Patent Application
20250161371
The present disclosure relates to regenerative compositions containing a mixture of a soluble fraction and insoluble fraction derived from one or more helminth eggs. The compositions can be used to promote wound healing and tissue repair in subjects in need of treatment thereof.
Parasitic worm infections affect billions of people worldwide, primarily in tropical developing regions, where they can cause morbidity and mortality (6-8). One such parasite, Schistosoma mansoni (S. mansoni), is a helminth that can infect and can cause severe damage to the liver and hepatic blood vasculature due in part to the immune responses against the eggs lodged in hepatic sinusoids. These responses to xenogeneic foreign bodies and their respective secretome lead to granuloma formation, and if left unresolved can result in fibrosis (9-12). While S. mansoni and other helminth infections can cause a variety of disease states, they also appear to promote a number of desirable features. For example, some helminth infections reduce the incidence of allergies and are associated with beneficial alterations in the microbiome (13, 14). Infection has also been associated with decreased symptom severity in auto-immune disease and even Alzheimer's disease symptoms, suggesting helminths may induce beneficial immunomodulation (15-17). More directly connecting helminth responses to tissue repair, Nusse et al. demonstrated that a Heligmosonoides polygyrus infection triggers epithelial stem cell niche reprogramming, suggesting a connection between helminths and tissue repair that is modulated by the immune response to the worm and its egg secretome (18, 19). Finally, helminth infection induces IL-4 receptor (IL-4R) signaling that downregulates IL-17A production to benefit rapid tissue repair after the initial infection (20).
Type 2 immunity is characterized by the influx of T helper type 2 (TH2) cells, eosinophils, type 2 innate lymphoid cells (ILC2), alternatively activated macrophages, and type 2 associated cytokines IL-4, IL-5, IL-9, and IL-13 (18, 21). Previous studies found that the egg secretome components of helminth (e.g., such as S. mansoni) and the soluble egg antigen (SEA) can stimulate type 2 immunity, recapitulating in part the immune response to infection. Evidence suggest that multiple components of SEA can contribute to the type 2 immune responses, in particular the glycoproteins Omega-1, IPSE/alpha-1 (IL-4 inducing principle of S. mansoni eggs), and the LewisX containing glycan LNFP III (22-24).
While the type 2 immune response is historically considered a protective response against parasitic worms, new perspectives suggest that helminths may be co-opting this immune response to repair the damage induced during infection by agonizing type 2 cytokines. For example, IL-4 secreted by eosinophils and TH2 cells can promote activation of other cell types required for skeletal muscle repair (4, 5). In the central nervous system, IL-4 secreting TH2 cells promote repair in the retina and spinal cord and IL-4 levels are positively associated with learning in Alzheimer's disease animal models (25-27). Although helminths drive type 2 responses, it is unknown whether helminth secretory agents can be formulated or engineered as safe and effective regenerative immunotherapies without the pathologies coincident with parasitic infections such as fibrosis and hepatic damage (28-30).
There is a need in the art for new regenerative immunotherapies that can stimulate a type 2 immune response to promote regeneration and repair of wounds and/or tissue in subjects in need of treatment thereof.
In one aspect, the present disclosure relates to a regenerative composition comprising a mixture of a soluble fraction and an insoluble fraction derived from one or more helminth eggs, wherein the composition is produced by a process comprising the steps of:
In some aspects the filtered soluble and filtered insoluble fraction is combined in a ratio of about 9:1 to about 6:1 to form a composition comprising a mixture of a soluble fraction and an insoluble fraction.
In one aspect, the helminth is Schistosoma mansoni (S. mansoni).
In another aspects, the soluble fraction comprises at least one helminth egg antigen. In yet further aspects, the at least one helminth egg antigen is one or more of omega-1, IPSE/alpha-1, secretory glycoprotein kappa-5, smp40, Histone H2A, fructose-bisphosphate aldolase, lacto-N-fucopentaose-III (LNFPIII), lacto-n-Neotetraose (LNnT), or any combinations thereof.
In other aspects, the insoluble fraction comprises at least one lipid. In yet further aspects, the at least one lipid is one or more of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), 5-hydroxyeicosatetraenoic (HETE), 15-HETE, linoleic acid, arachidonic acid, docosahexaenoic acid, 5S,18R-dihydroxy-6E,8Z,11Z,14Z,16E-eicosapentaenoic acid, 10S, 17S-dihydroxydocosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid, (4Z,7Z,10R,11E,13E,15Z,17S,19Z)-10,17-dihydroxydocosa-4,7,11,13,15,19-hexaenoic acid, lysophosphatidylcholine, or any combinations thereof.
In another aspect, in the above composition, the helminth eggs are at least about 90% homogenized.
In still yet another aspect, the composition further comprises an extracellular matrix. In some aspects, the composition is co-formulated with at least one extracellular matrix.
In still a further aspect, the composition is further incorporated into a gel or hydrogel. Specifically, the gel is a vitrified gel.
In still a further aspect, the above composition, when administered to a subject, increases IL-4 and decreases or reduces IL-17.
In yet another aspect, the present disclosure relates to a method of treating a wound or injury to a tissue in a subject, the method comprising the step of administering to the subject in need of treatment thereof, an effective amount of above-described composition. In some aspects, the subject is a human. In yet other aspects, the tissue with the wound and/or injury is muscle, an organ, cartilage, a ligament, skin, bone, nervous tissue, corneal tissue, a lens, or any combination thereof.
In yet a further aspect, the present disclosure relates to a kit containing the above-described composition.
Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other aspects “comprising,” “consisting of” and “consisting essentially of,” the aspects or elements presented herein, whether explicitly set forth or not.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
As used herein, the term “helminth” refers to a parasitic worm or nematode. Any helminth that induces a type 2 immune response in a subject can be used in the present disclosure. In some aspects, the helminth can be from the Ascaris species (e.g., such as Ascaris lumbricoides), Nippostrongylus brasiliensis, Enterobuis vermicularis, Trichuris trichiura, Ancylostroma duodenale, Necator americanus, Strongyloides stercoralis, and Trichinella spiralis. In other aspects, the helminth is a platyhelminth from trematodes and cestodes, such as Fasciolopsis, Echinostoma and Heterophyes species, Clonorchis sinensis, Oplishorchis viverrini, Opisthorchis felineus, Fasciola hepatica, Schistosoma species (e.g., Schistosoma mansoni), Diphyllobothrium species, Taenia saginata, Taenia solium and Hymenolepsis nana. In yet other aspects, the helminth is from Trichuris muris, Trichinella spiralis, Nippostronglylus prasiliensis, Heligmonsomoides polygyrus, Hymenolepsis nanan, Angiostrongylus species, Trichuris suis, Ascaris suum, Trichuris vulpis, Toxocara species and Pseudoterranova species. In still other aspects, the helminth is a filarial parasite or lung fluke. In yet other aspects, the helminth is Schistosoma mansoni.
As used herein, the phrases “insoluble fraction derived from one or more helminth eggs” or “insoluble portion derived from one or more helminth eggs” as used interchangeably herein, refer to one or more lipids derived or obtained from one or more helminth eggs. In some aspects, the one or more lipids are purified and/or filtered from the one or more helminth eggs. Examples of such lipids include one or more prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), 5-hydroxyeicosatetraenoic (HETE), 15-HETE, linoleic acid, arachidonic acid, docosahexaenoic acid, 5S,18R-dihydroxy-6E,8Z,11Z,14Z,16E-eicosapentaenoic acid, 10S,17S-dihydroxydocosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid, (4Z,7Z,10R,11E,13E,15Z,17S,19Z)-10,17-dihydroxydocosa-4,7,11,13,15,19-hexaenoic acid, lysophosphatidylcholine, or any combinations thereof.
In some aspects, the insoluble fraction derived from one or more helminth eggs comprises at least one of about 0.1% to about 20% PGD2, about 0.1% to about 20% PGE2, about 0.1% to about 20% of 5-hydroxyeicosatetraenoic acid (HETE), about 0.1% to about 20% of 15-HETE, about 0.1% to about 20% of at least one linoleic acid, about 0.1% to about 20% of arachidonic acid, about 0.1% to about 20% of docosahexaenoic acid, about 0.1% to about 20% of 5S,18R-dihydroxy-6E,8Z,11Z,14Z,16E-eicosapentaenoic acid, about 0.1% to about 20% of 10S,17S-dihydroxydocosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid about 0.1% to about 20% of (4Z,7Z,10R,11E,13E,15Z,17S,19Z)-10,17-dihydroxydocosa-4,7,11,13,15,19-hexaenoic acid, about 0.1% to about 20% of lysophosphatidylcholine, and any combinations thereof. In other aspects, the insoluble fraction derived from one or more helminth eggs and comprises about 0.1% to about 20% PGD2, about 0.1% to about 20% PGE2, about 0.1% to about 20% of 5-hydroxyeicosatetraenoic acid (HETE), about 0.1% to about 20% of 15-HETE, about 0.1% to about 20% of at least one linoleic acid, about 0.1% to about 20% of arachidonic acid, about 0.1% to about 20% of docosahexaenoic acid, about 0.1% to about 20% of 5S,18R-dihydroxy-6E,8Z,11Z,14Z,16E-eicosapentaenoic acid, about 0.1% to about 20% of 10S,17S-dihydroxydocosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid about 0.1% to about 20% of (4Z,7Z,10R,11E,13E,15Z,17S,19Z)-10,17-dihydroxydocosa-4,7,11,13,15,19-hexaenoic acid, and about 0.1% to about 20% of lysophosphatidylcholine.
“Promote,” “promotion,” and “promoting” refer to an increase in an activity, response, condition, disease, and/or other biological parameter. This can include, but is not limited to, the initiation of the activity, response, condition, and/or disease. This may also include, for example, a about a 5% increase in the activity, response, condition, and/or disease as compared to the native or control level. Thus, the increase can be about a 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of increase in between as compared to native or control levels.
“Regenerative” or “regeneration” as used herein refers to the renewal, re-growth, and/or restoration of a body or body part, such as, for example, a tissue, after injury or as a normal bodily process. In contrast to scarring, tissue regeneration involves the restoration of the body or body part to its original structural, functional, and physiological condition. In some aspects, the restoration can be partial or complete, meaning about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 90% or about 100% renewal, re-growth, or restoration, or any amount of restoration in between as compared to native and/or control levels. For example, in the case of a skin injury, tissue regeneration can involve the restoration of hair follicles, glandular structures, blood vessels, muscle, or fat. In the case of a brain injury, tissue regeneration can involve maintenance or restoration of neurons. In some aspects, tissue regeneration involves the recruitment and differentiation of stem cells to replace the damaged cells. As used herein, a “stem cell” refers to an undifferentiated cell found among differentiated cells in a tissue, or introduced from an external source for example, embryonic stem cells, adult bone marrow stem cells, that can renew itself and differentiate to yield the major specialized cell types of the tissue. The primary roles of stem cells in a living organism are to maintain and repair the tissue in which they are found. The phrase “stem cell differentiation” refers to the the process whereby an unspecialized cell (e.g., stem cell) acquires the features of a specialized cell such as a skin, neural, heart, liver, or muscle cell. In the case of a brain injury, tissue regeneration can involve the differentiation of stem cells into neurons.
As used herein, the phrases “soluble fraction derived from one or more helminth eggs” or “soluble portion derived from one or more helminth eggs” as used interchangeable herein refers to one or more egg antigens (e.g., proteins) derived or obtained from one or more helminth eggs. Examples of such antigens include one or more omega-1, IPSE/alpha-1, secretory glycoprotein kappa-5, smp40, Histone H2A, fructose-bisphosphate aldolase, lacto-N-fucopentaose-III (LNFPIII), lacto-n-Neotetraose (LNnT), or any combinations thereof.
In some aspects, the antigen is one or more of about 2% to about 50% of omega-1, about 2% to about 50% of about IPSE/alpha-1, about 2% to about 60% of secretory glycoprotein kappa-5, about 1% to about 40% of smp40, about 5% to about 70% of Histone H2A, about 10% to about 70% of fructose-bisphosphate aldolase, about 1% to about 50% of LNFPIII, about 1% to about 50% of LNnT, or any combinations thereof. In other yet aspects, the antigen is from about 2% to about 50% of omega-1, about 2% to about 50% of about IPSE/alpha-1, about 2% to about 60% of secretory glycoprotein kappa-5, about 1% to about 40% of smp40, about 5% to about 70% of Histone H2A, about 10% to about 70% of fructose-bisphosphate aldolase, about 1% to about 50% of LNFPIII, and about 1% to about 50% of LNnT.
“Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In some aspects, the subject may be a human or a non-human. In some aspects, the subject is a human. The subject or patient may be undergoing other forms of treatment. In some aspects, the subject is a human that may be undergoing other forms of treatment.
“Tissue” as used herein refers to groups of cells that have a similar structure and act together to perform a specific function. Examples of “tissue” include, muscle, an organ (e.g., brain, eye, heart, liver, pancreas, stomach, lung, large intestine, small, intestine, bladder, kidneys, reproductive organs, or any combinations thereof), cartilage, a ligament, skin, bone, nervous tissue, corneal tissue, a lens, or any combination thereof. In some aspects, the tissue is muscle. In other aspects, the tissue is an organ. In yet other aspects, the tissue is cartilage. In still yet other aspects, the tissue is a ligament. In yet other aspects, the tissue is skin. In still other aspects, the tissue is bone. In still other aspects, the tissue is nervous tissue. In still yet other aspects, the tissue is corneal tissue.
As used herein, “tissue injury” or “injury to a tissue” as used interchangeable herein, refers to an injury to a tissue that can result from, for example, a scrape, cut, laceration wound, crush wound, compression wound, stretch injury, bite wound, graze, bullet wound, explosion injury, body piercing, stab wound, burn wound, wind burn, sun burn, chemical burn, surgical wound, surgical intervention, medical intervention, host rejection following cell, tissue grafting, pharmaceutical effect, pharmaceutical side-effect, bed sore, radiation injury, cosmetic skin wound, internal tissue (e.g., organ) injury, disease process (e.g., asthma, cancer), infection, infectious agent, developmental process, maturational process (e.g., acne), genetic abnormality, developmental abnormality, environmental toxin, allergen, scalp injury, facial injury, jaw injury, foot injury, toe injury, finger injury, bone injury, sex organ injury, joint injury, excretory organ injury, eye injury, corneal injury, muscle injury, adipose tissue injury, lung injury, airway injury, hernia, anus injury, piles, ear injury, retinal injury, skin injury, abdominal injury, arm injury, leg injury, athletic injury, back injury, birth injury, premature birth injury, toxic bite, sting, tendon injury, ligament injury, heart injury, heart valve injury, vascular system injury, cartilage injury, lymphatic system injury, craniocerebral trauma, dislocation, esophageal perforation, fistula, nail injury, foreign body, fracture, frostbite, hand injury, heat stress disorder, laceration, neck injury, self mutilation, shock, traumatic soft tissue injury, spinal cord injury, spinal injury, sprain, strain, tendon injury, ligament injury, cartilage injury, thoracic injury, tooth injury, trauma, nervous system injury, aging, aneurism, stroke, digestive tract injury, infarct, ischemic injury, or any combination thereof.
By “treat” or “treatment” is meant a method of reducing the effects of a wound, injury, disease and/or condition. Treatment can also refer to a method of reducing the underlying cause of the wound, injury, disease and/or condition itself rather than just the symptoms. The treatment can be any reduction from native levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
In one aspect, the present disclosure relates a regenerative composition comprising a mixture of a soluble fraction and an insoluble fraction derived or obtained from one or more helminth eggs. The soluble fraction comprises at least one helminth egg antigen. The insoluble fraction comprises at least one lipid. In yet other aspects, one or both of the soluble fraction and the insoluble fraction are purified and/or filtered. In yet other aspects, the regenerative compositions comprise a mixture of a soluble fraction and an insoluble fraction derived or obtained from at least one egg derived from S. mansoni (such a composition is referred to herein as “rSEA”). It has been surprisingly discovered that the compositions of the present disclosure can be used to as pro-regenerative immunotherapies that can be used in promoting wound and/or tissue repair. It has also been found that when the regenerative compositions of the present disclosure are used in wound and/or tissue repair, the repaired wound and/or tissue exhibits reduced scar tissue and/or fibrotic tissue formation when compared to other compositions (such as extracellular matrix biomaterials) used for treating wounds and/or injuries to tissue. Moreover, the compositions of the present disclosure have been found increase IL-4 and decreases or reduces IL-17.
The helminth regenerative compositions comprising the mixture of a soluble fraction and an insoluble fraction is prepared using a unique process or method. Helminth eggs for use in the process can be obtained from any source known in the art. For example, the helminth eggs can be obtained from a commercially available source. Commercial sources of helminth eggs include, but are not limited to, Symmbio, Au Naturel, Biome Restoration, Ltd., etc. Alternatively, the eggs can be isolated from an animal infected with S. mansoni, such as a mouse, rat, rabbit, sheep, cow, cat, dog, cow, human, etc., using routine techniques known in the art.
The eggs are homogenized to produce a mixture using routine techniques known in the art (e.g., by using a homogenizer). In some aspects, the eggs are homogenized to greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99%. In some aspects, the eggs are 100% homogenized.
Once homogenized, the mixture is centrifuged using routine techniques known in the art. The centrifugation produces three different fractions or layers of the mixture: (a) a top lipid fraction or layer; (b) a middle soluble egg antigen fraction or layer; and (c) bottom egg shell fraction or layer. The bottom egg shell fraction or layer is then removed using routine techniques known in the art. The lipid fraction or layer and the soluble egg antigen fraction or layer is retained.
The lipid fraction or layer and the soluble egg antigen fraction or layer is then ultra-centrifuged using routine techniques known in the art. For example, the lipid fraction or layer and the soluble egg antigen fraction or layer can be ultra-centrifuged at 100,000×g for about 90 minutes at 4° C. The conditions under which the ultra-centrifugation occurs is not critical and can be modified or adjusted using routine techniques known in the art. The ultra-centrifugation produces an insoluble supernatant (e.g., fraction) that forms as a top layer followed by a soluble supernatant (e.g., fraction). The insoluble and soluble supernatants are harvested, separated or removed from each other using routine techniques known in the art, such as by use of a pipet. Each of the separated insoluble and soluble fractions are filtered using a sterile filter using routine techniques known in the art.
The filtered insoluble and soluble fractions are then combined in desired ratios to produce the helminth regenerative soluble egg antigen compositions of the present disclosure. In some aspects, the filtered insoluble and filtered soluble fractions are combined in a ratio sufficient to reduce a proinflammatory response in a subject. As used herein, the term “sufficient to reduce a proinflammatory response in a subject” refers to that when the composition of the present disclosure is administered to the subject, at least the subject's IL-17 and/or interfering gamma levels are reduced or decreased. In some aspects, the subject's IL-17 and/or interfering gamma levels are reduced or decreased by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%. In yet other aspects, the filtered soluble fraction is combined with the filtered insoluble fraction in a ratio of about 9:1 to about 6:1. In some aspects, the ratio is about 6:1, about 6:2, about 6:3, about 6:4, about 6:5, about 6:6, about 7:1, about 7:2, about 7:3, about 7:4, about 7:5, about 7:6, about 7:7, about 8:1, about 8:2, about 8:3, about 8:4, about 8:5, about 8:6, about 8:7, about 8:8, about 9.1.
A comparison of the process used to prepare the regenerative compositions of the present disclosure (such as rSEA) with the process used in the art to produce compositions containing helminth soluble egg antigens (SEA) is shown in
The regenerative compositions of the present disclosure have been found to stimulate or induce a type 2 immune response when administered to subjects suffering a wound and/or tissue injury and thus can be used to promote regeneration and repair of such wounds and/or tissue injuries. Thus, as discussed previously herein, these compositions can be used as pro-regenerative immunotherapies for wound and/or tissue repair in a subject in need of treatment thereof. Moreover, it has also been surprisingly discovered that when the compositions of the present disclosure are used for wound and/or tissue repair, the resulting repaired wound and/or tissue exhibits reduced scar tissue and/or fibrotic tissue formation when compared to other compositions used for treating wounds and/or injuries to tissue.
In addition to the mixture of a soluble fraction and an insoluble fraction derived from one or more helminth eggs, the compositions of the present disclosure can also contain at least one pharmaceutically acceptable carrier or excipient suitable for use in treating subjects (e.g., humans and/or animals) without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. The carrier or excipient would naturally be selected to minimize any degradation of the mixture of the soluble fraction and/or insoluble fraction and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Examples of pharmaceutically acceptable carriers include those selected from the group consisting of water, polyhydric alcohols including alkylene glycols, (particularly propylene glycol) and glycerol; alcohols such as ethanol and isopropanol; polyalkylene glycols such as polyethylene glycol; other ointment bases such as petroleum jelly, lanolin, dimethylformamide, ethylene glycol, tetrahydrofurfuryl alcohol, cyclohexane, cyclohexanone, acetone, ethylether, N-dodecylazocyclo-heptan-2-one, methyldecylsulfoxide, dimethylacetamide and diethylfoluamide; and mixtures thereof.
It should be appreciated that the above list is not exclusive as the present disclosure also encompasses the use of pharmaceutically acceptable carriers other than those specifically mentioned. In some aspects, the composition may further contain thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the pharmaceutically acceptable carriers and the mixture of the soluble fraction and the insoluble fraction.
In further aspects, the composition can further comprise any known or newly discovered substance that can be administered to a wound, tissue injury, and/or site of inflammation that include antimicrobial agents, antiinflammatory agents, anesthetics, and the like. For example, the compositions of the present disclosure can further comprise one or more of classes of antibiotics (e.g. Aminoglycosides, Cephalosporins, Chloramphenicol, Clindamycin, Erythromycins, Fluoroquinolones, Macrolides, Azolides, Metronidazole, Penicillin's, Tetracycline's, Trimethoprim-sulfamethoxazole, Vancomycin), steroids (e.g. Andranes (e.g. Testosterone), Cholestanes (e.g. Cholesterol), Cholic acids (e.g. Cholic acid), Corticosteroids (e.g. Dexamethasone), Estraenes (e.g. Estradiol), Pregnanes (e.g. Progesterone), narcotic and non-narcotic analgesics (e.g. Morphine, Codeine, Heroin, Hydromorphone, Levorphanol, Meperidine, Methadone, Oxydone, Propoxyphene, Fentanyl, Methadone, Naloxone, Buprenorphine, Butorphanol, Nalbuphine, Pentazocine), chemotherapy (e.g. anti-cancer drugs such as but not limited to Altretamine, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carrnustine, Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine, Diethylstilbesterol, Ethinyl estradiol, Etoposide, Floxuridine, Fludarabine, Fluorouracil, Flutamide, Goserelin, Hydroxyurea, Idarubicin, Ifosfamide, Leuprolide, Levamisole, Lomustine, Mechlorethamine, Medroxyprogesterone, Megestrol, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Paclitaxel, pentastatin, Pipobroman, Plicamycin, Prednisone, Procarbazine, Streptozocin, Tamoxifen, Teniposide, Vinblastine, Vincristine), anti-inflammatory agents (e.g. Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Decanoate; Deflazacort; Delatestryl; Depo-Testosterone; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lomoxicam; Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone; Mesterolone; Methandrostenolone; Methenolone; Methenolone Acetate; Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Nandrolone; Naproxen; Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin; Oxandrolane; Oxaprozin; Oxyphenbutazone; Oxymetholone; Paranyline Hydrochloride; Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin; Stanozolol; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Testosterone; Testosterone Blends; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin; Zomepirac Sodium), or anti-histaminic agents (e.g. Ethanolamines (like diphenhydramine carbinoxamine), Ethylenediamine (like tripelennamine pyrilamine), Alkylamine (like chlorpheniramine, dexchlorpheniramine, brompheniramine, triprolidine), other anti-histamines like astemizole, theophyline, loratadine, fexofenadine, Bropheniramine, Clemastine, Acetaminophen, Pseudoephedrine, Triprolidine).
Additionally, the compositions of the present disclosure may further comprise at least one extracellular matrix. In some aspects, the composition is co-formulated with at least one extracellular matrix. Examples of some extracellular matrices that can be used include PuraPly and PuraPlyAM from Organogenesis, Inc. and Cellvo™ from Stembiosys, AlloDerm Regenerative Matrix, Cymetra, Integra Dermal Regeneration Template, MiMidex materials (e.g., EPIFIX, EPICORD). These materials can also be combined with synthetic materials known in the art as well.
The compositions of the present disclosure can be in any form suitable for administration to a subject. Specifically, the compositions of the present disclosure can be contained or incorporated into one or more materials. For example, such materials can include materials used to treat wounds in which such materials are coated and/or impregnated with the compositions described herein. Examples of such materials which can be used to treat wounds include bandages, steri-strips, sutures, staples, grants (e.g., skin grafts) or any combinations thereof. For example, the material (e.g., bandage, steri-strip, suture, staple, graft, or combinations thereof) can be soaked, coated and/or dipped with or into the compositions described herein. Alternatively, the material can be coated with the composition described herein. Alternatively, the composition can be incorporated into the materials. For example, the composition can be incorporated into a hydrogel, such as a cross-linkable hydrogel system, such as the poly(lactic-co-glycolic acid) (PLGA) or polyurethane. In another aspect, the compsition can be incorporated into a vitrified gel (e.g., vitrigel). The hydrogel, vitrified gel, or hydrogel system can be fashioned into materials for treating wounds (e.g., bandage, steri-strip, suture, staple, graft, or combinations thereof). In yet another aspect, medical implants can be coated with the regenerative composition of the present disclosure. Such implants can be implanted into a subject for purposes of treatment. Examples of medical implants include, limb prostheses, breast implants, penile implants, testicular implants, artificial eyes, facial implants, artificial joints, heart valve prostheses, vascular prostheses, dental prostheses, facial prosthesis, tilted disc valve, caged ball valve, ear prosthesis, nose prosthesis, pacemakers, cochlear implants, and skin substitutes (e.g., porcine heterograft/pigskin, BIOBRANE, cultured keratinocytes).
The compositions of the present disclosure may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal, intramuscular injection or injection directly into the affected area requiring treatment, such as, but not limited to, muscle wound or arthritic joint. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
In some aspects, when the compositions are applied topically, for a local effect, the delivery of the composition is made to the skin but can also include the delivery of the compositions into the nose and nasal passages through one or both of the nares. Suitable forms include ointments, lotions, creams, gels (e.g., poloxamer gel), drops, suppositories, sprays, liquids, powders, sprayable liquids, liquids that may be applied using a roll-on device, lacquers, and sustained release matrices of transdermal delivery devices such as patches. Administration of the composition by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can used if desirable. Moreover, the compositions of the present disclosure can be administered, for example, in a microfiber, polymer (e.g., collagen), nanosphere, aerosol, lotion, cream, fabric, plastic, tissue engineered scaffold, matrix material, tablet, implanted container, powder, oil, resin, wound dressing, bead, microbead, slow-release bead, capsule, injectables, intravenous drips, pump device, silicone implants, or any bio-engineered materials.
In some aspects, the compositions of the present disclosure are formulated in such a way as to ensure that an effective amount of the mixture of the soluble fraction and insoluble fraction derived or obtained from one or more helminth eggs is present in the composition to elicit Type-2 immune response after administration or application thereof. More specifically, effective dosages and schedules for administering or application the compositions can be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration or application of the compositions are those sufficient enough to produce the desired effect in which the wound healing is achieved or effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary age, sex, weight and general condition of the subject, the severity of the wound being treated, the type of wound, the patient's immune state, route of administration or application, or whether other drugs are included in the regimen, and can be determined using routine techniques known in the art. The dosage can be adjusted by a clinician in the event of any counterindications. Dosages can vary, and can be administered or applied in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. The range of dosage largely depends on the application of the compositions herein, severity of condition, and its route of administration or application.
Following administration or application of the compositions of the present disclosure for promoting wound healing and/or tissue repair, the efficacy of the composition can be assessed in various ways well known to the skilled practitioner. For example, a person skilled in the art will understand that a composition disclosed herein is efficacious in promoting wound healing in a subject by observing that the composition can reduce scar tissue formation, reduce fibrotic tissue formation, improve tissue regeneration, and/or reduce inflammation in the subject following tissue injury using routine techniques known in the art.
The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing and/or aiding in the performance of the methods described below in Section 3. It beneficial for the kit components to be designed and adapted for use together in the below described methods. For example, if the kits are for promoting wound and/or tissue healing, the kit can contains one or more of the compositions of the present disclosure and in a pharmaceutically acceptable carrier. Such kits can also include gels, bandages, Millipore tapes, Medicated Q-tips, Sprays, props, Syrups, Liquids, Disposable tubes or pouches. The kits also can contain instructions for proper use and safety information of the product or formulation. The kits may contain dosage information based on the route of administration or application and method of administration or application as determined by a clinician.
In yet another aspect, the present disclosure relates to a method of promoting wound healing in a subject that has suffered an injury. In this aspect, the method comprises administering to the subject in need of such treatment one or more of the herein provided compositions in a pharmaceutically acceptable carrier. In yet another aspect, the present disclosure also provides method of treating a subject with tissue injury. In this aspect, the method comprises administering to the subject (e.g., having an injury and/or damage to a tissue) one or more of the herein provided compositions in a pharmaceutically acceptable carrier.
The methods provided herein can reduce scar tissue formation in a subject following wound and/or tissue injury. By “scar tissue” is meant the fibrous (fibrotic) connective tissue that forms at the site of injury or disease in any tissue of the body, caused by the overproduction of disorganized collagen and other connective tissue proteins, which acts to patch the break in the tissue. Scar tissue may replace injured skin and underlying muscle, damaged heart muscle, or diseased areas of internal organs such as the liver. Dense and thick, it is usually paler than the surrounding tissue because it is poorly supplied with blood, and although it structurally replaces destroyed tissue, it cannot perform the functions of the missing tissue. It is composed of collagenous fibers, which will often restrict normal elasticity in the tissue involved. Scar tissue may therefore limit the range of muscle movement or prevent proper circulation of fluids when affecting the lymphatic or circulatory system. Glial scar tissue following injury to the brain or spinal cord is one of the main obstacles to restoration of neural function following damage to the central nervous system. A reduction in scar tissue can be assessed by the population of cell types within the injured site. For example, a reduction in glial scar tissue can be estimated by an increased ratio of neuronal to astrocytic cells. A reduction in scar tissue formation can be measured by a simple measurement of scar width or area of scar tissue. In addition, histological assessments can be made about the restoration of structural complexity within healed tissue in comparison to normal tissue.
The methods provided herein can restore normal tissue mechanical properties such as tensile strength following tissue injury in a subject. “Tensile strength” refers to the amount of stress or strain required to break the tissue or wound. The tensile strength of treated wounds can be about 60, 65, 70, 75, 80, 85, 90, 95, 100% that of uninjured tissue within about 3 months after treatment. Thus, also provided is a method of restoring tissue mechanical properties, including increasing tensile strength of a healed injury to approach or reach that of normal uninjured tissue, in a subject comprising administering to the subject one or more of the compositions of the present disclosure in a pharmaceutically acceptable carrier.
The methods provided herein can improve tissue regeneration following tissue injury in a subject. The methods can enhance stem cell differentiation following tissue injury in a subject. Enhanced stem cell differentiation can be measured by providing a clinically acceptable genetic or other means of marking endogenous or engrafted stem cells and determining the frequency of differentiation and incorporation of marked stem cells into normal tissue structures.
Additionally, the methods provided herein can reduce inflammation in a subject. A reduction in inflammation can be measured by a reduction in the density of inflammatory cell types such as, for example, monocytes or astrocytes. A reduction in inflammation can be measured by a reduction in the density of inflammatory cell types such as, for example, neutrophils, mast cells, basophils, and monocytes. A reduction in inflammation can be calculated by an in vivo measurement of neutrophil activity. In addition, factors like frequency of mast cell degranulation or measurement of histamine levels or levels of reactive oxygen species can be used as measurements of reduction in inflammation. The level of inflammation can also be indirectly measured by checking for transcription levels of certain genes by qRT-PCR for example, for genes like, Interferon-alpha, -beta and -gamma, Tumor Necrosis Factor-alpha, Interleukine 1 beta, -2, -4, -5, -6, -8, -12, -18, -23, -27, CD4, CD28, CD80, CD86, MHCII, and iNOS. Measurement of pro-inflammatory cytokine levels in the tissues and or bodily fluids of the subject including plasma can measure a reduction in inflammation.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and aspects disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and aspects of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.
The present disclosure has multiple aspects, illustrated by the following non-limiting examples.
The material and methods for use in Example 2 is provided below.
Mice were housed and maintained in the Johns Hopkins Cancer Research Building animal facility in compliance with ethical guidelines outlined by the Animal Care and Use Committee (ACUC). All procedures performed on animals were approved by Johns Hopkins ACUC. Investigators involved with the studies were blinded whenever possible. All mice used in these studies shown below in Table 1 were maintained as Helicobacter negative.
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The VML injury was performed in female mice as a bilateral surgical removal of the quadriceps femoris as previously described. A unilateral longitudinal incision measuring approximately 1.5 cm in length was made in the epidermis, dermis, and the underlying fascia above the muscle. Using sterilized microdissection scissors, a 3 mm×4 mm×4 mm full thickness segment of skeletal muscle was resected from each hindlimb. The remaining defect space was filled with 50 μL to 75 μL of vehicle (1×DPBS or treatment). Immediately after treatment the epidermis and dermis were closed using a wound clipper with 7 mm sterile wound clips (Roboz, USA).
Post-traumatic osteoarthritis (PTOA) was induced in male mice by utilizing an anterior cruciate ACLT injury model in 10-week-old male C57BL/6j mice. SEA, rSEA, and various components were administered to the joint space of the operated knee via a 30-gauge needle or intraperitoneally. The joint cavity was opened in the sham group, but the ACL was not transected. Weight-bearing in mice was measured in the un-operated control animals and compared to ACLT animals receiving PBS control or rSEA therapy using an incapacitance tester (Columbus Instruments). The percentage if weight distributed on the ACLT limb was used as an index of joint discomfort in OA (55). The mice were positioned to stand on their hind paws in an angled box placed above the incapacitance tester so that each hind paw rested on a separate force plate. The force (g) exerted by each limb was measured. Three consecutive 3-sec readings were taken and averaged to obtain the mean score (64). To determine pain response times in post-injury and treated animals, mice were placed on an enclosed hotplate set to 55° C. The latency period for hind limb response (marked as jumping or paw-licking) was recorded as the response time before surgery and 4 weeks after surgery in all animal groups (55). At least three readings were taken per mouse and averaged to obtain the mean response time for each time point. After 4 weeks, animals were sacrificed, and mouse knees were fixed in 10% neutral-buffered formalin, decalcified for approximately 2 weeks in 10% EDTA at 4° C., step-wise dehydrated in EtOH, cleared in xylenes, and embedded in paraffin. 7 μm sections were taken throughout the joint, dried, and stained for proteoglycans with Safranin-O and Fast Green (Applied biosciences) per manufacturer's instructions. Evaluation of the cartilage damage was performed according to the Osteoarthritis Research Society International (OARSI) scoring system and was performed by blinded histological assessment the medial plateau of the tibia (65). Osteophytes on the tibial plateau were scored from 0 to 3, with 0 indicating no osteophytes or an osteophyte up to 100 μm in diameter; a score of 1 indicating an osteophyte measuring 100 μm to 200 μm in diameter; a score of 2 indicating an osteophyte of 200 μm to 300 μm in diameter; and a score of 3 indicating an osteophyte measuring more than 300 μm in diameter (64, 65).
All surgical procedures were performed under the guideline of the Johns Hopkins University Animal Care and Use Committee (ACUC). Male adult (8-12 weeks old) BALB/c mice, GATA1 KO mice, and IL4-IRES-eGFP (4get) mice were purchased from Jackson Labs. The corneal debridement wound was adapted with minor modifications from Stepp et al., 2014 (56). Mice were weighed and anesthetized with 90 mg/kg ketamine HCl (VetOne) with 10 mg/kg Xylazine HCl (VetOne) by injection. Proparacaine hydrochloride ophthalmic eye drops (Sandoz) were applied after the mice were sedated. The center area of the cornea was marked by a 1.5 mm biopsy punch, and the epithelium layer was removed within the area by a 1.5 mm flat blade (Fine Science Tools). After epithelium removal, a volume of 50 μL PBS solution with or without rSEA, were injected to the subconjunctival space of the wounded eye. After injection, drops of sterile PBS solution were applied to both wounded and unwounded eyes to keep the eyes moist until the mice were recovered from anesthesia. At 14 days post-surgery, the mice were euthanized, and the eye globes were collected. The picture of each globe was taken under surgical microscope (Nikon), and the scar areas and cornea areas were determined with ImageJ. The ratio was quantified as: scar ratio=As (scar area)/Ac (whole corneal area).
S. mansoni Egg Collection and Isolation of SEA
S. mansoni (from infected NMRI mice) reagents were provided by the NIAID Schistosomiasis Resource Center of the Biomedical Research Institute (Rockville, MD) through NIH-NIAID Contract HHSN272201700014I, supplied frozen at −80° C. Standard SEA was prepared according to standard operating procedures utilized by the center and based on Boros et. al (28). After thawing on ice in the dark, eggs were re-suspended in 4° C. 1×DPBS at a concentration of 100,000 eggs/mL and were homogenized on ice using a motorized pestle, or with a 2 mL dounce homogenizer (Kimble, USA). 95% to 100% of the eggs were disrupted, verified by visualization with a phase contrast microscope. The crude mixture was then centrifuged at 4° C. at 200×g for 45 minutes. The supernatant was retrieved and ultracentrifuged for 90 min at 100,000×g at 4° C. The entirety of the final supernatant was passed through a 0.22 μm sterile filter and stored at −80° C. Concentrations were determined using standard Bradford assays and the Qubit™ Protein Assay Kit (Invitrogen).
Isolation of rSEA Formulations
S. mansoni eggs were homogenized to isolate SEA as stated above with several modifications in the extraction process to generate rSEA. Initially the eggs are homogenized to 95% to 100%, verified by phase contrast microscopy. Centrifuged at 21,000×g for 45 minutes, then ultra-centrifuged at 100,000×g at 4° C. for 90 min. After ultra-centrifugation, an insoluble mixture that forms at the top layer is harvested and stored in sterile low-protein binding 1.5 mL Eppendorf tubes. The top half of the resulting soluble antigen volume is carefully removed by pipet, and sterile filtered using a low-protein binding 0.2 μm filter into a low-protein binding 1.5 mL tube. 900 μL of the soluble fraction is then combined with 100 μL of the lipid fraction that was sterile filtered using lipid 1.2 μm medical Supor disc filters (B Braun Medical, USA). The final mixture is then stored at −80° C. Concentrations were determined using standard Coomassie Bradford assays (ThermoFisher) and the Qubit Protein Assay Kit (Invitrogen).
Protein concentration of SEA and rSEA samples were measured prior to lipid analysis using a Qubit Protein Assay Kit (Invitrogen, Q33211). SEA or rSEA solutions at 2 mg/mL protein concentration were mixed 1:1 with chloroform, vortexed, and centrifuged at 15,000×g for 1 minute at 4° C. The organic phase of the resulting mixture was removed, dried with nitrogen gas, and resuspended in a 2:1:1 mixture of isopropyl alcohol/acetonitrile/water for LC-MS. Reversed-phase HPLC was performed with a C18 column (Phenomenex, 00D-4726-AN), MS was performed with a Bruker timsTOF Pro instrument, and post-run analysis was performed with Bruker MetaboScape software.
Preparation of Decellularized Extracellular Matrix from Porcine Small Intestines
Decellularized extracellular matrix (ECM) was produced from porcine small intestinal sub-mucosa (SIS) following procedures developed under Stephen Badylak and described in Keane, et al., with minor modifications (66). Fresh porcine small intestines were obtained from Wagner Meats (Maryland, USA), harvested from a five-year old animal. The tissues were thoroughly washed to remove debris and mechanically processed to remove mucosal, serosal, and muscular layers by scraping with sterile pyrogen-free plastics. The resulting tissue identified as SIS include the submucosa and basilar layers of the tunica mucosa, was treated using 0.1% peracetic acid (Sigma Aldrich) and 4% EtOH prepared type-1 sterile water in pyrogen-free plastics for 2 hrs while stirring. The ECM was then returned to neutral pH using serial washes of quality-1 water and sterile culture grade 1×DPBS. Upon return to neutral pH the samples were lash frozen in liquid nitrogen and lyophilized. All tissues were then cryo-milled in liquid nitrogen to particle mesh sizes approximately <400 μm and stored at −20° C. until use.
To enable delivery of rSEA, a vitrified ECM was utilized to combine the benefits of ECM biomaterials with rSEA for enhancing pro-regenerative outcomes. SIS-ECM vitrigels were verified to enable a measurable release of biological payloads while acting as an immunomodulator for immune type 2 responses. SIS-ECM is digested with 1 mg of pepsin (Sigma Aldrich) and 10 mg of ECM with 0.01 HCl in type-1 water, covered, and stirred for 48 hrs at room temperature. The working solution is then cooled on ice and neutralized with 1 mL of a 60 mM HEPES and 0.05 mM NaOH solution, and therapeutics like rSEA are added immediately after the neutralization is confirmed by a pH of 7. Gelation is then allowed to occur at 37° C. for 2 hrs and then placed into a 40° C. vitrification chamber for 7 days. Just before implantation, the vitrified gels are hydrated with 100 μL to 200 μL and trimmed into 2 mm×2 mm pieces.
Gene Expression Tissue Processing and qRT-PCR
Harvested tissues were immediately placed into RNALater for at least 24 hrs at 4° C., transferred into TRIzol reagent (Thermo Fisher Scientific), flash frozen, and stored in a −80° C. freezer. For mRNA isolation, samples were homogenized using a Bead Ruptor 12 (OMNI International) using the highest speed setting for 3 rounds of 15 secs with 2.8 mm ceramic beads (OMNI International). RNA was isolated from whole tissue using TRIzol reagent and chloroform extraction. RNA was purified using Qiagen's RNeasy PLUS kits (mini-kit and micro-kit), with gDNA eliminator columns. All qRT-PCR was performed using TaqMan Gene Expression Master Mix (Applied Biosystems) and TaqMan probes according to manufacturer's instructions. Briefly, 2.5 μg of mRNA was used to synthesize cDNA using Superscript IV VILO Master Mix (ThermoFisher Scientific) utilizing manufacturer guidelines with a C1000 Touch Thermocycler (Bio-Rad). The cDNA concentration was set to 100 ng/well (in a total volume of 20 μL qRT-PCR reaction). The qRT-PCR reactions were performed on the StepOne Plus Real-Time PCR System and software (Applied Biosystems, ThermoFisher Scientific), as TaqMan single-plex FAM-MGB assays, TaqMan Gene Expression Master Mix, using manufacturer recommended settings for quantitative and relative expression. All qRT-PCR reactions were performed in 96-well MicroAmp Fast Optical Plates (Life Sciences). For tissue samples, B2m, Rer1, Hprt, and Ppia were used as endogenous controls (reference housekeeping genes), with samples normalized to the most stable endogenous control. Samples were normalized to vehicle treated (saline) controls, unless otherwise stated. All qRT-PCR data was analyzed using the Livak Method, wherein ΔΔCt values are calculated and reported as relative quantification values (RQ), established by the result of the 2−ΔΔCt calculation (67). These results were further verified by analysis using the appliedbiosystems relative quantification online software (Thermo Fisher Scientific, ver. 2020.2.1-Q2-20-build4). RQ, same as fold-change (FC), values are represented by the geometric means with error bars representing the geometric standard deviation or by Log2 (FC) wherein the data are displayed linearly as means with the error bars representing standard deviation. All qRT-PCR assays were completed within the laboratory at Johns Hopkins. Table 2 shows the murine TaqMan gene expression assay probes used.
Tissue samples were obtained by cutting the quadriceps femoris muscle from the hip to the knee. Tissues were finely diced and digested for 45 min at 37° C. with 1.67 Wünsch U/mL (5 mg/mL) of Liberase TL (Roche Diagnostics, Sigma Aldrich) and 0.2 mg/mL DNase I (Roche Diagnostics, Sigma Aldrich) in RPMI-1640 medium supplemented with L-Glutamine and 15 mM HEPES (Gibco). The digested tissues were ground through 70 μm cell strainers (ThermoFisher Scientific) with excess RPMI-1640 (supplemented as before), and then washed twice with 1×DPBS. A discontinuous Percoll (GE Healthcare) density gradient centrifugation was used to enrich the leukocyte fraction (80%, 40%, and 20% layers) and to remove blood and debris from the muscle samples, centrifuged at 2,100×g for 30 min with the lowest acceleration, no brake, at room temperature. For intracellular staining, cells were stimulated for 4 hrs with Cell Stimulation Cocktail Plus Protein Transport Inhibitors (eBioscience) diluted in complete culture media (RPMI-1640 supplemented with 10% FBS, 15 mM HEPES, and 5 mM Sodium pyruvate). Cells were washed and surface stained, followed by fixation/permeabilization (Cytofix/Cytoperm, BD), and then stained for intracellular markers. Flow cytometry was performed using Attune N×T Flow Cytometer (ThermoFisher Scientific). Gating schemes are provided in
T cells (Live CD45+CD11b CD3+Singlets) and macrophages (Live CD45+CD11b+CD3-F4/80Hi Singlets) were sorted from quadriceps femoris muscles 1-week post-injury. Tissue processing is the same as described above for flow cytometry, but without Percoll isolation. Only viability and surface staining were performed for FACS, and these experiments were performed using a BD FACSAria Fusion SORP. The cell sort gating scheme is provided in
Dystrophin (rabbit anti-mouse monoclonal antibody, clone EPR21189, Abcam, dilution: 1:1000) was stained using tyramide signal amplification (TSA) method with Opal-650. Briefly, after blocking with bovine serum albumin, the first primary antibody was incubated at room temperature for 30 mins, followed by 30 mins of incubation with HRP polymer conjugated secondary antibody, and 10 mins of Opal-650. Slides were then counterstained with DAPI for 5 mins before being mounted using DAKO mounting medium. Imaging of the histological samples was performed on a Zeiss AxioObserver.Z2 and images were stitched on Zen Blue software.
Wounded corneas were collected from each experimental group (Saline vs. rSEA), 4-5 corneas were pooled for one “flow cytometry sample”. Cornea samples were processed similar to what is described in Ogawa, et al., with minor modification (68). Briefly, corneas in each group were digested in RPMI-1640 media containing 0.5 mg/mL Liberase TL (Sigma Aldrich)+0.2 mg/mL DNase I (Roche) for 45 min while gently rocking. Digested tissues were ground through 70 μm cell strainers and digestion stopped with FBS supplemented RPMI-1640. Cell suspensions were centrifuged, washed, and each cell pellet was resuspended in 200 μL 1×DPBS for staining and blocked with anti-mouse CD16/32 TruStain FcX (BioLegend) per manufacturer recommendations. The antibodies used were listed in Table 5 below.
Draining lymph nodes (submandibular lymph nodes) were collected and grinded through a 70 μm filter. Cells were collected after centrifugation and washing with 1×DPBS. Lymphocytes were stimulated for 4 hours with Cell Stimulation Cocktail, plus protein transport inhibitors (eBioscience), followed by staining of surface markers. After permeabilization and fixation of cells, cytokines IL-17A, and IFN-γ were stained for 4get mice and IL-17A, IL-4, and IFN-γ were stained for wild-type (WT) and GATA1 KO mice. The antibodies used are listed in Tables 6 and 7 below.
Dissected corneas were fixed in 100% methanol at −20° C. for 30 min, and permeabilized with PBS containing 0.25% Triton-X (PBST). The cornea samples were blocked with 1% goat serum+1% BSA in PBST for 30 mins and stained with rabbit anti-mouse αSMA (Abcam) overnight at 4° C. Following washing with PBST, corneas were stained with goat anti-rabbit 633 for 2 hrs at room temperature, and mounted flat in SlowFade Diamond Antifade Mountant (Thermo Fisher Scientific). Zeiss Apotome microscope was used for fluorescent imaging.
Data points for all in vivo experiments are biological replicates and were not randomly assigned. Investigators were not formally blinded to separation during experiments and outcome assessment, except for histological assessment for scoring. All experiments were independently replicated with similar results and trends at least twice, except sorted T cell and macrophages NanoString results in
All flow cytometry data was visualized, analyzed, and gated using FlowJo (version 10.7.1 for Windows, BD Life Sciences). Gating for positive populations utilized fluorescence-minus-one controls, and all reported populations for these studies are from events on-scale, singlets (using the diagonal gating of FSC-Height vs. FSC-Area cells, live (negative for amine-reactive dye stain), CD45-positive events, with negative/positive gating as shown in
Development of a Type 2 Immunotherapy from S. mansoni Eggs Antigens
The soluble egg antigen (SEA) extract from S. mansoni is composed of hundreds to thousands of proteins, glycoproteins, and lipids depending on the extraction protocol, and is well recognized in its ability to stimulate a type 2 immune response (31-34). It was first sought to determine if SEA could efficiently stimulate a type 2 immune response that would promote tissue repair without deleterious inflammation or fibrosis. S. mansoni eggs isolated from infected mice were mechanically disrupted and ultracentrifuged to remove insoluble components to isolate SEA as described by Boros, et al. (
To determine if SEA could be formulated to stimulate a dedicated type 2 regenerative immune response without increased interferon-dependent pathological inflammation, the SEA isolation was modified to extract specific layers of the ultracentrifuged egg supernatant. In particular, the SEA was further purified by isolating the lower density soluble fraction and combined it with the lipid portion (See,
rSEA Treatment Increases IL4-Expressing Eosinophils, TH2 T Cells, and Regulatory T Cells in a Volumetric Muscle Loss Injury
Since type 2 immune signals are critical for muscle injury repair, the kinetics and cellular sources of IL-4 were evaluated after rSEA treatment. A single treatment of rSEA at the time of injury increased Il4 gene expression in the muscle tissue 20- to 30-fold, relative to saline treated muscles, at one-week post-surgery (
Eosinophils are well recognized as a source of IL-4 after muscle injury the changes in these cell types after rSEA treatment was first evaluated using multiparametric flow cytometry. Eosinophils (CD11b+SiglecF+SSCHi), which depend on type 2 immune cytokines and chemokines such as IL-5 and eotaxin, were the most abundant CD11b+ cell population in the muscle 1-week post rSEA treatment, producing an 11-fold increase in the number of cells that remained significantly higher compared to saline treated controls at 3-weeks post-treatment (
At 1-week post-injury, bulk-RNA sequencing was performed on rSEA treated skeletal muscle and found a wide variety of differential gene expression relative to the saline treated muscles (
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A direct comparison of the transcriptional responses to SEA versus rSEA in muscle 1-week was also performed after treatment and found notable decreases among multiple inflammatory signatures after rSEA treatment, and it was confirmed that most of the type 2 transcriptional responses were alike (
Previous studies with ECM biomaterials demonstrated that TH2 cells are critical for the pro-regenerative response to ECM biological scaffolds so the T cell response to rSEA was further examined (4, 37, 38). At 1-week post-treatment, CD3+ T cell numbers increased in the muscle wound with CD3+CD4+ T cells increasing over 7-fold in the muscle with rSEA treatment compared to saline treated controls. Treatment with rSEA significantly increased TH2 (CD3+CD4+GFP+ (IL4+)) as a percentage of CD3+ cells in the muscle of 4get mice (
T cell gene expression changes with rSEA treatment was further characterized by sorting CD3+ T cells from 1-week post-injury muscle and analyzing gene expression signatures with the NanoString multiplex system. There were 20 genes significantly upregulated and downregulated in T cells with rSEA treatment (
In particular, rSEA treatment of the muscle wound significantly upregulated TH2 associated gene signatures including Il13, Gata3, and Cd4. Expression of Tnfrsf18, Ccr4, Il10, and Il10ra also significantly increased in the T cells after rSEA treatment suggesting a role for regulatory T cells (Tregs). Flow cytometry confirmed a significant increase in Tregs (CD3+CD4+Foxp3+) in the muscle with rSEA treatment compared to saline controls on days 5 and 7 post-injury (
The draining inguinal lymph nodes (iLNs) also revealed type 2 immune stimulation after rSEA treatment including a significant increase in IL4+CD4+ TH2 cell percentage in 4get mice (
rSEA Induces Alternatively Activated Macrophage Gene Expression in Muscle Wounds
Macrophages are another immune cell type that is central to tissue repair with alternatively activated macrophages associated with productive wound healing (40). The number of macrophages in the muscle tissue had no significant change with rSEA treatment (
The most strongly downregulated gene, Cysltr1, encodes for cysteinyl leukotriene receptor-1, a potent mediator of allergic inflammation. In contrast to an active helminth infection, rSEA treatment resulted in downregulation of genes such as Tlr2, Tlr4 and Jun in the sorted myeloid cells compared to saline treated controls. This suggests that the rSEA formulation can induce the regenerative components of the type 2 immune helminth response without the deleterious infection response component.
rSEA Stimulation of Type 2 Immunity Correlates with Increased Muscle Repair
IL-4 expressing eosinophils, TH2 cells and regulatory T cells are all associated with muscle repair after traumatic injury (4, 5, 39, 41). To assess whether rSEA stimulation of type 2 immunity also benefitted muscle repair, healing and fibrosis were assessed with histology and further expression analysis at early (1-week) and late (6-week) timepoints. At 1-week post-surgery time point, where it was found that broad type 2 immune stimulation, there was a significant increase in expression of genes associated with muscle satellite cell activation (Pax7, Myod1, Myf5, Myog, Mymk, and Areg) and myofiber fusion and development (Myh3, Myh8, and Myl2) with rSEA treatment compared to saline treated controls (
rSEA Treatment Decreases IL-17A Producing CD4+ and γδ+ T Cells
Schistosome eggs deposited in tissues can induce fibrosis over time and induce granuloma formation similar to the foreign body response. Furthermore, SEA has been used as a model for fibrosis when coated on glass, -sepharose, and -polystyrene beads in multiple tissues including liver and lung (28, 42). As fibrosis and granuloma formation are not desirable outcomes in tissue repair, it was therefore sought to further evaluate immunological features associated with fibrosis, specifically type 3 (17) immune cells (43, 44). Expression of type 3 immune-associated genes including Il23a, Rorc, Ptgs2 (COX2) and Il6 decreased in the muscle tissue 1-week after injury and rSEA treatment (
Release of rSEA from a Vitrified ECM Hydrogel Enhances the Type 2 Regenerative Response
Larger tissue defects may require a scaffold to enable cell migration and the repair of larger tissue volumes. Biological scaffolds derived from tissue ECM are used clinically for wound healing and reconstruction applications. Preclinical and clinical studies demonstrate that ECM materials can induce type 2 immune responses and promote tissue repair (37, 47, 48). Although the effects of a single dose of rSEA persist for weeks, the ability to incorporate rSEA into a biomaterial would provide a scaffold for larger tissue defects and deliver the immunotherapy over an extended period of time.
Biological scaffolds based on ECM can be processed into several material forms without compromising their regenerative capacity. Clinically formulations include powders, sheets, and hydrogels (49). Drugs can also be encapsulated into ECM hydrogels, although these gels are weak and quickly dissolve (38). To create more robust ECM hydrogels that can release proteins and lipids in a controlled manner, a vitrification process was applied that evaporates water in a controlled humidity and temperature so that the macromolecular assembly can occur while a drug or biologic is encapsulated (
The complex protein and lipid mixture of rSEA makes controlled release difficult to evaluate. Therefore, it was sought to evaluate rSEA-encapsulated vitrigels functionally by assessing their impact on wound healing and immune profile skewing. Particles of vitrified ECM, with or without encapsulated rSEA, were applied to the murine VML. A single dose of rSEA and rSEA-encapsulated vitrigel stimulated similar levels of Il4 in the muscle tissue at one week, however only the vitrigel+rSEA extended the increased Il4 expression to the 3-week time point compared to a single dose of rSEA, with a 30-fold increase in Il4 expression (
rSEA Promotes Healing in Articular Cartilage and Cornea Tissue Injury Models
A type 2 immune response and IL-4 expression is associated with repair in multiple tissues beyond muscle including liver, articular cartilage, the central nervous system, and skin (21, 26, 41, 52-54). To determine if rSEA could be broadly applied to promote regeneration in tissues beyond muscle, the therapeutic potential of rSEA in cartilage and cornea injury models was evaluated in male mice. For cartilage repair, the anterior cruciate ligament transection (ACLT) model was used that induces articular damage, loss of cartilage and development of osteoarthritis (55). rSEA intra-articular (IA) was injected two and three weeks after the ACLT injury (
Histological assessment of the articular joint structure and cartilage using Safranin-O staining for proteoglycans found that rSEA resulted in higher proteoglycan staining in the cartilage layer, improved tissue structure, and trended in higher levels of repair quality as measured by the semi-quantitative OARSI scoring system compared to vehicle controls (
The therapeutic potential of rSEA in a cornea wound that is similarly characterized by poor healing capacity and scar formation when damaged was evaluated. In the corneal debridement injury model, resident stromal cells are activated leading to fibrosis and scarring which results in limited vision (
The immune response in the cornea was then examined after wounding and rSEA treatment to see if an increased type 2 profile correlated with increased tissue repair similar to the muscle and cartilage. A few changes were observed in the immune cell populations in the cornea tissue as measured by flow cytometry which may be due to the small cell numbers even when multiple corneas are combined (
Since rSEA treatment increased eosinophil migration in other tissues it was hypothesized that eosinophils may be contributing to rSEA-mediated cornea repair. In the Δdb1GATA model that does not have eosinophils, the scar area significantly increased in size with or without rSEA treatment (
In this study, a pro-regenerative immunotherapy derived from fractionated helminth parasite egg antigens was designed and its ability to enhance wound healing and deter fibrosis post-traumatic injury across three injury models demonstrated. Taking the eggs from S. mansoni helminths, an alternative formulation from the soluble egg antigen, rSEA was derived. It was shown that the rSEA stimulated a type 2 immune signature in lymphoid cells and myeloid cells, further decreasing pro-inflammatory immune polarization, and later timepoints revealed decreased levels of fibrosis associated with inhibition of TH17 and γδ+IL-17A+ cells. Application of rSEA to muscle, cornea, and articular joint injuries generally improved tissue healing assessed by gene expression signatures, cell populations, and/or histological assessment. Controlled release of rSEA from a natural sourced and decellularized biomaterial hydrogel further promoted healing and regeneration of larger tissue volumes. The rSEA formulation described herein, particularly in the form of an ECM hydrogel, is therefore a regenerative immunotherapy with potentially broad application to tissue repair and homeostasis, though several vital questions remain for exploration in future work such as the optimal formulations and scaling of SEA to benefit healing, deleterious off-target effects, and immunotherapy-induced susceptibility to other pathogens during treatment.
A type 2 immune response is central to how the immune system responds to helminth infection. While the type 2 response has long been considered an anti-helminth response, it is also now believed that helminths may induce this anti-inflammatory immune signature to repair the damage caused to the host, thereby enhancing mutual survival. Recent studies highlight the importance of the context of expression of type 2 associated molecules that are important in dictating outcomes, such as amphiregulin, IL-13 and IL-33 (20). IL-4 and type 2 immunity is associated with tissue repair and healing in multiple tissue types including liver (52), bone (53), cartilage (48), muscle (4, 5), corneal (57), and nervous tissues (26, 54), suggesting that rSEA may be broadly applicable for tissue repair (18, 20, 21). Treatment with rSEA induced an immune profile that included eosinophils and TH2 cells producing significantly higher levels of IL-4, IL-5, and IL-13 protein or gene expression compared to injured tissue without treatment. The type of tissue where injury and treatment may impact which cells are responding to rSEA and promoting tissue repair. In these studies, application of rSEA to a cornea wound in the Δdb1GATA murine cornea injury model completely abolished repair. However, in skeletal muscle injuries the Δdb1GATA mice did not ablate pro-healing gene expression signatures induced by rSEA despite their well-recognized role in muscle tissue repair (5). This suggests that rSEA may activate multiple immune cell populations to promote repair that differ according to tissue type. It is also likely that rSEA influences stromal, stem, or progenitor cell populations in addition to immune cells as Helminth infections were shown to stimulate stem cells in the intestinal niche (19).
Sex differences in immune responses contribute to autoimmune disease, infection and vaccine responses. While sex differences in the immune response to tissue damage and contributions to repair are likely, these differences are not well-studied and currently remain largely unknown. In these studies, the rSEA response in a muscle injury in female mice and the articular and cornea injuries in male mice was tested so they could be compared to previous work in the respective fields (58-61). The rSEA treatment improved tissue repair in both male and female mice in their specific injuries.
It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.
Various changes and modifications to the disclosed aspects will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure, may be made without departing from the spirit and scope thereof.
The present application is a continuation of International Application No. PCT/US2023/016483 filed on Mar. 28, 2023, which claims priority to U.S. Application No. 63/324,908 filed on Mar. 29, 2022, the contents of which are herein incorporated by reference.
This invention was made with government support under W81XWH-19-1-0576 awarded by the Defense Health Agency, Medical Research and Development Branch, and AR076959 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63324908 | Mar 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2023/016483 | Mar 2023 | WO |
| Child | 18894513 | US |
