Double Blind, Randomized, Placebo-Controlled Assessment of the Efficacy of a Food Supplement in Reducing Hair Loss in Male Subjects

Vincenzo Nobile^1*, Enza Cestone¹, Gloria Roveda¹, Marta Pisati¹, Angela Michelotti¹ and Maurizia Dossena^2
*Correspondence: Vincenzo Nobile, Complife Italia Srl, San Martino Siccomario, Pavia, Italy, Email:

Keywords

Hair loss • Acute telogen effluvium • Food supplement • Telogen effluvium • Keratin • Venus hair fern extract

Introduction

Hair loss is a not life-threatening dermatological condition with some physical effects but with more severe psychosocial consequences. Hair symbolism has been and is still extensively researched by anthropologists, psychologists, and sociologists [1]. In 2005, Alfonso et al., surveyed 1536 European (Italy, Germany, France, Spain, and United Kingdom) men aged between 18 and 40 years old. Over 70% of these men reported hair as an important feature of image while 62% agreed that hair loss could affect selfesteem. Interestingly, even if in a small part of the interviewed, successful hair treatment resulted in psychosocial benefits [2]. For many people, hair is a central aspect of a daily grooming ritual and in contrast to other bodily transformations (e.g., weight loss, increase of muscle definition, face lift, etc.) does not require substantial time and effort [3]. The expressions “bad hair day” or “I have been tearing my hair out”, are a testimony in the common parlance of the psychological importance of hair. Male hair loss starts in the 20s, but it takes 15 to 25 years to go bald [4,5]. According to the American Hair Loss Association, by the age of 35, two-thirds of American men will have some degree of appreciable hair loss, and by the age of 50, approximately 50 to 85% of men will have significant hair thinning [4,6]. The prevalence of male pattern hair loss has been evaluated by an Australian team [7]. Among the 396 men and women over 20 years old examined by dermatologists, 98,6% of the men showed some level of bitemporal recession. The etiologies of hair loss in men are various but the 2 most common are acute Telogen Effluvium (aTE) and Androgenic Alopecia (AGA). Acute Telogen Effluvium (aTE), is a transient condition characterized by excessive and diffuse hair shedding (“effluvium”) for less than 6 months [8,9]. No racial predilections of telogen effluvium have been recognized. In subjects suffering from acute telogen effluvium, the hair shedding associated with the abrupt interruption of the anagen phase starts 2 to 3 months after the triggering event and lasts for less than 6 months (3 months average time). The identification of the triggering factor is difficult in one-third of the cases [10]. The triggering events of acute telogen effluvium include psychological causes, physical/ emotional stress, and pharmacological therapy [11]. Among these, severe infection, major surgery, severe trauma, hypothyroidism, crash dieting, low protein intake, malnutrition, heavy metal ingestion, iron/zinc deficiency, and seasonal variation (July to October) are the most common conditions associated with acute telogen effluvium [12]. Androgenic alopecia affects 80% of Caucasian men [13,14]. According to Hamilton's study in 1951, by the age of 30 years the mean prevalence was 30%, 40% in mid-forties, and this rate rises to 50% by the age of 50 in Caucasian men [15]. In studies from the US, Italy, Norway, and Australia similar results to Hamilton's study were reported [16-18]. Different processes are involved in the pathogenesis of androgenic alopecia, including: microinflammation of the folliculary bulge [19], abnormal sensitivity of follicules to androgens [20-23], dysregulation in arrector pili muscles [24-28] and genetics [29].

Interestingly there is mounting evidence that inflammation is central to the pathogenesis of all types of hair loss. Indeed, numerous triggers (genetic, hormonal, mental or nutritional) of hair loss induce inappropriate inflammatory responses and chronic inflammation at the level of the follicle is common to all hair loss conditions [30]. Controlled production of cytokines (the mediators of inflammation) is involved in hair cycle regulation. However, in the event of inflammation, overproduced cytokines like IL-1 and TNFα are known to induce premature catagen, liberate Reactive Oxygen Species (ROS), cause apoptosis, and further propagate inflammation [31-33]. In recent years, evidence of the beneficial role of food supplements in subjects affected by hair loss (including acute telogen effluvium from various aetiology) is growing [34-39]. In a survey administered to 177 dermatologists attending a national dermatology conference in Riyadh (Saudi Arabia), 62% of the respondents recommended vitamins and minerals for acute telogen effluvium.

In this study we tested the efficacy of a food supplement (Alline proMEN) containing a patented keratin (Keramax®), venus hair fern extract and a combination of 11 vitamins and two minerals in reducing hair loss and restoring hair density in men. The composition of this supplement addresses two of the central pathogenic factors of hair disorders: nutritional deficiencies and inflammation. Indeed, beside the classic vitamin and minerals approach, there is increasing evidence of the role of keratin as an effective supplement for improving hair conditions [40]. The role of keratin (alone or in combination with vitamins) in improving hair conditions in subjects with acute telogen effluvium has also been investigated by our team (unpublished data). Adiantum Capillus-veneris means hair of Venus, the goddess of love named by the ancient Greeks because she has a beautiful mane, "capillus" means "hair" and "veneris" comes from Venus [41]. Given its content of phenolic flavonoid compounds, it is not surprising that the Venus capillary has anti-inflammatory and antioxidant effects [42]. Finally, the efficacy of a Venus capillary extract was established in a murine model of androgenic alopecia [43].

Methods

Clinical evaluation

This was a multicentric study (3 sites), double-blind, placebocontrolled, parallel-group (with 1:1 balanced randomization), conducted in Italy by Complife Italia srl. The study protocol and the informed consent form were approved by the ‘Independent Ethical Committee for Non- Pharmacological Clinical trials’ during its meeting on 09 September 2020. All subjects provided written informed consent before initiation of any studyrelated procedures. No changes to treatment regimen or to methods were necessary after study starting. The study is registered at ClinicalTrials.gov, number NCT04884347.

Participants

The study included a total of 100 subjects with acute telogen effluvium. Eligible participants were all adult (age range 25-55 years old) Caucasian male subjects showing the clinical signs of acute telogen effluvium. The subjects were of good general health, with all scalp and hair type, with acute telogen effluvium (due to seasonal change, stress, fatigue, imbalanced diet, pollution), with a proportion of hair in telogen phase over 20%, had no alimentary/eating disorders or known medical history of metabolic syndrome. Exclusion criteria were acute, chronic or progressive illness liable to interfere with the study data, diagnosed hair disorder or diseases related to hair cycle, and inflammatory skin disease or progressive skin lesion on the scalp. The study further excluded subjects under systemic treatment affecting the hair growth taken for more than 4 consecutive weeks during the last 24 weeks, excessive and/or fluctuating hair shedding for more than 6 months, radiotherapy or chemotherapy and scalp surgery at any time during the study period. The complete list of the inclusion and exclusion criteria is reported in Table 1.

The study took place in Complife’s Italia facilities (Biella, Milan, and Pavia) from October 2020 to February 2021. Complife Italia is an independent testing laboratory for safety and efficacy assessment of cosmetics, food supplements and medical devices.

The study took place in Complife’s Italia facilities (Biella, Milan, and Pavia) from October 2020 to February 2021. Complife Italia is an independent testing laboratory for safety and efficacy assessment of cosmetics, food supplements and medical devices.

Intervention

Subjects were randomly assigned to receive a commercially available food supplement (Alline proMEN, Trenker Laboratoires, Thines Belgium) or a placebo product. The test food supplement contained a patented keratin (Keramax®), venus hair fern extract and a combination of 11 vitamins and two minerals; while the placebo product contained only excipients (Table 2). The frequency of product use was a tablet per day for a total period of use of 3 months. Subjects were asked to take the tablet during the meal with a glass of water. Both the active and placebo tablets were identical in appearance.

Outcomes

The primary end point with respect to efficacy in decreasing hair loss was the change from base-line of the number of hair in anagen and telogen phase (phototricogram) after 1 and 3 months product use. The secondary endpoints were hair radiance (colorimeter/spectrophotometer), hair mechanical properties (hair elongation and hair breakage force by dynamometer), and hair structure (SEM). Clinical evaluation (hair growth and hair radiance) and self-assessment were taken to integrate the instrumental measurements.

Phototricogram : Phototricogram was carried out as recommended by TrichoScan® supplier. Pictures were taken 48 hours after shaving. The phototricogram procedure involves: i) clipping of a scalp area two fingers width away from the parting on the receding hairline of the fronto-temporal region or on the vertex. A template (Ø 1.8 cm) is used; ii) removal of the clipped hair with sticky tape; iii) application of dye on the clipped scalp region; iv) removal of dye remnants by means of a swab and an alcohol solution; v) photograph taking. Anagen, telogen and hair density are automatically calculated by TrichoScan® software [44].

Hair structure by SEM: Hair structure was assessed by mean of scanning electronic microscopy (SEM). Proximal (near the scalp) hair was analysed and clinically scored (improvement vs. baseline). The scoring system was as follows: 1 no variation vs. baseline, 2 mild variations vs. baseline, 3 moderate variations vs. baseline, 4 strong variation vs. baseline.

Hair radiance: Hair radiance (ability to reflect the light) was measured using a spectrophotometer/colorimeter CM 700D (Konica Minolta) by means of the 8° gloss value.

Clinical analysis: Hair growth and radiance were assessed by the investigator on a 7-point Likert scale from -3 (greatly decreased/improved) to +3 (greatly increased/worsened).

Hair mechanical properties: The force at which the hair breaks (breakage force) and their elongation (hair elasticity) was evaluated by means of dynamometer reading (Tensolab 2512A, Mesdan Lab). The dynamometer reading is done on a single hair fiber. In total 10 hair fibers were measured. Measurements were carried out according to UNI EN ISO 5079:1998.

Self-assessment questionnaire: Subjects were asked to reply to a self-assessment questionnaire and to fill a day-by-day alimentar y diary.

Sample size

To detect a reduction of the percentage of anagen hair in 3 months with a two-sided 5% significance level and a power of 80%, a sample size of 50 patients per group was necessary, given an anticipated dropout rate of 10%. The recruitment and inclusion period were 1 month. Sample size was calculated using PASS11 statistical software (version 11.0.10 for Windows) running on Windows Server 2009 Standard 64-bit edition (Microsoft, USA). An official interim analysis (on December 4th, 2020) was performed after 1 month product use. The interim analysis was performed to monitor the product efficacy and safety. The outcomes analyzed in the interim analysis were: hair density, percentage anagen and telogen hair, hair radiance, and tolerability. No correction of the methods and/or to treatment regimen was performed after this interim analysis.

Randomization and blinding

Half of the test subjects were randomized to receive the test product and half of the test subjects were randomized to receive the placebo product. A restricted randomization list was created using PASS11 statistical software (version 11.0.10 for Windows) running on Windows Server 2009 Standard 64-bit edition (Microsoft, USA) by a biostatistician and stored in a safe place. The randomization sequence was stratified using “Efron’s biased coin” algorithm with a 1:1 allocation ratio. The allocation sequence was concealed from the study director in sequentially numbered, opaque, and sealed envelopes, reporting the unblinded treatment allocation (based on subject entry number in the study). A masked allocation sequence was prepared for the staff delivering the intervention based on the subject entry number in the study. An independent technician dispensed either the active or the placebo products according to the masked allocation sequence. The study adhered to established procedures to maintain separation between the investigator and its collaborators and the staff that delivered the intervention. The investigator, the study staff who obtained outcome measurements and the subjects participating in the study were not informed on the (masked) product group assignment. Staff members who delivered the intervention did not take outcome measurements.

Statistical methods

All the calculations were done using a Microsoft® Office 365 ProPlus (vers. 1902; build 11328.20468; Microsoft, USA) worksheet running on Microsoft® Windows 10 Pro (vers. 1903; build 18362.476; Microsoft, USA). Intragroup (vs. baseline) statistical analysis on parametric data was carried out using two-ways Student’s t-test for paired data; while intragroup statistical analysis on nonparametric data was carried out using Wilcoxon test. Intergroup (between treatments) statistical analysis on parametric data was carried out using two-way t test of Student for not paired data; while intergroup statistical analysis on nonparametric data was carried out using Mann-Whitney U test. Statistical analysis was carried out using NCSS10 statistical software (version 10.0.7 for Windows) running on Windows Server 2009 Standard 64-bit edition (Microsoft, USA). A p<0.05 was considered statistically significant. Statistical analysis output was reported as follows: *p<0.05, **p<0.01, and ***p<0.001.

Results

Participants, recruitment, and baseline characteristics

From October 2020 through February 2021, a total of 100 male subjects, meeting the inclusion criteria, were enrolled in three Complife’s centers (Figure 1). Of the 100 subjects, 50 were randomly assigned to the active product and 50 to the placebo product. The study population’s ethnicity was Caucasian. Demographic and baseline characteristics (Table 3) were similar across treatment arms, indicating an unbiased randomisation and the absence of covariates.

Subjects attended clinic visits at the time of randomization (baseline) and after 1 and 3 months of product use. Data analysis was per protocol and involved all the subjects who were randomly assigned. Subjects’ compliance to treatment was assessed by means of tablets count and was satisfactory. No deviations were observed in the treatment regimen. All the subjects were included in the safety analysis. Both the active and the placebo product were well-tolerated (neither objective or subjective tolerance reactions nor adverse events were reported).

Primary endpoints

The changes in hair count (hair density) are shown in Figure 2a. In the active treatment arm, the average number of baseline hair was 2 11.6 ± 2.5 hair/cm2, whereas the average number of hair was 220.7 ± 3.5 and 228.3 ± 2.8 after 1 and 3 months product use; respectively. The mean change over 3 months product use was +8.6%, which was statistically significant vs. baseline (p=0.004) and the placebo product (p=0.0195).

The changes in % telogen hair are shown in Figure 2b. In the active treatment arm, the average percentage of telogen hair was 25.2 ± 0.3, whereas the average percentage of telogen hair was 20.7 ± 0.6 and 12.0 ± 0.2 after 1- and 3-months product use; respectively. The mean change over 1 month product use was -4.6%; while the mean change over 3 months product use was -13.2%. The mean change of the percentage telogen hair was statistically significant both vs. baseline (p=0.0000 at 1 and 3 months) and the placebo product (p=0.0063 at 1 month and p=0.0000 at 3 months).

The changes in % anagen hair are shown in Figure 2c. In the active treatment arm, the average percentage of anagen hair was 74.8 ± 0.3, whereas the average percentage of anagen hair was 79.3 ± 0.6 and 88.0 ± 0.2 after 1- and 3-months product use; respectively. The mean change over 1 month product use was +4.6%; while the mean change over 3 months product use was +13.2%. The mean change of the percentage anagen hair was statistically significant both vs. baseline (p=0.0000 at 1 and 3 months) and the placebo product (p=0.0063 at 1 month and p=0.0000 at 3 months).

Secondary endpoints

The changes of the secondary endpoints are shown in Table 4. In the active treatment arm, hair radiance was improved by 15.4% and 24.0% after 1 and 3 months of product use. The variation of hair radiance was statistically significant both vs. baseline (p=0.0000) and the placebo product (p=0.0000) at 1 and 3 months. The hair breakage force and the hair elongation were statistically changed after 3 months of product use by +7.5 cN (p=0.0000 vs. baseline and placebo) and 5.3% (p=0.0000 vs. baseline and placebo); respectively. Clinical analysis confirmed a statistically significant improvement of hair growth, hair radiance and hair structure (Figure 3.), after 3 months product use, in the active treatment group vs placebo. The active product was perceived as effective by most of the subjects participating in the study (Figure 4).

Results

Participants, recruitment, and baseline characteristics

From October 2020 through February 2021, a total of 100 male subjects, meeting the inclusion criteria, were enrolled in three Complife’s centers (Figure 1). Of the 100 subjects, 50 were randomly assigned to the active product and 50 to the placebo product. The study population’s ethnicity was Caucasian. Demographic and baseline characteristics (Table 3) were similar across treatment arms, indicating an unbiased randomisation and the absence of covariates.

Subjects attended clinic visits at the time of randomization (baseline) and after 1 and 3 months of product use. Data analysis was per protocol and involved all the subjects who were randomly assigned. Subjects’ compliance to treatment was assessed by means of tablets count and was satisfactory. No deviations were observed in the treatment regimen. All the subjects were included in the safety analysis. Both the active and the placebo product were well-tolerated (neither objective or subjective tolerance reactions nor adverse events were reported).

Primary endpoints

The changes in hair count (hair density) are shown in Figure 2a. In the active treatment arm, the average number of baseline hair was 2 11.6 ± 2.5 hair/cm2, whereas the average number of hair was 220.7 ± 3.5 and 228.3 ± 2.8 after 1 and 3 months product use; respectively. The mean change over 3 months product use was +8.6%, which was statistically significant vs. baseline (p=0.004) and the placebo product (p=0.0195).

The changes in % telogen hair are shown in Figure 2b. In the active treatment arm, the average percentage of telogen hair was 25.2 ± 0.3, whereas the average percentage of telogen hair was 20.7 ± 0.6 and 12.0 ± 0.2 after 1- and 3-months product use; respectively. The mean change over 1 month product use was -4.6%; while the mean change over 3 months product use was -13.2%. The mean change of the percentage telogen hair was statistically significant both vs. baseline (p=0.0000 at 1 and 3 months) and the placebo product (p=0.0063 at 1 month and p=0.0000 at 3 months).

The changes in % anagen hair are shown in Figure 2c. In the active treatment arm, the average percentage of anagen hair was 74.8 ± 0.3, whereas the average percentage of anagen hair was 79.3 ± 0.6 and 88.0 ± 0.2 after 1- and 3-months product use; respectively. The mean change over 1 month product use was +4.6%; while the mean change over 3 months product use was +13.2%. The mean change of the percentage anagen hair was statistically significant both vs. baseline (p=0.0000 at 1 and 3 months) and the placebo product (p=0.0063 at 1 month and p=0.0000 at 3 months).

Secondary endpoints

The changes of the secondary endpoints are shown in Table 4. In the active treatment arm, hair radiance was improved by 15.4% and 24.0% after 1 and 3 months of product use. The variation of hair radiance was statistically significant both vs. baseline (p=0.0000) and the placebo product (p=0.0000) at 1 and 3 months. The hair breakage force and the hair elongation were statistically changed after 3 months of product use by +7.5 cN (p=0.0000 vs. baseline and placebo) and 5.3% (p=0.0000 vs. baseline and placebo); respectively. Clinical analysis confirmed a statistically significant improvement of hair growth, hair radiance and hair structure (Figure 3.), after 3 months product use, in the active treatment group vs placebo. The active product was perceived as effective by most of the subjects participating in the study (Figure 4).

Discussion

It’s nowadays clear that nutrition has an influence on hair loss [45-47]. This awareness fostered the food supplement industry in researching and developing products effectively helping the management of hair loss. In this trial we studied the efficacy of a commercially available food supplement (Alline proMEN) containing a patented keratin (Keramax®), venus hair fern extract and a combination of 11 vitamins and two minerals in men suffering from hair loss.

Conclusion

Although there is evidence in the literature of the efficacy for many of the ingredients used in the test product, to the best of our knowledge this is the first study reporting the benefits of their combined supplementation. While, most often, the food supplements efficacy is not investigated and their marketing claims relies on the literature on the ingredients, we preferred to test the finished product to give the real proof of product efficacy. In fact, ingredient claims referring to the properties of one or more specific ingredients shall not imply that the finished product has the same properties when the real product efficacy was not investigated. This is commendable and should be the preferred approach to avoid crowding the market with products of unknown efficacy.

Even in a reversible form of hair loss, the test product demonstrated to be effective in decreasing hair loss more quickly when compared to the placebo product. A quicker decrease of the percentage of telogen hair, in the real life, is related to a decrease of the time span in which subjects are worried about hair loss. Interestingly, the product was also effective in improving the hair mechanical properties and hair radiance. The improvement of these parameters was also correlated to an improvement of hair structure and was visible both to the investigator (clinical analysis) and to the subjects participating in the study (self-assessment que stionnaire).

In conclusion the oral supplementation with Alline proMEN for 3 months was effective in speeding up the resolution of the hair loss in men, in improving the hair physical and mechanical properties and was welltolerated. The product is then a safe and effective way to address hair loss in men.

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