US20020099008A1

US20020099008A1 - Method for stimulating hematopoiesis using tgf-alpha

Info

Publication number: US20020099008A1
Application number: US09/299,473
Authority: US
Inventors: Daniel R. Twardzik; Thomas S. Felker; Stefan L. Paskell
Original assignee: Stem Cell Pharmaceuticals Inc
Current assignee: Stem Cell Pharmaceuticals Inc
Priority date: 1999-04-26
Filing date: 1999-04-26
Publication date: 2002-07-25
Also published as: US6764683B1; EP1897441A1

Abstract

There is disclosed a novel genus of small peptides, much smaller than TGFα, was discovered as having TGFα biological activity and therefore are useful as pharmacologic agents for the same indications as full length TGFα polypeptide. There is further disclosed that TGFα and consequently the genus of small peptides disclosed herein, was found to have therapeutic activity to stimulate hematopoiesis in patients undergoing cytotoxic cancer chemotherapy and to act as a cytoprotective agent to protect a patient undergoing cancer cytotoxic therapy from gastrointestinal (GI) side effects, such as mucositis and otherwise support the barrier function of the GI tract when it is harmed by cytotoxic therapy.

Description

TECHNICAL FIELD OF THE INVENTION

The present invention provides a novel peptide that is derived from a loop or “lollipop” region of transforming growth factor alpha (TGF-α) and is biologically active for causing stem cells to proliferate and migrate. The present invention further provides a method for augmenting hematopoiesis, particularly trilineage hematopoiesis, and a method for suppressing immune functioning associated with autoimmune diseases, and a method for suppressing inflammatory responses mediated (in part) by excessive histamine release, comprising administering an effective amount of a TGF-α polypeptide or a fragment thereof, such as the lollipop region. The present invention further provides a method for treating or preventing mucositis and gastrointestinal side effects in patients undergoing cancer treatment, comprising administering an effective amount of a TGF-α polypeptide or a fragment thereof, such as the lollipop region.

BACKGROUND OF THE INVENTION

There are several disease treatments that could significantly benefit by having cells regenerate after injury or lesion formation, particularly in the CNS, in the immune system and in the gastrointestinal tract. The expression of growth factors and their receptors in the pre-implanted human embryo and maternal reproductive tract indicates that such factors influence growth and differentiation of embryonic cells in an autocrine and paracrine manner. Such growth factors are peptides that variously support survival, proliferation, differentiation, size and function of nerve cells and other lineages of cells. EGF (epidermal growth factor) is the first member found of the EGF family and characterized many years ago (Savage and Cohen, J. Biol. Chem. 247:7609-7611, 1972; and Savage et al., J. Biol. Chem. 247:7612-7621, 1972). Additional members of the EGF family have been found and they include vaccinia virus growth factor (VGF; Ventatesan et al., J. Virol. 44:637-646, 1982); myxomavirus growth factor (MGF; Upton et al. J. Virol. 61:1271-1275, 1987), Shope fibroma virus growth factor (SFGF; Chang et al., Mol. Cell. Biol. 7:535-540, 1987), amphiregulin (AR; Kimura et al., Nature 348:257-260, 1990), and heparin binding EGF-like factor (HB-EGF; Higashiyama et al., Science 251:936-939, 1991). A common structural feature of these polypeptides is the presence of six cysteine residues that form three disulfide cross links that support a conserved structure that binds to the EGF receptor.

Another member of the EGF family is TGFα and it also binds to the EGF receptor (Todaro et al., Proc. Natl. Acad. Sci. USA 77:5258-5262, 1980). TGFU stimulates the EGF receptor's tyrosine kinase activity and has many cellular functions, such as stimulating a mitogenic response in a wide variety of cell types. TGFα and EGF mRNAs reach their highest levels and relative abundance (compared to total RNA in the early postnatal period and decrease thereafter, suggesting a role in embryonic development. From a histological perspective, TGFα acts on numerous cell types throughout the body. The active form of TGFα is derived from a larger precursor and contains 50 amino acids. TGFα shares only a 30% structural similarity with the 53-amino acid form of EGF, but including conservation of all six cysteine residues. TGFα is highly conserved among species. For example, the rat and human polypeptides share about 90% homology as compared to a 70% homology as between the rat and human EGF polypeptide. The amino acid sequence of TGFα is shown in SEQ ID NO. 1. The sequence shows that a family consisting of vaccinia growth factor, amphiregulin precursor, betacellulin precursor, heparin binding EGF-like growth factor, epiregulin (rodent only), HUS 19878 and schwannoma derived growth factor have similar sequence motifs and can be considered as members of the same family based upon their shared cysteine disulfide bond structures.

TGFα is an acid and heat stable polypeptide of about 5.6 kDa molecular weight. It is synthesized as a larger 30-35 kDa molecular weight glycosylated and membrane-bound precursor protein wherein the soluble 5.6 kDa active form is released following specific cleavage by an elastase-like protease. TGFα binds with high affinity in the nanomolar range and induces autophosphorylation to transduce signal with the EGF receptor. TGFα is 50 amino acids in length and has three disulfide bonds to forms its tertiary configuration. All three disulfide bonds must be present for activity. TGFα is stored in precursor form in alpha granules of secretory cells. Moreover, the primary amino acid sequence is highly conserved among various species examined, such as more than 92% homology at the amino acid level as between human and rat TGFα polypeptides.

TGFα has been investigated extensively and has recently been identified as useful for treating a patient with a neurological deficit. This mechanism is thought to stimulate proliferation and migration of neural-origin stem cells to those site or lesions in a deficit. For example, Parkinson's Disease is characterized by resting tremor, rigidity, inability to initiate movement (akinesia) and slowness of movement (bradykinesia). The motor deficits are associated with progressive degeneration of the dopaminergic innervation to the nucleus accumbens and degeneration of noradrenergic cells of the locus ceruleus and serotonergic neurons of the raphe. Up to 80% of nigral dopamine neurons can be lost before significant motor deficits are manifest. TGFα (full polypeptide) was shown, when infused into rat brains, was useful for the treatment of neurodegenerative disorders. Intracerebroventricular (ICV) or intrastriatal infusions of TGFα induced neuronal stem cell proliferation, but degenerating or damaged or otherwise abnormal cells needed to be present to facilitate migration of the neuronal stem cells to a site of injury on a scale sufficient to impact recovery from an associated neurological deficit. Forebrain neural stem cells, that give rise to migrating progenitor cells that affect treatment and recovery from a neurological deficit disorder, are the migrating cells that affect treatment recovery from a neural deficit disorder (e.g., Parkinson's Disease, Huntington's Disease, Alzheimer's Disease and the like).

Neural stem cells have been found in subependyma throughout the adult rodent CNS (Ray et al. Soc. Neurosci. 22:394.5, 1996) and in the subependyma of adult human forebrain (Kirschenbaum et al., Cerebral Cortex 4:576-589, 1994). Thus, the discovery that TGFα stimulates proliferation of neural stem cells and promotes migration to a site of injury or deficit has led to its investigation for the treatment of a neurodegenerative disorder (Alzheimer's Disease, Huntington's Disease and Parkinson's Disease) or CNS traumatic injury (e.g., spinal chord injury), demyelinating disease, CNS inflammatory disease, CNS autoimmune disease (e.g., multiple sclerosis) or CNS ischemic disease (e.g., stroke or brain attack).

A CNS stem cell has the potential to differentiate into neurons, astrocytes and to exhibit replication of itself to provide a resource for self-renewal. Both neurons and glial cells seen to be derived from a common fetal precursor cell. In the vertebrate CNS, multipotential cells have been identified in vitro and in vivo. Certain mitogens, such as TGFα, can cause proliferation of CNS mutipotential cells in vitro and this is the basis for a procedure to harvest such cell, treat them ex vivo to stimulate proliferation in culture and then readminister such cells. Immunohistochemical analysis in the human brain supports the notion that TGFα is widely distributed in neurons and glial cells both during development and during adulthood. In mice genetically altered to lack expression of functioning TGFα, there was a decrease in neural progenitor cell proliferation in forebrain subependyma, providing evidence for TGFα as a proliferative factor for neural progenitor cells.

TGFα is found mainly in various neurons of the CNS during development and in the adult brain in the cerebral neocortex, hippocampus and striatum. It is also found in a few glial cells, primarily in the cerebral and cerebellar cortex areas. Northern blot analyses showed that TGFα and not EGF (epidermal growth factor) is the most abundant ligand that binds to the EGF receptor in the brain. TGFα mRNA levels were 15-170 times higher than EGF in cerebellum and cerebral cortex. TGFα also appears in germinal centers of the brain during neurogenesis and gliogenesis in the developing brain. In the midbrain, the distribution of TGFα overlaps with tyrosine hydroxylase mRNA and fetal dopaminergic neurons. In culture, TGFα enhanced survival and neurite outgrowth of neonatal rat dorsal ganglion neurons (EGF did not) and survival and differentiation of CNS neurons. TGFα induced proliferation of neural precursor cells of the murine embryonic mesencephalon and further induced a significant increase in the number of astroglia and microglia in fetal rat medial septal cells. TGFα increased glutamic acid decarboxylase activity and decreased choline actetyltransferase activity. Thus, TGFα acted as a general neuronal survival factor affecting both cholinergic and GABAergic neurons. In addition, TGFα is a mitogen for pluripotent brain stem cells. Forebrain subependyma contains nestin positive neural stem cells and their progeny, which are constitutively proliferating progenitor epithelial cells. A “knockout” mouse that was genetically engineered to delete the gene for TGFα showed a reduction in neuronal progenitor cells in the subependyma and a reduction in neuronal progenitors that migrate to the olifactory bulb. In vitro, TGFα promoted dopamine uptake in fetal rat dopaminergic neurons in a dose-dependent and time-dependent manner. TGFα selectively promoted dopaminergic cell survival, enhanced neurite length, branch number and the soma area of tyrosine hydroxylase immunopositive cells. The levels of TGFα were elevated in ventricular cerebrospinal fluid in juvenile parkinsonism and Parkinson's Disease and may represent a compensatory response to neurodegeneration. Further, TGFα prevented a striatal neuronal degeneration in an animal model of Huntington's Disease.

The mucosal epithelium of the intestine is in a continually dynamic state known as “epithelial renewal” in which undifferentiated stem cells from a proliferative crypt zone divide, differentiate and migrate to the luminal surface. Once terminally differentiated, they are sloughed from the tips of the villi. The turnover of the crypt-villus cell population is rapid and occurs every 24-72 hours. Continuous exfoliation of the cells at the villus tip is counterbalanced by ongoing proliferation in the crypt so that net intestinal epithelial mass remains relatively constant. The rapidly-proliferating epithelium of the gastrointestinal tract is extremely sensitive to cytotoxic drugs that are widely used in the chemotherapy of cancer. This “side effect” reduces the tolerated dose of such drugs as it can cause a breakdown of the GI barrier function and septic condition in a patient already immuno-compromised. This can also lead to life-threatening hemorrhage. Therefore, there is a need in the art for the development of products and delivery systems that stimulate the repair and rejuvenation of mucosal epithelium in the gastrointestinal tract to provide benefit to patient receiving chemotherapy and radiation therapy for cancer.

Therefore, there is a need in the art to find improved TGFα mimetic agents that are more economical to produce and are smaller (in terms of molecular weight). The present invention was made to address such a need.

SUMMARY OF THE INVENTION

The present invention is based upon two basic discoveries that have not been reported before in the extensive literature of TGFα . Firstly, a novel genus of small peptides, much smaller than TGFα, was discovered as having TGFα biological activity and therefore are useful as pharmacologic agents for the same indications as full length TGFα polypeptide. Secondly, TGFα and consequently the genus of small peptides disclosed herein, was found to have therapeutic activity to stimulate hematopoiesis in patients undergoing cytotoxic cancer chemotherapy and to act as a cytoprotective agent to protect a patient undergoing cancer cytotoxic therapy from gastrointestinal (GI) side effects, such as mucositis and otherwise support the barrier function of the GI tract when it is harmed by cytotoxic therapy.

The present invention provides a compound that acts as a TGFα mimetic, comprising at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

wherein X ₁is independently Val, Gly or Ala, wherein X₂is Try or Phe, wherein X₃is Arg or Lys, and wherein the two Cys moieties form a disulfide bond to create an 11-amino acid loop peptide. Preferably, at least one or more of the following seven amino acids are added to the C terminus Cys moiety from formula II:

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

wherein X ₄is Glu or Asp, wherein X₅is Leu or Ile, and wherein X₆is Asp or Glu. Preferably, X_1ais Val, X_1bis Gly and X_1cis Ala. Preferably, X₂is Tyr, and X₃is Arg. Most preferably, the loop peptide is 13 amino acids in length wherein X_1ais Val, X_1bis Gly, X_1cis Ala, and X₄is Gly.

The present invention further provides a pharmaceutical composition comprising a loop peptide in a pharmaceutically acceptable carrier, wherein the loop peptide compound comprises at least an 11 -membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

The present invention further provides a method for treating a neurodegenerative disease with an pharmaceutically active loop peptide, wherein the loop peptide comprises at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

The present invention further provides a method for treating a CNS disease or disorder, wherein the CNS disease or disorder is selected from the group consisting of CNS ischemia, spinal cord injury, MS, and retinal injury, comprising with an pharmaceutically active loop peptide, wherein the loop peptide comprises at least an 1 1-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

The present invention further provides a method for augmenting hematopoiesis during cytotoxic or immune-suppressing therapy, comprising administering a TGFα polypeptide or a pharmaceutically active loop peptide, or both, wherein the loop peptide comprises at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

wherein X ₄is Glu or Asp, wherein X₅is Leu or Ile, and wherein X₆is Asp or Glu. Preferably, X_1ais Val, X_1bis Gly and X_1cis Ala. Preferably, X₂is Tyr, and X₃is Arg. Most preferably, the loop peptide is 13 amino acids in length wherein X_1ais Val, X_1bis Gly, X_1cis Ala, and X₄is Gly. Preferably, the invention further comprises administering a second hematopoietic growth factor agent to stimulate more mature hematopoietic precursor cells, wherein the second hematopoietic growth factor is selected from the group consisting of erythropoietin, thrombopoietin, G-CSF (granulocyte colony stimulating factor), and GM-CSF (granulocyte macrophage colony stimulating factor).

The present invention further provides a method for treating or preventing mucositis of the gastrointestinal tract during cytotoxic or immune-suppressing therapy, comprising administering a TGFα polypeptide or a pharmaceutically active loop peptide, or both, wherein the loop peptide comprises at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

The present invention further provides a bifunctional compound that acts as a TGFα mimetic, comprising a compound from formula III:

Loop peptide N-terminus-linker-cyclic C₄H₈N₂-linker-Loop peptide N-terminus III

wherein the linker moiety is designed to link the N-terminus of the Loop peptide to a nitrogen atom of the ring C ₄H₈N₂and wherein the “loop peptide” comprises at least an I1 -membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

wherein X ₄is Glu or Asp, wherein X₅is Leu or Ile, and wherein X₆is Asp or Glu. Preferably, X_1ais Val, X_1bis Gly and X_1cis Ala. Preferably, the linker group is independently selected from the group consisting of substituted or unsubstituted C_1-6alkyl, substituted or unsubstituted C_2-6alkenyl, substituted or unsubstituted C_1-6alkoxy, xylenyl, wherein the substitutions are selected from the group consisting of oxo, epoxyl, hydroxyl, chloryl, bromyl, fluoryl, and amino Preferably, X₂is Tyr, and X₃is Arg. Most preferably, the loop peptide is 13 amino acids in length wherein X_1ais Val, X_1bis Gly, X_1cis Ala, and X₄is Gly.

The present invention further provides a method for treating inflammatory bowel disease, colitis, and Chron's Disease of the gastrointestinal tract, comprising administering a TGFα polypeptide or a pharmaceutically active loop peptide, or both, wherein the loop peptide comprises at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

The present invention further provides a method for treating an inflammatory reaction of autoimmune diseases, comprising administering a TGFα polypeptide or a pharmaceutically active loop peptide, or both, wherein the loop peptide comprises at least an 11-membered peptide compound from formula I:

N-X_1a-Cys-His-Ser-X_1b-X₂-X_1a-X_1b-X_1a-X₃- Cys COOH I

wherein X ₁is independently Val, Gly or Ala, wherein X₂is Try or Phe, wherein X₃is Arg or Lys, and wherein the two Cys moieties form a disulfide bond to create an 11-amino acid loop peptide. Preferably, the autoimmune diseases are selected from the group consisting of Type II (Juvenile) Diabetes, rheumatoid arthritis, lupus, and multiple sclerosis. Preferably, at least one or more of the following seven amino acids are added to the C terminus Cys moiety from formula II:

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of human TGFα polypeptide and its 50 amino acids arranged into three loops. [0040]
FIG. 2 shows a graph comparing TGFα biological activity of the three loop peptide regions of TGFα (see FIG. 1) wherein Loop A is amino acids 1-21 (starting at the N terminus), Loop B is amino acids 16 to 32 and Loop C is amino acids 33 to 50. Only Loop C showed significant TGFα activity as determined by cell proliferation and in a dose response fashion. [0041]
FIG. 3 shows a graph of mouse spleen weights that were treated with Cis Platinum (CP) at either 5 μg/g or 10 μg/g and with TGFα at concentrations of 10 ng/g or 50 ng/g. These data show that TGFα treatment caused a return to normal spleen weights despite CP treatment that reduced spleen weights significantly. [0042]
In FIG. 4, three panels of H&E-stained spleens are shown. Specifically, the top panel shows a CP-treated mouse spleen (10 μg/g) showing apoptotic cells (densely stained with fragments of nuclei) in the germinal center (GC). The T cells with the central arterial area show the absence of a marginal zone and much fewer erythrocytes and T cells in the perifolecular area (arrows). In the middle panel, a normal mouse spleen is shown (no CO and no TGFα) fixed in formalin showing an arteriole with T cells areas (arrow). A primary follicle and a second follicle are shown as containing a germinal center (GC). There is a presence of an erythrocyte rich (pink) perifollicular zone surrounding both a T cell and B cell compartments of white pulp. In the bottom panel, a mouse spleen treated with CP (10 μg/g) and TGFα (50 ng/g) shows an increased number of T cells and erythrocytes in the perifolicular zone (arrows). The T cell area contains lymph vessels in relation to arterioles. A germinal center (GC) is within the mantle zone. [0043]
In FIG. 5 there are three panels showing the histological examination of mouse intestines. In the top panel, CP (single ip dose of 10 μg/g) treated intestine is cross-sectioned and shows significant injury to the villi. Specifically, the villi are necrotic and the crypts are in irregular shapes. The tips of the crypts were losing their cellular integrity (arrows). In the middle panel is a cross section of a normal mouse GI tract (no CP and no TGFα) and shows a normal intestinal surface with villi having long and slender mucosal projections with a core of lamina propria covered by a luminal epithelial layer. A single row of intestinal crypt is found at the base of the mucosa. These crypts that lie between adjacent villi are surrounded by the same lamina propria that form the villous cores. Both columnar absorptive cells and globlet cells cover the villous surfaces. The globlet cells contain apical clear vacuoles. The bottom panel shows a cross section of a mouse intestine exposed both the CP (10 μg/g) and TGFα (50 ng/g). The intestinal structure is very similar to the normal intestinal structure. Specifically, the villus is long and slender. Both absorptive cells and globlet cells are visible at the surface of the villi. There is an abundant amount of globlet cells on the surface. [0044]
In FIG. 6, there are three panels shown at 160× magnification again corresponding to a CP-treated mouse in the top panel, a normal mouse in the middle panel and a CP treated and TGFα treated mouse in the bottom panel at the same doses as indicated for FIG. 5. In the top panel are injured villi with tips degenerating and necrotic (arrows). Red blood cells are observed in the damaged villi (arrows). The crypts (C) are in irregular shape and in various heights. The middle panel shows that the tips of the villi (arrows) are smooth and the nuclei of the enterocytes are observed throughout the villus. The crypts (C) are similar in height and regular in shape. The bottom panel has villi (arrows) appearing normal as in the middle panel. The crypts (C) also appear to be normal. [0045]
FIG. 7 shows three panels but the top and middle panels are CP (10 μg/g) treated without TGFα and the bottom panel is CP (10 μg/g) and 50 ng/g of TGFα. The panels are shown at higher magnification. In the top panel, the severely injured crypt surface from CP treatment shows cellular destruction due to necrosis. Red cells appear at the damaged surface to indicate intestinal bleeding. In addition, the middle panel of a CP-treated mouse shows a loss of brush border and very little of a glycocalyx or fuzzy coat. The interspersed globlet cells appear fewer in number (than normal) and are seen as necrotic. In the bottom panel, the effect of TGFα treatment shows protection of the villa surface (arrows). Specifically, the epithelial cells are normal appearing with extended brush borders. The nuclei are very densely stained and elongated. [0046]
The histological data is summarized in FIG. 8 that measured average crypt height of the three groups of mice. TGFα treatment (50 ng/g) was able to more-than-restore crypt height loss from CP treatment. [0047]
In FIG. 9, the three panels at 160× magnification are shown to correspond to normal intestine in the top panel, CP only treated (10 μg/g) in the middle panel and both CP (10 μg/g) and TGFα (50 ng/g) in the bottom panel. In the normal intestine (top panel), each villus extends from the luminal surface to the basal muscularis mucosal surface. Globlet cells are scattered and predominate in the base of the villus (arrows) whereas columnar absorptive cells line the luminal surface. In the middle panel, the alcian blue staining method shows that the villi contain a fewer number of globlet cells (than normal) (arrows). The injured absorptive and globlet cells are degenerating at the tip of the villi (arrows). Abundant secretory mucus material is stained in the luminal surface (arrows). In the bottom panel, there is an increased number of globlet cells scattered throughout the villi (arrows). The intestinal villi are in normal form with elongation. The majority of enterocytes are not alcian blue stained positive. The luminal plasma membranes of the villi (arrows) are well protected by TGFα treatment. [0048]
FIG. 10 shows that TGFα treatment not only increased the number of globlet cells but increased the number from CP treatment to a higher level than normal intestine. [0049]
FIG. 11 shows that TGFα treatment causes mast cells residing in the intestinal mucosal tissue and lamina propria to remain intact and thus not release histamine and other pro-inflammatory molecules. The bottom panel, by contrast, shows CP-treated mice who did not receive TGFα wherein there was a degranulation of mast cells and subsequent induction of inflammatory responses.[0050]

DETAILED DESCRIPTION OF THE INVENTION

Loop Peptide [0051]
TGFα is a polypeptide of 50 amino acids shown in FIG. 1. The TGFα polypeptide can be divided roughly into three loop regions corresponding roughly (starting at the N terminus) to amino acids 1-21, to amino acids 16-32, and to amino acids 33-50. Each of the three foregoing loop regions was investigated for TGFα-like biological activity, such as stimulation of cellular proliferation as measured by [0052] ³H thymidine incorporation of stem cells. As shown in FIG. 2, only the Loop C peptide (corresponding to amino acids 33-50) showed significant TGFα biological activity and is therefore a TGFα mimetic peptide. Therefore, in view of the fact that the loop peptide exhibits TGFα biological activity, data obtained with TGFα (50 amino acid polypeptide is predictive of activity of the loop peptide and similar loop peptides embodied in the genus of formula I with or without the addition of a “tail” region of formula II). These data predict activity for the loop peptide when activity is also shown for TGFU.
Pharmaceutical Composition and Formulations [0053]
The inventive pharmaceutical composition comprises a loop peptide in a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is suitable for the particular form of administration contemplated by the pharmaceutical composition. The term “carrier” is designed to mean any and all solvents, dispersion media, coatings, isotonic agents, antibacterial and antifungal agents designed to preserve a formulation from contamination, absorption agents and similar agents that are compatible with pharmaceutical administration irrespective of the route of administration. [0054]
The pharmaceutical formulations are made based upon the intended routes of administration. Specifically, those formulations that will be intended for a GI indication will likely be administered orally. In view of the peptide bonds present, such formulations will be made to pass through the stomach and protect the active compound from the low pH conditions of the stomach before there is a better chance for local activity in the villi of the small intestine and large intestine. The loop peptide formulations are intended for parenteral administration through some form of injection or for use in ex vivo culture media. Parenteral forms of administration include, for example, intravenous, intradermal, intramuscular, intraperitoneal for GI effects, injection directly into a target organ (e.g., brain) at the appropriate location, application in a biodegradable matrix to a site of CNS injury (e.g., spinal cord). [0055]
Solutions or suspensions useful in the pharmaceutical compositions that contains peptide components include sterile diluents such as water, saline, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic agents, plus an antibacterial or antifungal agent for preservation, antioxidants, chelating agents, buffer and agents that adjust tonicity for direct organ injections. Forms of pharmaceutical compositions include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions of dispersions. For intravenous injection or direct organ or peritoneal injections, suitable carriers include, for example, saline, bacteriostatic water, Cremophor, or phosphate buffered saline. The composition is formulated to preserve stability, be easily mixed and preserved against contamination. Isotonic agents, such as sugars or polyalcohols (e.g., glucose, fructose, mannitol, sorbitol and the like) or sodium chloride are used. Agents that delay target organ absorption can also be used and these include, for example, aluminum monostearate and gelatin. [0056]
Sterile injectable solutions can be prepared by incorporating the active agent (see formula I, formula II, or formula III and TGFα) in the required amount in an appropriate solvent and then sterilizing, such as by sterile filtration. Further, powders can be prepared by standard techniques such as freeze drying or vacuum drying. [0057]
In another embodiment, the active agent is prepared with a biodegradable carrier for sustained release characteristics for either sustained release in the GI tract or for target organ implantation (e.g., brain or spinal cord) with long term active agent release characteristics to the intended site of activity (such as a site of injury or neuronal degradation). Biodegradable polymers include, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acids, polylactic acids, collagen, polyorthoesters, and poly acetic acid. Liposomal formulation can also be used. [0058]
In addition, the active compound for the pharmaceutical composition needs to also be synthesized. If the compound is from formula I or formula II, a preferred means for synthesizing peptides of 13-18 amino acids in length is by direct peptide synthesis generally starting with the N-terminal amino acid and adding amino acids in the C terminal direction. Such small peptides can also be synthesized and later purified by standard recombinant techniques, but peptide of 18 amino acids in length are better synthesized from the amino acid building blocks directly. TGFα has bee made using recombinant techniques and is available as a laboratory reagent commercially. The bifunctional compounds of formula III are best synthesized with each loop peptide moiety synthesized and then added to the heterocyclic nitrogen atom using standard heterocyclic addition synthesis [0059]
Loop Peptide Mimics TGFα Neuroactive Therapeutic Activity [0060]
The neuroactive activity of the loop peptide is based upon the discovery that the loop peptide exhibits TGFα biological activity and can therefore stimulate CNS multipotent precursor cells to divide and migrate through the brain. This activity indicates that the loop peptide is effective to treat neurological deficits caused by a wide variety of diseases and injuries that each result in a neurological deficit in some specific area of the brain or specific kind of neuron. These include degenerative diseases, including the more common Alzheimer's Disease (AD), Parkinson's Disease (PD), and Huntington's Disease (HD), and the less common Pick's disease, progressive supranuclear palsy, striatonigral degeneration, cortico-basal degeneration, olivopontocerebellar atrophy, Leigh's disease, infantile necrotizing encephalomyelopathy, Hunter's disease, mucopolysaccharidosis, various leukodystrophies (such as Krabbe's disease, Pelizaeus-Merzbacher disease and the like), amaurotic (familial) idiocy, Kuf's disease, Spielmayer-Vogt disease, Tay Sachs disease, Batten disease, Jansky-Bielschowsky disease, Reye's disease, cerebral ataxia, chronic alcoholism, beriberi, Hallervorden-Spatz syndrome, cerebellar degeneration, and the like. [0061]
Further, injuries (traumatic or neurotoxic) that cause a loss of neuronal function can be treated by the loop peptide. Such injuries include, for example, gunshot wounds, injuries caused by blunt force, penetration injuries, injuries caused by surgical procedure (e.g., tumor removal, abscess removal, epilepsy lesion removal) poisoning (e.g., carbon monoxide), shaken baby syndrome, adverse reactions to medications, drug overdoses, and post-traumatic encephalopathy. Ischemia can further cause CNS injury due to disruption of blood flow or oxygen delivery that can kill or injure neurons and glial cells. Such injuries can be treated by administration of the loop peptide and include, for example, injuries caused by stroke, anoxia, hypoxia, partial drowning, myoclonus, severe smoke inhalation, dystonias, and acquired hydrocephalus. Developmental disorders that can be treated by the loop peptide include, for example, schizophrenia, certain forms of severe mental retardation, cerebral palsey, congenital hydrocephalus, severe autism, Downs Syndrome, LHRH/hypothalamic disorder, and spina bifida. The loop peptide can be further used to treat disorders affecting vision caused by the loss or failure of retinal cells and include, for example, diabetic retinopathy, serious retinal detachment (associated with glaucoma), traumatic injury to the retina, retinal vascular occlusion, macular degeneration, optic nerve atrophy and other retinal degenerative diseases. Injuries to the spinal cord can be treated by the loop peptide. Examples of spinal cord injuries are post-polio syndrome, amyotrophic lateral sclerosis, traumatic injury, surgical injury, and paralytic diseases. Demylinating autoimmune disorders can be treated by administration of the loop peptide and include, for example, multiple sclerosis. Lastly, the loop peptide can be used to treat neurological deficits caused by infection of inflammatory diseases, including, for example, Creutzfeldt-Jacob disease and other slow virus infectious diseases of the CNS, AIDS encephalopathy, post-encephalitic Parkinsonism, viral encephalitis, bacterial meningitis and other CNS effects of infectious diseases. [0062]
The loop peptide provides TGFα activity and therefor the present method of treating neurological deficit and injury disorders is based upon the biological activity of the loop peptide of formula I, formula II and formula III and the data available for TGFα that has been published. [0063]
Hematopoiesis [0064]
TGFα showed surprising activity in an in vivo model of general hematopoiesis when administered in conjunction with a potent cytotoxic agent Cis Platinum (CP). FIG. 3 shows a graph of mouse spleen weights that were treated with CP at either 5 μg/g or 10 μg/g and with TGFα at concentrations of 10 ng/g or 50 ng/g. These data show that TGFα treatment caused a return to normal spleen weights despite CP treatment that reduced spleen weights significantly. This in vivo experiment is a predictive model for hematopoiesis in humans as CP is a cytotoxic agent commonly used for cancer chemotherapy that is known to significantly reduce trilineage hematopoietic cells. Hematopoietic cells are red blood cell precursors, platelet precursors (megakaryocytes), and immune (white) blood cell precursors of various forms of T cells, B cells and macrophages. Moreover, platelet counts were higher in those mice injected with TGFα (and CP) as opposed to CP alone were such counts were significantly reduced from normal. [0065]
The experiment procedure dosed those animals to be treated with [0066] TGFα 4 hours prior to challenge with CP. A single dose of CP was administered. Additional doses (as indicated) of TGFα were made at 24 hours, 48 hours, 72 hours and 96 hours after the CP dose. All doses were made by IP injection. Controls were dosed with saline instead of either or both of CP and TGFα.
The animals were sacrificed about 4 hours after the last TGFα (or saline) dose. Key organs were removed and spleens were immediately weighed after a clean incision. All the relevant organs were placed in formalin, transported for histopathological analysis, mounted, sectioned, stained and observed. The results of this histological analysis of the spleens for hematopoietic effect and the GI tract (below) provide the surprising data of the effect of TGFα activity. [0067]
In FIG. 4, three panels of H&E-stained spleens are shown. Specifically, the top panel shows a CP-treated mouse spleen (10 μg/g) showing apoptotic cells (densely stained with fragments of nuclei) in the germinal center (GC). The T cells with the central arterial area show the absence of a marginal zone and much fewer erythrocytes and T cells in the perifolecular area (arrows). In the middle panel, a normal mouse spleen is shown (no CO and no TGFα) fixed in formalin showing an arteriole with T cells areas (arrow). A primary follicle and a second follicle are shown as containing a germinal center (GC). There is a presence of an erythrocyte rich (pink) perifollicular zone surrounding both a T cell and B cell compartments of white pulp. In the bottom panel, a mouse spleen treated with CP (10 μg/g) and TGFα (50 ng/g) shows an increased number of T cells and erythrocytes in the perifolicular zone (arrows). The T cell area contains lymph vessels in relation to arterioles. A germinal center (GC) is within the mantle zone. These in vivo data in a predictive model of hematopoiesis and confirmed by blinded histological analysis (the histologist/pathologist was blinded as to the treatment history of the coded tissues received) providing surprising evidence of the utility of peptides having TGFα activity to augment hematopoiesis following cytotoxic exposure. These data predict and provide a reasonable correlation that TGFα and the peptides of formula I, formula II and formula III are useful therapeutic agents for augmenting hematopoiesis following or during cytotoxic therapy, such as cancer treatment. Therefore, a useful method for treating cancer is to combine either TGFα or a peptide from formula I, formula II or formula III or combinations thereof with cytotoxic treatment regimens to reduce dose-limiting side effects. [0068]
Mucositis and Gastrointestinal Diseases [0069]
The small intestine comprises the duodenum, jejunum and ileum. It is the principal site for absorption of digestive products from the GI tract. Digestion begins in the stomach and is completed in the small intestine in association with the absorptive process. The intestinal mucosa surface is made up of numerous finger-like projections called villi. In addition, mucosa between the basis of the villi (crypts) is formed into the crypts. [0070]
TGFα or a peptide from formula I, formula II or formula III or combinations thereof are also useful for treating mucositis associated intestinal bleeding , dyspepsia associated with cytotoxic therapy and for improving the barrier function of the GI tract compromised by cytotoxic therapy. The in vivo experiment with seven groups of mice described above for hematopoietic effects noted in spleens also examined the GI tract of these treated mice. In FIG. 5 there are three panels showing the histological examination of mouse intestines. In the top panel, CP (single ip dose of 10 μg/g) treated intestine is cross-sectioned and shows significant injury to the villi. Specifically, the villi are necrotic and the crypts are in irregular shapes. The tips of the crypts were losing their cellular integrity (arrows). In the middle panel is a cross section of a normal mouse GI tract (no CP and no TGFα) and shows a normal intestinal surface with villi having long and slender mucosal projections with a core of lamina propria covered by a luminal epithelial layer. A single row of intestinal crypt is found at the base of the mucosa. These crypts that lie between adjacent villi are surrounded by the same lamina propria that form the villous cores. Both columnar absorptive cells and globlet cells cover the villous surfaces. The globlet cells contain apical clear vacuoles. The bottom panel shows a cross section of a mouse intestine exposed both the CP (10 μg/g) and TGFα (50 ng/g). The intestinal structure is very similar to the normal intestinal structure. Specifically, the villus is long and slender. Both absorptive cells and globlet cells are visible at the surface of the villi. There is an abundant amount of globlet cells on the surface. [0071]
In FIG. 6, there are three panels shown at 160× magnification again corresponding to a CP-treated mouse in the top panel, a normal mouse in the middle panel and a CP treated and TGFα treated mouse in the bottom panel at the same doses as indicated for FIG. 5. In the top panel are injured villi with tips degenerating and necrotic (arrows). Red blood cells are observed in the damaged villi (arrows). The crypts (C) are in irregular shape and in various heights. The middle panel shows that the tips of the villi (arrows) are smooth and the nuclei of the enterocytes are observed throughout the villus. The crypts (C) are similar in height and regular in shape. The bottom panel has villi (arrows) appearing normal as in the middle panel. The crypts (C) also appear to be normal. [0072]
FIG. 7 shows three panels but the top and middle panels are CP (10 μg/g) treated without TGFα and the bottom panel is CP (10 μg/g) and 50 ng/g of TGFα. The panels are shown at higher magnification. In the top panel, the severely injured crypt surface from CP treatment shows cellular destruction due to necrosis. Red cells appear at the damaged surface to indicate intestinal bleeding. In addition, the middle panel of a CP-treated mouse shows a loss of brush border and very little of a glycocalyx or fuzzy coat. The interspersed globlet cells appear fewer in number (than normal) and are seen as necrotic. In the bottom panel, the effect of TGFα treatment shows protection of the villa surface (arrows). Specifically, the epithelial cells are normal appearing with extended brush borders. The nuclei are very densely stained and elongated. [0073]
The histological data is summarized in FIG. 8 that measured average crypt height of the three groups of mice. TGFα treatment (50 ng/g) was able to more-than-restore crypt height loss from CP treatment. [0074]
An alcian blue staining method permits differentiation of two major cell types that are an absorptive cell and a globlet cell. The globlet cell mucus is stained a greenish blue color while the absorptive cells remain less stained. In FIG. 9, the three panels at 160× magnification are shown to correspond to normal intestine in the top panel, CP only treated (10 μg/g) in the middle panel and both CP (10 μg/g) and TGFα (50 ng/g) in the bottom panel. In the normal intestine (top panel), each villus extends from the luminal surface to the basal muscularis mucosal surface. Globlet cells are scattered and predominate in the base of the villus (arrows) whereas columnar absorptive cells line the luminal surface. In the middle panel, the alcian blue staining method shows that the villi contain a fewer number of globlet cells (than normal) (arrows). The injured absorptive and globlet cells are degenerating at the tip of the villi (arrows). Abundant secretory mucus material is stained in the luminal surface (arrows). In the bottom panel, there is an increased number of globlet cells scattered throughout the villi (arrows). The intestinal villi are in normal form with elongation. The majority of enterocytes are not alcian blue stained positive. The luminal plasma membranes of the villi (arrows) are well protected by TGFα treatment. The number of globlet cells were counted on the average unit length of intestine. These data are shown in FIG. 10. TGFα treatment not only increased the number of globlet cells but increased the number from CP treatment to a higher level than normal intestine. [0075]
Accordingly, these data show the effects of TGFα, and the loop peptides from formula I, formula II and formula III having therapeutic activity to treat or prevent mucositis associated with cytotoxic therapy and for inflammatory bowel diseases. Moreover, the histological effect showing that there was a prevention of mast cell degranulation (FIG. 11), provides additional data supporting the gastrointestinal applications for TGFα, and the loop peptide of formula I, formula II and formula III. [0076]
In addition, TGFα stimulated the proliferation of select immune cells (particularly of the T cell lineage) after administration to mice after immune-suppression of CP administration. The stimulated immune cells were phenotypically identified as CD4 positive T cells and double null CD4 negative CD8 negative T cell progenitors with characteristics of NK-1 cells. Thus, TGFα regulated immune functions and in particular defects in NK-1 cells. Therefore, these data predict that TGFα and the loop peptide of formula I, formula II and formula III will be effective in treating autoimmune diseases by mitigating over-inflammatory reactions. The in vivo activity of TGFα (FIG. 11) (and the loop peptide of formula I, formula II and formula III) to stimulate early T cell progenitors on the NK-1 type results in the release of TH-2 cytokines and this down regulates autoimmune phenomena. The stimulation of select immune cells, in particular cells of a T cell lineage, was seen consistently in the mice who received CP and TGFα (FIG. 11 for GI tract) in lymphoid tissue, Peyers Patches and the spleen. Further, recruitment of help via CD4 cells in some cases boosts immune system function in general. [0077]
In FIG. 11, TGFα administration prevented mast cell degranulation and subsequent histamine release. This is a parallel activity that is in addition to the gastrointestinal anti-inflammatory activity and prevention of mucositis of TGFα (and the loop peptide of formula I, formula II and formula III) described herein. [0078]
1 4 1 50 PRT Homo sapiens 1 Val Val Ser His Phe Asn Lys Cys Pro Asp Ser His Thr Gln Tyr Cys 1 5 10 15 Phe His Gly Thr Cys Arg Phe Leu Val Gln Glu Glu Lys Pro Ala Cys 20 25 30 Val Cys His Ser Gly Tyr Val Gly Val Arg Cys Glu His Ala Asp Leu 35 40 45 Asp Ala 50 2 18 PRT Homo sapiens 2 Val Cys His Ser Gly Tyr Val Gly Val Arg Cys Glu His Ala Asp Leu 1 5 10 15 Asp Ala 3 11 PRT Artificial Sequence Loop peptide 3 Xaa Cys His Ser Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 4 7 PRT Artificial Sequence Loop peptide 4 Xaa His Xaa Xaa Xaa Xaa Xaa 1 5

Claims

We claim:

1. A compound that acts as a TGFα mimetic, comprising at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

wherein X₁is independently Val, Gly or Ala, wherein X₂is Try or Phe, wherein X₃is Arg or Lys, and wherein the two Cys moieties form a disulfide bond to create an 11-amino acid loop peptide.

2. The compound of claim 1 wherein at least one or more of the following seven amino acids are added to the C terminus Cys moiety from formula II:

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

wherein X₄is Glu or Asp, wherein X₅is Leu or Ile, and wherein X₆is Asp or Glu.

3. The compound of claim 1 wherein X_1ais Val, X_1bis Gly and X_1cis Ala.

4. The compound of claim 2 wherein X₂is Tyr, and X₃is Arg.

5. The compound of claim 2 wherein the loop peptide is 13 amino acids in length, wherein X_1ais Val, X_1bis Gly, X_1cis Ala, and X₄is Gly.

6. A pharmaceutical composition comprising a loop peptide in a pharmaceutically acceptable carrier, wherein the loop peptide compound comprises at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

7. The pharmaceutical composition of claim 6 wherein at least one or more of the following seven amino acids are added to the C terminus Cys moiety from formula II:

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

8. The pharmaceutical composition of claim 6 wherein X_1ais Val, X_1bis Gly and X_1cis Ala.

9. The pharmaceutical composition of claim 7 wherein X₂is Tyr, and X₃is Arg.

10. The pharmaceutical composition of claim 7 wherein the loop peptide is 13 amino acids in length wherein X_1ais Val, X_1bis Gly, X_1cis Ala, and X₄is Gly.

11. A method for treating a neurodegenerative disease with an pharmaceutically active loop peptide, wherein the loop peptide comprises at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

12. The method for treating a neurodegenerative disease of claim 11 wherein at least one or more of the following seven amino acids are added to the C terminus Cys moiety from formula II:

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

13. The method for treating a neurodegenerative disease of claim 11 wherein X_1ais Val, X_1bis Gly and X_1cis Ala.

14. The method for treating a neurodegenerative disease of claim 12 wherein X₂is Tyr, and X₃is Arg.

15. The method for treating a neurodegenerative disease of claim 12 wherein the loop peptide is 13 amino acids in length wherein X_1ais Val, X_1bis Gly, X_1cis Ala, and X₄is Gly.

16. A method for treating a CNS disease or disorder, wherein the CNS disease or disorder is selected from the group consisting of CNS ischemia, spinal cord injury, MS, and retinal injury, comprising with an pharmaceutically active loop peptide, wherein the loop peptide comprises at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

17. The method for treating a CNS disease or disorder of claim 16 wherein at least one or more of the following seven amino acids are added to the C terminus Cys moiety from formula II:

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

18. The method for treating a CNS disease or disorder of claim 16 wherein X_1ais Val, X_1bis Gly and X_1cis Ala.

19. The method for treating a CNS disease or disorder of claim 17 wherein X₂is Tyr, and X₃is Arg.

20. The method for treating a CNS disease or disorder of claim 17 wherein the loop peptide is 13 amino acids in length wherein X_1ais Val, X_1bis Gly, X_1cis Ala, and X₄is Gly.

21. A method for augmenting hematopoiesis during cytotoxic or immune-suppressing therapy, comprising administering a TGFα polypeptide or a pharmaceutically active loop peptide, or both, wherein the loop peptide comprises at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

22. The method for augmenting hematopoiesis during cytotoxic or immune-suppressing therapy of claim 21 wherein at least one or more of the following seven amino acids are added to the C terminus Cys moiety from formula II:

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

23. The method for augmenting hematopoiesis during cytotoxic or immune-suppressing therapy of claim 21 wherein X_1ais Val, X_1bis Gly and X_1cis Ala.

24. The method for augmenting hematopoiesis during cytotoxic or immune-suppressing therapy of claim 22 wherein X₂is Tyr, and X₃is Arg.

25. The method for augmenting hematopoiesis during cytotoxic or immune-suppressing therapy of claim 22 wherein the loop peptide is 13 amino acids in length wherein X_1ais Val, X_1bis Gly, X_1cis Ala, and X₄is Gly.

26. The method for augmenting hematopoiesis during cytotoxic or immune-suppressing therapy of claim 21 further comprising administering a second hematopoietic growth factor agent to stimulate more mature hematopoietic precursor cells, wherein the second hematopoietic growth factor is selected from the group consisting of erythropoietin, thrombopoietin, G-CSF (granulocyte colony stimulating factor), and GM-CSF (granulocyte macrophage colony stimulating factor).

27. A method for treating or preventing mucositis of the gastrointestinal tract during cytotoxic or immune-suppressing therapy, comprising administering a TGFα polypeptide or a pharmaceutically active loop peptide, or both, wherein the loop peptide comprises at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

28. The method for treating or preventing mucositis of the gastrointestinal tract during cytotoxic or immune-suppressing therapy of claim 27 wherein at least one or more of the following seven amino acids are added to the C terminus Cys moiety from formula II:

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

29. The method for treating or preventing mucositis of the gastrointestinal tract during cytotoxic or immune-suppressing therapy of claim 27 wherein X_1ais Val, X_1bis Gly and X_1cis Ala.

30. The method for treating or preventing mucositis of the gastrointestinal tract during cytotoxic or immune-suppressing therapy of claim 28 wherein X₂is Tyr, and X₃is Arg.

31. The method for treating or preventing mucositis of the gastrointestinal tract during cytotoxic or immune-suppressing therapy of claim 28 wherein the loop peptide is 13 amino acids in length wherein X_1ais Val, X_1bis Gly, X_1cis Ala, and X₄is Gly.

32. A bifunctional compound that acts as a TGFα mimetic, comprising a compound from formula III:

wherein the linker moiety is designed to link the N-terminus of the Loop peptide to a nitrogen atom of the ring C₄H₈N₂and wherein the “loop peptide” comprises at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

33. The bifunctional compound that acts as a TGFα mimetic of claim 32 wherein at least one or more of the following seven amino acids are added to the C terminus Cys moiety from formula II:

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

34. The bifunctional compound that acts as a TGFα mimetic of claim 32 wherein X_1ais Val, X_1bis Gly and X_1cis Ala.

35. The bifunctional compound that acts as a TGFα mimetic of claim 32 wherein the linker group is independently selected from the group consisting of substituted or unsubstituted C_1-6alkyl, substituted or unsubstituted C_2-6alkenyl, substituted or unsubstituted C_1-6alkoxy, xylenyl, wherein the substitutions are selected from the group consisting of oxo, epoxyl, hydroxyl, chloryl, bromyl, fluoryl, and amino.

36. The bifunctional compound that acts as a TGFα mimetic of claim 33 wherein X₂is Tyr, and X₃is Arg.

37. The bifunctional compound that acts as a TGFα mimetic of claim 33 wherein the loop peptide is 13 amino acids in length wherein X_1ais Val, X_1bis Gly, X_1cis Ala, and X₄is Gly.

38. A method for treating inflammatory bowel disease, colitis, and Chron's Disease of the gastrointestinal tract, comprising administering a TGFα polypeptide or a pharmaceutically active loop peptide, or both, wherein the loop peptide comprises at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

39. The method for treating inflammatory bowel disease, colitis, and Chron's Disease of the gastrointestinal tract of claim 38 wherein at least one or more of the following seven amino acids are added to the C terminus Cys moiety from formula II:

—X₄-His-X_1c—X₄ 13 X₅—X₆—X_1c II

40. The method for treating inflammatory bowel disease, colitis, and Chron's Disease of the gastrointestinal tract of claim 38 wherein X_1ais Val, X_1bis Gly and X_1cis Ala.

41. The method for treating inflammatory bowel disease, colitis, and Chron's Disease of the gastrointestinal tract of claim 39 wherein X₂is Tyr, and X₃is Arg.

42. The method for treating inflammatory bowel disease, colitis, and Chron's Disease of the gastrointestinal tract of claim 39 wherein the loop peptide is 13 amino acids in length wherein X_1ais Val, X_1bis Gly, X_1cis Ala, and X₄is Gly.

43. A method for treating an inflammatory reaction of autoimmune diseases, comprising administering a TGFα polypeptide or a pharmaceutically active loop peptide, or both, wherein the loop peptide comprises at least an 11-membered peptide compound from formula I:

N—X_1a-Cys-His-Ser-X_1b—X₂—X_1a—X_1b—X_1a—X₃-Cys COOH I

44. The method for treating an inflammatory reaction of autoimmune diseases of claim 43 wherein the autoimmune diseases are selected from the group consisting of Type II (Juvenile) Diabetes, rheumatoid arthritis, lupus, and multiple sclerosis.

45. The method for treating an inflammatory reaction of autoimmune diseases of claim 43 wherein at least one or more of the following seven amino acids are added to the C terminus Cys moiety from formula II:

—X₄-His-X_1c—X₄—X₅—X₆—X_1c II

46. The method for treating an inflammatory reaction of autoimmune diseases of claim 43 wherein X_1ais Val, X_1bis Gly and X_1cis Ala.

47. The method for treating an inflammatory reaction of autoimmune diseases of claim 45 wherein X₂is Tyr, and X₃is Arg.

48. The method for treating an inflammatory reaction of autoimmune diseases of claim 45 wherein the loop peptide is 13 amino acids in length wherein X_1ais Val, X_1bis Gly, X_1cis Ala, and X₄is Gly.