BR112018068493B1 - USE OF ESTROMAL STEM CELLS - Google Patents

Abstract

Provided here are expanded, allogeneic, adipose tissue-derived stromal stem cells for use in the treatment of complex perianal fistulas in Crohn's Disease.

Description

FIELD OF INVENTION

[001] This invention relates to stem cells derived from adipose tissue for use in the treatment of refractory complex perianal fistulas in patients with Crohn's Disease.

HISTORY OF THE INVENTION

[002] Generally speaking, a fistula is an abnormal connection or passage between organs or vessels that do not connect in a normal way. Fistulas can develop in different parts of the body. For example, types of fistulas, named for the areas of the body in which they occur, include anorectal fistula or fistula in the anus or fecal fistula (between the rectum or other anorectal area and the skin surface), arteriovenous fistula or A-V fistula (between an artery and vein), biliary fistula (between the bile ducts to the skin surface, usually caused by gallbladder surgery), cervical fistula (abnormal opening in the cervix), craniosinusal fistula (between the intracranial space and a paranasal sinus), enteroenteral fistula (between two parts of the intestine), enterocutaneous fistula (between the intestine and the skin surface, namely, the duodenum or the jejunum or the ileum), enterovaginal fistula (between the intestine and the vagina), gastric fistula (between the stomach to the skin surface), metroperitoneal fistula (between the uterus and the peritoneal cavity), perilymphatic fistula (a laceration between the membranes between the middle and inner ears), pulmonary arteriovenous fistula (between an artery and vein in the lungs, resulting in shunting of blood ), rectovaginal fistula (between the rectum and vagina), umbilical fistula (between the navel and intestine), tracoesophageal fistula (between the breathing and feeding tubes) and vesicovaginal fistula (between the bladder and vagina). Causes of fistulas include trauma, complications from medical treatment, and illness.

[003] Treatment for fistulas varies depending on the cause and extent of the fistula, but generally involves surgical intervention. Several surgical procedures are commonly used, most commonly fistulotomy, insertion of a seton (a cord that is passed through the fistula path to keep it open for drainage), or an endorectal flap procedure (when healthy tissue is pulled from the inner side of the fistula to keep feces and other material from reinfecting the canal). Surgery for anorectal fistulas is not without side effects, including remission, reinfection, and incontinence.

[004] Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are the main causes for anorectal, enteroenteral and enterocutaneous fistulas. The reported incidence of fistula in Crohn's disease ranges from 17% to 50%. The management of fistulas in patients with Crohn's Disease continues to present an extremely challenging problem, as many of these fistulas do not respond to available treatments. These fistulas and their remission are a very painful complication that significantly reduces the quality of life of affected patients. Recent improvements in medical treatment (e.g., Infliximab® treatment) and specialized surgical management have decreased the need for complicated surgery. However, many patients are not cured. Failure of fistulas to heal is likely due to the suboptimal quality of tissues that have been affected by Crohn's disease. In fact, Crohn's fistulas provide a model system for wound healing under some of the worst possible conditions.

[005] Perianal fistulas are a common complication of Crohn's disease, A1 which are estimated to affect up to 28% of patients in the first two decades after diagnosis, A2, A3 particularly, those with colonic disease and rectal involvement. A4 They seriously compromise the patients' quality of life and cause considerable morbidity.A5, A6 Approximately, 70-80% of perianal fistulas are complex,A3, A7 and they are challenging to treat, as they are particularly refractory to conventional treatment strategies (antibiotics, immunosuppressants ) and anti-tumor necrosis factor (anti-TNF) therapies.

[006] Failure or intolerance to medical therapy can ultimately result in debilitating surgical approaches such as stoma surgery or prosthetic surgery.A19 Therefore, there remains a huge unmet need for alternative medical treatments.

[007] Mesenchymal stromal cells (MSCs) are non-hematopoietic stromal cells that are capable of differentiating into mesenchymal tissues, such as bone, cartilage, muscle, ligament, tendon and adipose. MSCs can be easily isolated from tissues such as bone marrow or adipose tissue and rapidly expanded in culture. WO-A-2006/136244 describes the treatment of fistulas using compositions containing stromal stem cells derived from adipose tissue. Adipose tissue-derived mesenchymal stromal cells are a promising new therapeutic approach, which may be useful for the treatment of complex perianal fistulas, due to their anti-inflammatory and tissue regeneration potential. A20-A22 Initial proof of concept has been previously obtained in an open-label, phase 1/2a clinical study of expanded, allogeneic, adipose-derived stem cells (eASC, Cx601) in 24 Crohn's Disease patients with complex perianal fistulas with 56.3% of patients experiencing complete closure of the opening external and the absence of collections measured by MRI of the treated fistula, 24 weeks after treatment.A23

[008] Complex perianal fistulas in Crohn's Disease are particularly challenging to treat and there remains a need for the establishment of clinically proven therapies for the treatment of complex perianal fistulas in Crohn's Disease.

SUMMARY OF THE INVENTION

[009] The invention relates to the provision of clinically proven therapies to treat refractory complex perianal fistulas in Crohn's Disease, based on the results of a multicenter, double-blind, placebo-controlled study that evaluated the efficacy and safety of eASC in 212 adults with Crohn's disease and refractory to treatment, draining complex perianal fistulas. The Examples here describe the first phase 3 placebo-controlled study evaluating the efficacy and safety of expanded allogeneic adipose tissue-derived stem cells alone or in addition to current medical therapy for treatment-refractory complex perianal fistulas in patients with Crohn's Disease. The results reported here are 24-week primary and secondary endpoints.

[010] New clinical study data surprisingly revealed that adipose tissue-derived stem cells treat complex multiple tract perianal fistulas especially effectively. The data show that the greatest numerical effect of cells was observed in patients who were receiving anti-TNF and immunosuppressive therapies at the time of fistula preparation. Additionally, clinical remission was achieved shortly after treatment, with the median time to clinical remission in the treatment group being 6.7 weeks. Similarly, the average time to response was 6.3 weeks. Improvement in the Perianal Disease Activity Index (PDAI) was significantly greater at weeks 6, 12, and 18. Overall, the data reveal that allogeneic eASCs are a surprisingly effective therapy for complex perianal fistulas in patients with Crohn's disease, so a A single administration is capable of providing a rapid and sustained therapeutic effect even in very complex fistulas, which are more difficult to treat, when previous drug therapy has failed.

[011] A first aspect of the invention provides expanded, allogeneic, adipose tissue-derived stromal stem cells for use in a method of treating a refractory complex perianal fistula in a patient having Crohn's Disease.

[012] A second aspect of the invention provides the use of expanded, allogeneic, adipose tissue-derived stromal stem cells in the manufacture of a medicament to treat a refractory complex perianal fistula in a patient having Crohn's Disease.

[013] A third aspect of the invention provides a method of treating a refractory complex perianal fistula in a patient having Crohn's disease, in a patient in need of such treatment, comprising the step of administering stromal stem cells derived from adipose tissue, allogenic, expanded to the fistula.

[014] Also disclosed here, among other things, are compositions containing stromal stem cells derived from adipose tissue. The adipose tissue-derived stromal stem cell-containing compositions described herein have a distinct phenotype and exhibit beneficial homogeneity of phenotype, thus making them more suitable for use in the treatment of fistulas. Compositions containing adipose tissue-derived stromal stem cells can be formulated with solutions or other substances to serve as pharmaceuticals or medical devices, for example, as sutures or adhesives. Furthermore, innovative methods of treating complex perianal fistulas using stromal stem cells derived from adipose tissue, as well as kits for their practice, are provided.

BRIEF DESCRIPTION OF FIGURES

[015] Figure 1 depicts the design of the Phase III clinical study. Cx601, expanded, allogeneic, adipose tissue-derived stem cells; ICF, free and informed consent form; MRI, magnetic resonance imaging.

[016] Figure 2 summarizes the Patient's disposition. Cx601, expanded, allogeneic, adipose tissue-derived stem cells; ITT, intention to treat; mITT, modified intention to treat; *Deep vein thrombosis, Crohn's crisis, intestinal obstruction, Crohn's disease, anal abscess (n=3); tFistula, proctalgia, anal abscess (n=4); ΦNo healing or worsening of symptoms; new course of antibiotics; new surgery in the perianal region; §Worsening of Crohn's disease requiring changes in therapy.

[017] Figure 3 presents the primary endpoint: Combined clinical and radiological remission* at week 24 in the ITT population (Panel A). Combined clinical and radiological remission* at week 24 in the mITT population (Panel B). Combined remission* by week 24, according to randomization stratification factors, i.e. Crohn's Disease treatments being received at the time of randomization, in the mITT population (Panel C). Cx601, expanded, allogeneic, adipose tissue-derived stem cells; IS, immunosuppressant; ITT, intention to treat; mITT, modified intention to treat; TNF, tumor necrosis factor. *Clinical assessment of closure of all treated external openings that were draining at baseline, and absence of collections > 2 cm from treated perianal fistulas in > 2 of 3 dimensions on centrally blinded MRI assessment at approximately week 24. Clinical assessment Closure was defined as absence of drainage despite non-invasive finger compression.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions:

[018] As used herein, the following terms and phrases shall have the meanings set forth below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains.

[019] The articles “one” and “one” refer to one or more than one (that is, at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[020] By “adipose tissue” we mean any fat tissue. Adipose tissue can be brown or white adipose tissue, derived from subcutaneous, omental/visceral, mammary, gonadal, or other adipose tissue sites. Typically, adipose tissue is subcutaneous white adipose tissue. These cells can compromise a primary cell culture or an immortalized cell line. Adipose tissue can be from any organism having fat tissue. Typically, adipose tissue is mammalian, more typically, adipose tissue is human. A convenient source of adipose tissue is from liposuction surgery, however, the source of adipose tissue or the method of isolating adipose tissue is not crucial to the invention.

[021] “Adipose tissue-derived stromal stem cells” or “ASCs” refer to mesenchymal stem cells that originate from the stromal fraction of adipose tissue, generally speaking, from human adipose tissue (hASCs).

[022] The term “adhesive” refers to any substance that joins or bonds surfaces together; for example, a glue.

[023] The term “biological medicinal product” should be considered to mean a protein or nucleic acid-based pharmaceutical substance for therapeutic use, which is typically produced by means other than direct extraction from a natural biological source (not designed ).

[024] The term “cellular composition” refers to a preparation of cells, this preparation may include, in addition to cells, non-cellular components, such as cell culture medium, for example, proteins, amino acids, nucleic acids, nucleotides, coenzyme, antioxidants, metals and the like. Furthermore, the cellular composition may have components that do not affect the growth or viability of the cellular component, but that are used to provide the cells in a particular format, for example, as a polymeric matrix for encapsulation or a pharmaceutical preparation.

[025] A “complex perianal fistula” is a perianal fistula having one or more of: (i) inter, trans, extra or high suprasphincteric origin; (ii) > 2 external openings; or (iii) associated collections.

[026] The complex perianal fistula can optionally have a maximum of 2 internal and 3 external openings. Furthermore, the complex perianal fistula may have been drained for at least 6 weeks prior to treatment, in accordance with the invention.

[027] The term “culture” refers to any growth of cells, organisms, multicellular entities, or tissue in a medium. The term “culture realization” refers to any method of achieving this growth, and can comprise multiple stages. The term “performing additional culture” refers to culturing a cell, organism, multicellular entity, or tissue at a particular stage of growth, then using another culture method to take said cell, organism, multicellular entity or tissue to another growth stage. A “cell culture” refers to the growth of cells in vitro. In this culture, cells proliferate, but do not organize themselves into tissue per se. A “tissue culture” refers to the maintenance or growth of tissue, for example, explants of a primordial organ or an adult organ in vitro, in order to preserve its architecture and function. A “monolayer culture” refers to a culture in which cells multiply in a suitable medium while primarily attached to each other and to a substrate. Furthermore, a “suspension culture” refers to a culture in which cells multiply while suspended in a suitable medium. Similarly, a “continuous flow culture” refers to the cultivation of cells or explants in a continuous flow of fresh medium to maintain cell growth, e.g. viability. The term “conditioned medium” refers to a supernatant, e.g., free from the cultured cells/tissue, resulting after a period of contact with the cultured cells, such that the medium has been altered to include certain paracrine factors and/or or autocrines produced by cells and secreted into the culture. A “confluent culture” is a cell culture in which all cells are in contact and thus the entire surface of the culture vessel is covered, and implies that the cells have also reached their maximum density, although confluence does not necessarily mean that the division will cease or that the population will not increase in size.

[028] The term “culture medium” or “medium” is recognized in the art, and refers, generally, to any substance or preparation used for the cultivation of living cells. The term “medium”, as used in reference to a cell culture, includes the components of the environment surrounding the cells. The media can be solid, liquid, gaseous or a mixture of phases and materials. Media include liquid growth media as well as liquid media that do not support cell growth. Media also include gelatinous media such as agar, agarose, gelatin, and collagen matrices. Exemplary gaseous media include the gaseous phase to which cells growing in a petri dish or other solid or semisolid support are exposed. The term “medium” also refers to material that is intended for use in a cell culture, even if it has not already come into contact with the cells. In other words, a nutrient-rich liquid prepared for bacterial culture is a medium. Similarly, a powdered mixture that, when mixed with water or other liquid, becomes suitable for cell culture, can be called “powdered medium”. “Defined media” refers to media that are made from chemically defined (commonly purified) components. “Defined media” do not contain poorly characterized biological extracts such as yeast extract and beef broth. “Rich environment” includes media that are intended to support the growth of most or all viable forms of a particular species. Rich media often include complex biological extracts. A “medium suitable for growth of a high-density culture” is any medium that allows a cell culture to reach an OD600 of 3 or more when other conditions (such as temperature and oxygen transfer rate) permit such growth. The term “basal medium” refers to a medium that promotes the growth of many types of microorganisms that do not require any special nutrient supplements. Most basal media generally comprise four basic chemical groups: amino acids, carbohydrates, inorganic salts, and vitamins. A basal medium usually serves as the basis for a more complex medium, to which supplements such as serum, buffers, growth factors, lipids, and the like are added. Examples of basal media include, but are not limited to, Eagle's Basal Medium, Minimum Essential Medium, Dulbecco's Modified Eagle's Medium, Medium 199, Ham's F-10 and Ham's F-12 Nutrient Blends, Mc Coy's 5A, Dulbecco's MEM/F-I 2, RPMI 1640, and Iscove's Modified Dulbecco's Medium (IMDM).

[029] The terms “comprises” and “comprising” are used in the inclusive meaning, in an open sense in which additional elements can be included.

[030] The term “Cx601” refers to a cell suspension in a buffered, aseptic saline solution containing expanded human adipose tissue-derived stem cells (eASCs) of allogeneic origin. These cells are provided in disposable vials without preservative agents. Cells are presented at a dose of 120 million cells (5 million cells/mL) for intralesional injection.

[031] The term “differentiation” refers to the formation of cells that express markers known to be associated with cells that are more specialized and closer to becoming differentiated cells and terminally incapable of division or further differentiation. For example, in a pancreatic context, differentiation can be seen to produce islet-like cell clusters containing an increased proportion of beta-epithelial cells that produce increased amounts of insulin. The terms “additional” or “further” differentiation refer to cells that are more specialized and closer to becoming terminally differentiated cells incapable of division or further differentiation than the cultured cells. The term “final differentiation” refers to cells that have become terminally differentiated cells incapable of division or further differentiation.

[032] The term “expanded”, as used here, when referring to cells, must be considered as having its common meaning in the art, namely, cells that have been proliferated in vitro. Methods for preparing eASCs are known in the art, for example as described in WO2007/039150. An ASC can be expanded to provide a cell population that retains at least one biological function of the ASC, typically the ability to adhere to a plastic surface, under standard culture conditions. The expanded population of cells may retain the ability to differentiate into one or more cell types.

[033] The term “fistula” refers to any abnormal passage or communication or connection, commonly between two internal organs or originating from an internal organ on the surface of the body. Examples of fistulas include, but are not limited to, anorectal fistula or anus fistula or fecal fistula, arteriovenous fistula or A-V fistula, biliary fistula, cervical fistula, craniosinusal fistula, enteroenteral fistula, enterocutaneous fistula, enterovaginal fistula, gastric fistula, perilymph metroperitoneal fistula, fistula pulmonary arteriovenous fistula, rectovaginal fistula, umbilical fistula, tracheoesophageal fistula and vesicovaginal fistula. The invention relates to perianal fistula.

[034] The term “including” is used here to mean “including, among others”. “Including” and “including, among others” are used interchangeably.

[035] “Marker” refers to a biological molecule whose presence, concentration, activity or phosphorylation state can be detected and used to identify the phenotype of a cell.

[036] “Mesenchymal stromal cells” (also referred to herein as “MSCs”) are multipotent stromal cells (i.e., they are cells that are capable of giving rise to multiple different types of cells), typically derived from connective tissue, and are cells non-hematopoietic. MSCs have the ability to differentiate into or relative to somatic cells, such as mesodermal cells (e.g., adipose, chondrocytes, osteoblasts) and, optionally, into or relative to endodermal and/or ectodermal cell types or lineages. Typically, cells have the ability to differentiate into or toward at least two or all cell types selected from the group consisting of adipocytic, chondroblastic, and osteoblastic lineages. ASCs are a type of MSC that are obtained from the stromal fraction of adipose tissue. MSCs and ASCs are adherent to plastic under standard culture conditions.

[037] A “plaster” is a bandage or covering applied to cover or protect a wound or other injury.

[038] A “patient”, “individual” or “host” to be treated by the method in question may mean a human or a non-human animal.

[039] The phrase “pharmaceutically acceptable” is used herein to refer to compounds, materials, compositions, and/or dosage forms that are, within the scope of common sense medical opinion, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable risk-benefit ratio.

[040] The phrase “pharmaceutically acceptable carrier”, as used herein, means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid reinforcement, diluent, excipient, or solvent encapsulation material, involved in loading or transporting the compound in question from one organ, or part of the body, to another organ, or part of the body. Each vehicle must be “acceptable” in the sense of being compatible with the other ingredients in the formulation and not aggressive to the patient. The term “phenotype” refers to the observable characteristics of a cell, such as size, morphology, protein expression, etc.

[041] The term “progenitor cell” refers to a cell that has the ability to create progeny that are more differentiated from each other. For example, the term may refer to an undifferentiated cell or cell differentiated to a short extent from final differentiation that is capable of proliferation and giving rise to more progenitor cells that have the ability to generate large numbers of mother cells that they can, in turn, give rise to differentiated or differentiable daughter cells. In a preferred embodiment, the term progenitor cell refers to a generalized mother cell whose descendants (progeny) generally specialize in different directions, by differentiation, for example, by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs over many cell divisions. A differentiated cell can derive from a multipotent cell that is itself derived from a multipotent cell, and so on. Although each of these multipotent cells can be considered stem cells, the variety of cell types each gives rise to can vary considerably. Some differentiated cells also have the ability to give rise to cells with greater development potential. This ability can be natural or can be induced artificially through treatment with various factors. By this definition, stem cells can also be progenitor cells, as well as the most immediate precursors to terminally differentiated cells.

[042] “Proliferation” refers to an increase in cell phone numbers. “Proliferating” and “proliferation” refer to cells undergoing mitosis.

[043] “Refractory” should be considered to mean not having significant clinical benefit when used in the treatment of a disease, for example, without significant improvement or improvement in symptoms.

[044] The term “remission” refers to the successful treatment of the fistula. “Clinical remission” is closure of all treated external openings that were draining at baseline despite noninvasive finger compression. Time to clinical remission is defined as the time from the start of treatment to the first evaluation of a patient with clinical remission. “Combined remission” is this clinical remission plus confirmation by MRI (magnetic resonance imaging) of the absence of collections larger than 2 cm in the treated perianal fistulas, in at least 2 of 3 dimensions. This is typically confirmed by blind central MRI reading. The absence of collections or abscesses is important because, if not cured, they will lead to a new fistula.

[045] The term “response” refers to the closure of at least 50% of all treated external openings that were draining at baseline despite non-invasive finger compression (i.e., as clinically assessed). Therefore, the response is met when 1 EO is closed if the number of EOs at the baseline date is 1 or 2, and it is met when 2 EOs are closed if 3 EOs are present at the baseline date. Time to response is defined as the time from the start of treatment to the first assessment of response.

[046] The term “relapse” refers to, in patients with Clinical Remission in the previous evaluation, new opening of any external openings treated with active drainage, as clinically evaluated, or the development of a perianal collection greater than 2 cm from the ) perianal fistula(s) treated, as confirmed by MRI.

[047] As used herein, the term “solution” includes a pharmaceutically acceptable vehicle or diluent in which the cells of the invention remain viable.

[048] The term “substantially pure”, in relation to adipose tissue-derived stem cell populations, refers to a population of adipose tissue-derived stem cell cells that are at least about 75%, typically at least at least about 85%, more typically at least about 90%, and even more typically at least about 95% pure, relative to the adipose tissue-derived stromal stem cells that make up the total cell population. Restated, the term "substantially pure" refers to an adipose tissue-derived stromal stem cell population of the present invention that contains less than about 20%, more typically less than about 10%, even more typically less than about 5% of lineage committed cells in the original non-amplified and isolated population for subsequent culture and amplification.

[049] “Support”, as used herein, refers to any device or material that can serve as a foundation or matrix for the growth of adipose tissue-derived stromal stem cells.

[050] The term “suture” refers to a thread or fiber or other fixation material that can be used to stitch a wound.

[051] A single “tract” fistula is one that has an internal opening and an external opening. A “multiple tract” fistula has more than one external opening and/or more than one internal opening. Therefore, a multi-tract fistula has different ramifications. Each external opening typically represents a tract.

[052] The term “treatment”, as used herein, refers to the repair of a fistula or wound, as well as the prevention of a fistula or wound from worsening or recurring.

[053] “Therapeutic agent” or “therapeutic” refers to an agent capable of having a desired biological effect on a host. Chemotherapeutic and genotoxic agents are examples of therapeutic agents that are generally known to be chemical in origin, as opposed to biological, or to cause a therapeutic effect by a particular mechanism of action, respectively. Examples of therapeutic agents of biological origin include growth factors, hormones and cytokines. A variety of therapeutic agents are known in the art and can be identified by their effects. Certain therapeutic agents are capable of regulating cell proliferation and differentiation. Examples include chemotherapeutic nucleotides, drugs, hormones, nonspecific (non-antibody) proteins, oligonucleotides (e.g., antisense oligonucleotides that bind to a target nucleic acid sequence (e.g., mRNA sequence)), peptides, and peptidomimetics.

2. Compositions containing adipose tissue-derived stromal stem cells

[054] The invention involves compositions containing adipose tissue-derived stromal stem cells with certain characteristics, such as a particular phenotype. For example, adipose tissue-derived stromal stem cells in a cellular composition of the invention can be characterized by marker expression cell surface area, size, glucose consumption, lactate production and cell production/viability. Still another aspect of the present invention relates to adipose tissue-derived stromal stem cell-containing compositions that include, as a cellular component, substantially pure preparations of adipose tissue-derived stromal stem cells having a particular phenotype, or the progeny thereof. Adipose tissue-derived stromal stem cell-containing compositions of the present invention include not only substantially pure populations of the progenitor cells, but may also include cell culture components, for example, culture media including amino acids, metals, coenzyme factors, as well as small populations of other stromal cells, for example, some of which may arise by subsequent differentiation of the cells of the invention. Furthermore, other non-cellular components may include those having the cellular component suitable for support under particular circumstances, e.g., implantation, e.g., continuous culture, or suitable for use as a biomaterial or pharmaceutical composition.

[055] In certain embodiments, compositions containing adipose tissue-derived stromal stem cells are produced using culture methods described in Section 3.

[056] The preferred ASCs are Cx601 cells. The proposed (but not confirmed) International Common Name for these cells is “Adalextemcel”.

[057] ASCs adhere to plastic under standard culture conditions.

[058] Expanded ASC (eASC) presents a fibroblast-like morphology in culture. Specifically, these cells are large and are morphologically characterized by a shallow cell body with few cellular projections that are long and thin. The nucleus is large and round with a prominent nucleolus, giving the nucleus a transparent appearance. Most eASCS present this axis-shaped morphology, but it is common for some of the cells to acquire polygonal morphologies (Zuk et al., 2002).

[059] Cx601 eASCs are positive for the surface markers HLA I, CD29, CD44, CD59, CD73, CD90 and CD105. One embodiment, therefore, provides a composition containing eASC wherein at least about 80%, at least about 90%, or at least about 95%, or typically at least about 96%, 97%, 98%, or 99% of eASCs express the surface markers HLA I, CD29, CD44, CD59, CD73, CD90 and CD105. Typically, at least about 90% of eASCs express the surface markers HLA I, CD29, CD44, CD59, CD73, CD90, and CD105. More typically, at least about 95% of eASCs express the surface markers HLA I, CD29, CD44, CD59, CD73, CD90, and CD105.

[060] CX601 eASCs are negative for HLAII, CD11b, CD11c, CD14, CD45, CD31, CD34, CD80 and CD86. One embodiment, therefore, provides an eASC-containing composition, in which less than about 5% of the eASCs express the surface markers HLAII, CD11b, CD11c, CD14, CD45, CD31, CD34, CD80 and CD86. More typically, less than about 4%, 3%, or 2% of eASCs express the surface markers HLAII, CD11b, CD11c, CD14, CD45, CD31, CD34, CD80, and CD86. In one embodiment, less than about 1% of eASCs express the surface markers HLAII, CD11b, CD11c, CD14, CD45, CD31, CD34, CD80 and CD86.

[061] In some embodiments, eASCs can express one or more (e.g., two or more, three or more, four or more, five or more, six or seven) of HLA I, CD29, CD44, CD59, CD73, CD90 and CD105. In some embodiments, the eASCs may not express one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or eight) of HLAII, CD11b, CD11c, CD14, CD45 , CD31, CD34, CD80. In some embodiments, eASCs express four or more of HLA I, CD29, CD44, CD59, CD73, CD90, and CD105 and do not express four or more of HLAII, CD11b, CD11c, CD14, CD45, CD31, CD34, CD80.

[062] Expanded human ASCs, according to certain embodiments of the invention, are described in DelaRosa et al (“Requirement of IFN-gamma-mediated indoleamine 2,3-dioxygenase expression in the modulation of lymphocyte proliferation by human adipose-derived stem cells .”; Tissue Eng Part A. 2009 Oct;15(10):2795-806. doi:10.1089/ten.TEA.2008.0630) and in Lopez-Santalla et al 2015 (“Human Adipose-Derived Mesenchymal Stem Cells Modulate Experimental Autoimmune Arthritis by Modifying Early Adaptive T Cell Responses.” STEM CELLS, 33: 3493-3503).

[063] In one embodiment (as described in Lopez-Santalla et al 2015), human adipose tissue aspirates from healthy donors were washed twice with phosphate-buffered saline and digested with 0.075% collagenase (Type I; Invitrogen). The digested sample was washed with 10% fetal bovine serum (FBS), treated with 160 mM NH4Cl to eliminate remaining erythrocytes, and suspended in the culture medium [Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS. The cells were cultured (2-3 • 104 cells/cm2) in the tissue culture flasks and expanded (37 °C, 5% CO2) with a change of culture medium every 3-4 days. The cells were transferred to a new flask (103 cells/cm2) when they reached 90% confluency. Cells were expanded to 12-14 doubling and frozen. Experiments were performed with cells from two adult male and two female donors in population doublings 12-14. ASCs were thawed from the same cryobanks and cultured before each experiment. ASCs were defined according to the International Society for Cellular Therapy criteria: being positive for HLA-I, CD73, CD90, and CD105 and negative for CD11b, CD14, CD31, CD34, and CD45.

[064] In another embodiment, (as described by DelaRosa et al 2009), lipoaspirates obtained from human adipose tissue from healthy adult donors were washed twice with PBS, and digested at 378 C for 30 min. with 18 U=mL of type I collagenase in PBS. One unit of collagenase releases 1 mM L-leucine equivalents from collagen in 5 h at 37°C, pH 7.5 (Invitrogen, Carlsbad, CA). The digested sample was washed with 10% fetal bovine serum (FBS), treated with 160 mM ClNH4, suspended in culture medium (DMEM containing 10% FBS) and filtered through a 40 mm nylon mesh. Cells were cultured (2-3104 cells=cm2) in tissue culture flasks and expanded at 378 C and 5% CO2, changing the culture medium every 7 days. The cells were transferred to a new culture flask (10008 cells=cm2) when the cultures reached 90% confluency. Cells were phenotypically characterized by their ability to differentiate into chondro, osteo and adipogenic lineages. Furthermore, hASCs were verified by staining with specific surface markers. hASCs were positive for HLA-I, CD90 and CD105, and negative for HLA-II, CD40, CD80, CD86 and CD34. A pool of six healthy donors (three men and three women, aged 35 to 47) was used in the study. Cells were used at passages 4-6.

[065] In some embodiments, ASCs (i) do not express APC-specific markers; (ii) do not express IDO constitutively; (iii) do not significantly express MHC II constitutively. Typically, IDO or MHC II expression can be induced by stimulation with IFN—Y.

[066] In some embodiments, ASCs may express one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more (e.g., up to 13)) of the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105. For example, ASCs may express one or more (e.g., two, three, or all) of the markers CD29, CD59, CD90, and CD105, e.g., CD59 and/or CD90.

[067] In some embodiments, the MSCs may not express one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more (e.g., up to 15)) of the markers Factor VIII, alpha-actin, desmin, S-100, keratin, CD11b, CD11c, CD14, CD45, HLAII, CD31, CD34, CD45, STRO-1, and CD133 , for example, MSCs do not express one or more (e.g., two, three, or all) of the markers CD34, CD45, CD31, and CD14, e.g., CD31 and/or CD34.

[068] In one embodiment, there is provided a composition containing stromal stem cells derived from adipose tissue, at least about 50%, at least about 60%, at least about 70%, at least about 80% , at least about 85%, at least about 90%, at least about 95%, or typically at least about 96%, 97%, 98%, or 99% of stem cells express the markers CD9, CD10, CD13 , CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and/or CD105. In certain embodiments of the compositions containing adipose tissue-derived stromal stem cells, less than about 15%, about 10%, about 5%, and typically about 4%, 3%, 2% or 1% of the cells. trunk express the markers CD34, CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 and/or CD133.

[069] In another embodiment, there is provided a composition containing stromal stem cells derived from adipose tissue, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80% , at least about 85%, at least about 90%, at least about 95%, or typically at least about 96%, 97%, 98%, or 99% of stem cells express the markers c-Kit, vimentin and/or CD90. In certain embodiments of the compositions containing adipose tissue-derived stromal stem cells, less than about 15%, about 10%, about 5%, and typically about 4%, 3%, 2% or 1% of the cells. trunk express the markers CD34, Factor VIII, alpha-actin, desmin, S-100 and/or keratin. A population of adipose tissue-derived stromal stem cells that express the markers c-Kit, vimentin and CD90 and do not express the markers CD34, Factor VIII, alpha-actin, desmin, S-100 and keratin are also provided.

[070] The phenotypic characterization of a cell population by surface markers can be carried out by staining individual cells (flow cytometry) or by making histological sections of the population in situ, made according to normal methods. The determination of the expression profile of surface markers by antibodies, the characterization of immunophenotype, can be direct, using a labeled antibody, or indirect, using a second antibody labeled in relation to the primary specific antibody of the cellular marker, thus achieving amplification signal. On the other hand, the presence or absence of antibody binding can be determined by different methods that include, among others, immunofluorescence microscopy and radiography. In a similar way, it is possible to monitor antibody binding levels using flow cytometry, a technique that allows fluorochrome levels to be correlated with the amount of antigens present on the cell surface specifically bound to the labeled antibodies. The differential expression of a series of surface markers in a cell population provides a method for identifying and isolating said population. Likewise, FACS (Fluorescence Activated Cell Separation) can typically be used.

[071] In certain embodiments, adipose tissue-derived stromal stem cell-containing compositions are suspensions of adipose tissue-derived stromal stem cells in various solutions or materials, for example, for use as pharmaceutical products or biomaterials, as described in more details below. In one embodiment, the cellular composition comprises a suspension of the subject adipose tissue-derived stromal stem cells in Ringer's solution and HSA. In another embodiment, the cellular composition comprises a suspension of the adipose tissue-derived stromal stem cells in question in a material, such as polymer, glue, gel, etc. Such suspensions can be prepared, for example, by sedimenting the adipose tissue-derived stromal stem cells in question from the culture medium and resuspending them in the desired solution or material. Cells can be pelleted and/or altered from the culture medium, for example, by centrifugation, filtration, ultrafiltration, etc.

[072] The concentration of the subject adipose tissue-derived stromal stem cells in the subject adipose tissue-derived stromal stem cell-containing compositions may be at least about 5 x 106 cells/mL, at least about 10 x 106 cells/mL, at least about 20 x 106 cells/mL, at least about 30 x 106 cells/mL, or at least about 40 x 106 cells/mL. Typically, the concentration is between about 1 x 106 cells/mL and 1 x 107 cells/mL, for example between about 5 x 106 cells/mL and 1 x 107 cells/mL. In the clinical study reported in the Examples, eASCs were administered at a concentration of 5 million cells/mL.

[073] Likewise, another aspect of the present invention pertains to the progeny of the adipose tissue-derived stromal stem cells in question, for example, cells that were derived from adipose tissue-derived stromal stem cells. Such progeny may include subsequent generations of adipose tissue-derived stromal stem cells, as well as lineage-committed cells generated by inducing differentiation of the adipose tissue-derived stromal stem cells in question following their isolation from the explant, e.g., induced in vitro. In certain embodiments, progeny cells are obtained after about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 population passages. parental. However, progeny cells can be obtained after any number of passages from the parental population.

[074] In certain embodiments, compositions containing adipose tissue-derived stromal stem cells of the invention will be provided as part of a pharmaceutical preparation, for example, a sterile preparation, free from the presence of unwanted viruses, bacteria and other pathogens, as well as pyrogen-free preparation. That is, for administration to humans, the compositions in question must meet the standards of sterility, pyrogenicity as well as general safety and purity required by the FDA's Office of Biological Standards.

[075] Expanded adipose tissue-derived stromal stem cells are allogeneic to the transplant host. Due to the difficulties in obtaining sufficient autologous stem cells, stromal stem cells derived from allogeneic donor adipose tissue constitute a valuable alternative source of stem cells for therapeutic use. It is known in the art that bone marrow stromal stem cells and adipose tissue-derived stromal cells do not elicit an allogeneic lymphocyte response in vitro and, consequently, donor-derived allogeneic adipose tissue-derived stromal stem cells could be used by any patient, regardless of MHC incompatibility.

[076] Methods of administering compositions containing adipose tissue-derived stromal stem cells to individuals, particularly human individuals, which are described in detail herein, include injection or implantation of the cells into target sites in the individuals, the cells can be inserted into a release device that facilitates the introduction by injection or implantation of cells into individuals. These delivery devices include tubes, e.g., catheters, for injecting cells and fluids into the body of a recipient individual. In a preferred embodiment, the tubes additionally have a needle, for example, a syringe, through which compositions containing adipose tissue-derived stromal stem cells can be introduced into the individual at a desired location. Compositions containing adipose tissue-derived stromal stem cells can be inserted into this delivery device, for example, a syringe, in different forms. For example, adipose tissue-derived stromal stem cell-containing compositions include adipose tissue-derived stromal stem cell compositions that are suspended in a solution or incorporated into a support matrix when contained in such a delivery device.

[077] Pharmaceutically acceptable vehicles and diluents include saline solution, aqueous buffer solutions, solvents and/or dispersion media. The use of these vehicles and diluents is well known in the art. The solution is typically sterile and fluid to an extent that allows easy exit from the syringe. Typically, the solution is stable under manufacturing and storage conditions and preserved against the contaminating action of microorganisms, such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. Solutions that are adipose tissue-derived stromal stem cell compositions of the invention can be prepared by incorporating adipose tissue-derived stromal stem cells as described herein in a pharmaceutically acceptable carrier or diluent and, as necessary, other ingredients enumerated above. , followed by filtered sterilization.

[078] Some examples of materials and solutions that can serve as a pharmaceutically acceptable vehicle include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar-agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic physiological solution; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[079] In certain embodiments, compositions containing stromal stem cells derived from adipose tissue further comprise an adhesive. In certain embodiments, the adhesive is a fibrin-based adhesive, such as fibrin gel or fibrin glue or polymer or fibrin-based adhesive, or other tissue adhesive or surgical glue, such as, for example, cyanoacrylate, collagen, thrombin, and polyethylene glycol. Other materials that can be used include, but are not limited to, calcium alginate, agarose, types I, II, IV or other collagen isoform, polylactic/polyglycolic acid, hyaluronate derivatives or other materials (Perka C. et al. (2000) J. Biomed. Res. 49:305-311; Res. 29:1147-1154; Hendrickson DA et al. In other embodiments, the adhesive is a liquid bandage, in which the adipose tissue-derived stromal stem cell-containing compositions of the method are mixed with the liquid bandage material. A “liquid bandage” is a solution comprising a compound, for example a polymeric material, which is applied to a wound with a spray or brush, followed by removal of the solvent by vaporization to provide a protective film on the wound.

[080] The adipose tissue-derived stromal stem cell-containing compositions of the invention can also be used to coat a support, for example, a medical device. For example, the support may be a suture or thread.

[081] The support can be coated with cells in any way, as known to one skilled in the art, for example, by immersion, spraying, painting, printing, etc.

[082] In one embodiment, the support is a suture, staple, absorbable thread, non-absorbable thread, natural thread, synthetic thread, monofilament thread or multifilament thread (also called braids). Preferred methods of preparing sutures and other scaffolds used to close wounds coated with adipose-derived stromal stem cells are disclosed in U.S. Patent Application No. 11/056,241 “Biomaterial for Suturing,” filed February 14, 2005, which order is incorporated herein by reference in its entirety. The adipose tissue-derived stromal stem cell-containing compositions disclosed herein represent innovative compositions that can be used with the methods disclosed in U.S. Patent Application No. 11/056,241.

[083] Furthermore, in any of the compositions containing stromal stem cells derived from adipose tissue, at least one therapeutic agent can be incorporated into the composition (although not necessary and can, optionally, be excluded). For example, a composition may contain an analgesic to help treat inflammation or pain at the fistula site, or an anti-infective agent to prevent infection of the site treated with the composition.

[084] More specifically, non-limiting examples of useful therapeutic agents include the following therapeutic categories: analgesics, such as non-steroidal anti-inflammatory drugs, opiate agonists and salicylates; anti-infective agents such as anthelmintics, antianaerobics, antibiotics, aminoglycoid antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, generic β-lactam antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, antimycobacterials, antimycobacterials antituberculosis, antiprotozoals, antiprotozoals antimalarials, antiviral agents, antiretroviral agents, scabicides, anti-inflammatory agents, corticosteroid anti-inflammatory agents, antipruritic/local anesthetics, topical anti-infectives, topical anti-infectives antifungals, topical anti-infectives antivirals; electrolyte and renal agents, such as acidifying agents, alkalinizing agents, diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics, osmotic diuretics, potassium-dispersing diuretics, thiazide diuretics, electrolyte replacements, and uricosuric agents; enzymes, such as pancreatic enzymes and thrombotic enzymes; gastrointestinal agents, such as antidiarrheals, antiemetics, gastrointestinal anti-inflammatory agents, salicylate gastrointestinal anti-inflammatory agents, anti-ulcer antacid agents, gastric acid pump inhibitor anti-ulcer agents, gastric mucosal anti-ulcer agents, H2 blocking anti-ulcer agents, cholelitholytic agents, digestives, emetics, laxatives and stool softening agents, and prokinetic agents; general anesthetics, such as inhalation anesthetics, halogenated inhalation anesthetics, intravenous anesthetics, barbiturate intravenous anesthetics, benzodiazepine intravenous anesthetics, and opiate agonist intravenous anesthetics; hormones and hormone modifiers, such as abortifacients, adrenal agents, adrenal corticosteroid agents, androgens, antiandrogens, immunobiological agents, such as immunoglobulins, immunosuppressants, toxoids and vaccines; local anesthetics such as amide local anesthetics and ester local anesthetics; musculoskeletal agents, such as anti-gout anti-inflammatory agents, corticosteroid anti-inflammatory agents, gold compound anti-inflammatory agents, immunosuppressive anti-inflammatory agents, non-steroidal anti-inflammatory drugs (NSAIDs), salicylate anti-inflammatory agents, minerals ; and vitamins, such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and vitamin K.

[085] Preferred classes of useful therapeutic agents from the above categories include: (1) general analgesics, such as lidocaine or derivatives thereof, and non-steroidal anti-inflammatory drug (NSAIDs) analgesics, including diclofenac, ibuprofen, ketoprofen and naproxen; (2) opiate agonist analgesics such as codeine, fentanyl, hydromorphone, and morphine; (3) salicylate analgesics such as aspirin (ASA) (enteric-coated ASA); (4) H1 blocker antihistamines such as clemastine and terfenadine; (5) anti-infective agents such as mupirocin; (6) anti-anaerobic anti-infectives, such as chloramphenicol and clindamycin; (7) anti-infective antifungal antibiotics, such as amphotericin b, clotrimazole, fluconazole and ketoconazole; (8) macrolide antibiotic anti-infectives such as azithromycin and erythromycin; (9) anti-infective generic β-lactam antibiotics, such as aztreonam and imipenem; (10) penicillin antibiotic anti-infectives, such as nafcillin, oxacillin, penicillin G and penicillin V; (11) anti-infective quinolone antibiotics, such as ciprofloxacin and norfloxacin; (12) anti-infective tetracycline antibiotics, such as doxycycline, minocycline and tetracycline; (13) anti-mycobacterial anti-tuberculosis anti-infectives, such as isoniazid (INH) and rifampin; (14) antiprotozoal anti-infectives, such as atovaquone and dapsone; (15) anti-malaria antiprotozoal anti-infectives, such as chloroquine and pyrimethamine; (16) antiretroviral anti-infectives, such as ritonavir and zidovudine; (17) antiviral anti-infective agents such as acyclovir, ganciclovir, alpha interferon and rimantadine; (18) topical antifungal anti-infectives, such as amphotericin B, clotrimazole, miconazole and nystatin; (19) antiviral topical anti-infectives, such as acyclovir; (20) electrolyte and renal agents such as lactulose; (21) loop diuretics such as furosemide; (22) potassium-dispersing diuretics such as triamterene; (23) thiazide diuretics such as hydrochlorothiazide (HCTZ); (24) uricosuric agents such as probenecid; (25) enzymes such as RNase and DNase; (26) antiemetics, such as prochlorperazine; (27) salicylate gastrointestinal anti-inflammatory agents such as sulfasalazine; (28) gastric acid pump inhibitor antiulcer agents such as omeprazole; (29) H2-blocking antiulcer agents such as cimetidine, famotidine, nizatidine, and ranitidine; (30) digestives, such as pancrelipase; (31) prokinetic agents, such as erythromycin; (32) ester local anesthetics such as benzocaine and procaine; (33) musculoskeletal corticosteroid anti-inflammatory agents such as beclomethasone, betamethasone, cortisone, dexamethasone, hydrocortisone and prednisone; (34) musculoskeletal anti-inflammatory immunosuppressants, such as azathioprine, cyclophosphamide and methotrexate; (35) musculoskeletal nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, ibuprofen, ketoprofen, ketorlac, and naproxen; (36) minerals, such as iron, calcium and magnesium; (37) vitamin B compounds such as cyanocobalamin (vitamin B12) and niacin (vitamin B3); (38) vitamin C compounds such as ascorbic acid; and (39) vitamin D compounds such as calcitriol.

[086] In certain embodiments, the therapeutic agent may be a growth factor or other molecule that affects cell differentiation and/or proliferation. Growth factors that induce final states of differentiation are well known in the art, and can be selected from any such factor that has been shown to induce a final state of differentiation. Growth factors for use in methods described herein may, in certain embodiments, be variants or fragments of a naturally occurring growth factor. For example, a variant can be generated by making conservative amino acid changes and testing the resulting variant in one of the functional tests described above or another functional test known in the art. Conservative amino acid substitutions refers to the ability to substitute residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine and isoleucine; a group of amino acids having aliphatic hydroxyl side chains are serine and threonine; a group of amino acids having amide-containing side chains are asparagine and glutamine; a group of amino acids having aromatic side chains are phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains are lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine.

[087] As those skilled in the art will appreciate, variants or fragments of polypeptide growth factors can be generated using conventional techniques, such as mutagenesis, including the creation of discrete point mutations, or by truncation. For example, the mutation may give rise to variants that retain substantially the same, or merely a subset of, the biological activity of a polypeptide growth factor from which it was derived.

3. Methods of Preparation of Compositions Containing Adipose Tissue-Derived Stromal Stem Cell

[088] Methods for isolating and culturing ASCs to provide eASCs and cell populations of the invention and compositions comprising cell populations of the invention are known in the art. Typically, methods for preparing compositions comprising cell populations comprise the following steps: (i) isolating ASCs from the stromal fraction of adipose tissue and selecting by adherence to a suitable surface, e.g., plastic (ii) expanding ASCs to provide populations cells of the invention comprising eASCs.

[089] Optionally, the cell populations of the invention can be cryopreserved during and/or subsequent to the expansion step (ii). Optionally, the phenotype of the cell populations of the invention can be evaluated during and/or subsequent to the expansion step (ii). Optionally, the cell populations of the invention can be isolated subsequent to the expansion step (ii) and resuspended in a pharmaceutically acceptable vehicle and/or diluents.

[090] ASCs can be obtained by any standard means in the art. Typically, said cells are obtained by dissociating cells from the tissue of origin (e.g., lipoaspirate or adipose tissue), typically by treating the tissue with a digestive enzyme, such as collagenase. The digested tissue material is then typically filtered through a filter of between about 20 microns to 1 mm. The cells are then isolated (typically by centrifugation) and cultured on an adherent surface (typically tissue culture plates or flasks). Such methods are known in the art and, for example, as disclosed in U.S. Patent No. 6777231. According to this methodology, liposuctions are obtained from adipose tissue and cells derived therefrom. In the cycle of this methodology, cells can be washed to remove contaminating residues and erythrocytes, preferably with PBS. Cells are digested with collagenase (e.g., at 37°C for 30 minutes, 0.075% collagenase; Type I, Invitrogen, Carlsbad, CA) in PBS. To eliminate remaining erythrocytes, the digested sample can be washed (e.g. with 10% fetal bovine serum), treated with ClNH4 160 mmol/L and finally suspended in complete DMEM medium (DMEM containing 10% FBS, glutamine 2 mmol/L and penicillin/streptomycin 1%). Cells can be filtered through a 40 μm nylon mesh.

[091] The cells were cultivated in a suitable tissue culture vessel, comprising a suitable surface for the adherence of ASCs, for example, plastic. Non-adherent cells are removed, for example, by washing in a suitable buffer, to provide an isolated population of adherent stromal cells (e.g. ASC). Cells isolated in this way can be cultured (preferably 2-3x104 cells/cm2) in tissue culture flasks and expanded at 37 °C and 5% CO2, changing the culture medium every 3-4 days. Cells are preferably detached from the adherent surface (e.g., by means of trypsin) and passed (“cultured”) to a new culture flask (1,000 cells/cm2) when cultures reach about 90% confluency.

[092] Cell isolation is preferably carried out under sterile or GMP conditions.

[093] In one embodiment, a method comprises: (a) collecting adipose tissue from an individual; (b) obtaining a cell suspension by enzymatic digestion; (c) sedimentation of the cell suspension and resuspension of the cells in a culture medium; (d) culturing the cells for at least about 10 days; and (g) expanding the cells by at least two culture passages.

[094] Adipose tissue-derived stromal stem cells are allogeneic, that is, not isolated from the adipose tissue of the individual to whom the final composition containing adipose tissue-derived stromal stem cells is to be introduced.

[095] In certain embodiments, the cells are cultured for at least about 15, at least about 20 days, at least about 25 days, or at least about 30 days. Typically, expansion of cells in culture improves the homogeneity of the cell phenotype of the cell population, so that a substantially pure or homogeneous population is obtained.

[096] In certain embodiments, the cells are expanded in culture for at least three culture passages or “cultured at least three times”. In other embodiments, the cells are cultured at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times. It is preferable that cells are cultured more than three times to improve the homogeneity of cell phenotype in the cell population. In fact, cells can be expanded in culture indefinitely as long as the homogeneity of the cellular phenotype is improved and the differential capacity is maintained.

[097] In certain embodiments, cells are multiplied in culture for at least three population doublings. In certain embodiments, the cells are expanded in culture for at least four, five, six, seven, eight, nine, ten, 15 or 20 population doublings. In certain embodiments, the cells are expanded in culture by less than seven, eight, nine, ten, 15 or 20 population doublings. In certain embodiments, the cells are expanded in culture by between about 5 and 10 population doublings. In certain embodiments, the cells are expanded in culture to between about 10 and 15 population doublings.

[098] In certain embodiments, cells are expanded in culture to between about 15 and 20 population doublings, for example, about 16 population doublings.

[099] Cells can be cultivated by any technique known for carrying out stem cell culture. A discussion of various culture techniques, as well as their expansion, can be found in Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, Wiley-Liss 2000. Cells can be expanded using culture flasks or bioreactors suitable for large-scale expansion. Bioreactors suitable for large-scale expansion of mesenchymal stromal cells are commercially available and may include 2D (i.e., substantially planar) and 3D expansion bioreactors. Examples of such bioreactors include, but are not limited to, a connector flow bioreactor, a perfusion bioreactor, a continuous stirred tank bioreactor, a fixed bed bioreactor. In certain embodiments, the cells are grown by monolayer culture. In one embodiment, cells are cultured and passaged as described in Example A below.

[0100] Any medium capable of supporting stromal cells in tissue culture can be used. Medium formulations that will support fibroblast growth include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), alpha modified Minimum Essential Medium (alpha.MEM), and Roswell Park Memorial Institute Medium 1640 (RPMI 1640 Medium), and the like. Typically, 0 to 20% Fetal Bovine Serum (FBS) or 1-20% Equine Serum will be added to the above medium in order to support the growth of stromal cells and/or chondrocytes. However, a defined medium could be used if the necessary growth factors, cytokines, and hormones in FBS for stromal cells and chondrocytes were identified and provided in appropriate concentrations in the growth medium. Means useful in the methods of the invention may contain one or more compounds of interest, including, among others, mitogenic antibiotics or stromal cell differentiating compounds. The cells will be grown at temperatures between 31 °C and 37 °C in a humidified incubator. The carbon dioxide content will be maintained between 2% and 10% and the oxygen content between 1% and 22%. Cells can remain in this environment for periods of up to 4 weeks.

[0101] Antibiotics that can be supplemented to the medium include, among others, penicillin and streptomycin. The concentration of penicillin in the chemically defined culture medium is about 10 to about 200 units per mL. The concentration of streptomycin in the chemically defined culture medium is about 10 to about 200 ug/mL.

[0102] Adipose tissue-derived stromal stem cells can be stably or temporarily transfected with a nucleic acid of interest using a plasmid, viral vector strategy or alternative. Nucleic acids of interest include, but are not limited to, those encoding gene products that enhance the production of extracellular matrix components found in the type of tissue being repaired, e.g., intestinal wall or vaginal wall.

[0103] Transduction of viral vectors carrying regulatory genes into stromal stem cells can be performed with viral vectors (adenovirus, retrovirus, adeno-associated virus, or other vector) purified by cesium chloride banding or other method in a multitude of infections (viral units:cell) between 10:1 to 2000:1. Cells will be exposed to virus in free serum or serum-containing medium in the absence or presence of a cationic detergent such as polyethyleneimine or Lipofectamine.TM. for a period of 1 hour to 24 hours (Byk T. et al. (1998) Human Gene Therapy 9:2493-2502; Sommer B. et al. (1999) Calcif. Tissue Int. 64:45-49).

[0104] Other suitable methods for transfecting vectors or plasmids into stem cells include lipid/DNA complexes, such as those described in U.S. Patent Nos. 5,578,475; 5,627,175; 5,705,308; 5,744,335; 5,976,567; 6,020,202; and 6,051,429. Suitable reagents include Lipofectamine, a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminocarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA). (Chemical Abstracts Registry name: N-[2-(2,5-bis[(3-aminopropyl)amino]-1--oxypentyl}amino)ethyl]-N,N-dimethyl-2,3-bis(9 -octadecenyloxy)- 1-propanaminium trifluoroacetate), and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) in membrane-filtered water. Exemplary is the Lipofectamine 2000 TM formulation (available from Gibco/Life Technologies #11668019). Other reagents include: FuGENETM 6 Transfection reagent (a mixture of lipids in non-liposomal form and other compounds in 80% ethanol, obtained from Roche Diagnostics Corp. #1814443); and LipoTAXITM transfection reagent (a lipid formulation from Invitrogen Corp., #204110). Stem cell transfection can be performed by electroporation, for example, as described in M. L. Roach and J. D. McNeish (2002) Methods in Mol. Biol. 185:1. Viral vector systems suitable for producing stem cells with stable genetic alterations can be based on adenoviruses and retroviruses, and can be prepared using commercially available viral components.

[0105] Transfection of plasmid vectors carrying regulatory genes to stromal stem cells can be introduced to cells in monolayer culture by use of calcium phosphate DNA precipitation or cationic detergent methods (Lipofectamine.TM., DOTAP) or in three-dimensional cultures by incorporating the plasmid DNA vectors directly into the biocompatible polymer (Bonadio J. et al. (1999) Nat. Med. 5:753-759).

[0106] For screening and detection of functional proteins encoded by these genes, viral or plasmid DNA vectors will contain a readily detectable marker gene, such as green fluorescent protein or beta-galactosidase enzyme, both of which can be screened by means histochemicals.

4. Treatment of complex perianal fistulas

[0107] The invention relates to the treatment of complex perianal fistulas in patients with Crohn's Disease, using expanded, allogeneic, adipose tissue-derived stromal stem cells. Adipose tissue-derived stromal stem cells typically comprise an adipose tissue-derived stromal stem cell-containing composition described herein, sometimes referred to as “Cx601”. However, other adipose tissue-derived stromal stem cell preparations may be used in the methods described herein, for example, such as those described in U.S. Patent Nos. 6,777,231 and 6,555,374 and U.S. Patent Application No. 11/ 065.461 “Identification and Isolation of Multipotent Cells from Non-Osteochondral Mesenchymal Tissue”, deposited on February 25, 2005.

[0108] The Examples show that allogeneic eASCs are a surprisingly effective therapy for complex perianal fistulas in patients with Crohn's Disease, whereby a single administration is capable of providing a rapid and sustained therapeutic effect, even in very complex fistulas, which are more difficult to treat. treat that did not respond to previous treatments. For example, benefit is typically seen in approximately 6 weeks, and this benefit is typically sustained through 24 weeks of therapy.

[0109] In one embodiment, a method of treating a complex refractory perianal fistula in a Crohn's Disease patient comprises injection of about 120 million allogeneic, intralesionally expanded adipose tissue-derived stromal stem cells into all fistula treatments. For the avoidance of doubt, in this embodiment, 120 million cells are administered to the patient in a single procedure, with each fistula tract receiving at least a proportion of this dosage. Approximately half the dose is injected into the tissue surrounding the internal opening or openings. The other half is injected into the fistula walls (no deeper than 2 mm), all along the fistula tract(s), making several microbubbles. The data in the Examples show that this single administration is capable of providing combined remission within 24 weeks or less, even within approximately 6 weeks or within approximately 8 weeks. Likewise, in one embodiment, the treatment consists of this single intralesional administration of 120 million eASCs.

[0110] Intralesional injection is typically performed as in the clinical study exemplified, whereby the cell suspension is injected through the internal opening(s) (with the syringe entering through the anus) and through the walls of the fistula tract ( with the syringe entering through the external opening of the fistula).

[0111] The patient can be male or female. The patient is typically an adult.

[0112] In one embodiment, any setons that may be present are first removed. Typically, a fistula is curetted before administration of eASCs. Internal openings can optionally be sutured. The eASCs are then administered with a long, thin needle. As noted above, approximately half the dose is injected into the tissue surrounding the sutured internal opening(s). The other half is injected into the fistula walls (at a depth of no more than 2 mm), all along the fistula tract(s), creating several microbubbles.

[0113] Prior to therapy, according to the invention, a pelvic MRI scan can be performed at screening to guide surgical procedures. Patients may also undergo fistula curettage and seton insertion as clinically indicated > 2 weeks prior to investigational product administration (Figure 1). If a seton is placed, it is removed immediately prior to administration of the investigational product.

[0114] The patient to be treated has Crohn's Disease, typically luminal Crohn's Disease. Typically, the patient has non-active or mildly active luminal Crohn's Disease, for example, as defined by a Crohn's Disease Activity Index (CDAI) of < 220A24. The patient may have had Crohn's disease for at least 6 months.

[0115] The patient has at least one complex perianal fistula. In one embodiment, the patient does not have a rectovaginal fistula, rectal stenosis, anal stenosis, active severe proctitis (defined by the presence of superficial or deep ulcers), deviating stomata, or an abscess or collections > 2 cm that were not resolved by the procedure. of surgical preparation.

[0116] Patients are refractory to at least one previous drug therapy. The prior therapy may be a drug or small molecule biological medicinal product. Typically, the patient has received said treatment for at least about 4, 6, 12, 18, 24 or 36 weeks and still has symptoms of active disease. In other words, even after treatment for at least about 4, 6, 12, 18, 24 or 36 weeks, the severity in the fistula was not improved. Prior therapy may be antibiotic therapy such as ciprofloxacin or metronidazole, immunosuppressive therapy (e.g., a purine analogue such as azathioprine or 6-mercaptopurine, a pyrminidine analogue such as fluorouracil, or a folic acid analogue such as methotrexate), or anti-TNF therapy, such as Adalimumab (Humira), Certolizumab (Cimzia), Etanercept (Enbrel), Golimumab (Simponi), or Infliximab (Remicade). Eligible patients are typically refractory (unresponsive) to at least one of the following treatments: after 1 month of antibiotics (e.g., ciprofloxacin, metronidazole); and/or after 3 months of immunosuppressants (e.g., azathioprine, 6-mercaptopurine); and/or induction or maintenance of anti-TNF therapies (e.g., infliximab, etanercept, adalimumab, Certolizumab, golimumab) at a stable dose.

[0117] In one embodiment, the patient is refractory to treatment with an immunosuppressant (e.g., azathioprine, 6-mercaptopurine) and a TNF-α inhibitor (e.g., infliximab or adalimumab).

[0118] A typical patient, therefore, has non-active luminal Crohn's Disease (CDAI < 220) diagnosed for at least six months, with a complex perianal fistula having 1 or 2 internal openings and 2 or 3 external openings, which has shown an inadequate response to one or more of the antibiotics, immunosuppressants or anti-TNF therapies. A fistula with 1 or 2 internal openings and 3 external openings is highly complex and, until now, has been very difficult to treat. In a further embodiment, at the time of treatment according to the invention, the patient is receiving (a) no anti-TNF therapy and no immunosuppressive therapy, or (b) an anti-TNF therapy and an immunosuppressive therapy. It is especially surprising that eASCs are able to significantly improve combined remission (at week 24) in patients receiving anti-TNF and immunosuppressive therapy, which are the best therapies currently available (see Figure 3C).

[0119] The complex perianal fistula has at least two external openings, for example 3 or more external openings. Some fistulas may have 4 or 5 external openings.

[0120] Complex perianal fistula typically has multiple tracts, at least two internal openings, or three external openings. The fistula may optionally comprise two or three of these features, for example: multiple tracts and at least two internal openings; multiple tracts and three external openings; at least two internal openings and three external openings; or multiple tracts, at least two internal openings and three external openings. In one embodiment, the complex perianal fistula contains no more than two internal openings and three external openings, e.g., the fistula consists of 1 or 2 internal openings and 2 or 3 external openings (e.g., 1IO/2EO, 1IO/3EO, 2IO/2EO, or 2IO/3EO).

[0121] After administration of eASC, patients may optionally be treated with antibiotics for no more than 4 weeks. Immunosuppressants and anti-TNFs can be maintained at stable doses throughout treatment, for example, until response, clinical remission or combined remission.

[0122] Patients who have not received prior treatment for perianal fistula creation Crohn's Disease, including antibiotics, and those who have undergone prior surgery for active fistula other than drainage or seton insertion are typically excluded. Patients are typically ineligible for treatment if they have received glucocorticosteroids within the 4 weeks immediately prior to eASC treatment. A course of steroids may be permitted to treat sudden increases in luminal disease during follow-up with an initial dose of 40 mg, recorded for a maximum of 12 weeks.

[0123] In certain embodiments, for example, where the first release of cells is insufficient, the method may further comprise an additional administration of the eASCS. Such additional administration may comprise: (c) delivering a second dose of at least about 20 x 106 cells, at least about 30 x 106, or at least about 40 x 106 adipose tissue-derived stromal stem cells, per For example, about 120 million cells, for example, in an adipose tissue-derived stromal stem cell-containing composition of the invention, to the internal orifice (optionally closed by suture) and the walls of the fistula.

[0124] In some embodiments, a method of treating a refractory complex perianal fistula in an individual comprises: (a) closing the internal os with a suture comprising allogeneic eASCs. Such cell-coated sutures in the subject adipose tissue-derived stromal stem cell-containing compositions are described in detail in U.S. Patent Application No. 11/056,241, filed February 14, 2005, which is incorporated herein by reference.

[0125] The methods may, in some embodiments, further comprise: deep scraping of at least one tract of the fistula; and/or filling said fistula tract with a material. In certain embodiments, the method may further comprise releasing at least about 10 x 106 adipose tissue-derived stromal stem cells, e.g., of a cellular composition in question, to the material. Typically, the material is a fibrin-based polymer or adhesive, such as fibrin glue or gel. In certain embodiments, the dose of at least about 10 x 106 adipose tissue-derived stromal stem cells is already encompassed within the material, for example, such that the material comprises the adipose tissue-derived stem cell-containing composition.

[0126] In a further embodiment, a method of treating a fistula in an individual comprises: (i) deep scraping of at least one fistula tract (ii) closing the internal orifice of the scraped tract with a suture

[0127] delivery of about 120 million expanded, allogeneic adipose-derived stromal stem cells to an internal opening (optionally closed by suture) and the walls of the fistula, e.g., in a composition containing adipose-derived stromal stem cell adipose tissue of the invention.

[0128] Additional cell administration is not necessary and can be optionally excluded. However, in certain embodiments, the method may further comprise: (iv) releasing a second dose of at least about 20 x 106 cells, at least about 30 x 106, or at least about 40 x 106 stromal stem cells derived from adipose tissue, for example, about 120 million eASCs, typically, to an internal opening closed by suture, for example, in an adipose tissue-derived stromal stem cell-containing composition of the invention.

[0129] Step (i) is typically performed by deep scraping of all fistula tracts to be treated, for example, a curettage needle is introduced into the fistula tract, and induced bleeding is produced by scraping the fistula walls. , in order to obtain natural fibrin that will fill the fistula tract. Previous clinical studies by the inventors suggest that natural fibrin produced by this scraping method is a preferred option compared to the use of artificial fibrin sealants, therefore, in a preferred embodiment of the method of the invention, the fistular tracts to be treated are not filled with this material.

[0130] Step (iv) is carried out by local release of cells, for example, a composition containing stromal stem cells derived from adipose tissue, by injection into the fistula walls along the fistula tract. For example, multiple injections of 10 million cells along 3 cm of the fistula tract.

[0131] The fistula can be accessed for surgical repair using any method known in the art, for example, through an incision, catheter, etc.

[0132] The methods described above may further comprise administering a therapeutic agent to the individual being treated, for example, systemically or locally at the suture site. In certain embodiments, the adipose tissue-derived stromal stem cells are formulated into an adipose tissue-derived stromal stem cell-containing composition that contains a therapeutic agent, as described above. In other embodiments, the therapeutic agent is administered separately, for example, simultaneously with the methods, before the method is performed, or after the method is performed. In some embodiments, the therapeutic agent is administered to the subject before, during, or after the methods are performed on the subject. Exemplary therapeutic agents are described above. In preferred embodiments, therapeutic agents for treating Crohn's Disease are administered to the subject. Exemplary therapeutic agents for Crohn's Disease are anti-inflammatory agents, such as agents comprising mesalamine, immunosuppressive agents, such as 6-mercaptopurine and azathioprine; biological agents such as infliximab (Remicade®), antibiotics and antidiarrheal agents such as diphenoxylate, loperamide and codeine.

[0133] Supportive treatment may be necessary. For example, immunosuppressants can be administered before, during and/or after treatment to prevent GVHD, according to methods known in the art. Prior to administration, the cells may also be modified to suppress an immune reaction of the individual to the cells or vice versa, according to methods known in the art.

[0134] The dosage of any therapeutic agent will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration and the form of the agent. Any of the formulations in question can be administered in a single dose or in divided doses. Dosages for therapeutic agents can be readily determined by techniques known to those skilled in the art or as set forth herein. Also, mixtures of more than one therapeutic agent can be administered, or multiple therapeutic agents administered in separate compositions.

[0135] The precise administration time and amount of any particular agent that will produce the most effective treatment in a given patient will depend on the activity, pharmacokinetics, and bioavailability of a particular compound, the patient's physiological condition (including age, sex, type and stage of the disease, general physical condition, responsiveness to a given dosage and type of medication), route of administration and the like. The guidelines presented here can be used to optimize treatment, for example, by determining the ideal timing and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the individual and adjusting the dosage and/or timing. .

[0136] While the individual is being treated, the patient's health can be monitored by measuring one or more of the relevant indices at predetermined times during a 24-hour period. Treatment, including supplement, quantity, times of administration and formulation, can be optimized according to the results of this monitoring. The patient may be reassessed periodically to determine the extent of improvement by measuring the same parameters, the first reassessment of this typically occurring at the end of four weeks of starting therapy, and subsequent reassessments occurring every four to eight weeks during therapy and, then every three months after that. Therapy may continue for several months or even years, with a minimum of one month being the typical length of therapy for humans. Adjustments to the amount(s) of agent administered and possibly the timing of administration can be made based on these reassessments.

[0137] The combined fistula remission exemplified here comprises clinical and radiological remission. Radiological assessment of remission is typically performed by MRI, which is a well-known technique. Other imaging techniques may alternatively be used, for example, endorectal ultrasound. These imaging techniques assess the existence of abscesses or collections.

[0138] Treatment can be started with smaller dosages that are less than the ideal dose of the compound. After this, the dosage can be increased by small increments until the ideal therapeutic effect is achieved.

[0139] The combined use of several therapeutic agents can reduce the dosage required for any individual component, because the onset and duration of the effect of the different components can be complementary. In this combined therapy, the different active agents can be released together or separately and simultaneously or at different times within the day.

[0140] Toxicity and therapeutic efficacy of the compounds in question can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 and ED50. Compositions that have high therapeutic indices are preferred. Although compounds that exhibit toxic side effects can be used, care must be taken to design a delivery system that focuses the agents to the desired site in order to reduce side effects.

[0141] Data obtained from cell culture tests and animal studies can be used in formulating a variety of dosages for use in humans. The dosage of any therapeutic agent, or alternatively any components thereof, is typically within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range, depending on the dosage form employed and the administration life used. For agents of the present invention, the therapeutically effective dose can be estimated initially from cell culture tests. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half an inhibition of symptoms) as determined in cell culture. This information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

5. Clinical response and remission

[0142] The allogeneic eASCs of the invention are surprisingly effective in treating refractory complex perianal fistulas in Crohn's Disease patients. The primary endpoint, Pooled Remission at Week 24, is statistically met; Cx601 is statistically superior to placebo. Both key secondary endpoints, Remission and Clinical Response at Week 24, meet threshold statistical significance (p=0.064 and p=0.054 in the ITT population; p=0.057 and 0.045 in the mITT population, respectively).

[0143] Data indicate that patients receiving a single administration of eASCs have a 30% greater chance of achieving clinical remission than placebo. The data also indicates that patients receiving eASCs have approximately 35% more chance of achieving a clinical response than placebo.

[0144] In certain embodiments, clinical remission is achieved within 24 weeks, typically within 18 weeks. Clinical remission can be achieved within 12 weeks or less, for example within 10 weeks, or within 8 weeks. Clinical remission can optionally be achieved after 6 weeks. The data shows that the mean time to clinical remission is approximately 2 times shorter with Cx601 than placebo (6.7 weeks vs. 14.6 weeks). Likewise, in one embodiment, clinical remission is achieved within 14 weeks.

[0145] In some embodiments, combined remission is achieved within 24 weeks, typically within 18 weeks. Combined remission may optionally be achieved within 12 weeks or less, for example within 10 weeks, or within 8 weeks. Combined remission can optionally be achieved after 6 weeks.

[0146] In certain embodiments, clinical response is achieved in 11 weeks or less. The data shows that the mean time to clinical response is also approximately 2 times shorter than placebo (6.3 weeks vs. 11.7 weeks). The time to clinical response may be, in certain embodiments, 10 weeks or less, 9 weeks or less, 8 weeks or less. The time to clinical response may optionally be 7 weeks or less, for example approximately 6 weeks.

[0147] These results are particularly surprising when it is noted that these improvements are observed after a single administration of eASCs.

[0148] The Perianal Disease Activity Index “PDAI” comprises five categories: discharge, pain, restriction of sexual activity, type of perianal disease and degree of induration. Each category is graded on a five-point scale, ranging from no symptoms (score 0) to severe symptoms (score 4). An improvement in the Perianal Disease Activity Index Score is observed to be significantly greater for eASCs than placebo at weeks 6, 12, and 18. Therefore, in certain embodiments, an improvement in PDAI is achieved within 6 weeks, within 12 weeks or within 18 weeks. The improvement in PDAI may optionally be an improvement of more than 1.5 PDAI points, optionally at least two PDAI points. The improvement in PDAI can optionally be a reduction (improvement) in each of the five components of the index.

6. Kits

[0149] In other embodiments, the invention contemplates kits including compositions containing stromal stem cells derived from adipose tissue and optionally instructions for their use. Kits comprising the pharmaceutical compositions and biomaterials of the present invention are also within the scope of the invention. Kit components can be packaged for manual or partial or fully automatic practice of the previous methods. These kits can have a variety of uses, including, for example, therapy, repair, biomaterial preparation, and other applications.

Exemplification

[0150] The invention, now being described generally, will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the present invention and are not intended to limit the invention.

PHASE III CLINICAL STUDY

[0151] The current study is the first placebo-controlled, phase 3 study evaluating the efficacy and safety of Cx601 alone or added to current medical therapy for treatment-refractory complex perianal fistulas in Crohn's Disease patients.

[0152] In general, the results demonstrate that allogeneic eASCs are indicated for the treatment of complex perianal fistula(s) in adult patients with non-active/mildly active luminal Crohn's Disease, when fistula(s) have (in) presented an inadequate response to at least one conventional or biological therapy.

Methods Study supervision

[0153] This phase 3, randomized, double-blind, parallel-group, placebo-controlled study (NCT01541579; EUDRACT 2011-006064-43) was conducted in 49 medical centers in 7 European countries and in Israel from July 2012 to July 2015. The protocol was approved by a central or local ethics committee. All patients gave free and informed consent.

[0154] The study was designed and implemented by the ADMIRE Steering Committee (see Supplementary Appendix). The sponsor collected data, which was analyzed by Chiltern International's Department of Biostatistics, and the sponsor, together with the Steering Committee, interpreted the data. All authors had full access to the data, agreed with the decision to submit the final manuscript for publication, and confirmed the accuracy and completeness of the reported data. The manuscript was drafted by the first author and all authors contributed to subsequent drafts.

Patients

[0155] Eligible patients were adults 18 years of age or older, had non-active or mildly active luminal Crohn's Disease for > 6 months, defined by a Crohn's Disease Activity Index (CDAI) of < 220A24, and had fistulas complex perianal openings (defined as one or more of the following: inter, trans, extra, or high suprasphincteric origin; > 2 external openings; or associated collections) with a maximum of 2 internal and 3 external openings, which had been draining for ^6 weeks prior to inclusion. Patients were excluded if they had rectovaginal fistulas, rectal and/or anal stenosis and/or active severe proctitis (defined by the presence of superficial or deep ulcers), diverting stomata, or an abscess or collections >2 cm that were not resolved by the surgical procedure. surgical preparation. Eligible patients had to be refractory to at least one of the following treatments documented as unresponsive after 1 month of antibiotics (ciprofloxacin, metronidazole) and/or after 3 months of immunosuppressants (azathioprine, 6-mercaptopurine) and/or induction or maintenance of anti-TNF therapies at a stable dose. Patients refractory to antibiotics alone represented < 25% of the total population.

[0156] After administration of the investigational product, patients could be treated with antibiotics for no more than 4 weeks. Immunosuppressants and anti-TNFs were maintained at stable doses throughout the study. Initiation or dose increases of these agents were not permitted.

[0157] Patients who had not received prior treatment for Crohn's Disease for perianal fistula development, including antibiotics, and those who had undergone prior surgery for active fistula other than drainage or seton insertion were also excluded. Patients were not eligible if they had received glucocorticosteroids within the previous 4 weeks. One course of steroid was permitted to treat sudden increases in luminal disease during follow-up with an initial dose of 40 mg measured for a maximum of 12 weeks.

Randomization and treatment

[0158] A pelvic MRI scan was performed at triage to guide surgical procedures and collections evaluated from central blind reading. In addition, patients underwent examination under anesthesia, fistula curettage, and seton insertion, as clinically indicated, >2 weeks before investigational product administration (Figure 1). If a seton was inserted, it had to be removed immediately before administration of the investigational product.

[0159] Patients were randomly assigned in a 1:1 ratio to Cx601 or placebo following the fistula preparation visit > 2 weeks prior to investigational product administration (Figure 1). Patients were stratified based on concomitant medication at randomization (anti-TNF and/or immunosuppressant or none). Treatments were assigned using a pre-established randomization list generated by the Department of Biostatistics, Linical.

[0160] Patients in the Cx601 group received a single injection of 120 million Cx601 delivered intralesionally to the fistula tracts. Isolation and expansion of Cx601 were performed as previously described.A23 Patients in the placebo group will receive an identical volume of saline. Study treatments were administered with a thin, long needle after removal of seton(s), if present, and fistula curettage. Half of the dose was injected into the tissue surrounding the sutured internal opening(s). The other half was injected into the fistula walls (no deeper than 2 mm) all along the fistula tract(s), making several microbubbles.

[0161] Blinding of treatments was not possible, since the cell suspension was clearly different from the physiological solution. The double-blind study design was maintained by the treatment being administered by an unblinded surgeon and a blinded gastroenterologist and radiologist evaluating response.

Study procedures and monitoring

[0162] Fistula closure was clinically evaluated at weeks 6, 12, 18 and 24 by the investigator examining the presence of spontaneous drainage and, after non-invasive finger compression on the treated external openings, A12 and was evaluated radiologically by MRI scanning blind pelvic, central reading at week 24; Central readers were provided with figures to identify treated fistulas, but were blinded to patient data, order of examinations and treatment received. Treatment-emergent adverse events (TEAEs) were assessed at all study visits. Severity of perianal Crohn's Disease was assessed at baseline and all study visits with the Perianal Disease Activity Index (PDAI).A25 Quality of life was assessed with the Inflammatory Bowel Disease Questionnaire (IBDQ)A26 at baseline and week 24 what was CDAI.A24

Efficacy and safety endpoints

[0163] The primary endpoint was combined remission at week 24 defined by clinical assessment of closure of all treated external openings that were draining at baseline, and the absence of collections > 2 cm from treated perianal fistulas in > 2 of 3 dimensions , confirmed by blinded central MRI reading (Bioclinics GmbH,Munich, Germany). Clinical assessment of closure was defined as the absence of drainage despite non-invasive finger compression.A12

[0164] There were two main secondary efficacy endpoints: clinical remission (i.e., closure of all treated external openings that were draining at baseline despite non-invasive finger compression) and response (i.e., closure of >50% of all treated external openings that were draining at baseline) by week 24. Other secondary efficacy endpoints included time to clinical remission, time to response, PDAI, CDAI, and IBDQ scores through week 24.

[0165] TEAEs were coded according to the Medical Dictionary for Regulatory Activities version 17.0.

Statistical analysis

[0166] The planned sample size to be screened was 278 patients, in order to randomize > 208 patients (104 for each group). The sample size was sufficient to detect a minimum 25% difference in the percentage of patients with combined remission between Cx601 and placebo (anticipated minimum combined remission was 50% for Cx601 and 25% for placeboA8, A12, A23, A27) with an alpha error of 0.025, and 80% power, and allowed for 20% of patients who discontinued the study.

[0167] Efficacy analyzes were conducted in the intention-to-treat (ITT) population that included all randomized patients, the modified ITT (mITT) population that included all randomized patients who received study treatment and had > 1 efficacy assessment. post-baseline efficacy, and the per-protocol (PP) population that included all patients treated without major protocol deviations. TEAEs were analyzed in the safety population defined as all patients who received study treatment.

[0168] The primary endpoint was analyzed using a stratified Cochran-Mantel-Haenszel test, adjusting for randomization strata (i.e., treatments for Crohn's Disease at randomization). The primary analysis was performed using the ITT population. We also provide results for the mITT population, as its definition more closely resembles the ITT population in other randomized clinical trials and provides a more reliable estimate of treatment effects. Missing data were imputed using the last observation carried forward (LOCF) method. A non-response/non-remission was assigned if a post-baseline MRI scan or clinical assessment was not performed by week 24. Non-response/non-remission was also assigned if a rescue event occurred before week 24. The effects of rescue events and missing efficacy data conventions were explored in sensitivity analyzes of the primary endpoint.

[0169] To address the issue of multiplicity, the two main secondary endpoints (remission and Clinical Response by week 24) were grouped into a long-term family, with a surveillance method using the HochbergA28 testing procedure to control for general type I error, with the primary efficacy endpoint acting as the mediator. Statistical significance was assessed with a two-sided type I error level of 0.05. No statistical adjustment was made for multiplicity for non-primary secondary endpoints. Percentages and treatment differences were expressed with 95% confidence intervals (CI) calculated with an asymptomatic Wald method. Time to remission and Clinical Response was analyzed with Kaplan-Meier estimates. Safety results were presented with descriptive statistics.

[0170] SAS System v9.1.3 or later was used for statistical analyzes (SAS Institute Inc., Cary, NC, USA).

Results

[0171] A total of 289 patients were screened, and of these, 212 were randomized to Cx601 or placebo (Figure 2). The baseline characteristics of the 2 groups were similar (Table 1). Most patients received > 1 treatment for Crohn's disease in the last 6 months. A greater proportion of patients in the Cx601 group had multi-tract fistulas compared to the placebo group (46.6% and 30.4%, respectively). A total of 171 patients (80.7%) completed the 24-week follow-up. During the study, 1 patient in the Cx601 group and 4 patients in the placebo group received steroids for sudden increase in Crohn's Disease.

Efficiency

[0172] A significantly greater proportion of patients treated with Cx601 achieved the primary endpoint of combined remission at week 24 vs. placebo in the ITT populations (49.5% and 34.3%, respectively; difference [97.5% CI]: 15.2% [0.2-30.3]; p=0.024]) and mITT (51.5% and 35.6%; 15.8% [0.5-31.2]; p=0.021; Figure 3A and B). These results were confirmed in the PP populations and in additional sensitivity analyzes (Table S1). The effect of Cx601 was greater than placebo across all 4 randomization strata, with the numerically greater effect of Cx601 being observed in patients who did not receive or received both anti-TNF and immunosuppressive therapies at randomization (treatment difference 33.1% and 20.0%, respectively; Figure 3C).

[0173] In the mITT population, a numerically greater proportion of patients in the Cx601 group vs. placebo achieved clinical remission (55.3% and 42.6%, respectively; difference [95% CI]: 12.8% [-0.8-26.4]; p=0.057) and had a response (68.9 % and 55.4%, respectively; 13.5% [0.3-26.7] p=0.045) around week 24.

[0174] The average time to clinical remission was approximately 2 times shorter with Cx601 vs. placebo (6.7 [95% CI, 6.4-11.9] weeks and 14.6 [11.9-22.9] weeks), as was the median time to response (6.3 [6. 0-6.6] weeks and 11.7 [6.7-12.9] weeks).

[0175] The improvement in PDAI with Cx601 in the mITT population was significantly greater than with placebo at 6 weeks (change from baseline [95% CI]: -1.0 [-1.7 to -0. 3]), week 12 (-1.2 [-2.0 to -0.4]), and week 18 (-1.2 [-2.0 to -0.3]), but not week 24 (-0.8 [1.8 to 0.2]). For IBDQ and CDAI total and subdomain scores, there were no significant differences between treatment groups (Table S2).

Security

[0176] The percentage of patients in the Cx601 and placebo groups who experienced TEAEs was similar (66.0% and 64.7%, respectively; Table 2). The most commonly reported TEAEs were proctalgia, anal abscess, and nasopharyngitis. A greater proportion of patients in the placebo group vs. Cx601 group showed treatment-related TEAEs (29.4% and 17.5%, respectively), the most common of which were anal abscess and proctalgia. Most TEAEs were mild or moderate in intensity. Fewer patients were withdrawn from the study due to TEAEs (4.9% and 5.9%), while a slightly higher proportion of patients in the Cx601 group experienced serious TEAEs (17.5% and 13.7%). Most common serious TEAE was anal abscess (Cx601: 8.7%; placebo: 6.9%). There were no deaths.

Discussion and Overview

[0177] This is the first large-scale, randomized, placebo-controlled clinical study of the treatment of complex perianal fistulas, refractory to therapy in patients with Crohn's Disease. In the difficult-to-treat study patient population who had complex perianal fistulas and failed conventional or biologic therapy, 1 in 2 patients treated with Cx601 alone or added to current medical therapy achieved combined clinical and radiological remission at week 24. This result was consistent across all statistical populations, although patients in the Cx601 group had more multiple tract fistulas. The study results therefore present that Cx601 offers treatment-refractory patients a first true closure alternative for complex perianal fistulas to radical surgical approaches.

[0178] The primary endpoint of combined clinical and radiological remission is stricter than that used in other randomized clinical trials of treatments for perianal fistulas in Crohn's disease, which have typically evaluated clinical responses (i.e., >50% reduction in number of fistulas draining) or clinical remission.A8, A12 This is the first large-scale randomized clinical trial to utilize clinical assessment of fistula closure and MRI assessment of absence of abscesses, as recommended in the European Crohn's and Colitis Organization guidelines. A29 [European Organization for Crohn's Disease and Colitis].

[0179] Secondary efficacy analyzes reinforce the clinical benefit of Cx601. With Cx601, time to remission and Clinical Response was rapid, occurring in half the time of the placebo group. The difference in clinical remission rates between Cx601 and placebo groups did not reach statistical significance, due to a considerably greater “placebo” effect (42.6%) than expected and observed in previous studies (13-19%). A12, A27 Placebo effects, according to randomization strata (i.e., highest with anti-TNF and concomitant immunosuppressant [46.7%] and lowest with no treatment [21.1%]), suggest that the greatest Placebo effect in this study may have been triggered by the use of concomitant medication. Surgical drainage and internal os closure may also have increased the increased placebo response. While beneficial improvements in PDAI scores were seen in the Cx601 group, there were no differences in IBDQ and CDAI scores between treatment groups. This was likely due to low CDAI (an inclusion criterion) and high IBDQ at baseline.

[0180] Safety data show that Cx601 was well tolerated in the study population in accordance with the results of the previous phase 1/2a study. A23 Treatment-related TEAEs occurred more frequently in the placebo group and thus may be related to natural evolution of the disease or surgical procedures in the study. The favorable safety profile may represent an advantage over anti-TNF therapies which are associated with several serious safety concerns resulting from drug immunogenicity and an increased risk of infections, including tuberculosis.A30, A31

[0181] The results of this study are encouraging, considering the need for new effective and well-tolerated treatment options for patients with Crohn's Disease and complex perianal fistulas. This study also has important implications for the care of Crohn's disease patients with treatment-refractory complex perianal fistulas, as many of them currently must undergo repeat surgery with the risk of damaging sphincter muscles, resulting in fecal incontinence. As a result, 12-38% of patients with perianal fistula-developing disease require proctectomy.A32 In contrast, administration of Cx601 is minimally invasive and can be performed in an outpatient setting.

[0182] The limitations of this clinical study were the exclusion of patients with more than 2 internal and 3 external openings, as well as those with previous surgery other than drainage and seton insertion. Furthermore, whether TEAEs were related to the surgical procedure has not been established.

[0183] In conclusion, Cx601 is an effective and safe therapy for complex perianal fistulas in Crohn's Disease that have failed to respond to conventional and/or biological treatments.

[0184] Table 1. Patient characteristics (safety population).

[0185] CD, Crohn's Disease; Cx601, expanded, allogeneic, adipose tissue-derived stem cells; TNF, tumor necrosis factor.

[0186] Data are averages (standard deviation) unless otherwise stated.

[0187] *Scores for Perianal Crohn's Disease Activity Index can range from 0 to 20; higher scores indicate more severe disease.

[0188] Crohn's Disease Activity Index scores can range from 0 to 600; higher scores indicate more severe disease.

[0189] ^Scores for the Inflammatory Bowel Disease Questionnaire can range from 32 to 224; higher scores indicate better quality of life.

[0190] Table 2. Treatment-emergent adverse events up to week 24 (safety population).

[0191] TEAE, treatment-emergent adverse event; Cx601, expanded, allogeneic, adipose tissue-derived stem cells; *In any treatment group.

[0192] Table S1. Support and sensitivity analyzes for primary endpoint: combined remission.*

[0193] CI, confidence interval; Cx601, expanded, allogeneic, adipose tissue-derived stem cells; ITT, intention to treat; LOCF, last observation carried forward; NA, not applicable; PP, according to protocol; TNF, tumor necrosis factor.

[0194] Rescue therapy is defined as corticosteroids at 40 mg prednisone equivalent for > 12 weeks; new anti-TNF therapy compared to baseline for > 8 weeks; new immunosuppressive therapy compared to baseline for > 12 weeks; or surgical intervention for the treated fistula.

[0195] *Clinical assessment of closure of all treated external openings that were draining at baseline, and the absence of collections > 2 cm from treated perianal fistulas in > 2 of 3 dimensions on centrally blinded MRI assessment at around week 24 .Clinical assessment of closure was defined as absence of drainage despite non-invasive finger compression.

[0196] tPP1 population includes those with important protocol deviations (Cx601, n=86; Placebo, n=84).

[0197] ^PP2 population includes PP1 patients who completed the week 24 visit (Cx601, n=76; Placebo,n=73).

[0198] Table S2. Crohn's Disease Activity Index (CDAI)* and Inflammatory Bowel Disease Questionnaire (IBDQ)+ patient-reported results: scores through week 24 (mITT population).

[0199] CI, confidence interval; Cx601, expanded, allogeneic, adipose tissue-derived stem cells; mITT, modified intention to treat.

[0200] Data are averages (standard deviation).

[0201] *Scores for Crohn's Disease Activity Index (CDAI) can range from 0 to 600; higher scores indicate more severe disease.

[0202] Inflammatory Bowel Disease Questionnaire (IBDQ) scores can range from 32 to 224; higher scores indicate better quality of life.

REFERENCES

[0203] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the knowledge of the art. These techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatment, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

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Equivalents

[0205] The present invention provides, among other things, methods and compositions for treating and preventing fistula. Although specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The appended claims are not intended to claim all such embodiments and variations and the full scope of the invention should be determined by reference to the claims, together with their full scope of equivalents and the specification, together with such variations.

Claims (7)

1. USE OF ESTROMAL STEM CELLS, derived from adipose tissue, allogeneic, expanded, characterized by being for the manufacture of a medicine to treat multiple tracts of a refractory complex perianal fistula in a patient with Crohn's Disease. 2. USE, according to claim 1, characterized in that the fistula comprises two internal openings and/or three external openings. 3. USE, according to any one of claims 1 or 2, characterized in that the fistula is refractory to one or more of (i) antibiotic, (ii) immunosuppressant and (iii) anti-TNF therapy. 4. USE, according to any one of claims 1 to 3, characterized by the patient: (a) having non-active or slightly active Crohn's Disease; and/or (b) Luminal Crohn's disease. 5. USE, according to any one of claims 1 to 4, characterized in that the fistula is monitored by magnetic resonance imaging. 6. USE, according to any one of claims 1 to 5, characterized in that the expanded, allogeneic, adipose tissue-derived stromal stem cells express four or more of the surface markers HLA I, CD29, CD44, CD59, CD73, CD90 and CD105; and do not express four or more of HLAII, CD11b, CD11c, CD14, CD45, CD31, CD34, CD80 and CD86. 7. USE, according to any one of claims 1 to 6, characterized in that the medicine is formulated to be used in a dose of 120 million cells in a single procedure.