Hemostatic compositions and therapeutic regimens

专利摘要:

公开号:ES2552842T9
申请号:ES08725771.3T
申请日:2008-02-19
公开日:2016-06-28
发明作者:Sergio Finkielsztein；John N. Vournakis
申请人:Marine Polymer Technologies Inc；
IPC主号:

专利说明:

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DESCRIPTION
Hemostatic compositions and therapeutic regimens
1. Field
The application refers in general to the field of hemostasis, including methods, compositions and devices that can be used to achieve hemostasis at a higher rate or at a reduced period. More specifically, the application describes hemostatic compositions that can be applied to wounds and therapeutic regimens for the treatment of wounds. The hemostatic compositions may comprise one or more biocompatible polymers or fibers, such as poly p-1 ^ 4-N-acetylglucosamine bandages. Optionally, the poly p-1 ^ 4-N-acetylglucosamine bandages comprise irradiated poly p-1 ^ 4-N-acetylglucosamine of reduced molecular weight and length.
2. Background
Wound healing, or wound repair, is the natural process of regeneration of the dermal and epidermal tissue of the body. After a wound occurs, a series of complex biochemical episodes in a closely organized cascade to repair the damage occur. These episodes overlap in time and can be artificially classified into independent stages: the inflammatory, proliferative, maturation and remodeling phases. In the inflammatory phase, bacteria and wastes are phagocytosed and eliminated, and factors that cause migration and division of cells that participate in the proliferative phase are released. The proliferative phase is characterized by angiogenesis (formation of new blood vessels from endothelial cells), fibroplasia, collagen deposition, granulation tissue formation, epithelialization and wound contraction. In the maturation and remodeling phase, the collagen is remodeled and realigned along lines of tension and the cells that are no longer needed are removed by apoptosis.
There are two types of wounds, open and closed. Open wounds are classified according to the object that caused the injury. For example, incisions or incised wounds (including surgical wounds) are caused by a clean, sharp-edged object such as a knife, a razor or a glass chip. Lacerations are irregular wounds caused by a blunt impact on soft tissue found on hard tissue {p. eg, the removal of the skin that covers the skull) or tearing of the skin and other tissues such as caused by childbirth. Abrasions or scratches are superficial wounds in which the upper layer of the skin (the epidermis) is scraped. Puncture wounds are caused by a sharp object to the skin, such as a nail or needle. Penetrating wounds are caused by an object, such as a knife as it enters the body. Bullet wounds are caused by a bullet or similar projectile that is directed (e.g., entry wound) and / or through the body (e.g., exit wound). In a medical context, all stab wounds and gunshot wounds are considered important wounds. Open wounds also include burn wounds caused by thermal, chemical or electrical injury.
Closed wounds include bruises (more often known as a bruise, caused by blunt force trauma that damages tissue under the skin), bruising (also called a bloody tumor, caused by damage to a blood vessel that in turn causes blood is collected under the skin), and crush injuries (caused by a large or extreme amount of force applied over a long period).
Chronic wounds are wounds that have not been able to proceed through an orderly and timely series of episodes to produce a lasting structural, functional and aesthetic closure. Many chronic wounds are wounds or cutaneous ulcers, caused by factors such as diabetes, venous stasis, arterial insufficiency, or pressure. Certain cutaneous wounds are burn wounds, caused by thermal, chemical or electrical injuries. Chronic wounds are the source of significant pain and suffering. If left untreated, they can cause life-threatening complications, reduce the recovery rate or worsen other health conditions. Intensive and effective treatment can help restore skin integrity, and avoid unwanted health problems. While these wounds are inflicted by different causes, the wound healing process and wound treatment strategies are similar in many ways.
Bedsores are a type of bedsores that can significantly reduce the quality of life and negatively affect the overall prognosis. Bedsores are localized areas of skin lesions that develop when soft tissue is compressed between a bony prominence and a hard surface for a long time. Bedsores usually develop when lying or sitting for a prolonged period without changing body posture. For those who are bedridden, bedsores are more likely to form on or around the heels, the hip bone and the lower back or coccyx. Bedsores can also develop in several other areas, including the spine, ankles, knees, shoulders and head, depending on the position of the patient. If left untreated, bedsores can degenerate into the stage of decomposition of epithelial tissue, with inflammation, bacterial infection and other serious complications. The body's response to infection often causes fever, chills, changes in mental state, rapid pulse and respiratory rate.
Temporary bandages, including interactive temporary bandages, are intended to provide supportive care until definitive closure can be achieved. It is expected that temporary bandages work
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as a barrier, just like human skin. Available wound dressings are effective to a certain extent, but suffer from significant deficiencies, such as the high frequency of bandage changes, wound drying or bandage adhesion, high treatment costs, the development of a reaction to foreign body, and a low rate of improvement, especially in elderly patients. The reaction to the foreign body begins as wound healing, including the accumulation of exudate at the site of the injury, the infiltration of inflammatory cells to debride the area, and the formation of granulation tissue. However, the persistent presence of a foreign body can inhibit complete healing. Instead of the reabsorption and reconstruction that occurs in wound healing, the reaction to the foreign body is characterized by the formation of giant cells in the foreign body, encapsulation of the foreign object and chronic inflammation. Encapsulation refers to the firm, generally avascular collagen layer deposited around a foreign body, effectively isolating it from the host's tissues. This response was developed as a measure of protection. The reaction to the foreign body can lead to chronic pain.
The healing time of a chronic wound can vary from a few weeks to a year, depending on the size and type of wound. Wound treatment involves many direct and indirect costs. According to the International Commitee on Wound Management (ICWM), wound dressings comprise only 10 percent to 15 percent of the total direct treatment cost (International Commitee on Wound Management, 1994, Wounds 6 (3): 94-100) Instead, a significant percentage of the total cost is attributed to taking care of providing salaries and personnel expenses (International Committee on Wound Management, 1994, Wounds 6 (3): 94-100).
There is a need in the technique of bandages for wounds that do not cause reactions to the foreign body or do so at a lower rate than traditional bandages. Such wound dressings need to be changed less frequently and reduce healing time to close, and therefore can result in a more effective treatment for the patient and a lower cost of care.
3. Compendium
The application is based, in part, on the inventors' discovery of improved wound healing and the reduced need for wound dressing changes when administered to wounds poly-p-1 - ^ 4-N-acetylglucosamine ("pGlcNAc" or "NAG") in a diabetic mouse model. These studies indicate that pGlcNAc can be used advantageously as a wound dressing without the need for frequent changes of bandages that require other bandages that are currently being used. Other polymers or fibers with characteristics similar to pGlcNAc can also be used in accordance with the methods described herein.
The features of improving wound healing of pGlcNAc together with the reduced need for bandage changes with pGlcNAc are especially pronounced when irradiating pGlcNAc to reduce its molecular weight and length. The inventors have discovered that irradiation reduces the molecular weight and length of pGlcNAc without affecting the microstructure of the fibers, so that the infrared spectrum of the irradiated pGlcNAc is substantially similar or equivalent to that of the non-irradiated pGlcNAc.
This reduced molecular weight pGlcNAc, referred to herein as "sNAG" (for short, N-acetyl glucosamine), has an improved activity by activating wound healing and no detectable reaction of foreign bodies in the treated animals. Therefore, this polymer or fiber is particularly useful for treating wounds with greater efficiency and reduced cost.
Accordingly, methods for treating a wound in a patient, preferably a human being, are described herein, said method comprising the topical application of a bandage to a wound in the patient, thereby treating the wound in the patient. . Preferably, the application is repeated every 3 to 35 days (a significant improvement over the methods currently used in the climate, in which the application is repeated every 2 days). The bandage materials are preferably polymers or fibers, as described in section 5.2 below. Preferably, the polymers or fibers are irradiated to improve their effectiveness, as described in section 5.3 below. Preferably, the bandage is a biocompatible and / or immunoneutra poly-p-1-4-N-acetylglucosamine bandage or a derivative thereof, as described in section
5.2.1 below. Alternative frequencies of changes or reapplications of bandages are described in section 5.4 below.
The invention relates to a poly-p-1-4-N-acetylglucosamine composition comprising poly-p-1-4-N-acetylglucosamine fibers, wherein (i) the mayone of the fibers have a shorter length of approximately 15 pm, where the fibers have been irradiated to reduce their length, and (ii) the composition (a) increases the metabolic rate of serum-deprived human umbilical cord vein endothelial cells in an MTT analysis and / or not pounds of serum-deprived human umbilical cord vein endothelial apoptosis in a trypan blue exclusion test, and (b) is not reactive when tested in an intramuscular implantation test. In certain embodiments, the composition increases the metabolic rate of serum-deprived human umbilical cord vein endothelial cells in an MTT analysis and does not free apoptosis to serum-deprived human umbilical cord vein endothelial cells in a trial of Trypan blue exclusion. In one embodiment, the fiber mayonnaise has a thickness or diameter of approximately 1-2 microns. In a
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embodiment, the mayone of the fibers have a thickness or diameter of about 1-2 pm. In one embodiment, at least 50% of the fibers are approximately 4 pm in length. In one embodiment, the fiber mayonnaise is less than about 10 pm in length. In a certain embodiment, the mayonnaise of the fibers is between approximately 2 to 15 pm in length. In certain embodiments at least 70% of N-acetylglucosamine monosaccharides of the poly-p-1-4-N-acetylglucosamine are acetylated. In certain embodiments, 100% N-acetylglucosamine monosaccharides of the poly-p-1-4-N-acetylglucosamine are acetylated.
In another aspect the invention relates to a method for producing a composition of poly-p-1 - ^ 4-N-acetylglucosamine, said method comprising irradiation of poly-p-1-4-N-acetylglucosamine fibers, such so that (i) the mayone of the irradiated fibers is less than about 15 pm in length, and (ii) the composition (a) increases the metabolic rate of endothelial cells of the human umbilical cord vein deprived of serum in an analysis with MTT and / or non-release of serum-deprived human umbilical cord vein endothelial apoptosis in a trypan blue exclusion test, and (b) is not reactive when tested in an intramuscular implantation test , thereby producing the composition of poly-p-1-4-N-acetylglucosamine. In certain embodiments, the poly-p-1-4-N-acetylglucosamine fibers are irradiated in the form of dry fibers, a dry fiber membrane or a dry lyophilized material. In one embodiment, the poly-p-1-4-N-acetylglucosamine fibers are irradiated by gamma irradiation at 500-2,000 kgy. In certain other embodiments, the poly-p-1-4-N-acetylglucosamine fibers are irradiated in the form of wet fibers. In one embodiment, the poly-p-1-4-N-acetylglucosamine fibers are formulated as a suspension, slurry or wet cake for irradiation. In one embodiment, the poly-p-1-4-N-acetylglucosamine fibers are irradiated by gamma irradiation at 100-500 kgy. In one embodiment, a poly-p-1-4-N-acetylglucosamine composition can be obtained by a method according to the invention.
The invention relates to the composition of poly-p-1-4-N-acetylglucosamine according to the invention for use in a method for treating a wound in a human being, wherein the method comprises the step of preparing a bandage to be applied topically to a wound in a human being who needs it. In one embodiment the method comprises the stage of preparing a bandage for repeated application every 5 to 35 days.
In another aspect, the invention relates to the composition of poly-p-1-4-N-acetylglucosamine according to the invention for use in a method for treating a wound in a human being, comprising: (a) applying topically a bandage to a wound in a human being that needs it, where the bandage comprises biocompatible poly-p-1-4-N-acetylglucosamine, and (b) repeat said application every 5 to 35 days, trying this mode the wound in said human being, where the human being is a diabetic, a smoker, a hemofflico, a person infected with HIV, an obese person, a person undergoing radiotherapy or a person with venous ulcer for stasis. In certain embodiments, the human being is a person with venous ulcer due to stasis. In one embodiment, the wound is a chronic wound. In certain embodiments, the chronic wound is a diabetic ulcer, a venous stasis ulcer, an ulcer due to arterial insufficiency or a pressure ulcer. In one embodiment, the chronic wound is a venous ulcer due to stasis. In other embodiments, the wound is a surgical wound or a burn wound.
In certain embodiments, the bandage of stage (a) is removed before stage (b). In certain other embodiments, the bandage of stage (a) is not removed before stage (b).
In certain embodiments, the poly-p-1-4-N-acetylglucosamine is a microalgae poly-p-1-4-N-acetylglucosamine. In certain embodiments, poly-p-1-4-N-acetylglucosamine is not a poly-p-1-4-N-acetylglucosamine from crustaceans.
In certain embodiments, at least 75% of the bandage consists of poly-p-1-4-N-acetylglucosamine. In one embodiment, the bandage comprises the composition according to the invention.
In certain embodiments, the deacetylated poly-p-1-4-N-acetylglucosamine comprises deacetylated poly-p-1-4-N-acetylglucosamine. In one embodiment, the deacetylated poly-p-1-4-N-acetylglucosamine is 20-70% deacetylated.
In embodiments of the invention, the mayone of the fibers is between about 2 to 15 mm in length. In further embodiments of the invention, the length of the fibers is determined by scanning electron microscopy analysis (SEM). In further embodiments of the invention, the infrared spectrum of the irradiated poly-p-1-4-N-acetylglucosamine fibers is substantially similar or equivalent to that of the non-irradiated poly-p-1-4-N-acetylglucosamine fibers. . In specific embodiments of the invention, the intramuscular implantation test is an implantation for 4 weeks in the paravertebral muscle tissue of a rabbit based on the Guidelines of the International Organization for Standardization.
4. Brief description of the figures
Fig. 1. Analysis of wound closure of wounds treated and not treated with pGlcNAc. Standardized photographs were taken on the day of the wound (day 0) and twice a week during follow-up. Wound contraction (C), reepithelialization (E) and open wound (O) were studied as a percentage of the area of the original wound.
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Fig. 2. pGIcNAc caused faster wound closure. (A) The application of the pGIcNAc patch for 1 hour caused faster reduction of the raw surface. (B) The 1 h group reached 90% wound closure faster than the Nt group. (C) The reepithelialization was accentuated after 1 h of the application of the patch. (D) The 1 h group presented accentuated reepithelialization on the 14th day. (E) The application of the patch for 24 h decreased wound contraction compared to untreated wounds. The average values and the standard deviations are shown. * p <0.01.
Fig. 3. Wound Watch staging system. (A) Ki-67 staining: the 1 h group showed increased cell proliferation on the 10th day compared to the other groups. (B) PECAM-1 staining: the 1 h group showed an increase in neovascularization on the 10th day compared to the other groups. (C) The graph combines the quantification of the two immunohistochemical markers, which visually shows the difference between the groups in the study. The 1 h group differs dramatically from both 24 h and NT groups. Average values and typical deviations are shown. * p <0.01.
Fig. 4. Effect of irradiation on the chemical and physical structure of pGlcNAc fibers. (A) Correlation between the molecular weight of pGlcNAc and the amount of irradiation. (B) Infrared (IR) spectrum of the non-irradiated pGlcNAc suspension (upper lmea), irradiated pGlcNAc suspension at 100 kGy (lower lmea), and irradiated pGlcNAc suspension at 200 kGy (middle lmea). (C) Scanning electron microscope (SEM) analysis of pGlcNAc. (D) Scanning electron microscopy (SEM) analysis of sNAG.
Fig. 5. Closure analysis of treated and untreated wound wound of reference of the sNAG membrane (irradiated pGlcNAc, see sections 5.3 and 6.2.1 below). Standardized photographs were taken days 0, 4, 7, 10, 14, 17, 21, 25, and 28. The contraction of the wound (C), the re-epithelialization (E) and the open wound (O) were studied in a percentage of the original area of the wound.
Fig. 6-10. Macroscopic photograph of a wound of mice treated with sNAG membrane and of a wound of an untreated mice of reference on day 0 (Fig. 6), 4 ° (Fig. 7), 10 ° (Fig. 8), 17 ° (Fig. 9) and 28 ° (Fig. 10).
Fig. 11-15. sNAG caused faster wound closure. The application of the sNAG membrane caused a faster reduction of the raw surface (Fig. 1 1). Mice treated with sNAG membrane achieved 50% (Fig. 12) and 90% (Fig. 13) of wound closure faster than untreated reference mice. Mice treated with sNAG membrane showed increased reepithelialization on the 4th, 7th and 10th days (Fig. 14). Mice treated with sNAG membrane showed greater wound contraction on the 14th and 17th days (Fig. 15).
Fig. 16-17. Histology of the wound edge in a mouse treated with sNAG membrane (Fig. 17) and a non-treated reference mouse (Fig. 16).
Fig. 18. Ki-67 staining: mice treated with sNAG membrane showed increased cell proliferation on the 10th day compared to untreated reference mice.
Fig. 19. PECAM-1 staining: mice treated with sNAG membrane showed increased neovascularization on the 10th day compared to untreated reference mice.
Fig. 20. Degree of reepithelialization of wounds of mice treated with sNAG membrane and of untreated reference mice measured on the 10th day.
Fig. 21. Degree of granulation tissue formation for mice treated with sNAG membrane and untreated reference mice measured on the 10th day.
Fig. 22. Histological analysis of untreated reference compared to wound treated with sNAG membrane on 10th day. Note that there is no reaction to the foreign body in mice treated with sNAG membrane.
Fig. 23. Reference untreated collagen staining compared to wound treated with sNAG membrane on 10th day. No encapsulations of the foreign body can be observed in mice treated with sNAG membrane.
Fig. 24. Endothelial cells (EC) of the human umbilical vein protected with pGlcNAc from cell death caused by serum deprivation. For each period (that is, at 24, 48 and 72 hours), the identity of each of the five bars (from left to right) is as follows: serum deprivation (SS), VEGF and pGlcNAc (NAG) a 50, 100 and 250 | jg / ml.
Fig. 25. pGlcNAc did not affect the metabolic rate. For each period (that is, at 24 and 48 hours), the identity of each of the four bars (from left to right) is as follows: serum deprivation (SS), VEGF and pGlcNAc (NAG) at 50 and 100 jg / ml
Fig. 26. pGlcNAc increased cell migration.
Fig. 27. pGlcNAc produced an increase in migration to fibronectin. The identity of each of the five bars (from left to right) is as follows: serum deprivation (SS), VEGF, pGlcNAc (NAG) at 50, 100 and 250 g / ml and NAG / VEGF.
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Fig. 28. pGIcNAc increased umbilical cord formation.
Fig. 29. PGlcNAc-induced effectors involved in cell motility (A) Treatment with pGlcNAc stimulated the expression of Ets1, metallothionema 2A (MT), Akt3 and Edg3. (B) Real-time PCR demonstrates that treatment with pGlcNAc produced approximately double Ets1. (C) The increase in Ets1 in the message was accompanied by increased protein expression as shown in the Western blot analysis.
Fig. 30. Induction by pGlcNAc of phospho-MAPK was a function of VEGFR2 (A) treatment with pGlcNAc resulted in a marked increase in phosphorylation of MAPK. (B) Induction by phosphorus-MAPK pGlcNAc was a function of VEGFR2.
Fig. 31. pGlcNAc not active to VEGFR2.
Fig. 32. Migration induced by pGlcNAc was a function of Ets1. (A) The inhibition of Ets1 activity in CE resulted in a marked decrease in EC migration in response to pGlcNAc. (B) The expression of the Ets1 protein decreases with the amount of dn-Ets mounting. (C) Expression levels resulting from Ets1 in CE were transfected with 2 amounts of RNAi containing plasmid directed against Ets1.
Fig. 33. Cellular motility caused by pGlcNAc requena integrin. (A) The results when antibodies directed against aVp3 or a5p1 integrin (CD49e) are used in fibronectin migration assays (the aVp3 receptor). (B) A similar experiment as in (A) using antibodies directed against aVp3 or a5p1 (CD49e) in vitronectin coated transwells.
Fig. 34. Cellular motility caused by pGlcNAc and activation of FAK with integrin intervention.
Fig. 35. pGlcNAc can activate an integrin pathway ^ Ets1 that leads to angiogenesis in a wound healing model. (A) The blocking of a5p1 integrin antibodies resulted in a reduction in the expression of Ets1 caused by pGlcNAc. (B) This inhibition of Ets1 expression using an a5p1 integrin block is repeated in the field of proteins.
Fig. 36. pGlcNAc caused the expression of VEGF and IL-1. (A) pGlcNAc increased the expression of both VEGF and IL-1. (B) The treatment of EC with its inhibitor blocked the induction of VEGF by Ets1 but had no effect on the induction of Ets1 by pGlcNAc.
Fig. 37. sNAG caused the expression of FGF1, FGFR3, stabilin, IFNg, CollagenA18 and CXCL9.
Fig. 38. sNAG increased cell migration. The identity of each of the four bars (from left to right) is as follows: serum deprivation (SS) and sNAG at 50 and 100 pg / ml.
Fig. 39. sNAG caused a marked increase in metabolic rate. The identity of each of the five bars (from left to right) is as follows: serum deprivation (SS), VEGF and sNAG at 50, 100 and 200 pg / ml.
Fig. 40. sNAG did not protect EC from cell death caused by serum deprivation. For each period (that is, at 24 and 48 hours), the identity of each of the five bars (from left to right) is as follows: serum deprivation (SS), VEGF and sNAG at 50, 100 and 200 pg / ml.
Fig. 41. sNAG caused the expression of VEGF and IL-1
5. Detailed description
Sections 6.1 and 6.2 below clearly demonstrate that the application of pGlcNAc and, in particular sNAG, to wounds in a diabetic mouse model for damaged wound healing significantly shortens the time of wound closure, reepithelialization of the wound and wound contraction Reepithelialization and granulation tissue have also increased in a shorter time for mice treated with sNAG membrane. The application of the sNAG membrane also resulted in the closure of all wounds in the treated animals in 28 days, compared with 85% in the untreated references. The immunohistological data are consistent with the increase in cell proliferation and the appearance of new blood vessels (angiogenesis) in mice treated with sNAG membrane. No response to the foreign body was observed throughout the study in any animal treated with the sNAG membrane.
These findings suggest that the pGlcNAc and sNAG membrane has clinical applications in the treatment of chronic wounds including diabetic ulcers, stasis venous ulcers, arterial insufficiency ulcers and bedsores. The membranes of pGlcNAc and sNAG also have clinical utilities in the treatment of other open wounds, such as, but not limited to, surgical wounds and burn wounds.
5.1 Injuries for treatment by the present methods and compositions
The methods and compositions described herein are useful for the treatment of wounds, such as open wounds caused by incision (i.e. incision or incised wound), loosening, abrasion, puncture, penetration,
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by firearm, burns, etc. The methods and compositions described herein are particularly useful for the treatment of chronic wounds or patients who cannot heal normally, or in an orderly and timely manner as in other human beings.
A chronic wound is a wound that has not been able to proceed through an orderly and timely series of episodes to produce a lasting structural, functional and aesthetic closure. Chronic wounds can be any wound that does not heal properly, including a surgical wound (e.g., a skin graft donor site), a skin ulcer (e.g., a diabetic ulcer, a stasis venous ulcer, an ulcer due to arterial insufficiency, or a pressure ulcer) or a burn injury.
The identification of the type of chronic wound being treated (eg, diabetic, venous stasis, arterial and decubitus insufficiency) can usually be determined by considering the patient's background and performing a physical exam. Objective tools to confirm the diagnosis may include Doppler ultrasound to qualify and quantify vascular insufficiency: arterial or venous (deep, superficial or mixed); transcutaneous oxygen tension measurements (tcpO2); index ankle / arm; filament tests to quantify sensory neuropamph; measurement of laboratory markers for diabetes mellitus; Histopathology of ulcer biopsies. In addition, there are widely accepted criteria used to classify ulcer stages (e.g., National Pressure Ulcer Advisory Panel (NPUAP) for Pressure Ulcers: NPUAP Classification, Wagner's Classification for foot ulcers). These methods are routinely put into practice by the person skilled in the art.
The identification of a patient that cannot be cured normally, or in an orderly and timely manner as in other human beings, can be determined routinely, for example, taking into account the patient's background and performing a physical examination. These determinations are routinely performed by the person skilled in the art.
5.2 Haemostatic composition materials
Hemostatic compositions are preferably formulated in wound dressings. Wound bandages can be made of any suitable natural or synthetic poffmeros or fibers. Examples of suitable poffmeros or fibers from which bandages can be prepared for the implementation of the methods and compositions described herein include cellulose, xanthan, polyamide, polyamides, polyimides, polyamide / imides, polylamide hydrazides, poffmeros. polyhydrazides, polyimidazoles, polybenzoxazoles, polyester / amide, polyester / imide, polycarbonate / amides, imides, polycarbonate / polysulfone / amides, polysulfone imides and the like, co-polymers and one of their mixtures. Other suitable classes of poffmeros or fibers include polyvinyldene fluorides and polyacrylonitriles. Examples of these poffmeros or fibers include those described in US Pat. RE 30,351 .; No. 4,705,540, No. 4,717,393; No. 4,717,394; No. 4,912,197; No. 4,838,900; No. 4,935,490; No. 4,851,505; No. 4,880,442; No. 4,863,496; No. 4,961,539; and European Patent Application No. 0 219 878. The poffmeres or fibers may include at least one of any of the cellulose, polyamide, polyamide, polyamide / imide or polyimide poffmeros. In certain embodiments, the poffmeros or fibers include polyaramides, polyester, urethane and polytetrafluoroethylene. In preferred embodiments, polymerized N-acetylglucosamine fibers or one of its derivatives are used. In a more preferred embodiment, the poffmero or fiber is poffmero or poly-N-acetylglucosamine fiber or one of its derivatives. In certain embodiments, the poffmero or poly-N- fiber
acetylglucosamine has a configuration (3-1-4). In other embodiments, the poly-N- or poffmero fiber
acetylglucosamine has an a-1-4 configuration.
In specific embodiments, the poffmero or fiber is chitin, chitosan, cellulose, nylon or PET (polyethylene terephthalate).
In a preferred embodiment, the poffmero or fiber is biocompatible and / or biodegradable. Biocompatibility can be determined by a variety of techniques, including, but not limited to procedures such as elution testing, intramuscular implantation or intracutaneous or generalized injection into animals. Such tests are described in US Pat. No. 6,686,342. Biodegradable poffmeros preferably degrade in about 1 day, 2 days, 5 days, 8 days, 12 days, 17 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days or 100 days after administration or implantation in a patient.
In certain aspects, the poffmero or fiber is immunoneutrous, because they do not cause an immune response.
In general, the poffmeros or fibers are not reactive in an intramuscular implantation test. In certain embodiments, the compositions comprise fibers as described in this section, wherein the fibers increase the metabolic rate of serum-deprived human umbilical cord vein endothelial cells in an MTT assay and / or do not release apoptosis. Endothelial cells of the human umbilical cord vein deprived of serum in an trypan blue exclusion test, and is not reactive when tested in an intramuscular implantation test. In certain embodiments, the fibers increase the metabolic rate of serum-deprived human umbilical cord vein endothelial cells in an MTT trial and do not free serum-deprived human umbilical cord vein endothelial cells from apoptosis in a serum assay. trypan blue exclusion, and is not reactive when tested in an intramuscular implantation test.
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In one embodiment, the hemostatic compositions comprise purified polymers or fibers, which may be approximately 100%, 99.9%, 99.8%, 99.5%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% pure. In a preferred embodiment, the polymers or fibers used in the compositions and methods described herein are 90-100% pure.
In certain embodiments, the polymer or fiber that is used in a wound dressing is not one or more of the following: an ionic synthetic hydrogel such as, but not limited to, crosslinked poly (AAn-acrylic acid) and poly (methacrylate of AAm-dimethylaminoethyl), poly (vimyl alcohol) (PVA), poly (ethylene glycol) (PEG), poly (N-vinyl pyrrolidone), poly (methoxy-PEG methacrylate). In certain embodiments, the polymer or fiber is not one or more of the following: a poly-L-amino acid, such as poly-L-lysine, poly-L-arginine, poly-L-glutamic acid, poly-L- histidine, poly-D-glutamic acid or a mixture thereof. In certain embodiments, the polymer or fiber is not one or more of the following: an alginate polymer, such as sodium alginate, calcium alginate, strontium alginate, barium alginate, magnesium alginate or any other alginate or one of their mixtures In certain embodiments, the polymer or fiber is not obtained from one or more of the following: a mollusk, a crustacean, insects, fungi or yeasts. In certain embodiments, the compositions do not comprise collagen fibers. In certain embodiments, the compositions do not comprise elastin fibers. In other embodiments, these polymers or fibers are included in the compositions.
The polymers described herein are generally in the form of fibers comprising polymers described herein. Therefore, the polymers described herein may be in the form of fibers. The fibers preferably have an average length of approximately 2, 3, 4, 5, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 microns determined by electron microscopy, or any interval between them (e.g., 10-150 microns, 20-100 microns or 50-120 microns on average). The fibers are preferably nanofibers, with average dimensions of 0.5 to 100 nm in thickness and / or diameter determined by electron microscopy. In specific embodiments, the nanofibers are approximately 0.5, 1, 2, 5, 10, 20, 50 or 100 nm thick and / or average diameter, or any interval between them (e.g., 0.5 -4 nm, 0.5-5 nm, 1-4 nm, 1-5 nm, 1-10 nm, 2-20 nm, 4-15 nm, 5-15 nm, 4-20 nm, 5-20 nm , 5-50 nm, etc.). In other embodiments, the fibers are preferably microfibers, with dimensions of approximately 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.75, 0.85 and 1 micron thick and / or average diameter, or any interval between them (e.g., 0.2-0.5 microns, 0.3-0.75 microns, 0.5-1 microns, and so on and on). As used herein, the term "approximately" is readily appreciated by those skilled in the art; generally, the term as used herein refers to a range of values 10% greater than and 10% less than the indicated value.
In specific embodiments, mayone (more than 50%, e.g., 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99, 9% or 100%) of the fibers are less than about 30, 25, 20, 15, 10, 5, 4, or 3 microns in length. In specific embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8% or 99.9% of the fibers are less than approximately 15 microns in length. In specific embodiments, all (100%) fibers are less than about 15 microns in length. In specific embodiments, mayone (more than 50%, e.g., 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99, 9%, or 100%) of the fibers are approximately 5, 4, 3, 2 or 1 microns in length. In specific embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, or 99.9% of the fibers are smaller. approximately 4 microns in length. In specific embodiments, all (100%) fibers are approximately 4 microns in length.
The fibers are preferably about 0.1, 0.5, 1, 2, 3, 4, 5, 8 or 10 microns in thickness or average diameter determined by electron microscopy, or any interval between them (e.g., 0 , 1-10 microns, 0.5-5 microns or 1-2 microns on average). In specific embodiments, mayone (more than 50%, e.g., 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99, 9%, or 100%) of the fibers have a thickness or diameter of approximately 1-2 microns. In specific embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, or 99.9% of the fibers have a thickness or diameter of approximately 1-2 microns. In specific embodiments, all (100%) fibers have a thickness or diameter of approximately 1-2 microns.
In certain embodiments, the mayone (more than 50%, e.g., 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99, 9%, or 100%) of the fibers are less than about 15 microns in length and have a thickness or diameter of about 1-2 microns.
In certain preferred embodiments, the polymers or fibers are formulated as wound dressings, which may be in the form of barriers, membranes or films. Alternatively, the polymers or fibers are added to bandage supports, such as barriers, membranes or films. A barrier, membrane or film can be supplied in a variety of typical sizes, which can be cut and sized for the area being treated. The support may be a conventional bandage material, such as a bandage or gauze to which a polymer or fiber is added or coated, before application to the patient. Alternatively, the polymer or fiber can be formulated as a barrier, membrane or film made of threads, microbeads, microspheres or microfibrils, or the composition can be formulated as a barrier forming mat. As noted herein, polymers can be present in the form of fibers. As such, throughout the report, whether or not explicitly mentioned, it is understood that any reference to embodiments referring to the polymers described herein, the polymers may be present in the form of fibers.
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In certain embodiments, at least 75%, at least 85%, at least 90% or at least 95% of a bandage is composed of one or more of the polymers listed above.
In certain aspects, a bandage does not contain a conventional bandage material, such as gauze or bandage. In said embodiments, the polymer itself is formulated as a wound dressing.
In one embodiment, the compositions described herein comprise more than one type of polymer (eg, poly-p-1-4-N-acetylglucosamine and cellulose).
In certain aspects, the polymer (eg, poly-p-1-4-N-acetylglucosamine or one of its derivatives) is the only active ingredient in a bandage. In other embodiments, the bandage comprises more active ingredients to promote wound healing, such as growth factors. In specific embodiments, the growth factor is PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PDGF-DD, FGF-1, FGF-2, FGF-5, FGF-7, FGF-10, EGF , TGF-a, (HB-EGF), anfirregulina, epirregulina, betacelulina, neurregulinas, epigen, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placenta growth factor (PLGF), angiopoietin-1, angiopoietin-2, IGF-I, IGF-II, hepatocyte growth factor (HGF) or macrophage stimulating protein (MSP).
However, in other aspects, a bandage does not comprise a significant amount of protein material. In specific embodiments, the protein content of a bandage is not greater than 0.1%, 0.5% or 1% by weight. In other embodiments, the protein content of a bandage is undetectable portincion with Coomassie.
A bandage may contain collagen, although in certain aspects a bandage does not contain collagen.
The bandage may also contain antimicrobial agents to prevent wound infection.
In a preferred embodiment, zinc is also included in a bandage. In addition to its antimicrobial properties, zinc also plays a role in wound healing (see Andrews et al., 1999, Adv. Wound Care 12: 137-8). Zinc is preferably added as a salt, such as zinc oxide, zinc sulfate, zinc acetate or zinc gluconate.
5.2.1 poly-p-1—4-N-acetylglucosamine ("pGlcNAc" or "NAG")
This section refers to US patents. No. 5,622,834 .; No. 5,623,064; No. 5,624,679; No. 5,686,115; No. 5,858,350; No. 6,599,720; No. 6,686,342; and No. 7,115,588 which describe in detail the structure of the poly-p-1-4-N-acetylglucosamine polymer.
In preferred embodiments, the poly-p-1-4-N-acetylglucosamine is obtained by a method comprising a) the treatment of a microalgae comprising a cell body and a poly-p-1 - »4-N polymer fiber - acetylglucosamine with a biological agent (such as fluorhydric) capable of separating the N-acetylglucosamine polymer fiber from the cell body for a sufficient time such that the poly-p-1-4-N-acetylglucosamine polymer fiber is released from the Cellular body; b) segregation of the poly-p-1-4-N-acetylglucosamine polymer fiber from the cell body; and c) removal of pollutants from the secreted poly-p-1-4-N-acetylglucosamine polymer fiber, so that poly-p-1-4-N-acetylglucosamine polymer is aflame and purified.
As used herein, derivatives of a poly-p1-4-N-acetylglucosamine polymer include: a semi-crystalline form of a poly-p1-4-N-acetylglucosamine polymer; a poly-p-1-4-N-acetylglucosamine polymer comprising about 50 to about 150,000 N-acetylglucosamine monosaccharides linked by covalent bonds in a p-1-4 configuration, and said polymer has a molecular weight of about 10,000 daltons at approximately 30 million daltons; a poly-p-1-4-N-acetylglucosamine polymer comprising about 50 to about 50,000 N-acetylglucosamine monosaccharides linked by covalent bonds in a p-1-4 configuration, and said polymer has a molecular weight of about 10,000 daltons at approximately 10 million daltons; a poly-p1-4-N-acetylglucosamine polymer comprises about 50 to about 10,000 N-acetylglucosamine monosaccharides linked by covalent bonds in a p-1-4 configuration, and said polymer has a molecular weight of about 10,000 daltons to about 2 million of daltons; a poly-p1-4-N-acetylglucosamine polymer comprising about 50 to about 4,000 N-acetylglucosamine monosaccharides linked by covalent bonds in a p-1-4 configuration, and said polymer has a molecular weight of about 10,000 daltons to about 800,000 daltons; a poly-p-1-4-N-acetylglucosamine semicrystalline polymer comprising at least one N-acetylglucosamine monosaccharide that is deacetylated, and wherein at least 10%, 20%, 30%, 40%, 50%, 60 %, 70%, 80%, 90% or 100% of said N-acetylglucosamine monosaccharides are acetylated; and a poly-p-1-4-N-acetylglucosamine semi-crystalline polymer comprising at least one N-acetylglucosamine monosaccharide that is deacetylated, and wherein at least 20-70% (or any range within the above embodiments) of said N-acetylglucosamine monosaccharides are deacetylated.
Derivatives of a poly-p-1-4-N-acetylglucosamine polymer also include compositions that are 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less poly-p-1-4-N-acetylglucosamine.
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Other poly-p-1— »4-N-acetylglucosamine derivatives can also be used in the composition described herein. For example, sulfated derivatives of poly-beta-1-4-N-acetylglucosamine, phosphorylated derivatives of poly-p-1-4-N-acetylglucosamine or nitrated derivatives of poly-p-1-4-N- can be used acetylglucosamine In addition, one or more of the monosaccharide units of the poly-p-1-4-N-acetylglucosamine may contain one or more sulfonyl groups, one or more O-acyl groups. In addition, one or more of the monosaccharides of the deacetylated poly-p-1-4-N-acetylglucosamine may contain an N-acyl group. One or more of the monosaccharides of the poly-p-1-4-N-acetylglucosamine or its deacetylated derivative may contain an O-alkyl group. One or more of the monosaccharide units of the poly-p-1-4-N-acetylglucosamine may be an alkaline derivative. One or more of the monosaccharide units of the deacetylated derivative of poly-p-1-4-N-acetylglucosamine may contain an N-alkyl group. One or more of the monosaccharide units of the deacetylated derivative of poly-p-1-4-N-acetylglucosamine may contain at least one deoxyhalogenated derivative. One or more of the monosaccharide units of the deacetylated derivative of poly-p-1-4-N-acetylglucosamine may form a salt. One or more of the monosaccharide units of the deacetylated derivative of poly-p-1-4-N-acetylglucosamine may form a metal chelate. Preferably, the metal is zinc. One or more of the monosaccharide units of the deacetylated derivative of poly-p-1-4-N-acetylglucosamine may contain an N-alkylidene group or an N-arylidene. The manufacturing methods of said derivatives are described in US Pat. No. 5,623,064.
5.3 Irradiation to reduce molecular weight and length
The polymers or fibers may be in polymers or fibers or membranes of polymer or fiber, both irradiated and dried, as described above. Alternatively, polymers or fibers can radiate when they are wet.
In preferred embodiments, the polymers or fibers are formulated in a suspension / slurry or wet cake for irradiation. Irradiation can be carried out before, simultaneously or after the formulation of polymers or fibers in a bandage. Generally, the polymer or fiber content of suspensions / slurries and wet cakes may vary, for example from about 0.5 mg to about 50 mg of polymer or fiber per ml of distilled water for grouts and from about 50 mg to about 1,000 mg of polymer or fiber per ml of distilled water for wet cake formulations. The polymer or fiber can be lyophilized first, frozen in liquid nitrogen and pulverized, to make it more susceptible to the formation of a suspension / slurry or wet cake. In addition, suspensions / grouts can be filtered to remove water such that a wet cake is formed. In certain aspects, the polymer or fiber is irradiated in the form of a suspension comprising approximately 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 15 mg, 18 mg, 20 mg, 25 mg or 50 mg of polymer or fiber per ml of distilled water, or any interval between the above embodiments (e.g., 1-10 mg / ml, 5-15 mg / ml, 2-8 mg / ml, 20-50 mg / ml, etc.). In other aspects, the polymer or fiber is irradiated in the form of a wet cake, comprising approximately 50-1,000 mg of polymer or fiber per ml of distilled water. In specific embodiments, the wet cake comprises about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 mg of polymer or fiber per ml of distilled water, or any interval between (e.g. , 100-500 mg / ml, 300-600 mg / ml, 50-1000 mg / ml, etc.).
The irradiation is preferably in the form of gamma radiation, electron beam radiation or x-rays. Two sources of irradiation are preferred: radioactive nuclides and electricity. In a specific embodiment, the radioactive nuclides are cobalt-60 and cesium-137. Both nuclides emit gamma rays, which are photons that contain no mass. Gamma rays have energies of 0.66 to 1.3 MeV. Using electricity, electrons are generated and accelerated to energies of up to 10 MeV or greater. When polymers or fibers are irradiated to reduce their size, a consideration to be taken is that the depth of penetration of materials with densities similar to water by 10 MeV electrons is limited to about 3.7 cm with exposure on one side or approximately 8.6 cm with two-sided exposure. The penetration depth decreases to lower electron energies. Electron energy can be converted into X-rays by placing a target metal (usually tungsten or so on) in the path of the electron beam. X-ray conversion is limited to electrons with energies of up to 5 MeV. X-rays are photons without mass and polymers or fibers similar to gamma rays can penetrate. There is only about 8% efficiency in the conversion of energy from electrons to x-ray energy. High power electron beam machines are needed in x-ray production facilities to take into account the low conversion efficiency.
Preferably, the irradiation is gamma irradiation.
The absorbed dose of radiation is the energy absorbed per unit weight of the product, measured in gray (gy) or kilogray (kgy). For polymers or dry fibers, the preferred absorbed dose is 500-2,000 kgy of radiation, more preferably 750-1,250 kgy of radiation, while for polymers or wet fibers, the preferred absorbed dose is approximately 100-500 kgy of radiation, even more preferably about 50-250 kgy of radiation.
The radiation dose can be described from the point of view of its effect on the length of the polymers or fibers. In specific embodiments, the radiation dose used preferably reduces the length of the polymer or fiber anywhere from about 10% to 90% of the initial length of the polymer or fiber, respectively. In specific embodiments, the average length is reduced by approximately 10%, by approximately
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20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, or approximately 90%, or any interval between ( e.g., 20-40%, 30-70%, and so on). Alternatively, the radiation dose used preferably reduces the length of the polymer or fiber anywhere from 30 to 100 microns. In specific embodiments, and depending on the length of the initial fiber, the average length of the polymer or fiber is reduced to less than about 15 microns, less than about 14 microns, less than about 13 microns, less than about 12 microns, less than approximately 11 microns, less than about 10 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than 2 microns, or less than 1 microns. In certain embodiments, the length of the mayone (more than 50%, e.g., 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8% , 99.9%, or 100%) of the polymers or fibers is reduced to no more than 40 microns, no more than about 30 microns, no more than about 20 microns, no more than about 15 microns, no more than about 10 microns microns, or not greater than about 5 microns. Any interval between the previous lengths are also included; for example, in certain embodiments, the irradiation of the polymers or fibers reduces the length of the mayone (more than 50%, e.g., 60%, 70%, 80%, 90%, 95%, 98%, 99 %, 99.5%, 99.8%, 99.9% or 100%) of the fibers anywhere between about 1 to 20 microns, between about 2 to 15 microns, between about 4 to 10 microns, and so on onwards.
The radiation dose can also be described from the point of view of its effect on the molecular weight of the polymer or fiber. In specific embodiments, the radiation dose used preferably reduces the molecular weight of the polymer or fiber anywhere from about 10% to 90% of the initial weight of the polymer or fiber. In specific embodiments, the average molecular weight is reduced by approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%. %, in about 80%, or in about 90%, or any interval between them (e.g., 20-40%, 30-70%, and so on in the future). Alternatively, the radiation dose used preferably reduces the molecular weight of the polymer or fiber anywhere from 1,000 to 1,000,000 daltons. In specific embodiments, and depending on the initial molecular weight, the average molecular weight of the polymer or fiber is reduced to less than 1,000,000 daltons, less than 750,000 daltons, less than 500,000 daltons, less than 300,000 daltons, less than 200,000 daltons, minus 100,000 daltons, less than 50,000 daltons, less than 25,000 daltons, less than 10,000 daltons, or less than 5,000 daltons. In certain embodiments, the average molecular weight is reduced to no less than 500 daltons, no less than 1,000 daltons, no less than 2,000 daltons, no less than 3,500 daltons, no less than 5,000 daltons, no less than 7,500 daltons, no less than 10,000 Daltons, no less than 25,000 Daltons, no less than 50,000 Daltons or no less than 100,000 Daltons. Any interval between the above average molecular weights is also included; for example, in certain embodiments, the irradiation of the polymer or fiber reduces the average molecular weight anywhere between 10,000 to 100,000 daltons, between 1,000 and 25,000 daltons, between 50,000 and 500,000 daltons, and so on thereafter.
In preferred embodiments, the irradiation used is gamma irradiation.
After irradiation, the grouts can be filtered and dried, and the wet cakes can be dried, to form bandages that are useful in the practice described herein.
5.3.1 Poly-p-1—4-N-acetylglucosamine shortened ("sNAG")
The poly-p-1-4-N-acetylglucosamine described in section 5.2 above is irradiated as described in section 5.3 above to reduce the length of its fibers to form poly-p-1 - »4 N-acetylglucosamine shortened, and thus reducing its molecular weight without affecting its microstructure. The infrared (IR) spectrum of the shortened poly-p-1-4-N-acetylglucosamine (sNAG) is substantially similar or equivalent to that of the non-irradiated poly-p-1-4-N-acetylglucosamine (NAG).
In certain embodiments, the mayone (more than 50%, e.g., 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99, 9% or 100%) of the sNAG fibers are less than 15 microns in length. In one aspect, the mayona of sNAG fibers has a thickness or diameter of approximately 1-2 microns. The fiber length can be measured by any method known to one skilled in the art, for example, by scanning electron microscopy (SEM).
In one aspect, sNAG increases the metabolic rate of endothelial cells (EC) of the human umbilical cord vein deprived of serum in an MTT assay. An MTT assay is a laboratory analysis and a standard colorimetric assay (an assay that measures color changes) to measure cell proliferation (cell growth). In summary, the yellow MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, a tetrazole) is reduced to form purple in the mitochondria of living cells. This reduction takes place only when mitochondrial reductase enzymes are active, and therefore the conversion can be directly related to the number of viable (live) cells. The metabolic rate of cells can be determined by other techniques commonly known to those skilled in the art.
In another aspect, sNAG does not free the apoptosis of serum-deprived EC in an exclusion test with blue
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trypan An trypan blue exclusion test is a dye exclusion test used to determine the number of viable cells present in a cell suspension. It is based on the principle that living cells have intact cell membranes that exclude certain dyes, such as trypan blue, eosin or propidium, while dead cells do not. The viability of the cells can be determined by other techniques commonly known to those skilled in the art.
In one aspect, an intramuscular implantation test is an ISO test of intramuscular implantation 4 weeks after implantation, as described in section 6.4.2 below.
sNAG (i) comprises fibers, wherein the mayone of the fibers is less than about 15 microns in length, and (ii) (a) increases the metabolic rate of serum-deprived EC in an MTT assay and / or does not free apoptosis of serum-deprived EC in a trypan blue exclusion test, and (b) is not reactive when tested in an intramuscular implantation test.
5.4 Methods of use of hemostatic compositions
Methods for treating a wound in patients are described herein. The methods generally comprise the application of a bandage to a wound in a patient, wherein the bandage comprises or consists of any of the polymers or fibers listed in sections 5.2 and 5.3 above.
The patient is preferably a mairnfero, even more preferably a human being.
In certain embodiments, the patient is an adult, a teenager or a child. In one embodiment, the patient is a human being over 45 years of age. In one embodiment, the patient is a woman who is not pregnant.
In certain embodiments, the marnffero is a ganadena mammal (e.g., a cow, a sheep or a pig) or a domestic animal (e.g., a cat or a dog).
In certain embodiments, methods are described herein for the treatment of a wound in patients who normally do not heal in an orderly and timely manner like other human beings. The patient is preferably a human being, such as a diabetic, a smoker, a hemofflico, a person infected with HIV, an obese person, a person undergoing radiotherapy or a person with stasis venous ulcer. In one aspect, the patient is a person with venous ulcer due to stasis. In certain aspects, the wound is a surgical wound or a burn. In certain other aspects, the wound is a chronic wound, such as a diabetic ulcer, a venous stasis ulcer, an ulcer due to arterial insufficiency or a pressure ulcer.
In certain embodiments, methods are described herein for the treatment of a chronic wound in patients, wherein the chronic wound is not healed in an orderly or timely manner. In certain embodiments, the wound is a chronic wound such as a diabetic ulcer, a venous stasis ulcer, an ulcer due to arterial insufficiency or a pressure ulcer. In one aspect, the chronic wound is a venous ulcer due to stasis.
The hemostatic compositions and methods described herein advantageously reduce the labor and cost of nursing care related to wounds, especially chronic wounds. Bandages currently available in the market require changes every two days or so; on the contrary, in these methods the bandages are changed or reapplied every 3-35 days. In specific embodiments, the bandages are changed or reapplied every 4-35 days, every 5-35 days, every 6-35 days or every 7-35 days. In a certain aspect, bandages are changed or reapplied every 3-10 days, every 4-14 days, every 5-12 days, every 7-14 days, every 7-28 days, every 7-21 days, every 14-28 days, or any interval between 3, 4, 5, 6, or 7 days at the lower end of every 8, 9, 10, 12, 14, 18, 21, 24, 28, 30 or 35 days in the upper end In one aspect, wound dressings are changed or reapplied only once at the beginning of treatment. In another aspect, wound dressings are changed or reapplied once a week during treatment. In another aspect, wound dressings are changed or reapplied every two weeks during treatment. In another aspect, the bandages are changed or reapplied every two weeks, three weeks or four weeks during treatment. The present methods reduce the frequency of wound dressing changes by at least 50%, and may reduce the frequency of wound dressing changes by 100%, 200%, 500% or even more. The specific frequency to change or reapply wound dressings may vary depending on the needs of the individual; that is, depending on the size, classification and responsiveness of the wound to the bandage. The frequency can be determined by typical thermal techniques, and does not have to be at fixed intervals. Rather, the frequency can be varied over time, depending on the needs of the individual. For example, as a chronic wound begins to heal, the frequency of changes or reapplications of wound dressings decreases.
In certain aspects, the wound dressing is applied to the wound, where it can remain until it is removed or biodegraded. More preferably, the wound dressing is reapplied (without removing the previous wound dressing) or changed (removing the previous bandage and applying a new bandage) at least once, but it can be reapplied or changed at frequencies. described above while the wound lasts.
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Preferably, the bandage is reapplied or changed at least twice, at least three times, at least four times or more. Therapeutic regimens can be performed for a period of a few weeks (e.g., 2-8 weeks) to several months (e.g., from 2-4 months to 6-12 months or more, and any interval between these).
Advantageously, the bandages also reduce the total healing time of the wound. For example, in the diabetic mouse model described in section 6 below, the pGlcNAc bandage reduced the time to get the wound closed a week faster than in the reference mice. Therefore, in addition to reducing the number of bandage changes for a given wound, the methods described herein are useful for reducing the time of wound closure. Accordingly, in certain aspects, methods are provided to reduce the wound closure time of a chronic wound, which comprise the topical application of a bandage to a chronic wound in a patient, wherein the bandage comprises or consists of one or more of the polymers listed in sections 5.2 and 5.3 above. Optionally, the application is repeated one or more times in accordance with the therapeutic regimens described in this section. In certain aspects, the wound closure time is reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%.
More preferably, the polymer from which a bandage is made is a biocompatible and / or immunoneutral polymer, including, but not limited to, poly-p-1 ^ 4-N-acetylglucosamine or one of its derivatives. In a preferred embodiment, the poly-p-1 ^ 4-N-acetylglucosamine or one of its derivatives is a microalgae poly-p-1-4-N-acetylglucosamine or one of its derivatives. In certain aspects, poly-p-1 ^ 4-N-acetylglucosamine or one of its derivatives does not come from shellfish or crustaceans.
The methods mentioned above can be used in conjunction with other conventional assistance procedures for chronic wounds. For example, the methods can be used in conjunction with one or more of the following treatments for cutaneous ulcer therapy: removal of necrotic or infected tissue (debridement); discharge; compression treatment for stasis venous ulcers; establishment of adequate blood circulation; maintenance of a moist environment in the wound; treatment of wound infection; wound cleaning; nutritional support, including glycemic control for patients with diabetic ulcers; intestinal and bladder care for individuals with bedsores at risk of contamination.
For burn wounds, typical care procedures that can be used in conjunction with the methods described herein include: hemodynamic resuscitation; comorbidities treatment; timely debridement and excision of burns; wound closure; wound infection treatment; pain control; nutritional support; measures to inhibit excessive scar formation; and rehabilitation, including the range of passive movement when burns line the joints.
5.5 Kits
In addition, a kit is provided comprising any of the bandage materials described above. The bandage is preferably contained within a sealed, waterproof, sterile package that facilitates the removal of the composition without contamination. The materials from which the containers can be made include aluminum foil, plastic, or other conventional material that is easily sterilized. The kit may contain a single bandage or several bandages, preferably where each is provided in a separate, waterproof and sterile package. The bandage may further comprise wound healing agents or antimicrobials, as described in sections 5.2 and 5.3 above.
In another embodiment, a container is provided that has two compartments. A first compartment contains the bandage, while the second compartment contains an active agent such as a growth factor or an antimicrobial agent. In the field or weather, the bandage can easily be immersed in the active agent applied subsequently to the wound.
A kit may comprise a note regarding FDA approval and / or instructions for use.
In addition, a kit designed for emergencies or military use may also contain disposable pre-sterilized instruments, such as scissors, bistun, clamp, tourniquet, elastic or inelastic bandages, or the like.
6. Examples
6.1 Example 1. Effects of the poly-N-acetylglucosamine (pGlcNAc) patch on wound healing in mice db / db.
6.1.1 Materials and Methods
Preparation of the pGlcNAc patch. PGlcNAc SyvekPatch ™ patch (Marine Polymer Technologies, Inc., Danvers, MA) consists of nanofibers obtained from microalgae produced as previously described (see Vournakis et al. U.S. Patent Nos. 5,623,064; and no. 5,624,679, the content of which is incorporated herein
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memory by reference in its entirety). In summary, microalgae were grown under conditions of a single bioreactor using a defined growth medium. After the collection of microalgae from high-density cultures, nanofibers were isolated by a step-by-step separation and purification procedure that produces batches of pure nanofibers suspended in water for injections (api). The fibers were patched by concentration and dried in an oven, and packed and sterilized by gamma irradiation. Average dimensions of nanofibers 20-50 nm x 1-2 nmx ~ 100 pm. The quality of each of the fiber batches was controlled using chemical and physical analysis parameters, and each batch meets strict purity criteria prior to release. The final lots were required to be substantially free of protections, metal ions and other components.
Wound model and study design. Homozygous, genetically diabetic, 8-12 week old male Lep / r-db / db (strain C57BL / KsJ-Leprdb) mice were used under an approved animal protocol at an accredited center Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The day before surgery, their hair was cut and shaved (Nair®, Church & Dwight Co., Princeton, NJ). On the day of the surgical intervention, the animals were weighed and anesthetized with 60 mg / kg of Nembutal (Pentobarbital). A 1.0 cm2 dorsal skin and fleshy area was removed and the wounds were photographed. The wounds were covered with the pGlcNAc patch for 1 hour (group 1 h, n = 15), 24 hours (group 24 h, n = 15), or left untreated (group NT, n = 15). All wounds were covered with semi-occlusive polyurethane bandages (Tegaderm®, 3M, St. Paul, MN). On the 10th and 21st days, 7-8 animals per group were sacrificed and the wounds were photographed, removed and fixed in 10% buffered neutral formalin solution. N = 15 per group were observed from the 1st day to the 10th, and n = 7-8 per group were observed from the 14th day to the 21st day.
Wound closure analysis. Three independent blind observers compared digital photographs taken twice a week with initial photographs at day 0 using planimetric methods. The wound closure was quantified by measuring the contraction (C), re-epithelialization (E) and the open wound (O) as a percentage of the area of the original wound. The sum of the wounded, reepithelized and open wound areas is equal to 100% of the original wound size (Fig. 1) (see Yannas I. Tissue and Organ Regeneration in adults. New York: Springer; 2001).
Cross sections of the central wound were incorporated into paraffin, sectioned and tinedon according to the routine protocols for Hematoxylin and Eosin (H&E). Panoramic digital images of the cross section of each wound were prepared using Adobe Photoshop CS Software (Adobe Systems Incorporated, San Jose, CA) in order to analyze the area and thickness of the granulation tissue with digital planimetna (Image J, NIH, Bethesda , MD).
Immunohistochemistry Paraffin incorporated sections were rehydrated and antigen recovery was carried out for the Ki-67 proliferation immunohistochemical marker by microwave in 10 mM sodium citrate (pH 6.0) for ten minutes. The frozen sections were fixed with acetone and the adhesion molecule of endothelial cells and platelets (PECAM-1) were stained for the immunohistochemical vascularization marker. The primary Ki-67 antibody (Lab Vision, Freemont, CA) was incubated for 1 hour at room temperature, while the primary PECAM-1 antibody (Pharmingen, San Jose, CA) was incubated at 4 ° C overnight. The PECAM-1 signal was intensified using the tyramide amplification system (Perkin Elmer, Boston, MA).
Quantification of the density of blood vessels. Digital color images of the wound sections were preprocessed before quantification to ensure uniform contrast of the positive areas of PECAM-1 in relation to the background. A positive staining mask was created using the color mask function of the Corel PhotoPaint v program. 10 (Corel Corporation, Ottawa, Ontario, Canada) taking samples of five different chromogen tones represented in positively stained areas. The areas of the masked vessels became pure black while the bottom became pure white. The black and white representations were used for the quantization zone in the IPLab software (BD Biosciences Bioimaging, Rockville, MD) applying the segmentation function. Tissue regions were defined by projecting the original H&E image onto the processed image. The density of blood vessels, quantified throughout the image, was expressed as the ratio of the area of the vessels to the total area of granulation tissue. Between 4 and 7 microscopic fields (20x) were used to assess the density of vessels for each wound and treatment modality.
Quantification of cell proliferation. Cell proliferation in wounds was analyzed using image analysis of sections taken with Ki-67 in a manner similar to the method of quantification of vessel density. High power digital images of wound sections taken with Ki-67 were used to measure the number of Ki-67 positive cells with respect to the total number of nuclei. The degree of proliferation was quantified in the entire wound section using 4-6 fields with a 20x magnification and was expressed as a ratio of proliferating nuclei (Ki-67 positive) to total nuclei.
Statistical Analysis Values were expressed in means ± typical deviation in the text and figures. One-way analysis of variance and specific tests with LSD were used to determine the significance of differences between treatment modes. A multifactor analysis was performed using Statistica v7.0 (Statsoft, Inc, Tulsa, OK).
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6.1.2 Results
Statistical Analysis Values are expressed as mean ± standard deviation in the text and figures. A form of variance analysis and ad hoc LSD tests were used to determine the significance of differences between treatment modes. A multifactor analysis was performed using Statistica v7.0 (Statsoft, Inc, Tulsa, OK).
Kinetics of wound healing altered with pGIcNAc patch. Treatment with pGlcNAc caused faster wound closure over time compared to the absence of treatment. The 1 h group showed a faster decrease (p <0.01) of the raw surface (open part of the wound), compared with the NT group on the 7th, 14th and 17th days (Fig. 2A). The 1-hour group also achieved an average closure of 90% in 16.6 days, which is nine days faster (p <0.01) than the NT group (25.6 days) (Fig. 2B). The 24 h group achieved 90% closure in 18.2 days (Fig. 2B).
The 1 h group showed increased reepithelialization (p <0.01), compared to the NT group on the 4th, 7th, 14th and 21st days (Fig. 2C, 2D). The 24 h group showed increased reepithelialization (p <0.01), compared to the NT group on the 4th and 21st days (Fig. 2C).
The 24 h group showed a decrease (p <0.01) in contraction, compared to the NT group on the 4th and 17th days; and decrease (p <0.01) of the contraction, compared with the group 1 h on the 7th day (Fig. 2E). The 1 h group showed a decrease (p <0.01) in contraction, compared to the NT group on the 14th day (Fig. 2E).
Higher density and proliferation of blood vessels with the pGlcNAc patch. 1 cm2, of total thickness, of untreated wounds in mouse db / db reached 50% closure between days 8-12 after the operation (Chan et al. Effect of recombinant platelet-derived growth factor (Regranex) on wound closure in genetically diabetic mice. J. Burn. Care Res. 2006; 27 (2): 202-5). The 10th day was chosen as the intermediate time point for staging wound healing after the treatments.
Proliferating cells were stained for Ki-67 (Fig. 3A). PECAM-1 (CD31) was selected to have endothelial cells and quantify the density of blood vessels in the granulation tissue (Fig. 3B). The 1 h group showed significant increases in blood vessel density and cell proliferation, compared to both the NT and 24 h groups (Fig. 3A, 3B). The results of PECAM-1 and Ki-67 were represented, together they give the visual correlation of angiogenesis and proliferation between different treatments (Fig. 3C).
The area and thickness of the granulation tissue were measured in microfotograffas with an increase of 4x (n = 7-8 per group) to assess the level of coverage for newly formed tissue in response to lesions and treatment modality, as shown then:
Average area of granulation tissue ±
typical deviation
(pixels)
Average thickness of granulation tissue ±
typical deviation
(pixels)
5.5x105 ± 5.3x105
3.5x102 ± 3.6x102
NT
1.1x106 ± 6.0x105
7.9x102 ± 3.9x102
1 hour
8.6x105 ± 2.7x105
5.7x102 ± 2.5x102
24 h
No significant differences were observed between the NT groups, 24 h and 1 h in the amount and distribution of granulation tissue.
Time of application of the response to the foreign body modulated by pGlcNAc patch. To study the effects of prolonged exposure of the wound base to insoluble fibers, the patch was initially left in place throughout the follow-up period (three weeks). The prolonged presence of the long insoluble fibers of the patch caused the formation of a response to the foreign body, characterized by an increase in the formation of granulation tissue and multinucleated giant cells. The application of the patch for 1 or 24 hours did not cause any reaction to the foreign body.
6.2 Example 2. Effects of the shortened poly-N-acetylglucosamine (sNAG) membrane on wound healing in the mouse db / db.
6.2.1 Materials and Methods
Preparation of the sNAG membrane. The sNAG membrane consists of nanofibers obtained from microalgae produced as described in section 6.1 above, in which the fibers are shortened by irradiation. In summary, the starting material contains 60 g of pGlcNAc suspension at a concentration of 1 mg / ml. The
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pGIcNAc suspension concentration was confirmed by filtering 5 ml on a 0.2 pm filter. 15 l of the pGlcNAc slurry containing 15 g of pGlcNAc was filtered until a wet cake was formed. The wet cake was then transferred to an aluminum foil bag, which is a container compatible with gamma radiation, and was subjected to gamma radiation of 200 kGy. Other irradiation conditions were tested to determine their effects on pGlcNAc compositions, as reflected in Fig. 4A.
Wound model and study design. The mouse model genetically described in section 6.1 above was used to test the effect of sNAG membranes on wound healing.
Analysis of wound closure. The wound closure analysis was performed substantially as described in section 6.1. The sum of the areas of counter-wounded, re-epithelialized and open wounds is equal to 100% of the original wound size (Fig. 5) (see Yannas I. Tissue and Organ Regeneration in adults. New York: Springer; 2001.).
Immunohistochemistry Immunochemistry was performed substantially as described previously in section 6.1.
Quantification of vessel density. The density of the vessels was quantified substantially as described previously in section 6.1.
Quantification of cell proliferation. The quantification of cell proliferation was carried out substantially as described previously in section 6.1.
Collagen staining. Wounds were tined for collagen content using routine methods.
Statistical analysis The statistical analysis of wound closure was performed substantially as described previously in section 6.1.
6.2.2 Results
Statistical analysis The statistical analysis of wound closure was performed substantially as described previously in section 6.1.
Effect of irradiation on pGlcNAc membranes. While irradiation reduces the molecular weight of pGlcNAc, irradiation does not affect the microstructure of the fibers. PGlcNAc was irradiated under different conditions: as a dry, lyophilized material; as a dry membrane; as a concentrated slurry (30:70 by weight by volume); and as a dilute suspension (5 mg / ml). A suitable molecular weight reduction (at a molecular weight of 500,000-1,000,000 daltons) was achieved at an irradiation dose of 1,000 kgy for dry polymer and 200 kgy for wet polymer (Fig. 4A).
The chemical and physical structure of the fibers was maintained during irradiation as verified by infrared spectrum (IR) (Fig. 4B), elementary analysis, and scanning electron microscopy (SEM) analysis. Microscopic observation of the irradiated fibers showed a decrease in particle length (Fig. 4C and 4D). The mayone of the fibers are less than about 15 pm in length, with an average length of about 4 pm.
Kinetics of wound healing altered with sNAG membrane. Macroscopic photograffas demonstrate that the wounds of mice treated with sNAG membrane healed faster than the wounds of untreated reference mice (see Fig. 6-10). Treatment with sNAG caused faster wound closure over time compared to the absence of treatment. Mice treated with the sNAG membrane had a faster decrease (p <0.05) of the raw surface (open part of the wound), compared to the reference mice without treating the 4th, 7th, 10th days. °, 14 °, 17 °, 21 ° and 25 ° (Fig. 11).
Mice treated with the sNAG membrane also achieved 50% average closure in a little more than 8 days, which is four days faster (p <0.01) than untreated reference mice (more than 12 days ) (Fig. 12).
Mice treated with the sNAG membrane also achieved a 90% average closure in less than 15 days, which is eight days faster (p <0.01) than untreated reference mice (approximately 23 days) (Fig .13).
On day 28, all (100%) wounds closed in mice treated with sNAG membrane, while only 85% wounds closed in untreated reference mice.
Mice treated with the sNAG membrane showed an increase (p <0.05) in reepithelialization, compared with the reference mice without treating days 14 and 17 (Fig. 14).
Mice treated with the sNAG membrane showed a decrease (p <0.01) in contraction, compared to untreated reference mice on the 4th, 7th and 10th day (Fig. 15).
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Higher density and proliferation of blood vessels with sNAG membrane. 1 cm2, of total thickness, of untreated wounds in mouse db / db reached 50% closure between days 8-12 after the operation (Chan et al. Effect of recombinant platelet-derived growth factor (Regranex) on wound closure in genetically diabetic mice. J. Burn. Care Res. 2006; 27 (2): 202-5). The 10th day was chosen as the intermediate time point for staging wound healing after the treatments.
The histology of the wound edge in an untreated reference mouse compared to a mouse treated with sNAG membrane is shown in Figs. 16 and 17, respectively. Proliferating cells were stained for Ki-67 (Fig. 18). PECAM-1 (CD31) was selected to have endothelial cells and quantify the density of blood vessels in granulation tissue (Fig. 19). Mice treated with sNAG membrane showed significant increases in blood vessel density and cell proliferation, compared to untreated reference mice (Fig. 16, 17).
The areas of reepithelialization and granulation tissue were measured in micrographs with an increase of 4x (n = 7-8 per group) to assess the level of coverage for newly formed tissue in response to the injury and treatment modality, as shown in Figs. 20 and 21.
Time of application of the response to the foreign body modulated by the sNAG membrane. To study the effects of prolonged exposure of the wound base to sNAG fibers, the membrane is left in place throughout the experiment (four weeks). Fig. 22 shows the lack of reaction to the foreign body at 10 days. On the other hand, no reaction to the foreign body was observed throughout the study in any of the mice treated with sNAG membrane.
Formation / construction of collagen modulated by the sNAG membrane. Collagen staining was observed in both treated and untreated mice with sNAG, but collagen bundles were denser and more uniform in treated mice, suggesting increased stimulation of wound fibroblasts and wound healing. more advanced in treated mice (Fig. 22).
6.3 Example 3. Effects of poly-N-acetylglucosamine (pGlcNAc) and sNAGs on the movement of endothelial cells (EC) and angiogenesis
6.3.1 Materials and Methods
Tissue culture, growth factors and transfection. Mixed endothelial cells (cell) of the human umbilical cord vein (Cambrex) from multiple donors were maintained at 37 ° C with 5% CO2 in basal endothelial medium 2 (Cambrex) enriched with EC 2 SingleQuots growth medium as Describe in Cambrex procedures. Serum deprivation was performed at 80-90% confluence in RPMI-1640 enriched with 0.1% fetal bovine serum (Gibco BRL) for 24 h followed by stimulation with VEGF 165 (20 ng / ml, R&D Systems) or with highly purified pGlcNAc nanofibers or sNAG nanofibers in sterile water (provided by Marine Polymer Technologies, Inc., Danvers, Mass., USA) with the amounts indicated in the text. For the inhibition using the VEGFR SU5416 inhibitor; (10 | jM; R&D Systems) the cells were pretreated for 15 minutes before stimulation with VEGF, pGlcNAc or sNAG.
The EC of the human umbilical cord vein was transfected using the Amaxa Nucleofector system in the procedures described by the manufacturer, obtaining transfection efficiencies of up to 80%. All transfections were controlled by expression of the green fluorescent protein (GFP) using a GFP expression vector (pFP-C1; Clontech) or an interference RNA (RNAi; Amaxa) directed against GFP. Plasmid RNAi directed specifically against Ets1 was purchased from Pandomics, Inc., and the dominant Ets negative assembly (dn-Ets) contains the Ets2 DNA binding domain cloned into a pcDNA3 expression vector.
Antibodies and Western blot analysis. The antibodies used for Western blot analysis are the following: anti-p85 subunit of PI3K (Upstate Biotechnology), anti-VEGFR2 phosphopedic (Cell Signaling), VEGFR2 (Santa Cruz), anti-p42 / p44 phosphopedic (Promega), and phospho-specific anti-VEGFR2 (BD Biosciences, Inc.), anti-p42 / p44 Erk1 / 2, anti-VEGFR2 and anti-Ets1 (Santa Cruz).
The treated cells were washed once with phosphate buffered saline (PBS) and lysed in 1 x RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, 40 mM NaF), enriched with complete protease inhibitors without EDTA (Roche) and 200 jM sodium ortho vanadate. Protein concentrations were determined by a bicincomic acid (Pierce) protein analysis resolved by SDS-PAGE and transferred to Immobilon-P polyvinylidene fluoride membranes (Millipore). Western analysis followed by conventional procedures. Proteins were visualized using the Luminol reagent (Santa Cruz).
Motility and cell proliferation assays. For "aranazos" wound closure trials, the ECs were grown to confluence in tissue culture plastic plates and a single 'wound' was produced using a pipette tip. Cells were incubated in serum-free media enriched with or without VEGF (20 ng / ml), pGlcNAc or sNAG in the amounts indicated in the text for 16-18 h. The cells were washed once with PBS, fixed for 10 min in methanol, stained with 0.1% crystal violet for 10 min and thoroughly rinsed with water. The essays
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In wounds, 10 magnification magnifications were photographed using an Olympus optical microscope equipped with digital image processing, and the migrated distance was measured.
For the modified transwell assays, transfected or non-transfected CEs were seeded in invasion chambers of 8 pm pore size previously coated with fibronectin or vitronectin at 20 pg / l (Sigma), 5 x 104 cells per chamber in 500 pl of media without serum and 500 pl of medium without serum were added to the well. VEGF (20 ng / ml), pGlcNAc or sNAG was added to the upper chamber. The cells were incubated for 12 h at 37 ° C in the presence of 5% CO2. Cells that did not migrate were removed by cleaning the top of each membrane with a cotton swab. Migrated cells were fixed in methanol for 10 min and stained with 0.1 mg / ml ethidium bromide in PBS. Migrated cells were counted using a Leica fluorescence microscope. Each test was performed in triplicate at least 3 independent times, and at least 6 fields were counted per transwell test.
For in vitro angiogenesis assays, EC were plated on plates in the Matrigel matrix with reduced growth factor (BD Laboratory) at 1.6 x 104 cells / 50 pl per well of a 96-well plate in serum-free medium in the presence or absence of VEGF (20 ng / ml), pGlcNAc or sNAG. Umbilical cord formation was evaluated for a maximum of 8 h after sowing. Cells were fixed and photographed when cells treated with VEGF, pGlcNAc and sNAG began to form cords, while the references retained a single layer of cells. The tests were performed in duplicate and repeated 2 independent times.
For the evaluation of cell proliferation / viability, 2 different assays were used: trypan blue exclusion by direct cell counts with a hemocytometer and an MTT assay [3- (4,5-dimethylthiazol-2-yl bromide) -2,5-diphenyltetrazolium] in the procedures described by the manufacturer (Promega).
Antibody block To block cell motility and signaling cells in which integrin acts as a mediator, EC was previously incubated for 15 min with blocking antibodies, at emphatically determined concentrations (1 pg / ml), directed against aVp3 or a5p1 (CD49e) acquired from Chemicon International or against the a5 subunit (Santa Cruz) before stimulation with pGlcNAc or sNAG. As a negative reference, normal rabbit serum was used. For the inhibition of cell migration using the aVp3 antibody, the transwells were previously coated with vitronectin (Sigma) instead of fibronectin (20 pg / pl). To block VEGFR activation, EC was previously incubated for 15 min with inhibitor, SU5416 (SU) at a concentration of 10 pg / ml.
Polymerase chain reaction with reverse transcription. For polymerase chain reaction with semiquantitative reverse transcription (RT-PCR), cDNA was synthesized from complete RNA (2-5 pg), isolated using STAT-60 RNA (Tel-Test, Inc.) in the procedures described by the manufacturer, with a Superscrpt First-Strand Synthesis Kit purchased from Gibco BRL using oligo (dT) following the manufacturer's instructions. The PCR reactions contain equal amounts of cDNA and 1.25 pM of the appropriate primer pair (Proligo, Inc.). The sequences of the primers are as follows: Ets1, 5'-TTCTCAGAGCCCAGCTTCAT-3 'direct, 5'- AAAGTTTGAATTCCCAGCCAT-3' reverse; direct metallothionema 2A, 5'-CAACCTGTCCCGACTCTAGC-3 '
reverse; S26, 5'-CTCCGGTCCGTGCCTCCAAG-3 'direct, inverse; VEGF, 5'-CTACCTCCACCATGCCAAGT-3 'direct,
reverse; IL-1, 5'-CTGCGCCAACACAGAAATTA-3 'direct,
reverse; IL-8,
AGGAGCAACTCCTGTCCTGA-3 '
CAGAGAATAGCCTGTCTTCAG-3
TGGTGATGTTGGCTCCTCA-3 '
ATTGCATCTGGCAACCCTAC-3 '
5'-TCGGATTTCACGATTTCTCC-3
direct,
5'
5'
5'
5'
5'-
Reverse GCTACAAGTGCGTCGTCAAA-3 '. Cycling conditions were: 94 ° C for 5 min; 20-35 cycles of 94 ° C for 1 min, 50-65 ° C (based on primer T m) for 1 min, 72 ° C for 1 min and 45 s + 2 s / cycle; 72 ° C for 7 min and cooled to 4 ° C. The number of cycles was determined emphatically that it was within the linear range of the test for each pair of primers used. All semiquantitative RTPCRs were performed in tandem with primers S26 as an internal reference. The products are introduced in 1-1.5% agarose gels (based on product size) and displayed on a BioRad Molecular Imaging System.
Real-time PCR was carried out using a Brilliant CYBR green quantitative PCR kit (QPCR) in combination with an Mx3000P real-time PCR system, both purchased from Stratagene. Real time was carried out in triplicate at least 2 independent times. Internal reference primers that detect the S26 subunit of ribosomal protein were used.
6.3.2 Results
6.3.2.1 pGlcNAc
pGlcNAc protected the EC from cell death caused by serum deprivation. To test whether pGlcNAc fibers have a direct effect on CE, serum-deprived CE cells were treated with VEGF or with different concentrations of pGlcNAc fibers. As shown in Fig. 24 at 48 h and 72 h after serum deprivation, compared to the total number of cells grown on plates (reference), there was a reduction of approximately double the number of cells after 48 hours 72 hours At 48 h, this decrease in the number of cells was read by adding VEGF or by adding pGlcNAc fibers at 50 or 100 pg / ml. At 72 h, the decrease in the number of cells is read by the addition of VEGF or to a large extent it is read by adding pGlcNAc fibers at a rate of 100 pg / ml. These results indicated that as VEGF, the
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pGIcNAc fiber treatment prevented cell death caused by serum deprivation.
pGIcNAc did not affect the metabolic rate. As shown in Fig. 25, pGlcNAc did not produce a higher metabolic rate measured by MTT assays, indicating that this polymeric material was not causing a noticeable increase in cell proliferation, but was getting rid of cell death by deprivation of serum.
pGlcNAc increased cell migration. To test whether the EC treatment with pGlcNAc fibers produced changes in cell motility, the "aranazo" wound closure test was used. The migration of cells in the injured area was significantly higher in the presence of pGlcNAc at both 50, 100 and 250 mg / ml. As shown in Fig. 26, wound closure was similar to that observed for treatment with VEGF. These results indicated that treatment with pGlcNAc produced an increase in the movement of the EC.
pGlcNAc caused an increase in migration to fibronectin. To determine whether this increase in cell motility correlates with an increase in cell invasion, EC motility was measured using transwell assays where the membranes were previously coated with the extracellular matrix protein, fibronectin. As shown in Fig. 27, treatment with pGlcNAc produced a triple increase in migration to fibronectin which increased by the addition of VEGF (4 times).
pGlcNAc increased umbilical cord formation. The stimulation of cell migration is a prerequisite for the increase of angiogenesis. To test, in vitro, if pGlcNAc was proangiogen, Matrigel assays were performed. Plates with EC in reduced Matrigel in growth factor under serum deprivation conditions were seeded and evaluated for umbilical cord formation in the presence or absence of VEGF or pGlcNAc fibers in 6 h. As shown in Fig. 28, treatment with both VEGF and pGlcNAc resulted in increased umbilical cord formation in Matrigel. These results indicate that pGlcNAc is proangiogen.
pGlcNAc produced effectors involved in cell motility. Complete RNA was isolated from serum-deprived EC (SS); SS treated with VEGF, pGlcNAc, Sphingosiing 1-phosphate (S1P), or Zn; sS pretreated with VEGF or S1P after treatment with pGlcNAc; SS pretreated with VEGF or pGlcNAc then following the treatment of the VEGFR inhibitor, SU5416 (Su). As shown in Fig. 29A, treatment with pGlcNAc stimulated the expression of the transcription factor Ets1, which is an important regulator of the movement of CE, and metallothionem 2A (MT), and Akt3 Edg3.As shown in Fig. 29B, real-time PCR indicates that Ets1 was caused approximately 2 times by treatment with pGlcNAc. This increase in Ets1 in message was accompanied by increased protein expression as shown in the Western blot analysis in Fig. 29C. Fig. 29A also indicated that the expression of MT stimulated by pGlcNAc was a function of VEGFR.
The induction by pGlcNAcc of phosphorus-MAPK was a function of VEGFR2. To test whether treatment with pGlcNAc resulted in the activation of the previously shown vias downstream of the VEGFR signaling, the ECs were treated with VEGF or pGlcNAc. As shown in Fig. 30A, treatment with pGlcNAc resulted in a marked increase in MAPK phosphorylation. To test whether this increase was a function of VEGFR2, the ECs were pretreated with the VEGFR inhibitor, after treatment with VEGF or pGlcNAc. The results indicated that the induction by phosphorus-MAPK pGlcNAc was a function of VEGFR2 (see also Fig. 30B).
VEGFR2 non-active pGlcNAc. To test if pGlcNAc activated VEGFR, a series of Western blots were performed using an antibody directed against the phosphorylated form of VEGFR2. As shown in Fig. 31, treatment with VEGF produced rapid phosphorylation of VEGFR, accompanied by the renewal of the total amounts of VEGFR2 protein, while pGlcNAc had no effect, either at these initial time points shown, or up to 6 h after treatment (data not shown).
Migration induced by pGlcNAc was a function of Ets. To test whether Ets1 is required for pGlcNAc-induced motility, Ets1 was inhibited using both a dominant negative approach as well as through RNAi. A dn-Ets assembly expressing the binding domain to the conserved Ets DNA was transfected in the EC. After 24 h to allow dn-Ets expression, the cells were evaluated for changes in cell migration to fibronectin in transwell assays after treatment with pGlcNAc. As shown in Fig. 32A, inhibition of Ets1 activity, as well as other family members expressed in CE resulted in a marked decrease in EC migration in response to pGlcNAc. As a reference for the activity of dn-Ets, Fig. 5B demonstrates that the transfection of increasing amounts of dn-Ets results in a decrease in the complete Ets1 protein. The expression of Ets1 can be controlled not only by another family member, but can also be self-regulated. The inhibition of Ets1 specifically by RNAi also resulted in a decrease in cellular motility caused by pGlcNAc on fibronectin (Fig. 32A, right side). As shown in Fig. 32B, in cells transfected with dn-Ets expression plasmid, the expression of the Ets1 protein decreased with the amount of plasmid increasing. As a reference for the RNAi experiment, Fig. 32C shows the expression levels resulting from Ets1 in CE transfected with 2 amounts of RNAi containing plasmid directed against Etsl. The expression of dn-Ets resulted in a more substantial reduction in the cell migration than the Ets1 RNAi, probably due to its blockade of other family members expressed in the EC. These findings support a function for Ets1 in the induction of cell motility by
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pGIcNAc.
Cellular motility caused by pGIcNAc requena integrin. To test whether the effects of pGIcNAc depend on integrin, blocking antibodies were used to interrupt the signaling in which the integrin in EC is mediated. The effect of these antibodies on cell migration caused by pGlcNAc was evaluated by Transwell assays. Fig. 33A shows the results using antibodies directed against aVp3 integrin or aspi (CD49e) in fibronectin migration assays (the aVp3 receptor). Fig. 33B shows a similar experiment using antibodies directed against aVp3 or aspi (CD49e) in vitronectin coated transwells. Antibody blocking of any integrin subtype resulted in the inhibition of cell motility caused by pGlcNAc in its related substrates. These results indicate that pGlcNAc stimulates cell motility by activating integrin. The results are also consistent with pGlcNAc that stimulates angiogenesis by activating integrin.
Cellular motility caused by pGIcNAc may involve the activation of FAK with the participation of integrin. FAK is phosphorylated in response to the grouping and activation of integrins. FAK is a key regulator of integrin and cell motility and invasion in which growth factor intervenes. To test the activation of integrin by pGlcNAc, EC with pGlcNAc fibers were treated for increasing amounts of time and changes in the phosphorylation level of FAK were tested. As shown in Fig. 34, treatment with pGlcNAc resulted in phosphorylation of FAK at 15 min of treatment. These results indicate that cellular motility caused by pGlcNAc may involve the activation of FAK through the participation of integrin.
pGlcNAc activates an integrin pathway ^ Etsl which leads to angiogenesis in a wound healing model. A function has been described for Etsi in the regulation of the transcription of a number of integrin subunits, placing Etsi upstream of the integrins. The discovery that the motility caused by pGlcNAc depends on both integrins and Etsi implies that Etsi can be regulated downstream of integrins. To confirm that the results of integrin activation in the regulation of Etsi expression, blocking antibodies directed against aspi (fibronectin receptor) or aVp3 (vitronectin receptor) were used to inhibit integrins or pGlcNAc. Fig. 35A demonstrates that the blocking of antibodies to integrin aspirates results in a reduction in Etsi expression caused by pGlcNAc. This inhibition of Etsi expression using a blockade of the integrin aspi is repeated in the field of protema (Fig. 35B). However, although the asp3 integrin antibody blocked the motility in vitronectin, it did not affect the expression of Etsi caused by pGlcNAc (Fig. 3s), indicating that the cellular motility caused by pGlcNAc in vitronectin may be independent of Etsi. Taken together, these results place Etsi downstream of certain integrins in the primary EC and indicate the potential specificity in integrin signaling with respect to Etsi expression in the primary EC. These results, therefore, indicate that pGlcNAc can activate an integrin ^ Etsi pathway that leads to angiogenesis in a wound healing model.
VEGF and IL1 expression caused by pGlcNAc. To test whether treatment with pGlcNAc caused the expression of growth factors or cytokines known to be secreted by activated CE, serum-deprived CE were treated with pGlcNAc for i2 h and changes in the expression of VEGF, IL-i and IL were evaluated. -8. As demonstrated by RT-PCR and QPCR (Fig. 36A), treatment with pGlcNAc resulted in increased expression of both VEGF and IL-i. These results also indicated that the EC response to pGlcNAc is specific since there were no changes in the expression of another interleukin, IL-8. To test the pGlcNAc-dependent induction of Etsi expression, a transcription factor known to be regulated by VEGF, secondary to the effect of pGlcNAc on VEGF expression, the activation of VEGFR was blocked using the pharmacological inhibitor, SUs4i6 (SU) , before treatment with pGlcNAc. As demonstrated by QPCR (Fig. 36B), EC treatment with this inhibitor blocked the induction of VEGF by Etsi but had no effect on the induction of Etsi by pGlcNAc.
Expression of FGF1 and FGFR3 caused by pGlcNAc. To test whether the pGlcNAc treatment caused the expression of factors related to angiogenesis, the ECs were treated with pGlcNAc and the changes in the expression of FGFi, FGF2, FGFRi, FGFR2, FGFR3, Stabilin, IFNg, ColagenoAi8 were evaluated. As the picture shows. 37, treatment with pGlcNAc resulted in an increase in the expression of Stabilin and CollagenAi8.
6.32.2 sNAG
Increase in cell migration by sNAG. To test whether treatment with EC sNAG fibers resulted in changes in cell motility, the "aranazo" wound closure test was used. Migration of the cells in the area of the wound increased significantly in the presence of sNAG both at and at 100 mg / ml. The wound closure was similar to that observed for treatment with pGlcNAc (see Fig. 38). These results indicated that sNAG treatment resulted in an increase in the movement of the EC.
sNAG caused a notable increase in metabolic rate. As measured by MTT tests, sNAG at so, 100 or 200 mg / ml resulted in a higher metabolic rate of EC than VEGF (Fig. 39).
sNAG did not protect the EC from cell death caused by serum deprivation. To test if sNAG fibers
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had a direct effect on EC, serum-deprived EC cells were treated with VEGF or with different concentrations of sNAG fibers. As shown in Fig. 40, at 48 h after serum deprivation, compared to the total number of cells grown on plates (reference), there was a reduction in the number of cells of approximately double. From this decrease in the number of cells, books were added by adding VEGF, but not by adding sNAG fibers at 50, 100 or 200 | jg / ml. These results indicated that unlike VEGF, treatment with sNAG fibers did not prevent cell death caused by serum deprivation.
VEGF and IL1 expression caused by sNAG. To test whether treatment with sNAG caused the expression of growth factors or cytokines that are known to be secreted by activated ECs and compare the effect with pGlcNAc, serum-deprived ECs were treated with pGlcNAc or sNAG for 12 h and the changes in the expression of VEGf, IL-1 and IL-8. As the picture shows. 41, treatment with sNAG resulted in increased expression of both VEGF and IL-1. These results also indicated that the EC response to sN AG is specific since there was no change in the expression of another interleukin, IL-8.
Expression of FGF1 and FGFR3 induced by sNAG. To test whether treatment with sNAG caused the expression of factors related to angiogenesis, the ECs were treated with sNAG and changes in the expression of FGF1, FGF2, FGFR1, FGFR2, FGFR3, Stabiline, IFNg, CollagenAl8 were evaluated. As demonstrated by RT-PCR (Fig. 37), treatment with sNAG resulted in an increase in the expression of FGF1 and FGFR3.
The above results demonstrate that both pGlcNAc and sNAG induced CE motility, and that both pGlcNAc and sNAG induce the expression of VEGF eIL-1.
The above results also show that sNAG increases the metabolic rate of serum-deprived EC in an MTT test and does not get rid of apoptosis of serum-deprived EC in a trypan blue exclusion test.
6.4 Example 4. Testing of sNAG with animals
6.4.1 Experimental product
An experimental product comprising sNAG produced as described above in section 6.2.1 was used. The experimental product was supplied sterile by Marine Polymer Technologies, Inc.
6.4.2 Biocompatibility tests - Elution test L929 MEM - ISO 10993-5
The biocompatibility of the experimental product was tested in mouse cells L929 mouse fibroblasts. No biological reactivity (Grade 0) was observed in L929 cells at 48 hours, after subsequent exposure to the experimental product. The observed cellular response obtained from the positive reference product (Grade 4) and the negative reference product (Grade 0) confirmed the suitability of the test system. Based on the criteria of the protocol, the experimental product is considered nontoxic and meets the requirements of the elution test, the International Organization for Standardization (ISO) standards 10993-5. See Table I below.
Table I
GRADES OF REACTIVITY
 Weather  Experimental product References
 Half  Positive Negative
 TO  B C A B C A B C A B C
 0 hours  0 0 0 0 0 0 0 0 0 0 0 0
 24 hours  0 0 0 0 0 0 0 0 0 3 3 3
 48 hours  0 0 0 0 0 0 0 0 0 4 4 4
 Grade  Reactivity Description of the reactivity zone
 0  no discrete intracytoplasmic granules; no cell lysis
 one  slight Less than 20% of the cells are round, slightly joined and without intracytoplasmic granules; sometimes lysed cells are present
 2  weak Less than 50% of the cells are round and devoid of intracytoplasmic granules; reduced cell lysis and empty areas between cells
 3  moderate
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Four. Five
 4  strong Less than 70% of cell layers contain rounded cells or are lysed
 Almost complete destruction of cell layers
6.4. 3 Intramuscular implantation test - ISO - 4 weeks of implantation
6.4.2.1 Materials and Methods
To evaluate the potential of the experimental product to cause local toxic effects, the Intramuscular Implantation Test - ISO - 4 weeks of implantation ("intramuscular implantation test") was used. In summary, the experimental product was implanted in the paravertebral muscle tissue of New Zealand White rabbits for a period of 4 weeks. The experimental product was then evaluated separately using two reference products: Positive reference surgicel (Johnson and Johnson, NJ) and high reference negative density polyethylene (negative reference plastic).
Preparation of the test and reference products. The experimental product measured approximately 1 mm wide and 10 mm long. The two reference products were prepared. The positive reference, Surgicel (C1), measured approximately 1 mm wide by 10 mm long and received sterile. The negative reference plastic (C2), measured approximately 1 mm wide by 10 mm in length and was sterilized by immersing it in 70% ethanol.
Pre-dose procedure. Each animal was weighed before implantation. The day of the test, the hair was cut on the backs of the animals and the loose hair was removed to the ford. Each animal was properly anesthetized. Before implantation, the area was cleaned with a solution of surgical preparation.
Administration of the dose. Four strips of experimental product were surgically implanted in each of the rabbit's paravertebral muscles, approximately 2.5 cm from the midline and parallel to the spine and approximately 2.5 cm between sf. Strips of experimental product were implanted on one side of the spine. Similarly, strips of positive reference product (Surgicel) were implanted in the contralateral muscle of each animal. Two negative reference strips (negative reference plastic) were implanted in the caudal zone (towards the tail) to the experimental product and to the reference C1 implant sites on each side of the spine (total of four strips). A total of at least eight strips of experimental product and eight strips of each reference product are required for evaluation.
Procedures after the dose. The animals were kept for a period of 4 weeks. The animals were observed daily during this period to ensure adequate healing of the implant sites and the toxic signs of toxicity. The observations include all the weather manifestations. At the end of the observation period, the animals were weighed. Each animal was sacrificed with an injectable barbiturate. Enough time was allowed for the tissue to be cut without bleeding.
Macroscopic observations. Paravertebral muscles in which they are implanted in the test or reference products were completely excised from each animal. Muscle tissue was removed by carefully cutting into sections around the implant sites with a scalpel and lifting the tissue. The tissues removed from implants were examined macroscopically, but without using excessive invasive procedures that could have altered the integrity of this tissue for histopathological evaluation. The tissues were placed in correctly labeled containers containing 10% neutral buffered formalin.
Histopathology ^ a. After formalin fixation, each of the implant sites was removed from the largest mass of tissue. The implant site, which contains the implanted material, was examined macroscopically. At each site the signs of inflammation, encapsulation, hemorrhage, necrosis and discoloration were examined using the following scale:
0 = Normal
1 = Small
2 = Moderate
3 = Strong
After macroscopic observation, the implant material was left in situ and a section of tissue containing the implant site was processed. Histological extensions of sections taken with hematoxylin and Toxikon eosin were prepared. Extensions were evaluated and scored by examination under an optical microscope.
Pathological evaluation of the effects of the implant. The following biological reaction categories were evaluated by microscopic observation for each area of the implant:
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1. inflammatory responses:
to. Polymorphonuclear leukocytes
b. Lymphocytes
C. Eosinophils
d. Plasma cells
and. Macrophages
F. Giant cells
g. Necrosis
h. Degeneration
2. Healing responses:
to. Fibrosis
b. Fatty infiltrate
Each response category was scored using the following scale:
0 = normal
0.5 = very light
1 = small
2 = moderate
3 = strong
The relative size of the affected area was scored by assessing the width of the area from the implant / tissue interface to the unaffected areas that have the characteristics of normal tissue and normal vascularization. The relative size of the affected area was scored using the following scale:
0 = 0 mm, no place
0.5 = up to 0.5 mm, very light
1 = 0.6 - 1.0 mm, small
2 = 1.1-2.0 mm, moderate
3 => 2.0 mm, marked
The intramuscular implantation test was performed based on the following references:
1. ISO 10993-6, 1994, Biological evaluation of medical devices - Part 6: Tests for local effects after implantation.
2. ISO 10993-12, 2002, Biological evaluation of sanitary devices - Part 12: Preparation of samples and reference materials.
3. ASTM F981-04 2004, Standard practice for the evaluation of the compatibility of biomaterials for surgical implants with respect to the effect of materials on muscles and bones.
4. F763-04 ASTM 2004, Standard practice for the short-term detection of implant materials.
5. ISO / IEC 17025, 2005, General Requirements for the competence of Testing and Calibration Laboratories.
The results of the intramuscular implantation test were evaluated based on the following criteria:
1. Calculated score: For each implanted site, a total score is determined. The average score of the test sites for each animal is compared with the average score of the reference sites for that animal. The average difference between the test and reference sites for all animals is calculated and the initial bioreactivity score is assigned as follows:
0-1.5 No reaction *
> 1.5-3.5 weak reaction
> 3.5-6.0 moderate reaction
> 6.0 strong reaction
5 * A negative calculation is presented as zero (0).
2. Modification of the punctuation: The observer of the pathology summarizes the level of bioreactivity calculated. Based on the observation of all the factors (e.g., relative size, response model, inflammatory versus resolution), the pathology observer can review the bioreactivity score. The justification for the modification to the punctuation is presented in the narrative report (A descriptive narrative report on the biocompatibility of the test material is provided by the pathology observer).
6.4.2.2 Results
The results indicated that the experimental product was not reactive when implanted for 4 weeks (Bioreactivity score of 0.2) compared to the positive reference Surgicel; and non-reactive (bioreactivity score of 0.0) compared to the negative reference of high density polyethylene (15 reference negative plastic).
Weather observation. Table II below shows the results of the macroscopic evaluation of the experimental product and the reference implant sites did not indicate significant signs of inflammation, encapsulation, hemorrhage, necrosis or discoloration in the 4-week period. Some test sites and the positive reference mayone, Surgicel, were not observed macroscopically and serial sections underwent microscopic evaluation.
 Tissue site  T1 T2 T3 T4 Test average C1-1 C1-2 C1-3 C1-4 Reference C1 average C2-1 C2-2 C2-3 C2-4 Reference C2 average
 Inflammation  0 NSF 0 NSF 0 NSF NSF NSF NSF N / A 0 0 0 0 0
 Encapsulation  0 NSF 0 NSF 0 NSF NSF NSF NSF N / A 0 0 0 0 0
 Hemorrhage  0 NSF 0 NSF 0 NSF NSF NSF NSF N / A 0 0 0 0 0
 Necrosis  0 NSF 0 NSF 0 NSF NSF NSF NSF N / A 0 0 0 0 0
 Decoloration  0 NSF 0 NSF 0 NSF NSF NSF NSF N / A 0 0 0 0 0
 Total  0 N / A 0 N / A N / A N / A N / A N / A 0 0 0 0
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Animal no .: 60961
 Tissue site  T1 T2 T3 T4 Test average C1-1 C1-2 C1-3 C1-4 Reference C1 average C2-1 C2-2 C2-3 C2-4 Reference C2 average
 Inflammation  NSF NSF NSF NSF N / A NSF NSF NSF NSF N / A 0 0 NSF 0 0
 Encapsulation  NSF NSF NSF NSF N / A NSF NSF NSF NSF N / A 0 0 NSF 0 0
 Hemorrhage  NSF NSF NSF NSF N / A NSF NSF NSF NSF N / A 0 0 NSF 0 0
 Necrosis  NSF NSF NSF NSF N / A NSF NSF NSF NSF N / A 0 0 NSF 0 0
 Discoloration  NSF NSF NSF NSF N / A NSF NSF NSF NSF N / A 0 0 NSF 0 0
 Total  N / A N / A N / A N / A N / A N / A N / A N / A 0 0 N / A 0
Animal no .: 60968
 Tissue site  T1 T2 T3 T4 Test average C1-1 C1-2 C1-3 C1-4 Reference C1 average C2-1 C2-2 C2-3 C2-4 Reference C2 average
 Inflammation  NSF NSF NSF NSF N / A NSF NSF NSF NSF N / A 0 0 0 0 0
 Encapsulation  NSF NSF NSF NSF N / A NSF NSF NSF NSF N / A 0 0 0 0 0
 Hemorrhage  NSF NSF NSF NSF N / A NSF NSF NSF NSF N / A 0 0 0 0 0
 Necrosis  NSF NSF NSF NSF N / A NSF NSF NSF NSF N / A 0 0 0 0 0
 Discoloration  NSF NSF NSF NSF N / A NSF NSF NSF NSF N / A 0 0 0 0 0
 Total  N / A N / A N / A N / A N / A N / A N / A N / A 0 0 0 0
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T = test zone (representative sections were submitted for microscopic evaluation)
C1 = Surgicel (Due to the nature of the material, representative sections were submitted for microscopic evaluation)
C2 = High density negative reference polyethylene (Negative reference plastic)
15 Score scale
0 = no reaction 2 = moderate reaction NSF = no site found
1 = weak reaction 3 = strong reaction N / A = not applicable
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Observations of the implantation site (microscopic). Table III below shows the results of the microscopic evaluation of the indicated implant sites of the experimental product without significant signs of inflammation, fibrosis, hemorrhage, necrosis or degeneration compared to each of the reference product sites. The bioreactivity score for the 4-week period (average of three animals) was 0.2, (C1 - Surgicel) and 0.0 (C2 - Negative reference plastic) indicating that there is no reaction compared to any of Reference implant sites. The pathologist noted that there was a moderate polymorphic and histiocytic infiltrate (macrophages) throughout the experimental product in situ that was not unexpected given the nature of the test material.
Table III
Macroscopic observations Implantation in 4 weeks
Animal no .: 60959
 Categories  Test sites ** Reference sites
 Reaction  T1 T2 T3 C1-1 C1-2 C1-3 C1-4 C2-1 C2-2 C2-3 C2-4
 Strange remains  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 Relative size of the affected area  0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
 * Polymorphs  0.5 0.5 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Lymphocytes  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Eosinophils  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Plasmocytes  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Macrophages  0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
 * Giant cells  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Degeneration  0.5 0.5 0.5 0.5 0.0 0.5 0.0 0.0 0.0 0.0 0.0
 * Necrosis  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Fibrosis  0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
 * Fatty infiltrate  0.0 0.0 0.5 0.0 0.5 0.0 0.5 0.5 0.5 0.0 0.5
 Total  1.5 2.0 2.5 1.5 1.5 1.5 1.5 1.5 1.5 1.0 1.5
T = Test zone C1 = Surgicel
C2 = High density negative reference polyethylene (Negative reference plastic) Score of the test animal (Average *) = 2.0 Score of the animal with C1 (Average *) = 1.5 Score of the animal with C2 (Average * ) = 1.4
Animal score (Average test score - Average score of C1) = 0.5 Animal score (Average test score - Average score of C2) = 0.6 * Used in the calculation of bioactivity score.
** No site found in T4.
 Categories  Test sites ** Reference sites **
 Reaction  T1 T2 T3 C1-1 C1-3 C1-4 C2-1 C2-2 C2-3
 Strange remains  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 Relative size of the affected area  0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
 * Polymorphs  0.0 0.0 0.5 0.5 0.0 0.5 0.5 0.5 0.5
 * Lymphocytes  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Eosinophils  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Plasmocytes  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Macrophages  0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
 * Giant cells  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Degeneration  0.5 0.5 0.5 0.5 0.5 0.0 0.0 0.0 0.0
 * Necrosis  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Fibrosis  0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
 * Fatty infiltrate  0.0 0.5 0.0 0.5 0.0 0.5 0.5 0.5 0.5
 Total  1.5 2.0 2.0 2.5 1.5 2.5 2.5 2.5 2.5
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T = Test zone C1 = Surgicel
C2 = High density negative reference polyethylene (Negative reference plastic) Test animal score (Average *) = 1.8 10 Animal score with C1 (Average *) = 2.2 Animal score with C2 (Average *) = 2.5
Animal score (Average test score - Average C1 score) = -0.4 Animal score (Average test score - Average C2 score) = -0.7 * Used in bioreactivity score calculation .
15 ** No sites found in T2, C1-2 or C2-4.
 Categories  Test sites ** Reference sites **
 Reaction  T1 T2 T3 T4 C1-1 C1-2 C1-3 C2-1 C2-2 C2-3 C2-4
 Strange remains  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 Relative size of the affected area  0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
 * Polymorphs  0.0 0.5 0.0 0.5 0.0 0.0 0.0 0.5 0.5 0.0 0.5
 * Lymphocytes  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Eosinophils  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Plasmocytes  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Macrophages  0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
 * Giant cells  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Degeneration  0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
 * Necrosis  0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
 * Fibrosis  0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
 * Fatty infiltrate  0.5 0.5 0.5 0.5 0.5 0.0 0.5 0.5 0.5 0.5 0.5
 Total  2.0 2.5 2.0 2.5 2.0 1.5 2.0 2.5 2.5 2.0 2.5
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C2 = High density negative reference polyethylene (Negative reference plastic) Score of the test animal (Average *) = 2.3 10 Score of the animal with C1 (Average *) = 1.8 Score of the animal with C2 (Average *) = 2.4
Score of the animal (Average score of the test - Average score of C1) = 0.5 Score of the animal (Average score of the test - Average score of C2) = -0.1 * Used in the calculation of bioreactivity score.
15 ** No sites found in C1-4.
C1 C2
Animal score 60759 = 0.5 0.6
Animal score 60961 = -0.4 -0.7 Animal score 60968 = 0.5 -0.1
20 Bioreactivity score = 0.2 = no reaction Bioreactivity score = -0.1 = no reaction
6.4.4 Intracutaneous Injection Test - ISO 10993-10
The potential of sodium chloride (NaCl) for 0.9% USP injectables and cottonseed oil (ASA) extracts of the experimental product to produce irritation after intradermal injection into New Zealand White rabbits was evaluated. The experimental product sites did not show a significantly greater biological reaction than the sites injected with the reference product. Based on the protocol criteria, the experimental product is considered an insignificant irritant and meets the requirements of ISO 10993-10. The results are shown below in Table IV.
Table IV
Skin reaction scores of the intradermal test 10 Extract in NaCl
 Animal n °  Vehicle Time Site punctuation figures (ER / ED)
 T-1 T-2 T-3 T-4 T-5 C-1 C-2 C-3 C-4 C-5
 0 hours + 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 61917  NaCl 24 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 48 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 72 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 0 hours + 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 61919  NaCl 24 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 48 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 72 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 Total  0.0 0.0
+ = Immediately after the injection, not used for the evaluation criteria.
Average total score * for the experimental product = 0.0 Total average score * for the reference product = 0.0
Difference between the average total score of the experimental product and the reference product = 0.0 - 0.0 = 0.0
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 Animal n °  Vetnculo Time Site punctuation figures (ER / ED)
 T-1 T-2 T-3 T-4 T-5 C-1 C-2 C-3 C-4 C-5
 0 hours + 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 61917  ASA 24 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 48 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 72 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 0 hours + 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 61919  ASA 24 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 48 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 72 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
 Total  0.0 0.0
+ = Immediately after the injection, not used for the evaluation criteria. Average total score * for the experimental product = 0.0 Total average score * for the reference product = 0.0
5 Difference between the total average score of the experimental product and the reference product = 0.0 - 0.0 = 0.0 ER = Erythema; ED = Edema; T = Test sites; C = Reference sites * Total mean score = Total erythema scores plus edema divided by 12
(2 animals x 3 punctuation periods x 2 punctuation categories)
6.4.5 Kligman maximization test - ISO 10993-10
10 Sodium chloride (NaCl) for USP 0.9% injectables and cottonseed oil (ASA) extracts from the experimental product did not cause any intradermal reaction in Hartley guinea pigs in the challenge test (0% sensitization), after a provocation phase. Therefore, as defined in the Kligman scoring system, this is a grade I reaction and the experimental product is classified as having weak allergenic potential. Based on the criteria of the protocol, a degree I sensitization rate is not considered significant and the experimental product meets the requirements of ISO 10993-10. The results are shown below in Table V.
Skin Exam Data
 Group  Animal n ° Sex 03 or LO CM Scores 26 ° dfa 27 ° d Percentage of sensitized animals Allergenic potential
 1 Male 0 0 0
 2 Male 0 0 0
 3 Male 0 0 0
 4 Male 0 0 0
 Product  5 Male 0 0 0 0% Weak
 experimental  6 Female 0 0 0
 (NaCl Extract)  7 Female 0 0 0
 8 Female 0 0 0
 9 Female 0 0 0
 10 Female 0 0 0
 11 Male 0 0 0
 12 Male 0 0 0
 13 Male 0 0 0
 Product  14 Male 0 0 0
 experimental  15 Male 0 0 0 0% Weak
 (ASA Extract)  16 Female 0 0 0
 17 Female 0 0 0
 18 Female 0 0 0
 19 Female 0 0 0
 20 Female 0 0 0
 21 Male 0 0 0
 22 Male 0 0 0
 Reference  23 Female 0 0 0 0% Weak
 negative (NaCl)  24 Female 0 0 0
 25 Female 0 0 0
 26 Male 0 0 0
 27 Male 0 0 0
 Reference  28 Female 0 0 0 0% Weak
 negative (ASA)  29 Female 0 0 0
 30 Female 0 0 0
 31 Male 2 1 0
 Reference  32 Male 2 2 1
 positive (DNCB)  33 Female 3 2 1 100% Extreme
 34 Female 3 2 1
 35 Female 3 3 2
 Sensitization rate  Class Grade
 0-8  I weak
 9-28  Mild II
 29-64  III Moderate
 65-80  IV Strong
 81-100  V Extreme
5 The test results are interpreted based on the percentage of sensitization observed.

权利要求:
Claims (29)
[1]
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
1. A poly-p-1-4-N-acetylglucosamine composition comprising poly-p-1-4-N-acetylglucosamine fibers, wherein (i) the mayone of the irradiated fibers have less than about 15 pm of length, and (ii) composition (a) increases the metabolic rate of serum-deprived human umbilical cord vein endothelial cells in an MTT analysis and / or non-pound apoptosis to endothelial cord vein cells human umbilical deprived of serum in a trypan blue exclusion test, and (b) is not reactive when tested in an intramuscular implantation test
[2]
2. The composition according to claim 1, wherein the mayone of the fibers has a thickness or diameter of 1-2 pm.
[3]
3. The composition according to claim 1, wherein the mayone of the fibers is less than about 10 pm in length.
[4]
4. The composition according to claim 1, wherein at least 50% of the fibers are approximately 4 pm in length.
[5]
5. The composition according to claim 1 or 2, wherein the mayone of the fibers is between approximately 2 and 15 pm in length.
[6]
6. The composition according to any one of claims 1 to 5, wherein at least 70% of N-acetylglucosamine monosaccharides of the poly-p-1-4-N-acetylglucosamine are acetylated.
[7]
7. The composition according to any one of claims 1 to 5, wherein 100% of N-acetylglucosamine monosaccharides of the poly-p-1-4-N-acetylglucosamine are acetylated.
[8]
8. A method for producing a poly-p-1-4-N-acetylglucosamine composition, said method comprises irradiation of poly-p-1-4-N-acetylglucosamine fibers, such that (i) the mayone of the irradiated fibers are less than about 15 pm in length, and (ii) the composition (a) increases the metabolic rate of endothelial cells of the human umbilical cord vein deprived of serum in an MTT analysis and / or not free of endothelial cell apoptosis of the human umbilical cord vein deprived of serum in a trypan blue exclusion test, and (b) is not reactive when tested in an intramuscular implantation test, thereby producing the composition of poly-p-1-4-N-acetylglucosamine.
[9]
9. The method according to claim 8, wherein the poly-p-1-4-N-acetylglucosamine fibers are irradiated as dry fibers, a dry fiber membrane or a dry lyophilized material.
[10]
10. The method according to claim 9, wherein the poly-p-1-4-N-acetylglucosamine fibers are irradiated by gamma irradiation at 500-2,000 kgy.
[11]
11. The method according to claim 8, wherein the poly-p-1-4-N-acetylglucosamine fibers are formulated as a suspension, a slurry or a wet cake for irradiation.
[12]
12. The method according to claim 11, wherein the poly-p-1-4-N-acetylglucosamine fibers are irradiated by gamma irradiation at 100-500 kgy.
[13]
13. A poly-p-1-4-N-acetylglucosamine composition that can be obtained by the process according to one of claims 8 to 12.
[14]
14. A composition of poly-p-1-4-N-acetylglucosamine as defined in any one of claims 1 to 7 or 13 for use in a method for treating a wound in a human being, wherein The method comprises the stage of preparing a bandage to be applied topically to a wound in a human being in need.
[15]
15. The composition for use according to claim 14, wherein the method comprises the step of preparing a bandage for repeated application every 5 to 35 days.
[16]
16. A composition of poly-p-1-4-N-acetylglucosamine as defined in any one of claims 1 to 7 or 13 for use in a method for treating a wound in a human being, wherein The method comprises the topical application of a bandage comprising said poly-p-1 - »4- N-acetylglucosamine composition to a wound in a human being in need and repeating the application every 5 to 35 days.
[17]
17. The composition for use according to claims 14 to 16, wherein the human being is a diabetic, a smoker, a hemofflic, a person infected with HIV, an obese person, a person undergoing radiotherapy or a person with ulcer venous stasis.
[18]
18. The composition for use according to claims 14 to 16, wherein the human being is a person with venous ulcer by stasis.
5
10
fifteen
twenty
25
[19]
19. The composition for use according to claims 14 to 18, wherein the wound is a chronic wound, a surgical wound or a burn wound.
[20]
20. The composition for use according to claim 19, wherein the chronic wound is a diabetic ulcer, a venous ulcer for stasis, an ulcer for arterial insufficiency or a pressure ulcer.
[21]
21. The composition for use according to claims 14 to 20, wherein the bandage is prepared to be removed before repeated application.
[22]
22. The composition for use according to claims 14 to 20, wherein the bandage is prepared so as not to be removed before repeated application.
[23]
23. The composition for use according to claims 14 to 22, wherein the poly-p-1 ^ 4-N-acetylglucosamine is a microalgae poly-p-1 ^ 4-N-acetylglucosamine or is not a poly-p -1 ^ 4-N-acetylglucosamine crustacean.
[24]
24. The composition for use according to claims 14 to 22, wherein at least 75% of the bandage consists of poly-p-1 ^ 4-N-acetylglucosamine.
[25]
25. A bandage comprising the composition according to any one of claims 1 to 7 or 13.
[26]
26. The composition according to claim 13, or the method according to claims 8 to 12, wherein the mayone of the fibers is between approximately 2 to 15 pm in length.
[27]
27. The composition according to any one of claims 1 to 7, 13 or 26, or the bandage according to claim 25, or the method according to any of claims 8 to 12 or 26, wherein the length of the fibers is determined by scanning electron microscopy (SEM).
[28]
28. The composition according to any one of claims 1 to 7, 13, 26 or 27, or the bandage according to the
claim 25 or 27, or the method according to any of claims 8 to 12, 26 or 27, wherein the spectrum
Infrared of the irradiated poly-p-1-4-N-acetylglucosamine fibers is substantially similar or equivalent to that of the non-irradiated poly-p-1-4-N-acetylglucosamine fibers.
[29]
29. The composition according to any one of claims 1 to 7, 13, 26 or 28, or the bandage according to the
claim 25, 27 or 28, or the method according to any of claims 8 to 12, or 26 to 28, wherein the
Intramuscular implantation test is a 4-week implantation in a rabbit's paravertebral muscle tissue based on the International Organization for Standardization of the Guidelines.

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US90182607P| true| 2007-02-19|2007-02-19|

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US901826P|2007-02-19|

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PCT/US2008/002172|WO2008103345A2|2007-02-19|2008-02-19|Hemostatic compositions and therapeutic regimens|

---