HK1240851B - Compositions for treating wounds - Google Patents

Description

Composition for treating wounds

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional patent application No. 62/063,793, filed on day 10, month 14, 2014, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to compositions and methods for treating wounds, particularly difficult to heal wounds such as leg ulcers, venous stasis ulcers, pressure ulcers, severe burns and large surgical wounds such as abdominoplasty and other types of surgical tissue flaps in diabetic patients.

Background

Diabetes is a worldwide epidemic. About 3.7 million people are reported to have diabetes and this number is increasing in every country. One of the most common and serious complications caused by diabetes is poor wound healing that most often occurs in high pressure areas of the foot surface such as under the big toe, the metatarsophalangeal joints, the top and ends of the toes, the middle and sides of the foot, and on the heel. Foot ulcers result from nerve damage that causes loss of sensation at such pressure points on the foot, resulting in extensive microtraumas, rupture of the overlying tissue, and eventual ulceration. In addition, this loss of sensation can result in minor abrasions or cuts that are not properly treated and ultimately lead to the formation of ulcers. A large percentage of diabetics will develop foot ulcers during their lifetime. Once a Diabetic Foot Ulcer (DFU) is formed, treatment can be difficult, particularly in view of the impaired healing environment due to the presence of neuropathy, vascular disease, altered neutrophil function, reduced tissue perfusion, and/or defective protein synthesis, all of which are often accompanied by diabetes.

There is a need for better treatment regimens for the formation of these chronic ulcers. DFU is the primary cause of amputation. The longer these wounds are maintained, the greater their size and depth and the chance of infection. As a result, these complications result in 80,000 amputations per year in the united states alone. This chronic pathology also seriously impairs the overall health of the patients, leading to further drastic deterioration of the health of these patients and additional costs for the healthcare system; its treatment doubles the medical care costs of affected diabetics.

The primary goal of DFU management is wound closure. Under current standards of care, DFU wound care focuses on thorough and repeated debridement, frequent inspection and bacterial control, relieving any pressure on the wound, and carefully controlling moisture balance to prevent maceration. However, effective DFU healing has not been consistently achieved by this approach, and the results can be heavily dependent on patient compliance. Therefore, adjuvant therapies have been developed to address DFU. Consensus reports on diabetic foot ulcer management suggest that for ulcers that show less than 50% healing at 4 weeks after good standard wound care, advanced treatments should be considered in order to accelerate wound healing and reduce complications. Such advanced treatments include negative pressure wound therapy, biological dressings, bioengineered skin equivalents, hyperbaric oxygen therapy, platelet rich plasma and growth factors.

However, only a few of these advanced wound care products have been demonstrated to accelerate DFU healing in prospective randomized enrollment trials, and some of their results have even been questioned from other studies. Products that have been studied in prospective randomized enrollment trials include becaplamine (b.c.)Smith and Nephew), a topical gel containing recombinant human platelet-derived growth factor B chain homodimer (rhPDGF-BB), biochoperone PDGF-BB (Adocia, Lyon, France), a topical spray containing molecules complexed with PDGF, two live skin equivalents: double-layer skin substitute (Organogenesis, inc., Canton, MA) and human fibroblast-derived dermal substitute(s) ((r)Shire, Pic, Dublin, Ireland), and vacuum assisted wound closure (KCI, San Antonio, TX). Other treatment modalities with less rigorous experimental data include collagen, platelet rich plasma, silver products, hyperbaric oxygen and electrical stimulation.

While some favorable results were obtained from prospective randomized registration trials for certain advanced wound care products, their overall benefits were disappointing as evidenced by the sustained high amputation rates. The following results are reported in the published meta-analysis of 35 randomized, controlled trials evaluating advanced treatment of diabetic foot ulcers:

platelet rich plasma did not improve diabetic ulcer healing compared to good standard wound care.

In the combined results from the three studies,biological skin equivalents showed insignificant improvement compared to standard care, with Dermagraft more favorable for ulcer healing (35% versus 24%).

It is reported that,the biological skin equivalent bilayers improved healing better than good standard wound care (55% versus 34%, p.001; 2 studies).

·5，rhPDGF-BB showed a percent improvement in healed ulcers compared to placebo or good standard wound care (58% versus 37%, p ═ 0.04; 7 studies).

·Negative pressure wound therapy improves healing more than good standard wound care (43% versus 29%, p)<0.05; 1 study).

For all studies, there was little or insufficient evidence associated with prolonged ulcer healing time.

Furthermore, in four studies, the incidence of complete wound closure was reported to be 50% or less (48%, 50%, 44% and 36%) for Regranex.

Such advanced therapies have not resulted in a consistently effective solution for treating DFU. Given their anxious clinical results and higher product costs compared to standard therapies, none of these advanced therapies have been widely used as a new standard of care for the treatment of DFU.

As noted above, one such advanced therapy is Regranex gel (Becaplerman), which consists of rhPDGF-BB at a concentration of 100 μ g/g in a sodium carboxymethyl cellulose gel. In particular, Regranex is formulated for multi-useNon-sterile, low bioburden, preserved sodium carboxymethylcellulose (CMC) -based topical gels and indicated for daily application to improve healing of chronic DFU over months. Regranex package insert (label) statement that it should be applied daily for up to 140 times a day over a 20 week period and if deemed appropriate by the physician to employ a dosage equal to about 0.006mg/cm²(6.25 μ g) of wound surface area, then longer.

Regranex remains the most advanced growth factor therapy for wound healing, which is evidenced by the following facts: although it was approved by the FDA 15 years ago, it is the only recombinant growth factor product approved by the FDA for the treatment of chronic wounds. In addition, since FDA approval, no one has successfully developed another formulation of Regranex (i.e., rhPDGF-BB). Although clinical and non-clinical data support its clinical applications, we consider Regranex to have a number of limitations, including: 1) daily application to the DFU by the patient is required, which requires the patient to change the wound dressing daily; 2) the dosage specified in the FDA approved instructions is low, about 0.006mg (6. mu.g)/cm²Wound surface area; 3) often inaccurate dosing results from the difficulty experienced by the patient visually inspecting and applying the gel from a tube (similar to a toothpaste tube) to a wound often located on the sole of the foot; 4) it is desirable to keep the product refrigerated (about 2-8 ℃); 5) regranex gel is not sterile; 6) patients require extended use-up to and possibly beyond about five months, 140 daily applications, and 7) the use of carboxymethyl cellulose (CMC) based topical gels, which lack the ability to provide a biological matrix for cell ingrowth.

Furthermore, Regranex is only limitedly recognized by the medical community as an effective treatment for DFU. After review of data from four clinical trials of regranox efficacy by the European Medicines Agency (EMA), the EMA concluded the following: the efficacy of the 30 μ g PDGF/g formulation was lower than that of the 100 μ g PDGF/g formulation, and the difference between the 100 μ g PDGF/g formulation and the 300 μ g PDGF/g formulation was small. EMA further concludes: the 100 μ g PDGF/g product formulation had only "limited" efficacy.

The effectiveness of the active ingredient in Regranex (i.e., rhPDGF-BB) in treating wounds has been questioned, perhaps due to the "limited" efficacy of Regranex. Park SA, Raghunnathan VK, Shah NM, Teixeira L, Motta MJ et al (2014) PDGF-BB Does Not Accelerate health in diabetes Mice with Splined Skin Wounds, PLoS ONE 9(8) e104447.doi:10.1371/journal. po. 0104447 reported results from studies using a controlled full thickness splint fixation in db/db Mice (type 2 diabetes mouse model). Two splint-fixed 8mm dorsal full-thickness wounds were introduced in db/db mice and treated topically once daily for 10 days with 30 μ l of 3 μ g PDGF-BB in a 5% PEG-PBS vehicle or an equal volume of vehicle. The study concluded the following: even if bioactive in vitro, PDGF-BB fails to accelerate wound healing in db/db mice using a splint-fixed wound model.

Although experts in the field question the effectiveness of the active ingredient rhPDGF-BB of Regranex, applicants believe that there are many reasons for the questionable efficacy of Regranex. First, Regranex is delivered to the wound site via a gel carrier. The formulation allows rhPDGF clearance from the site in minutes to hours. Secondly, although the gel carrier is biocompatible, it is believed that it does not provide a substrate for cellular and vascular ingrowth and may in fact be inhibitory to cellular growth and migration in the wound, potentially slowing the healing process and leading to poor healing. Third, reganex is non-sterile, stable only when stored at 2-8 degrees celsius (refrigerated), and must be applied daily to often inaccessible anatomical sites, all resulting in poor patient compliance; fourth, while clinical data show no difference between the 100 μ g/g formulation and the 300 μ g/g formulation, applicants believe that the concentration of growth factor in Regranex is too low for optimal cell recruitment and proliferation. The Regranex dose per square centimeter of wound surface area is only 6 μ g, and applicants believe the dose is too low for optimal cell recruitment and proliferation. Fifth and finally, despite its commercial application to patients over the last 15 years, the applicant believes that the growth factors contained in Regranex are not completely effective. The rhPDGF used in Regranex is recombinantly produced in yeast expression systems. When expressed in yeast, the protein is exported as a fully folded homodimeric protein, consisting of two antiparallel B chains held together by two interchain disulfide bonds. However, during fermentation, internal proteolysis (cleavage between residues Arg32 and Thr 33) and C-terminal truncation (Arg32 and Thr109) may occur. Internal proteolysis produces three possible forms of rhPDGF-BB: intact (both B strands are intact), single-stranded sheared (one B strand is sheared), and double-stranded sheared (both B strands are sheared). Cleavage also creates a new C-terminal site for further C-terminal truncation, and results in a very complex mixture of isoforms. Applicants believe that the non-intact isoforms of rhPDGF-BB included in Regranex are far less effective in treating DFU than the fully intact isoforms.

rhPDGF-BB is also used in orthopedic and periodontal indications where the healing environment and healing process are quite different from dermal wounds. Two such products include augmentation Bone Graft and GEM21S, both of which contain rhPDGF-BB as an active ingredient. GEM21S consists of a rhPDGF-BB solution and a synthetic particulate bone substitute, which was FDA approved in 2005 and its indication was to improve bone healing in chronic periodontal defects. The augmentation Bone Graft also consisted of rhPDGF-BB solution and synthetic particulate Bone substitute. Augment was FDA approved for improvement of foot and ankle fusion following a single implantation into a bone defect during surgery based on 434 patient critical clinical trials in the united states and canada. However, none of these products list dermal wounds, such as those of DFU as indications, and both products are intended to promote bone growth and fusion, cellular and physiological processes that are quite different from skin wound healing, using the product by a single intra-operative application. As with Regranex, GEM21S and the augmentation Bone Graft products must be stored refrigerated (about 2-8℃.), which detracts from user convenience and compliance.

In summary, poor patient outcomes leading to high amputation rates and conflicting scientific analyses indicate that there remains a need for more predictable, patient/user-friendly, and consistently effective methods and therapeutic compositions for promoting dermal wound healing, including treatment of DFU and other types of difficult to heal wounds.

Disclosure of Invention

The invention embodiments provided in the present disclosure are intended to be illustrative only and are intended to provide an overview of selected embodiments disclosed herein. The disclosure of the present invention, which is illustrative and optional, does not limit the scope of any claims, does not provide the full scope of the invention embodiments disclosed or encompassed herein, and should not be construed as limiting or restricting the scope of the disclosure or any claimed invention embodiments.

The present invention provides methods and compositions for treating or promoting healing of wounds, such as leg ulcers, venous stasis ulcers, pressure ulcers, severe burns, traumatic injuries, and large surgical wounds such as abdominoplasty and other types of surgical tissue flaps in diabetic patients. In some embodiments, the wound may extend into or deeper into the subcutaneous tissue, or the wound may be a diabetic foot ulcer.

Provided herein are improved formulations of rhPDGF-BB that simultaneously comprise a combination of the following improvements and benefits: 1) a carrier that facilitates maintenance of an effective dose of PDGF at the wound site for an extended period of time; 2) a carrier providing a substrate for cell attachment and vascular ingrowth; 3) sterile and therefore safer; 4) no refrigeration is necessary and therefore the patient is safer and easier to handle; 5) the frequency of application is lower than current therapy, preferably about once every week, which facilitates better patient compliance and ease of use; 6) having rhPDGF present at a concentration higher than existing formulations; and 7) contains a purer and more potent rhPDGF-BB preparation with fewer isoforms than some of the existing preparations. In certain embodiments of the invention, all of the above improvements and benefits are achieved simultaneously.

Provided herein are methods of treating a wound comprising applying a therapeutic composition to a wound surface, monitoring healing of the wound, and, if deemed necessary, periodically reapplying the therapeutic composition to the wound surface to effect healing. In some embodiments, the method further comprises debriding the wound to remove necrotic or infected tissue prior to applying the therapeutic composition, and covering the wound with a dressing after applying the therapeutic composition. In certain embodiments, the semi-occlusive or occlusive dressing and the dressing can be replaced periodically, such as with each reapplication of the therapeutic composition. The method of the present invention may also include the step of cleaning the wound with saline or a suitable antimicrobial wound cleanser and/or debridement chemical at the time of dressing change. The methods provided herein can also include treating a patient in the form of infection control or negative pressure wound therapy.

In some embodiments, the method further comprises forming a therapeutic composition by combining sterile PDGF with a sterile biocompatible matrix. The sterile PDGF may be a pre-formulated sterile PDGF solution, or it may be formed as part of a therapeutic procedure by reconstituting a lyophilized sterile powder containing PDGF with sterile water or a buffer solution. In some embodiments, the biocompatible matrix is a sterile porous matrix and may be selected from natural polymers such as collagen, gelatin, fibrin, alginate, cellulose, or fibronectin. Alternatively, the biocompatible matrix is a sterile porous matrix selected from synthetic polymers such as poly (DL-lactide-co-glycolide) (PLGA), poly (DL-lactide) (PDLA), poly (L-lactide) (PLLA), poly (e-caprolactone) (PCL), polyurethane or others. In some embodiments, the biocompatible matrix is a collagen sponge or a mixture of natural and synthetic polymers.

In some embodiments, the therapeutic composition is formed directly on the wound surface by first applying a matrix, such as a collagen sponge, to the wound surface and then applying the PDGF solution to the collagen sponge, or by first applying the PDGF solution to the wound surface and then applying a matrix, such as a collagen sponge, to the wound surface. In some embodiments, the therapeutic composition is formed by first reconstituting a lyophilized sterile powder comprising PDGF with sterile water or a buffer solution to form a sterile PDGF solution, and then aseptically adding the sterile PDGF solution to a sterile porous biocompatible matrix, such as a collagen sponge, such that the matrix is wetted with the PDGF solution.

According to the inventionIn another aspect, provided herein is a method of treating a dermal wound, comprising: debriding the wound; applying a therapeutic composition comprising recombinant platelet-derived growth factor BB (rhPDGF-BB) to a wound approximately once every 3 to 42 days for a treatment period of about 2 weeks to about 20 weeks, and wherein the first dose comprises at least about 10 μ g of rhPDGF/cm²Wound surface area; and covering the wound with a dressing after each application of the therapeutic composition. In some embodiments, the method may further comprise advising the patient to avoid applying pressure on the wound as it heals. In some embodiments, the cumulative total amount of rhPDGF-BB applied to the wound over the treatment period is less than about 25mg, or about 10mg, or about 5mg, or about 4mg, or about 3mg, or about 2mg, or about 1mg of rhPDGF-BB. In some embodiments, the method comprises applying the therapeutic composition to the wound once every 7 to 28 days, or once every 7 to 21 days, or once every 10 to 15 days, or once every 12 to 14 days. In some embodiments, each treatment comprises administering at least about 10 μ g of rhPDGF/cm²Wound surface area, or about 10 μ g of rhPDGF/cm²Wound surface area to about 5,000 μ g of rhPDGF/cm²Wound surface area.

The invention also provides a therapeutic composition comprising sterile PDGF and a biocompatible matrix that can be sterile and/or porous. In some embodiments, the sterile PDGF comprises a pre-formulated sterile PDGF solution, and in other embodiments, the sterile PDGF comprises a lyophilized sterile powder containing PDGF reconstituted with sterile water or a buffer solution.

In certain embodiments, the sterile PDGF included in the therapeutic compositions of the invention comprises a rhPDGF-BB solution containing about 0.05mg/ml to about 5mg/ml rhPDGF-BB. The rhPDGF-BB solution may be formed by combining a sterile powder containing lyophilized rhPDGF-BB with sterile water or saline to reconstitute the lyophilized rhPDGF-BB into a solution. In certain embodiments, the rhPDGF-BB can be produced by an e.coli (e.coli) expression system wherein at least about 80% of the rhPDGF-BB on a weight basis is undipped rhPDGF-BB, which can then be lyophilized. In certain embodiments, the lyophilized rhPDGF-BB is capable of being stored at about 20 ℃ to about 26 ℃ and still retaining at least 80% of the biological activity of the rhPDGF-BB for at least about six months or at least about one year, or is stored at about 16 ℃ to about 32 ℃ and still retaining at least 80% of the biological activity of the rhPDGF-BB for at least about six months or at least about one year.

In certain embodiments, the matrix comprised in the therapeutic composition of the invention may be selected from collagen, gelatin, fibrin, alginate, cellulose, chitosan (Chitan) or fibronectin. The matrix may provide a resorbable cell scaffold and may include a collagen sponge. In certain embodiments, the matrix has a pore size distribution of about 10 microns to about 2,000 microns, and/or an average pore size of about 50 microns to about 500 microns. In certain embodiments, a portion of the pores are interconnected or a majority of the pores are interconnected.

Also provided herein are therapeutic compositions comprising a rhPDGF-BB solution and a carrier, such as a matrix, wherein the ratio of rhPDGF-BB solution to matrix is about 4 μ l/cm³To about 40ml/cm³Alternatively, the ratio of rhPDGF-BB to matrix is about 1.2. mu.g PDGF/cm³Carrier to about 12mg PDGF/cm³And (3) a carrier. The rhPDGF-BB solutions disclosed herein may comprise about 0.05mg/ml to about 5mg/ml, or about 0.1mg/ml to about 1mg/ml, or about 0.2mg/ml to about 0.4mg/ml of rhPDGF-BB. The rhPDGF-BB solutions disclosed herein may comprise about 0.3mg/ml or about 0.5mg/ml or about 1.0mg/ml of rhPDGF-BB. In certain embodiments, at least about 80% or about 85% or about 90% or about 95% or about 97% of the rhPDGF-BB included in the PDGF solution or therapeutic composition is undipped rhPDGF-BB on a weight basis.

In certain aspects, a therapeutic composition is provided comprising a solution of rhPDGF-BB and a matrix, wherein at least about 20% of the rhPDGF-BB is entrapped within pores of the matrix such that when the composition is applied to a wound of a patient, the rhPDGF-BB is released over time as the matrix is absorbed by the patient's body. In certain embodiments, the therapeutic composition provides for the sustained delivery of rhPDGF-BB at the wound site with matrix resorption, and simultaneously provides a matrix for new cell and tissue ingrowth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials for use in the present disclosure are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Drawings

Figure 1 graphically shows the average wound area over time in three groups of animals included in the study, calculated by digital caliper measurements.

Figure 2 graphically illustrates the average wound area over time in three groups of animals included in the study, calculated using ImageJ software.

Figure 3 illustrates the wound area calculated using ImageJ software for each test animal on study day 0.

Figure 4 illustrates the wound area calculated using ImageJ software for each test animal on study day 7.

Figure 5 illustrates the wound area calculated using ImageJ software for each test animal on study day 14.

Figure 6 illustrates the wound area calculated using ImageJ software for each test animal on study day 21.

FIG. 7A is a graph showing the amount of wound healing (mm) at various time points for the Regranex group 1 and the rhPDGF/collagen group 3 using ImageJ software²) Cumulative number of doses.

Figure 7B illustrates the mean percentage of wound closure for each group over the treatment period.

Figure 8 includes a series of wound images showing the extent of healing of animals included in a study of the test compositions of the present invention over time.

Figure 9 includes a series of photomicrographs of a cross-section of the wound site from day 21 of three study animals.

Detailed Description

The present invention provides novel methods of treating dermal wounds such as Diabetic Foot Ulcers (DFUs), venous stasis ulcers, pressure ulcers, burns, traumatic injuries and major surgical wounds. The invention further provides novel bioactive therapeutic compositions for treating such wounds, novel methods of making bioactive dressings useful for treating wounds, and novel treatment regimens that improve patient compliance and wound healing.

The novel methods and therapeutic compositions according to the present invention will achieve equivalent or better efficacy in treating dermal wounds compared to existing products, and provide a better safety profile and improved patient compliance and convenience. The novel therapeutic compositions provided herein provide: 1) prolonged delivery of PDGF from each application to the wound, thereby eliminating the need for more frequent applications by the patient (e.g., daily or every other day of application of existing products); 2) physical materials such as collagen sponges, which can be applied to the wound once every several days as Band-Aid, improving patient compliance; 3) stability at room temperature, thereby eliminating the need for refrigeration of the product; 4) an aseptic product with improved safety compared to existing products; 5) a higher initial dose of PDGF compared to existing products, which better initiates the healing process thereby reducing the need for prolonged patient use; 6) the use of an improved carrier that not only maintains PDGF delivery but also simultaneously provides a biological scaffold and/or open porous matrix that promotes ingrowth of cells, blood vessels and new tissue, resulting in improved healing compared to existing products that lack the ability to provide a biological matrix for cell ingrowth; and 7) a purer and more potent rhPDGF-BB formulation containing fewer isoforms than the prior formulation. The novel process disclosed herein provides: 1) a higher initial dose of PDGF compared to existing products to better initiate the healing process, thereby reducing the need for prolonged use by the patient; and 2) treatment regimens that will promote improved patient compliance and convenience by reducing the number of regular applications of the therapeutic composition, perhaps as low as 1 to 6 applications compared to the 140 applications required by existing products.

I.Definition/naming

As used herein, open-ended terms such as "comprising," "including," "comprising," and the like, mean inclusion, unless otherwise specified.

Some embodiments herein relate to ranges of values. When a range of values is provided, unless otherwise stated, the range includes the endpoints of the range. Unless otherwise indicated, numerical ranges include all values and subranges therein as if such values and subranges were explicitly written out.

Some values herein are modified by the term "about". In some instances, the term "about" with respect to a reference value can include a range of values that is plus or minus 10% of the value. For example, an amount of "about 10" can include an amount of 9 to 11. In other embodiments, the term "about" with respect to a reference value can include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

As used herein, the articles "a" and "an" mean one or more unless explicitly stated otherwise.

Where methods and steps described herein indicate certain events occurring in a certain order, those of ordinary skill in the art will recognize that the ordering of certain steps may be changed, and that such changes are in accordance with the variations of the invention. Further, certain steps may be performed concurrently in a parallel process as well as performed sequentially when possible.

The abbreviations have the following meanings: "C" means celsius or celsius, from its usage, "μ L" or "uL" means microliter, "mL" means milliliter, "L" means liter, "mM" means millimeter, "nm" means nanometer, "mM" means millimolar concentration, "μ M" or "uM" means micromolar concentration, "M" means molar concentration, "mmol" means millimole, "μmol" or "uMol" means micromolar, "g" means gram, "μ g" or "ug" means microgram, "ng" means nanogram, "% w/v" means weight/volume percentage, "% v/v" means volume/volume percentage, "HPLC" means high performance liquid chromatography, "UPLC" means ultra performance liquid chromatography, and "GC" means gas chromatography.

The term "homology" refers to an optimal alignment of sequences (nucleotides or amino acids) which can be performed by computerized execution of an algorithm. For example, "homology" with respect to a polynucleotide can be determined by analysis using BLASTN version 2.0 with default parameters. "homology" with respect to polypeptides (i.e., amino acids) can be determined using default parameters using programs such as BLASTP version 2.2.2 that compare polypeptides or fragments (and may also compare nucleotide fragments) to be compared and determine the degree of amino acid identity or similarity between them.

The above description of sequence homology and methods are intended to be exemplary and it is recognized that this concept is well understood in the art. In addition, it is understood that the nucleic acid sequence may vary and still provide a functional enzyme, and such variations are within the scope of the present invention. The term "enzyme homologue" may also mean a functional variant.

As used herein, the term "carrier" is intended to broadly refer to any biocompatible substance that can serve as a delivery vehicle for PDGF, while the terms "matrix" and "scaffold" are used interchangeably to refer to a carrier that serves as a substrate for cell attachment and/or vascular ingrowth as the wound heals, and/or provides a means of capturing PDGF within its structure (e.g., through interconnected pores), thereby allowing for sustained or delayed or prolonged delivery of PDGF as the wound heals.

II.Novel method for treating wounds

The present invention provides a novel method of treating wounds. In one embodiment, a method of treating a wound comprises providing a therapeutic composition comprising a solution of PDGF incorporated in a biocompatible scaffold, matrix, or carrier, and applying the therapeutic composition to the wound. For example, a therapeutic composition comprising a PDGF solution incorporated into a biocompatible scaffold, matrix, or carrier can be applied topically to a wound. In some embodiments, the method of treating a wound comprises applying a therapeutic composition to the wound multiple times periodically over a period of weeks.

According to one aspect of the invention, a novel method of treatment for treating a wound comprises the steps of:

(1) debriding the wound as needed to remove necrotic or infected tissue;

(2) forming a therapeutic composition comprising sterile rhPDGF-BB and a sterile porous biocompatible carrier;

(3) applying a therapeutic composition comprising PDGF to the wound surface, wherein the carrier provides a substrate for cell attachment and vascular ingrowth as the wound heals;

(4) covering the wound with a dressing; and

(5) wound healing was monitored over the treatment period and steps (1) - (4) were repeated at treatment intervals of 3 or more days.

The novel method of treatment may further comprise preparing the novel therapeutic composition prior to applying the composition to the wound surface, wherein the composition comprises PDGF and a biomatrix. The method of making the composition may comprise:

(2a) reconstituting a lyophilized (freeze-dried) sterile PDGF powder with sterile water, saline, buffer or physiological solution to provide a specific safe and therapeutic concentration of PDGF; and

(1b) the sterile PDGF solution is removed from the vial (container) and aseptically added to the dry hydrophilic sterile matrix or patch so that the matrix or patch is wetted with the PDGF solution.

In some embodiments, the dressing is an occlusive or semi-occlusive dressing. In some embodiments, the repeating of steps (1) - (3) may further comprise the steps of: (A) removing the dressing and cleansing the wound with saline or a suitable antimicrobial wound cleanser prior to application of the therapeutic composition dressing, and (B) covering the wound with a new dressing after application of the therapeutic composition. In some embodiments, the novel bioactive therapeutic compositions described herein may be used in combination with other aspects of wound treatment including, for example, infection control, negative pressure wound therapy, and/or instructing a patient to avoid applying pressure at a wound site.

According to one aspect of the invention, a schedule is provided for periodic re-treatment of the wound, i.e. repeating steps (2) - (4) or periodic reapplication of the therapeutic composition to the wound. The actual number of retreatments and frequency of retreatments (i.e., treatment intervals) should be determined based on several factors, including the severity of the wound (e.g., grade, size, and depth of the wound), the extent to which the natural wound healing environment is compromised (e.g., vascularity of the site, metabolic state of the patient, ability to relieve pressure at the site, presence of infection, diabetic stage of DFU, extent of burn for burn), age of the patient, duration of the wound, and other co-morbidities such as smoking, obesity, uncontrolled blood glucose levels, patient compliance, and others. The number of retreatments and frequency of retreatment should be increased for more severe wounds or for wounds with a more impaired healing environment. Furthermore, the prescribed number of treatments and/or frequency of treatments can be adjusted during the treatment period according to the rate of healing of the wound, i.e., increasing the number of retreatments and/or increasing the frequency of retreatments for slower healing wounds, or decreasing the number of retreatments and/or decreasing the frequency of retreatments for faster healing wounds.

According to one aspect of the invention, the frequency of retreatment is at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or at least about 15 days, and so forth, up to at least about once every six weeks, or a combination thereof. According to another aspect of the invention, the frequency of retreatment is once every 2 to 42 days, or once every 3 to 42 days, or once every 2 to 28 days, or once every 3 to 28 days, or once every 2 to 7 days, or once every 3 to 7 days, or once every 4 to 21 days, once every 7 to 28 days, or once every 7 to 21 days, or once every 7 to 14 days, or once every 10 to 15 days, or once every 12 to 14 days. According to another aspect of the invention, the frequency of treatment is once every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 12 days, every 14 days, every 15 days, every 21 days, every 28 days, every 30 days, every 35 days, every 42 days, or a combination thereof.

According to another aspect of the invention, the frequency of retreatment is substantially the same over the treatment period and is at least about every 2 days, at least about every 3 days, at least about every 4 days, at least about every 5 days, at least about every 6 days, at least about every 7 days, at least about every 8 days, at least about every 9 days, at least about every 10 days, at least about every 11 days, at least about every 12 days, at least about every 13 days, at least about every 14 days, or at least about every 15 days, and so forth, up to at least about once every six weeks.

According to one aspect of the invention, the wound is retreated at least 1 time, at least 2 times, at least 3 times, at least 4 times or at least 5 times during the treatment period. According to another aspect of the invention, the wound is treated from 0 to 6 times, from 0 to 7 times or from 0 to 8 times during the treatment period. According to another aspect of the invention, the wound is treated 1 to 8 times, or 2 to 7 times or 3 to 6 times during the treatment period. According to another aspect of the invention, the wound is retreated 1, 2, 3, 4, 5, 6, 7, 8, 10 or 20 times during the treatment period. According to another aspect of the invention, the wound is retreated from 0 to 46 times, or from 1 to 46 times, or from 0 to 20 times, or from 1 to 20 times, or from 0 to 27 times, or from 1 to 27 times.

According to one aspect of the invention, the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is preferably greater than 0mg, but less than about 50mg, or less than about 25mg, or less than about 20mg, or less than about 15mg, or less than about 10mg, or less than about 5mg, or less than about 4mg, or less than about 3mg, or less than about 2mg, or less than about 1mg of rhPDGF-BB. In certain embodiments, the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is preferably between about 0.1mg to about 50mg, or between about 0.5mg to about 25mg, or between about 1mg to about 10mg, or between about 2.5mg to about 8mg, or between about 3mg to about 7mg, or between about 4mg to about 6 mg.

Various retreatments may involve either in terms of the exact amount of rhPDGF-BB applied to the wound (i.e., "absolute dose"), or in terms of the amount per square centimeter (cm)²) The same or different doses of rhPDGF-BB in terms of the amount of rhPDGF-BB applied to the wound area (i.e., the "area dose"). According to one aspect of the invention, an absolute dose of between about 10 μ g and about 50mg, or between about 10 μ g and about 25mg, or between about 10 μ g and about 20mg, or between about 10 μ g and about 15mg, or between about 10 μ g and about 10mg, or between about 10 μ g and about 5mg of rhPDGF-BB, or between about 10 μ g and about 5mg, is administered per treatmentAbout 10 μ g to about 1mg of rhPDGF-BB. According to another aspect of the invention, about 10 μ g PDGF/cm is administered per treatment²To about 1.0mg PDGF/cm²Or about 10. mu.g PDGF/cm²To about 0.5mg PDGF/cm²Or about 10. mu.g PDGF/cm²To about 0.25mg PDGF/cm²Or about 10. mu.g PDGF/cm²To about 0.1mg PDGF/cm²Or about 10. mu.g PDGF/cm²To about 0.05mg PDGF/cm²Area dose of (c). In certain embodiments, each treatment with rhPDGF-BB preferably is about 10 μ g to 1000 μ g PDGF/cm²Or from about 0.01mg to about 50mg PDGF/cm²Or from about 0.05mg to about 25mg PDGF/cm²Or from about 0.1mg to about 10mg PDGF/cm²Or from about 0.2mg to about 2mg PDGF/cm². In certain embodiments, at least about 10 μ g of rhPDGF/cm is administered per treatment²A wound surface area, or at least about 25 μ g of rhPDGF/cm²A wound surface area, or at least about 50 μ g of rhPDGF/cm²Wound surface area, or at least about 100 μ g of rhPDGF/cm²Wound surface area, or at least about 250 μ g of rhPDGF/cm²Wound surface area, or at least about 500 μ g rhPDGF/cm²Area dose of wound surface area. In certain embodiments, about 10 μ g of rhPDGF/cm is administered per treatment²Wound surface area to about 500. mu.g of rhPDGF/cm²Wound surface area, or about 10 μ g rhPDGF/cm²Wound surface area to about 100 μ g rhPDGF/cm²Wound surface area, or about 15 μ g rhPDGF/cm²Wound surface area to about 375 μ g rhPDGF/cm²Wound surface area, or about 30 μ g rhPDGF/cm²Wound surface area to about 190 μ g rhPDGF/cm²Wound surface area, or about 30 μ g rhPDGF/cm²Wound surface area to about 300. mu.g of rhPDGF/cm²Area dose of wound surface area.

According to one aspect of the invention, initial treatment with the composition of the invention may be the most important treatment. PDGF promotes the wound healing process through its effects on cell proliferation (mitogenesis) and directed cell movement (chemotaxis) and angiogenesis (the generation of new blood vessels). A number of cells have been shown to have receptors (binding sites) for PDGF, including connective tissue cells (skin)Skin, bone, cartilage, tendons and ligaments), vascular cells and cells of the nervous system. Cells having PDGF receptors respond by migrating to the wound site (where PDGF is present at elevated levels as a result of application of the therapeutic composition according to the present invention) and then proliferating after binding PDGF. Since PDGF receptors degrade soon after activation, cell proliferation is controlled and limited by the presence of locally available PDGF and by intercellular interactions that cause cells to travel from the proliferative phase of wound healing to the matrix deposition phase that ultimately results in complete healing. Therefore, a critical bolus of rhPDGF-BB must be applied during initial treatment to ensure that the patient's natural wound healing process is properly activated. Thus, according to the invention, the initial treatment comprises the application of a therapeutic composition comprising at least 10 μ g PDGF/cm²Wound surface area up to 5000. mu.g PDGF/cm²Wound surface area, or at least 20 μ g PDGF/cm²Up to 1000. mu.g PDGF/cm²Wound surface area, or at least 30 μ g PDGF/cm²Until 600. mu.g PDGF/cm²Wound surface area, or at least 40 μ g PDGF/cm²Up to 400. mu.g PDGF/cm²Wound surface area, or at least 50 μ g PDGF/cm²Up to 350. mu.g PDGF/cm²Wound surface area, or at least 60 μ g PDGF/cm²Until 300. mu.g PDGF/cm²Wound surface area, or at least 200 μ g PDGF/cm²Until 2000. mu.g PDGF/cm²Area dose of wound surface area. According to another aspect of the invention, the initial treatment comprises administering a therapeutic composition comprising at least 10 μ g PDGF/cm²Wound surface area, or at least 20 μ g PDGF/cm²Wound surface area, or at least 25 μ g PDGF/cm²Wound surface area, or at least 30 μ g PDGF/cm²Wound surface area, or at least 40 μ g PDGF/cm²Wound surface area, or at least 50 μ g PDGF/cm²Wound surface area, or at least 60 μ g PDGF/cm²Wound surface area, or at least 70 μ g PDGF/cm²Wound surface area, or at least 80 μ g PDGF/cm²Wound surface area, or at least 90 μ g PDGF/cm²Wound surface area, or at least 100 μ g PDGF/cm²Wound surface area, or at least 250 μ g PDGF/cm²Wound surface area, or at least 500 μ g PDGF/cm²Area dose of wound surface area.

According to another aspect of the invention, each treatment application is about 4. mu.l PDGF solution/cm³Carrier (which may be a matrix such as collagen sponge) to about 40ml PDGF solution/cm³Carrier, or about 0.1ml PDGF solution/cm³Carrier to about 30ml PDGF solution/cm³Carrier, or about 0.2ml PDGF solution/cm³Vehicle to about 20ml PDGF solution/cm³Carrier, or about 0.1ml PDGF solution/cm³Vehicle to about 10ml PDGF solution/cm³Carrier, or about 0.25ml PDGF solution/cm³Vehicle to about 5ml PDGF solution/cm³Carrier, or about 0.25ml PDGF solution/cm³Vehicle to about 2.5ml PDGF solution/cm³Carrier, or about 0.1ml PDGF solution/cm³Vehicle to about 1ml PDGF solution/cm³Carrier, or about 0.5ml PDGF solution/cm³Carrier to about 1.5ml PDGF solution/cm³And (3) a carrier. In certain embodiments, the PDGF solution contains about 0.3mg/ml of rhPDGF-BB.

According to another aspect of the invention, each treatment application is about 1.2 μ g PDGF/cm³Carrier to about 12mg PDGF/cm³Carrier, or about 30. mu.g PDGF/cm³Carrier to about 9mg PDGF/cm³Carrier, or about 60. mu.g PDGF/cm³Carrier to about 6mg PDGF/cm³Carrier, or about 75 μ g PDGF/cm³Carrier to about 3mg PDGF/cm³Carrier, or about 75 μ g PDGF/cm³Vector to about 1.5mg PDGF/cm³Carrier, or about 75 μ g PDGF/cm³Carrier to about 750 μ g PDGF/cm³Carrier, or about 120. mu.g PDGF/cm³Vector to about 600. mu.g PDGF/cm³Carrier, or about 150. mu.g PDGF/cm³Carrier to about 450. mu.g PDGF/cm³Carrier, or about 75 μ g PDGF/cm³Carrier to about 225. mu.g PDGF/cm³And (3) a carrier.

According to one aspect of the invention, the initial absolute PDGF treatment dose may be greater than the subsequent retreatment dose. The initial absolute PDGF treatment dose may be about 10%, about 20%, about 30%, about 40%, or about 50% or up to about 300% higher than each subsequent retreating PDGF dose.

According to one aspect of the invention, the method comprises storing PDGF at room temperature, typically 16 to 32 degrees celsius. Prior to use, it may be reconstituted with sterile water, saline, buffer or other physiological solution to form a solution having the desired PDGF concentration. The solution is then added to a carrier, preferably a cellular matrix (e.g., collagen sponge) having a desired porosity in a desired volume to wet out the matrix. The rhPDGF-soaked matrix is then applied to the wound surface. If the wound is an external wound, it is then covered with a wound dressing. The process may then be repeated according to the frequency and duration parameters described above until the wound is substantially healed.

III.Novel therapeutic compositions for the treatment of wounds

The present invention also provides novel therapeutic compositions for treating wounds comprising sterile PDGF incorporated into a biocompatible sterile carrier, matrix or scaffold. For example, the therapeutic composition may be applied topically to a wound to promote wound healing.

According to one aspect of the invention, a therapeutic composition is provided comprising a rhPDGF-BB solution and a carrier, preferably a biocompatible cell scaffold, wherein the rhPDGF-BB solution is disposed in or incorporated into the cell scaffold. In some embodiments, the rhPDGF-BB solution comprises about 0.05mg/ml to about 5mg/ml of rhPDGF-BB, or about 0.1mg/ml to about 1mg/ml of rhPDGF-BB, or about 0.2mg/ml to about 0.4mg/ml of rhPDGF-BB. According to one aspect of the invention, the rhPDGF-BB solution contains rhPDGF-BB at a concentration of about 0.05mg/ml, or about 0.1mg/ml, or about 0.2mg/ml, or about 0.25mg/ml, or about 0.3mg/ml, or about 0.35mg/ml, or about 0.4mg/ml, or about 0.5mg/ml, or about 0.6mg/ml, or about 0.7mg/ml, or about 0.8mg/ml, or about 0.9mg/ml, or about 1mg/ml, or about 2mg/ml, or about 3mg/ml, or about 4mg/ml, or about 5 mg/ml.

In some embodiments, the rhPDGF-BB solution is a pre-formulated sterile PDGF solution comprising elements described herein (e.g., PDGF concentration, sterile solution composition, etc.). In other embodiments, the rhPDGF-BB solution is formed at the time of use, preferably by combining a sterile solution (e.g., sterile water, saline, buffered solution, or physiological solution) with a sterile powder comprising or consisting essentially of lyophilized rhPDGF-BB. The sterile solution is used to reconstitute the lyophilized rhPDGF-BB. The lyophilized rhPDGF-BB is formed by lyophilizing under sterile conditions liquid rhPDGF-BB produced using a recombinant expression system described further below.

In another aspect of the invention, the rhPDGF may be incorporated into a carrier that is preferably a sterile, biocompatible, absorbable cell scaffold, and the PDGF-saturated carrier is then lyophilized to form a sterile, desiccated instrument that incorporates rhPDGF. Any known technique for lyophilizing recombinant proteins may be used to lyophilize rhPDGF-BB so long as it produces a sterile powder. The resulting lyophilized rhPDGF-BB powder is capable of being stored at room temperature and still maintaining at least about 80% of its biological activity for at least about 6 months, or at least about 1 year, or at least about 2 years, or at least about 3 years. The sterile lyophilized device may then be applied directly to the wound site or soaked with blood or other sterile solution prior to placement in the wound.

Because PDGF has a tendency to adhere to the surface of a container, such as a vial (especially at higher pH), achieving 100% reconstitution of lyophilized PDGF in a vial can be challenging. Thus, in certain embodiments, an additive may be included in the PDGF solution to lower its pH to below about 7, or below about 6, or below about 5, or below about 4 or below about 3. Additives that may facilitate reconstitution of lyophilized PDGF include salts, carrier proteins such as albumin, or low pH solutions such as dilute acetic acid or hydrochloric acid. However, if the PDGF solution is too acidic, it may negatively affect the biocompatible scaffold. Thus, in certain embodiments, the lyophilized PDGF is reconstituted in a solution having a pH of less than about 5, and once the PDGF is substantially completely reconstituted, a base solution is added to raise the pH of the PDGF solution to between about 6 to about 8, or to about 7, prior to its combination with the biocompatible scaffold. Such a pH adjustment step is particularly useful when the biocompatible scaffold is a collagen sponge.

The buffer solutions used to reconstitute the lyophilized rhPDGF-BB may include, but are not limited to, water, saline, carbonate, phosphate (e.g., phosphate buffered saline), histidine, acetate (e.g., sodium acetate), acidic buffers such as acetic acid and HCl, and organic buffers such as lysine, Tris buffers (e.g., Tris (hydroxymethyl) aminoethane), N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (HEPES), and 3- (N-morpholino) propanesulfonic acid (MOPS). Preferably, the buffer solution is sterile. The buffer may be selected for biocompatibility with PDGF and for its ability to prevent undesirable protein modification. The buffer may additionally be selected for compatibility with wound tissue. In one embodiment, a sodium acetate buffer is used. Buffers of varying molarity may be used, for example, from about 0.1mM to about 100mM, from about 1mM to about 50mM, from about 5mM to about 40mM, from about 10mM to about 30mM, or from about 15mM to about 25mM, or any molarity within these ranges. In some embodiments, an acetate buffer is used at a molarity of about 20 mM.

The rhPDGF-BB solution is combined with a carrier to form a therapeutic composition, as described above. The carrier may be a matrix or scaffold that serves as a substrate for cell attachment and/or vascular ingrowth as the wound heals and/or provides a means to capture PDGF within its structure (e.g., through interconnected pores), allowing for sustained or delayed or prolonged delivery of PDGF as the wound heals and the matrix or scaffold is resorbed by the body. In some embodiments, the carrier or matrix is a biocompatible, resorbable cell scaffold. The carrier or matrix may comprise a natural polymer such as collagen, gelatin, fibrin, alginate, cellulose, chitosan or fibronectin. The carrier or matrix may also comprise a synthetic biocompatible polymer selected from synthetic polymers such as poly (DL-lactide-co-glycolide) (PLGA), poly (DL-lactide) (PDLA), poly (L-lactide) (PLLA), poly (e-caprolactone) (PCL), polyurethane or others. The support or matrix may also be a mixture of such natural and synthetic polymers. In some embodiments, the matrix comprises a collagen or gelatin sponge, which may be a type 1 collagen sponge. The collagen sponge retains the rhPDGF at the wound site while providing a scaffold for cell growth, resulting in improved user-friendliness and faster and complete healing. In one aspect of the invention, the carrier or matrix (which may be a collagen sponge) has a porosity of about 10 microns to about 2mm, or about 50 microns to about 1000 microns, or about 100 microns to about 500 microns. The average pore size may be about 50 microns to about 500 microns, and wherein a majority of the pores are interconnected.

In some embodiments, the carrier or matrix material is bioresorbable. In one embodiment, the carrier or matrix material may be resorbed by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 90% or 100% within one month after it is applied to the wound. Bioresorbability will depend on: (1) the nature of the material (i.e., its chemical composition, physical structure, and size); (2) a location for placement of a material within the body; (3) the amount of material used; (4) metabolic state of the patient (diabetic/non-diabetic, smoker, old, etc.); and (5) the extent and/or type of wound being treated.

In one aspect of the invention, the rhPDGF-BB solution and carrier should be combined in suitable ratios to form a therapeutic composition having optimal efficacy in healing wounds, and in some embodiments, the rhPDGF-BB solution and carrier are at about 4 μ l PDGF solution/cm³Carrier (which may be a matrix such as collagen sponge) to about 40ml PDGF solution/cm³Carrier, or about 0.1ml PDGF solution/cm³Vehicle to about 30ml PDGF solution/cm³Carrier, or about 0.2ml PDGF solution/cm³Vehicle to about 20ml PDGF solution/cm³Carrier, or about 0.1ml PDGF solution/cm³Vehicle to about 10ml PDGF solution/cm³Carrier, or about 0.25ml PDGF solution/cm³Vehicle to about 5ml PDGF solution/cm³Carrier, or about 0.25ml PDGF solution/cm³Vehicle to about 2.5ml PDGF solution/cm³Carrier, or about 0.1ml PDGF solution/cm³Vehicle to about 1ml PDGF solution/cm³Carrier, or about 0.5ml PDGF solution/cm³Carrier to about 1.5ml PDGF solution/cm³The proportion of the carriers is combined.

In some embodiments, the rhPDGF-BB solution and carrier are administered at about 1.2 μ g PDGF/cm³Carrier to about 12mg PDGF/cm³Carrier, or about 30. mu.g PDGF/cm³Vector to about 9mg PDGF/cm³Carrier, or about 60. mu.g PDGF/cm³Carrier to about 6mg PDGF/cm³Carrier, or about 75 μ g PDGF/cm³Carrier to about 3mg PDGF/cm³Carrier, or about 75 μ g PDGF/cm³Carrier to about 1.5mg PDGF/cm³Carrier, or about 75 μ g PDGF/cm³Carrier to about 750 μ g PDGF/cm³Carrier, or about 120. mu.g PDGF/cm³Vector to about 600. mu.g PDGF/cm³Carrier, or about 150. mu.g PDGF/cm³Carrier to about 450. mu.g PDGF/cm³Carrier, or about 75 μ g PDGF/cm³Carrier to about 225. mu.g PDGF/cm³The proportion of the carriers is combined.

In one aspect of the invention, the carrier is a scaffold and the ratio of rhPDGF-BB/scaffold is such that when the rhPDGF-BB solution is combined with the scaffold, the scaffold is capable of entrapping at least about 20%, 30%, 40%, or 50% up to at least about 100% of the rhPDGF-BB within the pores of the scaffold, such that the rhPDGF-BB is released over time as the scaffold is absorbed by the patient's body, thereby providing controlled delivery of the rhPDGF-BB at the wound site over an extended period of time and simultaneously providing a matrix for new cell and tissue ingrowth. In some embodiments, the scaffold is capable of trapping about 20% to about 100%, or about 25% to about 95%, or 30% to about 90% of rhPDGF-BB within the pores of the scaffold. The above percentages of PDGF retention also apply to the retention of reconstituted lyophilized PDGF-BB.

Various amounts of rhPDGF-BB can be used in the therapeutic compositions of the present invention. According to one aspect of the invention, the total amount of rhPDGF-BB included in the therapeutic composition is less than 50mg, or less than 25mg, or less than 10mg, or less than 5mg, or less than 2.5mg, or less than 1 mg. According to another aspect of the invention, the total amount of rhPDGF-BB included in the therapeutic composition is about 50mg, or about 25mg, or about 10mg, or about 1.0mg, or about 0.5mg, or about 0.1 mg.

In embodiments of the invention, the concentration of PDGF may be determined by using an enzyme-linked immunoassay as described in U.S. patent nos. 6,221,625, 5,747,273, and 5,290,708, which are incorporated herein by reference, or any other assay known in the art for determining PDGF concentration. In embodiments of the invention, the PDGF concentration is less than about 10mg/g, or less than about 5mg/g, or less than about 1mg/g, or less than about 0.5mg/g, or less than about 0.1mg/g, or less than about 0.05 mg/ml. In another aspect of the invention, in an embodiment of the invention, the concentration of PDGF is about 0.05mg/g to about 5mg/g, or about 0.1mg/g to about 1mg/g, or about 0.25mg/g to about 0.5 mg/g.

The PDGF-BB used in the therapeutic compositions of the present invention can be derived from any source, such as natural, synthetic, or recombinant sources. According to one aspect of the invention, the PDGF is produced by recombinant DNA techniques. When PDGF is produced by recombinant DNA techniques, DNA sequences encoding a single monomer (e.g., the PDGF B chain) are inserted into the cultured cells for expression of the B chain monomer. The monomer is then extracted and isolated from the cell culture and refolded to form a biologically active homodimer (e.g., PDGF-BB), which can be further processed for additional purification. According to one aspect of the invention, the cultured cells are prokaryotic cells or E.coli cells. rhPDGF-BB produced by these recombinant techniques can be purified according to the techniques outlined in PCT No. wo 2005/077973 incorporated herein.

As described above, prior art recombinant DNA production methods have produced mixtures of rhPDGF-BB fragments. According to one aspect of the invention, substantially all of the rhPDGF-BB included in the therapeutic compositions described herein is an intact non-sheared strand. According to one aspect of the invention, the bacterial expression system is an e.coli expression system and the resulting protein is purified using reverse phase high performance liquid chromatography, gel filtration, or ion exchange chromatography, or some combination thereof, wherein the resulting rhPDGF-BB included in the purified protein composition is at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% uncleaved rhPDGF-BB on a weight basis.

In some embodiments, the rhPDGF-BB included in the therapeutic compositions of the present invention is rhPDGF-BB that comprises or consists essentially of an amino acid sequence having at least about 90%, about 92%, about 94%, about 96%, about 98%, about 99%, or about 100% homology to SEQ ID No.1, SEQ ID No.1 provides the following:

SEQ ID NO.1:

according to another aspect of the invention, the rhPDGF-BB included in the therapeutic composition of the present invention comprises or consists essentially of at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% undipped rhPDGF-BB on a weight basis. According to another aspect of the invention, the rhPDGF-BB included in the therapeutic compositions of the invention comprises or consists essentially of at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% of rhPDGF-BB that comprises or consists essentially of an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to SEQ ID No. 1.

In some embodiments, the components that make up the novel compositions of the present invention are provided in a kit. The kit may comprise three components:

a) a vial of sterile rhPDGF-BB lyophilized powder,

b) vials of sterile water, buffer, saline or physiological solution, and

c) and (3) a carrier.

The kit will be storable at room temperature for up to 3 years. In some embodiments, stored at 16 to 32 degrees celsius. In some embodiments, the powder contained in the kit comprises a predetermined amount of PDGF. In some embodiments, the amount of PDGF is consistent with the values provided herein. In some embodiments, the carrier is included in a blister pack containing a predetermined amount of carrier. In some embodiments, the amount of carrier is consistent with the values provided herein, and this type of carrier is consistent with the materials described herein.

In use, the rhPDGF-BB in the kit will be reconstituted with sterile water, saline, buffer, or physiological solution, and the carrier will be shaped to fit the size of the wound. After trimming the carrier to fit the wound, it was soaked with the rhPDGF solution so that the solution completely filled the interior pores of the carrier. The rhPDGF-saturated carrier would then be applied to the debrided wound and covered with a wound dressing. This process is repeated according to the schedule described above.

IV.Method for treating various types of wounds

The methods and compositions of the present invention are useful for treating a variety of wounds, including diabetic ulcers, pressure ulcers, neurological ulcers, vascular ulcers, burns, accidental acute wounds, and surgical wounds. Various wound classification systems exist and can be used to identify wounds for which the methods and compositions of the present invention are particularly useful therapeutically. Two such ulcer classification systems include the Wagner classification system (see, Wagner (1987) Orthopedics 10:163-72) and the university of Texas classification system (see, Lavery (1996) J Foot Ankle Surg 35: 528-31). The Wagner system rates wounds according to their depth and the presence of infection. It has five numerical levels:

stage 1: superficial diabetic ulcer

And 2, stage: enlargement of ulcer

Involving ligaments, tendons, joint capsules, or fascia

Without abscess or osteomyelitis

And 3, level: deep ulcers with abscesses or osteomyelitis

4, level: gangrene of partial forefoot

And 5, stage: large area gangrene of foot

The university of texas classification has four numerical ratings according to the depth of the wound. In addition, there are four letter ranks a to D associated with infection and ischemia. The classification system of the university of Texas comprises:

phases

Stage A: no infection or ischemia

And (3) stage B: the existence of infection

And C, stage: presence of ischemia

And (3) stage D: the presence of infection and ischemia

Grading

Level 0: epithelializing wounds

Stage 1: superficial wound

And 2, stage: wounds penetrating into tendons or sacs

And 3, level: penetrating a wound to a bone or joint

For example, a wound with a numerical rating of 3 and a letter rating of D would be a wound that penetrated to a bone or joint and was infected and ischemic. According to one aspect of the invention, the methods and compositions of the invention are used to treat wounds that are grade 2, 3 or 4 wounds under the Wagner classification system, or grade 1, 2 or 3 wounds (stage A, B, C or D) under the university of Texas classification system.

In some embodiments, the methods and compositions described herein can be used to treat wounds, such as leg ulcers, and in particular foot ulcers in diabetic patients. The methods and compositions of the present invention are particularly useful in treating non-healing diabetic ulcers of the lower extremities, which are about 50% non-healing after about 4 weeks of conventional therapy under the current standard of care as described in the background of the invention above.

In some embodiments, the compositions of the present invention are used to treat burns, abdominal wall arthroplasty (so-called "abdominoplasty"), healing after other types of plastic and orthopedic procedures, or wounds after amputation in combination with a 1:1.5 or 1:1.3 meshed stratified thick skin graft (meshing allows the graft to cover a wider area, but leaves a small opening that needs to heal).

V.Additional therapeutic ingredients

The therapeutic compositions of the present invention may contain additional therapeutic ingredients to further promote wound healing. In some embodiments, the PDGF-containing solution may further comprise additional ingredients, such as other bioactive agents. In other embodiments, the PDGF-containing solution may further comprise a cell culture medium, other stabilizing proteins such as albumin, antimicrobial agents, protease inhibitors [ e.g., ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (β -aminoethylether) -N, N' -tetraacetic acid (EGTA), aprotinin, epsilon-aminocaproic acid (EACA), etc. ] and/or other growth factors such as Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), Transforming Growth Factor (TGF), Keratinocyte Growth Factor (KGF), insulin-like growth factor (IGF), or other PDGF-like, including combinations of PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and/or PDGF-DD. In addition, bioactive agents that can be incorporated into the compositions of the invention in addition to PDGF can include organic molecules, inorganic materials, proteins, peptides, nucleic acids (e.g., genes, gene fragments, small inserted ribonucleic acids [ si-RNA ], gene regulatory sequences, nuclear transcription factors, and antisense molecules), nucleoproteins, polysaccharides (e.g., heparin), glycoproteins, and lipoproteins. Other non-limiting examples of biologically active compounds that may be incorporated into the compositions of the present invention are disclosed in U.S. patent application serial No. 11/159,533 (publication No. 20060084602), including, for example, anti-cancer agents, antibiotics, analgesics, anti-inflammatory agents, immunosuppressive agents, enzyme inhibitors, antihistamines, hormones, muscle relaxants, prostaglandins, trophic factors, growth factors, and vaccines.

Standard protocols and protocols for delivering additional bioactive agents are known in the art. Additional bioactive agents may be incorporated into the compositions of the present invention in an amount that allows for the delivery of a suitable dose of the agent to the wound site. In most cases, the dosage is determined using guidelines known to practitioners and appropriate for the particular agent in question. The amount of additional bioactive agent included in the compositions of the present invention may depend on such variables as the type and extent of the condition, the overall health of the particular patient, the formulation of the bioactive agent, the release kinetics, and the bioresorbability of the biocompatible scaffold. Standard clinical trials can be used to optimize the dosage and frequency of administration of any particular additional bioactive agent.

Examples

Example 1

The efficacy of a collagen wound dressing containing 0.3mg/ml recombinant human platelet-derived growth factor-BB (rhPDGF-BB) in the treatment of surgically induced full thickness wounds in mice (db/db) with diabetes caused by leptin receptor mutations was evaluated.

A. Design of research

Fifteen (15) male C57/B6(Leprdb) db/db mice, with an average starting body weight of 41.46g, were obtained from Jackson Laboratory (Bar Harbor, ME) strain code 000642. Animals were acclimated prior to study initiation. During this 3 day period, the animals were observed daily to eliminate underperforming animals.

During this study, all animals were housed individually in disposable cages under the same conditions. The study was conducted in an animal house supplied with HEPA filtered air at a temperature of 70F +/-5F and a relative humidity of 50% +/-20%. The animal house is set to maintain a minimum of 12 to 15 air changes per hour. The animal house was controlled with an automatic timer for a light/dark cycle of 12 hours on and 12 hours off (no transition light). Use ofAnd (5) bedding grass. The bedding is changed at least once every week. Cages, caps, bottles, etc. were washed with commercial detergent and allowed to air dry. Surfaces and materials introduced into the cabinet are disinfected using commercial disinfectants. The floor was cleaned daily and wiped with a minimum of two wipes of commercial detergent per week. The walls and cages were sponged once a minimum of diluted bleach solution every month. All cages were labeled with cage plates or labels with the appropriate information needed to identify the study, dose, animal number and treatment group. Temperature and relative humidity were recorded during the study and the record was kept. With sterile Purina5053 the rodent is eaten with food to feed the animals and provide sterile water for free drinking.

At the start of the study, fifteen (15) animals were randomized and prospectively divided into three (3) groups of five (5) animals each:

group 1-Regranex gel 0.01% rhPDGF-BB was applied daily for 21 days as specified in the package insert.

Group 2-collagen wound dressings in combination with buffer applied on days 0,7 and 14; and

group 3-collagen wound dressings containing 0.3mg/ml recombinant human platelet-derived growth factor-BB (rhPDGF-BB) were applied on days 0,7 and 14.

Each animal was identified by an ear tag corresponding to a separate number. On day 0, the average starting body weight was recorded to ensure that the average starting body weight was comparable between groups. Cage plates were used to identify each cage or label labeled with study number (LYN-01), treatment group number, and animal number.

Test and control collagen +/-PDGF preparations (described below) as surgical dressings were applied topically immediately after wound induction and replaced every seven (Q7) days. Sites of Regranex treatment were treated as specified in the product Instructions (IFU) contained in the product insert containing daily dosing as outlined below. Using Tegaderm^TMAll dressings were applied and held in place and secured in place outside the wound area with benzoin. At dressing changes, the wound area was rinsed with saline and the rinse collected and stored at-80 ℃ for future analysis of protease activity. For sites treated with collagen wound dressings, all non-adhered collagen was gently removed from the healed wound, the site was rinsed with saline, and the rinse solution was collected as described above. After removing the dressing and collecting the irrigation fluid, the wound was measured with calipers and photographed before reapplying the dressing/test article. All wound areas are in mm²Is recorded as a unit.

To take pictures to document wound healing, a camera is mounted on a tripod at an optimal position to ensure that all pictures are consistent. The ruler is placed so that it can be captured in the image to allow accurate estimation of lesion size. In addition to in vivo measurements of wound area, all wound photographs were analyzed using Image J software and wound area was traced and quantified at the end of the study.

Blood glucose levels were measured before study initiation and again before sacrifice on day 21 to confirm the diabetic status. At the end of the study, the wound sites were collected in 10% NBF and prepared for histopathology. The study design is summarized in table 1 below.

TABLE 1 study design

B. Test article and vehicle article

The topical formulation used in the study was Regranex gel (0.01% rhPDGF-BB in carboxymethyl cellulose gel) (group 1); collagen wound dressing wetted with rhPDGF-BB (group 3); and a collagen wound dressing wetted with saline (group 2). Tegaderm was used for all dressings^TMCovered and fixed with benzoin.

1. Dressing composition

a. Group 1rhPDGF doses

As described in the Regranex package insert, "approximately 0.25cm length of gel will need to be extruded from a 15 gram tube per square centimeter of ulcer surface area". Formula (II): (1x w) ÷ 4 ═ cm Regranex length. For a 1.5cmx1.5cm square wound: (1.5x1.5) ÷ 4 ═ 0.56cm Regranex length. As described in the Regranex package insert, "the weight of Regranex gel from a 15g tube is 0.25g/cm length". The regrainex was 0.01% rhPDGF-BB or 100 μ g/g regrainex. For a product length of 0.56cm, the weight of the product was 0.14g for a total dose of PDGF-BB of 14. mu.g. For sites treated with Regranex for 21 days, the maximum total dose for the duration of the study (assuming no change in open wound size from day 0) would be 14 μ g/day x21 days or 294 μ g PDGF-BB. However, on days 7 and 14, the open wound size was determined for all sites treated with Regranex and the amount of Regranex applied was recalculated using the above formula ((1xw) ÷ 4 ═ cm Regranex length).

b. Group 3rhPDGF and group 2 saline doses

The concentration of rhPDGF-BB used in this study was 0.3mg/ml or300. mu.g/ml. To not exceed the total dose of 294 μ g of PDGF-BB during this study (the same maximum study total dose as Regranex), a total of 0.98ml of 0.3mg/ml PDGF-BB was applied to the wound site over the 21 day study period. Assuming a total of 3 administrations (days 0,7 and 14), each administration will consist of about 327 μ 1PDGF-BB on collagen sponge, which represents a dose of about 98 μ g PDGFBB per administration (slightly greater than 7 times the initial individual dose at the site of Regranex treatment). This represents a total of 145. mu.1 (327. mu.1/2.25 cm) of open wound surface area per square centimeter²Open surface area).

The volume of 0.3mg/ml PDGF-BB (group 3) or buffer/sterile saline (group 2) applied to the new collagen sponge on days 7 and 14 was determined using the following formula:

145xcm²open wound surface area (length of open wound [ cm)]x width [ cm ]])。

c. Collagen sponge

All wounds were assessed and measured on days 7 and 14 as described above for group 1 treated sites to record open wound measurements for each individual site. For sites treated with collagen sponges (groups 2 and 3), after removing the dressing, gently rinsing the site, and recording findings including measurements and photographic files, the sponges were measured and trimmed to fit the open wound portion of the original wound.

C. Surgical operation

On day 0, animals were anesthetized with isoflurane. The hair on the back was cut off and the skin wiped with sterile solution. A 1.5x1.5cm square on the middle of the back of the animal was marked using a template and a full thickness wound corresponding to the template was created by excision of the skin and flesh membranes. A hot water circulation pad or equivalent is placed under the animal during surgery to maintain normal body temperature, and the animal recovers on a similar hot water circulation pad. Buprenorphine (0.06mg/kg) was administered by subcutaneous injection for 72 hours immediately after recovery from anesthesia and every 12 hours thereafter. After the mice regained consciousness, warmed Ringer solution (0.5mL) was administered by subcutaneous injection. Wounding of animals is performed under sterile conditions. The wound site was photographed and the length and width were measured with digital calipers immediately after the excision and daily thereafter. From day 0 to day 21, the test articles listed in table 1 were applied to the mice.

D. Results of the study

1. Survival of animals

Three animals died or were euthanized prematurely during the study (all animals were from group 1-Regranex). The first animal (animal No. 3) was found dead one day after surgery. The second animal (animal No. 1) was sacrificed on day 5 due to self-righting flank behind the disabled wound site. Animals No. 5 in group 1 were sacrificed on day 16 due to a weight loss of more than 20% compared to their initial weight. The following table 2 summarizes the animal deaths/sacrifices:

TABLE 2 summary of animal deaths/sacrifice

Day 1	Day 6	Day 16
			Group 1, animal number 3	Group 1, animal number 1	Group 1, animal No. 5
Death was found	Sacrifices and self-mutilations	Weight loss of more than 20%

2. Wound measurement

The wound area was measured with digital calipers and the length (L) and width (W) of each wound were recorded. The area of the wound was calculated using the following formula to calculate the area of the square, where a-LxW. Figure 1 shows the wound area for each group on day 0,7, 14 and 21. The peak wound area was recorded for all three groups on day 0, followed by a decrease in the average wound area on days 7, 14 and 21. All treatment groups showed a significant reduction in wound area during the course of the study.

To provide additional measurements and account for wounds that may not heal in the square (and therefore are not obtained in the formula used above), also by using ImageJ Software^TMThe interior of the wound is measured by tracing the interior of the wound margin. Figure 2 shows the wound area per animal obtained using this method for each treatment group. Figures 3-6 show the average wound area of all treatment groups to the evaluation day (day 0, day 7, day 14 and day 21) in a scatter plot to provide a more detailed evaluation of the individual measurements recorded on those days. For animals that died during this study, the last data point continues in fig. 3-6. Figure 7A highlights a key aspect of the invention, namely that positive results are obtained with fewer administrations of the therapeutic composition. FIG. 7A shows wound area reduction (mm) at each of the four time points (day 0, day 7, day 14, and day 21) for groups 1 and 3²) Cumulative number of treatments. Figure 7B shows the average percentage of wound closure during the course of this study for each group. For animals that died, the last data point was continued.

3. Clinical evaluation

The wound images were also evaluated clinically for possible differences in the degree of healing in re-epithelialization and granulation tissue formation. Fig. 8 shows representative images of the wounds from each animal at each time point for day 0 (fig. 8A), day 7 (fig. 8B), day 14 (fig. 8C), and day 21 (fig. 8D). The raw images from each treatment showed that group 3 (rhPDGF/collagen sponge group) resulted in a significant increase in granulation tissue formation and re-epithelialization compared to group 2 (collagen sponge control group treated with buffer). In addition, the wounds treated daily with Regranex (group 1) also showed better wound closure rates compared to group 2 (buffer control + collagen). Histopathological analysis of formalin-fixed wound area samples was also performed and further confirmed the accelerated wound healing caused by rhPDGF/collagen treatment and Regranex treated wounds compared to buffer control treated sponges. The pathology also indicates that re-epithelialization in rhPDGF/collagen treated wounds is even further improved than treatment with Regranex.

Representative samples of histopathological samples are provided in fig. 9, which includes a series of photomicrographs of sections of wound sites from three study animals on day 21, including group 1-animal 2 (fig. 9E and 9F), group 2-animal 2 (fig. 9A and 9B), and group 3-animal 1 (fig. 9C and 9D). For each set of micrographs, 2x magnification (fig. 9A, 9C, and 9E) and 10x magnification (fig. 9B, 9D, and 9F) are shown.

With respect to group 1, however, fig. 9E and 9F show 100% resurfacing of the wound on day 21, although some shearing due to fragility of the dermal-epidermal structure can be seen. Arrows 60 in fig. 9E indicate the approximate location of the left adjacent epidermis and the wound bed on the right. Fig. 9F provides a higher magnification image from the middle of the wound. 100% epidermal resurfacing was evident, and the presence of differentiated layers in the epidermis indicated its maturation. The new skin still contains high density of capillaries and new collagen formation is ongoing.

With respect to group 2, the micrograph shows that a portion of the collagen sponge remained in the wound bed for 21 days. In the wound margins, the sponge appears to impede resurfacing of the epithelium. Arrows 10 in fig. 9A indicate the wound edges. As shown in fig. 9A, some granulation tissue was formed under the collagen sponge. Referring to fig. 9B, arrows 20 and 30 indicate collagen sponge and granulation tissue, respectively. As shown, the collagen sponge exhibits adhesion in this region, although the degree of cell penetration is minimal. Typical granulation tissue appears to form under the sponge.

With respect to group 3, fig. 9C and 9D show the wound as 100% epidermal resurfacing. Arrow 40 in fig. 9C indicates the approximate edge of the wound bed, and region 50 is subcutaneous fat. Fig. 9D was taken in the middle of the wound and shows that all signs of the original collagen sponge disappeared. The wound was 100% resurfaced and well stratified, with the stratum corneum indicating maturation. The new skin showed evidence of new collagen production and decreased cell structure, indicating that the dermal tissue was maturing and the immature nature of the granulation tissue was regressing.

E. Conclusion of the study

The following conclusions were drawn from this study:

(1) in group 1 21 applications of Regranex were given, whereas in groups 2 and 3 only 3 applications of buffer/collagen or rhPDGF/collagen wound dressings were applied, respectively.

(2) Three animals from group 1(Regranex) were either found dead or had to be euthanized during the live part of the study.

(3) All treatment groups showed a reduction in wound area from day 0 to 21 as determined by both caliper measurements and wound tracing analyzed with Image J software. At sacrifice (day 21), Regranex treated wounds at 2/5, rhPDGF/collagen treated wounds at 3/5, and collagen dressing treated wounds at 0/5 healed.

(4) The raw images from each treatment showed that group 3 (rhPDGF/collagen) resulted in a significant acceleration of granulation tissue formation and re-epithelialization compared to the buffer-treated collagen wound dressing control group (group 2). In addition, wounds treated daily with Regranex (group 1) also showed better closure rates compared to control collagen sponge treated animals.

(5) Healing was best in wounds treated with three applications of rhPDGF/collagen (group 3) as assessed by wound re-epithelialization compared to 21 applications of Regranex (group 1) or three applications of saline-soaked collagen wound dressings.

(6) Application of rhPDGF/collagen three (3) times per week (group 3) accelerated wound closure, including granulation tissue formation and re-epithelialization, compared to the collagen wound dressing (group 2), and it showed at least as effective as the 21 daily doses of Regranex gel (group 1).

(7) rhPDGF/collagen is safe and effective, promoting better healing of diabetic wounds compared to commercially available collagen wound dressings. As evidenced by complete re-epithelialization, 3/5 rhPDGF/collagen treated wounds healed completely compared to 0/5 collagen wound dressing treated animals.

(8) As histologically confirmed, rhPDGF/collagen was safe and effective, promoting angiogenesis, granulation tissue formation and re-epithelialization, compared to commercially available collagen wound dressings.

(9) rhPDGF/collagen, a sterile product, is highly biocompatible as histologically confirmed.

(10) rhPDGF/collagen is easier to apply than Regranex gel, which will improve patient compliance.

(11) rhPDGF/collagen may be safer than regronex because animals receiving regronex have a high mortality rate that was not observed with rhPDGF/collagen or collagen wound dressings.

Example 2 prophetic

Studies were conducted to demonstrate the efficacy of the novel therapeutic compositions and the wound treatment methods described herein. The same study design outlined in example 1 was also used in this study, which included a db/db mouse model with five test groups-standard care group (saline-moistened gauze), Regranex group, collagen sponge group, and two groups utilizing therapeutic compositions comprising PDGF-BB and collagen sponge according to the present invention. However, as described in detail below, the frequency of dosing varied in this study. The study was also designed so that the total dose of PDGF delivered during the study was the same in the Regranex and collagen sponge/PDGF-BB groups.

A. Design of experiments

The experimental design was improved by the results of the study described in example 1, however, a higher number of animals per group (i.e. eight) and a longer duration of the study, i.e. 28 days, were expected. In addition, the test and control collagen +/-PDGF preparations were topically applied as wound dressings either immediately after wound induction and on about day 14 for a total of two applications (group 5) or for a total of four applications of the test preparations according to the novel compositions and methods of treatment described herein immediately after surgery and on days 7, 14 and 21 (see table X). Sites treated with negative control (saline-moistened gauze) and Regranex will undergo 28 daily topical applications as a wound dressing according to the prescribed instructions of use.

In this example, blood glucose levels were determined prior to study initiation and again prior to sacrifice on day 28 to confirm the diabetic state. In all other respects, the design of this study was the same as described in example 1. The details of this study are summarized in table 3 below.

TABLE 3 EXAMPLE 2 study design

Wound assessment and autopsy; # wound evaluation only.

Each animal was examined and survival recorded each day during the study period to assess possible visual differences in animal response between treatment groups. The rate of wound closure will be determined, as will the percentage of fully healed wounds at any given time point.

PDGF-BB dose calculation

The PDGF doses used in this study were designed to mimic the actual therapeutic doses according to the present invention (groups 4 and 5) or the actual therapeutic doses as specified according to the Regranex label (group 2). The dose for group 2 was determined in the same manner as for the Regranex group in example 1. For sites treated with Regranex for 28 days, the total dose of PDGF administered during this study was 14 μ g/day x28 days or 392 μ g of PDGF-BB. The dose can also be expressed as the amount of PDGF/area of original wound size ("area dose"), 6.22. mu.g/cm²Day or 174 mug/cm²PDGF-BB of (1).

For groups 4 and 5, PDGF solutions with PDGF concentrations of 0.3mg/ml or 300. mu.g/ml were used. To achieve a total dose of 392 μ g PDGF-BB (the same total study dose as the Regranex group) during this study period, the total will be reached over the 28 day study period1.307ml of PDGF solution was applied to the wound site. For sites receiving one dressing change every 7 days, there were a total of 4 applications (day 0, day 7, day 14, and day 21). Each administration consisted of 327. mu.1 PDGF-BB (or buffer only) on a collagen sponge. With respect to the dose of PDGF, each administration consisted of 0.3 μ g/μ lx326 μ 1 or 98ug PDGF-BB (approximately 7x Regranex single dose). For sites receiving only two doses, there were a total of 2 administrations on days 0 and 14. Each administration consisted of 654. mu.1 PDGF-BB on a collagen sponge. Each administration will consist of 0.3. mu.g/. mu.1x654. mu.1 or 196ug PDGF-BB (approximately 14 × Regranex single dose). These doses may also be expressed as the amount of PDGF/area of original wound size ("area dose"). For group 4, the PDGF dose was 43.5. mu.g/cm²Dose or 174. mu.g/cm²PDGF-BB of (1). For group 5, the PDGF dose was about 87. mu.g/cm²Dose or 174. mu.g/cm²I.e. with the same cumulative dose in all groups, but with a number of lower doses in groups 4 and 5.

Example 3 predictive

A randomized clinical trial was conducted to evaluate the effectiveness of various compositions of rhPDGF-BB and collagen in treating chronic diabetic foot ulcers compared to standard medical care consisting of moist wound healing to remove excess wound exudate, debridement of necrotic tissue, pressure relief, saline-wetted gauze, antibiotics (if needed), and wound dressing, and regronex. Table 4 below summarizes the study design. For each arm of the study (1-37), the product was applied at the dose and frequency shown in table 4 for up to 20 weeks or until the wound was completely closed. Regranex is applied according to its approved us label. The rhPDGF-BB/collagen composition was applied according to the procedure described in paragraph 50 above (steps 1-5).

The results of this study were measured as:

complete wound closure occurs.

Time to complete wound closure.

Percent reduction in total ulcer surface area per visit.

The number of ulcer recurrences observed at 12 weeks after wound healing.

Handling emergency adverse events (up to 52 weeks).

Inclusion criteria for this study included:

male or female aged 18 years or older with type 1 or type 2 diabetes

Patients with a single ulcer in the treated foot

Patients who are able and willing to provide informed consent

Patients who are able and willing to adhere to protocol interviews and procedures

Patients willing to use a relief method for the entire duration of the study

Full thickness plantar, lateral or dorsal ulcers of the extremity (below the ankle), excluding the inter-toe ulcers (webbed space), extending through the epidermis and dermis, but without involvement of the bones, tendons, ligaments or muscles (defined class IA according to the diabetes wound classification of the university of texas, or class 1 according to Wagner)

Chronic ulcers for at least six weeks despite proper wound care

After rapid debridement, the formula is adopted: length x width x0.8 ulcer area of 1 to 10cm²Including the two extremes

Infection or cellulitis well controlled prior to baseline visit (systemic anti-biotherapy)

Peripheral neuropathy as assessed by the Semmes-Weinstein monofilament test or by a biometer (bio-sensorer) (vibration perception threshold)

Ankle arm pressure index > 0.60 and < 1.3

Women with surgical sterilization, postmenopausal or consenting to full contraception and negative pregnancy test at screening

Non-lactation period

Exclusion criteria for this study included:

ulcer between toes

Ulcers of other etiology or origin: electrical, chemical or radiation injury, bedsore, vascular ulcer or Charcot's malformation

Foot of Charcot

Wounds originating from the amputation layer

Active ulcer infection assessed by clinical examination and radiography (if necessary). There is necrosis, suppuration or sinus that cannot be removed by debridement or controlled by standard wound care

Active osteomyelitis affecting the target ulcerated area

Poorly controlled diabetes mellitus (uncontrolled glycemia: HbAlc% > < 10%), renal failure (serum creatinine >3.0mg/dL), malnutrition (albumin <3.0g/dL or total protein <6.5g/dL)

Known connective tissue or malignant disease

Combination therapy with corticosteroids, immunosuppressive agents, radiotherapy or anti-cancer chemotherapy

Use of test drugs/devices or growth factors within 30 days

Local application of any prior wound care (antiseptic, antibiotic, debridement, enzyme) on the wound within 7 days

Revascularization within 8 weeks

Non-compliance to the regimen is expected (not feasible during compliance with the trial, therapy or wound care), or the investigator feels the patient unsuitable for any other reason

History of severe cerebrovascular events

TABLE 4 EXAMPLE 4 study design

Each rhPDGF/collagen sponge composition performs better than Regranex or standard of care in at least one outcome measure and/or achieves substantially the same outcome with less cumulative rhPDGF applied over the treatment period or with fewer treatments applied resulting in better patient compliance.

The embodiments, variations and sequences described herein are intended to provide an indication of the utility and versatility of the present invention. Other embodiments that do not provide all of the features and advantages set forth herein may also be utilized without departing from the spirit and scope of the present invention. Such modifications and variations are considered to be within the scope of the present invention.

Sequence listing

<110> Segmuir-Linqi

Leisley, Wiscany-Linqi

<120> composition for treating wounds

<130> LBIO-2015-01

<140> not obtained

<141> 2015-10-14

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 1

Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu Cys

1 5 10 15

Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp Arg

20 25 30

Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln Arg

35 40 45

Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr Gln

50 55 60

Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg Lys

65 70 75 80

Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu Ala

85 90 95

Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr

100 105

Claims (66)

1. A therapeutic composition consisting essentially of sterile recombinant human platelet-derived growth factor, a sterile porous matrix, and optionally additives to facilitate reconstitution of lyophilized PDGF, a buffer, and a stabilizing protein, wherein

(a) The recombinant human platelet-derived growth factor is recombinant human platelet-derived growth factor B chain homodimer complexed with a physiological solution, resulting in a therapeutic solution that produces 0.05mg/ml to 5mg/ml recombinant human platelet-derived growth factor B chain homodimer, wherein at least 80% of the recombinant human platelet-derived growth factor B chain homodimer is uncleaved recombinant human platelet-derived growth factor B chain homodimer;

(b) prior to forming the therapeutic solution, the recombinant human platelet-derived growth factor B chain homodimer is biostable and is capable of retaining at least 80% of its biological activity when stored at 16-32 ℃ for at least six months;

(c) said matrix consisting of a natural polymer or a synthetic polymer and comprising open pores that allow cell attachment and ingrowth into said pores and wherein said pores entrap at least 50% of said recombinant human platelet-derived growth factor within said pores, said pores having a pore size ranging between 50 μ ι η and 1mm or an average pore size between 50 μ ι η and 500 μ ι η; or wherein the matrix provides an open porous cell scaffold having a pore size distribution between 10 μm and 2000 μm, and wherein the pores entrap at least 50% of the recombinant human platelet-derived growth factor within the pores; and wherein the matrix is wetted with the therapeutic solution; and is provided with

(d) The ratio of the recombinant human platelet-derived growth factor to the matrix is 75 mug of platelet-derived growth factor/cm³Matrix to 225 μ g platelet-derived growth factor/cm³A substrate, wherein the substrate is a glass substrate,

wherein the matrix is collagen or gelatin.

2. The therapeutic composition of claim 1, wherein the matrix is a collagen sponge or a collagen wound dressing.

3. The therapeutic composition of claim 1, wherein the matrix is a collagen sponge.

4. The therapeutic composition of claim 1, wherein the therapeutic solution is formed from sterile recombinant human platelet-derived growth factor powder containing lyophilized recombinant human platelet-derived growth factor B chain homodimers, wherein at least 80% of the lyophilized recombinant human platelet-derived growth factor B chain homodimers are lyophilized uncleaved recombinant human platelet-derived growth factor B chain homodimers on a weight basis.

5. A therapeutic composition consisting essentially of:

(1) a recombinant human platelet-derived growth factor B chain homodimer solution comprising 0.05 to 5mg/ml recombinant human platelet-derived growth factor B chain homodimers and formed by combining:

a) a sterile powder comprising lyophilized recombinant human platelet-derived growth factor B chain homodimers, wherein on a weight basis at least 80% of the lyophilized recombinant human platelet-derived growth factor B chain homodimers are lyophilized uncleaved recombinant human platelet-derived growth factor B chain homodimers, and wherein the sterile powder is capable of being stored at 16 ℃ to 32 ℃ and still retaining at least 80% of the biological activity of the recombinant human platelet-derived growth factor B chain homodimers for at least one year, and

b) sterile water, saline, buffer or physiological solution, whereby the lyophilized uncleaved recombinant human platelet-derived growth factor B chain homodimer is reconstituted to a solution; and

(2) biocompatible, resorbable supports consisting of natural or synthetic polymers with an open porous structure,

wherein the vector is wetted by a solution of the recombinant human platelet-derived growth factor B chain homodimer; and the pores of said carrier retain at least 50% of said recombinant human platelet-derived growth factor, and

wherein said composition has a ratio of said recombinant human platelet-derived growth factor to said carrier of 75 μ g platelet-derived growth factor/cm³Vehicle to 225 μ g platelet-derived growth factor/cm³The carrier is suitable for treating wounds,

wherein the carrier is collagen or gelatin.

6. The therapeutic composition of claim 5, wherein the carrier is a collagen sponge or a collagen wound dressing.

7. The therapeutic composition of claim 5, wherein the carrier is a collagen sponge.

8. The therapeutic composition of claim 1 or 5, wherein the matrix of claim 1 or the carrier of claim 5 provides a porous cell scaffold having a pore size ranging from 50 μ ι η to 1 mm.

9. The therapeutic composition of claim 1 or 5, wherein the matrix of claim 1 or the carrier of claim 5 provides a porous cell scaffold having an average pore size of 50 microns to 500 microns.

10. The therapeutic composition of claim 1 or 5, wherein the matrix of claim 1 or the carrier of claim 5 provides a porous cell scaffold having an average pore size of 100 microns to 300 microns.

11. The therapeutic composition of claim 1 or 5, wherein a majority of the pores of the matrix of claim 1 or the carrier of claim 5 are interconnected.

12. The therapeutic composition of claim 1 or 5, wherein the recombinant human platelet-derived growth factor B chain homodimer solution is a pre-formulated sterile platelet-derived growth factor solution.

13. The therapeutic composition of claim 1 or 5, wherein the recombinant human platelet-derived growth factor B chain homodimer solution comprises 0.1 to 1mg/ml of recombinant human platelet-derived growth factor B chain homodimer.

14. The therapeutic composition of claim 1 or 5, wherein the recombinant human platelet-derived growth factor B chain homodimer solution comprises 0.2 to 0.4mg/ml of recombinant human platelet-derived growth factor B chain homodimer.

15. The therapeutic composition of claim 1 or 5, wherein the recombinant human platelet-derived growth factor B chain homodimer solution comprises about 0.3mg/ml of recombinant human platelet-derived growth factor B chain homodimer, and wherein about 0.3mg/ml represents a range of values of 0.3mg/ml plus or minus 10%.

16. The therapeutic composition of claim 1 or 5, wherein the recombinant human platelet-derived growth factor B chain homodimer solution comprises about 0.5mg/ml of recombinant human platelet-derived growth factor B chain homodimer, and wherein about 0.5mg/ml represents a range of values of 0.5mg/ml plus or minus 10%.

17. The therapeutic composition of claim 1 or 5, wherein the recombinant human platelet-derived growth factor B chain homodimer solution comprises about 1.0mg/ml of recombinant human platelet-derived growth factor B chain homodimer, and wherein about 1.0mg/ml represents a range of values of 1.0mg/ml plus or minus 10%.

18. The therapeutic composition of claim 4 or 5, wherein at least 85% of the lyophilized recombinant human platelet-derived growth factor B chain homodimers are lyophilized uncleaved recombinant human platelet-derived growth factor B chain homodimers on a weight basis.

19. The therapeutic composition of claim 4 or 5, wherein at least 90% of the lyophilized recombinant human platelet-derived growth factor B chain homodimers are lyophilized uncleaved recombinant human platelet-derived growth factor B chain homodimers on a weight basis.

20. The therapeutic composition of claim 4 or 5, wherein at least 95% of the lyophilized recombinant human platelet-derived growth factor B chain homodimers are lyophilized uncleaved recombinant human platelet-derived growth factor B chain homodimers on a weight basis.

21. The therapeutic composition of claim 4 or 5, wherein at least 97% of the lyophilized recombinant human platelet-derived growth factor B chain homodimers are lyophilized uncleaved recombinant human platelet-derived growth factor B chain homodimers on a weight basis.

22. The therapeutic composition according to claim 1 or 5, wherein the recombinant human platelet-derived growth factor B chain homodimers comprised in the therapeutic composition comprise at least 90% of recombinant human platelet-derived growth factor B chain homodimers comprising the amino acid sequence of SEQ ID No. 1.

23. The therapeutic composition according to claim 1 or 5, wherein the recombinant human platelet-derived growth factor B chain homodimers comprised in the therapeutic composition consist of at least 80% of recombinant human platelet-derived growth factor B chain homodimers consisting essentially of the amino acid sequence of SEQ ID No. 1.

24. The therapeutic composition of claim 5, wherein the ratio of the recombinant human platelet-derived growth factor B chain homodimer solution to the carrier is 0.1ml/cm³To 30ml/cm³。

25. The therapeutic composition according to claim 5, wherein the ratio of the recombinant human platelet-derived growth factor B chain homodimer solution to the carrier is 0.2ml/cm³To 20ml/cm³。

26. The therapeutic composition of claim 5, wherein the ratio of the recombinant human platelet-derived growth factor B chain homodimer solution to the carrier is 0.1ml/cm³To 10ml/cm³。

27. The therapeutic composition of claim 5, wherein the ratio of the recombinant human platelet-derived growth factor B chain homodimer solution to the carrier is 0.25ml/cm³To 10ml/cm³。

28. The therapeutic composition of claim 5, wherein the ratio of the recombinant human platelet-derived growth factor B chain homodimer solution to the carrier is 0.25ml/cm³To 5ml/cm³。

29. The therapeutic composition of claim 5, wherein the ratio of the recombinant human platelet-derived growth factor B chain homodimer solution to the carrier is 0.25ml/cm³To 2.5ml/cm³。

30. The therapeutic composition of claim 5, wherein the ratio of the recombinant human platelet-derived growth factor B chain homodimer solution to the carrier is 0.1ml/cm³To 1ml/cm³。

31. The therapeutic composition of claim 5, wherein the ratio of the recombinant human platelet-derived growth factor B chain homodimer solution to the carrier is 0.5ml/cm³To 1.5ml/cm³。

32. The therapeutic composition of claim 1 or 5, comprising recombinant human platelet-derived growth factor B chain homodimer produced by an E.coli expression system.

33. A therapeutic composition comprising a dry device comprising a lyophilized composition consisting essentially of recombinant human platelet-derived growth factor and a matrix, wherein

Said matrix consisting of a natural polymer or a synthetic polymer and comprising open pores that allow cell attachment and ingrowth into said pores, said pores having an average pore size between 50 microns and 500 microns, and said pores of said matrix retaining at least 50% of said recombinant human platelet-derived growth factor;

at least 80% of the recombinant human platelet-derived growth factor is uncut recombinant human platelet-derived growth factor and the desiccator is biostable such that the recombinant human platelet-derived growth factor contained therein retains at least 80% of its biological activity when stored at 16-32 ℃ for at least six months; and is provided with

Wherein said composition has a ratio of said recombinant human platelet-derived growth factor to said matrix of 75 μ g platelet-derived growth factor/cm³Matrix to 225 μ g platelet-derived growth factor/cm³The matrix is suitable for treating wounds,

wherein the matrix is collagen or gelatin.

34. The therapeutic composition of claim 33, wherein the matrix is a collagen sponge or a collagen wound dressing.

35. The therapeutic composition of claim 33, wherein the matrix is a collagen sponge.

36. Use of platelet-derived growth factor B chain homodimers for the preparation of a medicament suitable for topical administration for the treatment of a wound, wherein the medicament comprises a therapeutic composition according to claim 1 or 5, and wherein at least 10 μ g of platelet-derived growth factor B chain homodimers/cm are applied every 3-42 days when the medicament is used²The wound surface area is once.

37. The use of claim 36, wherein the therapeutic composition is formed by sterile addition of the sterile recombinant human platelet-derived growth factor B chain homodimer solution to the vector in a manner such that the matrix of claim 1 or the vector of claim 5 is wetted by the recombinant human platelet-derived growth factor B chain homodimer solution.

38. Use according to claim 36, wherein the matrix of claim 1 or the carrier of claim 5 is a collagen sponge or a collagen wound dressing.

39. The use according to claim 36, wherein the matrix of claim 1 or the carrier of claim 5 is a collagen sponge.

40. Use according to claim 36, wherein the matrix of claim 1 or the support of claim 5 provides a porous cell scaffold having a pore size in the range of 50 μm to 1 mm.

41. Use according to claim 36, wherein the matrix of claim 1 or the support of claim 5 provides a porous cell scaffold having an average pore size of from 50 microns to 500 microns.

42. Use according to claim 36, wherein the matrix of claim 1 or the support of claim 5 provides a porous cell scaffold having an average pore size of from 100 microns to 300 microns.

43. The use according to claim 40, 41 or 42, wherein a majority of the matrix of claim 1 or the carrier pores of claim 5 are interconnected.

44. The use of claim 36, wherein the solution of recombinant human platelet-derived growth factor B chain homodimers comprises 0.1 to 1mg/ml of recombinant human platelet-derived growth factor B chain homodimers.

45. The use of claim 36, wherein the recombinant human platelet-derived growth factor B chain homodimer solution comprises 0.2 to 0.4mg/ml of recombinant human platelet-derived growth factor B chain homodimer.

46. The use according to claim 36, wherein the solution of recombinant human platelet-derived growth factor B chain homodimers comprises about 0.3mg/ml of recombinant human platelet-derived growth factor B chain homodimers and wherein about 0.3mg/ml represents a range of values of 0.3mg/ml plus or minus 10%.

47. The use according to claim 36, wherein the solution of recombinant human platelet-derived growth factor B chain homodimers comprises about 0.5mg/ml of recombinant human platelet-derived growth factor B chain homodimers and wherein about 0.5mg/ml represents a range of values of 0.5mg/ml plus or minus 10%.

48. The use according to claim 36, wherein the recombinant human platelet-derived growth factor B chain homodimer solution comprises about 1.0mg/ml of recombinant human platelet-derived growth factor B chain homodimer, and wherein about 1.0mg/ml represents a range of values of 1.0mg/ml plus or minus 10%.

49. The use of claim 36, wherein the therapeutic solution is formed from sterile recombinant human platelet-derived growth factor powder comprising lyophilized recombinant human platelet-derived growth factor B chain homodimers, and wherein at least 85% of the lyophilized recombinant human platelet-derived growth factor B chain homodimers are lyophilized uncleaved recombinant human platelet-derived growth factor B chain homodimers on a weight basis.

50. The use of claim 36, wherein the therapeutic solution is formed from sterile recombinant human platelet-derived growth factor powder comprising lyophilized recombinant human platelet-derived growth factor B chain homodimers, and wherein at least 90% of the lyophilized recombinant human platelet-derived growth factor B chain homodimers are lyophilized uncleaved recombinant human platelet-derived growth factor B chain homodimers on a weight basis.

51. The use of claim 36, wherein the therapeutic solution is formed from sterile recombinant human platelet-derived growth factor powder comprising lyophilized recombinant human platelet-derived growth factor B chain homodimers, and wherein at least 95% of the lyophilized recombinant human platelet-derived growth factor B chain homodimers are lyophilized uncleaved recombinant human platelet-derived growth factor B chain homodimers on a weight basis.

52. The use of claim 36, wherein the therapeutic solution is formed from sterile recombinant human platelet-derived growth factor powder comprising lyophilized recombinant human platelet-derived growth factor B chain homodimers, and wherein at least 97% of the lyophilized recombinant human platelet-derived growth factor B chain homodimers are lyophilized uncleaved recombinant human platelet-derived growth factor B chain homodimers on a weight basis.

53. The use according to claim 36, wherein the recombinant human platelet-derived growth factor B chain homodimers comprised in the therapeutic composition comprise at least 90% of recombinant human platelet-derived growth factor B chain homodimers comprising the amino acid sequence of SEQ ID No. 1.

54. The use according to claim 36, wherein the recombinant human platelet-derived growth factor B chain homodimers comprised in the therapeutic composition consist of at least 80% of recombinant human platelet-derived growth factor B chain homodimers consisting essentially of the amino acid sequence of SEQ ID No. 1.

55. The use according to claim 36, wherein the ratio of the recombinant human platelet-derived growth factor B chain homodimer solution to the matrix of claim 1 or the vector of claim 5 is 0.25ml/cm³To 5ml/cm³。

56. The use according to claim 36, wherein the ratio of the recombinant human platelet-derived growth factor B chain homodimer solution to the matrix of claim 1 or the vector of claim 5 is 0.25ml/cm³To 2.5ml/cm³。

57. The use according to claim 36, wherein saidThe ratio of the B chain homodimer solution of the recombinant human platelet-derived growth factor to the matrix of claim 1 or the carrier of claim 5 is 0.1ml/cm³To 1ml/cm³。

58. The use according to claim 36, wherein the ratio of the recombinant human platelet-derived growth factor B chain homodimer solution to the matrix of claim 1 or the vector of claim 5 is 0.5ml/cm³To 1.5ml/cm³。

59. The use of claim 36, wherein the recombinant human platelet-derived growth factor B chain homodimer comprises a recombinant human platelet-derived growth factor B chain homodimer produced by an e.

60. The use of claim 36, wherein the wound is a chronic or acute wound.

61. The use of claim 36, wherein the wound is a diabetic foot ulcer.

62. The use of claim 36, wherein the wound is a venous stasis ulcer, a pressure ulcer, a burn, or a major surgical wound.

63. The use of claim 62, wherein the large surgical wound is an abdominal wall plasty or a surgical tissue valve.

64. Use of sterile recombinant human platelet-derived growth factor B-chain homodimer and a sterile porous biocompatible carrier in the manufacture of a therapeutic composition according to claim 1 or claim 5 for multiple applications at treatment intervals of 3 or more days over a treatment period in combination with a wound dressing to treat a wound from which necrotic or infected tissue has been removed.

65. The use of claim 64, wherein the matrix of claim 1 or the carrier of claim 5 is a collagen sponge or a collagen wound dressing.

66. The use of claim 64, wherein the matrix of claim 1 or the carrier of claim 5 is a collagen sponge.