The Clinical Evaluation of Serum WS Biotin, a Novel Encapsulated Form of D‐Biotin With Improved Water Solubility, for Anti‐Hair Shedding Applications. A Prospective Single‐Arm, Nonrandomized, Pretest−Posttest Study

ABSTRACT

Background and Aims

Hair shedding is an aesthetic concern increasingly being addressed by the cosmetic industry. Biotin has long been used as an oral supplement to promote hair growth and strength, without solid evidence supporting this practice. Recently, we have shown that encapsulated biotin has promising bioactivity in hair growth‐related parameters in vitro. For this reason, this study aimed at evaluating the association between the application of Serum WS Biotin, a novel water‐soluble form of D‐Biotin, and hair shedding and strength, after topical application for 3 months.

Methods

A cohort of 22 volunteers with moderate to excessive hair shedding was subjected to daily application of Serum WS Biotin for 3 months. Baseline and follow‐up (Days 30, 60, and 90 after the application started) Modified Pull Test (MPT), Modified Hair Wash Test (MHWT), and phototrichogrametry measurements were conducted on the study cohort.

Results

Hairs pulled through MPT were reduced by 43.0% at D30, 56.0% at D60, and 76.1% at D90. Regarding MHWT, the number of vellus hairs (< 3 cm) was significantly reduced by 90.6% at D30, 67.0% at D60, and 74.8% at D90. Concomitantly, pulled hairs > 3 cm significantly diminished by 69.4% at D60 and 60.0% at D90. Contrarily, a significant increase of 227.5% was recorded at D30. Last, the hair thickness median rose significantly by 7.5% at D30, 10.6% at D60, and 7.6% at D90. Consistently, hair thickness mean was nonsignificantly increased by 4.6% at D30, 18.0% at D60, and 17.8% at D90. Interestingly, results also provided a nonstatistically significant improvement in hair mass by 3.2% at D30 and 10.4% at D90.

Conclusion

The 3‐month application of Serum WS Biotin displays anti‐hair shedding and strengthening capabilities, showing good cutaneous acceptability and compatibility.

1. Introduction

Biotin, also called vitamin B7 or H, plays important roles in cell metabolism and, more recently, in gene expression, immune function, and cell proliferation [1]. Structurally, biotin is a heterocyclic compound with an appearance of white crystalline powder, based on a sulfur‐containing tetrahydrothiophene ring fused to a ureido group, functioning as an essential coenzyme in several metabolic processes, primarily related to the catabolism of amino acids, fatty acids, and carbohydrates. Biotin is considered essential for supporting many body structures, including the nervous system, liver, eyes, hair, skin, and nails [2], together with cellular growth and proliferation processes [3], and development [1]. Therefore, it is unsurprising that a deficiency in this crucial micronutrient results in a range of clinical issues, including growth retardation, neurological disorders, and dermatological problems related to skin rashes, hair loss, and brittle nails.

In nature, the biotin molecule (PKa = 4.5) exists in the form of eight stereoisomers, but only one of them, D‐biotin, occurs naturally and has full vitamin activity, being an essential cofactor for carboxylases in the intermediary metabolism [4]. Plant cells, together with some microorganisms, as specific bacteria and yeast, can synthesize biotin endogenously, unlike mammalian cells. Thus, mammals must obtain this molecule through dietary intake and bacterial synthesis in the gut. The daily biotin requirement (30 μg) is largely met with a regular diet; thus, the contribution of biotin synthesized by gut bacteria to the overall biotin status in humans remains uncertain [5]. Organ meats like liver and kidney, vegetables, egg yolks, milk, bananas, nuts and seeds, pork chops, salmon, or mushrooms, have been described as good sources of vitamin B7.

One of the main problems of biotin use lies in its low solubility in water and other pharmaceutically acceptable solvents [6], giving rise to poor bioavailability and reduced functionality when orally administered through food supplements, even though oral biotin is widely promoted for hair, skin, and nail health. This is probably one of the main reasons why scientific evidence supporting the clinical efficacy of biotin supplementation is limited. Previous research and reviews have indicated that biotin supplementation may benefit individuals with hair or nail conditions, such as brittle nail syndrome or uncombable hair syndrome. Nonetheless, there is a lack of robust evidence supporting its efficacy in healthy individuals without a predisposition to hair or nail deficiencies [7].

On the other hand, in vitro studies in human cell lines have clearly demonstrated the mechanism of action through which biotin could provide a beneficial effect on biological pathways related to hair strengthening and loss. Indeed, we demonstrated in previous studies that WS Biotin, a novel water‐soluble form of D‐Biotin in microcapsules, enhanced the expression of hair‐related keratins 40 (KRT40) and 85 (KRT85) after treatment in human hair follicle keratinocytes, as well as the expression of genes Vascular Endothelial Growth Factor (VEGF), Fibroblasts Growth Factor 7 (FGF7), and Insulin‐like Growth Factor 1 (IGF1) in human follicle dermal papilla cells [8]. KRT40 is a type I keratin, expressed in the hair cuticle, with a putative role in late hair differentiation, whereas KRT85 is a type II cuticular protein, whose function is to heterodimerize with type I keratins to form hair and nails [9]. On the other hand, FGF7 is a mediator of proliferation and differentiation in a wide variety of epithelial cells, including keratinocytes [10]. The upregulation of VEGF has been involved in several approaches leading to successful hair growth, either in vitro or in vivo [11, 12]. Furthermore, additional studies in animal models have demonstrated that IGF1 influences follicular proliferation, tissue remodeling, and the hair growth cycle, as well as follicular differentiation in vivo [13]. Notably, recent research has shown that IGF1 is regulated by androgens and its expression is significantly lowered in balding follicles compared with nonbalding ones. These findings highlight the crucial role of this growth factor in maintaining hair follicles [14].

Hair is one of the defining features of mammals, with crucial roles in heat retention, sexual dimorphism, attraction of mates, skin protection, and environmental sensing [15]. Several factors, including the increased prevalence of hair disorders, the rising aesthetic concerns, and the consumer preferences for noninvasive treatments, have brought the need for advanced cosmetic formulations aimed at fighting hair loss and weakness. In line with this, we previously developed Serum WS Biotin by applying a new microencapsulation technology, and demonstrated the enhanced water solubility of D‐Biotin in this formulation, simultaneously maintaining its bioactivity in in vitro tests [8].

Against this background, the present work aimed to assess the association between Serum WS Biotin application and hair loss and strength, after topical application in volunteers during 3 months.

2. Materials and Methods

2.1. Ethical Statement

The study protocol is in accordance with the guidance of the Scientific Committee on Consumer Safety (SCCS). It meets all international standards for research studies involving human subjects, Structure and Content of Clinical Study Reports from ICH Harmonized Tripartite Guideline, International Recommendations ICH Topic E6, European Parliament and Council Guideline 2001/20/CE, the Good Clinical Practices (ICH‐GCP), and the World Medical Association. It has been conducted pursuant to the Declaration of Helsinki (1964), with the amendments of Tokyo (1975), Venice (1983), Hong Kong (1989), South Africa (1996), Edinburgh (2000), Seoul (2008), and Fortaleza (2013). The participants read, signed, and dated the informed consent document to indicate their authorization to proceed and acknowledge their understanding of the contents. Additionally, all the volunteers included in this study had previously read, understood, and signed the Protection of Personal Data and Communication consent.

2.2. Study Population

The study cohort was composed of 22 volunteers (Supporting Information S1: Table 1) treated between October 2023 and January 2024, aged from 28 to 47 years (mean 43.3 ± 7.2), suffering from moderate to excessive hair shedding (grades 4–6 in the Sinclair Hair Shedding Scale, shown by Kovacevic and colleagues [16]) according to self‐reporting by participants (Supporting Information S1: Table 1), last participation in a clinical study for hair ending at least 4 months before the start of this study, being submitted to 15 days of wash‐out phase without any specific hair efficacy treatment (anti‐hair loss, strengthening…), and a willingness to comply with instructions. In contrast to Kovacevic et al., grade 4 was considered moderate hair shedding, based on our experience. Thus, this hair loss extent was included as it was considered useful to assess hair loss improvement. The exclusion criteria were subjects with a history of any form of skin cancer, melanoma, lupus, psoriasis, connective tissue disease, diabetes, or any disease that would increase risk associated with study participation, subjects with relevant cutaneous marks in the experimental area, which could interfere with the instrumental measurements or the clinical pictures (surgeries, scars, sunburns, etc.), subjects presenting allergy or reactivity to any of the components of the test product, or a product within the same or similar category than the tested one, individuals currently undergoing medical treatment that may mask or interfere with the test results, subjects with relevant hair aesthetic treatments (mesotheraphy, microneedling, platelet‐rich plasma, follicular unit extraction or follicular unit transplantation, laser or intense pulsed light removal, autologous procedures such as VieStem, etc.), on the experimental area upon their lifetime, females who were pregnant or lactating or had been pregnant or given birth within the 6‐month period immediately preceding the start of the study, and subjects with a forecast of change of routine or relevant way of life, during the period of study.

2.3. Test Sample and Product Application

A shampoo formulation (Serum WS Biotin), containing 1.2% w/w WS Biotin (0.55% free biotin), was prepared for the treatment. Each participant was provided with two samples of 100 mL.

The test product under experimentation in this project had previously been confirmed to have good cutaneous compatibility and acceptability in safety studies.

The experimental area was the whole hair scalp, and the product was applied on the hair root, according to the following instructions: “apply to the scalp, opening the hair in different sections to encourage the product to be applied on the root and not on the hair. Cover the entire head with a soft massage and wait a few minutes before resting the head on any surface, without rinsing off.” Around 4 mL per application was used, according to the consumption control (Supporting Information S1: Table 2). The first application and demonstration were conducted at the clinical facilities by the clinical researcher or the corresponding technician. The application was conducted at home once per day and at night.

Participants were instructed to wash hair between two and three times per week, noting down the day of each washing in the Volunteers' Diary, using the same neutral shampoo without additional anti‐hair loss effects during the whole period of treatment. Hair washing immediately after the application of the treatment was forbidden.

2.4. Instrumental Assessment of Efficacy

Fifteen minutes before every measurement, the volunteers were subjected to an acclimatization period under controlled temperature (23 ± 1°C) and controlled relative humidity (45 ± 10%). During the acclimatization period, the test area was not covered by clothes. Measurements were conducted on dry hair, without previous treatment application.

Pull Test and Modified Hair Wash Test (MHWT) were conducted at clinical facilities, according to the protocols explained below, at different timepoints. The Pull Test was repeated three times in four different areas of the scalp (frontal, vertex, parietal, and occipital), whereas MHWT was repeated twice to ensure a complete wash for all the volunteers, including those with long hair. Phototrichogrametry was conducted on four different areas of the scalp (frontal, vertex, parietal, and occipital) using TrichoScan® HD 4.0 Professional (DermoScan GmbH).

All the measurements were performed before the start of the treatment (D0), after 1 month of treatment (D30), after 2 months of treatment (D60), and after 3 months of treatment (D90). The study design is summarized in the flow chart depicted on Figure 1.

Figure 1.

Summarized workflow of the clinical study design.

2.5. Modified Pull Test (MPT)

The pull test is a commonly used clinical assessment noninvasive method to evaluate hair shedding and is often employed in anti‐hair loss clinical testing. It is also known as the “traction test,” “Sabouraud's sign,” or the “pull‐out sign” [17, 18]. Between 40 and 60 hairs were selected, and the bundle close to the scalp was held between the thumb, index finger, and middle finger (Supporting Information S1: Figure 1). Then, the bundle was firmly pulled on using slow traction as the fingers slide down the hair shaft, avoiding a fast and forceful tug, and the pulled hairs were counted. This protocol was repeated three times in four different areas of the scalp (frontal, vertex, parietal, and occipital), following always the same order in the same areas at each of the different visits. To maximize the potential effects of the treatment, individuals were requested to avoid shampooing for 5 days before each of the measurements at D0, D30, and D60.

Pull Test is usually conducted as a qualitative (positive or negative) method, with more than 10% hairs pulled being considered active hair shedding [19]. Here, to improve the reliability and reproducibility of the assay, the total number of pulled hairs was recorded as a quantitative measurement, and therefore, the methodology is referred to as MPT.

2.6. MHWT

The Wash Test or Hair Wash Test provides valuable insights into the ability of a product to minimize hair loss under conditions that simulate real‐world activities such as shampooing [20]. Individuals were requested to avoid shampooing for 5 days. After that, hair was thoroughly washed with neutral shampoo in a sink for 5 min and rinsed. This protocol was repeated twice to assure a complete wash (Supporting Information S1: Figure 2). The hairs trapped in a gauze used to cover the draining hole of the sink were collected in a plastic box containing blotting paper for 3–4 days. After drying, hairs were classified by length (>/< 3 cm) and manually counted. The global number of shed hairs indicates the severity of hair loss (telogen effluvium), while the percentage of vellus hairs indicates the severity of androgenetic alopecia [21].

2.7. Phototrichogrametry

TrichoScan® HD is a medical product for video dermoscopy to support the diagnosis of alopecia and to monitor the therapeutic progress of hair loss treatment [22, 23]. TrichoScan HD has been conceptualized for taking and archiving digital images with minimal time exposure, within the minimum possible required space, and with minimal requirements for the site. The recorded photographs were loaded into the TrichoScan software, which automatically proceeds with the analysis. There are two modes that can be used: TrichoScan and Trichogram. Here, for hair loss and strengthening, TrichoScan mode was used since Trichogram mode is mainly used for hair growth after clipping a specific region of hair, which was not the case in this specific study.

Phototrichogram measurements were performed in four different areas of the scalp (frontal, vertex, parietal, and occipital) opening the hair in different sections to encourage the image is obtained from the root and not from the hair. The average of the four different areas was represented and analyzed in the results. Hair was neither shaved nor cut before these measurements, as they are conducted for hair growth assessment, since we wanted to focus on hair loss and strength.

The following parameters were obtained by using the TrichoScan: Hair mass in mm/cm² (the cumulated thickness of hairs normalized for an area of 1 cm²), hair thickness mean in μm (the mean of the distribution of the hair thicknesses), and hair thickness median in μm (the median of the distribution of the hair thicknesses, being the median a more robust value, because especially short and long hairs are less important for statistical analyses than those in the middle), were obtained from the TrichoScan mode for each of the measurements.

Since all the measurements at the different timepoints were conducted following the same procedure for all the volunteers, the possibility of elliptical section of hair, which could introduce a deviation in the experiment, was considered random in the quantification, and therefore not to have a relevant impact on the trial results and conclusions.

2.8. Statistical Analysis

All 22 volunteers completing the study were considered to assess the efficacy of the test product. Parameter values in the experimental area of an individual at a given time point were normalized versus average baseline measurements of the same experimental area for the whole cohort. This way, the interindividual random variation is corrected, and the statistical power of the result is increased [24].

The individual results were expressed in percentage relative to baseline values (D0) for all the parameters studied. Mean ± Standard Error of the Mean (SEM) were calculated and shown in the graphs. * denotes statistical significance with p value < 0.05. ** denotes statistical significance with p value < 0.01. *** denotes statistical significance with p value < 0.001. **** denotes statistical significance with p value < 0.0001.

GraphPad Prism V10.1.2 software was used for statistical analysis. Data were statistically analyzed applying one‐way analysis of variance (ANOVA) with Dunnett's multiple comparisons test, since one‐way ANOVA is a technique that can be used to compare whether more than two sample means are significantly different or not (using the F distribution) [25, 26]. The normality of the data was determined through the D'Agostino and Pearson tests, the Anderson–Darling test, the Shapiro–Wilk test, and the Kolmogorov–Smirnov test. In light of these tests, normality was assumed for D0, D30, and D60 data.

ROUT (Robust regression and Outlier removal) method was used to identify outliers in the raw data, with the highest level of restriction (coefficient Q of 0.1%), to maintain the human intravariability inherent to clinical testing.

3. Results

3.1. The Application of Serum WS Biotin Greatly Reduces Hair Shedding

To assess the effect of Serum WS Biotin on the strength of hair‐scalp anchoring, we counted the number of pulled hairs using the MPT. Interestingly, the treatment with this formulation significantly reduced the number of hairs pulled with the MPT by 43.0 ± 7.6% after 1 month of treatment (D30), 56.0 ± 7.7% after 2 months of treatment (D60), and 76.1 ± 9.0% after 3 months of application (D90) (Figure 2, Supporting Information S1: Table 3). Overall, these data support the notion that topical application of Serum WS Biotin strongly reduces hair shedding and, thus, could be greatly beneficial to prevent hair loss.

Figure 2.

Hair strengthening effect of Serum WS Biotin in Modified Pull Test (MPT). Graphical representation of the number of hairs counted after three replicates of MPT, before (D0), after 1 month (D30), 2 months (D60), and 3 months of treatment (D90), with Serum WS Biotin, in the study cohort.

3.2. Serum WS Biotin Ameliorates Hair Retention in a Hair Wash Scenario

In a more physiologic and representative of the daily life approach, the number of hair fibers released in the MWHT was assessed. In this regard, results indicated that the treatment with Serum WS Biotin significantly reduced the number of vellus hairs (shorter than 3 cm) by 90.6 ± 13.4% after 1 month (D30), 67.0 ± 15.1% after 2 months (D60), and 74.8 ± 13.0% after 3 months of application (D90) (Figure 3A, Supporting Information S1: Table 4). Thus, in a condition of AGA, the application of this formulation would likely show remarkable benefits. Further research would help clarify this issue, as most individuals of the cohort were female.

Figure 3.

Hair strengthening effect after Modified Hair Wash Test (MHWT). Graphical representation of the number of hairs, shorter than 3 cm (A) and longer than 3 cm (B), counted after two replicates of MHWT before (D0), after 1 month (D30), 2 months (D60), and 3 months of treatment (D90), with Serum WS Biotin, in the study cohort.

Similarly, the treatment provoked a significant reduction of the number of hairs longer than 3 cm by 69.4 ± 21.1% after 2 months of treatment (D60) and 60.0 ± 22.8% after 3 months of application (D90), thus indicating a milder telogen effluvium after those periods of treatment. Conversely, after 1 month of treatment, a significant increase by 227.5 ± 58.5% was detected (Figure 3B, Supporting Information S1: Table 4).

The absolute values for hair loss at baseline (D0) indicated a mean average of hairs lost around 65, whereas the Sinclair Hair Shedding Scale classifies grades 4–6 with 200 and 750 hairs lost after washing. These differences are most likely due to the different hair wash procedures conducted within different laboratories since this protocol is very dependent on parameters like duration, number of repetitions, classification between shorter and longer hairs, and the manual protocol of the specific technician, among others.

3.3. Hair Density and Thickness are Improved Upon Serum WS Biotin Application

Once assessed the effects of the formulation in hair shedding and anchorage, the density and thickness of the hair fibers were assessed by phototrichogrametry. This technique revealed that the treatment with Serum WS Biotin significantly increased the hair thickness median by 7.5 ± 2.3% after 1 month (D30), 10.6 ± 3.0% after 2 months (D60), and 7.6 ± 2.7% after 3 months (D90) (Figure 4A). Consistently, hair thickness mean was also increased by 4.6 ± 2.7% at D30, 18.0 ± 9.8% at D60, and 17.8 ± 9.8% at D90, in a nonstatistically significant manner (Figure 4B). Interestingly, results also provided a nonstatistically significant improvement on hair mass by 3.2 ± 6.5% at D30 and 10.4 ± 12.1% at D90 (Figure 4C). Taken together, these results point toward a general improvement in hair density, mainly supported by the increase in hair diameter. Further studies with a longer duration would help clarify whether the application of Serum WS Biotin displays the ability to increase hair mass in a statistically significant manner.

Figure 4.

Effect of Serum WS Biotin on hair thickness and density. Graphical representation of the hair thickness median (A), hair thickness mean (B), and hair mass (C) using TrichoScan HD 4.0 Professional, before (D0), after 1 month (D30), 2 months (D60), and 3 months of treatment (D90), with Serum WS Biotin, in the study cohort.

4. Discussion

Biotin is an essential cofactor for some key metabolic pathways, with more recently discovered functions in other processes, such as proliferation or immune functions. A deficiency in this crucial micronutrient results in a range of clinical issues, including growth retardation, neurological disorders, and dermatological problems related to skin rashes, hair loss, and brittle nails, among others [27]. For this reason, clinical studies involving exogenous administration of biotin have focused so far on the treatment of the previously mentioned conditions, with data supporting the beneficial effect of this vitamin, particularly on hair loss, in cases with an underlying deficiency [28]. Nonetheless, to our knowledge, no interventions involving biotin administration, either topically or in healthy individuals, have been published so far.

Based on our previous in vitro findings suggesting potential benefits of Serum WS Biotin in the biology of hair follicles, this study aimed to evaluate the clinical efficacy of Serum WS Biotin against hair loss.

The results from our MPT revealed a pronounced and time‐dependent reduction in the number of hairs pulled, and a marked reduction in the number of hairs shed upon daily activities, such as hair washing. In the case of the sharp increase observed in hair fibers longer than 3 cm after 1 month, although greater hair loss during the first month of treatment, even working, is usually observed possibly due to anagen stage may be initiated while the telogen hair is still in place, further research is guaranteed to clarify the reason and mechanism underlying this phenomenon. A reasonable hypothesis would be that the treatment could induce an initial stress derived from exogen substances, which would eventually trigger a telogen effluvium scenario. After an adaptation period to the formulation (which would be longer than 1 month in light of our results), the anagen phase would be restarted in the follicles initially affected, with an improved anchoring to the hair scalp. Another possible scenario would be that our formulation could promote a massive anagen across the scalp and that initial hair shedding in long fibers would arise from the necessary telogen preceding the anagen. This hypothesis would be compatible with an enhanced expression in the dermal papilla of well‐known hair growth‐promoting genes VEGF, FGF7, and IGF1, as found in our previous in vitro tests [8].

Regarding phototrichogrametry, the data collected in this study clearly show an improvement in the thickness of hair fibers upon application of Serum WS Biotin from 1 month onwards. These results are compatible with our in vitro observations, which would support an enhanced keratinization via the upregulation of KRT40 and KRT85 [8], thus contributing to a thicker fiber. Concerning hair mass, although additional evidence would help clarify this issue, there was an increasing trend after long‐term application (3 months). Comparing with the in vitro results showing an increase in the expression of hair growth genes in follicle dermal papilla cells and an increasing trend in the proliferative ability of hair follicle keratinocytes [8], it cannot be discarded that a longer application would result in a statistically significant increased hair mass, derived from cell proliferation inside the hair follicle.

It is also important to remark that the study was conducted between October 10th, 2023 and January 12th, 2024. The mean weekly temperature in Valencia at baseline (Day 0) was 21°C, whereas at the end of the treatment (Day 60) it was 11°C, according to Weather Underground history (online). Since a placebo group was not included in the study, due to budget reasons, future research would be necessary to confirm that the beneficial effects observed are due to the application of Serum WS Biotin, and not due to seasonal changes‐related interference.

In conclusion, the data herein suggest beneficial effects and good cutaneous acceptability and compatibility of Serum WS Biotin as an active component in cosmetic formulations targeting hair loss improvement and strengthening.

Author Contributions

David González Fernández: conceptualization, writing – original draft, writing – review and editing, methodology, formal analysis, supervision, resources. Shaher Duchi: conceptualization, investigation, writing – review and editing, validation, project administration, resources, supervision. Lucía Fernández Gómez: investigation, methodology. Teresa León Sala: investigation, methodology. Akram Hajuj: investigation. Danit Molho: investigation. Nadeen Abo Saada: investigation. Daniel Molina Martínez: investigation, methodology, visualization, formal analysis, data curation. Alejandro Pérez‐Fernández: writing – original draft, writing – review and editing, visualization, supervision. Danny Goldstein: conceptualization, writing – review and editing, supervision, resources.

Conflicts of Interest

Serum WS Biotin is a commercial product of Tagra Biotechnologies. This study was partially conducted by personnel of and funded by Tagra Biotechnologies.

Transparency Statement

The lead authors, Alejandro Pérez‐Fernández and Danny Goldstein, affirm that this manuscript is an honest, accurate, and transparent account of the study being reported, that no important aspects of the study have been omitted, and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

Supporting information

Supporting information.

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article and its supporting materials.