JPH0586377B2 - - Google Patents

Description

Claim 1: A pharmaceutical composition for vasodilation, containing as an active ingredient a peptide having the following structure: [Table]. 2. The pharmaceutical composition of claim 1 for lowering blood pressure and/or increasing heart rate. 3. The pharmaceutical composition of claim 1 for use in treating hypertension. 4 Claim 1 for use in adjusting cardiovascular function
Pharmaceutical composition. FIELD OF THE INVENTION The present invention relates to peptides, pharmaceutical compositions, methods for producing peptides, genes encoding peptides, vectors containing this gene and host organisms transformed with this vector, and gene probes and antibodies for use in diagnosis. . BACKGROUND OF THE INVENTION The calcitonin gene system has been the subject of considerable research in recent years. In particular, studies of latcalcitonin gene expression have demonstrated the generation, by RNA processing, of apparently tissue-specific selective mRNA species to produce various peptide hormones from a single gene (Craig. R. K. et al. (1983), Genetic Engineering 4 , 57-125.
and Amara et al. (1982) Nature.
298, 240-244). Each mRNA species encodes a polyprotein that is divided as a result of post-translation processing and produces rat calcitonin or rat calcitonin gene-related peptide (rat CGRP). Rat CGRP is known to be widely distributed in discontinuous regions of the central and peripheral nervous systems, where it was found to have potential biological activity (Fitschier et al. (1983), Nature, 305 ). , 534-536). Pending European Patent Application EP-A ₁ −
No. 0070186 and EP-A ₁ -0070675 (R, K., Craig and I., Matsukintyre) describe a method for the production of human calcitonin and a carboxy-terminal peptide (katacalcin) designated PDN-21. Human calcitonin and katacalcin are produced as polypeptides encoded by human calcitonin mRNA (Craig et al. (1982) Nature, 295 , 345-347). The present inventors have discovered a human calcitonin gene region that is transcribed into human medullary carcinoma cells and located at the end of the 3' translated region of human calcitonin mRNA.
This region consists of a transcribed intronic region, a splice junction, an open reading frame encoding a previously unknown human peptide sequence, and a 3' untranslated region ending in a region of polyA residues. One of these peptides was apparently an analogue of rat CGRP, which has since been referred to as human calcitonin gene-related peptide (human
CGRP). SUMMARY OF THE INVENTION According to a first feature of the invention, human calcitonin gene-related peptides are provided. According to a second feature of the invention, there is provided a peptide having the structure: This novel peptide is formed as part of a polyprotein that is specifically cleaved in vivo within the secretory pathway by proteolytic enzymes that recognize flanking basic amino acid residues. This peptide (Tatemoto and Matsuto (1981) PNAS 78 , 6603-6607
PAF-37) was found to have potential biological effects in regulating cardiovascular function. In particular, this peptide was found to induce a decrease in blood pressure and increase heart rate and force. In a second aspect of the invention, the peptide is provided for use as a medicament, preferably for the treatment of hypertension. During the post-operative phase of major cardiac surgery (e.g. open heart bypass surgery), patients
It is common to respond to the stress of surgery with extensive vasoconstriction. This has the effect of raising the patient's blood pressure, making the heart strain. It has been found that the peptides of the invention act as antihypertensive agents and increase heart rate strength. These linking effects have potential applications in post-surgical treatment. Many people suffer from hypertension, and it is a high causative factor in the high incidence of heart disease. This peptide can be used for hypertension management. In a third aspect of the invention, there is provided a pharmaceutical composition comprising a peptide of structure () and a pharmaceutically acceptable excipient. The composition may be injectable. The pharmaceutical composition may be contained within, or form part of, a slow release composition or peptide system within the body. Such slow release forms are used for long-term hypertension control. Additionally, the present invention provides a method for treating hypertension, comprising administering an effective amount of a peptide of structure (). A third aspect of the invention also provides a method for making a pharmaceutical composition in which a peptide of structure () is combined with a pharmaceutically acceptable excipient. Peptides of structure () can be produced by various methods. This peptide can be produced by chemical peptide synthesis using techniques known in the art. Purification of peptides can be performed using ionic, reversed phase chromatography techniques. In another feature of the invention, the amino acid sequence of at least the following formula: [Table] (wherein R' is -H or an amino acid residue)
or a peptide that can be converted into an amide in vivo or in vitro. R′ is −
Gly−, −Gly−Arg−Arg−Arg−Arg or −Gly
-Lys-Lys-Arg may be used, but -Gly is preferable.
The carboxy-terminal residue R' can be enzymatically converted to the amide of the adjacent phenylalanine amino acid residue in vivo or in vitro. When R' is -Gly, suitable enzymes for this conversion are yeast carboxypeptidase Y or mammalian amidating enzymes (Platbury, A., F., Finney, M., D. A. and Smith, D. G. (1982) Nature (Nature), 298 , 686-688). Alternatively and preferably, the peptide is produced by recombinant DNA technology. As a fourth feature of the present invention, the intermediate peptide is a peptide according to the fourth feature of the present invention, and the intermediate peptide is obtained by culturing a host organism transformed with a vector containing a gene encoding an intermediate peptide. Then R′ is converted to amide, structure ()
The present invention provides a method for producing a peptide. The intermediate peptide may be a fusion protein comprising at least a portion of the protein produced in the transformed host organism and the amino acid sequence described in () above. Desirably, the fusion protein comprises a protein that is produced at high levels by the transformed host organism. Suitable proteins include at least in part chloramphenicol acetyltransferase (CAT) protein or at least in part β-galactosidase protein. The fusion protein preferably comprises a peptide having the amino acid sequence described in () above attached to the carboxy terminal group of the protein produced at high levels in the transformed host organism. The peptide may be linked to the protein via a selective chemical or enzymatically degradable linkage. This bond may be a methionine or glutamic acid residue. Methionine can be selectively cleaved by cyanogen bromide and glutamic acid can be cleaved selectively by sorghum-derived acid protease (EC3.4.23.14), sea urchin protease (EC3.4.24.12) or preferably Staphylococcus protease (EC3. 4.21.19) can be selectively cleaved (human CGRP
Sequence does not contain Met or Glu residues). The bond may be a lysine-arginine peptide difunctional group. Cleavage of this bond can be accomplished using mouse submandibular gland protease or preferably clostripan (EC 3.4.21.6). The peptides according to the first and second features of the present invention are produced in the body as polyproteins. This polyprotein white matter is processed in the body, and according to the first or second method of the present invention.
Cleave the amino and carboxy terminal groups leaving a characteristically amidated 37 amino acid peptide. As a sixth feature of the present invention, the intermediate peptide is obtained by culturing a eukaryotic host organism transformed with a vector containing a gene encoding an intermediate peptide comprising at least the following amino acid sequence: A method for producing a peptide having the structure () is provided, wherein the peptide is processed by a host organism and the peptide is then isolated. Preferably, the eukaryotic host organism is a tissue cultured mammalian cell line capable of containing proteolytic and amidating enzymes capable of producing structural peptides from polyproteins containing amino acid sequences. Desirably the intermediate peptide is produced as a fusion protein. In the processing of polyproteins, tetrapeptides are produced. This tetrapeptide also has biological activity, which is a feature of the present invention, and has the sequence of the following formula: Asp-Leu-Gln-Ala (). This peptide, designated PDA-4, was found to have biological activity in regulating cardiovascular function. In particular, this peptide was found to induce a decrease in blood pressure and increase heart rate. An eighth feature of the present invention is that the peptide of the sequence is used as a medicine, preferably for the treatment of hypertension. A ninth feature of the invention is to provide a pharmaceutical composition comprising a peptide of sequence () and a pharmaceutically acceptable excipient. The composition may be injectable. The pharmaceutical composition can be contained within or form part of a slow-release composition or peptide system in vivo. The composition may contain a combination of a peptide of structure and a peptide of sequence and a pharmaceutically acceptable excipient. A ninth feature of the present invention is to provide a gene (structure) encoding PAF-37, PAF-37-R' (structure), PDA-4 (structure), or a "non-processing" peptide (structure). Preferably,
The present invention is directed to the following amino acids (lower half of Figure 2) 1-
37,)-3 to -7,)+6 to +9 or)-7
A specific gene is provided that is described by nucleotides corresponding to ~+9 amino acids. The present invention also provides a nucleotide sequence substantially shown in FIG. 2 from nucleotides 1 to 1256. The invention further provides vectors containing any of these genes and host organisms transformed with the vectors. Suitable host organisms include bacteria (eg, E. coli), yeast (eg, Saccharomyces cerevisiae), and mammalian cells in tissue culture. Production of human CGRP is selectively mediated by tissue, mRNA
It is an organization based on processing (Edbruck, M.R. et al., Nature).
(1984) - raised). In some cells, such as those found in medullary thyroid carcinoma and lung cancer, human CGRP is produced at varying levels. Therefore, the presence of human CGRP can be diagnostic of abnormal tissue. Furthermore, the present invention
We will provide diagnostic reagents (including antibodies and gene probes) used to test CGRP gene expression and abnormal genetic tissue. As an eleventh feature of the invention, the invention provides a structural peptide, or portion thereof, containing an antigenic determinant (with a detectable label). The label may be an enzyme, a chromophore, a fluorophore or a chemiluminescent group. Most desirable, however, is a radioactive isotope, such as 125I, which binds to the histidine amino acid residue at position 5 of the peptide's amino acid sequence.
The labeled peptide is a structural peptide or 125I
It is preferable to include a portion thereof containing an optionally labeled tyrosine amino acid residue. The peptide moiety is preferably amino acids 25-37 (amidated phenylalanine) or amino acids 1-8. A twelfth feature of the present invention is to provide antibodies having specificity for antigenic determinants of the peptide structure. This antibody may be polyclonal or monoclonal. Antibodies can be labeled with a detectable label. The reagents of the eleventh and twelfth aspects of the invention may be those of immunoassays, preferably radioimmunoassays, those of peptide structures in samples, or those of immunocytochemistry of tissue parts. mRNA encoding PAF-37 or its mRNA
It is also possible to test for nucleotide sequences containing. Therefore, as the eleventh feature of the present invention, FIG.
Selected from the nucleotide sequences 1 to 1256 shown in
The purpose of the present invention is to provide a DNA hybrid probe having 15 or more nucleotide sequences. Probes can be immobilized on a solid phase or labeled with a detectable label. Such probes can be used to examine gene organization and gene expression using DNA or RNA blotting techniques, or to confirm hybrid cells in situ in tissue areas expressing genes. Preferred probes of the thirteenth aspect of the invention have sequences selected from the 1-725 nucleotide sequences shown in FIG. This desirable type of probe is directed against human calcitonin gene organization or expression.
It can be used to test human CGRP. In particular, this probe hybridizes to human CGRP mRNA and the human CGRP structural gene. Another preferred probe of the thirteenth feature of the invention consists of a sequence selected from the 726-1256 nucleotide sequence shown in FIG. This desirable type of probe can be used to test organisms for the human calcitonin CGRP gene. Specifically, this probe hybridizes to the intron region of the human CGRP structural gene. The invention will be explained by the following description with reference to the following drawings. FIG. 1 is a schematic diagram of overlapping cDNA sequences cloned in recombinant plasmids phTB3, phTB6 and phTB58. Figure 2 is the complete nucleotide sequence derived from recombinant plasmids phTB3, phTB6 and phTB58. Figure 3 shows plasmids pCT201, pCT202, pcT203
and a schematic diagram of the structure of pCAT-CGRP. FIG. 4 is a polyacrylamide gel showing the presence of a fusion protein of chloramphenicol acetyltransferase (CAT) and human CGRP in an extract of E. coli HB101 transformed with vector-pCAT-CGRP. Figure 5 shows E. coli transformed with pCAT-CGRP.
1 is a graph demonstrating the results of radioimmunoassay showing the presence of human CGRP antigenic determinant in protein extract of HB101. Figure 6 shows the drug response curves of heart rate and mean arterial pressure in rats (●=saline, ○=human
CGRP, △=rat CGRP, □=human CGRP + propranolol, ■=human CGRP + mepyramine +
cimetidine). Figure 7 is a tachograph trace demonstrating the effect of intravenously injected PDA-4 (and human CGRP) in rats on heart rate and mean arterial pressure. FIG. 8 shows the drug response curve of heart rate and force in guinea pigs. FIG. 9 is a graph demonstrating radioimmunoassay results showing the presence of human CGRP in medullary thyroid cancer tissue, plasma extracts of patients with medullary thyroid carcinoma, and production of CGRP by lung cancer cell lines. Figure 10 shows RNA blots demonstrating differential expression of calcitonin and human CGRP RNA in medullary thyroid carcinoma and lung carcinoma cell lines. FIG. 11 shows DNA blots demonstrating various organisms with genes homologous to the human CGRP gene when comparing DNA from human placental tissue with DNA from medullary thyroid carcinoma tissue or lymphocytes from a single patient. Pending European Patent Application No. EP-A1-
No. 0070675 describes the construction of a cDNA library using whole-cell poly(A)-containing RNA isolated from human medullary thyroid cancer tissue (Alison J. et al., Biochem J.).
(1981) 199 725-731) and nucleotide sequence analysis of the majority of human calcitonin mRNA cloned in two plasmids isolated from this library, phT-B3 and phT-B6 (Craig, R.K. et al. Nature (1982) 295 , 345-347). During this study, approximately 1600 bp of inserted cDNA
One recombinant plasmid containing the fragment phT-
B58 was identified through preliminary screening but was not analyzed further. This is because it was not thought to represent calcitonin mRNA since it is a very large molecule. Subsequent restriction enzyme analysis of the cDNA inserted into this plasmid (see Figure 1) revealed a position common to phTB3 and also to phT-B58.
The cDNA cloned within represents the sequence downstream from the pre-established sequence and represents human calcitonin mRNA.
It was pointed out that it shows a complete 3′ untranslated region of . Figure 1 shows the restriction sites separating the regions of the sequence used as sequence-specific hybrid probes and the relative positions of calcitonin, CGRP, and the common amino terminal peptide. The vertical dashed line indicates the Pst site separating the cDNA and plasmid sequences. phT−
Nucleotide sequence analysis of the entire cDNA sequence inserted into B58 was performed as previously described (Craig, R.K., Hall, L., Edbruck, M.R., Allison, G.I., and Matsukintyre, I. (1982), Nature. 295 , 345～
347) - This was done using the reference figure 2. The numbers compare the known nucleotide sequences of human and latcalcitonin and CGRA RNA transcripts with gaps introduced to maximize homology. The numbers immediately above the human nucleotide sequence indicate the relative number of nucleotides from the poly(A) tail cloned into phT B58.
The number on the human protein sequence is calcitonin or
Refers to the amino acid position relative to CGRP. Alternative or new amino acids or nucleotides present in the rat relative to the human sequence are shown immediately below the codon in question. Potential polyadenylation symbols are underlined. The arrow (↑) refers to the 3' end of mature calcitonin mRNA, and the (▲) mark points to the possible location of an additional intron in the human genome sequence. The dashed line indicates the region of the human sequence without the latcalcitonin gene. The inserted cDNA sequence is
It contains 1615 bp and ends with a poly(A) residue at the 3' end. Of these, the first 356 bp from the 5′ end are the same as those described above, and are part of human calcitonin mRNA encoding katacalcin.
AATAAA box and mature calcitonin mRNA
The entire 3′ untranslated region is shown, including the 12 nucleotides shown preceding the poly(A) tail.
Analysis of the remaining sequence showed that it contained a single open reading frame encoding 53 amino acids followed by a terminal codon. This is the splice junction receptor site (C) _o
NAG/G (Mt. S.M., Nucleic Acid Res.) (1982)
10 450-463) immediately 5' and an "intron"-like sequence of 645 nucleotides separated from the known calcitonin mRNA sequence by three adenosine residues. However, no recognizable donor splice junction was present between the intron-like sequence and the sequence known to be present in human calcitonin mRNA. The new open reading frame follows a region of 451 nucleotides containing 20 polyadenylene symbols, the first (AATAA) 26 bases downstream of the terminal codon and the second (AAAATTAAAAA) 18 bases before the terminal poly(A) region. located at the nucleotide. The coding sequence consists of a pair of basic amino acids (-1, -2), plus five amino acids (-3 to -7) at the amino terminus and a glycine residue (+1), four basic amino acids (+2 to +
5) and human CGRP (37 amino acid peptide) flanked at the carboxyl terminus by a tetrapeptide (+6 to +9). The presence of glycine reflects the in vivo requirement for amidated carboxyl-terminated phenylalanine (Bradbury A.F. et al., Nature (1982) 298 , 686-
688), 3786 calculations for amidated human CGRP
I became Mr. Comparison of the predicted human amino acid sequence with that of the rat (Amara et al., Nature (1982) 298 , 240-244) showed sequence conservation, with 7 amino acids changed from 53 amino acids and 4 It is located within human CGRP (1 alanine, 3 aspartate, 25 asparagine, 35 lysine), with the remaining three (-7, -6, -5) in the 5 amino acid amino-terminal leader sequence. Of the latter,
The first amino acid (arginine-7) was designated by the inventors based on the location of the splice junction and by homology to the lattocalcitonin gene. Significant sequence conservation is evident at the nucleotide level within the coding region, but is significantly reduced in the 3' non-coding region by comparison of the human sequence with the available rat CGRP mRNA sequence. Comparison of human CGRP amino acid sequence and other protein sequences (Wilber,
N.J.A. et al., PNAS (1983) 80 , 726-730)
showed no significant homology with other known peptides, including calcitonin (with the exception of rat CGRP). At most, nine comparable amino acids were identified by alignment of human CGRP with salmon calcitonin. Using cDNA fragments isolated from phT-B3 and phT-B58, the inventors cloned into phT-B58.
Restricted human genome cDNA exhibits partially processed polyadenylated RNA transcripts
Established by Southern method of DNA. In addition to the "introns" identified by nucleotide sequence analysis, one intron maps 3' to the CGRP coding sequence, and the others map to a genomic organization that closely resembles that of the rat calcitonin gene.
mapped to the 5' side of the calcitonin coding sequence, suggesting an organization (Rosenfelt M.G., Marmotsud G.G., Amara S.G., Stunson L.D., Sorchienko P.E., Riviere G.I.). (1983), Nature, 304 , 129-135). However, the “intron”-like sequences that separate the calcitonin exon and the CGRP coding sequence actually indicate genomic organization, and the phT-
1125bp Sph/PvuDNA containing calcitonin, intron and CGRP sequences isolated from B58
The fragment (see Figure 1) was obtained from Sph/Pvu-restricted human placenta, as determined by hybridization to an "intron" specific hybrid probe.
Electrophoresis is performed using genome fragments of the same size after DNA Southern analysis. Production of human CGRP by recombinant DNA technology To produce human CGRP by recombinant DNA technology, a fusion protein consisting of the active portion of chloramphenicol acetyltransferase protein (CAT) and the desired intermediate peptide is produced. I created a vector that can do this. (This vector is disclosed in pending International Patent Application No. PCT/GB84/00179, UK Patent Application No. 8413301, filed May 24, 1984). Plasmid is weakly resistant to chloramphenicol
The DNA of the R100R− plasmid mutant was digested with Pst1, and the single Pst1 fragment was then added to the plasmid pBR322.
(Iida et al. (1982) EMBOJ. 1 , 755-759). plasmid,
pBR322: Cn104 was obtained, with the last seven amino acid residues at the carboxy terminus removed by deletion.
CAT〓Coat with enzyme. This removal was due to a spontaneous in vivo mutation involving the insertional element IS1.
However, the resulting DNA molecule does not have a terminal codon at the end of the CAT structural gene. Therefore, the ribosome translated into protein and RNA was transcribed from the IS1 DNA until the phase terminal codon was matched. Net result is longer CAT than natural enzymes = protein 19
The last 26 amino acid residues are directed by the ISl DNA sequence. Since this structural gene also lacks suitable restriction sites useful in creating the desired fusion protein, a series of DNA manipulations were performed. The Pst1 restriction fragment containing the mutant CAT〓 gene described above was isolated from plasmid pBR322:Cn104 and ligated into the dephosphorylated Pst1 site of plasmid pAT153. Plasmid pAT/Cn104b (Fig. 3) was chosen because in this orientation both the CAT〓 and β-lactamase promoters are transcribed in the same direction. This cloning approach was primarily to construct a plasmid with a unique Tthlll restriction site. This cleavage site is derived from the 1Sl DNA attached to the end of the CAT structural gene and is located at the 19 amino acid codon of the 26 amino acid residue extension described above. Plasmid pAT/Cn104b was linearized with Tthlll and digested with BAL31 exonuclease.
Samples were taken at a series of time points and the reaction was stopped using excess EDTA. Any non-blunt ends generated by BAL31 digestion were filled in using Klenow fragment of DNA polymerase. These plasmid DNA molecules were then dephosphorylated using calf intestinal phosphanuse. Next, the sequence 5′−TCA
A kinased linker, R140, with GATCTGGAGCTCCAGATCTGA-3' was ligated to each Plasmid time point sample. After ligation, the plasmid DNA was digested with Sst restriction endonuclease and religated so that a unique linker was present on each plasmid. These sets of DNA molecules were transformed into E. coli DH1, and the fusion vector plasmid was added at 20 μg/m
It was selected on the basis that it grows significantly on L-agar containing chloramphenicol. A small scale plasmid preparation was made. single
A number of plasmids were isolated that had an Sst1 restriction site (derived from the linker DNA) and yielded relatively small DNA fragments when simultaneously digested with EcoR and Bgl. Plasmids determined by DNA sequence analysis
For pAB7, pAB8 and pAB19, linker DNA
was found to be attached to the 3' end of the CAT structural gene in each of the three open reading frames. Plasmids pAB7, pAB8 and pAB19 were each digested with restriction enzyme Sst and incubated with S ₁ exonuclease. After phenol/chloroform extraction and ethanol precipitation, these blunt-ended plasmid molecules were digested with Taq and ca.
A 750 base pair DNA fragment was isolated. These fragments contain complete CAT with Bgl sites in the three open reading frames.
Contains the fusion structural gene but lacks the CAT promoter. Then, these CAT〓 genes were put into a plasmid.
under the control of the trp promoter of pCT54 (Mtage et al., Proceedings National Academy of Sciences, USA Pro.
Natl. Acad. Sci. USA) 80 , 3671-3675, 1983).
This plasmid has the advantage of having a transcription terminator sequence, so high level expression is restricted to genes cloned upstream of this sequence and downstream of the trp promoter. Plasmid pCT54
Digested with EcoR and the 5' sticky ends were filled in using Klenow fragment of DNA polymerase. This molecule is restricted with the enzyme Cla1 and subsequently dephosphorylated,
A molecule receiving the CAT fusion vector gene cartridge isolated above was obtained. This molecule was ligated with a 3-fold molar amount of each CAT gene cartridge, and then E. coli HB101 was transformed into the chloramphenicol-resistant fusion vector plasmid pCT201,
pCT202 and pCT203 (Figure 3) were obtained. (In these three cases, the manipulation reshaped the EcoR site of pCT54). Plasmid vector pCT203 was cut with Hind. This yielded plasmid DNA with Hind sticky ends, which were then blunted with DNA polymerase. The generated plasmid DNA was then further cut with Bgl to obtain a DNA molecule with Bgl sticky ends and blunt ends. The generated DNA was ligated with the Bgl-Pvu fragment of plasmid phT-B58 (see Figures 1 and 3) to obtain a plasmid circular molecule. These plasmid molecules were transformed into E. coli HB101 cells, and E. coli HB101/pCAT-CGRP transformants were selected by growth on medium containing ampicillin (100 μg/m). By culture, E. coli
HB101/pCAT-CGRP cells were analyzed by SDS polyacrylamide gel electrophoresis and Commassie blue staining (Fig. 4a).
or after pulsing cells with S methionine ^for 1 min.
SDS polyacrylamide gel electrophoresis followed by autoradiography (Fig. 4b) produced a fusion protein with the expected size (Mtage G.S. et al., PNAS (1983)).
80, 3671-3675). In the pCAT-CGRP configuration,
A novel protein is obtained compared to CAT protein alone - Figures 4a, 4b, lanes 1, 2) - Figure 4
See a, 4b, lane 3. Lane M shows molecular weight marker proteins and their respective molecular weights. Evidence that the produced fusion protein contained CGRP protein sequences was determined by radioimmunoassay of HB101/pCAT-CGRP cell extracts. 500m
Cultured cells were taken, lysed with lysozyme/sodium deoxycholate mixture (5 m), and DN
The cells were treated with Aase at 5° C. for 30 minutes (Mtage G.S. et al., PNAS (1983), 80 , 3671-3675).
Equal volume of 0.1M Tris-HCl PH8.0, 0.1mM EDTA,
5% (v/v) glycol was added and the presence of human CGRP sequences was determined in the extracts. Radioimmunoassays were performed in a final volume of 400 μ in 0.05 M phosphate buffer PH7.4 (Gerges S.I. et al.
Journal Endocrinology (J.
Endocrinol.) 78 , 372-382). Antiserum was raised in rabbits against Tyr-(CGRP amino acids 25-37)-amide using known techniques.
For amino acids 25-37)-amide tracers,
Conjugated to chick ovalbumin (reitilin,
M., 1980, Method Enzymology (Meth.Enzymol.) 70 , 159-165). The tracer was iodinated using the Chloramine T method of Hunter and Greenwood. Figure 5 shows that the HB101/pCAT-CGRP protein extract displaced the tracer (5000 cpm) using a 1:10000 serum dilution with a displacement curve similar to that of the human CGRP standard (chemically synthesized). No displacement was observed using protein extracts predigested with trypsin (0.5 mg/m) overnight at 37°C, followed by trypsin inactivation with trasylol. Rabbit 1gG was quantitatively precipitated using goat anti-rabbit serum and pre-immunized rabbit serum was used as a carrier.
After adding 1μ, 125〓 tracer is bound (Craig R.K. et al., 1976, Biochem.J. 160 , 57-74),
Then, the precipitate 125% was quantified by γ-count.
This is human CGRP by HB101/pCAT-CGRP.
Demonstrate production of peptide sequences. The construction of the pCAT-CGRP plasmid was checked by Scal digestion. This is due to the expected amount
larger than the corresponding band obtained from pCT203
A DNA band was obtained on the gel. The composition of the plasmid was also checked by Hae digestion. phT−
The Bgl-Pvuc DNA fragment from B58 contains a Hae site that is not present in pCT203, and the gel showed a cDNA band of the expected size. The fusion protein produced by pCAT-CGRP consists of the amino acid sequence of CGRP with additional amino acid residues at the carboxyl and amino termini (Figure 1). The amino terminal has the sequence Lys-Arg immediately before CGRP.
including. This provides a clostripain cleavage site, which must be considered in view of potential clostripain sites within CGRP. Other unique cleavage sites can be used. Production of human CGRP by chemical synthesis; Human amide fragments thereof or analogs thereof;
CGRP and PDA-4 are Serotetsu 244-250
Synthesized by Bassroad, Slough, Berkshire, SLI, 407 UK or by Peninsula Laboratory Ltd, 61 Taylor Way, Belmont, California 94002 USA. Standard techniques of peptide synthesis can be used, for example Merrifield solid phase peptide synthesis or the so-called FMOC.
Operation (“Solid Phase Peptide Synthesis – Re-evaluation (Solid
Phase Peptide Synthesis-A
"Reassessment)" R.C. Sheepard - Monkular Endocrinology Edition, Matsukintyre and Zelke, Enzevier (1977) 43-56; E. Atherton et al., G.C.S.
Comm (JCSChem.Comm.) (1981) 1151~
1152; and G. Barney and R. B. Merrifield, The Peptides,
Yi Gross and Jie Jienhoufuer,
Academic Press, New York (1980) 3). Cardiovascular effects of human CGRP Using the amino acid sequence predicted by nucleotide sequence analysis of phT-B58, we
CGRP was chemically synthesized and purified using ion/reversed phase chromatography. final human
CGRP preparations are pure as judged by mass spectrometry;
A single ion was observed (Mr3786). The inventors have demonstrated that the cardiovascular effects of this preparation can be compared to synthetic rats of similar purity.
compared with CGRP preparation. Sprague Douri Illust (285-315g) 4
Groups of ~6 males were anesthetized with pentobarbitone (60 mg Kg ⁻¹ ip). Cannulae were inserted into the trachea, left carotid artery, and left jugular vein. Blood pressure is Statham
Recordings were made from the carotid artery on a glass polygraph with a P23ID pressure transducer, and mean arterial pressure was derived from the tracing. Heart rate was measured using a tachograph (glass model 7P4) guided by blood pressure signals. Human CGRP was either crudely produced from resin and then purified, or in the latter case obtained in purified form from the Peninsula Laboratory. Purified rat CGRP was of the same origin. All synthetic preparations were subjected to mass spectrometry (M-Scan) to confirm structure and purity before use. human CGRP, rat
CGRP, propanol hydrochloride (Sigma), mepyramine maleate (Sigma), cimetidine hydrochloride (SK&F), histamine diphosphate (Sigma) were dissolved in 0.9% w/v saline. All compounds were administered intravenously except mepyramine (SC). Saline (●), human CGRP (○)
and rat CGRP (△) were cumulatively administered to rats at 2 minute intervals in a volume of 0.1 m. The peak drop in mean arterial pressure (MAP) and heart rate (HR) 2 minutes later were measured. Administration of saline is MAP or
Both human CGRP and rat CGRP induced a dose-dependent decrease in MAP and an increase in HR without changing HR. 5 minutes before human CGRP, propranolol, 3.4μ
Mol Kg ^-1 (□) did not prevent the increase in heart rate. Mepyramine (12.4 μmol Kg ⁻¹ ) and cimetidine (59.5 μmol Kg ⁻¹ as a 30-minute infusion) (■) significantly reduced the hypotensive response to histamine (10 ⁻⁸
~10 ⁻⁵ mol Kg ⁻¹ ) did not significantly alter the hypotensive effect of human CGRP. Seven injections of saline over the course of the experiment did not significantly alter MAP or HR in the presence of propranolol or mepyramine and cimetidine. In rats anesthetized with pentobarbitone, intravenous human CGRP caused a rapid drug-related drop in blood pressure, reaching a maximum within 1 minute and reaching 0.28 nmol Kg ^-
A dose of ¹ showed a 50% reduction in blood pressure. This was not significantly different from the results obtained with rat CGRP (0.23 nmol Kg ^-1 ) in Figure 5 (p0.05, t-test).
Hypotension showed a small but significant relationship with increased heart rate. A decrease in blood pressure can be caused indirectly by basic peptides through the release of histamine (In Histamine and antihistamines (Goss, A. (1973)).
(by Schachter M.), Volume 1, Encyclopedia of Pharmacology (Int. Encyclopedia of Pharmacology)
and Therapeutics, Section 74, 25-43 Pergamon Press, Oxford) and Human
Since CGRP is a relatively basic peptide, the present inventors tested human CGRP with histamine.
_H1 -receptor antagonists mepyramine and histamine
The effect of pretreatment with the _H2 -receptor antagonist cimetidine was tested. The antagonist had no significant effect on the hypotensive response to human CGRP (Figure 6) or associated tachycardia (data not shown). It has also been suggested that through increased sympathetic drive, rat CGRP can increase heart rate (Fitzsia L.A., Kitsukawa D.O., Riviere G.I., Amara S.G.,
Evans, R. M., Rosenfeld, M. G., Bayle, D. D., & Brown, M. R. (1983), Nature, 305, 534-536). The inventor has determined that α-
Adrenoceptor antagonist propranolol (2log
After pretreatment with isoprenaline (in an amount sufficient to shift the isoprenaline drug-response curve to the right by units),
We investigated this possibility by intravenously administering human CGRP. Propranolol significantly reduced basal heart rate, but human CGRP continued to cause tachycardia;
Furthermore, compared to the saline control treatment (Figure 6), the limiting dose for this effect remained unchanged. These results indicate that the decrease in blood pressure and increase in heart rate caused by human CGRP are not indirectly mediated by the release of histamine or cetacolamine. The sensitive period to human CGRP was measured by single drug administration. 1 nmol Kg ^-1 of human CGRP reduced arterial pressure from 125±7.6 mmHg to 68.3±9.3 mmHg, and the time required to restore blood pressure to 50% was 3.8±0.5 minutes. The above-described study of the cardiovascular effects of intravenous POA-4 in rats anesthetized with phenobarbitone showed a small but significant effect. As can be seen in Figure 7, 7PDA-4 had 50-100 times lower potency than human CGRP. Therefore, 1μg/kilohuman
CGRP resulted in a sudden 50 mmHg decrease in mean arterial pressure and a 25-30 bpm increase in heart rate. on the other hand,
10μg/kg PDA-4 is 15-20mm of mean arterial pressure
It caused a decrease in Hg and an increase in heart rate by 15-20 bpm. The mechanism of action of PDA-4 was not tested. The cardiac effects of human CGRP were also studied in vitro to exclude the possible influence of reflexes or other factors that cause blood pressure to fall in vivo. Dunkin Hartley Guinea male (280-400
g) was killed by twisting the neck and blood was removed. The heart was taken out, the right atrium was chopped, and Krebs solution ₍ mM): NaCl118, KCl4.7, _CaCl2 2.5, _KH2PO4
1.18, NaHCO ₃ 25, MgSO ₄ 1.18 and glucose
11.1 at 34℃, 0.5g tension, 95:5
_O2 :bubbled with _CO2 . Atrial force and velocity were measured isometrically with a glass FT03 transducer and recorded on a glass polygraph. A single dose of human (●) or rat (▲) CGRP was randomly administered. In preliminary experiments, two sequential dose-response curves for CGRP showed reproducible results. Propranolol equilibrated for 30 minutes (○)
(300 nM) or cimetidine (100 μM) neutralized the effects of isoprenaline (3-30 nM) or histamine (500 nM), respectively, in the same experiment. These antagonists did not significantly attenuate the increased atrial velocity and force caused by CGRP. Antagonist alone did not significantly alter isolated atrium basal rate (187±9 b.min ^-1 ) or force (264±34 mg). In guinea pig right atrial preparations, human CGRP showed a concentration-dependent increase in contraction rate and force (Figure 8). Consistent with the results obtained in vivo, β-adrenoceptor-effective blocking concentrations of propranolol or the histamine H ₂ -receptor antagonist cimetidine did not alter the response to human CGRP.
It is interesting to note that rat CGRP is more effective than human CGRP in causing an increase in atrial force.
but with increasing atrial velocity, approximately 10
The capacity was doubled (Figure 8). The increase in atrial velocity or force produced by rat CGRP was not blocked by propranolol. Therefore, rat and human CGRP appears to act directly on isolated atria, and its action is independent of catecholamine and histamine receptor mechanisms. The inventor's research has demonstrated that the peripheral cardiovascular effects of human and rat CGRP in anesthetized rats are significantly different from those of rats administered peripherally to conscious animals.
Similar to what has been reported for CGRP, rat CGRP showed a significant increase in heart rate in the intentional preparation in our experiments, probably due to the blunting of cardiovascular reflexes by anesthesia (Morrison et al. J.A.
L., Walker H.A. & Richardson A.P. (1950) Arch.Int.Pharmacodyn. 82 ,
53-62). Antagonist studies have demonstrated that the vasodilatory effects of CGRP are not mediated by histamine or catecholamines. Thus, we demonstrate that CGRP acts directly on the cardiovascular system through a novel receptor mechanism and not through the release of other mediators. Similarly, the activity of CGRP on isolated atria that was unaffected by propranolol or cimetidine supports the concept of a CGRP receptor mechanism in cardiac tissue in addition to the vasculature. Reduced blood pressure in vivo and increased atrial contractile force in vitro in humans and rats
The similar abilities of CGRPs contrast with their varying abilities to increase the rate of atrial contraction. Results obtained in vitro indicate that rat CGRP has a desirable chronotropic effect (compared to inotropic), a property that was not evident in vivo, perhaps due to the different species used or the presence of anesthetics. It suggests. The inventors have demonstrated that PDA-4, when administered intravenously, has a hypotensive effect with simultaneous tachycardia. Acute toxicity test in rats Test method 40 rats (20 for treatment + 20 for control)
An acute toxicity test was conducted at . Human CGRP was administered by Sprague-1 at the doses shown in the table below.
Dawley's Charles River (CD) rats were dosed by a single intravenous injection via the tail vein. [Table] Results Immediately after administration, rats showed clinical changes.
Peripheral vasodilation was found in all animals, indicated by redness of the ears, limbs, tail, muzzle and abdomen, and this condition lasted for 4-5 hours. When human CGRP was administered intravenously to rats at doses up to 3040 μg/Kg, clear signs of pharmacological activity were found, but no obvious signs of toxicity were found. No deaths were observed during the subsequent 14-day observation period. At the end of this period, all animals were sacrificed and major organs were examined microscopically. Treatment with human CGRP had no effect on feed intake, body weight and major organ weights. Microscopic examination of the group treated with 1520 μg/Kg of human CGRP showed pale spleens in 4 of 5 males and 4 of 5 females. Although this change was unexpected, it was not considered to be of any significance. In particular, the phenomenon of the spleen becoming pale in color is
This did not occur in animals administered 3040 μg/Kg.
It has also been reported that it usually occurs depending on the age of the animal (approximately 8 weeks of age). Pathological examination of the lungs, liver, stomach, cecum, intestines, and oral cavity revealed no abnormal drug-related changes. From these results, it is clear that human CGRP has good safety when administered intravenously in a single dose up to 3040 μg/Kg. Diagnostic Applications Using the expected amino acid sequence for human CGRP in Figure 2, amidated CGRP and tyrosinated analogs;
- Amino acids 25-37) - Antibodies were raised in rabbits against the amide (Reitilin M (1980), Method.Enzym. 70 , 159-
165). Both demonstrated antigenicity as determined by their ability to bind 125〓Tyr-(CGRP-amino acids 25-37)-amide. As mentioned above, the present inventor has provided the following information: 1:
12500 serum dilution and 1800cpm in 400μ test
To perform a radioimmunoassay sensitive to Ing CGRP/tubes using the 125〓Tyr-(CGRP-amino acids 25-37)-amide tracer,
An antibody against Tyr-(CGRP-amino acids 25-37)-amide was used. Using this test (Figure 9),
This antibody is human calcitonin, tyrosinated PDA
-4, did not cross-react with katacalcin, Tyr-CGRP- (amino acids 1-8) or salmon calcytosine, but the antibody recognized the antigenic determinant in rat CGRP and when titrated with increasing amounts of human CGRP, the expected The inventors have demonstrated that a displacement curve is shown. Using this test, extracts from human medullary thyroid cancer tissue (Bennett H.P. et al.
1978, Biochem.J. 175 , 1139-1141) and tissue culture media derived from a human small cell carcinoma cell line (DMS153) and autopsy found to produce low levels of calcitonin. Liver metastasis (initial lung stage)
The present inventors identified the presence of human CGRP in a cell line derived from (Petstenzil et al. (1980) Cancer 45 , 906-918).
Tissue culture medium alone does not react with antiserum;
Media taken from DMS53 cells, a small cell carcinoma cell line known to produce high levels of calcitonin (Sorenson et al., 1981, Cancer 47 , 1289-1296), had only traces of human CGRP. . Parallel displacement curves were observed for MCT extract and DMS153 medium. Normal human plasma did not contain detectable amounts of CGRP within the range of the study, and plasma from some MCT patients did contain measurable amounts. The present inventor has developed the peroxidase-antiperoxidase method (Sternberger LA, 1979,
Immunocytochemistry 2nd edition,
Using immunostaining by J. A., Wiley & Son, NY), human CGRP-producing cells were isolated by immunocytochemistry at light levels in paraffin wax embedded with human medullary thyroid cancer tissue, and human CGRP-producing cells were isolated using immunocytochemistry. The diagnostic application of these antibodies was demonstrated. Total poly(A)-containing RNA was isolated from two different medullary thyroid carcinomas and DMS53 and 153 cell lines (Allison et al. (1981) Biochem.J. 199 , 725-731 and Hall et al.
1979, Nature 277 , 54-56). The distribution of calcitonin, CGRP and intron-specific transcripts (see Figure 1) was studied by RNA blotting including electrophoresis on a 1.1% (w/v) agarose gel followed by transfer to a Biodyne membrane for separation. (Taylor G.B. et al., 1984, Biochem Journal.
J.) 219 , 223-231). In another experiment, Bgl/Pst
32p-labeled calcitonin-specific cDNA fragment (Sp.Ac.3.2×10 ⁸ cpm/μg), Bgl/Pst
CGRP-specific cDNA fragment (Sp・Ac・7.9×10 ⁸
cpm/μg) and Bgl−Bgl “intron”
Specific cDNA fragment (Sp.Ac.2.8×10 ⁸ cpm/μg)
This membrane was probed using a 3-dimensional method - see Figure 1. After washing the filter, it was subjected to autoradiography. This result is consistent with RIA data, indicating that calcitonin and CGRP mRNA species are expressed at varying levels in cell lines and tumors, and that calcitonin and CGRP mRNA species are expressed at varying levels in cell lines and tumors.
It was established that the mRNA was probably processed from the normal transcript by various processing pathways (FIG. 10). In addition, the intron-specific sequence is the mature process calcitonin or CGRP mRNA.
It did not exist in the species. Normal placental DNA and patient MTC tissue DNA
After digesting the human CGRP gene sequence and blood lymphocyte DNA with restriction endonucleases (Fig.
organization). Therefore, each
Restrict 20 μg of DNA sample with BamHI and remove 1% fragments.
(w/v) Electrophoresed on agarose gel and separated by size, Gene Screen Plus membrane (NEN)
and probed with a labeled Bgl/PstCGRP-specific cDNA probe to a specific activity of 10 ⁸ cpm/μg. Hybridization is 50
overnight in 50% (v/v) formamide, 1% (w/v) SDS, 10% (w/v) dextran sulfate in mm Tris-HCPH7.5. Filters are sequentially 2 x SSC/RTP, 2 x SSC, 1%
(w/v) SDS, 65°C/30 min, and 0.1×SSC;
Washed at 65°C/15 minutes and then autoradiographed for 48 hours. This demonstrated a single major band (2.8 Kb) and minor band (2.6 Kb) of the hybrid in all DNA samples (Fig. 11), but
It was an additional minor band (3.0 Kb) in placental DNA. Therefore, using a CGRP-specific cDNA probe, we confirmed the gene rearrangement. In this case, the genes exhibit homology with the probe, as opposed to being homologous. These findings demonstrate the diagnostic value of sequence-specific probes in the study of genetic rearrangements in tumor tissue. This case demonstrates the potential of using the CGRP gene probe to study a family-specific type of combined restriction enzyme polymorphism in medullary thyroid cancer. SUMMARY The present invention regarding human calcitonin gene expression at the molecular level and the study of the cardiovascular activity of synthetic human CGRP confirms and extends work by others using the rat calcitonin gene as a model system (Amara S.G.; Jonas, V., Rosenfeld, M. G., Ong, E. S. & Evans, R. M. (1982), Nature, 298 , 240-244) (Rosenfeld, M. G., Marmotsd, G. I.・J.A., Amara S.G., Swanson L.
Davrieu, Souchenko P.E., Rivière G.I., Vert Davrieu &
Evans R.M. (1983) Nature, 304 , 129-135) Rosenfeld, M. G., Baer, D., & Brown, M. R. (1983) Nature, 305 , 534-536). The inventors identified CGRP mRNA sequences and calcitonin in human thyroid and lung cancer. The findings were identified using antibodies to human CGRP or its fragments, CGRP in lung cancer cell lines and medullary thyroid cancer tissue and plasma. Therefore, measurement of plasma CGRP levels, historical tissue testing using in-situ hybrid or immunocytochemical techniques, or testing of gene structure and expression using DNA or RNA blotting are of no use in the management of medullary thyroid cancer. (Hill C.S., Ibanets M.L., Samman N.A., Ahern M.G.A. & Clark R.L. (1973), Medicine, 52 ,
141-171), and in the management of lung cancer and diseases known to be associated with abnormal calcitonin gene expression, such as osteoporosis. The present invention's findings that the amidated human CGRP peptide PDA-4 has effects on the cardiovascular system, increasing cardiac contraction speed and force, and having a blood pressure lowering effect, suggest a role for this peptide in the clinical management of hypertension. Suggests. The present invention demonstrates that the peripheral effects of CGRP are independent of catecholamine β-receptors and histamine receptors. In view of the rapid onset of the peptide-induced hypotensive response, the effects of the present invention are consistent with the view that CGRP can act directly on the cardiovascular system through a new receptor mechanism that does not depend on other mediators.