ITMI20112438A1 - INJECTABLE FORMULATIONS OF CYTOKINES, GROWTH FACTORS, CHEMOTACTIC FACTORS, STAMINAL STIMULATION FATORS AND RNA FOR DERMA REGENERATION - Google Patents

Description

INJECTABLE FORMULATIONS OF CYTOKINES, GROWTH FACTORS, CHEMOTACTIC FACTORS, STIMULATION FACTORS AND RNA FOR THE REGENERATION OF DERMAâ €

The present invention relates to formulations containing a support consisting of a biomaterial and a composition comprising cytokines, growth factors, stem stimulating factors, RNA and chemotactic factors, useful as fillers in dermatology and plastic and aesthetic surgery.

State of the art

Wrinkles form with advancing age and begin to appear after the age of 25; are furrows that form in some parts of the body, in which the skin lining is subjected to continuous stress by the underlying muscle groups, such as occurs on the hands or in the face - facial muscles, under the eyelids, on the sides of the mouth and above the lips, on the forehead and near the nose. Wrinkles involve the skin: dermis and epidermis.

Wrinkles can be divided into senescence wrinkles and expression lines.

The wrinkles of senescence appear on the skin with the natural course of age, losing its elasticity and natural firmness. Expression lines appear on a relatively young epidermis and in any case not affected by the wrinkles of senescence.

With time, in fact, wrinkles become accentuated up to the dermis or the support structure of the skin, that deeper part of the skin and therefore the loss of elasticity of the skin tends to form this type of deep and marked wrinkle of about 0.005 mm in depth. . In addition to premature aging of the skin, this is associated with a sagging of the cheeks and an alteration of the elastic fibers.

Exposure of the skin to ultraviolet UV light can contribute approximately 2/3 to the aging of the skin.

Plastic surgery offers a possibility to remedy the imperfections caused by wrinkles. Today, plastic surgeons combat the effects of aging with various non-invasive methods such as augmentation of the soft tissues of the face with fillers. A large amount of fillers are on the market, each with its own chemical components, indications and effectiveness.

Since the late 19th century, injection of fat has been used to rebuild, but Illouz was the first in 1980 to re-inject fat obtained through liposuction. Fournier's modified technique, called microlipoextraction and injection, was in 1986 and represented a notable advance in cosmetic surgery. A study of the behavior of fat implanted in a rabbit showed that adipose tissue remains even after mechanical action on the cells during collection and injection. A study carried out by Skouge showed that the fat sample can replenish itself and become revascularized. Autologous tissue transplantation has been used in facial areas for the correction of wrinkles, for depressed or atrophic areas, for cheek and chin augmentation, for acne and trauma scars. It has also been used for the rejuvenation of the back of the hands, for breast augmentation, for defects caused by incorrect liposuction. The face is the area of choice because the rich vascularity supports the fragile adipose system.

Collagen implants, both animal and human, have long been used to augment soft tissue. Bovine collagen fillers have been the most popular injectable implants for about two decades. Later, bioengineered human collagen-based products became available in addition to fillers containing hyaluronic acid.

Fillers containing hyaluronic acid are the products of choice for soft tissue augmentation: they last longer, are less immunogenic and can be broken down by hyaluronidase. For these reasons, hyaluronic acid-based fillers are the most common today. However, the injection of hyaluronic acid-based fillers can cause both immediate and delayed complications, which can range from mild to severe. In recent years, the use of fillers based on chemically modified hyaluronic acid has spread.

All currently available fillers can induce adverse reactions. The rapidly biodegradable and reabsorbable substances can, for example, induce serious complications but normally disappear spontaneously in a few months. The slower biodegradable and non-absorbable fillers, on the other hand, can cause an increase in severe reactions with little or no tendency to spontaneous resolution. These reactions can also appear several years after the injection. There is therefore a need for new fillers that do not have the aforementioned disadvantages.

Description of the invention

According to current scientific literature, the therapeutic action of stem cells can be traced back to two mechanisms: differentiation of stem cells into resident cells and release of regenerative trophic factors by stem cells. The respective contributions of these two mechanisms remain to be clarified, even if it has been hypothesized that it is not the stem cells that transform into mature cells of the injured tissue, but that they transmit vital factors to this tissue, which can thus return to proliferate. and to differentiate, regenerating (Figure).

Stem cell therapy presents many problems related not only to costs and technical and application complications, but also to ethical and religious scruples.

Stem cell therapy is feasible only by injection or, in some cases, topically, and not by mouth. The stem cell supernatant in culture contains growth factors, cytokines, chemotactic factors, etc. believed to be responsible for the beneficial effect of stem cell therapy on tissue growth and / or repair.

The eventual use of vital factors that can be isolated from the supernatant of stem cells presents, however, not only the same ethical problems of the use of stem cells themselves but also very high costs.

It has surprisingly been found that the same active factors released by stem cells and therefore present in the supernatant of stem cell cultures are present in biological fluids and in some tissues of mammals. The best sources of these factors are mammalian serum, placenta, and colostrum.

The Table shows the qualitative and quantitative comparison between the factors present in the supernatant of stem cell cultures and the factors isolated from these new sources we call POOL OF MATERNAL FACTORS

(P.M.F.),

Table

P.M.F. Mesenchymal NAME (Pg / ml) stem cells (pg / ml) IL 1 ra IL- 1 receptor antagonist 29.05 0.0

IL-1b Interleukin-1b 0.09 0.0

IL -2 Interleuk -2 32.05 0.0

IL -4 Interleukin-4 0.17 21.12

IL -6 Interleukin -6 1.26 293.01 IL -8 Interleikin -8 0.71 359.56 IL -9 Interleukin-9 3.66 0.78

IL -10 Interleuk - 10 2.83 0.99

IL-12 Interleukin - 12 1.73 0.0

IL-15 Interleukin- 15 4.33 0.5

IL-17 Interleukin- 17 21.95 0.32 Eotaxin Eotax 5.01 0.0

INF-y Gamma -interferon 5.97 11.14 MCP-1 Monocyte chemotactic factor-1 4.12 11.23 PDGF Platelet derived growth factor 11.e 5 3.08 TNF Tumor necrosis factor 40.00 12.30

Vascular endotheliall growth 41.00 208.04 VEGF factor

HGF Hepatocyte growth factor 52.3 100.74 FGF Fibroblast growth factor 180.15 18.21

TGF- 64.00 51.32

beta1 transforming growyth factor

IGF-1 insulin-like growth factor 1.60 0.0

GM- Granulocyte / monocyte-colony 183.85 0.0 CSF stimulating factor

LIF leukemia inhibitory factor 15.20 27.32 SCF stem cell factor 10.55 4.54 SDF-1 stromal derived factor-1 41.75 28.8 NGF Nerve growth factor 8.33 0.0 BMP-2 Bone morphogenic protein 25.01 2.65

RNA 189 ng / ml 48 pg / ml

Presence of growth factors / cytokines in P.M.F. (1 mg / ml) and in the supernatant of Mesenchymal Stem Cells (1 million cells) It is evident (table) the higher concentration of almost all the factors present in P.M.F. compared to that present in the stem cell supernatant.

It is also evident the possibility of using even very high therapeutic concentrations of P.M.F., for example doses of 0.5 / 1 g topically or injected and doses up to 20/30 g orally, compared to the maximum therapeutic concentrations of cells used. stem cells that correspond to 1/2 million / kg and therefore to a maximum of 140 million per therapy and, therefore, to values of factors always of the order of picograms.

The object of the invention is therefore a compound (PMF) containing in very high concentration all the active factors present in the supernatant of stem cells but isolated, with simple extraction methods, from very cheap natural sources, easily available and without problems. ethical.

Furthermore P.M.F., isolated from biological fluids and mammalian tissues, also contains antibacterial factors (transferrin, lysozyme, lactoperoxidase, lactoferrin) and antibodies of the IgG and IgA classes. (P.M.F. Ab).

The absence of antibodies and antibacterials of the stem cell supernatant is another reason for the use of P.M.F. Ab in many therapeutic, topical and oral uses.

The serum shows the maximum peak of the factors in the last days before the birth, the colostrum in the first hours after the birth and no later than the 6th hour.

Already after 12 hours from the birth the factors decrease considerably, at 24 hours many of them are no longer measurable.

These factors are genetically very conserved in the various species and therefore it is possible to use on humans factors isolated from other mammal species such as cattle, horses, camels, marine mammals, etc. The factors are controlled with specific ELISA methods for the species, even if the interspecies cross reaction is very high as the factors are phylogenetically very conserved and, therefore, they are only qualitatively measurable even with ELISA used for different species (eg: human-bovine and viceversa).

A first source of the factors of the invention is the serum of mammals which, in the last days (5-15) before giving birth, is very rich in the active factors object of the invention compared to non-pregnant mammals or in the first months of pregnancy. A serum extraction method is described below, by way of example.

1 liter of blood is taken in 4 days for a total of 4 samples in order not to harm the animal, preferably bovine or equine.

The blood is serumed at room temperature for 24 h and then centrifuged to squeeze the clot.

The serum is recovered (about 30/40% of the total volume) to which are added, as antiseptic agents, 2.5% phenoxyethanol and 1% diazolidinyl-urea. The treated serum is then fractionated with the following steps.

Ultrafiltration 300'000 Da

The serum sample (frozen at -20 ° C) obtained by coagulation and centrifugation from mammalian blood is thawed at room temperature and diluted with 2 volumes of demineralized water. The solution obtained is ultrafiltered on a 300'000 Da polyethersulfone Millipore Pellicon Biomax tangential flow flat membrane at a Pi of 0.5 Ã · 1 bar, in a cold room at 4 ° C.

The retentate and a fraction corresponding to approximately 1:10 of the permeate are transferred to a 1000 Da regenerated cellulose Spectrum Spectrapor dialysis tube and dialysed against demineralized water.

Ultrafiltration 5'000 Da

The remaining permeate is ultrafiltered on a 5000 Da membrane. The 300000 Da ultrafiltration permeate is concentrated on a Millipore Pellicon Biomax cross flow flat membrane in polyethersulfone from 5000 to a Pi of 0.5 to 1 bar, in a cold room at 4 ° C.

The retentate is transferred to a 1000 Da regenerated cellulose Spectrum Spectrapor dialysis tube and dialyzed against demineralised water (preservatives are also eliminated with this dialysis). The mixture is then immediately freeze-dried.

A second source of the factors of the invention is the placenta. An extraction method is described below, by way of example.

Bovine or equine placentas are preferably used.

Homogenization

The placenta (frozen at â € “20 ° C) is thawed at room temperature, cut into small pieces, washed with abundant cold saline (0.9% NaCl) (4 ° C) and homogenized by means of a Siramm cutter in a buffer of lysis composed as follows: 50 mM Tris / HCl, 25 mM EDTA, 0.001% triton X-100 at pH 7.4. To the suspension obtained is added NaCl up to a concentration of 0.9%, and the preservatives (phenoxyethanol at 2.5% and diazolidinyl-urea at 1%). The suspension is stirred (magnetic stirrer) for 2 hours and kept static over night in a cold chamber at 4 ° C.

Centrifugation

The suspension is centrifuged at 13,000 rpm with a Sorvall RC6 centrifuge and SLA 15000 rotor for 45 minutes at 4 ° C. The supernatant of the centrifugation is recovered, pre-filtered under vacuum on Dicalite and on regenerated cellulose filters of 0.45 µm and 0.22 µm.

Ultrafiltration 300'000 Da

The filtered product is ultrafiltered on a 300'000 Da polyethersulfone Millipore Pellicon Biomax tangential flow flat membrane at a Pi of 0.5Ã · 1 bar, in a cold room at 4 ° C.

Ultrafiltration 5'000 Da

The 300000 Da ultrafiltration permeate is concentrated on a Millipore Pellicon Biomax cross flow flat membrane in polyethersulfone of 5000 at a Pi of 0.5Ã · 1 bar, in a cold room at 4 ° C.

The retentate is transferred into a 1000 Da regenerated cellulose Spectrum Spectrapor dialysis tube and dialyzed against demineralised water (therefore also the preservatives are eliminated) and then immediately freeze-dried.

A further source of the factors of the invention is colostrum. Bovine colostrum is preferably used due to the ease of supply and the quantities available. Colostrum from Holstein (Friesian) and Guernsey cows is particularly preferred. Such cows have been proven to produce colostrum with the highest concentration of active factors. The cows are preferably in the second or third calving. The colostrum is preferably collected between the 1st and the 6th hour from the birth as, in this period, there is the greatest concentration of active ingredients. In colostrum from the sixth hour after birth the active factors decrease rapidly (after 24 hours only 15% are present).

Collected colostrum is tested for tuberculosis, cytotoxicity on cell cultures, control of mycoplasma, prions and human and bovine viruses.

The colostrum in the mammary cistern is practically sterile, but once milked, despite all precautions, due to the high concentration of growth factors, its bacterial load rises very rapidly during freezing and thawing, rather slow processes given the ™ high density of colostrum in the first few hours.

The use of γ rays allows to obtain a sterile colostrum only if radiations greater than 10 Kgy are used which however destroy most of the active factors, and on the other hand this method does not prevent the formation of pyrogens whose use is inadvisable topically and prohibited injecting.

An innovative collection chain was therefore developed to obtain a sterile hypoallergenic compound, without preservatives and without pyrogens.

To the colostrum collected in sterile tanks (vacuum sterilized at 25 Kgy) antiseptic agents are added in such quantities as to guarantee sterility and the absence of pyrogens. Phenoxyethanol at a concentration of 2.5% and diazolidinyl urea at a concentration of 1% are preferably used.

The colostrum thus treated can, for short periods (max 30 days) not be stored frozen before the extraction processes of the active factors, with evident savings in industrial costs.

Active factors can be extracted with the following steps:

Centrifugation

Bovine colostrum is diluted 1:10 with demineralized water, NaCl is added until a concentration of 0.9% is obtained.

The suspension is centrifuged continuously at 8'000 rpm at a temperature of 20-25 ° C with an Alfa Laval centrifuge, discarding the pellet corresponding to the lipid part.

300,000 Da ceramic ultrafiltration

The product obtained from centrifugation is ultrafiltered on a ceramic membrane with a cut-off of 300,000 Da at a temperature of 20-25 ° C, recovering the permeate.

Ultrafiltration on 5000 Da

The permeate of the ultrafiltration 300'000 Da is concentrated on a spiral membrane wrapped by 5'000 Da in polyethersulfone at a temperature of 20-25 ° C and dialyzed against demineralized water up to a conductivity of the retentate of about 600 µs / cm <2> (preservatives are also eliminated with this dialysis).

Lyophilization

The 5'000 Da ultrafiltration retentate is filtered under vacuum on 0.2 µm regenerated cellulose Millipore filters, frozen at -20 ° C and freeze-dried.

Purification of P.M.F. Ab from antibodies

A saturated ammonium sulfate solution is added to a P.M.F. Ab isolated from either serum, placenta or colostrum under agitation and at a final ratio 1: 1 (v / v). The mixture is kept for two hours at room temperature or at 4 ° C for 12 hours, in a sterile environment and subsequently centrifuged at 18,000 rpm. The supernatant is then subjected to dialysis at 4 ° C against water, using membranes with a cut-off of 100 kDA. The fraction outside the dialysis membranes, devoid of the immunoglobulin component, is then lyophilized and stored at -20 ° C. Depletion of the immunoglobulin component is verified by SDS-PAGE analysis under reducing conditions and not using blue coomassie as a dye.

Alternatively or after the previous one, the purification involves the use of affinity columns functionalized with protein G. The solution of the active factors or the fraction previously precipitated with ammonium sulphate is eluted by a peristaltic pump at a flow of 1 ml / min through a protein G-functionalized column in a sterile environment. After 20 minutes of perfusion, the solution is dialyzed by ultrafiltration at high pressure to completely eliminate the preservatives and then lyophilized. A sterile powder is obtained, free of pyrogens, free of animal immunoglobulins, free of preservatives, hypoallergenic (casein and lactalbumin are responsible for over 95% of allergies to these animal components) of very high solubility and with the maximum possible concentration of factors active.

Whatever the source used, the presence of the same active factors measured with the ELISA is found.

The following are the active factors present in PMF and their main activities.

GROWTH FACTORS

Basic FGF (fibroblast growth factor): Regulates the growth and differentiation of cells of mesenchymal origin.

TGF-beta1 (transforming growth factor): Regulates growth, differentiation, adhesion, migration and other cellular functions.

IGF-1 (insulin-like growth factor): Regulates cell growth and development. NGF (nerve growth factor): Stimulates growth and functionally regulates structures of the nervous system and sensory organs.

PDGF (platelet-derived growth factor): Regulates the growth and differentiation of cells of mesodermal origin.

VEGF (vascular endothelial growth factor): Regulates the growth of endothelial cells and angiogenesis.

LIF STIMULATION FACTORS (leukemia inhibitory factor). Induction of hematopoietic stem cell differentiation, regulation of neuronal differentiation, epithelial-mesenchymal transition and maternal-fetal tolerance.

SCF (stem cell factor). Pleiotropic factor that acts on hematopoietic stem cells in adults.

SDF-1 (stromal derived factor-1). Chemotactic factor for stem progenitors expressing the CXCR4 ligand.

CYTOKINES WITH IMMUNOSTIMULANT ACTIVITY

IL-9: Stimulation of cell proliferation and block of apoptosis. IL-12: Stimulation of T lymphocytes and natural killer cells.

IL-15: Stimulation of T lymphocytes and natural killer cells.

INF-gamma (gamma interferon): Cytokine with antiviral, immunoregulatory and anti-tumor activity, powerful activator of macrophages.

TNF (tumor necrosis factor): Cytokine with multiple functions. Involved in proliferation, differentiation and apoptosis.

IL1-ra ANTI-INFLAMMATORY CYTOKINES (IL-1 receptor antagonist): Inhibition of the proinflammatory activity of IL-1.

IL10. Regulation of regulatory T cells, inhibition of T helper lymphocyte activity, stimulation of B lymphocytes.

CHEMOTACTIC FACTORS

Eotaxin: Recruitment of eosinophils.

IP-10: Recruitment of inflammatory cells.

MCP-1 (Monocyte chemotactic factor-1): Recruitment of inflammatory cells, particularly monocytes.

RNA: modulates target cells by inducing reprogramming at the genetic level.

The formulations of the invention are suitable for parenteral and / or intradermal administration, depending on the desired application and the specific organ damage to be treated. These formulations will contain the fraction obtainable as described above in unit dosages ranging from 0.5% to 5%.

The invention is described in greater detail in the following experimental part, given as an example.

IN VITRO TESTS

P.M.F. has been shown to significantly increase (> 100%) the proliferative capacity of human adipose-derived mesenchymal stem cells (hASCs) compared to control (10% FBS). P.M.F. used at two different concentrations (5 mg / ml and 1mg / ml), in the culture medium, it has shown that the effect is dose dependent and incubation time dependent.

LIVE TESTS

P.M.F. (100 γ) is mixed in 1 ml of non-cross-linked hyaluronic acid, also called â € œrivitalizingâ € hyaluronic acid, whose resistance to hyaluronidase is very limited. Hyaluronic acid or other vehicles, such as matrigel, are intended to release P.M.F. in a time of 15-20 days so as to allow the prolonged regeneration of the dermis and collagen over time. IN VIVO CELL PROLIFERATION

A mixture of P.M.F. is injected. of matrigel or subcutaneous hyaluronic acid in mice. After 7 days, cell and vascular proliferation is observed at the periphery of the amorphous support. After 20 days the amorphous support is totally colonized by cells. After another 30 days there is a noticeable formation of collagen.

MECHANICAL LESION OF THE PIG TAIL: INTRADERMAL THERAPY

Pig tail tying produces necrosis downstream of the tail itself. The experimental model involves the upstream injection of: 1) Physiological, 2) physiological P.M.F., 3) hyaluronic acid P.M.F.

After injection of physiological, upstream of the lesion, the tails fall in 24 hours. 15 days after the injection, upstream of the lesion, of physiological P.M.F., the tails do not fall, they begin to grow but not in optimal conditions; especially in the distal tracts to the lesion necrotic lesions are noted, there is no growth of the bristles. 15 days after the injection of hyaluronic acid P.M.F. upstream of the lesion, the tails returned perfectly normal.

The histological examination shows that in the treated animals no alterations of the nerve bundles are observed and the musculature, the dermis and the vascularization have returned to physiological conditions. The tail sections of untreated animals, in addition to having a diameter about half that of treated animals, show extensive sclerosis, with total atrophy of the neuro-muscular, vascular and dermal components. It is also noted that the arteries are completely blocked by intimal plaques.

Claims (8)

CLAIMS 1. Formulations containing a support consisting of a biomaterial and a composition comprising active factors selected from cytokines, growth factors, stem stimulating factors, RNA and chemo tactical factors isolated from serum, placenta or colostrum, useful as fillers in dermatology and plastic surgery and aesthetics. 2. Formulations according to claim 1 wherein the composition contains Basic FGF, TGF-beta1, IGF-1, NGF, PDGF, VEGF, LIF, SCF, SDF-1, IL-9, IL-12, IL-15, TNF , IL1-ra, IL10, Eotaxin, IP-10, MCP-1 and RNA (microRNA). 3. Formulations according to claim 1 or 2 wherein the active factors are isolated from mammalian serum. 4. Formulations according to claim 1 or 2 wherein the active factors are isolated from the placenta. 5. Formulations according to claim 1 or 2 wherein the active factors are isolated from colostrum. 6. Formulations according to one of claims 1 to 5 in which the biomaterials or supports suitable for the subcutaneous implant such as hyaluronic acid or analogues, collagen, synthetic polymers or copolymers such as polylactides, polyglycolides, polycaprolactones, matrigels, in situ gelling systems, organogels , hydrocolloids, possibly mixed together. 7. Formulations according to claim 6 wherein the biomaterial is non-cross-linked hyaluronic acid. 8. Formulations according to any one of claims 1 to 7 for use as a filler in dermatology and cosmetic surgery.