MDIK-15 Scientific Report

Pilot Study Evaluating the Efficacy of the New Drug MDIK-15 in Accelerating Cell Cycle of Mammalian cells
Abstract
The economic and psychological tolls of chronic, degenerative, and acute diseases are enormous. Many hopes were focused on human stem cells to cure these untreatable diseases, but there exists a widespread controversy over human embryonic stem cell research, while adult stem cells are rare and difficult to isolate and grow in the laboratory, which makes the therapy based on them very costly. MDIK-15, a new inexpensive regenerative medicine, was topically investigated on wounded and intact skin and its appendages. As a result, it regenerated the skin, enhanced nail growth three times more and lengthened the hair very quickly. It solved the problem of male pattern baldness. In wounds and burns, we found that MDIK-15 treated sites showed improved healing over a short period compared with conventional therapy by visual clinical assessment and by digital photographic measurements. Specifically, MDIK-15 possesses angiogenic activity and aids in the formation of healthy granulation tissue and re-epithelialization. As besides being an effective treatment it unexpectedly relieved pain and cleared infection with achieving a functional and aesthetically pleasing scar. This drug is rich of essential nutrients needed for protein synthesis, which augments their concentration in the interstitial fluid yielding in the increase of their penetration rate through the transmembrane proteins due to the pressure of number, which shortens the G1 phase then accelerates the cell cycle. MDIK-15, a regenerative drug, can be used to solve the problem of Alopecia and other cosmesis problem. It can cure fatal and debilitating diseases including chronic wounds and burns. Meanwhile, it holds the great promise in curing previously untreatable degenerative diseases including heart failure, cirrhosis, deafness, blindness, type I diabetes among others. Keywords: Cell cycle, cellular regeneration, G1 phase, MDIK-15, stem cell.
MDIK-15: Cell Cycle Accelerator
Introduction
The human body has limited potential to rejuvenate injured organs and tissues. Regenerative capacity is inversely related to complexity: in general, the more complex an organism is the less regeneration it is capable of. An old dream of scientists and physicians is to be able to rebuild spare parts to replace injured or diseased tissuesa notion that was once referred to as the field of science fiction. In spite of the extraordinary advances in the prevention, diagnosis and treatment of human diseases, devastating illnesses due to cell degeneration continue to deprive people of health, independence, and well-being. Research in human developmental biology has led to the discovery of human stem cells that could be used for cell-based therapies by differentiation into specific cell types [25]. From the perspective of the patient, stem cell research is still at a very early stage and continues to be largely a matter of basic research. It will be many years before there are effective methods of treatment based on stem cell transplantation. Hitherto, transplantation with blood stem cells has been the only established stem cell therapy. After nearly ten years of research, there are no approved treatments but only one human trial approved by US Food & Drug Administration in January 2009 using embryonic stem cells [22]. Today, donated organs and tissues are often used to replace ailing or destroyed tissue, but the need for transplantable tissues and organs far outweighs the available supply. A substantial obstacle to the success of transplantation of any cells, including stem cells and their derivatives, is the immune-mediated rejection of foreign tissue or cells by the recipients body [33]. In current stem cell transplantation procedures with bone marrow and
blood, success can hinge on obtaining a close match between donor and recipient tissues and on the use of immuno-suppressive drugs, which often have severe and life-threatening side effects such as infection and tumor growth. To ensure that stem cell-based therapies can be broadly applicable for many conditions and individuals, new means to overcome the problem of tissue and cells rejection must be found. Moreover, it will be essential that scientists are sure that stem cells have fully differentiated before they can use them for medical applications. If completely undifferentiated stem cells are implanted directly into an organism, they c a n cause a type of tumor called teratoma, which scientists have observed in experiments using mice [16]. There exists a widespread controversy over human embryonic stem cell research because, with the present state of technology, starting a stem cell line requires the destruction of a human embryo and/or therapeutic cloning. Many nations currently have moratoria on either ES cell research or the production of new ES cell lines. In addition, the availability of neural fetal tissue is very limited. Five to six aborted fetuses are needed to provide enough neural tissue to treat one Parkinsons patient. However, adult stem cells are difficult in purifying and culturing. Their plasticity is still unknown. A great deal of adult stem cell research has focused on clarifying their capacity to divide or self-renew indefinitely and their differentiation potential [4]. In mice, pluripotent stem cells are directly generated from adult fibroblast cultures. Unfortunately, many mice do not live long with stem cell organs [30]. Moreover, since stem cell research was to result in highly technological and expensive therapies, health insurers
might be treatments.
reluctant
to
fund
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Because of these hurdles another strategy currently under study is the addition of growth factors that aim to induce the patients own (stem) cells to repair the damaged organs without need to grow in culture and transplant them into the body which can modify the complicated and well organized cell cycle control system, then could perturb the organism function. However, it has been suggested that the growth of mammalian cells may be regulated by the availability of nutrients inside the cell [9, 17]. It is admitted that cells take time in G1 phase to accumulate nutrients needed for protein synthesis in S phase, which makes cell cycle longer. If the penetration rate of critical nutrients through the cytoplasmic membrane can be increased, then the cell cycle can be accelerated. For this reason, improved techniques are needed to properly increase nutrients uptake. This action is generally facilitated either by increasing the permeability of the plasma membrane or by elevating the nutrients concentration in the interstitial fluid. The relationship between biological uptake and nutrient concentration is of particular concern because nutrient uptake rate has been shown to increase with elevated concentrations due to the pressure of number. Unfortunately, the study of nutrition at the cellular level began to look almost hopelessly complex. Even though, significant role of complex media towards higher proliferation has been identified earlier, the unknown regulatory mechanisms could lead to improper interpretation of the data, obtained on cultivation with complex media. But advancement in
analytical techniques, has made possible to simulate the complex medium using chemically defined medium [35] leading to the understanding of the effects of individual components and its interaction. Hence, detailed knowledge on the metabolic process in terms of concentration level of nutrients could be effectively utilized for optimizing the external environment in order to facilitate the favorable condition for enhanced proliferation activity. It suggests that increasing nutrient concentration in tissue fluid would improve cell proliferation. However, nutrients in the skin can be increased either by increasing diffuse from blood plasma, or promoting the transdermal penetration. Thus topical application of nutrients should comprise a facilator to enhance their penetration through the stratum corneum. The present study aims to provide the unique properties of MDIK-15 as a regenerative nutrient-rich drug by accelerating cell cycle of mammalian cells.
Materials and Methods

Product Non-Confidential Description MDIK-15 is a new phytomedicine, semisynthesized from non toxic natural extractions by an accurate technique of work-up procedures before the final product is isolated. At room temperature, the final product is viscous but after special modification it can become wax with the same effects. At 37C it becomes liquid. The compound may be stored at room temperature. It should be protected from long light exposure. In these conditions, product stability and sterility can be assured for decades.
Figure 1. MDIK-15 The product is yellowish, odorless, sterile and preservative free. The pH of this drug is 6.3.
Objective In this first in-human pilot study, we focused on the safety and potency of MDIK-15 as a cell cycle accelerator by investigating its effect on some easy accessible organs. The skin and its appendages were these prominent target organs since they represent potent sources of cellular regeneration that can be simply diagnosed including nail, hair, and intact and wounded skin. IN VIVO HUMAN REGENERATION ASSAYS-, All individuals voluntarily participated in the following investigations and gave informed consent before the start of the corresponding assay. They were
informed of objectives, test protocols and the right to withdraw any time they wanted. The project did not involve any health risk for the volunteers. Indelible Ink Preparation100 g o f henna powder (TAG Cosmetics LTD, Omdurman, Sudan) was impasted in 3 mL of warm water (37 C) and 0.5 mL of Diluant Cellulosique (EL Yousr synthetic Industry, Sfax, Tunisia) just before use. This later is used as a catalyst to increase the intensity of the shade color and to speed the staining by quickly releasing the lawsone molecules from the henna past.
In Vivo MDIK-15's Effect on Human Intact Skin AssayClinical testing was performed on the intact skin of four healthy adult volunteers, ranging from 21 to 32 years old. At the beginning, to assess the skin irritation potential and tolerance of human skin to this novel finished product, we conducted the human single closed patch test on the upper outer arms in two sets. The first set was occluded 4 hours, if no sign of irritation occurred the second set would be further occluded for a total of 24 hours. After the required periods of skin contact, the remaining cream was removed and the skin was observed for any signs of irritation 1 hour after cream removal, then 24 and 48 hours afterwards. Later after insuring the product safety, approximately 0.1 g of MDIK-15 was once applied on the side of a cheek, whilst the other side remained as negative control. The application and control sites were assessed for evaluation of skin thickness and elastic properties at baseline before treatment and at one week interval over one month then on the second and third month post application. A further experiment was designed to assess the effect of MDIK-15 on the cell cycle of keratinocytes of the intact skin. A circular surface in the middle of each hand palm (4cm) of two volunteers among the four above participants (mean age 31.5 years) was applied with indelible ink for one hour then removed. Twenty-four hours later, 100 mg/cm of MDIK-15 was applied on the patch of the left hand while the right one served as negative control. The degree of stained red color was independently estimated by three observers every two days over one month after product application, at room temperature and under standard lighting conditions. Prior to measurement, the subject was placed in a horizontal position to obviate orthostatic effects having an influence on skin color and
hands were held on a flat surface directly in front of the observer. Responses were graded subjectively by each observer using an ordinal scale where 0= no fading, 1= slight fading, 2= intense fading, 3= more intense fading and 4= extreme fading, taking the baseline assessment and the surrounding unstained skin as minimum (0) and maximum (4) references respectively. The fading response results were calculated as a percentage of the average scores. Digital photographs of treated and control patch were taken at 1, 3, 7 and 10 days post MDIK-15 application. This assay aims to compare the red-brown stain contrast in both parts. The lawsone in the henna paste migrates into the outermost layer of the skin and makes staining. In this basal layer, during skin exfoliation, new cells push the older ones (stained cells) to the surface, which will be keratinized and shed. This process will be faster if the cell division is accelerated, yielding in an early henna color disappearance. During assessment, subjects were asked to evaluate subjectively the effect of treatment, specifically with regard to changes in texture and color of the skin. They were also questioned about changes in acne if present- and greasiness of the skin, as well to report any dermal sensation using predetermined descriptors of itch/pain and to score the sensation using visual analog scale (VAS). Photographs of reaction were taken using 10 mega pixels Panasonic camera (model DMC-FS62). In Vivo Human Nail Growth AssayIndelible ink was applied over nails and their eponychiums of both little fingers of six volunteers, ranging in age from 21 to 53 years, for an hour then removed to measure precisely the nail growth by measuring the rate over the unmarked nail plate between the level of distal end
of eponychium and proximal border of mark of indelible ink over nail. At least 24h after ink application, 2 mL/cm of MDIK-15 was applied between the lunula and the cuticle then gently massaged to reach the nail matrix of the left pinky which grows the slowest while the right one remained as negative control. Treated and control nails were digitally photographed using 10 mega pixels Panasonic camera (model DMC-FS62) every day over one week. Readings of nails growth were taken directly and from these photographs; every two photographs of each two successive days of a single finger were exactly superimposed, and the distance the mark on the nail had advanced was measured. The camera was prefocused on a 2 cm opening in a firmly mounted piece of board, and the finger was gently pressed upward against this frame. In Vivo Human Hair Growth AssayMDIK-15 (4 mL/cm) was once topically applied on the healthy head hair of two male volunteers as well on the forehead hair of a man manifesting a receding hairline. The mean age of subjects was 26.33 years (range: 23-32 years). Changes in hair diameter were used as an objective index of hair response to MDIK-15 since it is readily measurable and parallels changes in length. Measurement of hair length and weight were not assessed since they offer no advantage over hair diameter for the evaluation of hair growth. Several pieces of hair were collected at the outset of the study and 1month intervals for up to 6 months after the administration of MDIK15. Hair samples were collected by cutting close to the scalp. The shavings were mounted on a microscope slide and shaft diameter measurements were taken from each of ten separate hairs (to minimize the influence of hair shaft diameter variation along each hair shaft). The measurements were made with a Wild M20 microscope. The lower limit of
sensitivity at 10x magnification was 0.01 mm with a coefficient of variation 2.8%. To test the tensile strength of the hair, three typical terminal hairs were removed from each subject before and after 6 months of treatment and measured. At the end of this study, subjects were asked to evaluate subjectively the effect of the compound, specifically with regard to changes in texture and color of the hair and any change in the frequency of haircut. In Vivo Human Wound Healing Assay-
Based on the results presented herein, pilot case report studies have been initiated to evaluate wound healing potential of MDIK-15. Patients A total of 9 patients, aged 21-65 years including 5 traumatic injuries and 4 burns (see table I), were recruited. Characteristics of patients wounds are presented in the next section. Study Protocol In each case, unless mentioned, the use of MDIK-15 was once beyond the study period with an amount of 2
L/cm. The cream under study was applied locally on the wound. No other topical agents or any dressings were used through the duration of the study. Patients were not instructed to follow any special diet or modification of way of living. Every day details regarding the
condition of the wounds such as signs of wound infection, condition of surrounding unwounded tissues, discharge, smell, necrotic tissue and state of epithelialization was noted. Subjective factors such as pain and local irritation were regularly recorded. Allergies or other side effects were
noted whenever exist. Pain was assessed weekly in all patients using a visual analogue scale of 1 to 10 adapted from McCaffery and Beebe (1989) [13]. Progression towards healing was judged by the color of the tissue types present in the wound according to the Wound Healing Continuum (Gray et al. 2004) [8] and a reduction in wound size. Wounds and burns were observed for at least fifteen days focusing on the regeneration of basement membrane. Moreover a follow up was done for 6 months after the wound healing to record the frequency of scars keloids or contractures formation. Three wounds were digitally photographed then processed with Adobe Photoshop version 7.0.1 and the wounded areas were compared with reference areas. The test site lesion was measured using the perpendicular method described as measuring the longest measurement of the wound as the length regardless of the orientation,
and the longest measurement perpendicular to the length as the width. The depth was always measured at the deepest part of the wound. At screening, everyday evaluations and photographs were taken to assess changes to the target lesion. Wound measurements (cm) of length and width measured the nonepithelialized wound area. Global scores for % epithelialization and % granulation (Global Evaluation Scale: 0=complete healing of the lesion, 1=75% to 100% improvement, 2=50% to 75% improvement, 3=<50% improvement, 4=No change, 5=exacerbation of the lesion) documenting the overall change in the lesions status relative to its status at the screening visit were also taken at each evaluation visit.
At the completion of this trial, scars resulted from wounds treated by MDIK15 or standard-care, were assessed by three independent observers blinded to the method of skin closure using 10 point visual analog cosmesis scale (VACS).
Table I. Selected patient and wound baseline demographics
Age (years) Mean SD 35.56 12.39
Sex Male Female 6 3
Baseline wound size (cm) Injury Burn Mean SD Mean SD 1.28 0.17 11.3 10.91
Statistical Analysis Results of each assay are expressed as SD (standard deviation). The Students paired t-test was used to compare the significance between data points. All subjects acted as their own controls. Values of p<0.01 were considered statistically, significant differences.
Results
MDIK-15 Accelerates Human Wound Healing In a total of 9 patients (mean age 36.25 years, 62.5% male, 5 traumatic injuries and 4 burns) treated with MDIK-15, we were able to see all wounds closed completely in a very short time. Mean
time to healing was 11.75 ( 2.09) days in the MDIK-15 treated wounds (cf. figure 2). Time to healing did significantly change when compared with standardcare. The plot of estimated probability of complete healing for the MDIK-15 treated wounds is shown in Figure 4. The estimated probability of complete healing was significantly higher in the MDIK-15 treatment than in the standardized therapy which was not studied here. These results give an indication of how wound status shifted along MDIK-15 use towards granulation and epithelialization during treatment (see figure 8, 9 and 10). There were no adverse reactions or complications associated with the drug used during the course of treatment with excellent patient satisfaction due to a decrease in wound associated pain and ease of application. In the overall pain score, there was statistically significant pain relief in just few hours. At 3 h, there was considerable pain reduction by 50 %. Figure 3 shows the components of the overall pain score for the 10 h post drug application. Among the 8 subjects no
infections were evident. The patients tolerated the drug well. Although not statistically significant in this small preliminary study, positive trends toward greater improvement by MDIK-15 were also observed for wound length, wound area, global evaluation score for granulation, level of exudates and infection, and degree of erythema. Healing rates were unrelated to ethnicity, body mass index, exercise, alcohol consumption, or nicotine use. The 10 point visual analog cosmesis scale (VACS) assessment of scars resulted from wounds treated by MDIK15 or standard-care by three independent observers demonstrated good agreement for MDIK-15 treated wounds. There was significant difference observed between the two scars (Students matched paired test p>0.05). The absolute value trends consistently show higher values for wounds treated by MDIK-15 (table II). This section examines the various attributes listed above from clinical case perspective.
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24 21 min-|mean SD|-max Pain VAS (0-10) 18 15 12 9 6 3 0 10 9 8 7 6 5 4 3 2 1 0 0 1 2 3 4 5 6 Time (hour) 7 8 9 10
Figure 2. Box plot of healing time (days) for treated wounds by MDIK-15.
Figure 3. Plot analysis for time to pain relief for patients receiving MDIK-15 (mean SD in Bars)
Table II. Visual Analogue Cosmesis Scale (VASC) (110)
Standard-care treated wounds scar observer 1 6.87 0.93 observer 2 6.75 0.66 observer 3 6.87 0.60 Data presented as Means SD.
MDIK-15 treated wounds scar 8.62 0.69 8.12 0.59 8.00 0.75
p Value 0.004 0.001 0.007
0,8
probability of healing
0,6
0,4
0,2
0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time (day)
Figure 4. Mean probability of healing by MDIK-15 plotted daily throughout the study.
Bars are SEM.
We present the detail of the treated patients herein as we have completed data including digital photographs of six wounds. Case 1 A young man (21 years old) presented with a very painful wound (2.1 cm x 0.5 cm) on the proximal phalanx of his left index caused by a disc cutter at work. The lesion remained two weeks untreated until the skin appeared to increase in wetness and the presence of black exudate indicated bacterial colonization (figure 5a). MDIK-15 was
topically applied with the primary aim of reducing bacterial burden and to provide an ideal wound healing environment. After two weeks of treatment, the blackness had significantly subsided (figure 5b). Although this was not the most successfully healed wound, the use of MDIK-15 did significantly progress the wound towards healing until complete healing in twelve days. Wound was digitally photographed time dependently (1, 5, 12 and 30 days) post treatment using 1.3 megapixel camera of Nokia mobile 6230i (photo quality is not so optimal).
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1 day MDIK-15 treatment (14 days after injury), presence of black exudates on the distal phalanx c
st
5 day progress of the wound towards healing
th
12th day complete healing with subside of the blackness
6th month invisible scar
Figure 5. Small proximal phalanx wound healing in relation to MDIK-15 treatment. The blackness had subsided in two weeks and the wound has totally healed by the third week.
Case 2 This 31-year-old man was seriously injured in the proximal phalanx of his left thumb with a box cutter causing an incision painful wound which was approximately 2 cm long and deep into the muscle layers. The wound had initially appeared five days later without being treated. It was considered to have an increased bioburden but was not clinically infected. Local treatment with MDIK-15 achieved rapid healing and relieving of pain. The two edges adhered together in a very short time by unique use. At three days, this deep wound was completely healed.
Case 3 The right index of a 39-year-old man wounded recently following a traumatic injury catching from a motorcycle accident one week later. Though the wound was deep on the dorsal surface of the finger articulation exposing the tendon and bone, it had not responded to any previous treatment used. The wound was colonized due to pain description by the patient who asked to assess and treat the wound with MDIK-15. He had a good general health with no major underlying medical conditions. The patient, who was a hairdresser, was presently signed off work because he could not use the scissors. On MDIK-15 treatment initiation,
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the wound area measured 1.44 cm. After few days of treatment all necrotic / slough tissue had been debrided, the wound bed contained 100% healthy granulation tissue. One week later, the results were remarkably improved, the wound covered with 63 % epithelial tissue. Full healing was achieved within two weeks of MDIK15 application with neither visible scarring nor callus tissue. The subject continued then to be employed full-time. Case 4 A young man (30 years old) caught a road incident by his cycle causing three injuries on his right hand palm (0.5 cm,
figure 6.a) and on his both knees (0.7 cm on the right (figure 7.a) and 1.5 cm on the left (figure 8.a)). Wounds were treated five days after injuries for only once. The drug prevented infection and increased epithelialization and debridement until total healing by nine days for the wound on the hand palm and nineteen days for wounds located on the knees. Wounds were digitally photographed using at two days intervals until complete healing using 3 megapixel camera of I phone mobile 3GS.
MDIK-15 treatment four days after wounding (small open wounds) c
3rd day post treatment speed epithelialization of the wound edges d
5 day total close of the open wounds
th
9 day complete healing of the wounds
th
Figure 6. Case 4 right hand palm wound healing in relation to MDIK-15 treatment. The drug accelerated epithelialization, increased healing and prevented infection.
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MDIK-15 treatment 24 hours after wounding d
3 day post treatment e
rd
5 day f F
th
7th day (Mdik-15 second treatment) g
9th day h
11th day i
13 day (epithelialization)
th
15 day (total debridement)
th
2 months (barely visible scar)
nd
Figure 7. Case 4 right knee wound healing in relation to MDIK-15 treatment. The drug accelerated debridement, increased healing and prevented bacterial colonization.
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MDIK-15 treatment 24hours after wounding d
3rd day post treatment e
5th day f F
9th day h
11th day (debridement) i
13th day (epithelialization)
17th day (total debridement)
2nd months (pleasing scar)
Figure 8. Case 4 left knee wound healing in relation to MDIK-15 treatment. The drug accelerated debridement, increased healing and prevented bacterial colonization.
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Case 5 A 33-year-old male presented with an open wound located at the navicular of the carpals of his right hand. The wound was rectangle in shape measured 1.7 cm x 0.8 cm with a depth of 0.3 cm (figure 9 a). Wound was topically applied with MDIK-15 just after injury. The patient felt slight itching then gave a pain rating of 0 (no pain experienced). The aim of the treatment would be to promote granulation and to speed up closure. From the second day post treatment, there was evidence of epithelialization at the wound edges and the surrounding skin was healthy (figure 9 c, d and e). The size of the wound and the level of exudate were such that the addition of MDIK-15 would be very beneficial especially that the drug was initially applied while bleeding. By the 5th day, MDIK-15 was applied again to the wound. The rapid improvement in the wound was quite dramatic over the first two weeks post second treatment, with improvement in the wound bed which contained 100% healthy granulation tissue. The drug increased angiogenesis (figure 9g). The wound measured 0.8 cm x 0.4 cm and the depth had significantly decreased (figure 9j). This patient's wound responded well to treatment until
total closure with pleasing scar (figure 9k). As wound healed very quickly, photographs, with original x 3 enlargement, were taken at 24-hour intervals over the first ten days and then two months later post wounding using 10 mega pixels Panasonic digital camera (model DMC-FS62). Case 6 A 42-year-old house wife sustained a burn on her right hand following a fall of boiling water while cooking causing a superficial thickness burn (first degree). The wounded area became red and extremely painful (8 on the visual analog scale). The treatment objectives aimed to soften and promote autolysis to remove the eschar and to progress burn towards healing. MDIK-15 was topically applied on the wounded area one hour after burning. Softening of the eschar was seen from the first day and the patient experienced any pain more one half an hour following application. Within one week, total debridement had taken place, and there were visible signs of large areas of epithelialization until total closure by eleventh day, without forming any visible scar.
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MDIK-15 first treatment just after wounding d
1st day (24h post treatment) e
2nd day f
3rd day (a clean wound) g
4th day (sterile wound) h
5th day MDIK-15 second treatment (a moist environment) i
6 day (formation of new blood vessels) j
th
7 day (healthy granulation tissue) k
th
8 day (speed division and migration of basal keratinocytes) l
th
9 day (increased epithelialization)
th
2 month ( aesthetically pleasing scar)
nd
6 month (barely visible scar)
th
Figure 9. Navicular open wound healing in response to MDIK-15 treatment. MDIK-15 increased angiogenic activity and aids in the formation of healthy granulation tissue and re-epithelialization.
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Case 7 A 65-year-old woman with type 2 diabetes presented with very painful fullthickness burns (2 to 3 mm deep (second degree)) in her hand palms caused by a potent chemical agent. For two weeks the patient was provided standard of care therapy in treating the wound but in vain. Burns were then treated with MDIK-15 application where as in previous treatments, multiple medications were necessary for disinfection and closure with this patient. Stretches of epithelial tissue were noted and the wound was definitely cured in three days. Case 8 This subject, 29-year-old female, also had second degree burns (3 to 7 mm deep) on her hand palms secondary to the same chemical agent that had been present for 2 weeks and was deteriorating with conventional management. Her pain was regularly describing as 9 on the visual analog scale and she slept little at night as a result. After failed therapy with standard treatment, burning areas were topically applied with MDIK-15 for two successive days. The wound was responsive to the treatment as well, with significant improvement in the rate of healing as compared to prior treatment in this same patient using numerous other modalities. Pain levels became moderate and the patient was prescribed, co-codomal for pain. The reduction in pain improved the patient's quality of life and this, coupled with her wound's progression towards healing, enabled her to sleep well. Within few days, large areas of epithelial tissue were visible and one of her major wounds had reduced in size by 50%.
Within one week, the largest wound had become three times smaller with excellent granulation tissue present in the wound beds. Complete closure of the distal and dorsal wounds was achieved within twelve days. Case 9 A 30 year-old-man was burned with flaming sulfur on the thumb of his right hand (on the distal phalanx) causing a dangerous wound (2.5 cm x 1.2 cm) within formation of tremendous edema few hours later (cf. figure 10a). The patient also recorded his pain experience as 10 on the visual analogue scale. Treatment objectives were to debride the wound surface, reduce bioburden and promote healing. But firstly, MDIK-15 was applied few hours after burning to the edema that covered the wound site with the primary aim of reducing exudate and relieving pain. On the third day when the edema disappeared using a sterile needle, exudate levels significantly reduced (figure 10b). Within a week remarkable changes occurred with the wound bed consisting of 80% slough and 20% healthy granulation tissue (figure 10d) and the patient no longer had a pain (recorded as a 0 on the visual analogue scale). After ten days, the wound had completely debrided and granulation was very apparent with some contraction of the wound margins (figure 10f). The wound went on to heal successfully. Nineteen days later the wound had healed completely figure 10h). Healing progress was photographed time dependently (0, 3, 5, 7, 9, 10, 11, 19 and 60) days post treatment using 1.3 megapixel camera of Nokia mobile 6230i.
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MDIK-15 treatment 24hours after wounding (tremendous edema) d
3rd day post treatment, disappearance of the edema e
5th day f F
9th day the skin flap became necrotic h
10th day (total debridement) i
11 day (epithelialization)
th
19 day
th
2 months (barely visible scar)
nd
Figure 10. Case 9 Burn healing in relation to MDIK-15 treatment. The drug accelerated debridement, increased healing and prevented bacterial colonization.
MDIK-15 Enhances Nail Growth In the six subjects recruited in this assay, the treated fingernails began after 24 to 48 hours to grow faster than control. The average growth rate of treated and
control nails are presented in figure 11. From the first to second day after MDIK15 application, the growth rate of treated nails was faster than controls. From the third day, this difference became
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significantly more important. The maximum average growth of treated nails was recorded from the third to the fourth day, three times higher than control with approximately 0.32 cm a day. A photographed case of treated (left) and control (right) pinky nail is shown in figure 12. After 24 hours post treatment, the treated nail began to grow faster than control (figure 12 L1, R1). From the second to the third day, this difference became greater (figure 12 L2, R2 and L3, R3). By the fourth day, the unmarked nail plate of the treated pinky is at least three times longer than control (figure 12 L4, R4). Everyday this difference became more remarkable.
Without staining nails, we unexpectedly observed blanching of the free edges of the treated nail while pinker on the nail bed. Meanwhile, the plate became slightly thicker, therefore the grooves of the underneath surface of the plate along the length of the nail became slightly deeper and well interdigitate with the longitudinal ridges of the nail bed, securely attaching the two structures. When the hyponychium is shorter than normal, it quickly advanced to reach the distal end without exceed it whatever the amount of MDIK-15 was used. However, when there is an overgrown cuticle, this later started to reduce and disappear in at least one month to let a thin one remained on the lunula!
3,5
3,0 2,5 length (mm) MDIK-15
control
2,0
1,5 1,0
0,5
0,0 0 1 2 3 4 time (day) 5 6 7
Figure 11. MDIK-15 vs. control fingernail average growth measurements. From the first day, the treated nail (blue) grew faster than control (red). Everyday this difference became significantly greater.
Left pinky (MDIK-15 application)
Right pinky (control)
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L0
R0
Just after application (0 hour)
L1
R1
1st day (24 h later)
L2
R2
2 day
nd
L3
R3
3rd day
L4
R4
4 day
th
L5
R5
5th day
L6
R6
6th day
Figure 12. Growth of stained little fingernail in relation to MDIK-15 application vs. control. The treated nail (on the left) grew faster than control (on the right); the unmarked plate on the treated nail is nearly three times longer than control. Though this later also manifested a speed lengthening since it was touched by the drug remained on the treated nail when washing hands later after treatment.
L7
R7
7 day
th
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MDIK-15 Enhances Hair Growth The three participants who entered this study completed the protocol. Results for hair shaft diameter are presented in Figure 13. Hair diameter was increased significantly by MDIK-15 unique use. Compared to before treatment, mean changes after MDIK-15 application were three times more where a positive sign noted increase in hair diameter. Thus the benefit of MDIK-15 in inducing hair diameter is likely to exceed 70%. No regression in hair growth was found in the other man during the overall six months study period. While the already receding hair of the forehead started to appear again one by one in few months. When treated again, the lateral side regained more and stronger the lost hair. Thus hair density in the treated region
became higher as compared to before treatment. The difference was >50%. In table III, the mean hair tensile strength was increased by 71.87%. There was greater improvement in the tensile strength of treated hair by unique use of MDK-15. Subjective assessments of response to MDIK-15 are illustrated in Figure 14. They reported no side effects and expressed satisfaction with hair growth. A trend favoring benefit from the drug was denoted but this did not achieve statistical significance. However these encouraging results form the basis of a more extensive study of the treatment of different types of alopecia.
170 150
hair diameter/m
130 110 90 70 50 30 0 1 2
date/month
Figure 13. Human hair shaft diameter measurement in relation to MDIK-15 application. MDIK-15 has significantly increased hair diameter in the first month to reach an average of 70% greater than baseline measurement for more than five months post application.
21
3 worse no change better + better ++ better +++
worse no change better + better ++ better +++
Figure 14. Subjective assessment of MDIK-15 effect on hair growth. All volunteers were completely satisfied from drug results.
Table III. The increase of scalp hair tensile strength in relation to MDIK-15 treatment.
subject number subject 1 subject 2 subject 3
baseline measurement 3.5 4.3 2.7
6 months' measurement 5.9 6.8 5.1
change % 68.57 58.14 88.89
MDIK-15 Regenerates Human Skin All volunteers completed the present assay without erythema formation. Similarly, none of the volunteers developed irritation neither after the 4 hour MDIK-15 occlusion period, nor after the extended 24 hour test occlusion. However a slight itching was reported immediately after drug application which did not exceed 1 on the visual analogue scale and that was short-lived (1-2 min). Before application, no significant differences were observed in skin thickness between the control and the treated skin. However, a unique application of MDIK-15 showed an increase in skin thickness compared with the corresponding negative controls in all volunteers. This difference between the treated and the control group is
significant (P<0.01). The control side did not elicit any change in skin thickness measured. When applied on the stained skin, the color of the treated part A faded earlier than the control part B, as shown in figure 15. Figure 16 depicts the mean results of the visually-assessed stain-fading profiles for the three observers. All observers noted the total disappearance of the stain on the treated patch in less than two weeks after application. All volunteers reported consistent improvement in skin quality observed in the area treated with MDIK-15, together with improvement in skin elasticity and color with satisfactory cosmetic outcomes.
22
Left hand palm patch (treated)
Right hand palm patch (control)
A1
B1
1st day (24 h post MDIK-15 application)
A2
B2
3rd day
A3
B3
7 day
th
A4
B4
10
th
day
Figure 15. Glabrous skin patch fading in relation to MDIK-15 application vs. control. The red-brown stain disappeared earlier in the treated part (on left) than control (on the right), though this latter also manifested a rapid fading since it was also touched by a few amount of MDIK-15 remained on the treated patch when washing hands later after treatment.
23
100
% mean visual fading assessment
90 80 70 60 control treated
50
40 30 20 10 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
date/day Figure 16. Visually assessed stain-fading profiles of treated patch and control, obtained by the pool of three observers. The treated patch (red) disappeared earlier than the control one (blue).
Discussion
The speed lengthening of hyponichium, nails and hair exposed to MDIK-15 relative to controls as well the speed healing of wounds and burns without side effect suggest that this drug hastens the proliferation of mammalian cells until human conditions are right. It also unexpectedly relieved pain and cleared infections The mechanism by which MDIK-15 accelerates cell cycle remains to be more elucidated. Probably, MDIK-15 acts on the G1 phase of the cell cycle since it is rich of the critical nutrients. Because cells require growing and accumulating nutrients for protein synthesis in G1 phase before dividing, the standard cell cycle is generally quite long- 12 hours or more for fast growing cells. Hence it is generally accepted that nutrient availability is one of the key factors in regulating the rate of cell division [9, 17]. As much the nutrients uptake is increased as much the G1
phase is shortened and the cell cycle is accelerated. However there are two factors that can stimulate nutrients uptake; either increasing the permeability of plasma membrane or augmenting the concentration of nutrients in the interstitial fluid. This later would increase the penetration of these nutrients through the cytoplasmic membrane due to the pressure of number. Moreover in order to conserve resources a cell will only produce molecules according to the availability (i.e. the level and type of fuel that is available to a cell will determine the type of enzymes it needs to express from its genome for utilization). Receptors on the cell membrane's surface designed to be activated in the presence of specific fuel molecules communicate to the cell nucleus via a means of cascading interactions. In this way the cell is aware of the available nutrients and is able to produce only the molecules specific to that nutrient type. Hence if the concentration of needed nutrients is increased the nutrient sensing will
24
increase as a result with stimulating of faster nutrients uptake. Nutrients are diffused to the tissue fluid from blood plasma by hydrostatic pressure in the capillaries. However, results of clinical studies described here clearly showed that topical application of MDIK-15 has significant clinical benefits. MDIK-15 active molecules are likely to penetrate through the stratum corneum in measurable quantity to produce pharmacologic effects. Thus the primary mechanism by which the drug molecules can potentially exert their effect on the dermal matrix is by diffuse through skin cells followed by interaction with cells in the epidermis such as keratinocytes or traveling down hair follicles to reach the dermis layer then interacting with dermal cells such as fibroblasts. To penetrate the skin, MDIK-15 should have special mechanism to increase skin permeability then allows its molecules to penetrate through. Scientists believe that the skin is an effective barrier to all inorganic particles. It acts as a water resistant barrier so essential nutrients are not washed out of the body. Similarly nanoparticles of different materials have shown penetration limitations through the skin. Research confirms that nanoparticles larger than 40 nm do not penetrate the skin past the stratum corneum. [32] In order to successfully penetrate the skin, they must be 40 nm in diameter and smaller. [32] [27] [23] Alterations to the skin causing slightly damage would be the only way to increase permeability. [14] Therefore some effective methods have been developed to increase nanoparticle penetration including mechanical stressors, ultraviolet radiation, tape stripping, skin abrasion, chemical enhancement, and electroporation. [15, 20, 26] This drug has a special mechanism maybe a new molecule formed during the semi-synthesis. When studied, this mechanism can be used to stimulate glucose uptake at diabetic
patients. It can be added to many drugs to enhance the penetration of the active ingredients in the cells which would reduce their dose yielding in the reduction of the side effects especially in chemotherapy. It can also be used to deliver many drugs of dermatological effects topically which is safer. In addition it is proposed that a crucial increase in nutrient uptake is an upsurge in internal concentrations of nutrients resulting in the rise of cell size, which was manifested by increase of nail plate, hair shaft and skin thickness. To investigate the relationships between uptake and concentration and found that uptake rate increased with elevated nutrient concentration, the quantitative response of cells to nutrient enrichment can be measured with total cell counts. Nevertheless techniques used in this study for the evaluation of cell response to drug dosage are related to the in vivo increase of length and size of hair and nail as well to the thickness of the skin. In all cases studied, the transition point increased linearly with increasing the dose of the drug up to a maximum dose above which the nutrient did not enhance the growth anymore; the biological uptake became saturated at higher concentrations. However, no symptoms of overdose have been reported. On the other hand, after few days post single MDIK-15 application, drug effect remained relatively stable over 3 months for nutrient solutions, and then decreased up to cell maintenance levels in all cases (cf. figure 17). This early study did not yet evaluate whether this new drug acts on differentiated cells, adult stem cells or both.
25
max --
drug application
-1
intensity of drug effect
3 time/month
Figure 17. Duration of MDK-15 effect By single use of MDK-15, the cell proliferation increased abruptly to reach a constant rate for about three months then began decrease slowly to return normal after at maximum six months.
The promising field of regenerative medicine is to restore structure and function of damaged tissues and organs. It also creates solutions for organs that become permanently damaged. Therefore MDIK-15 holds the great promise to cure previously untreatable diseases due to cellular degeneration. It can intervene by topical application to accelerate burn and wound healing in an extremely remarkable way with achieving a functional and aesthetically pleasing scar. Over $25 billion is spent annually on wound care in the United States. Between 5 and 7 million Americans are affected each year by at least one form of chronic wound and the incidence of these wounds is increasing at approximately 10% a year. [10] The global wound care market totaled $7.2 billion in 2006, with a 10% growth rate. [5] MDIK-15 can solve the problem of male pattern baldness when topically applied
on the scalp. There are more than 40 million men in USA who suffer from Androgenic Alopecia. In some men, after years of exposure to the hormone dihydrotestosterone (DHT), this latter accumulates on the hair follicles reducing enough blood flow yielding in their shrinkage then they can produce only fine, short, light-colored hairs or none at all. MDIK-15 can nourish these follicles by topical application then thick and strong hairs on the balding a r e a can appear again without causing any side effect unlike the current products, which are not so effective. Moreover, it can only be used once to twice a year unlike Rogaine and Propecia that should be used twice a day otherwise all the saved or regrown hairs will quickly shed. In this early study, we contented with human skin and its appendages by topical application. However, it is generally accepted that nutrients of skin cells (keratinocytes, fibroblasts) are nearly common to all other human cells.
26
Therefore MDIK-15 holds the great promise in curing many other if not alldegenerative diseases. It can be evaluated on other (vital) organs by different routes of administration (oral, intravenous). For instance by insuring cellular regeneration it can intervene to cure certain degenerative diseases including heart failure, cirrhosis, deafness, blindness, type I diabetes among others. On the other hand, given the marked positive effects of nutrient enrichment upon cellular physiology, this new drug can be utilized to maintain nerve cell viability (i.e. maintenance energy) in case of neurodegenerative diseases such as Alzheimers which is characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions.
There is no potential conflict of interest.
References
1. Bertone AL. Management of exuberant granulation tissue. In: Booth LC, ed. Wound management. Vet Clin North Am Equine Pract. Philadelphia: WB Saunders Company, 1989;5:551562. 2. Bertone AL. Principles of wound healing. In: Booth LC, ed. Wound Management. Vet Clin North Am Equine Pract. Philadelphia: WB Saunders Company, 1989;5:449463. 3. Dry FW. The coat of the mouse (Mus Musculus). J Genet (1926) 16: 287 340. 4. Eicheler W, Happle R & Hoffmann R. 5 alpha-reductase activity in the human hair follicle concentrates in the dermal papilla. Arch Dermatol Res (1998) 290: 126132. 5. Epsicom Business Intelligence Ltd. 6. Gardner RL (2002). "Stem cells: potency, plasticity and public perception". Journal of Anatomy 200 (3): 27782. 7. Garlick JA, Taichman LB (1992) A model to study the fate of geneticallymarked keratinocytes in culture. J Dermatol 19: 797801. 8. Gray D. White R. Kingsley A. Cooper P (2004) Using the Wound Healing Continuum to identify treatment objectives. In applied Wound Management supplement: Part 2 Implementation. Wound UK, Aberdeen. 9. Holley, R. W., Proc. Natl. Acad. Sci. 69:2840 (1972). 10.http://www.infectioncontroltoday.com/ articles/231feat3.html Accessed March 25 2005. 11.Jindo T, Tsuboi R, Imai R, Takamori K, Rubin JS & Ogawa H. The effect of
Conclusion
MDIK-15 is a new regenerative medicine; it accelerates cell cycle of mammalian cells probably by shortening the G1 phase. Thus it can be used as a cosmetic product to regenerate the skin and to solve the problem of male pattern baldness. Meanwhile, it can intervene as a pharmaceutical drug to cure chronic and acute wounds and burns, by accelerating the healing process. Furthermore, it can also relieve pain earlier, clear infection, making the wound sterile, achieve a functional and aesthetically pleasing scar and is cost effective. By further studies, MDIK-15 can also be used to cure many other fatal and debilitating diseases due to cellular degeneration that would not otherwise be curable including heart failure, cirrhosis, deafness, blindness, Juvenile diabetes, spinal cord injury, Alzheimer's disease among others.
Disclosure Statement
27
hepatocyte growth factor/scatter factor on human hair follicle growth. J Dermatol Sci (1995) 10: 229232. 12.Juan, M.E., Lamuela-Raventos, R.M., de la Torre-Boronat, M.C. and Planas, J.M. (1999) Determination of transresveratrol in plasma by HPLC. Anal. Chem., 71, 747-50. 13.McCaffery, M., & Beebe A. (1989). Pain: Clinical manual for nursing practice St. Louis: C.V. Mosby. 14.Mortensen, L., Oberdorster, G., Pentland, A. and DeLoiuse, L. In Vivo Skin Penetration of Quantum Dot Nanoparticles in the Murine Model: The Effects of UVR. Nano Letters 2008;8(9):2779-2787 15.Mortensen, L., Zheng, H., Faulknor, R., De Benedetto, A., Beck, L., DeLouise, L.A. Increased in vivo skin penetration of quantum dots with UVR and in vitro quantum dot cytotoxicity. Colloidal Quantum Dots for Biomedical Applications IV 2009;7189:1605 16.National Academy of Sciences http://dels.nas.edu/bls/stemcells/worki ng-with-stem-cells.shtml. 17.Pardee, A.B., Natl. Cancer Inst. Monogr. 14:7 (1964). 18.Piazza GA, Ritter JL, Baracka CA. (1995) Lysophosphatidic acid induction of transforming growth factors and : modulation of proliferation and differentiation in cultured human keratinocytes and mouse skin. Exp. Cell. Res. 216:5164. 19.Pillai S, Cho S, Mahajan M, Frew L, Rawlings AV. (1996) Synergy between vitamin D precursor 25hydroxyvitamin D and short chain ceramides on keratinocyte proliferation and differentiation. J. Invest. Dermatol. Symp. Proc. 1:3943.
20.Prausnitz, M., Mitragotri, S. and Langer, R. Current Status and Future Potential of Transdermal Drug Delivery. Drug Discovery 2004 February;3:115-124 21.Relyea MJ, Miller J, Boggess D & Sundberg JP. Necropsy methods for laboratory mice: Biological characterization of a new mutation. In: Sundberg JP, Boggess D (eds)Systematic Approach to Evaluation of Mouse Mutations (1999) Boca Raton, FL: CRC Press p 5790. 22.Ron Winslow (2009). "First Embryonic Stem-Cell Trial Gets Approval from the FDA". The Wall Street Journal 23 January 2009. 23.Ryman-Rasmussen, J.P., Riviere, J.E. and Monteiro-Riviere, N.A. Penetration of Intact Skin by Quantum Dots with Diverse Physicochemical Properties. Toxicological Sciences 2006;91(1):159-165 24.Shimaoka S, Tsuboi R, Jindo T, Imai R, Takamori K, Rubin JS & Ogawa H. Hepatocyte growth factor/ scatter factor expressed in follicular papilla cells stimulates human hair growth in vitro. J Cell Physiol (1995) 165: 333 338. 25.Singec I, Jandial R, Crain A, Nikkhah G and Snyder EY. 2007. The leading edge of stem cell therapeutics. Annu. Rev. Med. 58: 313-328. 26.Sokolov, K., Follen M., Aaron, J., Pavlova, I., Malpica, A., Lotan, R., et al. Real-Time Vital Optical Imaging of Precancer Using Anti-Epidermal Growth Factor Receptor Antibodies Conjugated to Gold Nanoparticles. Cancer Research 2003 May;63:199 27.Sonavane, G., Tomoda, K., Sano, A., Ohshima, H., Terada, H. and Makino, K. In Vitro permeation of gold nanoparticles through rat skin and rat intestine: Effect of particle size.
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Colloids and Surfaces B: Biointerfaces 2008 August;65(1):1-10 28.Stashak TS. Principles of wound healing. In: Stashak TS, ed. Equine Wound Management. Philadelphia: Lea and Febiger, 1991:118. 29.Stashak TS. Principles of wound management and selection of approaches to wound closure. In: Stashak TS, ed. Equine Wound Management. Philadelphia: Lea and Febiger, 1991:3651. 30.Takahashi K, Yamanaka S (2006). "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors". Cell 126 (4): 66376. 31.Tredget EE, Falk N, Scott PG, Hogg AM, Burke J: Determination of 4hydroxyproline in collagen by gas chromatography/mass spectrometry. Anal Biochem 1990, 190:259265.
32.Vogt, A., Combadiere, B., Hadam, S., Stieler, K., Lademann, J., Schaefer, H., et al. 40nm, but not 750 or 1,500 nm, Nanoparticles Enter Epidermal CD1a+ Cells after Transcutaneous Application on Human Skin. Journal of Investigative Dermatology 33.Wu DC, Boyd AS, Wood KJ (2007). "Embryonic stem cell transplantation: potential applicability in cell replacement therapy and regenerative medicine". Front Biosci 12: 452535. 34.Youdim, K.A., Martin, A. and Joseph, J.A. (2000) Incorporation of the elderberry anthocyanins by endothelial cells increases protection against oxidative stress. Free Radic. Biol. Med., 29, 51-60. 35.Zhang J., Greasham R., Appl. Microbiol. Biotechnol. 51 (1999) 407.
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Addendum I (Equine Wound Healing by MDIK-15)

Animal and methods Animal- A Berber-Arab stallion, weighing 700 kg and aged 5 years was used for this study, which complied with the rules of Good Clinical Practice for the Conduct of Clinical Trials for Veterinary Medicinal Products in the European Union. This mature horse sustained a fullthickness, elliptical cutaneous wound on the right cheek underneath the medial angle of the eye. The location of the wound did not interfere with the normal daily activities of the horse, but since the wound seemed to be very painful due to infection colonization, the animal was rubbing it on the ground which made it bleeding with excessive exudates and the wound area had increased from its original size of 1.5 cm to 6 cm. The horse owner called a veterinarian who diagnosed the horse and gave some veterinarian medicines including DERMA SPRAID Antiseptic, Aluspray, Orcycline and Btadine. But the wound did not respond to these conventional veterinary treatments and remained getting worse over more than three months to reach an area of 8.41 cm (figure 19a). The owner, who asked to treat this wound with MDIK-15, gave informed consent prior to enrollment. Treatment- As before experiment initiation, the animal was housed in his native covered stall, fed on grain, grass hay and water ad libitum and had free access to grass pasture when allowed routine exercise outside for three hours daily. The horse was brought up, restrained and sedated by the animals owner before the procedures; the wound site was rinsed with clean water from a hosepipe to remove all scabs and debris then treated, diagnosed and/or photographed. The product was applied (200 L/cm) to the wound surface. The application of other agents in conjunction with the test product was not allowed during the trial. The wound was left to heal by second intention with no dressing. Subjective observations- The horse was kept under close observation for signs of discomfort and systemic illness over the first week and checked at regular intervals thereafter for detection of any long-term complication until three months after complete healing through reevaluation of stallion on farm and via telephone conversations with the owner. Meanwhile, at each visit, the wound was observed grossly for the following features: degree of exudate, hemorrhage, necrosis, maceration of the wound, neovascularization and infection (wound was classified as infected, if it showed clinical signs of inflammation).
30
The wound was also observed for formation of normal or exuberant granulation tissue (higher than the wound edges) during the study. Features were scored from 0 to 3 (0 = normal; 1= mild change; 2 = moderate change; 3 = marked change). The frequency of adverse events deemed possibly or probably related to treatment was assessed along the trial. Planimetry the cutaneous perimeter of the wound was traced manually using sterile transparent cellulose acetate and a wax pencil on day 0, 1, 2, 3, 4, and then at 5-day intervals until complete healing. Care was taken to differentiate and record epithelial growth from the full thickness wound edges. Complete wound healing was considered present when the defect had closed by the process of contraction and epithelialization. The height (measured in mm) and shape of the repair tissue within the scar were also estimated and recorded. The wound areas were calculated from the manual tracings using 2 linear measurements: wound length (maximum length of the wound in any direction) and wound width (maximum width of the wound perpendicular to its length). These measurements were multiplied to estimate wound area. The percentages of contraction and epithelialization of the treated wound, as well the time to healing between first treatment and complete healing, were evaluated. The cosmetic appearance was evaluated on the basis of subjective assessments and was not evaluated statistically.
Wound was digitally photographed on day 0, 1, 2, 3, 4, and 9, and then at 5day intervals between the first and second month post treatment initiation, using 10 mega pixels Panasonic digital camera (model DMC-FS62) for later evaluation. Results and Comments MDIK-15 (200 L/cm) was applied on an 8.41 cm wound on the cheek of a stallion that was reluctant to heal for more than three months. Part of the aim of this study was to investigate if MDIK-15 accelerates the cell division cycle of mammalian cells other than human cells by analyzing if the addition of this product speeds up the healing process in equine wound. Gross examination of the wound healing at two months showed great changes since first MDIK-15 treatment. Wound healed uneventfully; no clinical complications were observed. From the time the test drug was applied until the wound was completely epithelialized, wound exuded less fluid than did before enrollment and displayed any subsequent swelling in response to MDIK-15 treatment. Signs of moderate inflammation (redness, swelling, and discomfort during palpation) were observed prior to enrollment. However, these signs subsided within a week of MDIK-15 initiation. There was no evidence of inflammatory cells, lysis, or formation of cysts in the treated lesion. At nine days after treatment there was more obvious evidence of healing especially at the center of the lesion. A pinker, healthy tissue was well apparent and filled the defect to the level of the
31
surrounding. Several small vessels were also apparent on the surface of the treated wound. This was observed at the same time that epithelium was proliferating at a greater rate towards the center of the wound. Epithelium was first noted at the skin margins from day 6 after enrollment (figure 19g). The overall gross assessment score was significantly improved after one month post treatment; the large counterparts of the lesion had dense white connective tissue filling the defect (figure 18h), but the lesion's original outline was still evident. The speed with which normal granulation tissue formed was significant. However, the wound did not display exuberant granulation tissue (proud flesh) at any time during the study. Granulation tissue did not become excessive at any point of the treated site. Subjective evaluation of the wound on site and later from the photographs indicated noticeable differences between baseline and endpoint. Among the most obvious observation recorded was wound retraction and epithelialization. The percentage of contraction of wound undergoing MDIK-15 treatment that was fully filled with normal granulation tissue at visit 4 (day 3) was 21% of baseline wound size. This percentage rose
steadily with increasing time, ranging from 68% to 80% at visit 8 (day 21). The wound area determined from the acetate tracings are presented in Figure 18. There was substantial variation in wound size at each visit from the seventh day. Wound area decreased significantly over time. At visit 4 (day 14), the size in the MDIK-15 treated wound was 6.2 cm. By visit 9 (day 30), this value had changed to 3.8 cm. The time for complete healing of the wound was one month and 14 days. By one month and two weeks, wound had fully re-epithelialized (figure 19k). During this time, the animal sustained any adverse event that could be classified as possibly related to treatment. The wound displayed any evidence of strong antigenic reaction related to MDIK-15 treatment (vesiculation of the wound, maceration of the wound margins, or marked accumulation of exudate). The cosmetic evaluation indicated that the residual scar over the healed wound exhibited less fibrosis. Subjectively, treated wound appeared less edematous from the first days of the trial than before enrollment.
32
wound healing progression 9
8
7 wound area/cm 6
conventional veterinary treatment enrollment
MDIK-15 treatment initiation (day 0)
5
4 3 2 1 0 -60
-35
-10 time/day
15
40
Figure 18. Wound progression before and after MDIK-15 initiation. The wound was getting worse even with conventional veterinary treatment until MDIK-15 use when it started progressing towards healing
33
Before treatment signs of mild Inflammation (dry environment) d
first treatment (0h) (moist environment) e
1 day (24h post treatment) (debridement of dead tissue) f
st
2nd day wound photographed before cleaning g
3rd day (healthy granulation tissue) h
4th day (second treatment) i
9 day formation of healthy granulation tissue with epithelialization j i
th
1 month re-epithelialization and reappearance of thicker hair k
st
1 month and five days (wound size reduction) l
st
1 month and ten days contraction of the wound edges
st
1 month and 14 days complete healing with strong tensile strength
st
2 month small pleasing scar with formation of thicker hair
nd
Figure 19. Equine wound healing by MDIK-15. The test drug increased healthy granulation tissue formation, cleared infection, accelerated epithelialization and enhanced thick hair growth.
34
When equine wounds heal by second intention, hypergranulation tissue and delayed closure are common [2]. The contamination of many equine wounds, trauma sustained during wounding, and the relatively poor blood supply to the distal extremities further predispose to infection, hypergranulation tissue, and delayed healing [1,2,29]. An effective method of preventing this outcome is needed. MDIK-15 has previously been shown to promote the formation of healthy granulation tissue, clear infection and increase the rate of healing in human wounds. This study has shown that topical application of MDIK-15 is highly effective in preventing hypergranulation tissue and infection in equine wound and that it is superior to four current treatments in this regard. Besides, MDIK-15 was selected based on previous studies which showed that this drug added to wounds can protect the wound by clearing infection and aid to retrieve pain, suggesting a beneficial role for MDIK-15 in the healing process. Combined with the results of the current experiment, this confirms the improved quantity and quality of repair tissue in treated wounds. MDIK-15s beneficial effects on healing are attributed to its multiple effects on the disinfection, wound debridement, microcirculation (angiogenesis), formation of granulation tissue and epithelialization. Wound healing is classically divided into 4 overlapping periods, designated as the phases of inflammation, debridement, repair, and maturation [28]. Macrophages are essential for success during the debridement phase which can be enhanced by MDIK-15. It also stimulates epithelium, fibroblast and endothelial cell proliferation and migration, thereby promotes
epithelialization, angiogenesis and granulation tissue formation, all of which improve the wound healing. MDIK-15 thus promotes many of the processes that are important during the repair phase of healing, when migration and activity of fibroblasts and establishment of an adequate blood supply are important. Although a number of publications have focused on the treatment of problematic wounds, the pathogenesis of exuberant granulation tissue in horses continues to elude us. This trial showed that the rates of infection and hypergranulation tissue were lowest in the treated wound. The present study attempted to define the basis of MDIK-15 treated wound, as well as the relationship between MDIK15 and mammalian cell cycle acceleration. We recently documented the role of MDIK-15 in accelerating human cell cycle by investigating its effect on hair, nail and intact and wounded skin. We deemed it crucial to investigate its effect on other mammalian cells, thus we elected to use equine wound which delayed to heal as an example. The results of this study show that the contribution of MDIK-15 to equine wound healed faster. On the other hand, predictably, the lost hair on treated skin appeared faster, thicker and longer than the surrounding untreated hair (cf. figure 20). Previous studies have shown that MDIK-15 promotes human hair growth. As we have recently found (our unpublished data), the possible explanations of speed epithelialization include enhanced proliferation of epithelial cells and the speed growth of treated hair is related to the acceleration
35
of cell cycle by shortening the G1 phase due to the increase of nutrients uptake. MDIK-15 accelerates the cell cycle not only of human cells but also other
mammalian cells. The responses in the equine treated wound in the present experiment confirmed these observations.
One month after first treatment appearance of thick and strong hair. c
Three months and fifteen days after first treatment (the hair begun to appear less thick).
Figure 20. MDIK-15 treated hair in comparison with surrounding untreated one. The treated hair ( ) is significantly longer thicker and stronger than the untreated one ( ).
The field conditions under which this trial was performed imposed a number of constraints on the study design. Among these was the necessity for more treated and control groups. European Union
guidelines require that, when possible, new treatments be compared with positive control products that have a licensed claim for the indication under investigation. However the treatments
36
which were used with this equine wound before MDIK-15 initiation have both been shown to encourage wound healing in many species including horses but failed to treat this equine wound for more than three months. Moreover, the choice of assessment methods was also constrained by the study design. For example, wound infection was assessed by using a clinical score rather than biopsy. Biopsies would have yielded continuous data but would have carried the possibility of modifying the outcome (by increasing the risk of infection), since each biopsy would constitute a fresh insult to the wound. Biopsies would also have been ethically questionable, particularly in cases where multiple repetitions were required. The use of simple measurement methods may also have limited the ability of the study to distinguish prior to post treatments effects on other efficacy parameters (wound size, and speed of normal granulation tissue formation). In summary, topical administration of MDIK-15 to adult stallion at 200 L/cm
resulted in acceleration of equine wound healing that one would expect from previous report on human subjects, thereby confirming the biological activity of MDIK-15 on mammalian cells. There was no severe inflammation, or increased heat, pain or swelling which would indicate an adverse reaction of the test drug to the wound. Further, MDIK-15 can be done in a more economical form and in places without many resources. The use of this drug as an improved therapy for non-healing wounds in equine and in immunocompromised, diabetic or elderly individuals could provide quality healing of acute wounds that would be of significant personal, economic and social advantage. In conclusion, our data suggest that MDIK15 contributes to the acceleration of mammalian cell cycle Disclosure statement Special thank to the horse owner who helped sedating the horse when treating and taking photos.
37
Addendum II
Because of lack of materials, all the following experiments -written in the present tense- are planned but not yet performed. CELL PROLIFERATION ASSAYCells Treatment Method- Third passage keratinocytes (60-75% confluence) and fifth passaged fibroblasts are respectively resupended at a density of 3103 cells/cm in KGM or 1x107 in fibroblast growth medium (FGM), plated on glass coverslips and grown in a humidied 5% CO2 atmosphere at 37C. Medium is changed 3 times a week. For cell experiments, MDIK-15 is dissolved in phosphate buffered saline (PBS) on the day of the experiment to obtain the concentration of 3 M then added to each appropriate above-mentioned cell cultures. Control groups are treated with the same amount of PBS alone. MDIK-15 Cellular Uptake- HPLC analyses are carried out to ascertain the uptake of MDIK-15 by the cell lines. Cells are treated for at least 48 h with MDIK-15 (200 M). Cellular uptake is determined by high-performance liquid chromatography (HPLC), according to Youdim et al. [34], since this procedure allows to discriminate cytosolic from cell membrane content and is analyzed according to Juan et al. [12]. MTS assay- Cell proliferation is determined using the MTS (3-(4, 5dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2(4-ulfophenyl)2H-tetrazolium) assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA). To determine the linear range of the assay, keratinocytes are plated in triplicate in KGM at a concentration of 5x103 cells/well and fibroblast in FGM at a concentration of 3x107 in 96-well plates for 24 h. Subsequently, the cells are treated with various concentrations of MDIK-15 (1, 3, 5, and 7 M) dissolved in PBS or this later alone as a control and further cultured for 48 h. Then the MTS assay is performed according to the manufacturers protocol. All experiments are repeated at least three times. Results are expressed as the change in cell proliferation. Cell cycle analysis using Flow CytometerCell cycle analyses are performed by determining DNA content with propidium iodide (PI). Treated and control cells seeded at 5x105/mL in six- well-plates are grown for 48 hours, then trypsinized and washed twice with PBS. They are centrifuged at 100 x g for 5 minutes, resuspended with 200 L PBS, and cell pellets fixed in 2 mL of 70% ethanol at 4C for at least 1 hour. They are centrifuged at 100 x g for 5 minutes, then resuspended in 437 L PBS, treated with 13 L of 0.8 units/mL DNase-free RNase A, and stained with 40 g/mL propidium iodide at 37C for at least 30 minutes. The fluorescent characteristics of the cells are determined with a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and data are analyzed with Cylchred software (version Winmdi 2.7). Cell diameter measurement- The diameters of the treated cells are determined under a measuring microscope (MM-800/LMU, Nikon France S.A., Champigny sur Marne, France) at a magnification of 100. Measurements are taken with the help of E-MAX DS-V software which provides FOV (field-of-view) measurements. In each independent experiment, about 1000 cells are measured. CE Quantitation- Cornified envelope
38
(CE) content of cultured keratinocytes is determined as previously described [19]. Briefly, NHEKs are treated for one week with either CaCl2 o r MDIK-15 at the indicated concentrations and harvested in 2% SDS solution. After slight sonication (5 s), aliquots are removed for protein determination. The lysate is centrifuged at 12,000 rpm for 15 min and resuspended in 2% SDS/20 mM dithiothreitol (DTT) and boiled for 1 h, and the amounts of soluble cross- linked envelopes were quantified by spectrophotometry at 310 nm. IVLAssay- The amount of involucrin in NHEK cultures is determined as described elsewhere [18]. Briefly, following three days of treatment with either CaCl2 or MDIK-15 at the indicated concentrations, cells are harvested and homogenized. After microcentrifugation at 12,000 rpm for 15 min, supernatants are analyzed for involucrin using an enzyme-linked immunosorbent assay (ELISA) kit (Biomedical Technologies, Stoughton, MA, USA). In Vitro Wound Healing AssaySkin- equivalent cultures are prepared and wounded in vitro as previously described [7]. Briefly, the collagen matrix is constructed by adding NHDFs to neutralized Type I Collagen (Organogenesis, Canton, MA) at a final concentration of 2.5104 cells/mL. Then, 3 mL of this mixture is added to each 35 mm well insert of a 6-well plate and incubated for 7 days in media containing DMEM and 10% fetal calf serum until the collagen gel shows no further shrinkage. At the same time, NHEKs at a density of 5105 cells/well are seeded directly on a raised, mesa-like area is seen in the center of the contracted collagen gel. Cultures are submerged in low calcium epidermal growth media for 2 days, then for two other days in normal calcium epidermal growth media and raised to the air-liquid interface by
feeding from below at 37C in 5% CO2, 7 days after keratinocytes plating. To generate wounds, the skin-equivalent culture is removed from the insert membrane using 1.5 cm punch, and a 4 mm punch is used to incise the epidermis and collagen matrix. 100 L of PBS a l o n e o r c o n t a i n i n g 1 0 L MDIK-15 is put directly on top of the wound. The model wound healing is examined by means of an inverted phase-contrast microscope equipped with a digital camera until the wound is closed. The images of the model wounds are stored by an image analyzer (Q500MC; Leica, Cambridge, UK). Human Hair Follicles Ex-vivo Growth AssayHair follicles are isolated from occipital scalp skin samples of healthy human volunteers, with informed consent, by removing the epidermis and "plucking" the hair follicles with forceps from the dermis [4]. Whole hair follicles are placed in Williams E medium, supplemented with insulin (10 g per mL), L-glutamine (2 mM), penicillin (100 U per mL), and streptomycin (100 g per mL) (Gibco Invitrogen GmbH, Karlsruhe, Germany) in 24-well culture plates (Falcon, Franklin Lakes, New Jersey) incubated at 37C in 5% CO2 as previously described [11, 24]. Hair follicles from each donor are separated into groups and 0.1, 1, 10, or 100 ng per mL MDIK-15 in phosphate-buffered saline is added whereas control follicles received PBS vehicle alone. Hair follicle length is measured on day 0 using a Nikon inverted microscope with a digital camera and LuciaM software (version 2.995, Nikon GmbH, Dsseldorf, Germany) and at regular intervals after initiation of culture. Medium including MDIK-15 is replaced twice during the 8-d analysis period. Hair growth on day 8 of in vitro culture with MDIK-15 is compared with the control group using two-tailed t tests.
39
Animal Models-The present study is carried out with ethical committee approval. Eight- to twelve-week-old diabetic female BKS.Cg-m+/+ Leprdb mice (db/db+/+; 40-50 g, displaying similar metabolic perturbations as observed in human type II diabetes such as obesity, hyperglycemia, insulin resistance, and impaired wound healing), ten-week-old BALB/C-nude mice (4050g), 12-week-old NOD-SCID mice (40-50g), adult C57BL/6 mice, male C3H/HeN mice and Male Sprague-Dawley rats (180200 g) are housed in individual cages and allowed food and water ad libitum. They are raised under controlled temperature (22 1C), humidity (65%-70%), and daynight cycle (12:12-hr light:dark). 24 hours prior to the beginning of diabetic wound healing assay, the db/db (+/+) mice are clipped and the back skin below the shoulder blades is depilated under light ether anesthesia (shaving the hair is not recommended because MDIK-15 will quickly regrow it which may suffocate the diagnostic of the wounds) then cleaned with 3% H2O2 (hydrogen peroxide). Unless specified otherwise, each animal is anesthetized with 2 % Rompun solution (5 l/g) just before the start of the assay. In Vivo Proliferation And Tissue Preparation- Unanesthetized nude mice split into two groups of five mice each. The hairless mice are topically applied with MDIK-15 or PBS (vehicle) at a dose of 2 mL/cm. The hairless mice are sacrificed one week after the single treatment, and 4-mm punch biopsies of the treated part are taken and embedded in paraffin. Five crosssections are taken from each tissue and stained with H&E (hematoxylin and eosin). Stained slides are quantitatively characterized via digital image analysis using Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA). Images are captured through an
Olympus BH-2 microscope fitted with a MicroImage video camera (Boyertown, PA, USA). The area of the epidermis (Aep) and the length of basement membrane (Lba) are taken from at least ten series of random images on several slides to get a mean value for statistical comparison. Epidermal thickness is determined by the following formula: Aep/Lba. Diabetic And Non-diabetic Mice Wound Healing Assay- A dermal biopsy punch (Miltex GmbH, Ludwigshafen, Germany) is used to punch 8-mm full-thickness wound including the panniculus carnosus layer on the back skin below the shoulder blades of diabetic and nude mice after disinfecting this area with iodine. A wound placed in this area cannot be reached by the mouse and therefore prevents self-licking. In each animal type, mice are randomly divided into two groups of equal number. One group is treated with vehicle (phosphate-buffered saline, PBS) by topical application on the wounding area, while the other groups wounds are topically treated with MDIK-15. PBS and MDIK-15 are administered just after wounding and only once in all of the study period. Wounds from individual mice are digitally photographed every day, beginning on the day of wounding. For all measurements, wound area is quantified using Scion Image software. Three animals in each group are randomly selected, sacrificed and examined on days 5, 10 and 15. The complete wounds, including 2 mm of the wound margins, are harvested for biochemical analysis. The animal test evaluation focuses on the formation of granulation tissue, angiogenesis, and the regeneration of the basement membrane. Burn Healing Assay- Under deep anesthesia, nude mice are placed in a prone position. A full tissue burn wound
40
is made by electrocautery (Ellman Surgitron FFPF EMC Electrosurgical) on the back skin below the shoulder blades. The width of the pin is 0.3 cm; its length is 2 cm and is used at the cut modulation for 3 seconds. In these conditions burn corresponds to IIIAdegree in accordance with clinical classification of burns. Wounds of the half burned mice are topically applied with 2 mL/cm MDIK-15 and the other half with PBS (control). Every group consists of 20 animals. Treatment of animals begins immediately after burn induction. Randomization of experimental animals and treatment scheme are same as the abovementioned. Three animals from each group are sacrificed timedependently (1, 5, 10, 15 and 20 days after burn) and the wounds harvest using a 6-mm punch biopsy for biochemical analysis. Hydroxyproline Assay- The content of collagen in wounded and burned tissues is determined by mass spectrometric analysis for 4-hydroxyproline [31]. Samples obtained by biopsy are freezedried. Internal standard (N-methyl-Lproline) and 6 N HCl solution is added to wound tissue, and each sample is then hydrolyzed overnight at 115C. The O-butyl ester derivatives are prepared with 10% BF2-butanol for 30 minutes at 120C after drying the hydrolysate. Liquid chromatography (column: Eclipse XDBC18)/mass spectrometry analysis is performed on a Hewlett-Packard (series 1100, Atlanta, GA) mass selective detector monitoring the ions m/z 188. In Vivo Hair Growth Assay- The dorsal hair of anaesthetized fifty-day- old male C3H/HeN mice is gently cut short with a trimmer (shaving razors or hair depilatory creams are not used because they can perturb the hair cycle). MDIK15 (200 L/cm) is then applied topically on the test area for only one time. At different time points (1, 3, 5, 7, 10, 15
and 24 days) the mice are sacrificed and photographed using a digital camera. The fullthickness of the dorsal skin in the test area is excised and, after its reverse side is photographed, it is processed for paraffin embedding. The embedded skin samples are cut into 4 m sections using a microtome, stained with hematoxylin (blue) and observed under a microscope. The ratio of the hairy area versus the total test area is measured in the images of each mouse using ImageJ image analysis software and is processed for statistical evaluation. To characterize the effect of MDIK-15 in anagen growth phase of pelage hair follicles, eight mice aged 10 d old, when pelage hair follicles are in anagen of the first pelage coat generation, are irrigated by MDIK-15 or PBS by topical application (2 mL/cm). The mice are observed for a further 10 days and necropsied when 20 day old when generation of the first pelage coat is normally complete and pelage hair follicles of the dorsal skin are typically in telogen [3]. To characterize further the effects of MDIK-15 on hair follicles in aging C3H/HeN mice, a large number of mice all aged 246 days are shaved on their dorsal surface and only those mice exhibiting a uniform telogen stage skin are selected for use. The mice are topically applied with MDIK-15 or PBS (2 mL/cm) on the test area. They are observed for 24 days and all are necropsied aged 270 days. Dorsal skin samples, including the test and the surrounding area, are fixed in Fekete's acidalcoholformalin solution, paraffin embedded, sectioned at 6 M through the area of test, and stained with hematoxylin and eosin for histologic evaluation [21]. The size and distribution of hair follicles in the skin and the diameter of the hair shafts in each of the
41
four groups are then compared. Statistical analysis- Statistical differences are evaluated with Students t-test or ANOVA (analysis of variance between groups) using SigmaStat (SPSS,
Chicago, IL, USA). The data are presented as the mean S.D (standard deviation) from at least three independent experiments. A value of P<0.05 is considered statistically signicant.

MDIK-15 Scientific Report

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Pilot Study Evaluating the Efficacy of the New Drug MDIK-15 in Accelerating Cell Cycle of Mammalian cells

MDIK-15: Cell Cycle Accelerator

MDIK-15: Cell Cycle Accelerator

Materials and Methods

MDIK-15: Cell Cycle Accelerator

MDIK-15: Cell Cycle Accelerator

MDIK-15: Cell Cycle Accelerator

MDIK-15: Cell Cycle Accelerator

Table I. Selected patient and wound baseline demographics

Age (years) Mean SD 35.56 12.39

Sex Male Female 6 3

MDIK-15: Cell Cycle Accelerator

MDIK-15: Cell Cycle Accelerator

Table II. Visual Analogue Cosmesis Scale (VASC) (110)

p Value 0.004 0.001 0.007

Bars are SEM.

MDIK-15: Cell Cycle Accelerator

5 day progress of the wound towards healing

12th day complete healing with subside of the blackness

6th month invisible scar

MDIK-15: Cell Cycle Accelerator

MDIK-15 treatment four days after wounding (small open wounds) c

3rd day post treatment speed epithelialization of the wound edges d

5 day total close of the open wounds

9 day complete healing of the wounds

MDIK-15: Cell Cycle Accelerator

MDIK-15 treatment 24 hours after wounding d

3 day post treatment e

7th day (Mdik-15 second treatment) g

15 day (total debridement)

2 months (barely visible scar)

MDIK-15: Cell Cycle Accelerator

MDIK-15 treatment 24hours after wounding d

3rd day post treatment e

7th day (Mdik-15 second treatment) g

11th day (debridement) i

13th day (epithelialization)

17th day (total debridement)

2nd months (pleasing scar)

MDIK-15: Cell Cycle Accelerator

MDIK-15: Cell Cycle Accelerator

MDIK-15 first treatment just after wounding d

1st day (24h post treatment) e

3rd day (a clean wound) g

4th day (sterile wound) h

5th day MDIK-15 second treatment (a moist environment) i

6 day (formation of new blood vessels) j

7 day (healthy granulation tissue) k

8 day (speed division and migration of basal keratinocytes) l

9 day (increased epithelialization)

2 month ( aesthetically pleasing scar)

6 month (barely visible scar)

MDIK-15: Cell Cycle Accelerator

MDIK-15: Cell Cycle Accelerator

MDIK-15 treatment 24hours after wounding (tremendous edema) d

3rd day post treatment, disappearance of the edema e

7th day (Mdik-15 second treatment) g

9th day the skin flap became necrotic h

10th day (total debridement) i

2 months (barely visible scar)

MDIK-15: Cell Cycle Accelerator

MDIK-15: Cell Cycle Accelerator

Left pinky (MDIK-15 application)