WO1985001284A1 - Synthetic peptides with egf like activity and pharmaceutical compositions and methods of use - Google Patents
Synthetic peptides with egf like activity and pharmaceutical compositions and methods of use Download PDFInfo
- Publication number
- WO1985001284A1 WO1985001284A1 PCT/US1984/001459 US8401459W WO8501284A1 WO 1985001284 A1 WO1985001284 A1 WO 1985001284A1 US 8401459 W US8401459 W US 8401459W WO 8501284 A1 WO8501284 A1 WO 8501284A1
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- cys
- ser
- polypeptide
- egf
- tyr
- Prior art date
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- 238000000034 method Methods 0.000 title claims description 10
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to polypeptides havi epidermal growth factor (EGF)-li e activity, as well as to pharm ceutical compositions containing same as active ingredient a pharmaceutical processes using same.
- the peptide fractions an compositions according to the present invention activate EGF-sen sitive kinase and induce autophoshorylation of the EGF receptor and other endogenous membrane proteins. They stimulate thymidin incorporation into human foreskin fibroblasts and thus they hav effects like those of EGF, although of lesser potency.
- Epidermal growth factor (EGF) isolated from the sub maxillary glands of male mice is a single-chain polypeptide of 5 amino acids with three disulfide bonds which define three loope regions from residues 1-20, 14-31 and 32-53.
- EGF is a potent stimulator of cellular proliferation an inhibitor of gastric acid secretion. It binds to a diffusel distributed mobile membrane receptor which was identified to be 170,000 daltons glycoprotein. Upon EGF binding, the recepto cluster, mainly over coated regions of the plasma membrane, an the EGF-receptor complex become internalized by an energy depen dent process. Subsequently, the hormone and presumably also th receptor are degraded by lysosomal enzymes.
- EGF induces rapid reponse such as the uptake of ions and metabo lites, enhanced phosphorylation of various membrane protein including the autophosphorylation of the EGF-receptor itself; an induces changes in cell morphology and induces the reorganizatio of cyto ⁇ keletal elements. Delayed effects of EGF include th
- EGF and also human EGF, denoted urogastrone inhibits secretion of gastric acid i ⁇ vivo.
- a linear polypeptide which exhibits EGF activity.
- the polypeptide contains 10 amino acids out of the 53 amino acid sequence of the natural EGF polypeptide, namely that fragment between Cys (20) and Cys (31) of the EGF molecule, and contains the main receptor binding site of EGF.
- the affinity of the peptide according to the present invention is lower by a factor of about " 10,000 than the affinity of EGF towards the receptor molecule. It activates EGF sensitive kinase and exhibits also the other physiological effects of natural EGF, at a lower level of potency.
- the amino acid sequence between Cys (20) and Cys (31) of mouse EGF fulfills the structural requirements for occupancy of EGF receptors of the human epidermoid carcinoma cell line A-431 and human foreskin fibroblasts, activates EGF sensitive kinase, induces DNA synthesis and stimulates the clustering and internalization EGF receptors.
- polypeptide of the present invention may have an
- OMPI number of additional peptides on either side of the critic sequence although, for practical reasons, the total polypepti should not exceed about 25 peptide units.
- the present invention al3o comprehends the correspondi sequence between Cys (20) and Cys (31) of human EGF (ur gastrone) .
- Fig. 1 shows inhibition of the binding 1 nM I-E to human foreskin fibroblasts (HFF) by increasing concentration the following synthetic peptides: peptide 14 to 31 (•-•), peptid
- Fig. 2 shows inhibition of the binding of 125i-EGF t polyclonal rabbit anti-EGF antibodies by synthetic peptides
- Nonspecific binding wa determined with a 1500 fold excess of cold EGF and subtracte prior to calculations. Each value is rhe mean of duplicate deter minations.
- Fig. 3 shows enhancement of membrane protein phosphory lation by either EGF or synthetic peptides.
- the figure is auto radiography of NaDodSO /5-15% polyacrylamide gradient gel elec trophoretic analysis of shed membrane vesicle preparation using A
- Fig. 4 esablishes that synthetic peptides induce thy midine incorporation in quiescent human foreskin fibroblasts
- HFF by synthetic peptides 1 to 20 (o-o), 14 to 31 (•-•), 32 to 4 (____-_ ⁇ ), and 43 to 51 ( ⁇ -_i_) is shown.
- the dashed line indicates th level of thymidine incorporation achieved by 5 uM EGF in thi experiment. Cells were incubated at 37 ⁇ C for 18 hours with eithe peptides of EGF prior to a 4 hour pulse with (methyl- H) thy midine and determination of acid precipitable radioactivity.
- Fig. 5 shows induction of EGF receptor clustering in A 431 cells by synthetic peptides.
- the fluorescence micrographs o this frame show A-431 cells which were labelled with rhodamin anti-EGF-receptor monoclonal antibody (TL5) and subsequently wit buffer (A),EGF (10 ⁇ M, D),peptides 6 to 24 (240 ⁇ M, B), peptide 13 to 24 (270 ⁇ M, C), peptides 14 to 31 (480 ⁇ M, F), or peptide 20 to 31 (190 ⁇ M, E).
- buffer A
- EGF 10 ⁇ M, D
- peptides 6 to 24 240 ⁇ M, B
- peptide 13 to 24 270 ⁇ M, C
- peptides 14 to 31 480 ⁇ M, F
- peptide 20 to 31 190 ⁇ M, E.
- EGF iod radiolabelled EGF
- the activity is about 1 : 10,000 that of natural EGF. Acc dingly, it can be used as the active ingredient in pharmaceuti preparations which exhibit EGF activity.
- the basic active sequence is that of the 10 amino acid and although longer chains which include this sequence are activ their activity is not more pronounced than that of the 10-amin acid polypeptide.
- the present invention comprehends all su longer chain fragments having EGF-like activity containing the 1 amino-acid polypeptide sequence, up to a total length of about peptide units. Above about 25 peptide with the synthesis becom difficult and expensive. Theoretically, even longer polypeptid are encompassed by the present invention as long as the acti fragment is present.
- the add tional peptide units will substantially follow the natural E sequence.
- the following polypeptide corresponding to Cys (1 - Cys (31) of natural EGF is a particularly preferred such fra ment: Cys-Leu-Asn-Gly-Gly-Val-Cys-Met-His-Ile-Glu-Ser-Leu-As Ser-Tyr-Thr-Cys.
- the mEGF-(21-30) fragment has been found to be the active fragment of mouse EGF, the corresponding fragment of human EFG (urogastrone) will also be the active fragment.
- the present invention also comprehends the hEGF-(20-30) fragment: Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala.
- the hEGF-(20-31) frag ⁇ ment with Cys with at both ends is the preferred fragment.
- any other EGF-like hormones of other species having fragments which correspond to the Cys-Met-His-Ile-Glu-Ser- Leu-Asp-Ser-Tyr-Thr-Cys fragment of mEGF or the Cys-Met-Tyr-Ile- Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys fragment of hEGF can be simulated as far as EGF activity is concerned, by synthesizing a polypeptide including at least the sequence between the two Cys peptide units of such fragment in the same manner as discussed above for mEGF.
- the synthetic polypeptides of the present invention defined above are of value as active ingredient in pharmaceutical compositions. Due to the pronounced inhibition of the secretion of gastric acid by same, they are of value in anti-ulcer drugs and in methods for treating ulcers.
- polypeptides of the present invention can be used for other purposes, such as defleecing agents for sheep.
- the preferred effective concentration of the peptide in the blood serum is a minimum of 50 ⁇ M, preferably an effective concen ⁇ tration of 50 - 200 ⁇ M.
- the composition is administered orally.
- the composi- tion is preferably administered intravenously.
- Tha olypeptides of the present invention may be mulated into pharmaceutical or veterinary compositions using ventional pharmaceutically acceptable excipients in a manner k in the art.
- novel polypeptides may be prepared by the so phase Merrifield method, P. Amer. Chem. Soc, 85, 2149-2 (1963). Specific details of experimental materials and meth for the production of such peptides are set forth in the pub cation of Komoriya et al, Proc. Nat'l Acad. Sci. USA, 81, 13 * 51 (1984), the entire contents of which are hereby incorporated reference.
- the 12-amino acid polypeptide [(Acm)Cys ' ]mEGF-( 31)(Ac ⁇ t, S-acetamidomethyl) was synthesized according to Merrifield solid-phase method using a Beckman model 990B pept synthesizer. Anhydrous hydrogen fluoride treatment of pept resins for 1 hr at 0°C in the presence of 10% (vol/vol) anis and 1% ethanedithiol effected the cleavage from resin and deprotection of peptides.
- the peptides were purified by gel f tration chromatography on Sephadex G-10 and G-25 (fine) elu with 1 M acetic acid, followed by ion-exchange chromatography DEAE-cellulose or CM-cellulose as appropriate. Purity of synthetic peptides was monitored by high-performance liquid chr atography on Waters Associates ⁇ Bondapak C. g analytical colu (0.39 x 30 cm), in water/acetonitrile gradients of 10-40% c taining 0.1% CF COOH. Where further purification was necessa reverse-phase liquid chromatography on ⁇ Bondapak C. g colu (0.78 x 30 cm) was performed with the same elution system. peptide concentrations and amino acid compositions were determi after acid hydrolysis in constant-boiling HCl containing tr
- Fig. 4 establishes that synthetic peptides induce th midine incorporation in quiescent human foreskin fibroblasts.
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Abstract
A polypeptide which exhibits EGF activity includes ten amino acids of the natural EGF polypeptide, namely that sequence corresponding to the fragment between Cys (20) and Cys (31) of mEGF. The polypeptide may be formulated into a pharmaceutical or veterinary composition and used, for example, in the treatment of gastric ulcer or for defleecing sheep.
Description
- I -
SYNTHETIC PEPTIDES WITH EGF LIKE ACTIVITY AND PHARMACEUTICAL COMPOSITIONS AND METHODS OF USE
Field of the Invention The present invention relates to polypeptides havi epidermal growth factor (EGF)-li e activity, as well as to pharm ceutical compositions containing same as active ingredient a pharmaceutical processes using same. The peptide fractions an compositions according to the present invention activate EGF-sen sitive kinase and induce autophoshorylation of the EGF receptor and other endogenous membrane proteins. They stimulate thymidin incorporation into human foreskin fibroblasts and thus they hav effects like those of EGF, although of lesser potency.
Background of the Invention Epidermal growth factor (EGF) isolated from the sub maxillary glands of male mice is a single-chain polypeptide of 5 amino acids with three disulfide bonds which define three loope regions from residues 1-20, 14-31 and 32-53.
EGF is a potent stimulator of cellular proliferation an inhibitor of gastric acid secretion. It binds to a diffusel distributed mobile membrane receptor which was identified to be 170,000 daltons glycoprotein. Upon EGF binding, the recepto cluster, mainly over coated regions of the plasma membrane, an the EGF-receptor complex become internalized by an energy depen dent process. Subsequently, the hormone and presumably also th receptor are degraded by lysosomal enzymes. During these event EGF induces rapid reponse such as the uptake of ions and metabo lites, enhanced phosphorylation of various membrane protein including the autophosphorylation of the EGF-receptor itself; an induces changes in cell morphology and induces the reorganizatio of cytoβkeletal elements. Delayed effects of EGF include th
- UR__Λ OMPI WIPO
activation of cytoplasmic enzymes and the stimulation of DNA syn¬ thesis. In addition EGF (and also human EGF, denoted urogastrone) inhibits secretion of gastric acid iτ vivo.
Hollenberg and Gregory, Molec. Pharmacol., 17, 314-320 (1980) proteolytically shortened the EGF molecule and concluded that when up to six terminal amino acids were removed from the 53 amino acid chain, the remaining polypeptide still retained mito- genic potency, though with a lower binding affinity towards the membrane receptor. Furthermore, it has been reported that EGF cleaved at the 21-position ethionyl residue lacks mitogenic activity while still binding to the EGF receptor (Nature, vol. 278, p. 835 (1978)).
Summary of the Invention According to the present invention, there is provided a linear polypeptide which exhibits EGF activity. The polypeptide contains 10 amino acids out of the 53 amino acid sequence of the natural EGF polypeptide, namely that fragment between Cys (20) and Cys (31) of the EGF molecule, and contains the main receptor binding site of EGF. The affinity of the peptide according to the present invention is lower by a factor of about "10,000 than the affinity of EGF towards the receptor molecule. It activates EGF sensitive kinase and exhibits also the other physiological effects of natural EGF, at a lower level of potency. Thus, the amino acid sequence between Cys (20) and Cys (31) of mouse EGF fulfills the structural requirements for occupancy of EGF receptors of the human epidermoid carcinoma cell line A-431 and human foreskin fibroblasts, activates EGF sensitive kinase, induces DNA synthesis and stimulates the clustering and internalization EGF receptors.
As long as the ten peptide sequence defined above is present, the polypeptide of the present invention may have an
OMPI
number of additional peptides on either side of the critic sequence, although, for practical reasons, the total polypepti should not exceed about 25 peptide units.
The present invention al3o comprehends the correspondi sequence between Cys (20) and Cys (31) of human EGF (ur gastrone) .
Brief Description of the Drawings
» The attached figures illustrate the biological activi of synthetic polypeptides of the present invention compared wi polypeptides lacking such activity.
125 Fig. 1 shows inhibition of the binding 1 nM I-E to human foreskin fibroblasts (HFF) by increasing concentration the following synthetic peptides: peptide 14 to 31 (•-•), peptid
20 to 31 («-»), peptide 32 to 48 (D-D), peptide 43 to 51 (Δ-Δ) and peptide 1 to 20 (o-o). Non-specific binding was determine with a 200 fold excess of cold EGF and it did not exceed 20% o the total binding. Results are the average of . two independen experiments performed in duplicates.
Fig. 2 shows inhibition of the binding of 125i-EGF t polyclonal rabbit anti-EGF antibodies by synthetic peptides
Rabbit anti-EGF antibodies were bound to polyvinylchloride plate by immobilized goat anti-rabbit antibodies and incubated with 3. jiM 125I-EGF and the following synthetic polypeptides: a) peptide
1 to 20 (430 μι" ) , b) peptides 6 to 24 (70 μt ) , c) peptides 13 t
24 (160 ;ιM), d) peptides 14 to 31 (200 μM ) , e) peptides 20 to 3
(170 >ιM), f) peptides 25 to 42 (490 μtt ) , g) peptides 32 to 42 (46 μM), and h) peptides 43 to 51 (165 jiM ) . Nonspecific binding wa determined with a 1500 fold excess of cold EGF and subtracte
prior to calculations. Each value is rhe mean of duplicate deter minations.
Fig. 3 shows enhancement of membrane protein phosphory lation by either EGF or synthetic peptides. The figure is auto radiography of NaDodSO /5-15% polyacrylamide gradient gel elec trophoretic analysis of shed membrane vesicle preparation using A
431 cells incubated with [/- 32p]ATP and buffer alone (lanes and G), EGF (33 nH, lane A), peptides 1 to 20 (350 μM, lane B peptides 14 to 31 (350 μM , lane C), peptides 20 to 31 (125 μM lane I), peptides 25 to 42 (100 μM, lane E), peptides 32 to 4 (210 μM, lane F), and peptides 43 to 51 (210 μM, lane H).
Fig. 4 esablishes that synthetic peptides induce thy midine incorporation in quiescent human foreskin fibroblasts
3 Enhancement of (methyl- H) thymidine incorporation in quiescen
HFF by synthetic peptides 1 to 20 (o-o), 14 to 31 (•-•), 32 to 4 (___-_■), and 43 to 51 (Δ-_i_) is shown. The dashed line indicates th level of thymidine incorporation achieved by 5 uM EGF in thi experiment. Cells were incubated at 37βC for 18 hours with eithe peptides of EGF prior to a 4 hour pulse with (methyl- H) thy midine and determination of acid precipitable radioactivity.
Fig. 5 shows induction of EGF receptor clustering in A 431 cells by synthetic peptides. The fluorescence micrographs o this frame show A-431 cells which were labelled with rhodamin anti-EGF-receptor monoclonal antibody (TL5) and subsequently wit buffer (A),EGF (10 μM, D),peptides 6 to 24 (240 μM, B), peptide 13 to 24 (270 μM, C), peptides 14 to 31 (480 μM, F), or peptide 20 to 31 (190μM, E). Magnification:x.
Detailed Description of Preferred Embodiments
Various parts and sequences of the natural EGF polypep
- 1 ' - r.
tide were prepared and tested for their activity. Extens experiments indicated that the smallest polypeptide sequence wh retains EGF activity is the 10-amino acid polypeptide: Met-H Tle-Glu-Ser-Leu-Asp-Ser-Tyr-Thr. This polypeptide sequen occurring between Cys (20) and Cys (31) of natural EGF obtai from the submaxillary glands of male mice, may be in linear f or, particularly when the Cys units are added to either end the of, in cyclic form. This polypeptide is physiologically act and was found to induce changes in the morphology and icrofi ents of human A-431 cells. It displaces the binding of iod radiolabelled EGF, and induces DNA synthesis in human fibrob^as The activity is about 1 : 10,000 that of natural EGF. Acc dingly, it can be used as the active ingredient in pharmaceuti preparations which exhibit EGF activity.
The basic active sequence is that of the 10 amino acid and although longer chains which include this sequence are activ their activity is not more pronounced than that of the 10-amin acid polypeptide. The present invention comprehends all su longer chain fragments having EGF-like activity containing the 1 amino-acid polypeptide sequence, up to a total length of about peptide units. Above about 25 peptide with the synthesis becom difficult and expensive. Theoretically, even longer polypeptid are encompassed by the present invention as long as the acti fragment is present.
Preferably, if the polypeptide chain is to be extend beyond the minimum of ten peptides from EFG-(21-30), the add tional peptide units will substantially follow the natural E sequence. The following polypeptide corresponding to Cys (1 - Cys (31) of natural EGF is a particularly preferred such fra ment: Cys-Leu-Asn-Gly-Gly-Val-Cys-Met-His-Ile-Glu-Ser-Leu-As Ser-Tyr-Thr-Cys.
As the mEGF-(21-30) fragment has been found to be the active fragment of mouse EGF, the corresponding fragment of human EFG (urogastrone) will also be the active fragment. Thus, the present invention also comprehends the hEGF-(20-30) fragment: Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala. The hEGF-(20-31) frag¬ ment with Cys with at both ends is the preferred fragment.
Similarly, any other EGF-like hormones of other species having fragments which correspond to the Cys-Met-His-Ile-Glu-Ser- Leu-Asp-Ser-Tyr-Thr-Cys fragment of mEGF or the Cys-Met-Tyr-Ile- Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys fragment of hEGF can be simulated as far as EGF activity is concerned, by synthesizing a polypeptide including at least the sequence between the two Cys peptide units of such fragment in the same manner as discussed above for mEGF.
The synthetic polypeptides of the present invention defined above, are of value as active ingredient in pharmaceutical compositions. Due to the pronounced inhibition of the secretion of gastric acid by same, they are of value in anti-ulcer drugs and in methods for treating ulcers.
They are also of value for other pharmaceutical uses as indicated by the physiological activities set out above.
Furthermore, the polypeptides of the present invention can be used for other purposes, such as defleecing agents for sheep.
When used for such pharmaceutical or veterinary pur¬ poses, the preferred effective concentration of the peptide in the blood serum is a minimum of 50 μM, preferably an effective concen¬ tration of 50 - 200μM. For the anti-ulcer utility the composition is administered orally. For the defleecing utility the composi-
tion is preferably administered intravenously.
Tha olypeptides of the present invention may be mulated into pharmaceutical or veterinary compositions using ventional pharmaceutically acceptable excipients in a manner k in the art.
The novel polypeptides may be prepared by the so phase Merrifield method, P. Amer. Chem. Soc, 85, 2149-2 (1963). Specific details of experimental materials and meth for the production of such peptides are set forth in the pub cation of Komoriya et al, Proc. Nat'l Acad. Sci. USA, 81, 13*51 (1984), the entire contents of which are hereby incorporated reference.
Examples The 12-amino acid polypeptide [(Acm)Cys ' ]mEGF-( 31)(Acπt, S-acetamidomethyl) was synthesized according to Merrifield solid-phase method using a Beckman model 990B pept synthesizer. Anhydrous hydrogen fluoride treatment of pept resins for 1 hr at 0°C in the presence of 10% (vol/vol) anis and 1% ethanedithiol effected the cleavage from resin and deprotection of peptides. The peptides were purified by gel f tration chromatography on Sephadex G-10 and G-25 (fine) elu with 1 M acetic acid, followed by ion-exchange chromatography DEAE-cellulose or CM-cellulose as appropriate. Purity of synthetic peptides was monitored by high-performance liquid chr atography on Waters Associates μBondapak C.g analytical colu (0.39 x 30 cm), in water/acetonitrile gradients of 10-40% c taining 0.1% CF COOH. Where further purification was necessa reverse-phase liquid chromatography on μBondapak C.g colu (0.78 x 30 cm) was performed with the same elution system. peptide concentrations and amino acid compositions were determi after acid hydrolysis in constant-boiling HCl containing tr
C
amounts of phenol at 110βC for 24 hr.
Also synthesized in a similar manner was [Ala ]mEG ((14-31) as well as the following additional comparison polype tides: [Alal4]mEGF-(l-20); mEGF-(32-48) ; mEGF-(43-51
C(Acm)Cysl4]mEGF-(6-24); [(Acm)Cysl4]mEGF-(13-24) ; mEGF-(3
31 42); and [(Acm)Cys )mEGF-(25-42) .
The binding capacity of these synthetic peptides to E receptors was tested by determining the inhibition of the 125
EGF to EGF receptors on human foreskin fibroblasts (HFF).» T results are given in Fig. 1. Binding to plasma membrane recepto
125 ws monitored by; the inhibition of I-EGF binding to conflue monolayers of human fibroblasts. Two peptides, namely Cys(1
125 - Cys(31) and Cys(20) - Cys(31) inhibit the binding of I-E
4 membrane receptors. The binding is about 3 x 10 fold low than that of EGF towards the receptor.
In Figure 2 there are presented the results of t binding activity of the various peptides to anti-EGF antibodi using solid phase radioimmunoassay.
The enhancement of membrane protein phosphorylation either EGF or any of various synthetic test fragments is shown Fig. 3.
Fig. 4 establishes that synthetic peptides induce th midine incorporation in quiescent human foreskin fibroblasts.
The induction of EGF receptor clustering in A-431 cel by the various synthetic peptides tested is shown in fig. 5. On the fragments -in accordance with the present invention show sign ficant clustering.
Also synthesized was the cyclic mEGF-(20-31) and 31y Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr-Cys. The latter corres ponds to mEGF-(20-31) with Gly substituted for the initial Cy unit. These also tested positive for EGF-like activity in th receptor binding assay.
It is to be understood that the present invention is no limited to the embodiments disclosed which are illustrativel offered and that modifications may be make without departing fro the invention. •
Claims
1. A polypeptide having EGF-like activity and having n more than about 25 peptide units therein, wherein said polypeptid includes a ten-amino acid sequence of a natural EGF-like hormone, which sequence corresponds to that sequence between Cys (20) an Cys (31) of natural mEGF.
2. A polypeptide in accordance with claim 1, wherei said sequence comprises Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr.
«
3. A polypeptide in accordance with claim 1, includin the sequence Cys-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr-Cys o Gly-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr-Cys.
4. A polypeptide in accordance with claim 1, wherei said sequence comprises Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala.
5. A polypeptide in accordance with claim 1, includin the sequence Cys-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys.
6. A polylpeptide in accordance with claim 1, con sisting essentially of the sequence: Cys-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr-Cys Gly-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr-Cys or Cys-Leu-Asn-Gly-Gly-Val-Cys-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr- Thr-Cys.
7. A polypeptide in accordance with claim 1, wherein the peptide units thereof correspond substantially to the sequence of a fragment of the natural EGF-like hormone containing said te amino acid sequence.
8. A polypeptide in accordance with claim 1, wherei
V-flP said natural EGF-like hormone is mEGF or hEGF.
9. A polylpeptide in accordance with claim 8, con sisting essentially of the sequence: Cys-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr-Cys Gly-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr-Cys or Cys-Leu-Asn-Gly-Gly-Val-Cys-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr- Thr-Cys.
10. A pharmaceutical composition for treating condition treatable by natural EGF, comprising a polypeptide in accordanc with claim 1 and a pharmaceutically acceptable excipient.
11. A pharmaceutical composition for treating condition treatable by natural EGF, comprising a polypeptide in accordanc with claim 8 and a pharmaceutically acceptable excipient.
12. A method for the treatment of conditions treatabl by natural EGF, comprising administering an effective amount of polypeptide in accordance with claim 1.
13. A method for the treatment of conditions treatabl by natural EGF, comprising administering an effective amount of polypeptide in accordance with claim 8.
14. A method for the inhibition of the secretion o gastric acid comprising orally administering an effective amoun of a polypeptide in accordance with claim 1.
15. A method for defleecing sheep comprising adminis tering an effective amount of a polypeptide in accordance wit claim 1 and then removing the fleece.
O PI
Applications Claiming Priority (2)
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IL69719A IL69719A0 (en) | 1983-09-14 | 1983-09-14 | Synthetic peptides with egf like activity |
IL69719 | 1983-09-14 |
Publications (1)
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WO1985001284A1 true WO1985001284A1 (en) | 1985-03-28 |
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PCT/US1984/001459 WO1985001284A1 (en) | 1983-09-14 | 1984-09-14 | Synthetic peptides with egf like activity and pharmaceutical compositions and methods of use |
Country Status (4)
Country | Link |
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EP (1) | EP0156899A1 (en) |
AU (1) | AU3439084A (en) |
IL (1) | IL69719A0 (en) |
WO (1) | WO1985001284A1 (en) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4743679A (en) * | 1986-02-24 | 1988-05-10 | Creative Biomolecules, Inc. | Process for producing human epidermal growth factor and analogs thereof |
US5087616A (en) * | 1986-08-07 | 1992-02-11 | Battelle Memorial Institute | Cytotoxic drug conjugates and their delivery to tumor cells |
WO1992003476A1 (en) * | 1990-08-13 | 1992-03-05 | Board Of Regents, The University Of Texas System | Bioactive egf peptides for promotion of tissue regeneration and cancer therapy |
US5447100A (en) * | 1991-02-08 | 1995-09-05 | United States Surgical Corporation | Apparatus for calendering sutures in orthogonal directions |
US5472702A (en) * | 1987-08-26 | 1995-12-05 | United States Surgical Corporation | Sterilization of growth factors |
US5904716A (en) * | 1995-04-26 | 1999-05-18 | Gendler; El | Method for reconstituting cartilage tissue using demineralized bone and product thereof |
WO2003053457A1 (en) * | 2001-12-21 | 2003-07-03 | Zakrytoe Aktsionernoe Obschestvo 'russkie Biotehnologii' | Polypeptide, the conjugate thereof containing doxorubicine and a pharmaceutical composition based thereon |
EP2093241A2 (en) | 2008-01-29 | 2009-08-26 | Confluent Surgical Inc. | Bioabsorbable block copolymer |
JP2010505941A (en) * | 2006-10-10 | 2010-02-25 | ケアジェン シーオー エルティーディー | Peptide having epidermal growth factor activity and use thereof |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989001489A1 (en) * | 1987-08-10 | 1989-02-23 | Commonwealth Scientific And Industrial Research Or | Control of angiogenesis and compositions and methods therefor |
WO1995002411A1 (en) * | 1993-07-16 | 1995-01-26 | Mallinckrodt Veterinary Limited | Stabilised polypeptide growth factor formulation at low ph |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3917824A (en) * | 1973-03-28 | 1975-11-04 | Ici Ltd | Pharmaceutical compositions containing epidermal growth factor or closely related derivatives thereof for inhibiting the secretion of acidic gastric juice in warm blooded animals |
US4418691A (en) * | 1981-10-26 | 1983-12-06 | Massachusetts Institute Of Technology | Method of promoting the regeneration of tissue at a wound |
-
1983
- 1983-09-14 IL IL69719A patent/IL69719A0/en unknown
-
1984
- 1984-09-14 AU AU34390/84A patent/AU3439084A/en not_active Abandoned
- 1984-09-14 WO PCT/US1984/001459 patent/WO1985001284A1/en unknown
- 1984-09-14 EP EP84903725A patent/EP0156899A1/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3917824A (en) * | 1973-03-28 | 1975-11-04 | Ici Ltd | Pharmaceutical compositions containing epidermal growth factor or closely related derivatives thereof for inhibiting the secretion of acidic gastric juice in warm blooded animals |
US4418691A (en) * | 1981-10-26 | 1983-12-06 | Massachusetts Institute Of Technology | Method of promoting the regeneration of tissue at a wound |
Non-Patent Citations (3)
Title |
---|
Nature, Vol. 278, April 1979, SCHECHTER, Local Aggregation of Hormone-Receptor Complexes is Required for Activation by Epidermal Growth Factor, pages 835-838 * |
The Journal of Biological Chemistry, Vol. 23, December 1972, SAVAGE, Jr., The Primary Structure of Epidermal Growth Factor, pages 7612-7621 * |
The Journal of Biological Chemistry, Vol. 248, No. 23, November 1973, SAVAGE, Jr., Epidermal Growth factor, pages 7669-7672, fig. 1, page 7670 * |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5004686A (en) * | 1986-02-24 | 1991-04-02 | Creative Biomolecules, Inc. | Process for producing human epidermal growth factor and analogs thereof |
US4743679A (en) * | 1986-02-24 | 1988-05-10 | Creative Biomolecules, Inc. | Process for producing human epidermal growth factor and analogs thereof |
US5087616A (en) * | 1986-08-07 | 1992-02-11 | Battelle Memorial Institute | Cytotoxic drug conjugates and their delivery to tumor cells |
US5472702A (en) * | 1987-08-26 | 1995-12-05 | United States Surgical Corporation | Sterilization of growth factors |
WO1992003476A1 (en) * | 1990-08-13 | 1992-03-05 | Board Of Regents, The University Of Texas System | Bioactive egf peptides for promotion of tissue regeneration and cancer therapy |
US5540773A (en) * | 1991-02-08 | 1996-07-30 | United States Surgical Corporation | Apparatus for calendering and coating/filling sutures |
US5447100A (en) * | 1991-02-08 | 1995-09-05 | United States Surgical Corporation | Apparatus for calendering sutures in orthogonal directions |
US5904716A (en) * | 1995-04-26 | 1999-05-18 | Gendler; El | Method for reconstituting cartilage tissue using demineralized bone and product thereof |
WO2003053457A1 (en) * | 2001-12-21 | 2003-07-03 | Zakrytoe Aktsionernoe Obschestvo 'russkie Biotehnologii' | Polypeptide, the conjugate thereof containing doxorubicine and a pharmaceutical composition based thereon |
US7368431B2 (en) | 2001-12-21 | 2008-05-06 | Zakrytoe Aktsionernoe Obschestvo “Russkie Biotechnologll” | Polypeptide, the conjugate thereof with doxorubicine and a pharmaceutical composition based thereon |
JP2010505941A (en) * | 2006-10-10 | 2010-02-25 | ケアジェン シーオー エルティーディー | Peptide having epidermal growth factor activity and use thereof |
JP4942818B2 (en) * | 2006-10-10 | 2012-05-30 | ケアジェン シーオー エルティーディー | Peptide having epidermal growth factor activity and use thereof |
EP2093241A2 (en) | 2008-01-29 | 2009-08-26 | Confluent Surgical Inc. | Bioabsorbable block copolymer |
Also Published As
Publication number | Publication date |
---|---|
IL69719A0 (en) | 1983-12-30 |
EP0156899A1 (en) | 1985-10-09 |
AU3439084A (en) | 1985-04-11 |
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