Therapeutic effects of growth factor cocktail (CellcurinTM) containing FGF5s (fibroblast growth factor 5 short) and NMN (nicotinamide mononucleotide) in patients with androgenetic alopecia: A split study

Background: Growth Factor Cocktail (GFC), which contains growth factors and cytokine, has been gradually gaining attention as a new treatment for hair loss. Several studies reported the effectiveness of GFC on hair regrowth via microneedles; however, studies regarding formulations with fibroblast growth factor 5 short (FGF5s) and nicotinamide mononucleotide (NMN) have not been conducted. Objective: This study aimed to investigate the effect of treatment with microneedle technology by adding FGF5s and NMN to GFC in patients with androgenetic alopecia (AGA). Methods: In this study, AGA patients were treated six times for a total of 3 months at 2-week intervals from the first visit. The scalp was divided into two sections at the time of treatment; the right side was treated with GFC with FGF5s and NMN, and the left were treated with normal saline, with microneedles to a depth of 0.8 mm. A total of 20 patients (11 males and 9 females) were included in the study. Clinical photographs and phototrichogram were performed before and 2 weeks after treatment to determine the final treatment effect. Results: The phototrichogram images of the scalp treated with GFC for 3 months showed increased hair density and diameter. In contrast, there was no change in hair density and diameter in the scalp treated with normal saline. Conclusion: GFC with FGF5s and NMN with microneedling is an effective and safe treatment of androgenetic alopecia. Future studies with more thorough investigations in controlled clinical settings are warranted. *Correspondence to: Byung In Ro, M.D., Ph.D., Invited Professor, Department of Dermatology, Myongji Hospital, Hanyang University College of Medicine, Goyang 10475, Korea, Tel: +82-31-810-7050; Fax: +82-31-810-7057; E-mail: drro@mjh.or.kr


Introduction
Androgenetic alopecia (AGA) is a disease of the scalp that is common form of hair loss in both men and women. Recently, the oral administration of finasteride and dutasteride and topical application of minoxidil have been commonly used as treatment [1][2][3]. Furthermore, surgical methods such as scalp skin auto-implantation and follicle autograft have been implemented [1][2][3]. Recently, several studies on various cytokines contributing to the growth and differentiation of hair have been conducted, biochemical therapies different from conventional treatments for alopecia have been emerging. Accordingly, a growth factor cocktail (GFC) containing several growth factors and cytokines is currently being introduced. In addition to studies on GFC efficacy, we reported on the therapeutic effect of GFC formulations with FGF9 that promote the onset of growth within the hair cycle. The core aspect of this treatment is the transdermal absorption of GFC through a microneedle, which displays a therapeutic effect via absorption through the hair follicle. Fibroblast growth factor 5 short (FGF5s) prolongs the growth process by inhibiting the transition of the anagen to the catagen, while nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide+ (NAD+), is involved in cellular energy metabolism and helps promote hair growth. In this Therapeutic effects of growth factor cocktail (Cellcurin TM ) containing FGF5s (fibroblast growth factor 5 short) and NMN (nicotinamide mononucleotide) in patients with androgenetic alopecia: A split study

Parameters measurement
A tattoo point was applied to the right and left side of the scalp of each patient before treatment to compare hair growth. A phototrichogram (Folliscope ® 2.8; Lead M, Seoul, Korea) was taken from a fixed area marked with a tattoo on both sides to measure hair density and diameter at the baseline, and at the end of the treatment. An unbiased investigator, unaware of the study, counted the number of hairs and the hair diameter by using phototrichogram images.

Statistical analysis
All data are expressed as mean ± standard deviation. Wilcoxon matched-pairs signed-rank test was performed using STATA/SE ver.12 (Stata Corp LP, College Station, TX, USA) to test the effectiveness of the treatment on the same patients. The Student's t-test was conducted to analyze the differences between the effect of the treatment on the right and left sides of the scalp. The statistical significance was calculated for p-values <0.05.

Patient characteristics
Twenty out of 24 patients aged 22-47 years (mean, 35.9±6.5 years) completed a 12-week treatment course. The patients comprised: 11 male patients with a mean age of 37.5±6.4 years, and male pattern hair loss type Ⅱ (2 patients), type Ⅲ (6 patients), type Ⅳ (3 patients) according to the Hamilton-Norwood classification; 9 female patients with a mean age of 33.9±5.9 years, and female pattern hair loss type Ⅰ(6 patients), type Ⅱ (3 patients) according to the Ludwig scale (Table 1).

Treatment efficacy and adverse effects
The measurement was taken before and after treatment to compare the density and thickness of hair, respectively. In the phototrichogram images, the density and thickness of the hair increased on the right side of the scalp ( Figure 2). The change of hair density and diameter between the right and left sides after treatment are shown in Table 2. On the right side of the scalp, hair density increased from 182.5±13.1/cm 2 to 188.7±13.4/cm 2 (p-value<0.0001) and hair diameter increased from 62.1±9.3 µm to 64.05±9.2 µm (p-value<0.0112). On the left side, there were no significant differences; from 181.9±16.4/cm 2 to 181.9±15.8/cm 2 (p-value=0.3650) in hair density, and from 62.1±8.3 µm to 62.4±8.7 µm (p-value=0.4076) in hair diameter ( Figure 3). In addition, there were no adverse reactions related to the treatment except for minimal pain.

Discussion
Androgenetic alopecia is the most common type of progressive loss of terminal hairs on the scalp in a characteristic distribution [4]. AGA is an androgen-dependent disease, and dihydrotestosterone (DHT) is thought to have the greatest effect on follicular changes [5]. DHT is a metabolite of testosterone and has a higher affinity for androgen receptors than testosterone. DHT acts on the dermal papilla of the hair where the androgen receptor and type 2 5-reductase are frequently   Table 2. Statistical analysis of the changes in hair density and diameter between baseline and 2 weeks after treatment expressed, which induces the miniaturization of hair follicles and converts terminal hair into vellus hair [6]. Although the cause of female pattern hair loss is still unclear, studies have shown that it is related to the male pattern hair loss, which is also partially contributed to by hormone imbalance [7]. In men, hair loss starts from the frontal line, followed by vertex thinning, and finally complete baldness, which falls under the Hamilton-Norwood classification. Female pattern hair loss appears as hair loss in the centro-parietal region while the frontal hairline is maintained and is classified as the Ludwig type. Currently, the most commonly used FDA-approved drugs for the treatment of hair loss are finasteride and topical minoxidil, while medical device based treatment includes low-level laser light therapy (LLLT), and meta-analysis for the effect of the treatment for each method showed a significantly higher therapeutic effect than the placebo group [1].
Hair follicles arise in developing skin due to a complex set of interactions that are likely to be mediated by diffusible, cell-and matrix-bound factors. Various hypotheses such as epithelial-mesenchymal interaction, extrafollicular growth-modulating signals such as hormones, growth factors, and other dietary changes, are introduced to the hair follicle cycle mechanism [8]. We have conducted several studies to date, focusing on the effect of various hair growth factors on hair follicles to treat hair loss. In this study, 7 growth factors (IGF-1, stable aFGF, SCF, KGF-2, FGF9, noggin peptide, and protaetide) that contribute to the growth of the previous hair were used in the form of basic solutions with the addition of FGF5s and NMN. IGF-1 has been shown to increase the number of hair follicles and prolong the growth phase [9]. Furthermore, it stimulates the proliferation of follicle cells in the anagen phase and downregulates the expression of transforming growth factor β1(TGF-β1) in hair follicles in a dosedependent manner, thus preventing hair follicles from entering the catagen state [9]. SCF has been demonstrated to play a significant role in skin and hair pigmentation by activating melanocytes through the c-kit pathway [10]. Cultured dermal papilla cells obtained from patients with AGA secrete less SCF than normal cells, owing to the inhibition of SCF production by androgens in the dermal papilla [10]. KGF has been reported to protect the epidermis and hair follicles from cell death induced by ultraviolet irradiation, chemotherapeutic drugs, or cytotoxic agents [10]. FGF has been implicated in the control of epidermal and mesenchymal cell function, and it is likely that they also affect the proliferation and differentiation of cells in the cutaneous appendages during development [11]. Immunolocalization of the basic FGF (bFGF) adjacent to areas of proliferation in developing and in mature follicles suggests that this factor may regulate the mitotic activity of epithelial-derived cells; while acidic FGF (aFGF); appears in the differentiating cells of the follicle bulb and thereby might participate in the formation of structural components of the follicle or fiber [11]. FGF9 has been reported to be a triggering factor for Wnt expression and as a hair follicle regenerator [12]. Noggin is an antagonist of bone morphogenetic protein (BMP), which suppresses the transition of telogen to anagen [13]. Overexpression of noggin enlarges the size of anagen hair follicles [13]. FGF5s is a growth factor that promotes hair growth by blocking the activity of FGF5, which inhibits hair growth. It has been confirmed that FGF5s has an action that delays the anagen phase and upregulates factors important for hair growth such as IGF-1 and noggin [14][15][16]. NMN is a precursor of NAD+, and NAD+dependent signaling plays an important role in the catabolic reactions and energy production in the cell [17]. NAD+ is an essential pyridine nucleotide that plays major roles in several critical biological processes, including oxidative phosphorylation and adenosine triphosphate (ATP) production, and the synthesis of cholesterol, fatty acids, and steroids [18].
Lee et al. [19] reported the effects of GFC with microneedling on short-term treatment in women with female pattern hair loss over a 5-weeks period. Our previous studies confirmed that GFC treatment effectively results in hair growth and thickening, and that the effect differed in accordance with the microneedle depth. Hair density increased by 17.5% in the scalp injected with GFC including bFGF, vascular endothelial growth factor (VEGF), KGF-2, SCF, IGF-1, superoxide dismutase-1 (SOD-1), and FGF9 at a depth of 0.8 mm and 4% in the scalp treated using normal saline [20]. Ro et al. [21] reported that the density and thickness of the hair significantly increased after applying a solution containing IGF-1, bFGF, VEGF, SCF, KGF-2, noggin peptide, and SOD-1 to a depth of 0.5 mm. Ro et al. [22] applied GFC including IGF-1, bFGF, VEGF, SCF, KGF-2, noggin peptide, SOD-1, FGF9, and FGF5s for 24 weeks using iontophoresis instead of microneedling and reported that the number of hairs significantly increased. Compared to the previous studies [20,21], FGF5s and NMN  were added to the GFC, and the changes in the density of the hair were not significant in this study.
Recently, it has been suggested that microneedling facilitates the penetration of drugs and is one mechanism by which it promotes hair growth. The micro-injury caused by microneedling leads to minimal superficial bleeding and causes a wound healing cascade that releases of various growth factors, such as the platelet-derived growth factor (PGF), which transforms the growth factor-alpha and beta (TGF-α and TGF-β), connective tissue activating protein, connective tissue growth factor, and fibroblast growth factor (FGF) [23]. Repeated microneedle stimulation induces hair growth and increases Wnt3α, β-catenin, VEGF, Wnt10b mRNA and protein expression in a murine model [23]. The microneedle needs to be deep enough to penetrate the skin barrier for enhanced drug delivery, but also short enough to cause minimal skin injury and pain. Through previous studies, it was found that the appropriate depth is 0.5~0.8 mm; and therefore, the deepest depth in this range was selected in this study to obtain the maximum effect [20,23,24]. However, we observed no significant difference in hair density and thickness in the left scalp that was treated with normal saline.
The results of this study were less effective than the results from previous studies that used GFC with VEGF, which strongly induced perifollicular vascularization during the anagen phase. Since microneedling increases VEGF, the hair growth effect was greater when VEGF was used. In addition, the effect on the left scalp was minimal, which could be attributed to improper or weak transmission of power from the clinician to the scalp. Therefore, it can be concluded that the power of microneedling was also minor in the right scalp, and thus the hair density did not increase significantly in both the right and left sides. In addition, the hair density of participants in this study was higher than in previous studies, which suggests that there may have been a selection bias in our study. Therefore, further randomized controlled studies are needed. Other significant limitations of our study are the relatively small number of enrolled patients and the short study period. Moreover, the microneedling procedure may have errors depending on the skill of the clinician; the intensity level of the stimulations by the microneedle may have varied depending on the clinician's power. Eventually, studies with various injection depths and with controlled clinical settings are needed to determine the most effective microneedle depth.
Drugs used in AGA treatment have been reported to trigger various side effects. Finasteride deteriorates sexual function and contributes to erectile dysfunction, decreased libido, and ejaculatory dysfunction [25]. In addition, topical minoxidil has minor side effects, including contact dermatitis, irritant dermatitis and hypertrichosis of the face [26].
Several studies have demonstrated the usefulness of LLLT. Proposed mechanisms include the acceleration of cellular mitosis, the stimulation of hair follicle stem cells or follicular keratinocytes, effects to increased adenosine triphosphate production and cellular activity, and anti-inflammatory effects [27]. In our opinion, this therapy accompanied by GFC treatment seems to be an interesting option with few side effects for the treatment of AGA.

Conclusion
In this study, GFC with FGF5s and NMN is an effective treatment for hair regrowth and thickness in patients with AGA. Since this treatment has few adverse effects, it can be used for patients who are difficult to treat due to their sensitivity to side effects or corresponding contraindication to the drugs. Besides, the treatment has the advantage of being in combination with finasteride, minoxidil, or LLLT. This study confirmed the effectiveness of the treatment with GFC, which could be used in addition to AGA. However, additional studies involving many patients will be needed to confirm the effects of the treatment more accurately, and further evaluation will help determine the exact mechanism of the treatment.