a third enzymatically neutral substance called AA and finasteride 10 nM were performed on human follicles dermal papillae cells (Table I). In-vitro tests evaluated the capability of the different T able I.— Different combination and concentration of molecules analyzed in the in-vitro test. molecules testosterone 100 nm arg 0.008% Arg 0.04% ZnGl 0.008% ZnGl 0.04% ZnGLArg 0.008% ZnGLArg argaa 0.008% ArgAA 0.04% ZnGlAA 0.008% ZnGlAA 0.04% ZnGlArgAA 0.008% ZnGlArgAA 0.04% finasteride 10 nm Arg: Arginine; ZnGl: zinc gluconate; AA: enzymatically neutral substance. COPYRIGHT © 2022 EDIZIONI MINERVA MEDICA This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies (eith or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other means which may to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is not permitted. It is not permi cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo, or other proprietary information of th roSSI neW ComBInatIon of moleCuleS for aga anD te 80 ItalIan Journal of Dermatology anD Venereology february 2022 tions occurred at baseline (T0), after 6 weeks (T1) and at the end of the therapy (T2). At each visit, pull test was performed to evaluate the eventual decrease of telogen hairs with consequent increase of anagen hairs. In addi - tion, systematic trichoscopic photographs were taken on three selected scalp regions with a video-dermoscope at 50× magnification. On 20 randomly selected patients (10 from the lotion A group and 10 from the lotion B group), we also performed noninvasive phototrichograms with trichoscan ® HD (DermoScan GmbH; Regensburg, Ger - many), with the aim of monitoring the following param - eters: • total number of hairs; • total hair density/cm 2 ; • vellus hair density/cm 2 ; • terminal hair density/cm 2. Investigators had to perform an objective assessment of products efficacy, based on pull test and trichoscopy. Fur - thermore, a subjective assessment of the efficacy of prod - ucts, as well as of their cosmetical qualities, was realized with a satisfaction survey submitted to all 40 individuals at t1 and t2. Statistical analysis Comparisons between treatments was performed using Wilcoxon test and Student’s t- test. P value lower than 0.05 was considered statistically significant. Results In-vitro test studying the capability of inhibiting the 5-ar enzyme showed that the triple combination ZNGL-ARG- ylglycerin, polysorbate 20, ellagic acid, phenoxyethanol, vanillin butyl ether, menthol, octenidine HCl, sodium ben - zoate, parfum, sodium hyaluronate, magnesium stearate). lotion B corresponded to a placebo solution containing mainly water and glycol. Inclusion criteria of the study are summarized in table II. Patients undergoing the topical treatment with lotion a comprised 13 AGA and 7 TE individuals of both female and male gender, whose age ranged between 29 and 72 years old (median age: 51). Patients applying lotion B comprised 13 aga and 7 te female and male individuals, which were between 28 and 69 years old (median age: 55). All 40 individuals were scrupulously informed on how apply the examined products. 10 days before starting the topical treatment with lotion a or B, patients were instruct- ed to use a delicate shampoo for 2-3 times per week. Then, patients started the treatment, with an evening alternate- day topical application of the examined lotions (A or B) on the entire scalp (frontal/temporal/vertex region). Therapy duration was 23 consecutive weeks. Follow-up examina - T able II.— Inclusion criteria of our sample. Inclusion criteria Both female and male gender; age 18 to 70 years; Fitzpatrick skin type: I, II, III or IV; persistent hair loss (telogen effluvium) for over a month, with no specific underlying diseases associated (anemia, dysthyroidism, hypoproteinemia, autoimmune or inflammatory disorders) or Androgenetic Alopecia (AGA) (type II-IV of Hamilton-Norwood Scale for male AGA; type I-II of Ludwig classification for female AGA); no other concomitant therapies. figure 1.—5-alpha reductase enzymat- ic assay in dermal papilla. testosterone 100 nM was used as positive control. The values reported in the graphics are the average of three different experi - ments, each of them performed in trip- licate. The bars marked with asterisks represent statistically significant values (*P≤0.05; **P≤0.01; ***P≤0.0001). Arg: Arginine; ZnGl: zinc gluconate; AA: enzymatically neutral substance. 5-alpha reductase activity 5-alpha reductase activity (% to control = 100) 120 100 80 60 40 20 0 Testosteron 100nM Untreated Untreated Control Arg 0,008% Arg 0,04% ZnGl 0,008% ZnGl 0,04% ZnGLArg 0,008% ZnGLArg 0,04% ArgAA 0,008% ArgAA 0,04% ZnGIAA 0,008% ZnGIAA 0,04% ZnGIArgAA 0,008% Finasterid e 10 nM 20,36 100,00 44,75 42,40 64,35 48,62 25,36 40,05 36,23 49,59 28,67 35,07 21,27 18,33 ZnGIArgAA 0,04% 50,23 COPYRIGHT © 2022 EDIZIONI MINERVA MEDICA This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies (either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo, or other proprietary information of the Publisher. neW ComBInatIon of moleCuleS for aga anD te roSSI Vol. 157 - no. 1 ItalIan Journal of Dermatology anD Venereology 81 AA had a strong statistically significant inhibitory activity on 5-AR of dermal papillae cells (Figure 1). Furthermore, in-vitro 5-AR gene expression was evaluated. However, both ZNGL and ARG showed a weak activity on reducing 5-AR gene expression. Thus, the examined substances have a mechanism of action similar to finasteride, causing the direct inhibition of 5-AR with no influences on its gene expression. Concerning the in-vivo study on our sample of 40 indi - viduals, statistically significant decrease in removed hairs after pull test was evident after 6 and 23 weeks of therapy with lotion a, compared to baseline. on the other hand, the decrease was not significant in patients undergoing topical treatment with lotion B (Table III). Concerning the trichoscopic analysis, a remarkable im - provement of hair thickness and hair regrowth was evident after 6 and 23 weeks of treatment with lotion A, while no evident changes in hair thickness and regrowth were ob - served with lotion B, as also subjectively assessed by the investigators. Several parameters were measured with phototricho - grams. Firstly, a progressive statistically significant in - crease in total hair number was present after 6 (+15.83%) and 23 weeks (+52.52%) of therapy with lotion A, while no significant variations were observed after 6 and 23 weeks of therapy with lotion B (Table IV). A statistically significant improvement of hair density was also observed T able III.— Number of total hairs removed after pull test at T0, T1 (6 weeks) and T2 (23 weeks). lotions time Average±SD Variation (%) lotion a t0 20.80±4.05 t1 16.45±3.17 -20.91* t2 14.35±3.12 -31.01* lotion B t0 19.95±1.82 t1 19.70±2.49 -1.25 t2 19.25±2.77 -3.51 Variation compared to t0 is expressed as percentage. *P value <0.05. SD: standard deviation. T able IV.— Number of total hairs measured by phototrichogram at T0, T1 (6 weeks) and T2 (23 weeks). lotions time Average±SD Variation (%) lotion a t0 106.15±13.62 t1 122.95±15.32 +15.83* t2 161.90±25.74 +52.52* lotion B t0 104.70±12.51 t1 105.00±11.27 +0.29 t2 105.40±10.61 +0.67 Variation compared to t0 is expressed as percentage. *P value <0.05. SD: standard deviation. T able V.— Total hair density (number of total hairs/cm 2 ) meas - ured by phototrichogram at T0, T1 (6 weeks) and T2 (23 weeks). lotions time Average±SD Variation (%) lotion a t0 185.97±21.36 t1 214.96±27.43 +15.59* t2 245.35±29.91 +29.91* lotion B t0 187.35±12.99 t1 188.85±14.91 +0.80 t2 187.75±14.57 +0.21 Variation compared to t0 is expressed as percentage. *P value <0.05. SD: standard deviation. T able VI.— Vellus hair density (number of vellus hairs/cm 2) meas - ured by phototrichogram at T0, T1 (6 weeks) and T2 (23 weeks). lotions time Average±SD Variation (%) lotion a t0 24.14±3.92 t1 25.92±4.15 +7.37* t2 30.22±6.57 +25.19* lotion B t0 25.29±3.36 t1 25.65±3.26 +1.42 t2 25.67±3.76 +1.50 Variation compared to t0 is expressed as percentage. *P value <0.05. SD: standard deviation. T able VII.— Terminal hair density (number of terminal hairs/ cm 2) measured by phototrichogram at T0, T1 (6 weeks) and T2 (23 weeks). lotions time Average±SD Variation (%) lotion a t0 119.47±12.41 t1 133.84±17.64 +12.03* t2 177.05±32.67 +48.20* lotion B t0 120.21±13.47 t1 120.78±11.57 +0.47 t2 120.80±12.91 +0.49 Variation compared to t0 is expressed as percentage. *P value <0.05. SD: standard deviation. in patients undergoing the treatment with lotion A after 6 (+15.59%) and 23 weeks (+29.91%), as opposed to pa - tients applying lotion B (Table V). Concerning the density of vellus hair, lotion B treatment did not induce significant changes, while lotion a treatment was associated with a statistically significant progressive increase of this pa - rameter after 6 (+7.37%) and 23 weeks (+25.19%) (Table VI). Moreover, the density of terminal hair presented a statistically significant increase after 6 (+12.03%) and 23 weeks (+48.20%) of lotion A treatment, while no signifi - cant variations were reported with lotion B (Table VII). In group A patients, anagen hairs removed after pull test were significantly increased after 6 and 23 weeks (respectively COPYRIGHT © 2022 EDIZIONI MINERVA MEDICA This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies (eith or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other means which may to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is not permitted. It is not permi cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo, or other proprietary information of th roSSI neW ComBInatIon of moleCuleS for aga anD te 82 ItalIan Journal of Dermatology anD Venereology february 2022 Discussion In this double-blind randomized, monocentric, placebo- controlled study we evaluated the efficacy of an alternative topical treatment for female and male aga and te. to date, therapeutic options for the management of hair loss conditions, such as aga and te, are partially limited and not definitive. Systemic finasteride is nowadays the most efficient product for the treatment of male aga. It inhibits the type-II 5α-reductase, which catalyzes the conversion of testosterone (T) to the more active form of dihydrotestos - terone (DHT), responsible of hair miniaturization. 3 However, finasteride may be associated with several ad - verse events, such as decreased libido, erectile disfunction, reduced spermatic volume, gynecomastia and depression. Even in the female population, systemic finasteride may cause sexual alterations, and its assumption is contraindi- cated during pregnancy because of the risk of feminization of the male fetus. 3 In consideration of finasteride side effects, several sub - stances have been tested over the years with the aim of finding alternative therapeutic options. In our study, the active substances tested were zinc glu - conate (ZNGL) and arginine gluconate/ascorbate (ARG), which resulted to be good alternative products inhibiting the 5-ar enzyme. Zinc is an essential trace element, which must be sup - plied through diet. It is involved in several biological func - tions, such as Dna synthesis, hormone regulation, enzy- matic reactions, gene expression and cell proliferation. In particular, zinc is involved as a structural element and regulatory factor and has a role in important functional ac- tivities within the hair follicle. 9 In fact, alopecia is a well-known sign of zinc deficien - cy and zinc supplementation is associated with hair re- growth. 10 Arginine is a semi essential amino acid involved in nu - merous biological activities. In particular, arginine is the precursor for nitric oxide (NO) synthesis in animal cells. In blood vessels, NO released by endothelial cells causes smooth muscle relaxation and consequent vasodilation. no is an important molecule in trichology because of its role as vasodilator in the stimulation of hair growth. NO is also involved in opening potassium channels, the activity mechanism of minoxidil. 11, 12 To test the activity of ZNGL and ARG, in-vitro tests on human follicles dermal papillae cells were performed. the aim of these tests was to evaluate the presence of a syner - gic action between these two substances. +5.27% and +22.24%) (Table VIII), while telogen hairs were statistically decreased after 6 (-6.80%) and 23 weeks (-28.71%) (Table IX). No significant variations of anagen and telogen hair removed after pull test were registered in the placebo group. Objective assessment of products efficacy performed by investigators and subjective assessment made by patients were also evaluated. After 6 weeks of therapy with lotion A, investigators observed a sufficient product efficacy in 40% of patients, a good efficacy in 40% of cases and a great efficacy in 5% of individuals. Concerning lotion B, investigators indicated a poor efficacy in 80% of patients, and a sufficient efficacy in 20% of individuals after 6 weeks. After 23 weeks of treatment with lotion A, investi - gators observed a sufficient product efficacy in 5% of pa - tients, a good efficacy in 60% of cases and a great efficacy in 30% of individuals. Concerning lotion B, investigators indicated a poor efficacy in 60% of patients, a sufficient efficacy in 15% and good efficacy in 25% of individuals after 23 weeks. Patients assessment indicated a good efficacy of lotion A in 45% and 35% of cases after 6 and 23 weeks respec - tively. Concerning lotion B, a good efficacy was reported only in 5% of cases after 6 weeks and in 25% of cases after 23 weeks. T able VIII.— Number of anagen hairs removed after pull test at T0, T1 (6 weeks) and T2 (23 weeks). lotions time Average±SD Variation (%) lotion a t0 56.35±4.94 t1 59.32±6.21 +5.27* t2 68.88±7.83 +22.24* lotion B t0 57.66±5.60 t1 58.30±6.38 +1.11 t2 58.44±6.79 +1.35 Variation compared to t0 is expressed as percentage. *P value <0.05. SD: standard deviation. T able IX.— Number of telogen hairs removed after pull test at T0, T1 (6 weeks) and T2 (23 weeks). lotions time Average±SD Variation (%) lotion a t0 43.65±4.94 t1 40.68±6.21 -6.80* t2 31.12±7.83 -28.71* lotion B t0 42.34±5.60 t1 41.70±6.38 -1.51 t2 41.56±6.79 -1.84 Variation compared to t0 is expressed as percentage. *P value <0.05. SD: standard deviation. COPYRIGHT © 2022 EDIZIONI MINERVA MEDICA This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies (eith or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other means which may to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use is not permitted. It is not permi cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, logo, or other proprietary information of th neW ComBInatIon of moleCuleS for aga anD te roSSI Vol. 157 - no. 1 ItalIan Journal of Dermatology anD Venereology 83 Conclusions Based on our results, the combination of ARG and ZNGL tested in our study could represent a good therapeutic op- tion for the treatment of aga and te and it might repre- sent a valid alternative to finasteride. References 1. Piraccini Bm, alessandrini a. androgenetic alopecia. g Ital Dermatol Venereol 2014;149:15–24. 2. Cousen P, messenger a. female pattern hair loss in complete androgen insensitivity syndrome. Br J Dermatol 2010;162:1135–7. 3. rossi a, magri f, D’arino a, et al. Efficacy of Topical Finasteride 0.5% vs 17α-Estradiol 0.05% in the Treatment of Postmenopausal Female Pattern Hair Loss: A Retrospective, Single-Blind Study of 119 Patients. Dermatol Pract Concept 2020;10:2020039. 4. Rossi A, Cantisani C, Scarnò M, Trucchia A, Fortuna MC, Calvieri S. Fin - asteride, 1 mg daily administration on male androgenetic alopecia in different age groups: 10-year follow-up. Dermatol Ther (Heidelb) 2011;24:455–61. 5. Sato A, Takeda A. Evaluation of efficacy and safety of finasteride 1 mg in 3177 Japanese men with androgenetic alopecia. J Dermatol 2012;39:27–32. 6. Bennardo L, Del Duca E, Dastoli S, Schipani G, Scali E, Silvestri M, et al. Potential applications of topical oxygen therapy in dermatology. Der- matol Pract Concept 2018;8:272–6. 7. Hughes EC, Saleh D. Telogen effluvium. Treasure Island, FL: Stat - Pearls Publishing; 2020. 8. nistico S, tamburi f, Bennardo l, Dastoli S, Schipani g, Caro g, et al. Treatment of telogen effluvium using a dietary supplement containing Boswellia serrata, Curcuma longa, and Vitis vinifera: results of an obser - vational study. Dermatol Ther (Heidelb) 2019;32:e12842. 9. Dhaher SA, Yacoub AA, Jacob AA. Estimation of Zinc and Iron Lev - els in the Serum and Hair of Women with Androgenetic Alopecia: case- control Study. Indian J Dermatol 2018;63:369–74. 10. almohanna Hm, ahmed aa, tsatalis JP, tosti a. the role of Vitamins and Minerals in Hair Loss: A Review. Dermatol Ther (Heidelb) 2019;9:51–70. 11. Yazdani-Arazi SN, Ghanbarzadeh S, Adibkia K, Kouhsoltani M, Hamishehkar H. Histological evaluation of follicular delivery of arginine via nanostructured lipid carriers: a novel potential approach for the treat - ment of alopecia. Artif Cells Nanomed Biotechnol 2017;45:1379–87. 12. Vidal VP, Chaboissier MC, Lützkendorf S, Cotsarelis G, Mill P, Hui CC, et al. Sox9 is essential for outer root sheath differentiation and the formation of the hair stem cell compartment. Curr Biol 2005;15:1340–51. firstly, an in-vitro test to study the capability of inhibit- ing the 5-AR enzyme has been performed. ARG and ZNGL were tested: 1) individually; 2) together; and 3) together combined with a third enzymatically neutral substance called aa and turned out to be a strong “booster.” the test showed that the triple combination ZNGL-ARG-AA had the strongest statistically significant inhibitory activity on 5-AR of dermal papillae cells. furthermore, 5-AR gene expression was evaluated. However, both ZNGL and ARG showed a weak activity on reducing 5-AR gene expression. thus, the examined sub- stances have a mechanism of action similar to finasteride, causing the direct inhibition of 5-AR with no influences on its gene expression. In our in-vivo study on 40 individuals, after 23 weeks of therapy with lotion A, great results were evident. The pull test performed on patients under treatment with lotion a showed a statistically significant decrease in removed hair (-31.01%). Furthermore, lotion A was associated with hair regrowth and thickening, as observed trichoscopically by the investigators. Moreover, lotion A was associated with a good cosmetic acceptability (for both patients and inves - tigators). Concerning phototrichograms, all objective parameters evaluated (total number of hairs; hair density/cm 2 ; vellus hair density/cm 2 ; terminal hair density/cm 2 ; hair thickness) showed better results in the lotion a group when compared with the placebo group. no side effects were reported in patients treated with lotion a. Limitations of the study a study limitation is represented by the small sample size. Further studies with larger sample are needed to confirm our results. Conflicts of interest.— The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. Authors’ contributions .—Alfredo Rossi and Leonardo Celleno have given substantial contributions to manuscript conception and supervision, Francesca magri, marco Di fraia, gemma Caro and maria Caterina fortuna to in-vivo study and manuscript writing, marco Piacentini to in-vitro experiments. all authors read and approved the final version of the manuscript. History.— Article first published online: April 21, 2021. - Manuscript accepted: January 18, 2021. - Manuscript revised: December 18, 2020. - Manuscript received: November 3, 2020. COPYRIGHT © 2022 EDIZIONI MINERVA MEDICA This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies (eith or systematically, either printed or electronic) of the Article for any purpose. 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