WO2006122162A2 - Procede et composition pour traiter la mucosite - Google Patents

Procede et composition pour traiter la mucosite Download PDF

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Publication number
WO2006122162A2
WO2006122162A2 PCT/US2006/018014 US2006018014W WO2006122162A2 WO 2006122162 A2 WO2006122162 A2 WO 2006122162A2 US 2006018014 W US2006018014 W US 2006018014W WO 2006122162 A2 WO2006122162 A2 WO 2006122162A2
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WIPO (PCT)
Prior art keywords
mucositis
peptide
protein
amp77
therapy
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PCT/US2006/018014
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English (en)
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WO2006122162A3 (fr
Inventor
F. Gary Toback
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The University Of Chicago
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Publication date
Priority claimed from EP05010217A external-priority patent/EP1595547A3/fr
Application filed by The University Of Chicago filed Critical The University Of Chicago
Priority to EP06770157A priority Critical patent/EP1885390B1/fr
Priority to AT06770157T priority patent/ATE526983T1/de
Priority to JP2008511298A priority patent/JP5058980B2/ja
Priority to US11/913,928 priority patent/US8017576B2/en
Priority to AU2006244080A priority patent/AU2006244080C1/en
Priority to CA2643736A priority patent/CA2643736C/fr
Priority to MX2007013982A priority patent/MX2007013982A/es
Publication of WO2006122162A2 publication Critical patent/WO2006122162A2/fr
Publication of WO2006122162A3 publication Critical patent/WO2006122162A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis

Definitions

  • Antrum Mucosal Protein (also called gastrokine-1) is an 18 kDa protein expressed in mucosal epithelial cells of the gastric antrum. Within stomach cells the protein is found in vesicles together with mucins just under the lumenal cell surface. AMP -18 is secreted onto the stomach lining as a component of the viscoelastic gel that coats the mucosal surface.
  • the native protein and a 21-mer peptide comprising amino acids 77-97 both function as mitogens and motogens for gastrointestinal (GI) epithelial cells.
  • AMP77-97 peptide also acts on the tight junctions (TJs) (FIG.
  • Mucositis is an inflammatory and ulcerative injury of the mouth, throat or GI tract most commonly caused by chemo- or radiation therapy for cancer. This disease has its onset when chemotherapy or radiation disrupts the mucosal surface of the mouth and other portions of the GI tract, affecting both the epithelial layer and the underlying connective tissue. In severe cases, oral mucositis can be extremely painful, preventing the patient from eating, and requiring hospitalization for hydration, narcotics for pain, and/or total parenteral nutrition. Pain resulting from mucositis is so severe that it is often cited by cancer patients as the primary reason for discontinuing treatment.
  • Mucositis can also be life-threatening because oral ulcerations can permit the entry of bacteria into the bloodstream, a situation which can be fatal in a patient already immune- compromised by treatment for cancer.
  • mucositis affects 15-40 percent of patients receiving standard-dose chemotherapy and 76-100 percent of patients receiving higher doses of chemotherapy for bone marrow transplantation (FIG. 7). Mucositis also affects virtually all patients receiving radiation therapy for head and neck cancer, as well as patients receiving radiation along the GI tract.
  • esophagitis or esophageal mucositis
  • Mucositis afflicts over 400,000 patients a year in the US, and the incidence is growing as the need for radiation and chemotherapy treatments grows. This represents a potential annual market of greater than $800 million in the US.
  • KepivanceTM is the known mitogenic protein keratinocyte growth factor (KGF) that must be administered intravenously.
  • KepivanceTM is approved for a single indication which comprises only 4% of the total mucositis population, i.e., treatment of mucositis resulting from pre-conditioning regimens (chemotherapy and radiation) in stem-cell transplant patients.
  • peptide compositions derived from AMP- 18 protein surprisingly reduce mucositis in mammals.
  • peptide compositions, including dosage and administration modes enablingly demonstrate that AMP- 18 protein derived peptides reduce the intensity of mucositis and/or delays the onset of mucositis.
  • dislcosures in the present application and the priority application expressly demonstrate that specific peptides derived from a gastrokine, e.g., AMP- 18, are therapeutically effective to treat, reduce the intensity, delay the onset, and minimize the duration of mucositis in mammals.
  • AMP-18 protein and its derived peptides act as mitogens and motogens.
  • AMP-18 protein includes purified protein and recombinant protein.
  • AMP-18-derived peptides includes AMP-18-derived peptides from human, mice, and pig.
  • AMP77-97 peptides include AMP58-99 (KKTCIVHKMKKEVMPSIQSLDALVKEKKLQGKGPGGPPPKGL), AMP84-97 (KKLQGKGPGGPPPK) , AMP84-101 (KKLQGKGPGGPPPKGLMY), AMP77-101 (LDALVKEKKLQGKGPGGPPPKGLMY), and mouse AMP-derived peptide77-99 (LDTMVKEQKGKGPGGAPPKDLMY).
  • the amino acid positions, e.g., 77-97 refer to the amino acid positions of the mature/processed form of AMP- 18 protein from human or from mouse or pig as indicated.
  • AMP77-97 peptide's effects include mitogenic and motogenic functions.
  • AMP77-97 peptide stimulates healing of injured mucosal surfaces by its actions to facilitate restitution and cell proliferation after injury, and enhance formation of new tight junctions between cells to repair the mucosal barrier.
  • AMP77-97 peptide provides a useful treatment of diseases and abnormal conditions of the GI tract, including oral mucositis which continues to be a significant unmet medical need.
  • AMP77-97 peptide is effective in alleviating mucositis, for example cancer-therapy induced mucositis.
  • AMP 77-99 peptide derived from murine AMP- 18 protein is also suitable to treat mucositis.
  • the AMP77-97 peptide prevented decreases in TJ proteins and epithelial integrity seen after treatment of human colonic Caco2 (C2) cell monolayers with the oxidant monochloramine, which disrupts intercellular junctions (FIG. 2; C 3 D). These cells were exposed to low-calcium medium (3 ⁇ M) for 1 hr to disrupt TJs 5 and then allowed to recover in medium containing AMP77-97 peptide, epidermal growth factor (EGF) or keratinocyte growth factor (KGF-I) which are known epithelial cell mitogens that might facilitate recovery of disrupted TJs. The results in FIG. 3 show that only treatment with AMP77-97 peptide facilitated rapid recovery of TJs between the cells.
  • EGF epidermal growth factor
  • KGF-I keratinocyte growth factor
  • AMP77-97 peptide displays an effect on these epithelial cell TJs not seen with either EGF or KGF.
  • AMP77-97 peptide facilitates the formation of TJs by enhancing translocation of several proteins (protein kinase C-zeta, Par6, Cdc42, ECT2 and Par3) from the cytosol into TJ domains.
  • AMP77-97 peptide When administered subcutaneously to mice, AMP77-97 peptide increased TJ protein components in intestinal, but not kidney or liver tissue. Thus, AMP77-97 peptide, in addition to promoting the repair of mucosal surfaces via cell growth and migration, protects existing TJs in GI epithelial cells and thereby preserves mucosal barrier function and structure. Treatment of C2 cells in culture with AMP77-97 peptide has also been shown to preserve transepithelial electrical resistance, a measure of barrier function which is a marker for mucosal integrity of the GI tract. This effect was accompanied by stabilization of perijunctional actin molecules via the p38 MAP kinase/hsp27/actin pathway, a signal-transduction pathway that is activated by AMP77-97 peptide.
  • compositions including AMP-18 protein and peptides derived from
  • AMP-18 protein are suitable to treat mucositis.
  • a pharmaceutical composition includes a therapuetically effective amount of a peptide derived from AMP- 18 and a pharmaceutically acceptable carrier, wherein the peptide is effective in treating mucositis.
  • a therepeutically effective amount corresponds to about 1-100 mg/kg of body weight.
  • the pharmaceutical composition includes a peptide that has an amino acid sequence selected from LDALVKEKKLQGKGPGGPPPK (AMP77-97), KKTCIVHKMKKEVMPSIQSLDALVKEKKLQGKGPGGPPPKGL (AMPSS-PP), KKLQGKGPGGPPPK (AMP84-97) , KKLQGKGPGGPPPKGLMY(AMP84-101), LDALVKEKKLQGKGPGGPPPKGLMY(AMP77-101), and mouse AMP-derived peptide LDTMVKEQKGKGPGGAPPKDLMY(AMP77-99) .
  • the mucositis includes oral mucositis. Mucositis is induced by a therapy to treat cancer that includes radiation therapy, chemo therapy or a combination of chemo and radiation therapies.
  • the pharmaceutical composition can be administered parenterally. Suitable modes of administration includes oral administration, intravenous administration, subcutaneous administration, topical administration, transdermal administration, intraperitoneal administration, transmucosal administration and nasal administration. D0016] A method to reduce or delay the onset of mucositis includes the steps of:
  • a method to alleviate cancer-therapy induced mucositis includes:
  • a method to repair injuries to the gastric mucosa includes:
  • a modified peptide may be produced by the following method:
  • a pharmaceutical composition includes at least one growth stimulating peptide and a pharmaceutically acceptable carrier.
  • a method to stimulate growth of epithelial cells in the gastrointestinal tract of mammals includes:
  • a method to stimulate migration of epithelial cells after injury to the gastrointestinal tract of mammals includes:
  • a method for cell protection of damaged epithelial cells and the tight junctions between them in the gastrointestinal tract of mammals includes:
  • the damaged cells may form an ulcer.
  • a method to prevent or reduce in a subject in need thereof recurrences of gastric and duodenal infections, inflammation and ulceration caused by H. pylori includes the steps of:
  • agent includes nucleic acids, proteins, protein fragments, peptides, synthetic peptides, peptidomimetics, analogs thereof, small molecules, inhibitors, and any chemical, organic or bioorganic molecule capable of affecting protein-protein interaction or a cellular process.
  • AMP-18 derived peptides means peptides, modified peptide sequences with amino acid substitutions or amino acid analogs or amino acid deletions compared to a corresponding region in AMP-18, and peptidomimetics that correspond to a particular region in AMP-18 protein.
  • the AMP 18-derived peptides can range from about 5-50 amino acids in length or about 5-20 amino acids in length or about 5-10 amino acids in length.
  • the AMP-18 -derived peptides can also be modified to affect their lipophilicity to enhance peptide delivery to mucosal epithelial cells of the mouth and GI tract.
  • the peptides can be synthesized ("synthetic peptides”) or can also be produced through recombinant techniques (“recombinant peptide”). These peptides can also be engineered to increase their in vivo stability without significantly affecting their efficacy to promote cell growth and migration, enhance formation and assembly of tight junction proteins, and mediate cell protection.
  • a "peptide derivative” is used to mean a molecule having an amino acid sequence of a region of AMP-18 protein or of an AMP-18 protein homolog, but additionally having at least one chemical modification of one or more of its amino acid side groups, ⁇ -carbon atoms, terminal amino group, or terminal carboxylic acid group.
  • a chemical modification includes adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications at amino acid side groups include acylation of lysine, ⁇ -amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine.
  • Modifications of the terminal amino include the des-amino, N-Io was alkyl, N-di-lower alkyl, and N-acyl modifications.
  • Modifications of the terminal carboxy group include the amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications.
  • a lower alkyl is a C1-C4 alkyl.
  • one or more side groups, or terminal groups may be protected by protective groups known to the ordinarily-skilled protein chemist.
  • the ⁇ - carbon of an amino acid may be mono-or di-methylated.
  • FIG. 1 is a shematic illustration of tight junctions and their associated proteins.
  • FIG. 2 shows the effects of AMP77-97 peptide on occludin visualized by confocal microscopy in C2 cells under control conditions and following oxidant injury. Occludin immunoreactivity in a control cell monolayer (panel A) formed a uniform band outlining the cell junctions that was more intense than in the cytoplasm. When cells were exposed to AMP77-97 peptide for 18 hr (panel B), occludin appeared to be relatively more abundant in the TJs and less in the cytoplasm than in control cells.
  • FIG. 3 shows that AMP77-97 peptide, but not epidermal growth (EGF) or keratinocyte growth factor (KGF) facilitates formation of new tight junctions in human colonic epithelial cells after their disruption by exposure to low-calcium medium.
  • EGF epidermal growth
  • KGF keratinocyte growth factor
  • a growth factor at a known mitogenic concentration was added to the medium, and 2 hours later its effect on tight junction formation was assessed by confocal microscopy.
  • Vehicle, AMP peptide (8 ⁇ g/ml), KGF-I (100 ng/ml), and EGF (100 ng/ml) were compared.
  • Arrows indicate persistent separation of cells with TJ disruption and apparent loss of ZO-I in cultures exposed to vehicle, KGF-I, or EGF.
  • AMP peptide facilitated recovery of ZO-I and the formation of intact tight junctions.
  • FIG. 4 shows the effects of AMP77-97 peptide on radiation-induced tongue damage in the mouse.
  • AMP77-97 peptide 25 mg/kg, s.c.
  • Tissue from control irradiated mice treated with vehicle (saline) show substantial epithelial and connective tissue cell loss in both compartments compared to normal mouse tongue.
  • tissue from animals treated with AMP77-97 peptide exhibits a thickened hyperkeratotic epithelial surface and a largely intact connective tissue compartment.
  • FIG. 5 shows scoring system for mucositis in the hamster cheek pouch.
  • FIG. 6 shows the effects of AMP77-97 peptide on the number of animal days with mucositis scores of 3 or greater. The level of clinically significant mucositis was defined by presentation with open ulcers on the buccal mucosa after radiation (a score of 3 or greater). The total number of days in which a hamster exhibited an elevated score was summed and expressed as a percentage of the total number of days scored. The asterisk denotes that treatment with AMP- 18 peptide (40 mg/kg) resulted in a statistically significant lower mucositis score when compared to the vehicle (PBS) using a Chi-squared test (PO.001).
  • FIG. 7 shows causes of mucositis during cancer therapy.
  • CRC colorectal cancer
  • NHL non-Hodgkins lymphoma
  • NSCLC non-small-cell lung cancer.
  • Peptide compositions derived from AMP-18 protein surprisingly reduce mucositis in mammals.
  • the present disclosure and the priority application provide data to enablingly demonstrate that specific peptides derived from a gastrokine, e.g., AMP-18, are therapeutically effective to treat, reduce the intensity, delay the onset, and minimize the duration of mucositis in mammals.
  • peptide compositions, including dosage and administration modes enablingly demonstrate that AMP- 18 protein derived peptides reduce the intensity of mucositis and/or delays the onset of mucositis.
  • AMP77-97 peptide acts in cell culture as a mitogen and a motogen, and appears to protect against injury to tight junctions between mucosal epithelial cells (FIG. 1).
  • AMP77-97 peptide is a therapeutic agent in oral mucositis.
  • administration of AMP77- 97 peptide reduced the duration and extent of ulcerative lesions on the tongue and buccal mucosa thereby significantly accelerating recovery.
  • AMP77-97 peptide is useful in treating a wide range of diseases of the GI tract such as esophagitis, peptic ulcer disease, and inflammatory bowel disease (ulcerative colitis, Crohn's disease) (IBD).
  • IBD inflammatory colitis, Crohn's disease
  • inflammatory reactions compromise barrier functions such as vectorial transport of nutrients, water and electrolytes, and allow bacterial toxins and pro-inflammatory molecules to pass from the gut lumen into the intestinal submucosa, causing considerable morbidity and mortality (patient population ⁇ 1 million in the US).
  • Current therapies for these latter diseases include steroids and other anti-inflammatory agents which target unknown immune causes of disease.
  • AMP77-97 peptide has the potential to be a therapy for preventing injury or speeding recovery from multiple types of GI insults as evidenced by AMP77-97 peptide delaying appearance of blood in the stool and reducing weight loss in mice in which acute colitis was induced by dextran sulfate sodium.
  • FIG. 2 shows the effects of AMP77-97 peptide on occludin as visualized by confocal microscopy in C2 cells under control conditions and following oxidant injury.
  • Occludin immunoreactivity in a control cell monolayer (panel A) formed a uniform band outlining the cell junctions that was more intense than in the cytoplasm.
  • panel B When cells were exposed to AMP77-97 peptide for 18 hrs (panel B), occludin appeared to be relatively more abundant in the TJs and less in the cytoplasm than in control cells.
  • KKTCIVHKMKKEVMPSIQSLDALVKEKKLQGKGPGGPPPKGL AMP58-99
  • KKLQGKGPGGPPPK AMP84-97
  • LDALVKEKKLQGKGPGGPPPKGLMY(AMP77-101) mouse AMP-derived peptide LDTMVKEQKGKGPGGAPPKDLMY(AMP77-99) also suitable for use in treating mucositis based on their mitogenic and motogenic functions. Effects of these peptides can be tested using the mouse biomodels disclosed herein.
  • Derivatives and analogs of AMP-18 peptides may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described herein.
  • Derivatives or analogs of the nucleic acids or proteins of the invention include, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel et al., (1993).
  • Peptides and other compositions disclosed herein can be administered via any suitable means.
  • the peptide compositions may be diluted in saline or any suitable buffer and administered directly intravenously,subcutaneously, topically, and/or intraperitoneally.
  • compositions disclosed herein may be via any route known to be effective by the physician of ordinary skill. Peripheral, parenteral administrations, and topical administrations are suitable. Dosage of the AMP-18-derived peptides depends on the efficacy of the peptide, stability of the peptide in vivo, mode of administration, body weight, age of the patient and other factors that are commonly considered by a skilled artisan. For example, dosage of a AMP-18-derived peptide drug can range from about 0.1-10.0 mg /kg body weight or from about 10-20 mg/kg body weight or from about 0.5-50 mg/kg body weight or from about 10.0-70.0 mg/kg body weight.
  • Parenteral administration is commonly understood in the medical literature as the injection of a dosage form into the body by a sterile syringe.
  • Peripheral parenteral routes include intravenous, intramuscular, subcutaneous, and intraperitoneal routes of administration. Intravenous, intramuscular, and subcutaneous routes of administration of the compositions disclosed herein are suitable.
  • the peptides disclosed herein can be combined with phosphate buffered saline (PBS) or any suitable pyrogen-free pharmaceutical grade buffer that meets FDA standard for human subject administration.
  • PBS phosphate buffered saline
  • pharmaceutically acceptable carrier includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives and the like, as suited to the particular dosage form desired.
  • Remington (2000) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.
  • Solutions or suspensions of the compositions described herein can also include a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; chelating agents, such as EDTA; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose.
  • a parenteral preparation of the compositions can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic, in accordance with standard practice in the field.
  • the compositions disclosed herein can be stored as a lyophilized sterile powder in vials prior to reconstitution and the unreconstituted product may be stored at -20°C.
  • Example 1 Effect of AMP77-97 peptide in mouse model of mucositis 0042]
  • the capacity of the AMP77-97 peptide to induce epithelial thickening of oral mucosal surfaces in mice and hamsters was studied to determine if these animals could be used to evaluate the potential of AMP77-97 peptide in the treatment of oral mucositis.
  • mouse AMP77-97 peptide 25 mg/kg was administered once daily subcutaneously before and after radiation (days -5 to 10; radiation on day 0). Histological analysis of the tongue revealed that administration of AMP77-97 peptide reduced signs of mucositis in both the epithelial cell layer (FIG. 4, dark layer in slides under "Epithelium") and in connective tissue, relative to saline-treated controls (FIG. 4, note cellularity of "Connective tissue”). The evaluation was performed on day 10 when mucositis injury is at its peak. This effect on connective tissue supported that AMP77-97 peptide has beneficial effects on multiple cell and tissue types damaged by radiation. There was no evidence of any toxicity associated with AMP77-97 peptide treatment.
  • AMP77-97 peptide was tested in a hamster mucositis model in which radiation is used to injure the mucosal surface of the cheek pouch.
  • AMP77-97 peptide (0-40 mg/kg) was administered by subcutaneous injection once daily starting five days before radiation (day -5) and continued until day 15, excluding day of radiation (day 0). Scoring in this model uses the same objective assessments of damage as the World Health Organization (WHO) scoring system used clinically.
  • WHO World Health Organization
  • peptide-treated animals showed a significant reduction in the number of days with a mucositis score of 3 or higher (mucosal ulceration) compared to vehicle-treated animals (PO.001), thereby providing strong evidence of a protective effect.
  • Hamsters treated with the vehicle (PBS) in the control group had a score of 3 or higher on 32.1% of the animal days evaluated, whereas the group treated with AMP77-97 peptide had a significant reduction with only 14.1% of animal days in this group having a score of 3 or higher (PO.001) (FIG. 6).
  • the data disclosed herein points to multiple therapeutic targets for AMP-18/AMP peptide. These include treatment of IBD by: (a) preventing or decreasing the frequency and intensity of acute exacerbations of this episodic disease by the AMP77-97 peptide's cell protective effect, and (b) speeding recovery of the colonic mucosal epithelium after_an attack of disease occurs, i.e., a benefit inferred from the mitogenic and motogenic (wound healing) effects observed in cell culture and murine models of colitis.
  • the cell protective, mitogenic and motogenic effects of AMP77-97 peptide also result in a therapeutic role in cancer-therapy induced mucositis of the GI tract as often occurs during chemotherapy and/or radiation therapy.
  • Mucositis occurs in this setting because the therapeutic protocol is designed to destroy proliferating cancer cells, but may also damage rapidly growing cells that line the mouth, throat, or GI mucosa at any point along its entire length.
  • Injury and/or destruction of the protective mucosal epithelium can result in life-threatening infection which puts the patient at risk for gut-derived sepsis and death.
  • Evidence has been obtained in mice and cell culture which suggests therapeutic benefits of AMP77-97 peptide in the treatment of gut-derived sepsis (cell protection), gastritis and gastric ulcers (cell protection, mitogenesis, restitution), and infection with H. pylori (growth inhibition of the organism) in humans.
  • AMP77-97 peptide on renal epithelial cells (MDCK line) in culture disclosed herein also predict therapeutic role for the peptide in patients with acute renal failure.
  • the cell protective, mitogenic, and motogenic effects of AMP77-97 peptide and recombinant human AMP- 18 (rhAMP-18) offer multiple therapeutic strategies to prevent and/or limit disruption of epithelial barrier function and structure, and also speed regeneration after mucosal injury in gut and kidney.
  • AMP- 18 The synthesis of AMP- 18 is confined to lumenal mucosal lining epithelial cells of the gastric antrum of humans and other mammals. Inside cells the protein is co-localized with mucins in secretion granules, and appears to be secreted into the mucus overlying the apical plasma membrane. Recombinant human AMP-18 prepared in E. coli exerts its mitogenic effect at a concentration an order of magnitude lower than growth-promoting synthetic peptides derived from the center of the mature protein.
  • Peptide 77-97 the most potent mitogenic AMP-derived peptide identified to date, is amino acid sequence-specific, and appears to be cell-type specific as it does not stimulate growth of fibroblasts or HeLa cells. Mitogenesis by specific AMP77-97 peptides appears to be mediated by a cell surface receptor because certain peptides that are not active mitogens can competitively inhibit, in a concentration-dependent manner, the growth-stimulating effects of peptide 58- 99 and antrum cell extracts. AMP- 18 and its derived peptides exhibit diverse effects on stomach and intestinal epithelial cells making it likely they play a critical role in repair after gastric mucosal injury.
  • AMP-18 likely plays a role in physiological and pathological processes such as wound healing in the gastric mucosal epithelium in vivo as may occur in gastritis secondary to NSAID drugs, other pharmaceutical agents and alcohol, ulcer disease, and the consequences of H. pylori infection and inflammation.
  • AMP-18 Localization of AMP-18:
  • the antisera to AMP-18 have proven to be excellent histochemical probes, reacting strongly with sections of the mouse gastric antrum region but not with tissue from the gastric fundus, duodenum or intestine, confirming the results of the immunoblots.
  • the preirnmune sera give negligible reactions even at much higher concentration.
  • the AMP-18 protein appears to be concentrated in mucosal epithelial cells lining the stomach lumen, although lesser signals in cells deeper in the tissue and along the upper crypt regions suggest that cells may begin to express the protein as they migrate toward the lumenal layer.
  • a function for AMP-18 is that it is a growth factor at least partly responsible for the maintenance of a functional mucosal epithelium in the pyloric antrum and possibly elsewhere in the stomach.
  • stomach epithelial cell lines were not immediately available, but kidney epithelial cell systems (Kartha et al., 1992; Aithal et al., 1994; Lieske et al., 1994) were used.
  • a fractionated antrum mucosal cell extract was used for these experiments.
  • the gel was cut into 2-mm slices and each slice was extracted with 3% acetonitrile in phosphate-buffered saline containing 1% BSA. The extract supernatants were assayed for mitogenic activity. The results indicated that one slice containing protein in the 16-19 kDa range possessed growth-promoting activity. Significantly, this growth response was blocked by the immune but not the pre-immune sera. Taken together with the relatively low sedimentation rate of the protein, these findings provide additional evidence to support the conclusion that AMP-18 is an epithelial cell mitogen and that it functions as a monomer or possibly a homotypic dimer. It also implies that the structure of the protein is such that it can readily reacquire a native conformation after the denaturing conditions of SDS-gel electrophoresis.
  • EGF 50 ng/ml
  • untreated mouse antrum extract (10 ⁇ g/ml) or heated, dialyzed pig extract (10 ⁇ g/ml) exhibited additive stimulation of mitogenesis; up to 74% increase in cell number above the quiescent level; the greatest stimulation observed so far for any factor using the BSC-I cell assay.
  • AMP-18 and EGF initiate proliferation by acting on different cell surface receptors. It also implies that AMP- 18 growth factor activity might normally collaborate with other autocrine and paracrine factors in the maintenance or restitution of the epithelium.
  • AMP- 18 is secreted at and possibly acts upon the apical face (i.e., stomach lumenal face) of the epithelial cell layer while other factors (for which EGF may serve as an example) act from the basal surface.
  • the human AMP- 18 sequence lacking the 20 amino acid signal peptide and containing a His6-tag was also expressed in bacteria.
  • Pichiapastoris Smong the simple eukaryotes, the budding yeast P. pastoris is useful as an expression system for production and secretion of functional recombinant proteins (Romanos et al, 1992; Cregg et al, 1993). In this system, secretion of the foreign protein may utilize either its own signal peptide or the highly compatible yeast mating-type alpha signal. This organism will correctly process and secrete and at least partially modify the AMP- 18 protein. Vectors for constitutive and regulated expression of foreign genes are developed in Pichia (Sears et al, 1998).
  • these vectors contain either the high expression constitutive glyceraldehyde-3 -phosphate dehydrogenase (GAP) or the methanol-regulated alcohol oxidase promoter (AOXl).
  • GAP glyceraldehyde-3 -phosphate dehydrogenase
  • AOXl methanol-regulated alcohol oxidase promoter
  • Bac ⁇ lovirus/insect cells IAn alternative, frequently successful, non-mammalian eukaryotic expression system is that using recombinant Baculovirus, such as Autographa californica, in an insect cell culture system. As with Pichia, a large repertoire of convenient vectors are available in this system, containing both glutathione S-transferase (GST)-and His6-tags (Pharmingen). Transfections are carried out into Spodoptera frugiperda (Sf) cells; these cells can be slowly adapted to protein-free medium to favor the purification of secreted proteins.
  • GST glutathione S-transferase
  • His6-tags Pharmingen
  • an endogenous signal peptide does not function in these cells, secretion of foreign proteins can also be forced using vectors containing the viral gp67 secretion signal upstream of the cloning site.
  • Recombinant proteins can be expressed at levels ranging from 0.1-50% total cell protein. Some protein modifications may be more favored in this insect cell system relative to yeast, but still may not duplicate the mammalian system. It appears that the insect expression system would be somewhat more onerous than Pichia, and not entirely substitute for expression in mammalian cells.
  • the human AMP- 18 sequence lacking the 20 amino acid signal peptide and containing a His6-tag was expressed in Baculovirus. ,00059] Mammalian cells.
  • pcDNA3 contains a polylinker cloning site flanked by the strong constitutive cytomegalovirus (CMV) promoter and a SV40 polyA signal (Invitrogen). Laboratory experience is that 60- 90% transient transfection levels can be achieved. To this end, PCR amplification of the human preAMP cDNA clone is performed with oligonucleotides that contain the initiation codon and native ribosome binding site (Kozak sequence) as well as suitable restriction enzyme linkers for correct orientation into pcDNA3.
  • CMV cytomegalovirus
  • amphipathic helices the conserved cystine (C) residues and the basic amino acids doublets, which may be cleavage sites, are attractive targets.
  • the mitogenesis assay is routine and replicable, and enables "mutants” to be characterized as fast as they are constructed. Dominant negative (or positive) "mutants" are as significant as mutations exhibiting simple loss of function, because these imply interactions with other factors including possible cell receptors.
  • Biochemical and immunoaffinity fractionation of expressed and native gastrokine proteins In the case of some of the expressed forms of AMP-18, the recombinant protein contains peptide tags that will permit the rapid purification of soluble protein. The presence of these tags, if they do not severely interfere with the protein's normal functions, also permit analysis of interactions with other relevant macromolecules. His6-tags permit purification by binding the recombinant proteins to Ni-NTA resin beads (Janknecht et ah, 1991 ; Ni-NTA resin from Qiagen).
  • the tagged protein is bound with greater affinity than most antigen-antibody complexes and can be washed rigorously before the Nj 2+ -histidine chelation complex is disrupted by excess imidazole to release the purified protein.
  • GST-tagged recombinant proteins are purified on glutathione-agarose, washed and then eluted with reduced glutathione (Smith and Johnson, 1988). As with all the proposed expression systems, each protein preparation may be tested at the earliest possible stage for its growth factor activity. )0064] Conventional fractionation procedures are used to achieve the desired purity, particularly in the case of the isolation of the natural protein from tissue.
  • Pig antrum mucosa is a preferred starting point for the latter, using initial centrifugation and heat- treatment protocol, followed by a size-exclusion column: BioGel P60 is suitable, given the evidence that the 18 kDa protein exists, most probably as a monomer in the extracts.
  • the eluant is loaded on an immunoaffinity matrix created by crosslinking anti-AMP antibodies purified on HiTrap Protein A to CNBr-activated Sepharose 4B (Pharmacia). Further modification of the immnoaffinity matrix may be helpful, either by extension of the linker to the matrix, which has proven useful in the past (Aithal et ah, 1994), or by crosslinking the antibody to immobilized protein-A.
  • active protein can be recovered by SDS- gel elution, active protein may also be recovered from the antigen-antibody complexes. Further fractionation could be achieved by C8 reversed-phase high-performance liquid chromatography (HPLC) column. A final step is the use of the SDS-gel elution technique with confirmation of identity by N-terminal sequencing. In all of these steps the immunodetectable AMP- 18 and the growth factor activity should fractionate together.
  • AMP-18 related synthetic peptides AMP-18 may be precursor to one or several bioactive peptides. Synthetic peptides provide a convenient avenue to explore the function of a protein; peptides may mimic aspects of the function or antagonize them. If a peptide either duplicates or inhibits the protein's activity, then it suggests the identity of functional domains of the intact protein, and also provides the possibility of synthesizing specifically tagged probes to explore protein-cell interactions.
  • D-amino acids in the peptides (in normal or reverse order) may stabilize them to degradation while permitting retention of biological function.
  • tags that facilitate studies of the nature, tissue distribution and number of cellular receptors. Such tags include His-6 biotin or iodinated tyrosine residues appended to the peptide sequence (several of the bioactive peptides have a naturally occurring tyrosine at the C-terminus).
  • Non-transformed monkey kidney epithelial cell line BSC-I and other epithelial cell lines were used to assess effects on growth. In general, conditions were chosen for each line such that cells are grown to confluence in plastic dishes in supplemented growth medium with minimal calf (or fetal) serum for growth (Lieske et ah, 1991); BSC-I cells become confluent at 10 6 /60-mm dish with 1% calf serum. At the start of the growth assay the medium on the confluent culture was aspirated and replaced with fresh medium with minimal serum to maintain viability (0.01% for BSC-I) cells.
  • AMP-18 preparations were added to the culture medium and 4 days later the cell monolayer was rinsed, detached with trypsin, and the cells were counted using a hemocytometer. Determination of the capacity of AMP-18 to initiate DNA synthesis was measured by the incorporation of [ 3 H]thymidine (Toback, 1980); to confirm the DNA synthesis assay, autoradiograms of leveled cells were counted (Kartha and Toback, 1985).
  • the protein AMP- 18 is expressed in the antrum mucosa and to a lesser extent in the adjacent corpus mucosa.
  • both antrum extracts and the active synthetic peptides stimulate proliferation of most simple epithelial cell lines.
  • Many transformed stomach lines derived from human cancer patients are available from various sources, but most of these do not exhibit growth control. For example, a gastric AGS adenocarcinoma cell subline from Dr.
  • Duane Smoot (Howard University College of Medicine) showed a greater degree of contact inhibition, and responded well to AMP-18 and its derived peptides. These cells do not naturally synthesize AMP-18. Similar responses were observed with the non- transformed rat IEC intestinal epithelial cells (provided by Dr. Mark Musch, Dept. Medicine, University of Chicago); the latter show excellent epithelial cell characteristics in culture (Quaroni et al, 1979; Digass et al, 1998).
  • AMP-derived peptide in phosphate-buffered saline (PBS) was administered at 0 to 80 mg/kg by daily subcutaneous injection. starting five days before (day -5) radiation, and 15 days afterwards (day +15)
  • a control group consisted of irradiated animals dosed with vehicle (PBS) on days -5 to +15. Neither AMP77-97 peptide or vehicle was given on the day radiation was administered.
  • different amounts of AMP peptide were administered before and after administration of radiation to define an optimal dosing schedule for AMP- 18 -derived peptides in this acute radiation induced model of mucositis in the hamster cheek pouch.
  • Mucositis Evaluation The progression of mucositis was monitored daily for both models. Every other day starting on day 6 postirradiation, animals were anesthetized using inhalation anesthesia, and the left buccal pouch was everted and photographed. At the conclusion of the study's clinical phase, film was developed, and resulting photos were randomly numbered and then scored in blinded fashion by two observers. A 0-5 scoring system was used that applied the following numerical score to buccal lesions: 0, normal mucosa; 1, erythema and vasodilation; 2, severe erythema and vasodilation, with erosion of superficial aspects of mucosa leaving denuded areas with decreased stippling of mucosa.
  • Amino acids 77-97 (LDALVKEKKLQGKGPGGPPPK) are highlighted by double underlines. The amino acid positions correspond to the mature protein. The first twenty amino acids correspond to the signal peptide and is shown by a single underline.

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Abstract

La présente invention concerne des procédés et des compositions à base de peptides dérivés d'AMP-18, qui font la preuve de façon validante que les compositions à base de peptides dérivés d'AMP-18 atténuent de façon surprenante la mucosite chez les mammifères. L'invention concerne notamment des procédés et des compositions qui traitent la mucosite, qui retardent la survenue ou la durée de la mucosite, y-compris les mucosites induites par les thérapies anticancéreuses. Les peptides AMP 77-97 dérivés de la protéine AMP-18 humaine atténuent les mucosites induites par les thérapies anticancéreuses.
PCT/US2006/018014 2005-05-10 2006-05-09 Procede et composition pour traiter la mucosite WO2006122162A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
EP06770157A EP1885390B1 (fr) 2005-05-10 2006-05-09 Utilisation des derivees du facteur de croissance amp-18 pour le traitement de la stomatitis
AT06770157T ATE526983T1 (de) 2005-05-10 2006-05-09 Verwendung von peptiden aus dem wachstumsfaktors amp-18 zur behandlung von stomatitis
JP2008511298A JP5058980B2 (ja) 2005-05-10 2006-05-09 粘膜炎を処置するための増殖因子ａｍｐ−１８に由来するペプチドの使用
US11/913,928 US8017576B2 (en) 2005-05-10 2006-05-09 Methods and compositions to treat mucositis
AU2006244080A AU2006244080C1 (en) 2005-05-10 2006-05-09 Use of peptides derived from the growth factor AMP-18 for the treatment of mucositis
CA2643736A CA2643736C (fr) 2005-05-10 2006-05-09 Procede et composition pour traiter la mucosite
MX2007013982A MX2007013982A (es) 2005-05-10 2006-05-09 Uso de peptidos derivados del factor de crecimiento amp-18 para el tratamiento de la mucositis.

Applications Claiming Priority (4)

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EP05010217.7 2005-05-10
EP05010217A EP1595547A3 (fr) 2004-05-11 2005-05-10 Utilisation du facteur de croissance AMP pour le traitement des lésions gastrointestinales et de la insuffisance renale aigue
US79017706P 2006-04-07 2006-04-07
US60/790,177 2006-04-07

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110158983A1 (en) * 2008-03-05 2011-06-30 Newell Bascomb Compositions and methods for mucositis and oncology therapies
WO2013030512A1 (fr) 2011-08-30 2013-03-07 Nutrialys Medical Nutrition Sa Utilisation de compositions a faible teneur en polyamines dans la prevention ou le traitement des effets indesirables lies a un traitement anti-cancereux
EP2709619A2 (fr) * 2011-05-16 2014-03-26 Cellceutix Corporation Composés utilisés dans le traitement de la mucosite
US9889120B2 (en) 2016-01-14 2018-02-13 Vicus Therapeutics, Llc Combination drug therapies for cancer and methods of making and using them
US10166232B2 (en) 2006-12-29 2019-01-01 Innovation Pharmaceuticals Inc. Arylamide compounds and compositions and uses thereof
EP3628314A4 (fr) * 2017-04-19 2020-10-28 Otsuka Pharmaceutical Factory, Inc. Agent anti-inflammatoire
US11771694B2 (en) 2020-06-05 2023-10-03 Innovation Pharmaceuticals Inc. Arylamide compounds for treatment and prevention of viral infections

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WO2002078640A2 (fr) 2001-03-29 2002-10-10 University Of Chicago Regulation de la croissance et de la reparation de tissus gastro-intestinaux par des gastrokines et des inhibiteurs
US20050031582A1 (en) 2001-03-29 2005-02-10 Toback F. Gary Control of growth and repair of gastro-intestinal tissues by gastrokines and inhibitors

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2002078640A2 (fr) 2001-03-29 2002-10-10 University Of Chicago Regulation de la croissance et de la reparation de tissus gastro-intestinaux par des gastrokines et des inhibiteurs
US20050031582A1 (en) 2001-03-29 2005-02-10 Toback F. Gary Control of growth and repair of gastro-intestinal tissues by gastrokines and inhibitors

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10166232B2 (en) 2006-12-29 2019-01-01 Innovation Pharmaceuticals Inc. Arylamide compounds and compositions and uses thereof
US20110158983A1 (en) * 2008-03-05 2011-06-30 Newell Bascomb Compositions and methods for mucositis and oncology therapies
EP2709619A4 (fr) * 2011-05-16 2015-04-22 Cellceutix Corp Composés utilisés dans le traitement de la mucosite
EP2709619A2 (fr) * 2011-05-16 2014-03-26 Cellceutix Corporation Composés utilisés dans le traitement de la mucosite
US9155738B2 (en) 2011-05-16 2015-10-13 Cellceutix Corporation Compounds for use in treatment of mucostitis
US9457027B2 (en) 2011-05-16 2016-10-04 Cellceutix Corporation Compounds for use in treatment of mucostis
RU2606128C2 (ru) * 2011-05-16 2017-01-10 Селлсьютикс Корпорейшн Соединения для применения в лечении мукозита
US9795575B2 (en) 2011-05-16 2017-10-24 Innovation Pharmaceuticals Inc. Compounds for use in treatment of mucositis
US10206894B2 (en) 2011-05-16 2019-02-19 Innovation Pharmaceuticals Inc. Compounds for use in treatment of mucositis
US10603294B2 (en) 2011-05-16 2020-03-31 Innovation Pharmaceuticals Inc. Compounds for use in treatment of mucositis
WO2013030512A1 (fr) 2011-08-30 2013-03-07 Nutrialys Medical Nutrition Sa Utilisation de compositions a faible teneur en polyamines dans la prevention ou le traitement des effets indesirables lies a un traitement anti-cancereux
US9889120B2 (en) 2016-01-14 2018-02-13 Vicus Therapeutics, Llc Combination drug therapies for cancer and methods of making and using them
EP3628314A4 (fr) * 2017-04-19 2020-10-28 Otsuka Pharmaceutical Factory, Inc. Agent anti-inflammatoire
US11771694B2 (en) 2020-06-05 2023-10-03 Innovation Pharmaceuticals Inc. Arylamide compounds for treatment and prevention of viral infections

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