Intradermal injections of a hair growth factors formulation, for enhancement of human hair regrowth – Safety & Efficacy Evaluation in a First-in-Man Pilot Clinical Study

Rinky Kapoor, MD1*,

Debraj Shome, MD, FRCS, FACS, MBA2*

1Consultant Dermatologist & Director, The Esthetic Clinics, Mumbai, India.

2Consultant Facial Plastic Surgeon & Director, The Esthetic Clinics, Mumbai, India.

Running title: Efficacy of hair growth factors formulation for hair regrowth

Conflict of Interests: The authors have no conflict of interests in any of the materials & methods utilized in this paper.

Funding: Nil.

Word Count: (Excluding title page, abstract, references, figure and table captions): 3807 words.

*Corresponding author:

Dr. Debraj Shome, Consultant Facial Plastic Surgeon,

The Esthetic Clinics,

Kandivali, Mumbai, India

Email: debraj.shome@theestheticclinic.com,

Phone: +91-22-43344600 

Abstract:

Background: Research has shown the efficacy of hair growth factors in hair regrowth. We describe the intra-dermal injections of a recombinant, bioengineered, hair formulation, containing growth factors, into the scalp skin, for enhancement of hair regrowth & evaluate its efficacy.

Objectives: The objective of this study was to assess the efficacy & safety of the hair growth factor formulation, in reducing hair loss & enhancing hair growth.

Materials & methods: This was an open label, prospective, single arm interventional pilot study, in which 1000 patients were given intradermal injections of a hair formulation, into the scalp skin. The formulation contains Vascular Endothelial Growth Factor, Basic Fibroblast Growth Factor, Insulin like Growth Factor, Keratinocyte Growth Factor, Thymosin β4 & Copper tripeptide 1; suspended in a sterile injectable vehicle. Intradermal injections of this hair formulation, were injected into the scalp, once every 3 weeks, for a total of 8 such sessions. Hair pull test was performed, before every session. Videomicroscopic & global images were taken at baseline, fourth session, eighth session, & 2 months after the completion of the 8 sessions. Relevant safety assessments through physical examination, questionnaires & appropriate laboratory examination were conducted throughout the study.

Results: Significant reduction in hair fall was seen in 83% patients, on hair pull test. Videomicroscopic image evaluation showed that most patients had a decrease in the number of vellus hairs, increase in number of terminal hairs & increase in shaft diameter. 75% patients believed that the hair injections were aiding the treatment of their hair loss, & it was also beneficial in post-hair transplant patients. At one year, a statistically significant increase in total hair count (P=0.002) continued to be seen. Treatment was well tolerated.

Conclusions: Intra-dermal injections of this hair formulation may be a promising option for treating male as well as female patterns of hair loss.

Key words: Alopecia; Hair Loss; Growth Factors; Hair growth; Novel formulation 

Introduction:

The most common cause of baldness or hair loss (95%) is Androgenetic alopecia.1 The current surgical, medical & cosmetic interventions are limited in approach & success. 2 There are several growth factors which have been found to stimulate or inhibit different stages in the hair growth cycle. Various growth factors studied for hair follicle growth are vascular endothelial growth factor (VEGF) 2, epidermal growth factor (EGF), insulin 1-like growth factor (IGF), Fibroblast growth factor (FGF) 3, Wingless-related integration site (Wnt), noggin, Keratinocyte Growth Factor, Copper Tripeptides, & more. These growth factors can be safe, cheap, & non-allergenic tools, in the management of alopecia. 4, 5, 6

So, we have prepared a bioengineered, recombinant formulation, consisting of a combination of growth factors, administered intradermally, capable of preventing hair loss & stimulating hair growth. Before conducting this pilot human study, the formulation was tested on animals to establish the concentration of the multiple ingredients, the safety & the efficacy. 7, 8

Therapeutically acceptable ranges of the growth factors used are as below:

  1. Vascular endothelial growth factor (Human oligopeptide-11) – 0.01mg/L – 100mg/L
  2. Basic fibroblast growth factor (Human oligopeptide-3) – 0.01mg/L – 100 mg/L
  • Insulin like growth factor (Human oligopeptide-2) – 0.01mg/L – 100mg/L
  1. Copper tripeptide 1 – 0.1mg/L – 500 mg/L
  2. Keratinocyte growth factor – 0.01mg/L – 100 mg/L
  3. Thymosin β4 – 0.005mg/L – 100 mg/L

In a pre-clinical trial experiment, 20 C3H mice were divided into 4 groups of 5 mice each  The backs of the C3H mice, (60 days old, telogen hair growth phase) were closely clipped on day 1 with an electric clipper. 4 different solutions were prepared, each containing different proportions of IGF 1, VEGF, bFGF, KGF, Thymosin β4 and copper tripeptide-1, in distilled water, labeled as solutions 1 through 4. Each group of mice received intradermal injection (i.e. infiltrated under the skin) of the sterile solution (group 1 received solution 1, group 2 received solution 2, group 3 received solution 3, and group received solution 4) of the respective pharmaceutical composition at 2 locations within the clipped back areas of the mice. Injection at 2 locations provided 2 test locations within the clipped area of each mouse. A separate group of 5 saline injected mice (0.1 ml of saline per mouse) served as controls.(Table 1)

Following injection of the above intradermal pharmaceutical composition, indications of hair growth were seen within 10 days. The first visual signs were darkening of the skin in a circular region surrounding the injection site. The size of this region is generally dose dependent, increasing with an increase in dose to a certain extent. The 0.1 ml injections used in this experiment produced a circle of hair growth measuring approximately 0.5 cm2 to 5 cm2 in diameter. Active hair growth occurred between 14-20 days of the injection, with a maximum effect seen on day 30. Both the number of mice growing hair at the injection site and the diameter of hair growth region were determined on day 21. A positive response was observed with respect to the number of mice exhibiting hair growth at the injection sites compared to the total number of mice injected in the study. The results of this experiment are presented in Table 2 (The day of onset is the day at which hair follicle pigmentation was first observed).

Solution 3 comprising 0.0002 mg/0.1 ml of IGF1, 0.0002 mg of bFGF, 0.0005 mg of VEGF and 0.0001 mg of KGF, 0.001 mg of copper tripeptide and 1×10-6mg of Thymosin β4 within distilled water, gave the best response. Increase in concentration of the ingredients beyond that, in solution 4, did not give any significant benefit in terms of number of mice growing hair or the diameter of hair growth region.

This hair Formulation showing the best results, called QR 678 by us, is relatively free of untoward effects as demonstrated by animal studies done to evaluate the safety & efficacy. This formulation, with the concentrations of individual ingredients so pre decided, was then used in the human trial described in this paper. A QR Code is a code used in medicine derived from “Quick Response“. 678 in Morse Code signifies “there is no answer”. This formulation has been named QR 678 to signify a “Quick Response to a disease which earlier had no answer” i.e. to Alopecia.

Table 1: Groups of C3H mice, injected with different concentrations of the growth factor formulation.

 VEGF (mg/L)bFGF (mg/L)IGF -1 (mg/L)Cu tripeptide 1 (mg/L)KGF (mg/L)Thymosin β4 (mg/L)
Solution 10.010.010.010.10.010.005
Solution 221150.50.001
Solution 35221010.01
Solution 415553020.1

Materials & methods:

This is an open label, prospective, single arm interventional pilot study, in which 1000 patients were treated for hair loss. The study was approved from the Institutional Review Board. Written, informed consents were obtained from all the patients. 

Inclusion Criteria: Indian men & women, 20-60 years of age. Patients had Male pattern / Female pattern hair loss (Male – Norwood Hamilton grades II to V & Females – Ludwig all 3 grades), which had not responded to 1 year or more of conventional medical therapy topical minoxidil (2% in case of females & 5% in case of males) along with oral 1 mg finasteride for males (We specifically excluded any patients who had recently started or stopped these medications to avoid the confounding effects that this might have created) or those who had recent post hair transplant hair loss (had undergone hair transplant surgeries, within a year before study enrollment).

Patients had to agree not to change their hairstyle or use hair color throughout the study. Patients with diabetes, hypertension, & hypercholesterolemia underwent regular monitoring of their relevant parameters. All medications which affect hair growth were required to be withdrawn 6 months before the study & were not permitted during the study.

Exclusion Criteria: We excluded patients with a hair loss history of less than 6 months. Patients with a history of multifactorial or serious drug allergy, history of or suspected malignancy, severe seborrheic dermatitis of the scalp, pregnant & lactating women were excluded from the study. 

Preparation & physicochemical characterization of the growth factor formulation (QR 678)

In a preferred embodiment, the present formulation utilizes an intradermal pharmaceutical formulation, which includes the growth factors in the concentrations, as given in the detailed formulation in Table 3, additionally along with vitamins, minerals, nucleic acids & amino acids, diluents &/or carriers along with pharmaceutically acceptable diluents &/or carriers.

The present pharmaceutical formulation as described herein above is formulated for intradermal injection to the treatment area. Suitable vehicles for injection include, but are not limited to saline & distilled water. 

Table 2: Results in the groups of mice, injected with different concentrations of the growth factor formulations.

CompositionNumber of animals growing hairDay of onsetArea of hair growth around the injection site
Solution 12/510< 1 cm diameter
Solution 23/510> 1 cm diameter
Solution 35/510> 1 cm diameter
Solution 44/510> 1 cm diameter
Solution 50NANA

Preparation of the formulation:

5 mg of VEGF, 2 mg of Basic FGF, 2 mg of IGF, 10 mg of Copper tripeptide 1, 1mg of KGF & 0.01mg of Thymosin β4 additionally with pharmaceutically/ cosmetically acceptable & appropriate dose of vitamins, minerals, amino acids & nucleic acids is added to 1 litre of distilled water. The formulation is then biologically sterilized & bottled into vials of 5 millilitre each. The formulation of the present invention is stable & can be stored at room temperature (below 25°C). 

Table 3: Final Composition of novel formulation (QR 678).

S NoIngredientsQuantity
1Vascular endothelial growth factor0.01mg/L – 100mg/L

 

2Basic fibroblast growth factor0.01mg/L – 100 mg/L
3Insulin like growth factor0.01mg/L – 100mg/L
4Copper tripeptide 10.1mg/L – 500 mg/L
5Keratinocyte growth factor0.01mg/L – 100 mg/L
6Thymosin β40.005mg/L – 100 mg/L
7VitaminsVitamin A , Vitamin B1, Vitamin B2 , Vitamin B3 , Vitamin B5 ,Vitamin B6 , Vitamin B7 , Vitamin B12 , Vitamin C , Vitamin E , Vitamin I , Vitamin K
8MineralsCalcium, Sodium, Potassium, Magnesium
9Nucleic acids, Essential and non-essential amino acids

Injections of the formulation to the scalp:

Multiple, tiny, virtually painless, intradermal injections, of the solution, were administered into the scalp skin. The injections were given using an insulin syringe, with a 31G needle. These injections were given in all areas of visible hair thinning & alopecia. A total of 1.5 ml of solution was injected per session by the nappage technique in the affected areas, once in three weeks, for 8 such sessions. Approximately 60 – 70 injections are given per session to the scalp skin in the areas of hair thinning, intradermally by the nappage technique, each injection spaced 1 cm apart, the total volume per injection being 0.02 ml.

Scalp Assessment & Evaluation:

At each visit, patients had to undergo safety assessments, which included physical examination & administration of a non-leading questionnaire about adverse experiences. Hair pull test was performed before every session, by an independent observer. The “hair pull test” was performed 3 times by the same clinician, wherein a bundle of approximately 50-60 hair are grasped between the thumb, index, & middle finger & pulled from the base close to the scalp. The hair is firmly tugged away from the scalp, & the extracted hair is counted in every session. Periodic laboratory evaluation was done, along with Global photographic assessment & video microscopic assessment to know the condition of hair growth. Subjective analysis was performed by administering the hair growth questionnaire comprising of seven questions, four relating to efficacy of treatment & three to satisfaction with the appearance of scalp hair. The final evaluation was done at 2 months after the 8th session.

Investigator Assessments:

  • Hair pull test: Assessment to determine improvement in hair loss was done before every session, with the Hair pull test. (Figure 1).

Global photographic assessment: Standardized clinical photographs of the head for clinical assessment were taken at session 1, 4, 8, & 2 months

after session 8. The vertex & superior frontal areas of the scalp were photographed using a standardized technique. Photographs were assessed by 3 independent dermatologists, who graded each image from 0 to 10, where 0 represented no growth & 10 indicated full, thick hair growth. The scores were averaged & compared before & after.

  • Videomicroscopic assessment: Videomicroscopic photographs were taken with proscope digital hand held camera, at fixed positions on the central scalp, 15 cm & 20 cm posterior to the glabella. At each fixed position, images were taken through both ¼ cm & ½ cm windows to calculate hair counts per cm2 (Figure 2 A & 2B). All videomicroscopic images were analyzed for changes in vellus hair count, terminal hair count & hair shaft diameter, using specialized software (Trilogic Company, Moscow, Russia; Tricho science version 1.5 has been available since 2008 through Merz Pharmaceuticals, Frankfurt, Germany).                                            Paired t-testing of the data was performed using Graphpad Software, an online calculator for statisticians (http://www.graphpad.com/quickcalcs/ttest1.cfm). P < 0.05 represents significant difference.
  • Patient self-assessments: Patients completed a validated, hair growth questionnaire comprising seven questions, four relating to efficacy of treatment & three to satisfaction with appearance of scalp hair. 12 (Table 4)
  • Safety assessments: Medical history of the patient was recorded at the screening visit & a complete physical examination was performed. Safety assessments included physical examination & non-leading questioning about adverse experiences at each visit, as well as periodic laboratory evaluations.

Laboratory evaluations:

Haematology & serum biochemical analysis were performed at baseline, sessions 4 & 8 & then 2 months post the 8th session.

(a)

(b)

 

Figure 2. AShows a photograph of ¼ cm cutout of videomicroscopic images showing vellus hair count (in red) and terminal hair count (in green). 2B: Shows a photograph of ¼ cm cutout of videomicroscopic image showing assessment of mean hair shaft diameter. All measurements shown were multiplied by a factor of 2.77 for conversion to microns.

Table 4. Questionnaire administered to patients, to evaluate efficacy & satisfaction with the QR 678 treatment.

Sr.
No
QuestionPossible responses
1Since the start of the study, I can

see my bald spot getting smaller

Strongly agree › Strongly
disagree
2Because of the treatment I have
received since the start of the study,
the appearance of my hair is :
A lot better › A lot worse
3Since the start of the study, how?
would you describe the growth of
your hair
Greatly increased › Greatly
decreased
4Since the start of the study, how
effective do you think the treatment
has been in slowing down your hair
loss?
Very effective › Not effective at
all
5Compared to the beginning of the
study, which statement best
describes your satisfaction with the
appearance of :a) the hairline at the front of your
head?
b) the hair on top of your head ?
c) your hair overall ?
 

 

Very satisfied › Very
dissatisfied
Very satisfied › Very
dissatisfied
Very satisfied › Very
dissatisfied

 

ResultsThis study was conducted from May 2008 to May 2016. In this study 1000 (680 males & 320 females) patients were included. The age of the patients were from 20-60 years. There were a total of 250 patients withdrawn from the study.

  • The primary factor for patient drop out seems to be the long duration of the treatment and the necessity of treatments every 3 weeks. This is further compounded by the fact that India is a very large country and our patients came from cities, which were very far from Mumbai, where the trial center was located. Hence, we had included a large number of patients to begin with, anticipating this issue of drop out.
  • Some patients realized after a few sessions that they were finding it difficult to commute every 3 weeks, for the sessions.
  • In case of some professionals, their sudden professional commitments entailed outstation travel.
  • Some women got pregnant.
  • Some participants could not resist the desire to chemically color the hair or treat the hair, with straightening, etc, during the study period. These patients had to be removed from the study as they digressed from the inclusion criteria & requirements of no chemical procedures to be performed on the hair during the study duration. 

Hair Pull Test:

Before treatment, the average number of hair pulled out was 10. After 4 sessions, the average number of hair pulled out was 3 (which is a negative pull test), in almost 83% of patients & the pull test remained negative henceforth, suggesting a reduction in hair fall, which is apparent around the 4th session (Figure 1).

Video Microscopic Pictures:

As seen in Figure 2.

Vellus hair counts: Vellus hair counts for each patient, were taken at 15 cm from the glabella, are depicted in Figure 3 (only 7 patients shown here). Overall, 86% had a decrease in the number of vellus hairs, while remaining patients had an increase. Paired t-testing indicated that, on average, the patients after 4 sessions had 8.57 fewer vellus hairs & 11.57 fewer vellus hairs after 8 sessions, than at baseline. This was statistically significant.  Vellus hair counts, taken at 20 cm from the glabella, are depicted in Figure 4. 71% patients had a decrease in the number of vellus hairs, while remaining patients had an increase. Paired t-testing indicates that, on average, the patients after 4 sessions had 3.29 fewer vellus hairs, & after 8 sessions had 7.29 few vellus hairs than at baseline. Again, this was statistically significant (p<0.05) (Table 5). 

Figure 3. Vellus hair counts at 15 cm from the glabella after fourth session (shown for seven patients) X-axis = patient serial number; Y-axis = number of vellus hairs/cm2 (series 1 = blue = baseline; series 2 = red = after four sessions; series 3 = green = after eight sessions).

 

Figure 4. Vellus hair counts at 20 cm from glabella after fourth session (shown for seven patients) (X-axis = patient serial number; Y-axis = number of vellus hairs/cm2) (series 1 = blue = baseline; series 2 = red = after four sessions; series 3 = green = after eight sessions).

Table 5. Vellus and terminal hair counts and hair shaft diameters, post QR 678 hair injection treatment.

 
Mean at baseline
Mean after 4 sessions
Mean after 8 sessions
delta
p – value
Vellus hair counts at 15 cm
52.43
43.86
40.86
-11.57
0.003131
Vellus hair counts at 20 cm
48.86
45.57
41.57
-7.29
0.006474
Terminal hair counts at 15 cm
64.14
71.71
78.71
+ 14.57
0.004183
Terminal hair counts at 20 cm
82.14
88.29
90.29
+ 8.15
0.004441
Hair shaft diameter at 15 cm (μm)
27.98
28.98
30.98 
+3
0.006361
Hair shaft diameter at 20 cm (μm)
29.74
30.71
32.77
+ 3.03
0.005161

Terminal hair counts: Terminal hair counts for each patient, was taken at 15 cm from the glabella, are depicted in Figure 5. 80% patients had an increase in the number of terminal hairs, while others patients had a decrease. Paired t-testing indicates that, on average, the patients after 4 sessions had 7.57 more terminal hairs, & after 8 sessions had 14.57 more terminal hairs than at baseline. This was statistically significant. Terminal hair counts for each patient, taken at 20 cm from the glabella, are depicted in Figure 6. 70% patients had an increase in the number of terminal hairs, while other patients had a decrease. Paired t-testing indicates that, on average,

Figure 5. Terminal hair counts at 15 cm from glabella after four sessions (shown for seven patients) (X-axis = patient serial number; Y-axis = number of terminal hairs/cm2) (series 1 = blue = baseline; series 2 = red = after four sessions; series 3 = green = after eight sessions).

 

Figure 6. Terminal hair counts at 20 cm from glabella after four sessions (shown for seven patients) (X-axis = patient serial number; Y-axis = number of terminal hairs/cm2) (series 1 = blue = baseline; series 2 = red = after four sessions; series 3 = green = after eight sessions).

the patients after 4 sessions had 6.14 more terminal hairs, & after 8 sessions had 8.15 more terminal hairs than at baseline (Table 5).

Hair shaft diameter: The average hair shaft diameter for each patient, taken at 15 cm from the glabella, is depicted in Figure 7. 57% patients had an increase in the width of their hairs, while remaining patients had a decrease. Paired t-testing indicated that after 4 sessions, patients had an average hair shaft diameter that was 1.0 μm wider, & 3 μm wider after 8 sessions than at baseline. This was statistically significant. The average shaft diameter, taken at 20 cm from the glabella, is depicted in Figure 8. Paired t-testing indicated that after 4 sessions patients had an average hair shaft diameter that was 0.97 μm wider, & after 8 sessions average hair shaft diameter was 3.03 um wider than at baseline. This was statistically significant. (Table 5)

Subjective evaluation of clinical photographs was provided by three blinded reviewers. All images were randomized prior to grading, so the reviewers did not know which was before or after. The results of the clinical photograph evaluation are provided in Table 6.There was an increased score for 71% patients, a decreased score for 10% patients, & no change in score for 19% patients. Figures 9, 10, 11 & 12 show the representative global photographs of patients at baseline & after 4 & 8 sessions of treatment.

Finally an overall opinion of the patients was assessed. It was observed that 75% patients believed it was helping treat their hair loss, 20% patients did not see any benefit & remaining 5% were not sure. The proportion of patients reporting improvement from baseline after 8 sessions is depicted in Figure 13 A & B.

Figure 7. Hair shaft diameter at 15 cm from glabella after four sessions (shown for seven patients) (X-axis = patient serial number; Y-axis = hair shaft diameter in micrometer) (series 1 = blue = baseline; series 2 = red = after four sessions; series 3 = green = after eight sessions).

 

Figure 8. Hair shaft diameter at 20 cm from glabella after four sessions (shown for seven patients) (X-axis = patient serial number; Y-axis = hair shaft diameter in micrometer) (series 1 = blue = baseline; series 2 = red = after four sessions; series 3 = green = after eight sessions).

Table 6. Results of the clinical photograph evaluation.

Patient
Reviewer 1
Revie

er 2

Reviewer 3
Mean score
Delta
1.

Before

After

 

4

7

 

4

7

 

6

7

 

4.67

7.00

 

Increase

2.

Before

After

 

9

9

10

10

10

10

9.67

9.67

same
3.

Before

After

4

5

6

6

7

4

5.67

5.00

decrease
4.

Before

After

7

7

5

5

7

7

6.33

6.33

same
5.

Before

After

5

6

6

7

5

6

5.33

6.33

Increase
6.

Before

After

8

8

7

8

7

9

7.33

8.33

Increase
7.

Before

After

5

5

5

5

6

7

5.33

5.67

Increase

The treatment was effective in improving the appearance of scalp hair & slowing the loss of hair in men & women with patterned hair loss. Improvement in hair growth with therapy was evident as early as after 4 sessions for all measured endpoints. At one year, a statistically significant increase in total hair count (P=0.002) continued to be seen.

In order to understand over what distance the QR 678 injections caused hair growth, the scalp was averaged into 48 quadrants, as described by Zimber et al. 2 We found that the QR 678 injections’ effect on hair growth was limited to 1-2 mm around the injection sites. This is consistent with other publications demonstrating minimal diffusion of the growth factors, when injected in to the scalp skin. 2, 3, 7, 8, 9, 10

Total 15% patients reported occasional slight itching of the scalp. Tolerable pain was experienced by most

Figure 9. Photographs pre- and post eight sessions of treatment of a representative male patient.

Figure 10. Photographs pre- and post eight sessions of treatment of a representative male patient.

patients, while the injections were administered. There were no deaths or serious drug related adverse experiences & no drug related adverse experiences which resulted in discontinuation of the study medication during the study. The diabetics, hypertensives, & hypercholesteremic patients included in the study had been monitored & there was no significant change in the biochemical values due to the injections.

Figure 11. Photographs pre- and post eight sessions of treatment of a representative post-hair transplant male patient.

Figure 12. Photographs pre- and post eight sessions of treatment of a representative female patient.

Figure 13. The proportion of patients reporting improvement from baseline after eight sessions (A and B).

The findings of this study suggest that the beneficial clinical effects of this therapy are similar in men & women, across different age groups, & in patients irrespective of the presence of metabolic disorders like diabetes, hypertension, hypercholesterolemia etc.The differences in results across gender groups  are not statistically significant.  Moreover, results indicate that therapy was also effective in controlling hair loss in 14 post hair transplant patients. There was a negative correlation between the duration & stage of hair loss, & the degree of improvement.

Discussion:

We have developed a novel formulation which may be a good treatment option in our armamentarium to fight hair loss. Improvement in hair growth with therapy was evident as early as after 4 sessions, for all measured endpoints. After 8 sessions, global photographs showed improvement from baseline for 71% patients, a decreased score for 10% patients, & no change in score for 19% patients. At one year, a statistically significant increase in total hair count (P=0.002) continued to be seen. In our study, we have shown the benefits of a combination of specific growth factors in stimulating hair growth. Several researchers have studied the effects of various individual growth factors on hair growth.

VEGF, essential for angiogenesis & vascular permeability, may be responsible for maintaining proper vasculature around the hair follicle, during the anagen growth phase. 9 KGF is highly capable of counteracting chemotherapy induced alopecia & it is one of the components of our formulation. 11 IGF-I is critically involved in promoting hair growth by regulating cellular proliferation & migration during the development of hair follicles. IGF-I has been reported to prevent the follicle from developing catagen- like status. 13, 14 Thymosin B4 promotes hair growth in various rat & mice models, including a transgenic thymosin B 4 overexpressing mouse, by influencing follicle stem cell growth, migration, differentiation, & protease production. 15 The bFGF has been found to promote hair growth by inducing the anagen phase in resting hair follicles & has been considered to be a potential hair-growth promoting agent. 16 The effects of L alanyl L histidyl L lysine Cu2+ (AHK-Cu) copper tripeptide on human hair growth ex vivo & cultured dermal papilla cells was investigated & shown to promote the growth of human hair follicles. 17

Platelet rich plasma (PRP), which contains growth factors, has been studied as a new method for hair regrowth in pattern hair loss. Multiple studies have concluded that PRP injections are a simple & feasible treatment option for androgenetic alopecia, with high overall patient satisfaction. 18, 19, 20, 21, 22 However, it is a tedious procedure, as the patient’s blood needs to be drawn in every session, requires special equipment, & there is no standardization of the method of preparation, the effective dose, the duration of treatment & the long term safety profile, apart from the fact that it is also an expensive procedure.

On performing a detailed literature review, we came across only one small study using growth factors as combination treatment, to treat hair loss. 23 The study was carried out in eleven Korean women (mean age, 41.36±2.43 years), with Female Pattern Hair Loss. The major components of the topical solution used for treatment were bFGF (2.5 µg/ mL), IGF-1 (1 µg/mL), VEGF (2.5 µg/mL), stem cell factor (2.5 µg/mL), KGF-2 (2.5 µg/mL), superoxide dismutase-1 (5 µg/mL) & Noggin (2.5 µg/mL). The effects of the topical application of growth factors were suggested to result from the effective penetration afforded by microneedle therapy. 23 Ours is a much larger study, with specific growth factors used through a simple intra-dermal injection technique & does not require microneedle use, which is very painful for patients.

Total 15% patients reported occasional slight itching of the scalp for a few hours post the injections. Tolerable pain was experienced by most patients while the injections were administered. There were no deaths or serious drug related adverse experiences & no drug related adverse experiences which resulted in discontinuation of the study medication during the trial. The diabetics, hypertensives, & hypercholesteremic patients included in the study had been monitored with respective tests after 4 sessions & after 8 sessions, & there was no significant change in the biochemical values due to the injections of novel formulation.

A major limitation of this study is the lack of a control group. This was a pilot study and since the results preliminarily seem encouraging, randomized controlled trials are currently underway.

Several factors may have biased the reviewers’ blinded analysis of the images. In studies of minoxidil 24 & finasteride 25, tattooing has been used to identify the exact location of the scalp that is being monitored. Likewise, trimming the hairs in areas of the scalp undergoing treatment, can provide greater accuracy in analyzing results. Phototrichogram, which involves both the above, seems to be more suitable for clinical trials. However, it is not diagnostic, is tedious, time consuming and subjective and requires expertise. 26As this was an independent study, where no patients received monetary reimbursement, it was difficult to convince them to allow us to tattoo their scalps. Unfortunately, we were also not able to convince our female patients to let us trim their hair, especially because their hair was already thinning.

Trichoscan is a digitized phototrichogram that combines standardized epiluminescence microscopy with automatic digital image analysis for the measurement of human hair.The software quantifies the hair parameters within one operation.The use of trichoscan initially involves shaving a scalp area, dyeing it after 3 days, taking a digital photograph and analyzing the data using a software. Processing of photographs is speedy and results are reproducible. 26 However, use of the trichoscan needs clipping of hair in the study area and dyeing it , which was not feasible in our study. Also, there have been recent disputes regarding the accuracy of Trichoscan and many physicians have observed that the Trichoscan software is error prone and not precise. 26

Canfield methodology uses a stereotactic positioning device for global photography on which the patient’s chin and forehead are fixed, and on which a given camera and flash device are mounted.It assures that the view , magnification and lighting are the same at consecutive study visits. 26

In our resource limited settings, we did not have access to the device, however, our investigators had a separate room dedicated for global photography, where the lighting and the distance were maintained throughout the study. We photographed the scalp at fixed positions of 15 cm & 20 cm from the glabella and used videomicroscopic assessment and analysis of photographs as the objective assessment test.

More studies are needed to fully assess the role of this formulation containing a combination of growth factors (the QR 678), in hair growth. Whether more sessions will give added benefit, does the time interval between 2 sessions make a difference, if it would help in other forms of alopecia also for example post chemotherapy hair loss, alopecia areata etc. are some unanswered questions at present. More randomized, single & multicentric trials with large number of patients will be required to prove the validity of these results, to obtain these answers & to get more robust statistical results.

In conclusion, this pilot study represents the first independent study of a unique combination of growth factors & hair growth. The findings of this study suggest that this formulation of multiple growth factors (QR 678) is safe & efficacious in treating male as well as female pattern hair loss & alopecia.

References:

1) Rogers NE, Avram MR. Medical treatments for male & female pattern hair loss. J Am Acad Dermatol. 2008; 59: 547-66.

2) Zimber MPZiering CZeigler FHubka M, at al. Hair regrowth following a Wnt- & follistatin containing treatment: safety & efficacy in a first-in-man phase 1 clinical trial. J Drugs Dermatol. 2011; 10:1308-12.

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Table Captions:

 

Table 1: Composition of different solutions with varied concentrations of growth factors that were injected in the backs of C3H mice.

Table 2: Results of the experiment on C3H mice to determine the solution with the best concentration of growth factors

Table 3: Formulation of novel solution

Table 4: Patient Questionnaire for efficacy & satisfaction with the treatment

Table 5: Vellus & Terminal hair counts & hair shaft diameters on videomicroscopic analysis

Table 6: The results of the clinical photograph evaluation

 

Figure Legends:

Figure 1: Hair Pull test results.

Figure 2A: Shows a photograph of 1/4 cm cut-out of videomicroscope images showing vellus hair count (in red) & terminal hair count (in green) & 2B: Shows a photograph of ¼ cm cut-out of videomicroscope image showing assessment of mean hair shaft diameter. All measurements shown were multiplied by a factor of 2.77 for conversion to microns.

Figure 3: Vellus hair counts at 15 cm from the glabella after 4th session (shown for 7 patients) X axis = patient serial number; Y axis = number of vellus hairs/cm2 (Series 1 = Blue = baseline, series 2 = red = after 4 sessions, series 3 = Green= after 8 sessions)

 

Figure 4: Vellus hair counts at 20 cm from glabella after 4th session (shown for 7 patients) (X axis = patient serial number; Y axis = number of vellus hairs/cm2) (Series 1 = Blue = baseline, series 2 = red = after 4 sessions, series 3 = Green= after 8 sessions)

 

Figure 5: Terminal hair counts at 15 cm from glabella after 4 sessions (shown for 7 patients) (X axis = patient serial number; Y axis = number of terminal hairs/cm2) (Series 1 = Blue = baseline, series 2 = red = after 4 sessions, series 3 = Green= after 8 sessions)

 

Figure 6: Terminal hair counts at 20 cm from glabella after 4 sessions (shown for 7 patients) (X axis = patient serial number; Y axis = number of terminal hairs/cm2) (Series 1 = Blue = baseline, series 2 = red = after 4 sessions, series 3 = Green= after 8 sessions)

 

Figure 7: Hair shaft diameter at 15 cm from glabella after 4 sessions (shown for 7 patients) (X axis = patient serial number; Y axis = hair shaft diameter in micrometer) (Series 1 = Blue = baseline, series 2 = red = after 4 sessions, series 3 = Green= after 8 sessions)

 

Figure 8: Hair shaft diameter at 20 cm from glabella after 4 sessions (shown for 7 patients) (X axis = patient serial number; Y axis = hair shaft diameter in micrometer) (Series 1 = Blue = baseline, series 2 = red = after 4 sessions, series 3 = Green= after 8 sessions)

Figure 9: Photographs pre & post 8 sessions of treatment of a representative male patient.

Figure 10: Photographs pre & post 8 sessions of treatment of a representative male patient.

Figure 11: Photographs pre & post 8 sessions of treatment of a representative post hair transplant male patient.

Figure 12: Photographs pre & post 8 sessions of treatment of a representative female patient.

Figure 13: The proportion of patients reporting improvement from baseline after 8 sessions (A & B).