Morphogen-induced modulation of inflammatory response
The present invention is directed to methods and compositions for alleviating tissue destructive effects associated with the inflammatory response to tissue injury in a mammal. The methods and compositions include administering a therapeutically effective concentration of a morphogen or morphogen-stimulating agent sufficient to alleviate immune cell-mediated tissue destruction.
This application is a continuation-in-part of (1) U.S. Ser. No. 753,059, filed Aug. 30, 1991, which is a continuation-in-part of U.S. Ser. No. 667,274, filed Mar. 11, 1991, (2) U.S. Ser. No. 752,764, filed Aug. 30, 1991, which is a continuation-in-part of U.S. Ser. No. 667,274 and [Atty. Docket No. CRP-068] filed on even date herewith.
FIELD OF THE INVENTIONThe present invention relates generally to a method for modulating the inflammatory response induced in a mammal following tissue injury. More particularly, this invention relates to a method for alleviating immune-cell mediated tissue destruction associated with the inflammatory response.
BACKGROUND OF THE INVENTIONThe body's inflammatory response to tissue injury can cause significant tissue destruction, leading to loss of tissue function. Damage to cells resulting from the effects of inflammatory response e.g., by, immune-cell mediated tissue destruction, has been implicated as the cause of reduced tissue function or loss of tissue function in diseases of the joints (e.g., rheumatoid and osteo-arthritis) and of many organs, including the kidney, pancreas, skin, lung and heart. For example, glomular nephritis, diabetes, inflammatory bowel disease, vascular diseases such as atheroclerosis and vasculitis, and skin diseases such as psoriasis and dermatitis are believed to result in large part from unwanted acute inflammatory reaction and fibrosis. A number of these diseases, including arthritis, psoriasis and inflammatory bowel disease are considered to be chronic inflammatory diseases. The damaged tissue also often is replaced by fibrotic tissue, e.g., scar tissue, which further reduces tissue function. Graft and transplanted organ rejection also is believed to be primarily due to the action of the body's immune/inflammatory response system.
The immune-cell mediated tissue destruction often follows an initial tissue injury or insult. The secondary damage, resulting from the inflammatory response, often is the source of significant tissue damage. Among the factors thought to mediate these damaging effects are those associated with modulating the body's inflammatory response following tissue injury, e.g., cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), and oxygen-derived free radicals such as superoxide anions. These humoral agents are produced by adhering neutrophilic leukocytes or by endothelial cells and have been identified at ischemic sites upon-reperfusion. Moreover, TNF concentrations are increased in humans after myocardial infarction.
A variety of lung diseases are characterized by airway inflammation, including chronic bronchitis, emphysema, idiopathic pulmonary fibrosis and asthma. Another type of lung-related inflammation disorders are inflammatory diseases characterized by a generalized, wide-spread, acute inflammatory response such as adult respiratory distress syndrome. Another dysfunction associated with the inflammatory response is that mounted in response to injury caused by hyperoxia, e.g., prolonged exposure to lethally high concentrations of O2-(95-100% O2). Similarly, reduced blood flow to a tissue (and, therefore reduced or lack of oxygen to tissues), as described below, also can induce a primary tissue injury that stimulates the inflammatory response.
It is well known that damage occurs to cells in mammals which have been deprived of oxygen. In fact, the interruption of blood flow, whether partial (hypoxia) or complete (ischemia) and the ensuing inflammatory responses may be the most important cause of coagulative necrosis or cell death in human disease. The complications of atherosclerosis, for example, are generally the result of ischemic cell injury in the brain, heart, small intestines, kidneys, and lower extremities. Highly differentiated cells, such as the proximal tubular cells of the kidney, cardiac myocytes, and the neurons of the central nervous system, all depend on aerobic respiration to produce ATP, the energy necessary to carry out their specialized functions. When ischemia limits the oxygen supply and ATP is depleted, the affected cells may become irreversibly injured. The ensuing inflammatory responses to this initial injury provide additional insult to the affected tissue. Examples of such hypoxia or ischemia-are the partial or total loss of blood supply to the body as a whole, an organ within the body, or a region within an organ, such as occurs in cardiac arrest, pulmonary embolus, renal artery occlusion, coronary occlusion or occlusive stroke.
The tissue damage associated with ischemia-reperfusion injury is believed to comprise both the initial cell damage induced by the deprivation of oxygen to the cell and its subsequent recirculation, as well as the damage caused by the body's response to this initial damage. It is thought that reperfusion injury may result in dysfunction to the endothelium of the vasculature as well as injury to the surrounding tissue. In idiopathic pulmonary fibrosis, for example, scar tissue accumulates on the lung tissue lining, inhibiting the tissue's elasticity. The tissue damage associated with hyperoxia injury is believed to follow a similar mechanism, where the initial damage is mediated primarily through the presence of toxic oxygen metabolites, followed by an inflammatory response to this initial injury.
Similarly, tissues and organs for transplantation also are subject to the tissue destructive effects associated with the recipient host body's inflammatory response following transplantation. It is currently believed that the initial destructive response is due in large part to reperfusion injury to the transplanted organ after it has been transplanted to the organ recipient.
Accordingly, the success of organ or tissue transplantation depends greatly on the preservation of the tissue activity (e.g., tissue or organ viability) at the harvest of the organ, during storage of the harvested organ, and at transplantation. To date, preservation of organs such as lungs, pancreas, heart and liver remains a significant stumbling block to the successful transplantation of these organs. U.S. Pat. No. 4,952,409 describes a superoxide dismutase-containing liposome to inhibit reperfusion injury. U.S. Pat. No. 5,002,965 describes the use of ginkolides, known platelet activating factor antagonists, to inhibit reperfusion injury. Both of these factors are described as working primarily by inhibiting the release of and/or inhibiting the damaging effects of free oxygen radicals. A number of patents also have issued on the use of immunosuppressants for inhibiting graft rejection. A representative listing includes U.S. Pat. Nos. 5,104,858, 5,008,246 and 5,068,323. A significant problem with many immunosuppressants is their low therapeutic index, requiring the administration of high doses that can have significant toxic side effect.
Rheumatoid and osteoarthritis are prevalent diseases characterized by chronic inflammation of the synovial membrane lining the afflicted joint. A major consequence of chronic inflammatory joint disease (e.g., rheumatoid arthritis) and degenerative arthritis (e.g., osteoarthritis) is loss of function of those affected joints. This loss of function is due primarily to destruction of the major structural components of the joint, cartilage and bone, and subsequent loss of the proper joint anatomy. As a consequence of chronic disease, joint destruction ensues and can lead to irreversible and permanent damage to the joint and loss of function. Current treatment methods for severe cases of rheumatoid arthritis typically include the removal of the synovial membrane, e.g., synovectomy. Surgical synovectomy has many limitations, including the risk of the surgical procedure itself, and the fact that a surgeon often cannot remove all of the diseased membrane. The diseased tissue remaining typically regenerates, causing the same symptoms which the surgery was meant to alleviate.
Psoriasis is a chronic, recurrent, scaling skin disease of unknown etiology characterized by chronic inflammation of the skin. Erythematous eruptions, often in papules or plaques, and usually having a white silvery scale, can affect any part of the skin, but most commonly affect the scalp, elbows, knees and lower back. The disease usually occurs in adults, but children may also be affected. Patients with psoriasis have a much greater incidence of arthritis (psoraitic arthritis), and generalized exfoliation and even death can threaten afflicted individuals.
Current therapeutic regimens include topical or intralesional application of corticosteroids, topical administration of keratolytics, and use of tar and UV light on affected areas. No single therapy is ideal, and it is rare for a patient not to be treated with several alternatives during the relapsing and remitting course of the disease. Whereas systematic treatment can induce prompt resolution of psoriatic lesions, suppression often requires ever-increasing doses, sometimes with toxic side effect, and tapering of therapy may result in rebound phenomena with extensions of lesions, possibly to exfoliation.
Inflammatory bowel disease (IBD) describes a class of clinical disorders of the gastrointestinal mucosa characterized by chronic inflammation and severe ulceration of the mucosa. The two major diseases in this classification are ulcerative colitis and regional enteritis (Crohn's Disease). Like oral mucositis, the diseases classified as IBD are associated with severe mucosal ulceration (frequently penetrating the wall of the bowel and forming strictures and fistulas), severe mucosal and submucosal inflammation and edema, and fibrosis (e.g., scar tissue formation which interferes with the acid protective function of the gastrointestinal lining.) Other forms of IBD include regional ileitis and proctitis. Clinically, patients with fulminant IBD can be severely ill with massive diarrhea, blood loss, dehydration, weight loss and ever. The prognosis of the disease is not good and frequently requires resection of the diseased tissue.
Therefore, an object of the present invention is to provide a method for protecting mammalian tissue, particularly human tissue, from the damage associated with the inflammatory response following a tissue injury. The inflammatory reaction may be in response to an initial tissue injury or insult. The original injury may be chemically, mechanically, immunologically or biologically related. Another object is to provide methods and compositions for protecting tissue from the tissue destructive effects associated with chronic inflammatory diseases, including arthritis (e.g., reheumatoid or osteoarthritis), psoriatic arthritis, psoriasis and dermatitis, inflammatory bowel disease and other autoimmune-diseases.
Another object of the invention is to provide methods and compositions for enhancing the viability of mammalian tissues and organs to be transplanted, including protecting the transplanted organs from immune cell-mediated tissue destruction, such as the tissue damage associated with ischemia-reperfusion injury, such as can occur upon initiation of blood flow after transplantation of the organ in the recipient host.
Another object of the invention is to provide a method for alleviating tissue damage associated with ischemic-reperfusion injury in a mammal following a deprivation of oxygen to a tissue in the mammal. Other objects of the present invention include providing a method for alleviating tissue damage associated with ischemic-reperfusion injury in a human which has suffered from hypoxia or ischemia following cardiac arrest, pulmonary embolus, renal artery-occlusion, coronary occlusion or occlusive stroke, as well as tissue damage associated with a surgical or other aggressive clinical procedure. Still another object is to provide a method for alleviating tissue damage associated with hyperoxia-induced injury in a human following exposure to lethally high oxygen concentrations.
Still another object of the invention is to provide a method for modulating inflammatory responses in general, particularly those induced in a human following tissue injury.
These and other objects and features of the invention will be apparent from the description, drawings and claims which follow.
SUMMARY OF THE INVENTIONThe present invention provides a method for alleviating the tissue destructive effects associated with activation of the inflammatory response following tissue injury. The method comprises the step of providing to the affected tissue a therapeutically effective concentration of a morphogenic protein (“morphogen”, as defined herein) upon tissue injury or in anticipation of tissue injury, sufficient to substantially inhibit or reduce the tissue destructive effects of the inflammatory response.
In one aspect, the invention features compositions and therapeutic treatment methods that comprise the step of administering to a mammal a therapeutically effective amount of a morphogenic protein (“morphogen”), as defined herein, upon injury to a tissue, or in anticipation of such injury, for a time and at a concentration sufficient to inhibit the tissue destructive effects associated with the body's inflammatory response, including repairing damaged tissue, and/or inhibiting additional damage thereto.
In another aspect, the invention features compositions and therapeutic treatment methods for protecting tissues and organs from the tissue destructive effects of the inflammatory response which include administering to the mammal, upon injury to a tissue or in anticipation of such injury, a compound that stimulates in vivo a therapeutically effective concentration of an endogenous morphogen within the body of the mammal sufficient to protect the tissue from the tissue destructive effects associated with the inflammatory response, including repairing damaged tissue and/or inhibiting additional damage thereto. These-compounds are referred to herein as morphogen-stimulating agents, and are understood to include substances which, when administered to a mammal, act on cells of tissue(s) or organ(s) that normally are responsible for, or capable of, producing a morphogen and/or secreting a morphogen, and which cause the endogenous level of the morphogen to be altered. The agent may act, for example, by stimulating expression and/or secretion of an endogenous morphogen.
As embodied herein, the term “ischemic-reperfusion injury” refers to the initial damage associated with oxygen deprivation of a cell and the subsequent damage associated with the inflammatory response when the cell is resupplied with oxygen. As embodied herein, the term “hyperoxia-induced injury” refers to the tissue damage associated with prolonged exposure to lethally high doses of oxygen, e.g., greater than 95% O2, including the tissue damage associated with the inflammatory response to the initial toxically high oxygen concentration. Accordingly, as used herein, “toxic oxygen concentrations” refers to the tissue damage associated with the injury induced by both lethally low oxygen concentrations of oxygen (including the complete lack of oxygen), and by lethally high oxygen concentrations. The expression “alleviating” means the protection from, reduction of and/or elimination of undesired tissue destruction, particularly immune cell-mediated tissue destruction. The tissue destruction may be in response to an initial tissue injury, which may be mechanical, chemical or immunological in origin. The expression “enhance the viability of” tissues or organs, as used herein, means protection from, reduction of and/or elimination of reduced or lost tissue or organ function as a result of tissue death, particularly immune cell-mediated tissue death. “Transplanted” living tissue includes both tissue transplants, (e.g., as in the case of bone marrow transplants, for example), and tissue grafts. Finally, a “free oxygen radical inhibiting agent” means a molecule capable of inhibiting the release of and/or inhibiting the tissue damaging effects of free oxygen radicals.
In one embodiment of the invention, the invention provides methods and compositions for alleviating the ischemic-reperfusion injury in mammalian tissue resulting from a deprivation of, and subsequent reperfusion of, oxygen to the tissue. In another embodiment, the invention provides a method for alleviating the tissue-destructive effects associated with hyperoxia. In still another embodiment of the invention, the invention provides methods and compositions for maintaining the viability of tissues and organs, particularly transplanted tissues and organs, including protecting these tissues and organs from ischemia-reperfusion injury. In still another embodiment, the invention provides methods for protecting tissues and organs from the tissue destructive effects of chronic inflammatory diseases, such as arthritis, psoriasis, dermatitis, including contact dermatitis, IBD and other chronic inflammatory diseases of the gastrointestinal tract, as well as the tissue destructive effects associated with other, known autoimmune diseases, such as diabetes, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and other autoimmune neurodegenerative diseases.
In one aspect of the invention, the morphogen is provided to the damaged tissue following an initial injury to the tissue. The morphogen may be provided directly to the tissue, as by injection to the damaged tissue site or by topical administration, or may be provided indirectly, e.g., systemically by oral or parenteral means. Alternatively, as described above, an agent capable of stimulating endogenous morphogen expression and/or secretion may be administered to the mammal. Preferably, the agent can stimulate an endogenous morphogen in cells associated with the damaged tissue. Alternatively, morphogen expression and/or secretion may be stimulated in a distant tissue and the morphogen transported to the damaged tissue by the circulatory system.
In another aspect of the invention, the morphogen is provided to tissue at risk of damage due to immune cell-mediated tissue destruction. Examples of such tissues include tissue grafts and transplanted tissue or organs, as well as any tissue or organ about to undergo a surgical procedure or other clinical procedure likely to either inhibit blood flow to the tissue or otherwise induce an inflammatory response. Here the morphogen or morphogen-stimulating agent preferably is provided to the patient prior to induction of the injury, e.g., as a prophylactic, to provide a cyto-protective effect to the tissue at risk.
The morphogens described herein are envisioned to be useful in enhancing viability of any organ or living tissue to be transplanted. The morphogens may be used to particular advantage in lung, heart, kidney, liver and pancreas transplants, as well as in transplantation and/or grafting of skin, gastrointestinal mucosa, bone marrow and other living tissues.
Where the patient suffers from a chronic inflammatory disease, such as diabetes, arthritis, psoriasis, IBD, and the like, the morphogen or morphogen-stimulating agent preferably is administered at regular intervals as a prophylactic, to prevent and/or inhibit the tissue damage normally associated with the disease during flare periods. As above, the morphogen or morphogen-stimulating agent may be provided directly to the tissue at risk, for example by injection or by topical administration, or indirectly, as by systemic e.g., oral or parenteral administration.
Among the morphogens useful in this invention are proteins originally identified as osteogenic proteins, such as the OP-1, OP-2 and CBMP2 proteins, as well as amino acid sequence-related proteins such as DPP (from Drosophila), Vgl (from Xenopus), Vgr-1 (from mouse, see U.S. Pat. No. 5,011,691 to Oppermann et al.), GDF-1 (from mouse, see Lee (1991) PNAS 88:4250-4254), all of which are presented in Table II and Seq. ID Nos.5-14), and the recently identified 60A protein (from Drosophila, Seq. ID No. 24, see Wharton et al. (1991) PNAS 88:9214-9218.) The members of this family, which include members of the TGF-β super-family, of proteins, share substantial-amino acid sequence homology in their C-terminal regions. The proteins are translated as a precursor, having an N-terminal signal peptide sequence, typically less than about 30 residues, followed by a “pro” domain that is cleaved to yield the mature sequence. The signal peptide is cleaved rapidly upon translation, at a cleavage site that can be predicted in a given sequence using the method of Von Heijne ((1986) Nucleic Acids Research 14:4683-4691.) Table I, below, describes the various morphogens identified to date, including their nomenclature as used herein, their Seq. ID references, and publication sources for the amino acid sequences for the full length proteins not included in the Seq. Listing. The disclosure of these publications is incorporated herein by reference.
The OP-2 proteins have an additional cysteine residue in this region (e.g., see residue 41 of Seq. ID Nos. 7 and 8), in addition to the conserved cysteine skeleton in common with the other proteins in this family. The GDF-1 protein, has a four amino acid insert within the conserved skeleton (residues 44-47 of Seq. ID No. 14) but this insert likely does not interfere with the relationship of the cysteines in the folded structure. In addition, the CBMP2 proteins are missing one amino acid residue within the cysteine skeleton.
The morphogens are inactive when reduced, but are active as oxidized homodimers and when oxidized in combination with other morphogens of this invention (e.g., asheterodimers). Thus, as defined herein, a morphogen is a dimeric protein comprising a pair of polypeptide chains, wherein each polypeptide chain comprises at least the C-terminal six cysteine skeleton defined by residues 43-139 of Seq. ID No. 5, including functionally equivalent arrangements of these cysteines (e.g., amino acid insertions or deletions which alter the linear arrangement of the cysteines in the sequence but not their relationship in the folded structure), such that, when the polypeptide chains are folded, the dimeric protein species comprising the pair of polypeptide chains has the appropriate three-dimensional structure, including the appropriate intra-or inter-chain disulfide bonds such that the protein is capable of acting as a morphogen as defined herein. Specifically, the morphogens generally are capable of all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells, including the “redifferentiation” of transformed cells. In addition, it is also anticipated that these morphogens are capable of inducing redifferentiation of committed cells under appropriate environmental conditions.
In one preferred aspect, the morphogens of this invention comprise one of two species of generic amino acid sequences: Generic Sequence 1 (Seq. ID No. 1) or Generic Sequence 2 (Seq. ID No. 2); where each Xaa indicates one of the 20 naturally-occurring L-isomer, α-amino acids or a derivative thereof. Generic Sequence 1 comprises the conserved six cysteine skeleton and Generic Sequence 2 comprises the conserved six cysteine skeleton plus the additional cysteine identified in OP-2 (see residue 36, Seq. ID No. 2). In another preferred aspect, these sequences further comprise the following additional sequence at their N-terminus:
Preferred amino acid sequences within the foregoing generic sequences include: Generic Sequence 3 (Seq. ID No. 3), Generic Sequence 4 (Seq. ID No. 4), Generic Sequence 5 (Seq. ID No. 30) and Generic Sequence 6 (Seq. ID No. 31), listed below. These Generic Sequences accommodate the homologies shared among the various preferred members of this morphogen family identified in Table II, as well as the amino acid sequence variation among them. Specifically, Generic Sequences 3 and 4 are composite amino acid sequences of the following proteins presented in Table II and identified in Seq. ID Nos. 5-14: human OP-1 (hOP-1, Seq. ID Nos. 5 and 0.16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), and GDF-1 (from mouse, Seq. ID No. 14.) The generic sequences include both the amino acid identity shared by the sequences in Table II, as well as alternative residues for the variable positions within the sequence. Note that these generic sequences allow for an additional cysteine at position 41 or 46 in Generic Sequences 3 or 4, respectively, providing an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and contain certain critical amino acids which influence the tertiary structure of the proteins.
wherein each Xaa is independently selected from a group of one or more specified amino acids defined as follows: “Res.” means “residue” and Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser or Lys); Xaa at res.7=(Asp or Glu); Xaa at res.8=(Leu or Val); Xaa at res.11=(Gln, Leu, Asp, His or Asn); Xaa at res.12=(Asp, Arg or Asn); Xaa at res.14=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Leu or Gln); Xaa at res.23=(Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr, Asp or Gln); Xaa at res.28=(Glu, Lys, Asp or Gln); Xaa at res.30=(Ala, Ser, Pro or Gln); Xaa at res.31=(Phe, Leu- or Tyr); Xaa at res.33=(Leu or Val); Xaa at res.34=(Asn, Asp, Ala or Thr); Xaa at res.35=(Ser, Asp, Glu, Leu or Ala); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn or Ser); Xaa at res.39=(Ala, Ser or Gly); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile or Val); Xaa at res.45=(Val or Leu); Xaa at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at res.49=(Val or Met); Xaa at res.50 (His or Asn); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52=(Ile, Met, Asn, Ala or Val); Xaa at res.53=(Asn, Lys, Ala or Glu); Xaa at res.54=(Pro or Ser); Xaa at res.55=(Glu, Asp, Asn, or Gly); Xaa at res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at res.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp; Xaa at res.59=(Lys or Leu); Xaa at res.60=(Pro or Ala); Xaa at res.63=(Ala or Val); Xaa at res.65=(Thr or Ala.); Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa at res.68=(Asn, Ser or Asp); Xaa at res.69=(Ala, Pro or Ser); Xaa at res.70=(Ile, Thr or Val); Xaa at res.71=(Ser or Ala); Xaa at res.72=(Val or Met); Xaa at res.74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr or Leu); Xaa at res.76=(Asp or Asn); Xaa at res.77=(Asp, Glu, Asn or Ser); Xaa at res.78=(Ser, Gln, Asn or Tyr); Xaa at res.79=(Ser, Asn, Asp or Glu); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile or Val); Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln or His); Xaa at res.86=(Tyr or His); Xaa at res.87=(Arg, Gln or Glu); Xaa at res.88 (Asn, Glu or Asp); Xaa at res.90=(Val, Thr or Ala); Xaa at res.92.=(Arg, Lys, Val, Asp or Glu); Xaa at res.93=(Ala, Gly or Glu); and Xaa at res.97=(His or Arg);
wherein each Xaa is independently selected from a group of one or more specified amino acids as defined by the, following: “Res.” means “residue” and Xaa at res.2=(Lys or Arg); Xaa at res.3=(Lys or Arg); Xaa at res.4=(His or Arg); Xaa at res.5=(Glu, Ser, His, Gly, Arg or Pro); Xaa at res.9=(Ser, Asp or Glu); Xaa at res.11=(Arg, Gln, Ser or Lys); Xaa at res.12=(Asp or Glu); Xaa at res.13=(Leu or Val); Xaa at res.16=(Gln, Leu, Asp, His or Asn); Xaa at res.17=(Asp, Arg, or Asn); Xaa at res.19=(Ile or Val); Xaa at res.20=(Ile or Val); Xaa at res.23=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.25=(Tyr or Phe); Xaa at res.26 (Ala, Ser, Asp, Met, His, Leu, or Gln); Xaa at res.28=(Tyr, Asn or Phe); Xaa at res.31=(Glu, His, Tyr, Asp or Gln); Xaa at res.33=Glu, Lys, Asp or Gln); Xaa at res.35=(Ala, Ser or Pro); Xaa at res.36=(Phe, Leu or Tyr); Xaa at res.38=(Leu or Val); Xaa at res.39=(Asn, Asp, Ala or Thr); Xaa at res.40=(Ser, Asp, Glu, Leu or Ala); Xaa at res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met, Phe, Gly or Leu); Xaa at res.44=(Ala, Ser or Gly); Xaa at res.45=(Thr, Leu or Ser); Xaa at res.49=(Ile or Val); Xaa at res.50=(Val or Leu); Xaa at res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser); Xaa at res.54=(Val or Met); Xaa at res.55=(His or Asn); Xaa at res.56=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.57=(Ile, Met, Asn, Ala or Val); Xaa at res.58=(Asn, Lys, Ala or Glu); Xaa at res.59=(Pro or Ser); Xaa at res.60=(Glu, Asp, or Gly); Xaa at res.61=(Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at res.62=(Val, Ala or Ile); Xaa at res.63=(Pro or Asp); Xaa at res.64=(Lys or Leu); Xaa at res.65=(Pro or Ala); Xaa at res.68=(Ala or Val); Xaa at res.70=(Thr or Ala); Xaa at res.71=(Gln, Lys, Arg or Glu); Xaa at res.72=(Leu, Met or Val); Xaa at res.73=(Asn, Ser or Asp); Xaa at res.74=(Ala, Pro or Ser); Xaa at res.75=(Ile, Thr or Val); Xaa at res.76=(Ser or Ala); Xaa at res.77=(Val or Met); Xaa at res.79=(Tyr or Phe); Xaa at res.80=(Phe, Tyr or Leu); Xaa at res.81=(Asp or Asn); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at res.83=(Ser, Gln, Asn or Tyr); Xaa at res.84=(Ser, Asn, Asp or Glu); Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile or Val); Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln or His); Xaa at res.91=(Tyr or. His); Xaa at res.92=(Arg, Gln or Glu); Xaa at res.93=(Asn, Glu or Asp); Xaa at res.95=(Val, Thr or Ala); Xaa at res.97=(Arg, Lys, Val, Asp or Glu); Xaa at res.98=(Ala, Gly or Glu); and Xaa at res.102=(His or Arg).
Similarly, Generic Sequence 5 (Seq. ID No. 30) and Generic Sequence 6 (Seq. ID No. 31) accommodate the homologies shared among all the morphogen protein family members identified in Table II. Specifically, Generic Sequences 5 and 6 are composite amino acid sequences of human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), and GDF-1 (from mouse, Seq. ID No. 14), human BMP3, (Seq. ID No. 26), human-BMP5 (Seq. ID No. 27), human BMP6 (Seq. ID No. 28) and 60(A). (from Drosophila, Seq. ID Nos. 24-25), The generic sequences include both the amino acid identity shared by these sequences in the C-terminal domain, defined by the six and seven cysteine skeletons (Generic Sequences 5 and 6, respectively), as well as alternative residues for the variable positions within the sequence. As for Generic Sequences 3 and 4, Generic Sequences 5 and 6 allow for an additional cysteine at position 41 (Generic Sequence 5) or position 46, (Generic Sequence 6), providing an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and containing certain critical amino acids which influence the tertiary structure of the proteins.
wherein each Xaa is independently selected from a group of one or more specified amino acids defined as follows: “Res.” means “residue” and Xaa at res.2=(Tyr or Lys); Xaa at res.3 Val or Ile); Xaa at res.4=(Ser, Asp-or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or Ala); Xaa at res.7=(Asp, Glu or Lys); Xaa at res.8=(Leu, Val or Ile); Xaa at res.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.12 (Asp, Arg, Asn or Glu); Xaa at res.14=(Ile or Val); Xaa at res.15 (Ile or Val); Xaa at res.16 (Ala or Ser); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.19 (Gly or Ser), Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Gln, Leu or Gly); Xaa at res.2-3 (Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr, Asp, Gln or Ser); Xaa at res.28 (Glu, Lys, Asp, Gln or Ala); Xaa at res.30=(Ala, Ser, Pro, Gln or Asn); Xaa at res.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu, Val or Met); Xaa at res.34=(Asn, Asp, Ala, Thr or Pro); Xaa at res.35=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn, Ser or Lys); Xaa at res.39=(Ala, Ser, Gly or Pro); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu or Ile); Xaa at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at res.48=(Leu or Ile); Xaa at res.49=(Val or Met); Xaa at res.50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val or Leu); Xaa at res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Ser or Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val or Lys); Xaa at res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at res.57=(Val, Ala; or Ile); Xaa at res.58 (Pro or Asp); Xaa at res.59=(Lys, Leu or Glu); Xaa at res.60=(Pro or Ala); Xaa at res.63 (Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa at res.68=(Asn, Ser, Asp or Gly); Xaa at res.69 (Ala, Pro or Ser); Xaa at res.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or Pro); Xaa at res.72=(Val, Met or Ile); Xaa at res.74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or Leu); Xaa at res.77=(Asp, Glu, Asn or Ser); Xaa at res.78=(Ser, Gln, Asn, Tyr or Asp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile, Val or Asn); Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln, His or Val); Xaa at res.86=(Tyr-or His); Xaa at res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu or Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg, Lys, Val, Asp or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser); Xaa at res.95=(Gly or Ala) and Xaa at res.97 (His or Arg).
wherein each Xaa is independently selected from a group of one or more specified amino acids as defined by the following: “Res.” means “residue” and Xaa at res.2=(Lys, Arg, Ala or Gln); Xaa at res.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or Gln); Xaa at res.5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr); Xaa at res.7=(Tyr or Lys); Xaa at res.8=(Val or Ile); Xaa at res.9=(Ser, Asp or Glu); Xaa at res.11=(Arg, Gln, Ser, Lys or Ala); Xaa at res.12=(Asp, Glu, or Lys); Xaa at res.13=(Leu, Val or Ile); Xaa at res.16=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.17=(Asp, Arg, Asn or Glu); Xaa at res.19 (Ile or Val); Xaa at res.20=(Ile or Val); Xaa at res.21=(Ala or Ser); Xaa at res.23=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.24=(Gly or Ser); Xaa at res.25=(Tyr or Phe); Xaa at res.26=(Ala, Ser, Asp, Met, His, Gln, Leu, or Gly); Xaa at res.28=(Tyr, Asn or Phe); Xaa at res.31=(Glu, His, Tyr, Asp, Gln or Ser); Xaa at res.33=Glu, Lys, Asp, Gln or Ala); Xaa at res.35=(Ala, Ser. Pro, Gln or Asn); Xaa at res.36=(Phe, Leu or Tyr); Xaa at res.38=(Leu, Val or Met); Xaa at res.39=(Asn, Asp, Ala, Thr or Pro); Xaa at res.40=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met, Phe, Gly or Leu); Xaa at res.43=(Asn, Ser or Lys); Xaa at res.44=(Ala, Ser. Gly or Pro); Xaa at res.45=(Thr, Leu or Ser); Xaa at res.49=(Ile, Val or Thr); Xaa at res.50=(Val, Leu or Ile); Xaa at res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser); Xaa at res.53=(Leu or Ile); Xaa at res.54=(Val or Met); Xaa at res.55=(His, Asn or Arg); Xaa at res.56=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.57=(Ile, Met, Asn, Ala, Val or Leu); Xaa at res.58=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.59=(Pro, Ser or Val); Xaa at res.60=(Glu, Asp, Gly, Val or Lys); Xaa at res.61=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at res.62=(Val, Ala or Ile); Xaa at res.63=(Pro or Asp); Xaa at res.64=(Lys, Leu or Glu); Xaa at res.65=(Pro or Ala); Xaa at res.68=(Ala or Val); Xaa at res.70=(Thr, Ala or Glu); Xaa at res.71=(Gln, Lys, Arg or Glu); Xaa at res.72=(Leu, Met or Val); Xaa at res.73=(Asn, Ser, Asp or Gly); Xaa at res.74=(Ala, Pro or Ser); Xaa at res.75=(Ile, Thr, Val or Leu); Xaa at res.76=(Ser, Ala or Pro); Xaa at res.77=(Val, Met or Ile); Xaa at res.79=(Tyr or Phe); Xaa at res.80=(Phe, Tyr, Leu or His); Xaa at res.81=(Asp, Asn or Leu); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at res.83=(Ser, Gln, Asn, Tyr or Asp); Xaa at res.84=(Ser, Asn, Asp, Glu or Lys); Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile, Val or Asn); Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln, His or Val); Xaa at res.91=(Tyr or His); Xaa at res.92=(Arg, Gln, Glu or Pro); Xaa at res.93=(Asn, Glu or Asp); Xaa at res.95=(Val, Thr, Ala or Ile); Xaa at res.97-=(Arg, Lys, Val, Asp or Glu); Xaa at res.98=(Ala, Gly, Glu or Ser); Xaa at res.100=(Gly or Ala); and Xaa at res.102=(His or Arg).
Particularly-useful sequences for use as morphogens in this invention include the C-terminal domains, e.g., the C-terminal 96-102-amino acid residues of Vgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A, CBMP-2B, GDF-1 (see Table II, below, and Seq. ID Nos. 5-14), as well as proteins comprising the C-terminal domains of 60A, BMP3, BMP5 and BMP6 (see Seq. ID Nos. 24-28), all of which include at least the conserved six or seven cysteine skeleton. In addition, biosynthetic constructs designed from the generic sequences, such as COP-1,3-5, 7, 16; disclosed in U.S. Pat. No. 5,011,691, also are useful. Other sequences include the inhibins/activin proteins (see, for example, U.S. Pat. Nos. 4,968,590 and 5,011,691). Accordingly, other useful sequences are those sharing at least 70% amino acid sequence homology or “similarity”, and preferably 80% homology or similarity with any of the sequences above. These are anticipated to include allelic and species variants and mutants, and biosynthetic muteins, as well as novel members of this morphogenic family of proteins. Particularly envisioned in the family of related proteins are those proteins exhibiting morphogenic activity and wherein the amino acid changes from the preferred sequences include conservative changes, e.g., those as defined by Dayoff et al., Atlas of Protein Sequence and Structure; vol. 5 Suppl. 3, pp. 345-362, (M. O. Dayoff, ed., Nat'l BioMed. Research Fdn., Washington, D.C. 1979). As used herein, potentially useful sequences are aligned with a known morphogen sequence using the method of Needleman et al. ((1970) J. Mol. Biol. 48:443-453) and identities calculated by the Align program (DNAstar, Inc.). “Homology” or “similarity” as used herein includes allowed conservative changes as defined by Dayoff et al.
The currently most preferred protein sequences useful as morphogens in this invention include those having greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the conserved six cysteine skeleton of hOP1 (e.g., residues 43-139 of Seq. ID No. 5). These most preferred sequences include both allelic and species variants of the OP-1 and OP-2 proteins, including the Drosophila 60A protein. Accordingly, in another preferred aspect of the invention, useful morphogens include active proteins comprising species of polypeptide chains having the generic amino acid sequence herein referred to as “OPX”, which accommodates the homologies between the various identified species of OP1 and OP2 (Seq. ID No. 29).
The morphogens useful in the methods, composition and devices of this invention include proteins comprising any of the polypeptide chains described above, whether isolated from naturally occurring sources, or produced by recombinant DNA or other synthetic techniques, and includes allelic and species variants of these proteins, naturally occurring or biosynthetic mutants thereof, as well as various truncated and fusion constructs. Deletion or addition mutants also are envisioned to be active, including those which may alter the conserved C-terminal cysteine skeleton, provided that the alteration does not functionally disrupt the relationship of these cysteines in the folded structure. Accordingly, such active forms are considered the equivalent of the specifically described constructs disclosed herein. The proteins may include forms having varying glycosylation patterns, varying N-terminal family of related proteins having regions of amino acid sequence homology, and active truncated or mutated forms of native or biosynthetic proteins, produced by expression of recombinant DNA in host cells.
The morphogenic proteins can be expressed from intact or truncated cDNA or from synthetic DNAs in procaryotic or eucaryotic host cells, and purified, cleaved, refolded, and dimerized to form morphogenically active compositions. Currently preferred host cells include E. coli or mammalian cells, such as CHO, COS or BSC cells. A detailed description of the morphogens useful in the methods, compositions and devices of this invention is disclosed in copending U.S. patent application Ser. No. 752,764, filed Aug. 30, 1991, and Ser. No. 667,274, filed Mar. 11, 1991, the disclosure of which are incorporated herein by reference.
Thus, in view of this disclosure, skilled genetic engineers can isolate genes from cDNA or genomic libraries of various different species which encode appropriate amino acid sequences, or construct DNAs from oligonucleotides, and then can express them in various types of host cells, including both procaryotes and eucaryotes, to produce large quantities of active proteins capable of protecting tissues and organs from immune cell-mediated tissue destruction, including substantially inhibiting such damage and/or regenerating the damaged tissue in a variety of mammals, including humans.
The foregoing and other objects, features and advantages of the present invention will be made more apparent from the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 4(A and B) are schematic representations of morphogen inhibition of early mononuclear phagocytic multinuclearization in vivo;
It now has been surprisingly discovered that the morphogens defined herein are effective agents in alleviating the tissue destructive effects associated with the body's inflammatory response to tissue injury. In particular, as disclosed herein, the morphogens are capable of alleviating the necrotic tissue effects associated with the ensuing inflammatory responses that occur following an initial tissue injury.
When tissue injury occurs, whether caused by bacteria, trauma, chemicals, heat, or any other phenomenon, the body's inflammatory response is stimulated. In response to signals released from the damaged cells (e.g., cytokines), extravascularization of immune effector cells is induced. Under ordinary circumstances these invading immune effector cells kill the infectious agent and/or infected or damaged cells (through the release of killing substances such as superoxides, performs, and other antimicrobial agents stored in granules), remove the dead tissues and organisms (through phagocytosis), release various biological response modifiers that promote rapid healing and covering of the wound (quite often resulting in the formation of fibrotic scar tissue), and then, after the area is successfully healed, exit from the site of the initial insult. Once the site is perceived to be normal, the local release of inflammatory cytokines ceases and the display of adhesion molecules on the vessel endothelium returns to basal levels. In some cases, however, the zeal of these interacting signals and cellular systems, which are designed to capture and contain very rapidly multiplying infectious agents, act to the detriment of the body, killing additional, otherwise healthy, surrounding tissue. This additional unnecessary tissue death further compromises organ function and sometimes results in death of the individual. In addition, the resulting scar tissue that often forms can interfere with normal tissue function as occurs, for example, in idiopathic pulmonary fibrosis, IBD and organ cirrhosis.
The vascular endothelium constitutes the first barrier between circulating immune effector cells and extravascular tissues. Extravasation of these circulating cells requires that they bind to the vascular endothelial cells, cross the basement membrane, and enter insulted tissues e.g., by phagocytosis or protease-mediated extracellular matrix degradation. Without being limited to a particular theory, it is believed that the morphogens of this invention may modulate the inflammatory response in part by modulating the attachment of immune effector cells to the luminal side of the endothelium of blood vessels at or near sites of tissue damage and/or inflammatory lesions. Because the method reduces or prevents the attachment of immune effector cells at these sites, it also prevents the subsequent release of tissue destructive agents by these same immune effector cells at sites of tissue damage and/or inflammatory lesions. Because attachment of immune effector cells to the endothelium must precede their extravascularization, the method also prevents the initial or continued entry of these cells into extravascular sites of tissue destruction or ongoing inflammatory lesions. Therefore, the invention not only relates to a method to reduce or prevent the immune cell-mediated cellular destruction at extravascular sites of recent tissue destruction, but also relates to a method to prevent or reduce the continued entry of immune effector cells into extravascular sites of ongoing inflammatory cascades. As will be appreciated by those skilled in the art, the morphogens of this invention also may be contemplated in mechanisms for disrupting the functional interaction of immune effector cells with endothelium where the adhesion molecules are induced by means other than in response to tissue injury.
One source of tissue damage follows cell exposure to toxic oxygen concentrations, such as the tissue damage following ischemic-reperfusion tissue injury (oxygen deprivation), and following hyperoxia injury (lethally high oxygen concentrations). Accordingly, the process of the present invention provides a method for alleviating the tissue damage induced by ischemic-reperfusion injury or hyperoxia-induced injury comprising the step of administering to the afflicted individual a therapeutic amount of a morphogen prior to, during, or after damage to the affected tissue. Where the toxic oxygen concentrations may be deliberately or unavoidably induced, as by a surgical or clinical procedure, the morphogen preferably is administered prior to induction.
In addition, the morphogens described herein, in contrast to fibrogenic growth factors such as TGF-β, stimulate tissue morphogenesis and do not stimulate fibrosis or scar tissue formation (see Example 9, below.) Accordingly, in addition to inhibiting the tissue destructive effects associated with the inflammatory response, the morphogens further enhance the viability of damaged tissue and/or organs by stimulating the regeneration of the damaged tissue and preventing fibrogenesis.
The morphogens described herein also can inhibit epithelial cell proliferation (see Example 10, below.) This activity of the morphogens also may be particularly useful in the treatment of psoriasis and other inflammatory diseases that involve epithelial cell populations.
Provided below are detailed descriptions of suitable morphogens useful in the methods and compositions of this invention, as well as methods for their administration and application, and numerous, nonlimiting examples which 1) illustrate the suitability of the morphogens and morphogen-stimulating agents described herein as therapeutic agents for protecting tissue from the tissue destructive effects associated with the body's inflammatory response; and 2) provide assays with which to test candidate morphogens and morphogen-stimulating agents for their efficacy.
I. Useful Morphogens
As defined herein a protein is morphogenic if it is capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new, organ-specific tissue and comprises at least the conserved C-terminal six cysteine skeleton or its functional equivalent (see supra).
Specifically, the morphogens generally are capable of all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells, including the “redifferentiation” of transformed cells. Details of how the morphogens useful in the method of this invention first were identified, as well as a description on how to make, use and test them for morphogenic activity are disclosed in U.S. Ser. No. 667,274, filed Mar. 11, 1991 and U.S. Ser. No. 752,764, filed Aug. 30, 1991, the disclosures of which are hereinabove incorporated by reference. As disclosed therein, the morphogens may be purified from naturally-sourced material or recombinantly produced from procaryotic or eucaryotic host cells, using the genetic sequences disclosed therein. Alternatively, novel morphogenic sequences may be identified following the procedures disclosed therein.
Particularly useful proteins include those which comprise the naturally derived sequences disclosed in Table II. Other useful sequences include biosynthetic constructs such as those disclosed in U.S. Pat. No. 5,011,691, the disclosure of which is incorporated herein by reference (e.g., COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16).
Accordingly, the morphogens useful in the methods and compositions of this invention also may be described by morphogenically active proteins having amino acid sequences sharing 70% or, preferably, 80% homology (similarity) with any of the sequences described above, where “homology” is as defined herein above.
The morphogens useful in the method of this invention also can be described by any of the 6 generic sequences described herein (Generic Sequences 1, 2, 3, 4, 5 and 6). Generic sequences 1 and 2 also may include, at their N-terminus, the sequence
Table II, set forth below, compares the amino acid sequences of the active regions of native proteins that have been identified as morphogens, including human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-23), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No. 10), BMP3. (Seq. ID No. 26), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), GDF-1 (from mouse, Seq. ID Nos. 14, 32 and 33), 60A protein (from Drosophila, Seq. ID Nos. 24 and 25), BMP5 (Seq. ID No. 27) and BMP6 (Seq. ID No. 28). The sequences are aligned essentially following the method of Needleman et al. (1970) J. Mol. Biol., 48:443-453, calculated using the Align Program (DNAstar, Inc.) In the table, three dots indicates that the amino acid in that position is the same as the amino acid in hOP-1. Three dashes indicates that no amino acid is present in that position, and are included for purposes of illustrating homologies. For example, amino acid residue 60 of CBMP-2A and CBMP-2B is “missing”. Of course, both these amino acid sequences in this region comprise Asn-Ser (residues 58, 59), with CBMP-2A then comprising Lys and Ile, whereas CBMP-2B comprises Ser and Ile.
**Between residues 56 and 57 of BMP3 is a Val residue; between residues 43 and 44 of GDF-1 lies the amino acid sequence Gly—Gly-Pro—Pro.
As is apparent from the foregoing amino acid sequence comparisons, significant amino acid changes can be made within the generic sequences while retaining the morphogenic activity. For example, while the GDF-1 protein sequence depicted in Table II shares only about 50% amino acid identity with the hOP1 sequence described therein, the GDF-1 sequence shares greater than 70% amino acid sequence homology (or “similarity”) with the hOP1 sequence, where “homology” or “similarity” includes allowed conservative amino acid changes within the sequence as defined by Dayoff, et al., Atlas of Protein Sequence and Structure vol.5, supp.3, pp. 345-362, (M. O. Dayoff, ed., Nat'l BioMed. Res. Fd'n, Washington D.C. 1979.)
The currently most preferred protein sequences useful as morphogens in this invention include those having greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the conserved six cysteine skeleton of hOP1 (e.g., residues 43-139 of Seq. ID No. 5). These most preferred sequences include both allelic and species variants of the OP-1 and OP-2 proteins, including the Drosophila 60A protein. Accordingly, in still another preferred aspect, the invention includes morphogens comprising species of polypeptide chains having the generic amino acid sequence referred to herein as “OPX”, which defines the seven cysteine skeleton and accommodates the identities between the various identified mouse and human OP1 and OP2 proteins. OPX is presented in Seq. ID No. 29. As described therein, each Xaa at a given position independently is selected from the residues occurring at the corresponding position in the C-terminal sequence of mouse or human OP1 or OP2 (see Seq. ID Nos. 5-8 and/or Seq. ID Nos. 16-23).
II. Formulations and Methods for Administering Therapeutic Agents
The morphogens may be provided to an individual by any suitable means, preferably directly (e.g., locally, as by injection or topical administration to a tissue locus) or systemically (e.g., parenterally or orally). Where the morphogen is to be provided parenterally, such as by intravenous, subcutaneous, intramuscular, intraorbital, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, buccal, rectal, vaginal, intranasal or by aerosol administration, the morphogen preferably comprises part of an aqueous solution. The solution is physiologically acceptable so that in addition to delivery of the desired morphogen to the patient, the solution does not otherwise adversely affect the patient's electrolyte and volume balance. The aqueous medium for the morphogen thus may comprise normal physiologic saline (9.85% NaCl, 0.15M), pH 7-7.4. The aqueous solution containing the morphogen can be made, for example, by dissolving the protein in 50% ethanol containing acetonitrile in 0.1% trifluoroacetic acid (TFA) or 0.1% HCl, or equivalent solvents. One volume of the resultant solution then is added, for example, to ten volumes of phosphate buffered saline (PBS), which further may include 0.1-0.2% human serum albumin (HSA). The resultant solution preferably is vortexed extensively. If desired, a given morphogen may be made more soluble by association with a suitable molecule. For example, association of the mature dimer with the pro domain of the morphogen keeps the morphogen soluble in physiological buffers. In fact, the endogenous protein is thought to be transported in this form. Another molecule capable of enhancing solubility and particularly useful for oral administrations, is casein. For example, addition of 0.2% casein increases solubility of the mature active form of OP-1 by 80%. Other components found in milk and/or various serum proteins also may be useful.
Useful solutions for parenteral administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences (Gennaro, A., ed.), Mack Pub., 1990. Formulations may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Formulations for direct administration, in particular, may include glycerol and other compositions of high viscosity to help maintain the morphogen at the desired locus. Biocompatible, preferably bioresorbable, polymers, including, for example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, lactide, and glycolide polymers and lactide/glycolide copolymers, may be useful excipients to control the release of the morphogen in vivo. Other potentially useful parenteral delivery systems for these morphogens include ethylene-vinyl acetate copolymer particles, osmotic pumps, implant able infusion systems, and liposomes. Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or cutric acid for vaginal administration.
Suppositories for rectal administration also may be prepared by mixing the morphogen or morphogen-stimulating agent with a non-irritating excipient such as cocoa butter or other compositions which are-solid at room temperature and liquid at body temperatures.
Formulations for topical administration to the skin surface may be prepared by dispersing the morphogen or morphogen-stimulating agent with a dermatologically acceptable carrier such as a lotion, cream, ointment or soap. Particularly useful are carriers capable of forming a film or layer over the skin to localize application and inhibit removal. For topical administration to internal tissue surfaces, the morphogen may be dispersed in a liquid tissue adhesive or other substance known to enhance adsorption'to a tissue surface. For example, hydroxypropylcellulose or fibrinogen/thrombin solutions may be used to advantage. Alternatively, tissue-coating solutions, such as pectin-containing formulations may be used.
Alternatively, the morphogens described herein may be administered orally. Oral administration of proteins as therapeutics generally is not practiced as most proteins are readily degraded by digestive enzymes and acids in the mammalian digestive system before they can be absorbed into the bloodstream. However, the morphogens described herein typically are acid stable and protease-resistant (see, for example, U.S. Pat. No. 4,968,590.) In addition, at least one morphogen, OP-1, has been identified in mammary-gland extract, colostrum and 57-day milk. Moreover, the OP-1 purified from mammary gland extract is morphogenically active. Specifically, this protein induces endochondral bone formation in mammals when implanted subcutaneously in association with a suitable matrix material, using a standard in vivo bone assay, such as is disclosed in U.S. Pat. No. 4,968,590. Moreover, the morphogen also is detected in the bloodstream. Finally, soluble form morphogen, e.g., mature morphogen associated with the pro domain, is morphogenically active. These findings indicate that oral and parenteral administration are viable means for administering morphogens to an individual. In addition, while the mature forms of certain morphogens described herein typically are sparingly soluble, the morphogen form found in milk (and mammary gland extract and colostrum) is readily soluble, probably by association of the mature, morphogenically active form with part or all of the pro domain of the intact sequence and/or by association with one or more milk components. Accordingly, the compounds provided herein also may be associated with molecules capable of enhancing their solubility in vitro or in vivo.
The compounds provided herein also may be associated with molecules capable of targeting the morphogen or morphogen-stimulating agent to the desired tissue. For example, an antibody, antibody fragment, or other binding protein that interacts specifically with a surface molecule on cells of the desired tissue, may be used. Useful targeting molecules may be designed, for example, using the single chain binding site technology disclosed, for example, in U.S. Pat. No. 5,091,513.
As described above, the morphogens provided-herein share significant sequence homology in the C-terminal active domains. By contrast, the sequences typically diverge significantly in the sequences which define the pro domain. Accordingly, the pro domain is thought to be morphogen-specific. As described above, it is also known that the various morphogens identified to date are differentially expressed in the different tissues. Accordingly, without being limited to any given theory, it is likely that, under natural conditions in the body, selected morphogens typically act on a given tissue. Accordingly, part or all of the pro domains which have been identified associated with the active form of the morphogen in solution, may serve as targeting molecules for the morphogens described herein. For example, the pro domains may interact specifically with one or more molecules at the target tissue to direct the morphogen associated with the pro domain to that tissue. Accordingly, another useful targeting molecule for targeting morphogen to a tissue of interest is part or all of a morphogen pro domain. For example, part or all of the pro domain of GDF-1, may be used to target a morphogen to nerve tissue. Alternatively, part or all of the pro domains of OP-1 or CBMP2 may be used to target a morphogen to bone tissue, both of which proteins are found naturally associated with bone tissue.
The morphogens described herein are useful for providing neuroprotective effects to alleviate neural pathway damage associated with the body's immune/inflammatory response to an initial injury to nerve tissue. As used herein, a “neural pathway” describes a nerve circuit for the passage of electric signals from a source to a target cell site and includes both the central nervous system (CNS) and peripheral nervous system (PNS). The pathway includes the neurons through which the electric impulse is transported, including groups of interconnecting neurons, the nerve fibers formed by bundled neuronal axons, and the glial cells surrounding and associated with the neurons. An inflammatory response to nerve tissue injury may follow trauma to nerve tissue, caused, for example, by an autoimmune (including autoantibody) dysfunction, neoplastic lesion, infection, chemical or mechanical trauma, or other disease. An exemplary nerve-related inflammatory disease is multiple sclerosis. Neural pathway damage also can result from a reduction or interruption, e.g., occlusion, of a neural blood supply, as in an embolic stroke, (e.g. ischemia or hypoxia-induced injury), or by other trauma to the nerve or surrounding material. In addition, at least part of the damage associated with a number of primary brain tumors also appears to be immunologically related. Application of the morphogen directly to the cells to be treated, or providing the morphogen to the mammal systemically, for example, intravenously or indirectly by oral administration, may be used to alleviate and/or inhibit the immunologically related response to a neural injury. Alternatively, administration of an agent capable of stimulating morphogen expression and/or secretion in vivo, preferably at the site of injury, also may be used. Where the injury is to be induced, as during surgery or other aggressive clinical treatment, the morphogen or agent may be provided prior to induction of the injury to provide a neuraprotective effect to the nerve tissue, at risk.
Where the morphogen is intended for use as a therapeutic to alleviate tissue damage associated with an immune/inflammatory condition of the central nervous system (CNS) an additional problem must be addressed: overcoming the so-called “blood-brain barrier”, the brain capillary wall structure that effectively screens out all but selected categories of molecules present in the blood, preventing their passage into the brain. The blood-brain barrier may be bypassed effectively by direct infusion of the morphogen or morphogen-stimulating agent into the brain. Alternatively, the morphogen or morphogen-stimulating agent may be modified to enhance its transport across the blood-brain barrier. For example, truncated forms of the morphogen or a morphogen-stimulating agent may be most successful. Alternatively, the morphogen or morphogen-stimulating agent may be modified to render it more lipophilic, or it may be conjugated to another molecule which is naturally transported across the barrier, using standard means known to those skilled in the art, as, for example, described in Pardridge, Endocrine Reviews: 7:314-330 (1986) and U.S. Pat. No. 4,801,575. A more detailed description of morphogens for use in treating inflammatory, conditions in nerve tissue, including a model for evaluating morphogen transport across the blood brain barrier is disclosed in U.S. Ser. No. 922,813, the disclosure of which is incorporated herein by reference.
Finally, the morphogens or morphogen-stimulating agents provided herein may be administered alone or in combination with other molecules known to be beneficial in the treatment compositions and methods described herein, including, but not limited to anticoagulants, free oxygen radical inhibiting agents, salicylic acid, vitamin D, and other antiinflammatory agents. Psoriais treatments also may include ultra-violet light treatment, zinc oxide and retinoids.
The compounds provided herein can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable nontoxic excipients and carriers. As noted above, such compositions may be prepared for parenteral administration, particularly in the form of liquid solutions or suspensions; for oral administration; particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols.
The compositions can be formulated for parenteral or oral administration to humans or other mammals in therapeutically effective amounts, e.g., amounts which provide appropriate concentrations for a time sufficient to alleivate the tissue destructive effects associated with the inflammatory response, including protecting tissue in anticipation of tissue damage.
As will be appreciated by those skilled in the art, the concentration of the compounds described in a therapeutic composition will vary depending upon a number of factors, including the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration. The preferred dosage of drug to be administered also is likely to depend on such variables as the type and extent of progression of the tissue damage, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound excipients, and its route of administration. In general terms, the compounds of this invention may be provided in an aqueous physiological buffer solution containing about 0.001% to 10% w/v compound for parenteral administration. Typical dose ranges are from about 10 ng/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.1 μg/kg to 100 mg/kg of body weight per day. Optimally, the morphogen dosage given in most cases is between 0.1-100 μg of protein per kilogram weight of the patient. No obvious morphogen induced pathological lesions are induced when mature morphogen (e.g., OP-1, 20 μg) is administered daily to normal growing rats for 21 consecutive days. Moreover, 10 μg systemic injections of morphogen (e.g., OP-1) injected daily for 10 days into normal newborn mice does not produce any gross abnormalities.
In administering morphogens systemically in the methods of the present invention, preferably a large volume loading dose is used at the start of the treatment. The treatment then is continued with a maintenance dose. Further administration then can be determined by monitoring at intervals the levels of the morphogen in the blood.
Where tissue injury is induced-deliberately as part of, for example, a surgical procedure, the morphogen preferably is provided just prior to, or concomitant with induction of the trauma. Preferably, the morphogen is administered prophylactically in a surgical setting.
Alternatively, an effective amount of an agent capable of stimulating endogenous morphogen levels may be administered by any of the routes described above. For example, an agent capable of stimulating morphogen production and/or secretion from cells of affected tissue or a transplanted organ may be provided to a mammal, e.g., by direct administration of the morphogen to the tissue or organ. A method for identifying and testing agents capable of modulating the levels of endogenous morphogens in a given tissue is described generally herein in Example 15, and in detail in copending U.S. Ser. No. 752,859, filed Aug. 30, 1991, the disclosure of which is incorporated herein by reference. Briefly, candidate compounds can be identified and tested by incubating the compound in vitro with a test tissue or cells thereof, for a time sufficient to allow the compound to affect the production, i.e., the expression and/or secretion, of a morphogen produced by the cells of that tissue.
For purposes of the present invention, the above-described morphogens effective in alleviating ischemic-reperfusion injury (or the agents that stimulate them, referred to herein collectively as “therapeutic agent”) are administered prior to or during the restoration of oxygen (e.g., restoration of blood flow, reperfusion.) Where treatment is to follow an existing injury, the therapeutic agent preferably is administered as an intravenous infusion provided acutely after the hypoxic or ischemic condition occurs. For example, the therapeutic agent can be administered by intravenous infusion immediately after a cerebral infarction, a myocardial infarction, asphyxia, or a cardiopulmonary arrest. Where ischemia or hypoxia is deliberately induced as part of, for example, a surgical procedure where circulation to an organ or organ system is deliberately and/or transiently interrupted, e.g., in carotid enterectomy, coronary artery bypass, grafting, organ transplanting, fibrinolytic therapy, etc., the therapeutic agent preferably is provided just prior to, or concomitant with, reduction of oxygen to the tissue. Preferably, the therapeutic agent is administered prophylactically in a surgical setting.
Similarly, where hyperoxia induced-injury already has occurred, the morphogen is administered upon diagnosis. Where hyperoxia may be induced an, for example, during treatment of prematurely newborn babies, or patients suffering from pulmonary diseases such as emphysema, the therapeutic agent preferably is administered prior to administration of oxygen (e.g., prophylactically).
III. EXAMPLES Example 1 Identification of Morphogen-Expressing TissueDetermining the tissue distribution of morphogens may be used to identify different morphogens expressed in a given tissue, as well as to identify new, related morphogens. Tissue distribution also may be used to identify useful morphogen-producing tissue for use in screening and identifying candidate morphogen-stimulating agents. The morphogens (or their mRNA transcripts) readily are identified in different tissues using standard methodologies and minor modifications thereof in tissues where expression may be low. For example, protein distribution may be determined using standard Western blot analysis or immunofluorescent techniques, and antibodies specific to the morphogen or morphogens of interest. Similarly, the distribution of morphogen transcripts may be determined using standard Northern hybridization protocols and transcript-specific-probes.
Any probe capable of hybridizing specifically to a transcript, and distinguishing the transcript of interest from other, related transcripts may be used. Because the morphogens described herein share such high sequence homology in their active, C-terminal domains, the tissue distribution of a specific morphogen transcript may best be determined using a probe specific for the pro region of the immature protein and/or the N-terminal region of the mature protein. Another useful sequence is the 3′ non-coding region flanking and immediately following the stop codon. These portions of the sequence vary substantially among the morphogens of this invention, and accordingly, are specific for each protein. For example, a particularly useful Vgr-1-specific probe sequence is the PvuII-SacI fragment, a 265 bp fragment encoding both a portion of the untranslated pro region and the N-terminus of the mature sequence (see Lyons et al. (1989) PNAS 86:4554-4558 for a description of the cDNA sequence). Similarly, particularly useful mOP-1-specific probe sequences are the BstX1-BglI fragment, a 0.68 Kb sequence that covers approximately two-thirds of the mOP-1 pro region; a StuI-StuI fragment, a 0.2 Kb sequence immediately upstream of the 7-cysteine domain; and the Ear1-Pst1 fragment, an 0.3 Kb fragment containing a portion of the 3′untranslated sequence (See Seq. ID No. 18, where the pro region is defined essentially by residues 30-291.) Similar approaches may be used, for example, with hOP-1 (Seq. ID No. 16) or human or mouse OP-2 (Seq. ID Nos. 20 and 22.)
Using these morphogen-specific probe's, which may be synthetically engineered or obtained from cloned sequences, morphogen transcripts can be identified in mammalian tissue, using standard methodologies well known to those having ordinary skill in the art. Briefly, total RNA is prepared from various adult murine tissues (e.g., liver, kidney, testis, heart, brain, thymus and stomach) by a standard methodology such as by the method of Chomczyaski et al. ((1987) Anal. Biochem 162:156-159) and described below. Poly (A)+ RNA is prepared by using oligo (dT)-cellulose chromatography (e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.). Poly (A)+ RNA (generally 15 μg) from each tissue is fractionated on a 1% agarose/formaldehyde gel and transferred onto a Nytran membrane (Schleicher & Schuell). Following the transfer, the membrane is baked at 80° C. and the RNA is cross-linked under UV light (generally 30 seconds at 1 mW/cm2). Prior to hybridization, the appropriate probe is denatured by heating. The hybridization is carried out in a lucite cylinder rotating in a roller bottle apparatus at approximately 1 rev/min for approximately 15 hours at 37° C. using a hybridization mix of 40% formamide, 5× Denhardts, 5×SSPE, and 0.1% SDS. Following hybridization, the non-specific counts are washed off the filters in 0.1×SSPE, 0.1% SDS at 50° C.
Examples demonstrating the tissue distribution of various morphogens, including Vgr-1, OP-1, BMP2, BMP3, BMP4, BMP5, GDF-1, and OP-2 in developing and adult tissue are disclosed in co-pending U.S. Ser. No. 752,764, and in Ozkaynak, et al., (1991) Biochem. Biophys. Res. Commn. 179:116-123, and Ozkaynak, et al. (1992) (JBC, in press), the disclosures of which are incorporated herein by reference. Using the general probing methodology described herein, northern blot hybridizations using probes specific for these morphogens to probe brain, spleen, lung, heart, liver and kidney tissue indicate that kidney-related tissue appears to be the primary expression source for OP-1, with brain, heart and lung tissues being secondary sources. OP-1 RNA also was identified in salivary glands, specifically rat parotid glands, using this probing methodology. Lung tissue appears to be the primary tissue expression source for Vgr-1, BMP5, BMP4 and BMP3. Lower levels of Vgr-1 also are seen in kidney and heart tissue, while the liver appears to be a secondary expression source for BMP5, and the spleen appears to be a secondary expression source: for BMP4. GDF-1 appears to be expressed primarily in brain tissue. To date, OP-2 appears to be expressed primarily in early embryonic tissue. Specifically, northern blots of murine embryos and 6-day post-natal animals shows abundant OP2 expression in 8-day embryos. Expression is reduced significantly in 17-day embryos and is not detected in post-natal animals.
Example 2 Active Morphogens in Body FluidsOP-1 expression has been identified in saliva (specifically, the rat parotid gland, see Example-1), human blood serum, and various milk forms, including mammary gland extract, colostrum, and 57-day bovine milk. Moreover, and as described in U.S. Ser. No. 923,780, the disclosure of which is incorporated herein, by reference, the body fluid-extracted protein is morphogenically active. The discovery that the morphogen naturally is present in milk and saliva, together with the known observation that mature, active OP-1 is acid-stable and protease-resistant, indicate that oral administration is a useful route for therapeutic administration of morphogen to a mammal. Oral administration typically is the preferred mode of delivery for extended or prophylactic therapies. In addition, the identification of morphogen in all milk forms, including colostrum, suggests that the protein may play a significant role in tissue development, including skeletal development, of juveniles.
2.1 Morphogen Detection in Milk
OP-1 was partially purified from rat mammary gland extract and bovine colostrum and 57 day milk by passing these fluids over a series of chromatography columns: (e.g., cation-exchange, affinity and reverse phase). At each step the eluant was collected in fractions and these were tested for the presence of OP-1 by standard immunoblot. Immunoreactive fractions then were combined and purified further. The final, partially purified product then was examined for the presence of OP-1 by Western blot analysis using OP-1-specific antisera, and tested for in vivo and in vitro activity.
OP-1 purified from the different milk sources were characterized by Western blotting using antibodies raised against OP-1 and BMP2. Antibodies were prepared using standard immunology protocols well known in the art, and as described generally in Example 15, below, using full-length E. coli-produced OP-1 and BMP2 as the immunogens. In all cases, the purified OP-1 reacted only with the anti-OP-1 antibody, and not with anti-BMP2 antibody.
The morphogenic activity of OP-1 purified from mammary gland extract was evaluated in vivo essentially following the rat model assay described in U.S. Pat. No. 4,968,590, hereby incorporated by reference. Briefly, a sample was prepared from each OP-1 immunoreactive fraction of the mammary gland extract-derived OP-1 final product by lyophilizing a portion (33%) of the fraction and resuspending the protein in 220 μl of 50% acetonitrile/0.1% TFA. After vortexing, 25 mg of collagen matrix was added. The samples were lyophilized overnight, and implanted in Long Evans rats (Charles River Laboratories, Wilmington, Mass., 28-35 days old). Each fraction was implanted in duplicate. For details of the collagen matrix implantation procedure, see, for example, U.S. Pat. No. 4,968,590, hereby incorporated by reference. After 12 days, the implants were removed and evaluated for new bone formation by histological observation as described in U.S. Pat. No. 4,968,590. In all cases, the immunoreactive: fractions-were osteogenically active.
2.2 Morphogen Detection in Serum
Morphogen may be detected in serum using morphogen-specific antibodies. The assay may be performed using any standard immunoassay, such as Western blot (immunoblot) and the like. Preferably, the assay is performed using an affinity column to which the morphogen-specific antibody is bound and through which the sample serum then is poured, to selectively extract the morphogen of interest. The morphogen then is eluted. A suitable elution buffer may be determined empirically by determining appropriate binding and: elution conditions first with a control (e.g., purified, recombinantly-produced morphogen.) Fractions then are tested for the presence of the morphogen by standard immunoblot, and the results confirmed by N-terminal sequencing. Preferably, the affinity column is prepared using monoclonal antibodies. Morphogen concentrations in serum or other fluid samples then may be determined using standard protein quantification techniques, including by spectrophotometric absorbance or by quantitation of conjugated antibody.
Presented below is a sample protocol for identifying OP-1 in serum. Following this general methodology other morphogens may be detected in body fluids, including serum. The identification of morphogen in serum further indicates that systemic administration is a suitable means for providing therapeutic concentrations of a morphogen to an individual, and that morphogens likely behave systemically as endocrine-like factors. Finally, using this protocol, fluctuations in endogenous morphogen levels can be detected, and these altered levels may be used as an indicator of tissue dysfunction. Alternatively, fluctuations in morphogen levels may be assessed by monitoring morphogen transcription levels, either by standard northern blot analysis as described in Example 1, or by in situ hybridization, using a labelled probe capable of hybridizing specifically to morphogen “mRNA”, and standard RNA hybridization protocols-well-described in the art and described generally in Example 1.
OP-1 was detected in human serum using the following assay. A monoclonal antibody raised against mammalian, recombinantly produced OP-1 using standard immunology techniques well described in the art and described generally in Example 15, was immobilized by passing the antibody over an agarose-activated gel (e.g., Affi-Gel™, from Bio-Rad Laboratories, Richmond, Calif., prepared following-manufacturer's instructions) and used to purify OP-1 from serum. Human serum then was passed over the column and eluted with 3M K-thiocyanate. K-thiocyanante fractions then were dialyzed in 6M urea, 20 mM PO4, pH 7.0, applied to a C8 HPLC column, and eluted with a 20 minute, 25-50% acetonitrile/0.1% TFA gradient. Mature, recombinantly produced OP-1 homodimers elute between 20-22 minutes. Fractions then were collected and tested for the presence of OP-1 by standard immunoblot using an OP-1 specific antibody as for Example 2.A.
Administered or endogenous morphogen levels may be monitored in the therapies described herein by comparing the quantity of morphogen present in a body fluid sample with a predetermined reference value, for example, to evaluate the efficiency of a therapeutic protocol, and the like. In addition, fluctuations in the level of endogenous morphogen antibodies may be detected by this method, most likely in serum, using an antibody or other binding protein capable of interacting specifically with the endogenous morphogen antibody. Detected fluctuations in the levels of the morphogen or endogenous antibody may be used, for example, as indicators of a change in tissue status. For example, as damaged tissue is regenerated and the tissue or organ's function returns to “normal” and, in the absence of additional tissue damage, lower doses of morphogen may be required, and a higher level of circulating morphogen antibody may be measured.
Example 3 Effect of Morphogen after the Onset of the Ischemic ProcessThe cardioprotective effect of morphogens following ischemic-reperfusion injury in a mammal can readily be assessed in a rat model. In this example, morphogen (e.g., OP-1) is administered just prior to the onset of the ischemic process in experimentally-induced myocardial infracted rats, essentially following the method of Lefer, et al. (1990) Science 249:61-64 and (1992) J. Mol. Cell. Cardiol. 24: 385-393, the disclosures of which are hereby incorporated by reference. Briefly, loss of myocardial tissue function following ischemia and reperfusion is assayed by measuring loss of myocardial creatine kinease activity (CK) and loss of endothelium-dependent vasorelaxation function (see Example 4, below).
In a first group of ether-anesthetized rats, the left coronary artery was occluded just proximal to the first main branch with a silk ligature to induce a myocardial infarction (MI). The ligature was removed 10 minutes after occlusion to allow for coronary reperfusion. This first group is referred to herein as the “myocardial infarcted”. (MI) group. A second group of rats underwent the same procedure except that the coronary artery was not occluded, and thus no myocardial infarction occurred. The second group of rats is referred to herein as the “sham myocardial infarcted group” (SHAM MI).
The first group of rats, the MI group of rats, further was divided into three sup-groups. 2 μg of morphogen (OP-1) were injected intravenously into the first sub-group of MI rats 10 minutes after ligature, immediately before reperfusion; into the second sub-group of MI rats 20 μg of OP-1 were injected intravenously 10 minutes after ligature and immediately before reperfusion; and into the third sub-group of MI rats (control) was injected vehicle only, e.g., 0.9% NaCl, as for the OP-1 treated rats.
Twenty-four hours later, the hearts were removed from all of the rats and the levels of creatine kinase (CK) from the left ventricle (the infarcted region) and from the interventricular septum (the control nonischemic region) were determined by standard means. By comparing the difference in CK activities in both regions, the amount of CK activity lost from the infarcted region was used as an index of cardiac cellular injury to the infarcted region.
As shown in
The loss of CK activity by the subgroup of MI rats which received 2 μg of OP-1 just before reperfusion showed some protection as compared with the control MI rats which received injections of vehicle alone, when the levels from both subgroups are measured against, and compared to, the levels obtained for the SHAM MI control. Significant cardioprotection was observed in the subgroup of MI rats which received 20 μg of OP-1 immediately before reperfusion as compared with the control MI rats which received injections of vehicle alone, when the levels from both subgroups are measured against, and compared to, the levels contained within the SHAM MI control.
These data indicate that OP-1 offers significant cardiac protection when administered after ischemia and before reperfusion.
A variation of this example also may be performed providing morphogen to the animal prior to induction of ischemia. The experiments may be performed both in normal and immune-compromised rats to assess the cardioprotective effects of morphogen administered prior to ischemia.
Example 4 Vasodilation of Myocardial Infarcted Cardiac Tissue Treated with MorphogenCertain vasodilators like acetylcholine (ACh) and adenosine diphosphate (ADP, an immune mediator) exert their vasodilation activity only in the presence of intact endothelium, which is stimulated to release a substance termed endothelium-derived relaxing factor (EDRF). If the endothelium is injured so that EDRF is not released, no vasodilation occurs in response to these endothelium-dependent agents. In contrast, several other vasodilators including nitroglycerine (NTG) and nitroprusside, are endothelium-independent dilators, as they dilate blood vessels directly.
The present example demonstrates the ability of OP-1 to prevent the loss of cardioendothelium-dependent relaxation (EDR) activity in the coronary microvasculature following reperfusion of ischemic myocardium, and their ability to reduce myocardial injury 24 hours after morphogen treatment, Briefly, 2 or 24 hours after morphogen treatment ischemia-reperfusion injury is induced in isolated rat hearts, the reperfused hearts are are vasodilated with either ACh or NTG. In the absence of morphogen treatment, injured tissue should inhibit ACh-induced vasodilation, but not NTG-induced vasodilation. Morphogen treatment in expected to enhance ACh-induced vasodilation in the reperfused hearts.
Accordingly, 48 adult male Sprague-Dawley rats (250-330 g) were divided into eight groups of 6 rats each. Twelve rats were subjected to sham myocardial infarcts (SHAM MI) as described in Example 3. The hearts of the remaining 36 rats were isolated as follows: one set of twelve rats was-injected intravenously with OP-1 24 hours prior to isolation of the heart; another set of rats was injected intravenously with 20 μg of OP-0.1 2 hours prior to isolation of the heart; the final group of rats was injected with vehicle only (e.g., 0.9% NaCl.). The rats then were anesthetized with pentobarbital sodium (35 mg/kg, intraperitonial); their hearts were isolated and perfused by the Langendorff method at a constant flow (15 ml/min) with oxygenated Krebs-Henseleit solution (Aoki et al. (1988) J. Pharmacol. 95:35).
Each group of rats then were divided into two subgroups of six rats each. Twenty minutes before reperfusion, coronary vasodilator response was measured by inducing constriction with 0.05 μmol U-44619 (9,11-methanoepoxyprostaglandin H2) followed by a vasodilating agent 3 minutes later: subgroup one—15 nmol ACh; subgroup 2—15 nmol NTG and the increase in coronary perfusion pressure (CPP) level measured as an indication of vasodilation. When CPP levels returned to normal, the hearts were subjected to ischemia by reducing coronary infusion to 15% of control flow for 30 minutes, then reestablishing normal flow, i.e., reperfusion, for an additional 20 minutes.
The vasodilator reponse then was remeasured by constriction and administration of vasodilating agent as described above.
The results of these experiments are shown in
The role of neutrophil adherence in endothelium dysfunction and the cardioprotective effects of morphogens in modulating this activity can be assessed using a standard polymorphonuclear neutrophil (PMN) adherence assay such as described in Lefer et al., (1992) J. Mol. Cell. Cardiol. 24: 385-393, disclosed hereinabove by reference. Briefly, segments of superior mesenteric artery were isolated from rats which had either been treated with morphogen (OP-1, 20 μg) or 0.9% NaCl, 24 h prior to isolation of the artery. The segments were cleaned, cut into transverse rings of 1-2 mm in length, and these were subsequently cut open and incubated in K-H solution at 37° C., pH 7.4. Neutrophils were prepared and fluorescently labelled using standard procedures (e.g., leukocytes were isolated from rats essentially following the procedure of Pertroft et. al. (1968) Exp Cell Res 50: 355-368, washed in phosphate buffered saline (PBS), purified by gradient centrifugation; and labelled by the method of Yuan et. al. (1990) Microvasc Res 40: 218-229.
Labelled neutrophils then were added to open ring baths and activated with 100 nM leukotriene B4 (LTB4). Rings were incubated for 20 minutes and the number of neutrophils adhering to the endothelial surface then determined visually by fluorescent microscopy.
As shown in
Other tissues seriously affected by ischemic-reperfusion injury-include neural tissue, renal tissue and lung tissue. The effect of morphogens on alleviating the ischemic-reperfusion injury in these tissues may be assessed using methodologies and models known to those skilled in the art, and disclosed below. Similarly, a methodology also is provided for assessing the tissue-protective effects of a morphogen on damaged lung tissue following hyperoxia injury.
For example, the rabbit embolic stroke model provides a useful method for assessing the effect of morphogens on tissue injury following cerebral ischemia-reperfusion. The protocol disclosed below is essentially that of Phillips et al. (1989) Annals of Neurology 25:281-285, the disclosure of which is herein incorporated by reference. Briefly, white New England rabbits (2-3 kg) are anesthesized and placed on a respirator. The intracranial circulation then is selectively catheterized by the Seldinger technique. Baseline cerebral angiography then is performed, employing a digital substration unit. The distal internal carotid artery or its branches then is selectively embolized with 0.035 ml of 18-hour-aged autologous thrombus. Arterial occlusion is documented by repeat angiography immediately after embolization. After a time sufficient to induce cerebral infarcts (15 minutes or 90 minutes), reperfusion is induced by administering a bolus of a reperfusion agent such as the TPA analogue Fb-FB-CF (e.g., 0.8 mg/kg over 2 minutes).
The effect of morphogen on cerebral infarcts can be assessed by administering varying concentrations of morphogens, e.g., OP1, at different times preceding or following embolization and/or reperfusion. The rabbits are sacrificed 3-14 days post embolization and their brains prepared for neuropathological examination by fixing by immersion in 10% neutral buffered formalin or at least 2 weeks. The brains then are sectioned in coronal plane at 2-3 mm intervals, numbered and submitted for standard histological processing in paraffin, and the degree of neutral tissue necrosis determined visually.
The renal-protective effects of morphogens on renal ischemia-reperfusion injury readily can be assessed using the mouse model disclosed by Oueliette, et al. (1990), J. Clin. Invest. 85:766-771, the disclosure of which is hereby incorporated by reference. Briefly, renal ischemia is induced surgically in 35-45 days old out-bred Swiss male mice by performing a standard right nephrectomy, and occluding the artery to the left kidney with a microaneurism clamp for 10-30 minutes. Morphogen then may be provided parentally at various times prior to or following, occlusion and/or reperfusion. The effects of morphogen then may be assessed by biological and histological evaluation using standard techniuques well known in the art.
The tissue protective effects of morphogen on tissue exposed to lethally high oxygen concentrations may be assessed by the following procedure. Adult rats (275-300 gms) first are provided with morphogen (e.g., hOP1) or vehicle only, and then are exposed to 0.96-98% oxygen essentially as described by Rinaldo et al (1983) Am. Rev. Respir. Dis. 130:1065, to induce hyperoxia. Animals are housed in plastic cages (38 cm×48 xm×21 cm). A cage containing 4-5 animals is placed in a 75 liter water-sealed plexiglass chamber. An atmosphere of 96-98% oxygen then is maintained by delivery of O2 gas (liquid O2). Gas flow through the chamber is adjusted to maintain at least 10 air changes/hr., temperature at 22±1° C., minimal levels of condensation within the cage, and carbon dioxide concentration of <0.5% as measured with a mass spetrophotometric medical as analyzer.
At the end of 72 hours all survivors are observed at room air for 1.5 hours and at longer time periods to assess degree of respiratory distress and cyanosis induced by the initial insult and subsequent immune insult and subsequent immune cell-mediated damage. The number of survivors at the end of the challenge is recorded and the treated groups compared with the untreated control group by chi-square test of proportions. Several of the surviving animals for each group are randomly chosen for histological processing of lung tissue.
Lung tissue for histological processing is fixed by infusion of 10% buffered formalin through a tracheal cannula at a constant pressure of 20 cm H2O. After fixation for 24-48 hours, sections from each lobe are cut and subsequently stained with hematoxylin and eosin. Coded slides then are examined, preferably in a double-blind fashion for evidence of pathological changes such as edema, interstitial cellularity, and inflammatory response.
Example 7 Morphogen Inhibition of Cellular and Humoral Inflammatory Response Morphogens described herein inhibit multinucleation of mononuclear phagocytic cells under conditions where these cells normally would be activated, e.g., in response to a tissue injury or the presence of a foreign substance. For example, in the absence of morphogen, an implanted substrate material (e.g., implanted subcutaneously) composed of, for example, mineralized bone, a ceramic such as titanium oxide or any other substrate that provokes multinucleated giant cell formation, rapidly becomes surrounded by multinucleated giant cells, e.g., activated-phagocytes stimulated to respond and destroy the foreign object. In the presence of morphogen however, the recruited cells remain in their mononuclear precursor form and the matrix material is undisturbed.
In addition, the morphogens described herein also suppress antibody production stimulated in response to a foreign antigen in a mammal. Specifically, when bovine bone collagen matrix alone was implanted in a bony site in a rat, a standard antibody response to the collagen is stimulated in the rat as determined by standard anti-bovine collagen ELISA experiments performed on blood samples taken at four week intervals following implantation (e.g., between 12 and 20 weeks.) Serum anti-collagen antibody titers, measured by ELISA essentially following the procedure described by Nagler-Anderson et al, (1986) PNAS 83:7443-7446, the disclosure of which is incorporated herein by reference, increased consistently throughout the experiment. However, when the matrix was implanted together with a morphogen (e.g., OP-1, dispersed in the matrix and adsorbed thereto, essentially as described in U.S. Pat. No. 4,968,590) anti-bovine collagen antibody production was suppressed significantly. This ability of morphogen to suppress the humoral response is further evidence of morphogen utility in alleviating tissue damage associated with autoimmune diseases, including autoantibody diseases, such as rheumatoid arthritis.
Example 8 Morphogen protection of Gastrointestinal Tract Mucosa from Ulceration and InflammationOral mucositis is a gastrointestinal tract inflammatory disease which involves ulcerations of the mouth mucosa as a consequence of, e.g., radiation therapy or chemotherapy. While not typically a chronic disease, the tissue destructive effects of oral mucositis mirror those of chronic inflammatory diseases such as IBD. The example below demonstrates morphogen efficacy in protecting the oral mucosa from oral mucositis in a hamster model, including both inhibiting inflammatory ulceration and enhancing regeneration of ulcerated tissue. Details of the protocol can be found in Sonis, et al., (1990) Oral Surg. Oral Med. Oral Pathol 69: 437-443, the disclosure of which is incorporated herein by reference. Based on these data, the morphogens described herein should be efficacious in treating chronic inflammatory diseases including IBD, arthritis, psoriasis and psoriatic arthritis, multiple sclerosis, and the like.
Golden syrian hamsters (6-8 wks old, Charles River Laboratories, Wilmington, Mass.) were divided into 3 test groups: Group 1, a placebo (e.g., saline) control, and a morphogen-low dose group (100 ng) and a morphogen high dose group (1 μg), Groups 2 and 3, respectively. Morphogen dosages were provided in 30% ethanol. Each group contained 12 animals.
Beginning on day 0 and continuing through day 5, Groups 2 and 3 received twice daily morphogen applications. On day 3, all groups began the mucositis-induction procedure. 5-fluorouracil (60 mg/kg) was injected intraperitoneally on days 3 and 5. On day 7, the right buccal pouch mucosa was superficially irritated with a calibrated 18 gauge needle. In untreated animals, severe ulcerative mucositis was induced in at least 80% of the animals by day 10.
For each administration of the vehicle control (placebo) or morphogen, administration was performed by first gently drying the cheek pouch mucosa, then providing an even application over the mucosal surface of the vehicle or morphogen material. A hydroxypropylcellulose-based coating was used to maintain contact of the morphogen with the mucosa. This coating provided at least 4 hours of contact time.
On day 12, two animals in each group were sacrificed for histological studies. The right buccal pouch mucosa and underlying connective tissue were dissected and fixed in 10% formalin using standard dissection and histology procedures. The specimens were mounted in paraffin and prepared for histologic examination. Sections then were stained with hematoxylin and eosin and were examined blindly by three oral pathologists with expertise in hamster histology and scored blind against a standard mucositis panel. The extent of atrophy, cellular infiltration, connective tissue breakdown, degree of ulceration and epithelialization were assessed.
The mean mucositis score for each group was determined daily for each experimental group for a period of 21 days by photography and visual examination of the right buccal cheek pouch. Differences between groups were determined using a standard ‘t’ test, e.g., the Students' ‘t’ test. In addition, data was evaluated between groups by comparing the numbers of animals with severe mucositis using Chi Square statistical analysis. The significance of differences in mean daily weights also was determined.
The experimental results are presented in
The morphogens described herein induce tissue morphogenesis of damaged or lost tissue. The ability of these proteins to regenerate new tissue enhances the anti-inflammatory effect of these proteins. Provided below-are a series of in vitro experiments demonstrating the ability of morphogens to induce migration and accumulation of mesenchymal cells. In addition, the experiments demonstrate that morphogens, unlike TGF-β, do not stimulate fibrogenesis or scar tissue formation. Specifically, morphogens do not stimulate production of collagen, hyaluronic acid (HA), or metalloproteinases in primary fibroblasts, all of which are required for fibrogenesis or scar tissue formation. By contrast, TGF-β, a known-inducer of fibrosis, but not of tissue morphogenesis, does stimulate production of these fibrosis markers.
Chemotaxis and migration of mesenchymal progenitor cells were measured in modified Boyden chambers essentially as described by Fava, R. A. et al (1991) J. Exp. Med. 173: 1121-1132, the disclosure of which is incorporated herein by reference, using polycarbonate filters of 2, 3 and 0.8 micron ports to measure migration of progenitor neutrophils, monocytes and fibroblasts Chemotaxis was measured over a range of morphogen concentrations, e.g., 10−20 M to 10−12M OP-1. For progenitor neutrophils and monocytes, 10−18-10−17M OP-1 consistently induced maximal, migration, and 10−14 to 10−13M OP-1 maximally induced migration of progenitor fibroblasts. In all cases the chemotactic activity could be inhibited with anti-OP-1 antibody. Similar migration activities also were measured and observed with TGF-β.
The effect of morphogen on fibrogenesis was determined by evaluating fibroblast production of hyaluronic acid (HA), collagen, collagenese and tissue inhibitor of metalloproteinases (TIMP).
Human fibroblasts were established from explants of infant foreskins and maintained in monolayer culture using standard culturing procedures. (See, for example, (1976) J. Exp. Med. 144: 1188-1203.) Briefly, fibroblasts were grown in maintenance medium consisting of Eagle's MEM, supplemented with nonessential amino acids, ascorbic acid (50 μg/ml), NaHCO3 and HEPES buffers (pH 7.2), penicillin (100 U/ml), streptomycin (100 μg/ml), amphotericin B (1 μg/ml) and 9% heat inactivated FCS. Fibroblasts used as target cells to measure chemotaxis were maintained in 150 mm diameter glass petri dishes. Fibroblasts used in assays to measure synthesis of collagen, hyaluronic acid, collagenase and tissue inhibitors of metalloproteinases (TIMP) were grown in 100 mm diameter plastic tissue culture petri dishes.
The effects of morphogen on fibroblast production of hyaluronic acid, collagens, collagenase and TIMP were determined by standard assays (See, for example, Posttethwaite et al. (1989) J. Clin. Invest. 83: 629-636, Posttethwaithe (1988) J./ Cell Biol. 106: 311-318 and Clark et al (1985) Arch. Bio-chem Biophys. 241: 36-44, the disclosures of which are incorporated by reference.) For these assays, fibroblasts were transferred to 24-well tissue culture plates at a density of 8×104 cells per well. Fibroblasts were grown confluency in maintenance medium containing 9% FCS for 72 h and then grown in serum-free-maintenance medium for 24 h. Medium was then removed from each well and various concentrations of OP-1 (recombinantly produced mature or soluble form) or TGF-β-1 (R&D Systems, Minneapolis) in 50 μl PBS were added to triplicate wells containing the confluent fibroblast monolayers. For experiments that measured production of collagenase and TIMP, maintenance medium (450 μl) containing 5% FCS was added to each well, and culture supernatants were harvested from each well 48 h later and stored at −70° C. until assayed. For experiments that assessed HA production, maintenance medium (450 μl) containing 2.5% FCS was added to each well, and cultures grown for 48 h. For experiments that measured fibroblast production of collagens, serum-free maintenance medium (450 μl) without non-essential amino acids was added to each well and cultures grown for 72 h. Fibroblast production of HA was measured by labeling newly synthesized glycosaminoglycans (GAG) with [3H]-acetate the last 24 h of culture and quantitating released radioactivity after incubation with hyaluronidase from Streptomyces hyalurolyticus (ICN Biochemicals, Cleveland, Ohio) which specifically degrades hyaluronic acid. Production of total collagen by fibroblasts was measured using a collagenase-sensitive protein assay that reflects [3H]-proline incorporation the last 24 h of culture into newly synthesized collagens. Collagenase and TIMP protein levels in fibroblast cultures supernatants was measured by specific ELISAs.
As shown in
This example demonstrates the ability of morphogens to inhibit epithelial cell proliferation in vitro, as determined by 3H-thymidine uptake using culture cells from a mink lung epithelial cell line (ATCC No., CCL 64), and standard mammalian cell culturing procedures. Briefly, cells were grown to confluency in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 0.200 units/ml penicillin, and 200 μg/ml streptomycin, and used to seed a 48-well cell culture plate at a cell density of 200,000 cells per well. When this culture became confluent, the media was replaced with 0.5 ml of EMEM containing 1% FBS and penicillin/streptomycin and the culture incubated for 24 hours at 37 C. Morphogen test samples in EMEM containing 5% FBS then were added to the wells, and the cells incubated for another 18 hours. After incubation, 1.0 μCi of 3H-thymidine in 10 μl was added to each well, and the cells incubated for four hours at 37 C. The media then was removed and the cells washed once with ice-cold phosphate-buffer saline and DNA precipitated by adding 0.5 ml of 10% TCA to each well and incubating at room temperature of 15 minutes. The cells then were washed three times with ice-cold distilled water, lysed with 0.5 ml 0.4 M NaOH, and the lysate from each well then transferred to a scintillation vial and the radioactivity recorded using a scintillation counter (Smith-Kline Beckman).
The results are presented in Table-III, below. The anti-proliferative effect of the various morphogens tested was expressed as the counts of 3H-thymidine (x 1000) integrated into DNA, and were compared with untreated cells (negative control) and TGF-β (1 ng), a local-acting factor also known to inhibit epithelial cell proliferation. COP-5 and COP-7 are biosynthetic constructs that previously have been shown to have osteogenic activity, capable of inducing the complete cascade resulting in endochondral bone formation in a standard rat bone assay. (see U.S. Pat. No. 5,011,691.) The morphogens significantly inhibit epithelial cell proliferation. Similar experiments, performed with the morphogens COP-16, bOP (bone-purified osteogenic protein, a dimeric protein comprising CBMP2 and OP-1), and recombinant OP-1, also inhibit cell proliferation bOP and COP-16 also induce endochondral bone formation (see U.S. Pat. No. 4,968,590 and 5,011,691.)
The following example provides a rat adjuvant induced arthritis model for demonstrating morphogen efficacy in treating arthritis and other systemic inflammatory diseases. Rat adjuvant-induced arthritis induces a systemic inflammatory disease with bone and cartilage changes similar to those observed in rhematoid arthritis, but in an accelerated time span (see, for example, Pearson (1964) Arth. Rheum. 7:80). A detailed description of the protocol is provided in Walz, et al., (1971) J. Pharmac. Exp. Ther. 178: 223-231, the disclosure of which is incorporated herein by reference.
Briefly, Sprague-Dawley female rats (e.g., Charles River Laboratories, Wilmington, Mass.) are randomized into 3 groups: control; morphogen, low dose (e.g., 1-10 μg/kg weight per day) and morphogen, high dose (e.g., 10-20 μg/kg weight per day), referred to as Groups 1, 2, and 3, respectively.
Adjuvant arthritis is induced in all three groups by injection of 0.05 ml of a suspension of 1.5% dead Mycobacterium butyricum in mineral oil into the subplantar surface of the right hand paw. On Day 18 after adjuvant injection, the limb volumes of both hind limbs are determined. In the absence of morphogen treatment, a systemic arthritic condition is induced in the rats by this time, as determined by rats with significant swelling of the uninjected hind limbs (<2.3 ml, volume measured by mercury displacement). Subsequent determinations of paw edema and x-ray scores are made on the uninjected hind limb. Rats in Group 2 and 3 also are dosed orally daily, beginning on Day 1, with morphogen. Limb volumes are recorded on Days 29 and 50 after adjuvant injection and edema determined by volume difference compared to Day 18. The uninjected hind limb on each-rat was x-rayed on Day 50 and the joint damage assayed on an arbitrary scale of 1 to 10 (1=no damage, 10=maximum damage). Data on differences between control and treated groups (Day 29 edema, Day 50 edema and Day 50 x-ray scores) are analyzed by using a standard “t-test”. Morphogen-treated rats show consistently reduced joint damage (e.g., decreased in edema and in x-ray scores) as compared with untreated control rats.
As another, alternative example, Groups 2 and 3 are dosed daily with morphogen beginning on Day 18 and continuing through Day 50 to demonstrate the efficacy of morphogens in arthritic animals.
Example 12 Morphogen Inhibition of Localized EdemaThe following example demonstrates morphogen efficacy in inhibiting a localized inflammatory response in a standard rat edema model. Experimental rats (e.g., Long-Evans from Charles River Laboratories, Wilmington, Mass.) are divided into three groups: Group 1, a negative control, which receives vehicle alone; Group 2, a positive control, to which is administered a well-known characterized anti-inflammatory agent (e.g., indomethacin), and Group 3, to which morphogen is provided.
Groups 2 and 3 may be further subdivided to test low, medium and high doses (e.g., Group 2: 1.0 mg/kg, 3.0 mg/kg and 9.0 mg/kg indomethacin; Group 3: 0.1-5 μg; 5-20 μg, and 20-50 μg of morphogen). Sixty minutes after indomethacin or morphogen is provided to the rats; of Group 2 or 3 (e.g., as by injection into the tail vein, or by oral gavage) inflammation is induced in all rats by a sub-plantar injection of a 1% carrageenin solution (50 μl) into the right hind paw. Three hours after carrageenin administration paw thickness is measured as an indication of edema (e.g., swelling) and induced inflammatory response to the injected carrageenin solution.
Significant swelling is evident in untreated rats by three hours after carrageenin injection. Inflammation also is measured by histology by standard means, following euthanasia e.g.: the right hind paw from each animal is removed at the ankle joint and weighed and foot pad tissue is fixed in 10% neutral buffered formalin, and slides prepared for visual examination by staining the prepared tissue with hematoxylin and eosin.
The morphogen-treated rats show substantially reduced edema induction following carrageenin injection as compared with the untreated rats.
Example 13 Morphogen Treatment of Allergic EncephalomyelitisThe following example demonstrates morphogen efficacy in treating experimental allergic encephalomyelitis (EAE) in a rat. EAE is a well-characterized animal model for multiple sclerosis, an autoimmune disease. A detailed description of the protocol is disclosed in Kuruvilla, et al., (1991) PNAS 88:2918-2921, the disclosure of which is incorporated herein by reference.
Briefly, EAE is induced in rats (e.g., Long-Evans, Charles River Laboratories, Wilmington, Mass.) by injection of a CNS tissue (e.g., spinal cord) homogenate in complete Freund's adjuvant (CFA) on days −44, −30 and 0 (last day of immunization), by subcutaneous injection to three sites on the animal's back. Morphogen is administered daily by interperitoneal injection beginning on day −31. Preferably, a series of morphogen dose ranges is evaluated (e.g., low, medium and high) as for Example 12, above.) Control rats receive morphogen vehicle only (e.g. 0.9% NaCl or buffered saline). Rats are examined daily for signs of disease and graded on an increasing severity scale of 0-4.
In the absence of morphogen treatment, significant neurological dysfunction (e.g., hind and fore limb weakness, progressing to total hind limb paralysis) is evident by day +7 to +10. Hematology, serum chemistry profiles and histology are performed to evaluate the degree of tissue necropsy using standard procedures. Morphogen treatment significantly inhibits the neurological dysfunction normally evident iii an EAE animal. In addition, the histopathological markers typically associated with EAE are absent in the morphogen-treated animals.
Example 14 Morphogen Treatment of Collagen-Induced ArthritisThe following example demonstrates the efficacy of morphogens in inhibiting the inflammatory response in a collagen-induced arthritis (CIA) in a rat. CIA is a well-characterized animal model for rheumatoid arthritis, an autoimmune disease. The protocol disclosed is essentially that disclosed in Kuruvilla et al., (1991) PNAS 88:2918-2921, incorporated by reference hereinabove. Briefly, CIA is induced in experimental rats (e.g., Long-Evans, Charles River Laboratories, Wilmington), by multiple intradermal injection of bovine Type II collagen (e.g., 100 μg) in CFA (0.2 ml) on Day 1. Animals are divided into two groups: Group 1, control animals, which receive vehicle alone, and Group 2: morphogen-treated animals, which, preferably, are subdivided into low, medium and high dose ranges, as described for Example 13, above. Morphogen is administered daily (e.g., by tail vein injection) beginning at different times following collagen injection, e.g., beginning on day 7, 14, 28, 35 and 42. Animals are evaluated visually and paw thickness and body weight is monitored throughout the experiment. Animals are sacrificed on day 60 and the proximal and distal limb joints, and ear, tail and spinal cord prepared for histological evaluation as described for Examples 12 and 13, above. In a variation of the experiment, morphogen may be administered for prescribed periods, e.g., five day periods, beginning at different times following collagen injection (e.g., on days 0-4,7-11, 14-18, 28-32.)
In the absence of morphogen treatment, an arthritic condition typically is induced by 30 days post collagen injection. In morphogen-treated animals, CIA is suppressed and the histopathological changes typically evidenced in control CIA-induced animals are absent: e.g.; accumulations of activated mononuclear inflammatory cells and fibrous connective tissue. In addition, consistent with the results in Example 7, above, serum anti-collagen antibody titers are suppressed significantly in the morphogen-treated animals.
Example 15 Screening Assay for Candidate Compounds which Alter Endogenous Morphogen LevelsCandidate compound(s) which may be administered to affect the level of a given morphogen may be found using the following screening assay, in which the level of morphogen production by a cell type which produces measurable levels of the morphogen is determined with and without incubating the cell in culture with the compound, in order to assess the effects of the compound on the cell. This can be accomplished by detection of the morphogen either at the protein or RNA level. A more detailed description also may be found in U.S. Ser. No. 752,861, incorporated hereinabove by reference.
15.1 Growth of Cells in Culture
Cell cultures of kidney, adrenals, urinary bladder, brain, or other organs, may be prepared as described widely in the literature. For example, kidneys may be explanted from neonatal or new born or young or adult rodents (mouse or rat) and used in organ culture as whole or sliced (1-4 mm) tissues. Primary tissue cultures and established cell lines, also derived from kidney, adrenals, urinary, bladder, brain, mammary, or other tissues may be established in multiwell plates (6 well or 24 well) according to conventional cell culture techniques, and are cultured in the absence or presence of serum for a period of time (1-7 days). Cells may be cultured, for example, in Dulbecco's Modified Eagle medium (Gibco, Long Island, N.Y.) containing serum (e.g., fetal calf serum at 1%-10%, Gibco) or in serum-deprived medium, as desired, or in defined-medium (e.g., containing insulin., transferring glucose, albumin, or other growth factors).
Samples for testing the level of morphogen production includes culture supernatants or cell lysates, collected periodically and evaluated for OP-1 production by immunoblot analysis (Sambrook et al., eds., 1989, Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), or a portion of the cell culture itself, collected periodically and used to prepare polyA+ RNA for RNA analysis. To monitor de novo OP-1 synthesis, some cultures are labeled according to conventional procedures with an 35S-methionine/35S-cysteine mixture for 6-24 hours and then evaluated to OP-1 synthesis by conventional immunoprecipitation methods.
15.2 Determination of Level of Morphogenic Protein
In order to quantitate the production of a morphogenic protein by a cell type, an immunoassay may be performed to detect the morphogen using a polyclonal or monoclonal antibody specific for that protein. For example, OP-1 may be detected using a polyclonal antibody specific for OP-1 in an ELISA, as follows.
1 μg/100 μl of affinity-purified polyclonal-rabbit IgG specific for OP-1 is added to each well of a 96-well plate and incubated at 37° C. for an hour. The wells are washed four times with 0.167M sodium borate buffer with 0.15 M NaCl (BSB), pH 8.2, containing 0.1% Tween 20. To minimize non-specific binding, the wells are blocked by filling completely with 1% bovine serum albumin (BSA) in BSB and incubating for 1 hour at 37° C. The wells are then washed four times with BSB containing 0.1% Tween 20. A 100 μl aliquot of an appropriate dilution of each of the test samples of cell culture supernatant is added to each well in triplicate and incubated at 37° C. for 30 min. After incubation, 100 μl biotinylated rabbit anti-OP-1 serum (stock solution is about 1 mg/ml and diluted 1:400 in BSB containing 1% BSA before use) is added to each well and incubated at 37° C. for 30 min. The wells are then washed four times with BSB containing 0.1% Tween 20. 100 μl strepavidin-alkaline (Southern Biotechnology Associates, Inc. Birmingham, Ala., diluted 1:2000 in BSB containing 0.1% Tween 20 before use) is added to each well and incubated at 37° C. for 30 min. The plates are washed four times with 0.5M Tris buffered Saline (TBS), pH 7.2. 50 μl substrate (ELISA Amplification System Kit, Life Technologies, Inc., Bethesda, Md.) is added to each well incubated at room temperature for 15 min. Then, 50 μl amplifier (from the same amplification system kit) is added and incubated for another 15 min at room temperature. The reaction is stopped by the addition of 50 μl 0.3 M sulphuric acid. The OD at 490 nm of the solution in each well is recorded. To quantitate OP-1 in culture media, a OP-1 standard curve is performed in parallel with the test samples.
Polyclonal antibody may be prepared as follows. Each rabbit is given a primary immunization of 100 ug/500 μl E. coli produced OP-1 monomer (amino acids 328-431 in SEQ ID NO:5) in 0.1% SDS mixed with 500 μl Complete Freund's Adjuvant. The antigen is injected subcutaneously at multiple sites on the back and flanks of the animal. The rabbit is boosted after a month in the same manner using incomplete Freund's Adjuvant. Test bleeds are taken from the ear vein seven days later. Two additional boosts and test bleeds are performed at monthly intervals until antibody against OP-1 is detected in the serum using an ELISA assay. Then, the rabbit is boosted monthly with 100 μg of antigen and bled (15 ml per bleed) at days seven and ten after boosting.
Monoclonal antibody specific for a given morphogen may be prepared as follows. A mouse is given two injections of E. coli produced OP-1 monomer. The first injection contains 100 μg of OP-1 in complete Freund's adjuvant and is given subcutaneously. The second injection contains 50 μg of OP-1 in incomplete adjuvant and is given intraperitoneally. The mouse then receives a total of 230 μg of OP-1 (amino acids 307-431 in SEQ ID NO:5) in four intraperitoneal injections at various times over an eight month period. One week prior to fusion, both mice are boosted intraperitoneally with 100 μg of OP-1 (307-431) and 30 μg of the N-terminal peptide (Ser293-Asn309-Cys) conjugated through the added-cysteine to bovine serum albumin with SMCC crosslinking agent. This boost was repeated five days (IP), four days (IP), three days (IP) and one day (IV) prior to fusion. The mouse spleen cells are then fused to myeloma (e.g., 653) cells at a ratio of 1:1 using PEG 1500 (Boeringer Mannheim), and the cell fusion is plated and screened for OP-1-specific antibodies using OP-1 (307-431) as antigen. The cell fusion and monoclonal screening then are according to standard procedures well described in standard texts widely available in the art.
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims
1-48. (canceled)
49. A method for enhancing recovery of central nervous system function in a mammal, comprising administering an effective amount of a morphogen to a mammal afflicted with a central nervous system injury selected from ischemia or trauma, wherein said morphogen induces tissue-specific morphogenesis in said mammal and comprises a pair of folded polypeptides, each having an amino acid sequence having at least 70% homology with or 60% identity to the C-terminal seven-cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: 5.
50. A method for enhancing recovery of central nervous system function in a mammal, comprising administering an effective amount of a morphogen to a mammal afflicted with a central nervous system injury selected from ischemia or trauma, wherein said morphogen induces tissue-specific morphogenesis in said mammal and comprises a pair of folded polypeptides, each having an amino acid sequence selected from:
- (a) Generic Sequence 3 defined by SEQ ID NO: 3;
- (b) Generic Sequence 4 defined by SEQ ID NO: 4;
- (c) Generic Sequence 5 defined by SEQ ID NO: 30; or,
- (d) Generic Sequence 6 defined by SEQ ID NO: 31.
51. A method for enhancing recovery of central nervous system function in a mammal, comprising administering an effective amount of a morphogen to a mammal afflicted with a central nervous system injury selected from ischemia or trauma, wherein said morphogen is human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP-2A, BMP-2B, DPP, Vgl, Vgr-1, BMP-3, BMP-5, or BMP-6.
52. The method claim 49, wherein said amino acid sequence is that of the C-terminal seven-cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: 5.
53. The method of claim 49, wherein said morphogen is complexed with at least one pro-domain peptide comprising an N-terminal 18 amino acid peptide selected from N-termini of the pro domains of OP-1, OP-2, 60A, GDF-1, BMP-2A, BMP-2B, DPP, Vgl, Vgr-1, BMP-3, BMP-5, or BMP-6.
54. The method claim 49, wherein said morphogen is noncovalently complexed with at least one solubility-enhancing fragment of a pro-domain polypeptide selected from the pro-domains of naturally-occurring morphogens.
55. The method of claim 54, wherein said morphogen is complexed with a pair of said fragments.
56. The method of claim 49, wherein said morphogen comprises the amino acid sequence of SEQ ID NO: 5.
Type: Application
Filed: Jun 9, 2004
Publication Date: May 5, 2005
Inventors: Thangavel Kuberasampath (Medway, MA), Roy Pang (Etna, NH), Hermann Oppermann (Medway, MA), David Rueger (Hopkinton, MA), Charles Cohen (Medway, MA), Engin Ozkaynak (Milford, MA), John Smart (Weston, MA)
Application Number: 10/865,514
