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

AbstractThe 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.

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MDIK-15: Cell Cycle Accelerator

IntroductionThe 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

MDIK-15: Cell Cycle Accelerator

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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 MethodsProduct 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.

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MDIK-15: Cell Cycle Accelerator

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.

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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

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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 2L/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

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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=