IE47611B1 - Polypeptides comprising a tetrapeptide segment,pharmaceutical compositions thereof,and processes for the preparation thereof - Google Patents
Polypeptides comprising a tetrapeptide segment,pharmaceutical compositions thereof,and processes for the preparation thereofInfo
- Publication number
- IE47611B1 IE47611B1 IE2423/78A IE242378A IE47611B1 IE 47611 B1 IE47611 B1 IE 47611B1 IE 2423/78 A IE2423/78 A IE 2423/78A IE 242378 A IE242378 A IE 242378A IE 47611 B1 IE47611 B1 IE 47611B1
- Authority
- IE
- Ireland
- Prior art keywords
- amino acid
- polypeptide
- amino
- resin
- gly
- Prior art date
Links
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- 230000008569 process Effects 0.000 title claims description 14
- 238000002360 preparation method Methods 0.000 title description 7
- 239000008194 pharmaceutical composition Substances 0.000 title 1
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- 238000004519 manufacturing process Methods 0.000 claims abstract description 14
- 125000003412 L-alanyl group Chemical group [H]N([H])[C@@](C([H])([H])[H])(C(=O)[*])[H] 0.000 claims abstract description 13
- 125000002842 L-seryl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])O[H] 0.000 claims abstract description 13
- 125000003301 D-leucyl group Chemical group N[C@@H](C(=O)*)CC(C)C 0.000 claims abstract description 11
- 125000000722 D-seryl group Chemical group N[C@@H](C(=O)*)CO 0.000 claims abstract description 11
- 125000003440 L-leucyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C(C([H])([H])[H])([H])C([H])([H])[H] 0.000 claims abstract description 11
- 125000003580 L-valyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(C([H])([H])[H])(C([H])([H])[H])[H] 0.000 claims abstract description 11
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- 125000004077 D-glutamic acid group Chemical group [H]N([H])[C@@]([H])(C(=O)[*])C([H])([H])C([H])([H])C(N([H])[H])=O 0.000 claims abstract description 9
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- 125000000769 L-threonyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])[C@](O[H])(C([H])([H])[H])[H] 0.000 claims abstract description 8
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1021—Tetrapeptides with the first amino acid being acidic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
- C07K7/062—Serum thymic factor
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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
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- Chemical & Material Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
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- General Chemical & Material Sciences (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Peptides Or Proteins (AREA)
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Abstract
Biologically active polypeptides containing the following polypeptide segment: -ALA-LYS-SER-GLN- are disclosed. Biological activity is generally retained upon substitution of an amino acid radical selected from L- asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl, L-leucyl, L-alamyl, L- seryl, sarcosyl, 2-methylalonyl, D- asparagyl, D-glutamyl, D-threonyl, D- valyl, D-leucyl, D-alamyl and D-seryl for either or both of L-alanyl in the first position and L-seryl in the third position, which polypeptides are capable of induction of the differentiation of T-lymphocytes, as measured by the acquisition of the thymic differentiation antigen Th-1, as well as B-lymphocytes as measured by the acquisition of the differentiation antigen Bu-1. The polypeptides are thus useful in thymic function and immunity areas such as in treatment for congential absence of thymus. Also provided are methods of manufacture of the polypeptides, therapeutic compositions, and methods of use of the polypeptides.
Description
The present invention relates generally to novel polypeptide segments and polypeptides, to methods for the preparation thereof, and uses thereof.
Many polypeptides have been isolated from various 5 tissues and organs (including the blood) of animals. Certain of these polypeptides are related to immune function in the body, such as, the various immune globulins and the thymic hormone thymopoietin. The isolation and synthesis of several of these polypeptides is described in United States Patent Nos. 4,002,602 and 4,002,740.
Until the past decade, little was known about the thymus, although it is now understood that the thymus is one of the organs principally responsible for immune function in mammals and birds. It is now realised, however, that the thymus is a compound organ with both epithelial (endocrine) and lymphoid (immunological) components and thus the thymus is involved in the immune functions of the body. The thymus consists of an epithelial stroma derived from the third branchial arch and lymphocytes derived from stem cells originating in - 3 haemopoietic tissues, Goldstein, et al., The Human Thymus, Heinemann, London, 1969. Lymphocytes are differentiated within the thymus and leave as mature thymus-derived cells, called T cells, which circulate to the blood, lymph, spleen and lymph nodes. The induction of stem cell differentiation within the thymus appears to be mediated by secretions of the epithelial cells of the thymus.
It has been known for some time that the thymus is connected with the immune characteristics of the body and, therefore, great interest has been shown in substances which have been isolated from the thymus. In this regard, there have been published in recent years a relatively large body of articles based on scientific work relating to materials which are present in bovine thymus. Pertinent publications may be found, for example in, The Lancet, July 20, 1968, pp. 119-122; Triangle, Vol. II, No. 1, pp. 7-14, 1972; Annals of the New York Academy of Sciences, Vol. 183, pp. 230-240, 1971; and Clinical and Experimental Immunology, Vol. 4, No. 2, pp. 181-189, 1969; Nature, Vol. 247, pp. 11-14, 1974; Proceedings of the National Academy of Sciences USA, Vol. 71, pp. 1474-1478, 1974; Cell, Vol. 5, pp. 361-365 and 367-370, 1975; Lancet, Vol. 2, pp. 256-259, 1975; Proceedings of the National Academy of Sciences USA, Vol. 72, pp. 11-15, 1975; Biochemistry, Vol. 14, pp. 2214-2218, 1974; Nature, Vol. 255, pp. 423-424, 1975.
A second class of lymphocytes having immune function are the B lymphocytes or B cells. These are differentiated in the Bursa of Fabricius in birds and by an as-yet-unidentified organ in mammals. T-cells and B-cells cooperate in many aspects of immunity. See, for example, articles in Science, 193, 319 (July 23, 1976) and Cold Spring Harbor Symposia on Quantitative Biology, Vol. XLI, 5 (1977).
A nonapeptide material has recently been isolated from porcine serum by J. F. Bach, et al. and identified as facteur thymique serique (FTS). The isolation of this material and its structure are disclosed in C, R. Acad. Sc.
Paris, t. 283 (November 29, 1976), Series D-1605 and Nature 266, 55 (March 3, 1977). The structure of this nonapeptide has been identified as H-GLX-ALA-LYS-SER-GLN-GLY-GLY-SER-ASNOH, where GLX represents either glutamine or pyroglutamic acid. The material where GLX is glutamine or pyroglutamic acid has been synthesized. In these articles, Bach disclosed that his nonapeptide FTS selectively differentiated T cells (and not B cells) by use of an E rosette assay. Bach, therefore, concluded that his material was a thymic hormone. Recently, a more thorough investigation of the activity of this nanapeptide by the present Applicant disclosed that FTS differentiated both T cells and B cells and was, therefore, more like ubiquitin in its activity than thymopoeitin. Brand, Gilmour and Goldstein, Nature, 269:597 (1977).
It has now been discovered that a synthesized 420 amino acid polypeptide segment of this FTS nonapeptide possesses many of the characteristics of the nonapeptide discussed in the above publications.
Accordingly the present invention provides a polypeptide having the capability of including the differentiation + + of both Th-1 T-lymphocytes and Bu-1 B-lymphocytes, which polypeptide has the following amino acid sequence: R-X-LYS-Y-GLN-R' wherein X and Y are each natural or non-natural amino acid residues selected from L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl, L-leucyl, L-alanyl, L-seryl, sarcosyl, 2methylalanyl, D-asparagyl, D-glutamyl, D-threonyl, D-valyl, D-leucyl, D-alanyl, and D-seryl and R and R' are each selected fran R R' Hydrogen OH —Cy alkyl nh2C6~C12 aryl NHR? Cy—C2Q alkaryl N(R?)2C7—C20 —Cy alkanoyl OR 7 C2—C? alkenyl GLY-OH C2—Cy alkenyl GLY-GLY-OH H-GLN H-SAR wherein R^ is —Cy alkyl, C2—C? alkenyl, C.,—Cy alkynyl, C6-C20 aryl, C7~C2O aralkyl, and C7-C2O alkaryl and the pharmaceutically acceptable salts thereof; further provided 4. that the polypeptide induces the differentiation of both Th-1 T-lymphocytes and Bu-l+B-lymphocytes in the chicken induction assay test (as hereinafter described) at a concentration of one ng/ml or less.
Specific examples of the combinations of R and R' which may be incorporated into the polypeptides of the invention are: R R' CHgCO OH ch3 OH ch3co nh2 H och3 H oc2h, phenyl ohC2H50C2H!C2H5 nh2 H-GLN oh A particular polypeptide of this invention is the novel biologically active polypeptide having the following formula: R-ALA-LYS-SER-GLN-R' wherein R and R' are as above defined.
The biological activity of this polypeptide segment is generally retained upon substitution of a natural or non-natural amino acid residue for either or both of: 1) L-alanyl in the first position; and 2) L-seryl in the third position. Certain of these substituted polypeptide segments are strikingly potent. Terminal subsitution of the subject polypeptide segments yields the polypeptides of the invention.
Also provided is a procedure for the preparation of the polypeptide segments and polypeptides of the invention by solid phase peptide synthesis, as well as therapeutic compositions containing the polypeptides.
In the principal embodiment of the present invention, there is provided a biologically active polypeptide which has the following formula: I. R-ALA-LYS-SER-GLN-R' wherein R and R' are as above defined.
Since the biological activity of the subject polypeptide segment is generally retained upon substitution of certain natural or non-natural amino acid residue for either or both of: 1) alanyl in the first position; and 2) seryl in the third position, the principal embodiment of the present invention further includes biologically active polypeptides which have the following formula: IA.
R-X-LYS-Y-GLN-R’ 611 wherein X, Y, R and R* are each as above defined. Majority of such substitutions of X and Y groups will allow retention of biological activity, it is possible that certain natural or non-natural amino acid residues will interfere with the folding of the molecule (as disscussed more fully below) and thus substantially eliminate the biological activity. Such activity-destroying substituents are specifically excluded from the scope of the present invention.
Whether a particular substitution allows reaction of the biological activity of the polypeptide may be readily + estabilised by testing it for differentiation of Th-1 Tlymphocytes and Bu-1+ B-lymphocytes in the chicken induction assay described below. Compounds which are specifically active in nanogram (ng)/milliliter (ml) concentrations (about one ng/ ml or less) in this assay are considered to be biologically active.
The polypeptide of the present invention contains terminal substituents on the 4-amino acid sequence. These terminal substituents must not substantially affect the biological activity of the peptide as measured by the ability to 4- + induce the differentiation of Th-1 T-lymphocytes and Bu-1 B-lymphocytes in the chicken induction assay described below.
It is believed that the activity requirements of the peptide molecule are generated by its stereochemistry, that is, by the particular, folding of the molecule. In this regard, it should be understood that polypeptide bonds are not rigid but flexible, and polypeptides may exist as sheets or helices. As a result, the entire molecule is flexible and will fold in a certain way. In the present invention, it has been discovered that the novel tetrapeptides probably fold in a similar manner to the corresponding tetrapeptide segment in the natural nonapeptide in that they exhibit the same biological characteristics. For this reason, the tetrapeptide segments may be terminally substituted by various functional groups so long as the substituents do not substantially affect the biological activity or interfere with the natural folding" of the molecule.
The ability of the molecule to retain its biological activity and natural folding is clearly illustrated by the fact that the tetrapeptide segments of this invention exhibit the same biological characteristics as the natural 9-amino acid peptide disclosed as FTS by J.F. Bach in the above disclosed articles. Xn this nonapeptide, one tetrapeptide sequence of this invention may be identified within the molecule but only in combination with the other amino acids des10 cribed therein. Since the tetrapeptide segments of this invention provide the same biological activity as the nonapeptide FTS, it is clear that the amino acids and peptide chains substituted on the terminal amino acid residues of the tetrapeptide segment do not effect the biological character15 istics thereof.
It will be understood that R and R' in Formula 1 must be substituents that do not substantially affect the biological activity of the peptide and the substituents R and R’ which have been found to be suitable in this respect are as defined above.
One preferred embodiment of the invention is that wherein X is L-alanyl, Y is L-seryl, R is hydrogen and R' is OH. This preferred embodiment may be symbolized chemically as H2N—CH—CONH——CH—CONH—CH—CONH—CH—COOH CH, (CH,). CH, I 4 I 2 NH2 oh CONH2 H-ALA-LYS-SER-GLN-OH A second preferred embodiment is a polypeptide having a polypeptide having the following amino acid sequence: R-X-LYS-Y-GLN-R1 611 wherein X and Y are each independently D-ALA or SAR and R and R" are each selected from: R R Hydrogen OH —Cy alkyl NH2C6^12 aryl nhr7 Cy—C20 alkaryl N(R7)2 C7—C2Q aralkyl 0R? C1—Cy alkanoyl C2—Cy alkenyl GLY-OH C2—C? alkynyl GLY-GLY-OH H-GLN GLY-GLY-SER-OH H-SAR GLY-GLY-SER-ASN-OH is —C? alkyl, C2—C7 alkenyl C2—Cy alk Cg—C2Q aryl, C?—C2Q aralkyl, and —C2Q alkaryl, and the pharmaceutically acceptable salts thereof.
A more preferred embodiment is that wherein X is sarcosyl, Y is sarcosyl or D-alanyl, R is hydrogen, and R is NH2.
Also included within the scope of the invention are the pharamceutically acceptable salts of the polypeptides. As acids which are able to form salts with the polypeptides, there may be mentioned inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid or phosphoric acid, and organic adds such as formic add, acetic add, propionic add, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid or sulfanilic acid, for instance.
Throughout the present application, the amino acid components of the peptide are identified by abbreviations for convenience. These abbreviations are as The D-amino acids are -indicated by placing D e.g. follows. before the abbreviation, by D-ALA.
Amino Acid L-Alanine L-Aspartic Acid L-Asparagine L-Serine L-Glutamic Acid L-Glutamine L-Leucine L-Lysine L-Threonine Glycine L-Valine Sarcosine 2-Methylalanine The polypeptides of , D-alanine is represented Abbreviated Designation ALA ASP ASN SER GLU GLN LEU LYS THR GLY VAL SAR 2-Me-ALA this invention are 4-amino acid peptides (and their substituted derivatives) which have been found to exhibit characteristics similar to the 9-amino acid polypeptide FTS isolated from porcine blood as disclosed in the above-referenced Bach, et al., articles. The peptides of this invention are particularly characterized in their ability to induce the differentiation of T-precursor cells as well as B-precursor cells. Certain of the subject polypeptides are active in a concentration as low as one picogram (pg)/ntl in the chicken induction assay discussed below.
It has been found that the polypeptides of this invention induce the differentiation of immunocyte-precursor cells in vitro in the same way as the nonapeptides disclosed by Bach. Thus, the polypeptides of this invention have been found to induce the differentiation of both T-precursor cells, as measured by the acquisition of the thymic differentiation antigen Th-1 as well as B-precursor cells, as measured by the acquisition of the differentiation antigen Bu-1. Stated another way, the subject polypeptides have the capability of inducing differentiation of both Th-1+ T-lymphocytes and Bu-1+ B-lymphocytes.
It has also been found that the subject polypeptides increase the capability of in vivo production of cytotoxic lymphocytes upon stimulation by allogenic antigens. That is, administration of the subject polypeptides to, e.g., rats,promotes the production of cytotoxic lymphocyte precursors as measured by an in vitro assay of rat spleen cells. Since the generation of cytotoxic lymphocytes directly corresponds to the extent of graft rejection in allogenic graft vs host reaction, the above finding is further support for the immunologic utility of the subject polypeptides.
To provide an understanding of the importance of the differentiating biological characteristics of the polypeptides of this invention, it should be noted that the function of the thymus in relation to immunity may be broadly stated as the production of thymus-derived cells, or lymphocytes, which are called T cells. T cells form a large proportion of the pool of recirculating small lymphocytes. T cells have immunological specificity and are directly involved in cell-mediated immune responses (such as homograft responses), as effector cells. T cells however, do not secrete humoral antibodies. These antibodies are secreted by cells (termed B cells) derived directly from the bone marrow independently of the thymic influence. However, for many antigens, B cells require the presence of appropriately reactive T cells before they can produce antibodies. The mechanism of this process of cell cooperation is not yet completely understood.
From this explanation, it may be said that in operational terms, the thymus is necessary for the development of cellular immunity and many humoral antibody responses and it affects these systems by inducing, within the thymus, the differentiation of haemopoietic stem cells to T cells. This inductive influence is mediated by secretions of the epithelial cells of the thymus, that is, the thymic hormones.
Further, to understand the operation of the thymus and the cell system of lymphocytes, and the circulation of lymphocytes in the body, it should be pointed out that stem cells arise in the bone marrow and reach the thymus by the blood stream. Within the thymus, stem cells become differentiated to immunologically competent T cells, which migrate to the blood stream and, together with B cells, circulate between the tissues, lymphatics, and the blood stream.
The cells of the body which secrete antibody (B cells) also develop from haemopoietic stem cells, but their differentiation is not determined by the thymus. In birds, they are differentiated in an organ analogous to the thymus, called the Bursa of Fabricius. In mammals, no equivalent organ has been discovered and it is thought that these cells differentiate within the bone marrow.
Hence, they are termed bone marrow-derived cells or B cells. The physiological substances dictating this differentiation remain completely unknown.
As pointed out above, the polypeptides of this invention are therapeutically useful in the treatment of humans and animals. Since the new polypeptides have the capability of inducing the differentiation of lymphopoietic stem cells originating in the haemopoietic tissues to both thymus-derived lymphocytes (T cells) and immunocompetent B cells which are capable of involvement in the immune response of the body, the products of this invention are considered to have multiple therapeutic uses. Primarily, since the compounds have the capability of carrying out certain of the indicated functions of the thymus, they have application in various thymic function and immunity areas. A primary field of application is in the treatment of DiGeorge Syndrome, a condition in which there is a congenital absence of thymus. Injection of one of the subject polypeptides, as further set out below, will overcome this deficiency. Another application is in agammaglobulinemia, which is due to a defect of the putative B cell differentiative hormone of the body. Injection of one of the subject polypeptides will overcome this defect. Since the subject polypeptides are extremely active at low concentrations, they are useful in augmenting the collective immunity of the body in that they increase therapeutic stimulation of cellular immunity and humoral immunity and are thereby useful in the treatment of diseases involving chronic infection in vivo, such as fungal or mycoplasma infections, tuberculosis, leprosy, acute and chronic viral infections, and the like. Further, the subject peptides are considered to be useful in any area in which cellular or humoral immunity is an issue and particularly where there are deficiencies in immunity such as in the DiGeorge Syndrome mentioned above. Further, because of the characteristics of the polypeptides, they have in vitro usefulness in inducing the development of surface antigens of T cells, in inducing the development of the functional capacity to achieve responsiveness to mitogens and antigens, and cell collaborativity in enhancing the ability of B cells to produce antibodies. They have in vitro usefulness in inducing the development of B cells as measured by the development of surface receptors for complement. The subject peptides are also useful in inhibiting the uncontrolled proliferation of lymphocytes which are responsive to ubiquitin (described in Applicant's United States Patent No. 4,002,602). An important characteristic of the subject polypeptides is their in vivo ability to restore cells with the characteristics of T cells and also their in vivo ability to restore cells with the characteristics of B cells. They are, therefore, useful in the treatment of relative or absolute B cell deficiencies as well as relative or absolute T cell deficiencies, whether or not these deficiencies are due to deficiencies in the tissue differentiating B cells or the thymus, respectively, or to some other cause.
A further important property of the polypeptides of this invention is that they are highly active in very low concentrations. Thus, it has been found that the polypeptides are active in concentrations of 1 ng/ml, while certain strikingly potent polypeptides (H-SAR-LYS-D-ALA-GLN-NI^ and H-SAR-LYS-SAR-GLN-NH2) are active in concentrations ranging from 0.1 pg/ml. The present invention thus includes a therapeutic composition comprising a therapeutically effective amount of a polypeptide of the invention together with a pharmaceutical acceptable diluent or carrier. The carrier may be any of the well-known carriers for this purpose including normal saline solutions, preferably with a protein diluent such as bovine serum albumin to prevent adsorptive losses to glassware at these low concentrations. The polypeptides of this invention are generally active at a range of above 1 ug/kg of body weight, e.g. up to 10 ug/kg, while certain strikingly potent polypeptides are active from 0.1 ng/kg of body weight. For the treatment of DiGeorge Syndrome, the polypeptides may be administered at a rate of 1 to 100 ug/kg of body weight, while the strikingly potent polypeptides may be administered at a rate of 1 to 100 ng/kg of body weight. Generally, the same range of dosage amounts may he used in treatment of the other conditions or diseases mentioned. While the above discussion hae been given with respect to parenteral adminitration, it should be understood that oral administration is also possible at dosage ranges generally 100 to 1000 times greater than those for injection.
The polypeptides of this invention were prepared using the concepts similar to those described by Merrifield as reported in Journal of American Chemical Society, 85, pp. 2149-2154, 1963. The synthesis involved the stepwise addition of protected amino acids to a growing peptide chain which was bound by covalent bonds to a solid resin particle. By this procedure, reagents and by-products were removed by filtration and the recrystallization of intermediates were eliminated. The general concept of this method depends on attachment of the C-terminal amino acid of the chain to a solid polymer by a covalent bond and the addition of the succeeding amino acids one at a time in a stepwise manner until the desired sequence is assembled. Einally, the peptide is removed from the solid support and protective groups removed. Ihis method provides a growing peptide chain attached to a caipletely insoluble solid particle so that it is in a convenient form to be filtered and washed free of reagents and by-products.
In a particular aspect the present invention provides a method for manufacture of a polypeptide having the amino acid sequence H-X-LYS-Y-GTH-CH, wherein X and Y are each independently selected fran L-seryl, L-alanyl, L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl, L-leucyl, D-alanyl, D-seryl, D-asparagyl, D-glutamyl, D-threonyl, D-valyl, D-leucyl, sarcosyl, and 2-methyl-alanyl, which process catprises attaching L-glutamine having a protected amino group, to an insoluble resin polymer by convalent banding; removing the a-amino protecting group fran the Lglutamine moiety, reacting with a Y-amino acid having a protected a-amino group to couple the Y amino acid to the L-glutamine-resin; removing the α-amino protecting group fran the Y amino acid moiety, reacting with Llysine having a protected a-amino group to couple L-lysine to the Y-amino acid-L-glutamine-resin; removing the α-amino protecting group frctn the L-lysine moiety, reacing with an X amino acid having a protected a-amino group to couple the X amino acid to the L-lysine-Y-amino acid L-glutamine-resin reactive side chains on the amino 'acid reactants being protected during the reactions and removing all protecting groups and the resin fran the peptide.
In a further aspect the present invention provides a method for manufacture of a polypeptide having the amino acid sequence H-X-LYS-Y25 GIN-Nt^, vSierein X and Y are each independently selected fran L-seryl, Lalanyl, L-asparagyl, L-glutamyl, L-threcxiyl, glycyl, L-valyl, L-leucyl, D-alanyl, D-seryl, D-asparagyl, D-glutamyl, D-threcnyl, D-valyl, D-leucyl, sarcosyl, and 2-methylalanyl, which process catprises attaching L-glutamine having a protected amino group, to an insoluble resin polymer hy covalent banding; removing the a -amino protecting group fran the L-glutamine moiety, reacting with a Y amino acid having a protected α-amino group to couple the Y amino acid to the L-glutamine-resin; removing the a -amino protecting group fran the Y amino acid moiety, reacting with L-lysine having a protected α-amino group to couple L-lysine to the Y-amino acid-L-glutamine35 resin; removing the a -amino protecing group fran the L-lysine moiety, reacting with an X amino acid having a protected α-amino group to couple the X amino acid to the L-lysine-Y-amino acid L-glutamine-resin, cleaving the resin fran the peptide with amtcnia in dimathylformamide under amidating conditions, any reacting side chains on the amino acid reactants being protected during the reactions, and removing all protecting groups.
In a still further aspect the present invention provides a method for manufacture of a polypeptide having the amino acid sequence H-X-LYSY-GtN-NHj, wherein X and Y are each independently selected from L-seryl, L-alanyl, L-asparagyl, L-glutanyl, L-threcnyl, glycyl, L-valyl, L-leucyl, D-alanyl, D-seryl, D-asparagyl, D-glutarryl, D-threcnyl, D-valyl, D-leucyl, sarcosyl, and 2-methylalanyl, which process ccnprises amidating L-glutamine having a protected amino group, to a benzhydxylamlne resin polymer by covalent bending; removing the α-amino protecting group from the L-glutamine moiety, reacting with a Y amino acid having a protected a-amino group to couple the Y amino acid to the L-glutamine-resin; removing the α-amino protecting group fran the Y amino acid moiety, reacting with L-lysine having a protected α-amino group to couple L-lysine to the Yamino acid-L-glutamine-resin; removing the α-amino protecting group fran the L-lysine moiety, reacting with an X amino acid having a protected α-amino group to couple the X amino acid to the L-lysine-Y-amino acid Lglutamine-resin, any reactive side chains on the amino acid being protected during the reactions, and removing all protecting groups and the resin from the peptide. lh yet a further aspect the present invention provides a method for manufacture of the polypeptide of sequence R-X-LYS-Y-GIN-R' wherein X and Y are each selected from Irseryl, L-alanyl, L-asparagyl, L-glutanyl, L-threcnyl, glycyl, L-valyl, L-leucyl, D-alanyl, D-seryl, D-asparagyl, Dglutanyl, D-threcnyl, D-valyl, D-leucyl, sarcosyl, and 2-methyl-alanyl and R and R' are each selected frcm :- R R' Hydrogen OH Cj—Cy alkyl NH2 Cg-C^ aryl NHRy £7-^20 alkary·1· N(R ) Cy—C20 aralkyl <*7 C^—Cy alkanoyl C2—Cy alkenyl C2—C7 alkynyl wherein Ry is Cj-~-Cy alkyl, C2—Cy alkenyl, C2—Cy alkynyl, Cg—C2Q aryl, Cy—C2Q aralkyl, and Cy—C2Q alkaryl, which process comprises attaching Lglutamine having a protected amino group, to an insoluble resin polymer by covalent bending; removing the α-anino protecting group frcm the L47611 glutamine iroiety, reacting with a Y amino acid having a protected a -amino group to couple the Y amino acid to the Ir-glutamine-resin; removing the α-amino protecting group from the Y amino acid moiety, reacing with Llysine having a protected α-amino group to couple L-lysine to the Y-amino acid-Irglutamine-resin; removing the α-amino protecting group frcm the L-lysine moiety, reacing with an N-R substituted X amino acid having a protected α-amino group to couple the N-R substituted X amino acid to the L-lysine-Y-amino acid L-glutamine-resin, cleaving the resin fran the peptide with an acid(R'=€H), anmcnia (R* =5®^), a primary amine of formula NHjR? (R^fflR?) a secondary amine of formula or an alcohol of formula HOR^ (R'=0R^), any reactive side chains an the amino add being protected during the reactions, and removing all protecting groups.
In carrying out the processes of the inventicn, the amino adds may be attached to any suitable polymer which merely has to be readily separable firm the unreacted reagents. The polymer may be insoluble in the solvents used or may be soluble in certain solvents and insoluble in others. The polymer should have a stable physical form permitting ready filtration. It must contain a functional group to which the first protected amino acid can be firmly linked ty a covalent bend. Various insoluble polymers sdtable for this purpose are those such as cellulose, polyvinyl alcohol, polymethacrylate and sulfonated polystyrene but in the synthesis of this inventicn, there was generally used achlorcnethylated copolymer of styrene and divinylbenzene. Polymers which are soluble in organic solvents while being insoluble in aqueous solvents may also be used. Che such polymer is a polyethylene/glyool having a molecular weight of about 20,000, which is soluble in nethylene chloride but insoluble in water, lhe use of this polymer in peptide synthesis is described in F. Bayer and M. Mutter, Nature 237 , 512 (1972) and references ccntained therein.
The various functional groups cn the aminp acid which were active, but which were not to enter into the reactions, were protected by conventional protecting groups as used in the polypeptide art throughout the reaction. Thus, the functional group cn lysine was protected hy protecting groups which could be ranoved cn carcplsticn of the sequence wri-thout adversely affecting the polypeptide final product. In the synthesis fluorescamine was used to determine if coupling was complete hy an indication of positive fluorescence (see Felix, et al., Analyt, Biochem., 52, 377, 1973). If complete coupling wras not indicated, the coupling was repeated with the same protected amino acid before deprotecticn.
The C-terminal amino acid may be attached to the polymer in a variety of well-known ways. Summaries of methods for attachment to halomethyl resins are given in Horiki, et al., Chem Letters, pp 165-168 (1978) and Gisin, Helv. Chim. Acta, 56, 1476 (1973), and references given therein.
The general procedure involved initially esterifying L-glutamine, protected on its amino groups, to the resin in absolute alcohol containing an amine. The coupled amino acid resin was then filtered, washed with alcohol and water and dried. The protecting group on the α-amino group of the glutamine amino acid (e.g., t-BOC, i.e., t-butyloxycarbonyl), was then removed. The resulting coupled amino acid resin, having the free amino group, was then reacted with a protected L-serine, preferably alphat-BOC-O-benzyl-L-serine to couple the L-serine. The reactions were then repeated with protected L-lysine and L-alanine until the complete molecule was prepared. The sequence of reactions was carried out as follows: Resin | a-Rj^-Gln-OH a-R^-Gln-Resin Remove a-amino protecting group H.-Gln-Resiri 4 «-R-^-Ser-Gln-Res in Remove a-amino protecting group H-Ser-Gln-Resin R, R. a-R^-Lys-Ser-Gln-Resin Remove a-amino protecting group I 3 H-Lys-Ser-Gln-Resin α-R.-Ala-OH R, R,34, ,2 a-R^-Ala-Lys-Ser-Gln-Resin Remove all protecting groups and resin H-Ala-Lys-Ser-Gln-OH In the above sequence of reactions Rj is a protecting group of the α-amino group and R2 and R^ are protecting groups on the reactive side chains of the L-serine and L-lysine, respectively, which are not affected or re10 moved when R^ is removed to permit further reaction. Preferably, in the above intermediate pentapeptide resin, the term R^ stands for a protective grouping such as t-butyloxycarbonyl, R2 stands for benzyl or substituted benzyl (e.g., 4 chlorobenzyl), and R^ stands for substituted benzyloxycarbonyl (e.g., 2,6-dichlorobenzyloxycarbonyl). The resin is any of the resins mentioned above as being useful in the process.
After the final intermediate was prepared, the peptide resin was cleaved to remove the R^, R2» and R3 tecting groups thereon and the resin. The protecting groups were removed by conventional means, e.g., by treatment with anhydrous hydrogen fluoride, and the resulting free peptide was then recovered.
As pointed out above, in conducting the process, it is necessary to protect or block the amino groups in order to control the reaction and obtain the products desired. Suitable amino protecting groups which may be usefully employed include salt formation for protecting strongly-basic amino groups, or urethane protecting sub30 stitutes such as £-methoxy benzyloxycarbonyl and t-butyloxycarbonyl. It is preferred to utilize t-butyloxycarbonyl (BOC) or t-amyloxycarbonyl (AOC) for protecting the aamino group in the amino acids undergoing reaction at the carboxyl end of the molecule, since the BOC and AOC (t-amyloxycarbonyl) protecting groups are readily removed following such reaction and prior to the subsequent step (wherein such α-amino group itself undergoes reaction) by relatively mild action of acids (e.g., trifluoroacetic acid), which- treatment does not otherwise affect groups used to protect other reactive side chains. It will thus be understood that the α-amino groups may be protected by reaction with any material which will protect the amino groups for the subsequent reaction(s) but which may later be removed under conditions which will not otherwise affect the molecule. Illustrative of such materials are organic carboxylic acid derivatives which will acylate the amino group.
In general, any of the amino groups can be protected by reaction with a compound containing a grouping of the formula: II r4— 0 — c — wherein R^ is any grouping which will prevent the amino group from entering into subsequent coupling reactions and which can be removed without destruction of the molecule.
Thus, R^ is a straight or branched chain alkyl which may be unsaturated, preferably of 1 to 10 carbon atoms, and preferably halo- or cyano-substituted; aryl, preferably of 6 to 15 carbons; cycioalkyl, preferably of 5 to 8 carbon atoms; aralkyl, preferably of 7 to 18 carbon atoms; alkaryl, preferably of 7 to 18 carbon atoms; or heterocyclic, e.g., isonicotinyl. The aryl, aralkyl and alkaryl moieties may also be further substituted as by one or more alkyl groups of 1 to about 4 carbon atoms.
Preferred groupings for R include t-butyl, t-amyl, tolyl, xylyl and benzyl. Highly preferred specific amino-protecting groups include benzyloxycarbonyl; substituted benzyloxycarbonyl, wherein the phenyl ring is substituted by one or more halogens, e.g., Cl or Br; nitro; lower C^-C? alkoxy, e.g., methoxy; lcwar C^-Ct, alkyl; t-butyloxycarbonyl, t-anylaxycarbonyl; cyclohexyloxycarbonyl; vinyloxycarbonyl; adamantyloxycarbonyl; and biphenyliscprcpcaycarbcnyl.
Other protecting-gtoups which can be used include isonicotinyloxycarbonyl, phthaloyl, g-tolylsulfonyl and formyl.
In conducting the general process of the invention, the peptide is built by reaction of the free α-amino group with a compound possessing protected amino groups. For reaction or coupling, the compound being attacked is activated at its carboxyl group so that the carboxyl group can then react with the free ct-amino group on the attached peptide chain. To achieve activation the carboxyl group can be converted to any reactive group such as an ester, anhydride, azide, or acid chloride. Alternately, a suitable coupling reagent . may be added during the reaction. Suitable coupling reagents are disclosed, e.g., in Bodanszky, et al. Peptide Synthesis, Interscience, second edition, 1976, chapter five, including carbodiimides (e.g., dicyclocarbodiimide) and carbonyIdiimidizole.
It should also be understood that during these reactions, the amino acid moieties contain both amino groups and carloxyl groups and usually one grouping enters into the reaction while the other is protected.
Prior to the coupling step, the protecting group on the alpha or terminal amino group of the attacked peptide is removed under conditions which will not substantially affect other protecting groups, e.g., the group on the epsilon-amino of the lysine molecule. The preferred procedure for effecting this step is mild acidolysis, as by reaction at room temperature with trifluoroacetic acid.
As may be appreciated, the above-described series of process steps results in the production of the tetrapeptide of Formula III as follows: III. H-ALA-LYS-SER-GLN-OH This tetrapeptide contains one tetrapeptide segment of this invention necessary for biological activity . The substitution of certain natural or nan-natural amino acid residue for either or both of L-alanyl and L-seryl may be effected by replacing either or hoth of alanine and serine by the appropriately protected natural or non-natural amino acid in the above synthetic scheme, thus yielding the tetrapeptide of the following formula: IIIA. H-X-LYS-Y-GLN-OH wherein X and Y are as previously described. The substituted tetrapeptide of Formula II, wherein the terminal amino acid groups may be further substituted as described above, may then be prepared by reaction of the tetrapeptide of Formula (IIIA) or the protected peptide resin precursor with suitable reagents to prepare the desired derivatives. Reactions of this type such as acylation, esterificatien and amidaticn are, of course, veil-known in the art. Further, other amino acids defined under -R, R' and R above that is amino acid groups which do not affect the biological activity of the tetrapeptide sequence, may be added to either end of the peptide chain by the same sequence of reactions by which the tetrapeptide itself was synthesized.
While the solid phase technique of Merrifield has been used to prepare the subject polypeptides, it ie clearly contemplated that classical techniques described in, for example, M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience, 1966, may also be employed.
Identity and purity of the subject peptides were determined by such well-known methods as thin layer chromatography, electrophoresis or amino acid analysis. . 24 The following Examples are presented to illustrate the invention, but it is not to be considered as limited thereto. In the Examples, and throughout the specification, parts ; ratios and percentages are by weight unless otherwise indicated.
EXAMPLE I In preparation of the polypeptide of this invention, the following materials were purchased commercially: Alpha-BOC-L-Glutamine-o-nitrophenyl-ester Alpha-B0C-s-2-chloro-benzyloxycarbonyl-L-lysine Alpha-BOC-O-benzyl-L-serine Alpha-BOC-L-Alanine.
In these reagents, BOC is t-butyloxycarbonyl.
Sequenal grade reagents for amino acid sequence determinations, dicyclohexyl carbodiimide, fluorescamine, and the resin were also purchased commercially. -The resin used was a polystyrene divinyl benzene resin, 200-400 mesh size (U.S. Standard sieve series) containing. 1% divinyl benzene and .75 nM of chloride per gram of resin.
In preparation of the polypeptide, 2 mmoles of α-BOC-L-Glutamine were esterified to 2 mmoles of chloromethylated resin in absolute ethyl alcohol containing litM triethylamine for 24 hours at 80°C. The resulting amino acid resin ester was filtered, washed with absolute ethyl alcohol and dried. Thereafter, the other α-BOC-amino acids were similarly coupled to the deprotected α-amino group of the peptide-resin in the correct sequence to result in the polypeptide of this invention using equivalent amounts of dicyclohexyl carbodiimide. After each coupling reaction, an aliquot of resin was tested with fluorescamine and if positive fluorescence was found, coupling was taken to be incomplete and was repeated with the same protective amino acid. As a result of the several coupling reactions, the intermediate tetrapeptide-resin was prepared.
This peptide-resin was cleaved and the protective groups removed in a Kel-F cleavage apparatus (Peninsula Laboratories, Inc.) using anhydrous hydrogen fluoride at • 47611 0°C for 60 minutes with 1.2 ml anisole per gram peptideresin as scavenger. The peptide mixture was washed with anhydrous diethyl ether and extracted with aqueous acid. Hie extract was lyophilised and the peptide was chromatographed on P-6 Bio-Gel in 1 N acetic acid. The resulting polypeptide was determined to be 94% pure and was determined to have the following sequence: H-ALA-LYS-SER-GLN-OH For identification, thin layer chromatography and electrophoresis were performed as follows.
Thin layer chromatography was performed on a 30 ug sample on silica gel (Brinkman Silica Gel with fluorescent indicator, 20 x 20 cm, 0.1 mm thick) using the following eluents.
Rj^: n-butanol: pyridine: acetic acid:water; 30:15:3:12 2 Rf : ethyl acetate:pyridine:aeefcic acid:water; 5:5:1:3 R 3 f : ethyl acetate:n-butanol:acetic acid:water: 1:1:1:1 Electrophoresis was performed on 100 ug sample on Whatman 3 mm paper (11.5 x 56.5 cm) using a pH 5.6 pyridine-acetate buffer solution and 1000 volts potential for one hour. Whatman is a Registered Trade Mark.
Spray reagents for both thin layer chromatography and electrophoresis were Pauly and Ninhydrin.
The following results were obtained: R,3- = 3 1 immobile, Rf = immobile, and R^ = 0.336. Electrophoresis resulted in a migration of 9.4 cm toward cathode.
EXAMPLE IX To determine the activity and characteristics of the tetrapeptide produced in Example I, the following chicken induction assay was employed. This assay is described in greater detail in Brand, et al., Science, 193 319-321 (July 23, 1976) and references contained therein.
Bone marrow from newly-hatched chickens was selected as a source of inducible cells because it lacks ψ χ an appreciable number of Bu-1 or Th-1 cells. Pooled , Α7β1ί cells from femur and tibiotarsus of five newly-hatched chicks of strain SC (Hy-Line) were fractionated by ultracentrifugation on a five-layer discontinuous bovine serum albumin (BSA) gradient. Cells from the two lighter layers were combined, washed, and suspended for incubation at a concentration of 5 x 10® cells per milliliter with the appropriate concentration of test polypeptide in KPMI 1630 medium supplemented with 15 mM hepes, 5 percent γ-globulin-free fetal calf serum, deoxyribonuclease (14 to 18 unit/ml), heparin (5 unit/ml), penicillin (100 unit/ml) and streptomycin (100 ug/ml). Controls were incubated with BSA (1 μg/ml) or medium alone. After incubation, the cells were tested in the cytotoxicity assay using chicken Cl and guinea pig C2 to C9 complement fractions as described in the reference article. The proportion of Bu-1+ or Th-1+ cells in each layer was calculated as a cytotoxicity index, 100 (a-b)/a, where a and b are the percentages of viable cells in the complement control and test preparation, respectively. The percentage of cells induced was obtained by subtracting the mean values in the control incubations without inducing agents (usually 1 to 3 percent) from those of the test inductions.
The specificity of the action of the test polypeptide and its similarity to ubiquitin were demonstrated by the inhibition of induction of Bu-1+ B cells and Th-1+ T cells by the test polypeptide upon addition of ubiquitin in a concentration of 100 ug/ml. This high dose of ubiquitin inactivates the ubiquitin receptors and thus prevents the induction of cells by any agent which acts through these receptors.
As a result of this assay, it was discovered that the tetrapeptide of Example I displayed biological activity similar to that of ubiquitin in inducing the differentiation of both Th-1+ T and Bu-1+ B lymphocytes in ng/ml concentrations.
EXAMPLE XII A. The assay of Example II was repeated, using as the test polypeptide one of the following: H-GLN-ALA-LYS-SER-GLN-GLY-GLY-SER-ASN-OH H-GLN-ALA-LYS-SER-GLN-OH H-SAR-ALA-LYS-SER-GLN-OH In each case, biological activity similar to that of ubiquitin was observed.
B. The assay of Example II was repeated, using as the test polypeptide one of the following: h-sar-lys-d-ala-gln-nh2 h-sar-lys-sar-gln-nh2 h-d-ala-lys-d-ala-gln-nh2 In each case, biological activity similar to that of ubiquitin was observed. For the first of these polypeptides, this activity was observed in the range of concentration from 1 pg/ml to 100 pg/ml<- For the second polypeptide, activity was observed at a concentration as low as 0.1 pg/ml.
EXAMPLES IV - VI Using the reaction techniques described hereinabove for preparing substituted polypeptides, these are prepared polypeptides of the following formula: R-X-LYS-Y-GLN-R' These peptide amides were prepared on a benzhydrylamine resin by solid phase synthesis techniques known in the art EXAMPLE NUMBER R X ' Y R' IV H SAR D-ALA NH2 IVA H SAR SAR nh2 V H D-ALA D-ALA nh2 VA H D-ALA SAR nh2 VI H SAR 2-Me-ALA nh2 VIA H SAR SAR OH The polypeptides prepared in Examples iv-Vl retain the biological activity as described herein for the active polypeptide segment.
For identification, thin layer chromatography and electrophoresis were performed as follows.
Thin layer chromatography was performed on 20 ug samples on silica gel (Kieselgel, 5 x 20 cm) using as eluent n-butanol:acetic acid:ethyl acetate:water in proportions of 1:1:1:1 (Rf1) and on cellulose 6064 (Eastman, x 20 cm) using as eluent n-butanol:pyridine:acetic acid: water in proportions of 15:10:3:12 (Rf ).
Electrophoresis was performed on 50 ug samples on Whatman No. 3 paper (5.7 x 55 cm) using a pH 5.6 pyridine-acetate buffer solution and 1000 volts potential for one hour. The compounds migrate toward the cathode.
Spray reagents for both thin layer chromatography and electrophoresis were Pauly and Ninhydrin.
The following results were obtained (both Rf values and electrophoresis are LYS-ASP-VAL-TYR-OH): given relative to H-ARGElectrophoresis migration purity 2.07 98% 1.78 98% 2.10 98% chromatography and Example R 2 Rf IV 0.44 0.68 IVA 0.88 0.60 V 0.56 0.71 Following the thin : electrophoresis procedure of Example I, the following results are obtained for the compound of Example VI: H-l = 3 1 (0.155), Rf = immobile, Rf = 0.265 and electrophoresis migration is 13.1 cm toward cathode.
EXAMPLE VII The tetrapeptide resins having protected LYS and SER prepared as in Examples I and VIA are each acylated by reaction with acetic anhydride under acetylating conditions, followed by removal of the protecting groups and the resin, to prepare the following acylated derivatives: ch3co-ala-lys-SER-GLN-OH CH3CO-SAR-LYS-SAR-GLN-OH EXAMPLE VIII The protected tetrapeptide resins prepared as in Examples I and VIA are each transesterified from the resin fay reaction with sodium methoxide in methyl alcohol under transesterification conditions, followed by removal of the protecting groups, to prepare the esterified derivatives of the following formulas: H-ALA-LYS-SER-GLN-OCH3 h-sar-lys-sar-gln-och3 EXAMPLE IX The protected tetrapeptide resins prepared as in Examples I and VIA are each cleaved from the resin with diethyl amine under reaction conditions known in the art, followed by removal, of the protecting groups, to prepare the following amino substituted derivatives: H-ALA-LYS-SER-GLN-N(CjHg)2 h-sar-lys-sar-gln-n(c2h5)2 EXAMPLE X Following the methods of Examples I and VIA but substituting for the ALA or SAR used to add the N-terminal amino acid residue, an equivalent amount of suitably protected Ν-α-ethyl-L-alanine or Ν-α-ethyl-sarcosine, respectively, there are prepared the following: c2h5-ala-lys-ser-gln-oh C2H5-SAR-LYS-sar-gln-oh EXAMPLE XI Cleaving the protected resin tetrapeptides formed in Example X from the resin using ammonia in dimethylformamide under amidation conditions, followed by removal of the protecting groups yields peptide amides of the formulas: C2H5-ALA-LYS-SER-GLN-NH2 C2H5-SAR-LYS-SAR-GLN-NH2 EXAMPLE XII The protected acetylated tetrapeptide resins prepared as in Example VII are each reacted with anmonia in dimethylformamide under amidation conditions, followed by removal of the protective groups, to prepare the following peptide amides: ch3co-ala-lys-ser-gln-nh2 CH3CO-SAR-LYS-SAR-GLN-NH2 EXAMPLES XIII - XXVI Using the reaction techniques described hereinabove for the lengthening of the polypeptide chain, the following polypeptides are prepared which contain the active amino acid sequence but which are substituted on the terminal amino and carboxylic groups R and R' to provide the polypeptide of formula: R-ALA-LYS-SER-GLN-R' which is substituted by the amino acids given in the following Table as indicated.
EXAMPLE NUMBER R 51 XIII H-GLN OH XIV H-SAR OH XV a GLY-OH XVI H GLY-GLY-OH XVII H-GLN GLY-OH XVIII H-GLN GLY-GLY-OH XIX H-SAB GLY-OH XX H-SAR GLY-GLY-OH The polypeptide derivatives prepared in Examples IV-'XX retain the biological activity as described herein for the active polypeptide segment.
Following the thin layer chromatography and electrophoresis procedure of Example I for the compound of Examples XXII and tained: •XIV, the following results are ob- R 2Rf immobile immobile R 3Rf 0.303 0.186 electrophoresis migration toward cathode Example XIII XIV immobile immobile 7.4 cm 8.3 cm EXAMPLE XXI To further illustrate the utility of the subject polypeptides, this example describes a microculture assay for estimating the frequencies of the cytotoxic lymphocytes produced upon stimulation by allogenic antigens.
The frequencies of cytotoxic precursors between control animals and animals injected with various concentrations of the drug were compared by a limiting dilution assay.
Materials and Methods Mice Inbreed C57 BL/6J (female, 8 weeks) were obtained from Jackson Laboratory, Bar Harbor, Maine.
Inbreed DBA/2J (male or female) were also obtained from Jackson Laboratory, Bar Harbor, Maine.
Media Phosphate buffered saline (PBS), RPMI 1640, fetal calf serum (FCS) - (.lot number R776116) , N-2-hydrexyethylpiperazine-N'-2-ethanesulfonic aoid (HEPES) buffer were obtained from Gibo, Grand Island; 2-mercaptoethanol was from Eastman Kodak, Rochester, N.Y. Cells were washed with PBS and cultured in RPMI containing 10% FCS, 10 mM HEPES buffer and 5xlO-SM 2-mercaptoethanol.
Drug treatment The test animals (C57 BL/6J) were injected (i.v. or i.p.) with various concentrations of H-SAR-LYS-SARGLN-NH2 (the drug; identification no. GO40) in 0.2 ml volume 24 hours before they were sacrificed for the experiments.
Cell preparations Cell suspensions from spleens of C57 BL/6J mice or DBA/2J mice were prepared by mincing the organ and pressing them through a wire mesh (60 gauge) with the plunger of a 5 c.c. syringe into a falcon petri dish (falcon 3002, 15x60 mm). The cell suspensions were allowed to stand at room temperature for 10 minutes to let the big chunks of tissue settle. The cell suspensions were then transferred to 15 ml Corning centrifuge tubes (Corning 25310) and spun for 10 minutes at 1500 RM in the Beckman TJ-6 centrifuge. Beckman is a Registered Trade Mark. All cell suspensions were washed at least three more times with PBS. After the third wash.,,. the responder20 (C57 BL/6J) cells were resuspended in culture medium and counted in the Coulter (Coulter is a Registered Trade Mark) counter. DBA/2J (stimulator calls) were resuspended in HMI to 10 cells/ml. 30 pg of itiitoiycin C was added to each· ml of the DBA/2J spleen cells and the mixtures were incubated at 37° for 30 minutes. After mitomycin C treatment, the spleen cells were washed three times with PBS to remove any excess mitomycin C. The DBA cells were then resuspended in the culture medium and counted in the Coulter counter.
Mixed lymphocyte cultures (MLC) MLC were set up in microtiter trays (Linbro Chemicals, New Haven, Conn., IS-MVC-96). Each tray contained 96-v bottom wells. The outside wells surrounding the edge of the plate were not used for cell culture but filled with PBS to avoid evaporation from the culture wells. 60 samples were set up in each V-bottom tray. Usually each tray contained three responder cell concentrations (20 replicates of each) and one stimulator cell concentration. The responder cells were usually 5 5 suspended to concentrations of 7.5x10 , 5x10 and 2.5x10® per ml and 0.1 ml was added to each well. The stimulator cell concentrations used were 106, 2.5x10®, 5x10® per ml, also 0.1 ml was added to each well. The same stimulator cell concentration was used throughout the whole plate. Control plate containing only responder cells with no stimulator cells was also set up for estimating background stimulation due to the medium. The cells were cultured for six days at 37®C in a humidified incubator containing 5% CO2· Target cells The target cell used in the cytotoxic assay was a DBA mastocytoma cell line P815. The cell line was maino tained by routine passage through DBA/2J mice. 5x10 P815 cells were used for each passage and the tumor cells from the peritoneal cavity of the carriers were used four to five days after passage. The tumor cells from the peritoneal cavity were washed three times with PBS and then labeled with Cr at a concentration of 100 uci per 10? cells. Labeling was done for an hour at 37°c in a humidified incubator.
The labeled target cells were then washed for three times with PBS to remove any excess label.
Cytotoxic assay After six days of culture, 0.1 ml of medium was removed from each well without disturbing the cell pellet.
Then, using an automatic micropipet (MLA pipet), 100 4 51 pi of target cells, containing 2.5x10 Cr -labeled target cells were pipetted into each well, resuspending the cell pellet during the process (a new pipet tip has to be used for each well). The miorotiter trays were then spun at room temperature at 1000 RPM for seven minutes in the Sorvall GLC-2B. The trays were then incubated for four hours at 37°C. 100 microliters of supernatant were then removed into Gamma counting tubes (Amershen 196271). The tubes were then counted in the Beckman Gamma Counter (Beckman 310). The tubes were usually counted for one minute.
Determination of the frequencies of the precursors of cytotoxic lymphocytes (CLP) The limiting dilution analysis is an all or none response'assay described by the Poisson probability distribution. The probability of a non-response is given by the zero order term Po=e where δ = frequency of CLP and N = the number of lymphocytes per well. Thus a plot of the logarithm of the proportion of non-responding cultures vs cell dose should yield a straight line with a slope of -δ, the frequency of CLP.
In the Example, the background chromium release (spontaneous release) from 20 wells containing just responder cells (C57 BL/6J) with no stimulator cells were averaged. Test wells were scored as positive if their counts were greater than the mean spontaneous value by more than 2.07 standard deviations (P<0.05). The spontaneous lysis usually ranged from 9-15% of the totalcounts incorporated into the tar-6N get cells. According to the Poisson equation Po=*e , when Po=e~''· = 0.37 (corresponds to 37% non-responding cultures), δ = 1/N, thus the reciprocal of the responding cell number corresponding to 37% non-responding cultures is the CLP frequency. Usually the number of cells per well and their corresponding value for per25 cent non-responders were fitted into the computer which compute the best fit regression line through these points and the number of cells per well which correspond to 37% non-responding cultures, the reciprocal of that value is the frequency of the CLP.
Results The frequencies of cytotoxic lymphocyte precursors for each stimulator cell concentration were plotted as a function of drug dose. The mean and standard deviation of the twenty replicates were also computed for com35 parison. The three stimulator cell concentrations used were 10 (suboptimal stimulation), 2.5x10 (optimal stimulation) and 5x10^ (over optimal stimulation) . It was found that the test drug promoted the production of cytotoxic lymphocyte precursors at concentrations of from 1 pg/mouse to 100 ng/mouse (equivalent to 50 pg/kg to 5 ug/kg body weight) in the presence of suboptimal stimulator cell concentrations. The test drug is therefore acting as an immuno-regulator at these concentrations to increase the cellular immune response of the treated mice.
Claims (43)
1. CLAIMS:IA polypeptide having the capability of inducing the + + differentiation of both Th-1 T-lymphocytes and Bu-1 B-lymphocytes, which polypeptide has the following amino acid sequence R-X-LYS-Y-GLN-R' wherein X and Y are each natural or non-natural amino acid residues selected from L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl, L-leucyl, L-alanyl, L-seryl, sarcosyl, 2methylalanyl, D-asparagyl, D-glutamyl, D-threonyl, D-valyl, 10 D-leucyl, D-alanyl, and D-seryl and R and R' are each selected from R Hydrogen 0 χ -Cy alkyl C 6 -C 12 aryl Cy-C 2Q alkaryl Cy-C 2Q aralkyl -Cy alkanoyl C 2 -Cy alkenyl C 2 -Cy alkynyl H-GLN H-SAR R' OH nh 2 NHRy N(Ry) 2 GLY-OH GLY-GLY-OH wherein R ? is C 1 -Cy alkyl, C 2 -Cy alkenyl, C 2 -Cy alkynyl, Cg-C 2Q aryl, Cy-C^^ aralkyl, and C ? -C 2Q alkaryl, and the 25 pharmaceutically acceptable salts thereof; further provided that the polypeptide induces the differentiation of both Th-1 T-lymphocytes and Bu-1 + B-lymphocytes in the chicken induction assay test (as hereinbefore described ) at a concentration of one ng/ml or less.
2. A polypeptide as claimed in claim 1 wherein R is 5 hydrogen and R' is OH.
3. A polypeptide as claimed in claim 1 wherein R is CH^ CO- and R' is OH.
4. A polypeptide as claimed in claim 1 wherein R is CH 3 and R' is OH. 10 5. A polypeptide as claimed in claim 1 wherein R is H and
5.R' is NH 2
6. A polypeptide as claimed in claim 1 wherein R is H and R' is N(C 2 H 5 ) 2<
7. A polypeptide as claimed in claim 1 wherein R is 15 CHjCO- and R' is NH 2 ·
8. A polypeptide as claimed in claim 1 wherein R is H and R' is -OCH.,.
9. R' A polypeptide as is OC 2 H 5 . claimed in claim 1 wherein R is H and
10. A polypeptide as claimed in claim 1 wherein R is phenyl and R' is -OH.
11. A polypeptide as claimed in claim 1 wherein R is C 2 H 5 and R' is OC 2 Hg. ί
12. A polypeptide as claimed in c.’/irn 1 wherein R is C 2 H 5 and R' is -NH 2 · f I i
13. A polypeptide as claimed in ί claim 1 wherein R is f HGLN and R' is -OH. t I
14. A polypeptide as claimed in / claim 1 wherein R is 10 H-SAR and R' is -OH. ,?
15. A polypeptide as claimed im claim 1 wherein R is I hydrogen and R' is GLY-OH.
16. A polypeptide as claimed i- claim 1 wherein R is f hydrogen and R' is GLY-GLY-OH. / I 15
17. A polypeptide as claimed'in claim 1 wherein R is H GLN and R' is GLY-OH. t
18. A polypeptide as clairin in claim 1 wherein R is H-GLN and R' is GLY-GLY-OH. \ » f f ί i
19. A polypeptide as claimed in claim 1 wherein R is H-SAR and R' is GLY-OH.
20. A polypeptide as claimed in claim 1 wherein R is H-SAR and R' is GLY-GLY-OH. 5. 21. A polypeptide having the following amino acid sequence:
21.H-ALA-LYS-SER-GLN-OH and the pharmaceutically acceptable salts thereof.
22. A polypeptide having the following amino acid 10 sequence: R-X-LYS-Y-GLN-R wherein X and Y are each independently D-ALA or SAR and R and R' are each selected from: R R Hydrogen OH C^—Cy alkyl NH 2 C 6~ C 12 aryl NHRy Cy—C 2Q alkaryl N(Ry) 2 C ? —C 2Q aralkyl ORy C^—Cy alkanoyl C 2 —Cy alkenyl GLY-OH C 2 —Cy alkynyl GLY-GLY-OH R R H-GLN GLY-GLY-SER-OH H-SAR GLY-GLY-SER-ASN-OH wherein R ? is —C ? alkyl, C 2 —C ? alkenyl C 2 —C? alkynyl, 5 Cg—Ο 2 θ aryl, C?-C 2Q aralkyl, and ^7 - ^20 alkaryl, and the pharmaceutically acceptable salts thereof.
23. A polypeptide having the following amino acid sequence: h-sar-lys-sar-gln-nh 2 6. 10 and the pharmaceutically acceptable salts thereof.
24. A polypeptide having the following amino acid sequence: h-sar-lys-d-ala-gln-nh 2 and the pharmaceutically acceptable salts thereof.
25. 15 25. A therapeutic composition comprising a therapeutically effective amount of a polypeptide as claimed in any one of claims 1 to 21 together with a pharmaceutically acceptable carrier or diluent.
26. A therapeutic composition as claimed in claim 25 8. 20 wherein the therapeutically effective amount of the polypeptide is in the range of from 1 to 100 μg/kg body weight.
27. A therapeutic composition comprising a therapeutically effective amount of polypeptide as claimed in claim 22 or claim 23 together with a pharmaceutically acceptable carrier or diluent.
28. A therapeutic composition as claimed in claim 27 wherein the therapeutically effective amount of the polypeptide is in the range of from 0.1 ng/kg to lOyg/kg body weight.
29. A method for manufacture of a polypeptide having the amino acid sequence H-X-LYS-Y-GLN-OH, wherein X and Y are each independently selected from L-seryl, L-alanyl, L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl, L-leucyl, D-alanyl, D-seryl, D-asparagyl, D-glutamyl, D-threonyl, D-valyl, D-leucyl, sarcosyl, and 2-methyl-alanyl, which process comprises attaching L-glutamine having a protected amino group, to an insoluble resin polymer by covalent bonding; removing the a -amino protecting group from the L-glutamine moiety, reacting with a Y amino acid having a protected α-amino group to couple the Y amino acid to the L-glutamine-resin; removing the a-amino protecting group from the Y amino acid moiety, reacting with L-lysine having a protected α-amino group to couple L-lysine to the Y-amino acid-L-glutamine-resin; removing the α-amino protecting group from the L-lysine moiety, reacting with an X amino acid having a protected a-amino group to couple the X amino acid to the L-lysine-Y-amino acid L-glutamine-resin, any reactive side chains on the amino acid reactants being protected during the reactions, and removing all protecting groups and the resin from the peptide.
30. A method as claimed in claim 29 wherein the resin polymer is cellulose, polyvinylalcohol, polymethylmethacrylate, sulfonated polystyrene, or a chloromethylated copolymer of styrene and divinylbenzene.
31. A method as claimed in claim 29 or claim 30 wherein X and Y are both sarcosyl.
32. A method for manufacture of a polypeptide having the amino acid sequence H-X-LYS-Y-GLN-NH 2 , wherein X and Y are each independently selected from L-seryl, L-alanyl, L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl, L-leucyl, D-alanyl, D-seryl, D-asparagyl, D-glutamyl, D-threonyl, D-valyl, D-leucyl, sarcosyl, and 2-methylalanyl, which process comprises attaching L-glutamine having a protected amino group, to an insoluble resin polymer by covalent bonding; removing the a-amino protecting group from the L-glutamine moiety, reacting with a Y amino acid having a protected a-amino group to couple the Y amino acid to the L-glutamine-resin; removing the a-amino protecting group from the Y amino acid moiety, reacting with Llysine having a protected α-amino group to couple L-lysine to the Y-amino acid-L-glutamine-resin; removing the α-amino protecting group from the L-lysine moiety, reacting with an X amino acid having a protected α-amino group to couple the X amino acid to the L-lysine-Y-amino acid L-glutamine-resin, cleaving the resin from the peptide with ammonia in dimethylformamide under amidating conditions, any reactive side chains.47611 on the amino acid reactants being protected during the reactions, and removing all protecting groups.
33. A method as claimed in claim 32 wherein the resin polymer is cellulose, polyvinylalcohol, polymethylmethacrylate, sulfonated polystyrene, or a ohloromethylated copolymer of styrene and divinylbenzene.
34. A method as claimed in claim 32 or claim 33 wherein X and Y are both sarcosyl.
35. A method for manufacture of a polypeptide having the amino acid sequence H-X-LYS-Y-GLN-NHj, wherein X and Y are each independently selected from L-seryl, L-alanyl, L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl, L-leucyl, D-alanyl, D-seryl, D-asparagyl, D-glutamyl, D-threonyl, D-valyl, D-leucyl, sarcosyl, and 2-methylalanyl, which process comprises amidating L-glutamine having a protected amino group, to a benzhydrylamine resin polymer by a covalent bonding? removing the α-amino protecting group from the L-glutamine moiety, reacting with a Y amino acid having a protected α-amino group to couple the Y amino acid to the L-glutamine-resin; removing the α-amino protecing group from the Y amino acid moiety, reacting with L-lysine having a protected α-amino group to couple L-lysine to the Y-amino acid-L-glutamine-resin? removing the α-amino protecting group from the L-lysine moiety, reacting With an X amino acid having a protected α-amino group to couple the X amino acid to the L-lysine-Y-amino acid L-glutamine-resin, any reactive side chains on the amino acid being protected during the reactions, and removing all protecting groups and the resin from the peptide.
36. A method as claimed in claim 35 wherein X and Y are both sarcosyl.
37. A method for manufacture of the polypeptide of seguence R-X-LYS-Y-GLN-R' wherein X and Y are each selected from L-seryl, L-alanyl, L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl, L-leucyl, D-alanyl, D-seryl, D-asparagyl, D-glutamyl, D-threonyl, D-valyl, D-leucyl, sarcosyl, and 2-methyl-alanyl and R and R' are each selected from:— R R' Hydrogen OH —Cy alkyl nh 2 C 6~ C 12 aryl NHRy Cy—C 2Q alkaryl N(Ry) Cy—C 2Q aralkyl ORy C^—Cy alkanoyl C 2 —-Cy alkenyl C 2 —Cy alkynyl wherein Ry is —Cy alkyl, C 2 —C ? alkenyl, C 2 —Cy alkynyl, Cg—C 2Q aryl, Cy—C 2Q aralkyl, and Cy—C 2Q alkaryl, which process comprises attaching L-glutamine having a protected amino group, to an insoluble resin polymer by covalent bonding; removing the α-amino protecting group from the Lglutamine moiety, reacting with a Y amino acid having a pro45 tected α-amino group to couple the Y amino acid to the Lglutamine-resin; removing the α-amino protecting group from the Y amino acid moiety, reacting the L-lysine having a protected α-amino group to couple L-lysine to the Y-amino acid5 L-glutamine-resin; removing the α-amino protecting group from the L-lysine moiety, reacting with an N-R substituted X amino acid having a protected α-amino group to couple the N-R substituted X amino acid to the L-lysine-Y-amino acid L-glutamine-resin, cleaving the resin from the peptide with an 10 acid (R'=0H), ammonia (R'=NH 2 ), a primary amine of formula NH 2 R ? (R'=NHRy) a secondary amine of formula NH(Ry) 2 /fe'=N(Ry)/ or an alcohol of formula HORy (R'=ORy), any reactive side chains on the amino acid being protected during the reactions, and removing all protecting groups. 15
38. A method as claimed in claim 37 wherein the resin polymer is cellulose, polyvinylalcohol, polymethylmethacrylate, sulfonated polystyrene, or a chloromethylated copolymer of styrene and divinylbenzene.
39. A method as claimed in claim 29 substantially as 20 hereinbefore described.
40. A method as claimed in claim 32 substantially as hereinbefore described.
41. A method as claimed in claim 35 substantially as hereinbefore described.
42. A method as claimed in claim 37 substantially as hereinbefore described.
43. A polypeptide whenever produced by a method as claimed in any one of claims 29 to 42.
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US85849677A | 1977-12-08 | 1977-12-08 | |
US94053178A | 1978-09-08 | 1978-09-08 | |
US05/960,550 US4232008A (en) | 1978-11-17 | 1978-11-17 | Tetrapeptides and methods |
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CA (1) | CA1120031A (en) |
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IT (1) | IT1110889B (en) |
NL (1) | NL7812004A (en) |
NO (1) | NO149631C (en) |
NZ (1) | NZ189101A (en) |
PT (1) | PT68883A (en) |
SE (1) | SE444687B (en) |
YU (1) | YU41322B (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4215112A (en) * | 1979-03-14 | 1980-07-29 | Ortho Pharmaceutical Corporation | Tripeptides and methods |
US4215111A (en) * | 1979-03-14 | 1980-07-29 | Ortho Pharmaceutical Corporation | Peptides having ubiquitin-like activity |
CA1156220A (en) * | 1979-04-26 | 1983-11-01 | George Heavner | Method and composition for preparation of h-sar-lys-sar-gln-nh.sub.2 |
US4426324A (en) * | 1979-09-28 | 1984-01-17 | Hoffmann-La Roche Inc. | Immunopotentiating peptides |
JPS5711950A (en) | 1980-06-25 | 1982-01-21 | Kureha Chem Ind Co Ltd | Peptide and its synthesis |
FR2546164B1 (en) * | 1983-05-16 | 1987-07-17 | Centre Nat Rech Scient | NOVEL PEPTIDE DERIVATIVES, THEIR PREPARATION AND THEIR APPLICATION AS ELASTASE INHIBITORS |
FR2741076B1 (en) | 1995-11-15 | 1998-01-30 | Rech De Pathologie Appliquee S | PEPTIDE CONJUGATES DERIVED FROM THERMAL HORMONES, THEIR USE AS MEDICAMENTS AND COMPOSITIONS CONTAINING THEM |
RU2210382C1 (en) * | 2002-07-09 | 2003-08-20 | Терентьев Александр Александрович | Peptide with immunoregulating property and composition based on thereof |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2544348C3 (en) * | 1975-10-03 | 1979-12-13 | Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V., 3400 Goettingen | L-leucine-13-motiline, process for its preparation and agent containing the same |
FR2423481A2 (en) * | 1978-04-21 | 1979-11-16 | Anvar | Polypeptide analogues of serum thymus factor - with thymus hormonal or antagonistic activity |
FR2391994A1 (en) * | 1977-05-25 | 1978-12-22 | Anvar | Polypeptide analogues of serum thymus factor - with thymus hormonal or antagonistic activity |
CH637111A5 (en) * | 1977-05-25 | 1983-07-15 | Anvar | POLYPEPTIDE COMPOUNDS WITH THERMAL OR ANTAGONIST ACTIVITY AND METHODS OF SYNTHESIS THEREOF. |
-
1978
- 1978-12-06 NZ NZ189101A patent/NZ189101A/en unknown
- 1978-12-07 DK DK554078A patent/DK149595C/en not_active IP Right Cessation
- 1978-12-07 GR GR57816A patent/GR65013B/en unknown
- 1978-12-07 NO NO784128A patent/NO149631C/en unknown
- 1978-12-07 FI FI783769A patent/FI67368C/en not_active IP Right Cessation
- 1978-12-07 DE DE19782853002 patent/DE2853002A1/en not_active Withdrawn
- 1978-12-07 FR FR7834508A patent/FR2411174A1/en active Granted
- 1978-12-07 PT PT68883A patent/PT68883A/en unknown
- 1978-12-07 ES ES475857A patent/ES475857A1/en not_active Expired
- 1978-12-07 GB GB7847642A patent/GB2014581B/en not_active Expired
- 1978-12-07 SE SE7812614A patent/SE444687B/en not_active IP Right Cessation
- 1978-12-07 IT IT52239/78A patent/IT1110889B/en active
- 1978-12-07 CA CA000317547A patent/CA1120031A/en not_active Expired
- 1978-12-07 IE IE2423/78A patent/IE47611B1/en unknown
- 1978-12-07 IL IL56150A patent/IL56150A/en unknown
- 1978-12-08 CH CH1256678A patent/CH642058A5/en not_active IP Right Cessation
- 1978-12-08 NL NL7812004A patent/NL7812004A/en not_active Application Discontinuation
- 1978-12-08 JP JP15116378A patent/JPS5498719A/en active Granted
- 1978-12-08 YU YU2880/78A patent/YU41322B/en unknown
Also Published As
Publication number | Publication date |
---|---|
DE2853002A1 (en) | 1979-06-13 |
DK554078A (en) | 1979-06-09 |
CA1120031A (en) | 1982-03-16 |
CH642058A5 (en) | 1984-03-30 |
GB2014581B (en) | 1982-05-19 |
PT68883A (en) | 1979-01-01 |
FR2411174B1 (en) | 1984-05-25 |
FI783769A (en) | 1979-06-09 |
ES475857A1 (en) | 1980-01-16 |
IT1110889B (en) | 1986-01-06 |
DK149595C (en) | 1987-03-23 |
FI67368C (en) | 1985-03-11 |
GR65013B (en) | 1980-06-12 |
NL7812004A (en) | 1979-06-12 |
NO149631B (en) | 1984-02-13 |
JPS6327360B2 (en) | 1988-06-02 |
IE782423L (en) | 1979-06-08 |
DK149595B (en) | 1986-08-04 |
FR2411174A1 (en) | 1979-07-06 |
SE7812614L (en) | 1979-06-09 |
SE444687B (en) | 1986-04-28 |
NZ189101A (en) | 1984-07-06 |
YU41322B (en) | 1987-02-28 |
IT7852239A0 (en) | 1978-12-07 |
IL56150A0 (en) | 1979-03-12 |
FI67368B (en) | 1984-11-30 |
GB2014581A (en) | 1979-08-30 |
YU288078A (en) | 1983-02-28 |
IL56150A (en) | 1982-02-28 |
NO149631C (en) | 1984-05-23 |
NO784128L (en) | 1979-06-11 |
JPS5498719A (en) | 1979-08-03 |
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