Calcitonin peptides, and gene related pharmaceutical compositions

This invention relates to human gene related peptide pharmaceutical compositions containing the peptide and the method of treating hypertension with said pharmaceutical composition.

FIELD OF THE INVENTION 
This invention relates to peptides, pharmaceutical compositions, processes 
for producing the peptides, genes coding for the peptides, vectors 
including the genes and host organisms transformed with the vectors, 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 on rat calcitonin gene expression 
have demonstrated the generation, by RNA processing, of alternative mRNA 
species in an apparently tissue specific manner to produce different 
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 cleaved by post translational 
processing events to yield either rat calcitonin or rat calcitonin gene 
related peptide (rat CGRP). The rat CGRP is known to be widely distributed 
in discrete regions of the central and peripheral nervous system where it 
has been shown to have potent biological activity (Fisher et al (1983) 
Nature, 305, p 534-536). 
In copending European patent applications EP-A1-0070186 and EP-A1-0070675 
(R. K. Craig and I. MacIntyre) there are described the production of human 
calcitonin and a carboxy terminal peptide designated PDN-21 (Katacalcin). 
Human calcitonin and katacalcin are formed as a polyprotein encoded by the 
human calcitonin mRNA (Craig et al (1982) Nature, 295, p 345-347). 
We have now discovered a region of the human calcitonin gene which is 
transcribed in human medullary carcinoma cells, and is located distal to 
the 3' translated region of the human calcitonin mRNA. The region 
comprises a transcribed intron region, a splice junction, an open reading 
frame which encodes previously unknown human peptide sequences, and a 3' 
untranslated region terminating with a tract of poly A residues. One of 
the peptides is apparently an analogue of rat CGRP, which analogue we 
refer to hereinafter as human calcitonin gene related peptide (human 
CGRP). 
SUMMARY OF THE INVENTION 
According to a first aspect of the invention, we provide human calcitonin 
gene related peptide. 
According to a second aspect of the present invention we provide a peptide 
having the structure 
##STR1## 
This novel peptide is formed as part of a polyprotein which is specifically 
cleaved in vivo within the secretory pathway by proteolytic enzymes which 
recognise flanking basic amino acid residues. The peptide (which has been 
designated PAF-37 in accordance with the terminology of Tatemoto and Mutt 
(1981) PNAS 78 p 6603-6607) has been found to have a potent biological 
effect in the modulation of cardiovascular function. In particular the 
peptide has been shown to induce hypotension and to increase heart beat 
rate and force. 
In a second aspect of the invention the peptide is provided for use as a 
pharmaceutical, preferably for use in treatment of hypertension. It is 
common, in the post operative stages of major heart surgery (for example 
in open heart bypass operations), for the patient to react to the stress 
of the operation by extensive vasoconstriction. This has the effect of 
increasing the patient's blood pressure, thus straining the heart. The 
peptide of the invention has been shown to act as a hypotensive agent and 
to increase the force of heart beat. These combined effects make it of 
potential use as a post-operative treatment. A large percentage of people 
suffer from hypertension and it is a strong contributory factor in the 
high incidence of heart failure. The peptide may be used in the management 
of hypertension. 
In a third aspect of the invention we provide a pharmaceutical composition 
comprising a peptide of the structure (I) and a pharmaceutically 
acceptable excipient. Preferably the composition is an injectable 
composition. The pharmaceutical composition may be contained within, or 
form part of, a system for the controlled slow release of the composition 
or the peptide in or into the body. Such a controlled slow release system 
is of use in the long term management of hypertension. 
We further provide a method of treatment of hypertension comprising 
administering an effective amount of the peptide of the structure (I). We 
also provide a process for preparing a pharmaceutical composition 
according to the third aspect of the invention comprising the step of 
bringing a peptide of the structure (I) into association with a 
pharmaceutically acceptable excipient. 
The peptide of structure (I) may be produced by a variety of processes. 
The peptide may be produced by chemical peptide synthesis using techniques 
well known in the art. A number of commercial concerns now specialise in 
the custom synthesis of peptides. Purification of the peptide may be 
conducted using the techniques of ion and reverse phase chromatography. 
In a fourth aspect of the invention we provide a peptide comprising at 
least the amino acid sequence as follows: 
##STR2## 
wherein R' is -H or an amino acid residue or a peptide convertible into 
amide in vivo or in vitro. R' may be -Gly-, -Gly-Arg-Arg-Arg-Arg or 
-Gly-Lys-Lys-Arg but is preferably -Gly. The carboxy terminal residue R' 
may be converted enzymically in vivo or in vitro to an amide on the 
adjacent phenylalanine amino acid residue. A suitable enzyme for this 
conversion when R' is -Gly is yeast carboxypepdidase Y, or the mammalian 
amidation enzyme. (Bradbury, A. F., Finnie, M. D. A. & Smyth, D. G. (1982) 
Nature, 298, 686-688). 
Alternatively and preferably the peptide is produced by a recombinant DNA 
technique. 
In a fifth aspect of the invention we provide a process for the preparation 
of a peptide of the structure (I) comprising the steps of culturing a host 
organism transformed with a vector including a gene coding for an 
intermediate peptide wherein the intermediate peptide is a peptide 
according to the fourth aspect of the invention to produce the 
intermediate peptide and converting R' to amide. 
Preferably the intermediate peptide is a fusion protein comprising at least 
a portion of a protein produced in a transformed host organism and the 
amino acid sequence defined as (II) above. Desirably the fusion proteins 
include a protein produced at a high level by a transformed host organism. 
Suitable such proteins include at least a portion of a chloramphenicol 
acetyltransferase (CAT) protein or at least a portion of the 
.beta.-galactosidase protein. Preferably the fusion protein comprises a 
peptide having the amino acid sequence defined as (II) above linked to the 
carboxy terminus of the protein produced at high levels in a transformed 
host organism. Preferably the peptide is linked to the protein through a 
linkage capable of selective chemical or enzymic cleavage. The linkage may 
be a methionine or glutamic acid amino acid residue. Methionine may be 
selectively cleaved by cyanogen bromide and glutamic acid may be 
selectively cleaved by using an acid protease from Sorghum (EC 3.4.23.14), 
sea urchin hatching protease (EC 3.4.24.12) or, preferably, staphylococcal 
protease (EC 3.4.21.19). (The human CGRP sequence contains no Met or Glu 
residues). The linkage may be a lysine-arginine peptide diradical. 
Cleavage of this linkage may be achieved using a mouse sub-maxillary gland 
protease or, preferably, clostripain (EC 3.4.21.6). 
The peptide of the first and second aspects of the invention is produced as 
a polyprotein in the body. The polyprotein is processed by the body to 
cleave amino and carboxy terminus groups leaving the amidated 37 amino 
acid peptide of the first or second aspect of the invention. In a sixth 
aspect of the invention we provide a process for the production of peptide 
of the structure (I) comprising the steps of culturing a eucaryotic host 
organism transformed with a vector including a gene coding for an 
intermediate peptide including at least the amino sequence 
##STR3## 
to produce the intermediate peptide, allowing processing of the 
intermediate peptide by the host organism and isolating the peptide. 
Preferably the eucaryotic host organism is a tissue cultured mammalian 
cell line containing proteolytic and amidating enzymes capable of 
producing the peptide of structure I from a polyprotein including amino 
acid sequence III. Preferably the intermediate peptide is produced as a 
fusion protein. 
In the processing of the polyprotein a tetrapeptide is produced. This 
tetrapeptide also has biological activity, and is also a feature of the 
invention, having the following sequence: 
EQU Asp-Leu-Gln-Ala (IV) 
This peptide is denoted PDA-4 and has been found to have biological effect 
in the modulation of cardiovascular function. In particular the peptide 
has been shown to induce hypotension and to increase rate of heart beat. 
In an eighth aspect of the invention we provide a peptide of sequence IV 
for use as a pharmaceutical, preferably for use in the treatment of 
hypertension. 
In a ninth aspect of the invention we provide a pharmaceutical composition 
comprising a peptide of the sequence (IV) and a pharamceutically 
acceptable excipient. Preferably the composition is an injectable 
composition. The pharmaceutical composition may be contained within, or 
form part of, a system for the controlled slow release of the composition 
or peptide in or into the body. The composition may contain a combination 
of the peptides of structure I and the peptide of sequence IV and a 
pharmaceutically acceptable excipient. 
In a tenth aspect of the invention we provide a gene coding for PAF-37 
(structure I), PAF-37-R' (structure II) PDA-4 (structure IV) or an 
`unprocessed` peptide (structure III). Preferably we provide the specific 
genes defined by the nucleotides corresponding to the following amino 
acids the lower half of FIG. 2; i) 1 to 37 inclusive ii) -3 to -7 
inclusive iii) +6 to +9 inclusive or iv) amino acids -7 to +9 inclusive. 
We also provide nucleotide sequence substantially as shown in FIG. 2 from 
nucleotides 1 to 1256 inclusive. We further provide a vector including any 
of these genes and a host organism transformed with such a vector. 
Suitable host organisms include bacteria (e.g. E. coli.), yeasts (e.g. 
Saccharomyces cerivisiae) and mammalian cells in tissue culture. 
The production of human CGRP is tissue dependent due to an alternative, 
tissue mediated, mRNA processing. (Edbrooke, M. R. et al Nature (1984) - 
submitted). In some cells, such as those found in medullary thyroid 
carcinoma and lung carcinoma, human CGRP is produced in varying levels. 
The presence of human CGRP may therefore be diagnostic of abnormal tissue. 
We further provide therefore diagnostic reagents (with antibodies and gene 
probes) for use in the assay of human CGRP gene expression and abnormal 
gene organisation. 
In an eleventh aspect of the invention we provide a peptide of the 
structure I, or a portion thereof including an antigenic determinant, 
wherein the peptide or the portion thereof has a detectable label attached 
thereto. The label may be an enzyme, a chromophore, a fluorophore or a 
chemiluminescent group. Most preferably however the label is a radiactive 
isotope, for example .sup.125 I attached to the histidine amino acid 
residue at position 10 in the amino acid sequence of the peptide. 
Preferably the labelled peptide comprises the peptide of structure I or a 
portion thereof which further includes a tyrosine amino acid residue 
optionally labelled with .sup.125 I. Preferably the portions of the 
peptide are from amino acid 25 to 37 inclusive (amidated phenylalamine) or 
from amino acid 1 to 8 inclusive. 
In a twelth aspect of the invention we provide an antibody having 
specificity for an antigenic determinant of the peptide of structure I. 
The antibody may be a polyclonal or monoclonal antibody. The antibody may 
be labelled with a detectable label. 
The reagents of the eleventh and twelth aspects of the invention allow for 
the immunoassay, preferably the radioimmunoassay, for the peptide of 
structure I in a sample, or for the immunocyto chemical localisation in 
tissue sections. 
It is also possible to assay for mRNA coding for PAF-37 or the nucleotide 
sequence including such mRNA. 
In an eleventh aspect of the invention therefore we provide a DNA 
hybridisation probe comprising a sequence of 15 or more nucleotides 
selected from the nucleotide sequence from 1 to 1256 as shown in FIG. 2 of 
the accompanying drawings. The probe may be immobilised on a solid phase, 
capable of immobilisation on a solid phase or may be labelled with a 
detectable label. 
Such probes may be used to examine gene organisation or gene expression by 
DNA or RNA blotting techniques or to identify by in situ hybridisation 
cells in tissue sections expressing the gene. 
A preferred probe of the thirteenth aspect of the invention comprises a 
sequence selected from the nucleotide sequence from 1 to 725 inclusive as 
shown in FIG. 2. A probe of this preferred type may be used to examine 
human CGRP as opposed to human calcitonin gene organisation or expression. 
In particular the probe will hybridise to human CGRP mRNA and to the human 
CGRP structural genes. 
An alternative preferred probe of the thirteenth aspect of the invention 
comprises a sequence selected from the nucleotide sequence from 726 to 
1256 inclusive as shown in FIG. 2. A probe of this preferred type may be 
used to examine the organisation of the human calcitonin CGRP gene. In 
particular, the probe will hybridise to an intron region of the human CGRP 
structural gene.

In our copending European published application EP-A1-0070675 there is 
described the construction of a cDNA library using total cellular 
poly(A)-containing RNA isolated from human medullary thyroid carcinoma 
tissue (see also Allison J. et al Biochem J. (1981) 199 p 725-731) and the 
nucleotide sequence analysis of the greater part of the human calcitonin 
mRNA cloned within two plasmids isolated from this library, phT-B3 and 
phT-B6 (see also Craig, R. K. et al Nature (1982) 295 p 345-347). During 
this study, one recombinant plasmid phT-B58 containing an inserted cDNA 
fragment of about 1600 bp was identified during preliminary screening but 
not analysed further since it appeared too large to represent the 
calcitonin mRNA. Subsequent restriction enzyme analysis of the cDNA 
inserted in this plasmid (see FIG. 1) showed sites in common with phTB3 
and moreover indicated that the cDNA cloned within phT-B58 represented 
sequence downstream from sequence previously established to represent the 
complete 3' untranslated region of the human calcitonin mRNA. FIG. 1 shows 
restriction sites separating regions of sequence subsequently used as 
sequence specific hybridisation probes and the relative positions of 
calcitonin, CGRP and the common amino terminal peptide. Vertical broken 
lines denote Pst I sites separating cDNA and plasmid sequences. Nucleotide 
sequence analysis of the entire cDNA sequence inserted into phT-B58 was 
performed using the method previously described (Craig, R. K., Hall, L., 
Edbrooke, M. R., Allison, J. & MacIntyre, I. (1982) Nature, 295, 345-347) 
- see FIG. 2. The figure compares known nucleotide sequence of human and 
rat calcitonin and CGRP RNA transcripts with gaps introduced to maximise 
homologies. Numbers immediately above the human nucleotide sequence denote 
the relative number of nucleotides from the poly(A)tail cloned into 
phTB58. Numbers above the human protein sequence refer to amino acid 
position relative to calcitonin or CGRP. Alternative or additional amino 
acids or nucleotides present within rat relative to human sequence are 
shown immediately below the codons in question. Potential polyadenylation 
signals are underlined. A thin arrow indicates the 3' end of the mature 
calcitonin mRNA, whilst heavy arrows () indicate the probable position of 
additional introns in the human genomic sequence. Broken lines show 
regions of human sequence absent from the rat calcitonin gene. The 
inserted cDNA sequence contained 1615 bp, terminating with a tract of 
poly(A) residues at the 3' terminus. Of these, the first 356 bp from the 
5' end were identical to those previously described, and represented part 
of the human calcitonin mRNA encoding Katacalcin and the whole of the 3' 
untranslated region including the AATAAA box and 12 nucleotides previously 
shown to precede the poly(A) tail in the mature calcitonin mRNA. Analysis 
of the remaining sequence showed it to contain a single open reading frame 
encoding 53 amino acids followed by a termination codon. This was preceded 
immediately on the 5' side by a splice junction acceptor site (C).sub.n 
NAG/G (Mount, S. M. Nucleic Acid. Res. (1982) 10 p 450-463) and a further 
645 nucleotides of `intron`-like sequence separated from previously known 
calcitonin mRNA sequence by three adenosine residues. However no 
recognisable donor splice junction was present between the intron-like 
sequence and sequence known to be present in the human calcitonin mRNA. 
The novel open reading frame was followed by a tract of 451 nucleotides 
containing two polyadenylation signals, the first (AATAA) 26 bases 
downstream of the termination codon, and the second (AAAATTAAAAA) 
positioned 18 nucleotides before the terminating poly(A) tract. The coding 
sequence comprised human CGRP (a peptide of 37 amino acids) flanked at the 
amino terminal end by paired basic amino acids (-1, -2) and a further five 
amino acids (-3 to -7), and at the carboxyl end by a glycerine residue 
(+1) four basic amino acids (+2 to +5) and a tetrapeptide (+6 to +9). The 
presence of the glycerine reflects the requirement in vivo for an amidated 
carboxyl terminal phenylalanine (see Bradbury, A. F. et al nature (1982) 
298 p 686-688) resulting in a calculated Mr of 3786 for the amidated human 
CGRP. Comparison of the predicted human amino acid sequence with that of 
the rat (Amara et al Nature (1982) 298 p 240-244) shows sequence 
conservation, with seven amino acid changes out of the 53 amino acids, 
four within the human CGRP (alanine 1; aspartic acid 3; asparagine 25; 
lysine 35), the remaining three (-7, -6, -5) residing in the 5 amino acid 
amino terminal leader sequence. Of the latter, the first amino acid 
(arginine -7) we have assigned on the basis of the position of the splice 
junction and by analogy with the rat calcitonin gene. Significant sequence 
conservation is apparent at the nucleotide level within the coding region, 
but is markedly reduced in the 3' non-coding region on comparison of the 
human sequence with available rat CGRP mRNA sequence. Comparison of the 
human CGRP amino acid sequence with other protein sequences (Wilbur, N. J. 
et al PNAS (1983) 80, p 726-730) revealed (rat CGRP apart) no significant 
homology with other known peptides including the calcitonins. At best nine 
matched amino acids were identified by alignment of human CGRP with salmon 
calcitonin. 
Using isolated cDNA fragments from phT-B3 and phT-B58 we have established 
by Southern Blotting of restricted human genomic DNA that the cDNA cloned 
into phT-B58 represents a partially processed polyadenylated RNA 
transcript. In addition to the `intron` identified by nucleotide sequence 
analysis, an intron has been mapped to the 3' side of the CGRP coding 
sequence, and others to the 5' side of the calcitonin coding sequence 
suggesting a genomic organisation very similar to that of the rat 
calcitonin gene (Rosenfeld, M. G., Mermod, J--J., Amara, S. G., Swanson, 
L. W., Sawchenko, P. E., Rivier, J., Vale, W. W. & Evans, R. M. (1983) 
Nature, 304, 129-135). However, the `intron` like sequence separating the 
calcitonin exon and CGRP coding sequence truly represents the genomic 
organisation, since a 1125 by SphI/PvuII DNA fragment which includes 
calcitonin, intron and CGRP sequence isolated from phT-B58 (see FIG. 1) 
electrophoreses with a genomic fragment of identical size after Southern 
Blot analysis of human placental DNA restricted with SphI/PvuII as 
determined by hybridisation to an `intron` specific hybridisation probe. 
Production of human CGRP by recombinant DNA techniques 
In order to produce human CGRP by a recombinant DNA technique, vectors 
capable of producing a fusion protein comprising an active portion of a 
chloramphenicol acetyltransferase protein (CAT) and a desired intermediate 
peptide were produced. (The vectors are described generally in copending 
International patent application PCT/GB 84/00179 and in British patent 
application 8413301 filed May 24, 1984). 
A plasmid had been isolated by Pst1 digestion of the DNA of a weakly 
chloramphenicol resistant R100 R-plasmid mutant and subsequent ligation of 
a single Pst1 fragment into the Pst1 site of plasmid pBR322 (Iida et al 
(1982) EMBO J. 1, 755-759). The plasmid, pBR322: Cm104, was obtained and 
encodes a CAT.sub.I enzyme that has had the last seven amino acid residues 
of the carboxy terminus removed by deletion. The removal was due to a 
spontaneous in vivo mutation which involved the insertion element IS1. 
However, the resulting DNA molecule has no termination codon at the end of 
the CAT.sub.I structural gene. The ribosome, therefore, translates into 
protein the RNA transcribed from the IS1 DNA until it meets an in phase 
termination codon. The net result is a CAT.sub.I protein nineteen amino 
acid residues longer than the native enzyme in which the last twenty-six 
amino acid residues are directed by the IS1 DNA sequence. This structural 
gene also lacks any suitable restriction sites which would be useful to 
create a desirable fusion protein so a series of DNA manipulations were 
performed. 
A Pst1 restriction fragment containing the mutant CAT.sub.I gene outlined 
above was isolated from plasmid pBR322: Cm104 and ligated into the 
dephosphorylated Pst1 site of plasmid pAT153. The plasmid pAT/Cm104b (FIG. 
3) was chosen since in this orientation both the CAT.sub.I and 
.beta.-lactamase promoters transcribe in the same direction. This cloning 
maneouvre was primarily to construct a plasmid which carries a unique 
Tth111I restriction site. This cleavage site is derived from the IS1 DNA 
which was joined to the end of the CAT.sub.I structural gene and lies in 
the nineteenth amino acid codon of the twenty-six amino acid residue 
extension described above. 
Plasmid pAT/Cm104b was linearised with Tth111I and digested with BAL31 
exonuclease. Samples at a series of time points were withdrawn and the 
reaction was stopped using excess EDTA. Any non-flush ends created by the 
BAL31 digestion were filled in using the Klenow fragment of DNA polymerase 
I. These plasmid DNA molecules were then dephosphorylated using calf 
intestinal phosphatase. Next a kinased linker, R140 with the sequence 
EQU 5'-TCAGATCTGGAGCTCCAGATCTGA-3' 
was ligated to each plasmid time point sample. After ligation the plasmid 
DNAs were digested with SstI restriction endonuclease and re-ligated to 
ensure that only one linker was present in each plasmid. 
These sets of DNA molecules were then transformed into E. coli DH1 and 
fusion vector plasmids were selected on the basis of vigorous growth on 
L-agar containing 20 .mu.g/ml chloramphenicol. 
Small scale plasmid preparations were performed. A number of plasmids which 
carried a single Sst1 restriction site (derived from the linker DNA) and 
which also generated a comparatively small DNA fragment when 
simultaneously digested with EcoR1 and Bg1II were isolated. DNA sequence 
analysis revealed that in plasmid pAB7, pAB8 and pAB19 the linker DNA had 
been attached to the 3' end of the CAT.sub.I structural gene in each of 
the three reading frames. 
Plasmids pAB7, pAB8 and pAB19 were each digested with restriction enzyme 
SstI and incubated with S1 exonuclease. After phenol/chloroform extraction 
and ethanol precipitation these blunt-ended plasmid molecules were 
digested with TaqI and DNA fragments of approximately 750 base pairs were 
isolated. These fragments contain the entire CAT.sub.I fusion structural 
genes with Bg1II sites in three reading frames but lack the CAT.sub.I 
promoter. 
These CAT.sub.I genes were then put under the control of the trp promoter 
of plasmid pCT54 (Emtage et al, Proc Natl Acad Sci USA 80, 3671-3675, 
1983). This plasmid also has the advantage of having a transcription 
terminator sequence so that high level expression is limited to the gene 
cloned upstream of this sequence and downstream of the trp promoter. 
Plasmid pCT54 was digested with EcoR1 and the 5' cohesive ends were filled 
in using the Klenow fragment of DNA polymerase I. Subsequent restrictions 
of this molecule with the enzyme C1a1 followed by dephosphorylation 
created a molecule which would accept the CAT.sub.I fusion vector gene 
cartridges isolated above. Ligation of this molecule with a 3-fold molar 
excess of each the CAT.sub.I gene cartridges followed by transformation of 
E coli HB101 gave the chloramphenicol resistant fusion vector plasmids 
pCT201, pCT202 and pCT203 (FIG. 3). (NB in all three cases the 
manipulation result in the reformation of the EcoR1 site of pCT54). 
The plasmid vector pCT 203 was cut with Hind III. This gave plasmid DNA 
having Hind III sticky ends which were then blunted with DNA polymerase. 
The resultant plasmid DNA was then further cut with Bg1 II to yield DNA 
molecules having a Bg1 II sticky end and a blunt end. The resultant DNA 
was then ligated with the Bg1 II-Pvu II fragment of plasmid phT-B58 (see 
FIGS. 1 and 3) to give circular plasmid molecules. These plasmid molecules 
were then transformed into E. coli HB101 cells and E. coli HB101/pCAT-CGRP 
transformants were selected by growth on medium containing ampicillin (100 
ug/ml). On culturing, the E. coli HB101/pCAT-CGRP cells produced a fusion 
protein having the predicted size, as judged by SDS polyacrylamide gel 
electrophoresis and Commassie Blue staining (FIG. 4a), or by SDS 
polyacrylamide gel electrophoresis followed by autoradiography (FIG. 4b) 
after pulsing cells for 1 min. with .sup.35 S methionine (see Emtage, J. 
S. et al PNAS (1983) 80, p 3671-3675 ). Thus in the pCAT-CGRP constructs a 
novel protein is produced see - Fig. 4(a), 4(b) Lanes 1,2) compared with 
the CAT protein alone - FIG. 4(a),4(b) Lane 3. Lane M shows molecular 
weight marker proteins and their respective molecular weights. 
Evidence that the fusion protein produced, contains CGRP protein sequence 
was determined by radioimmunoassay of HB101/pCAT-CGRP cell extracts. Cells 
from a 500 ml culture were harvested and lysed in a lysozyme/sodium 
deoxycholate mixture (5 ml), then treated with DNase for 30 mins. at 
5.degree. C. (Emtage J. S. et al PNAS (1983), 80, p 3671-3675). An equal 
volume of 0.1M Tris HCl pH 8.0, 0.1 mM EDTA, 5% (v/v) glycol, was then 
added, and the presence of human CGRP sequence determined in the extract. 
Radioimmunoassay was carried out in 0.05M phosphate buffer pH 7.4 in a 
final volume of 400 ul (see Girgis, S. I. et al J. Endocrinol 78, 
372-382). Antiserum was raised in rabbits, using the known techniques, 
against Tyr-(CGRP amino acids 25-37)-amide and conjugated to chick 
ovalbumin (Reichlin, M.1980, Meth.Enzymol 70 159-165) and against .sup.125 
I Tyr-(CGRP amino acids 25-37)-amide tracer. 
The tracer was iodinated using the Chloramine T method of Hunter and 
Greenwood. It can be seen from FIG. 5 that 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 
synthesised). No displacement was observed using protein extract which had 
been previously digested overnight with trypsin (0.5 mg/ml) overnight at 
37.degree. C. followed by inactivation of the trypsin with trasylol. Goat 
anti rabbit serum was used to quantitatively precipitate rabbit IgG and 
bound .sup.125 I tracer after the addition of 1 ul of pre-immune rabbit 
sera as a carrier (Craig R. K. et al (1976 Biochem J. 160 p 57-74 and the 
precipitated .sup.125 I quantitated by .gamma.-counting. This demonstrates 
the production of human CGRP peptide sequence by HB101/pCAT-CGRP. 
The construction of the pCAT-CGRP plasmid was checked by ScaI digestion - 
this gave a DNA band on a gel that was bigger by the predicted amount, 
than the corresponding band obtained from pCT203. The plasmid construction 
was also checked by HaeII digestion. The Bg1II-Pvu II cDNA fragment from 
phT-B58 contains an HaeII site that is not present in pCT203 and the gels 
showed a cDNA band of the expected size. 
The fusion protein produced by pCAT-CGRP comprises the amino acid sequence 
of CGRP with additional amino acid residues at the carboxyl terminal and 
the amino terminal (see FIG. 1). The amino terminal includes the sequence 
Lys-Arg immediately preceding CGRP. This provides a site for clostripain 
cleavage, although case must be taken in view of potential clostripain 
sites within the CGRP. Other unique cleavage sites may be used. 
Production of human CGRP by chemical synthesis 
Human CGRP in its amidated form fragments or analogues thereof, and PDA-4 
were synthesised by Celltech Limited 244-250 Bath Road, Slough, Berkshire 
SL1 4D7, United Kingdom or by Peninsula Laboratories Inc., 61 Taylor Way, 
Belmont, Calif. 94002 U.S.A. The standard techniques of peptide synthesis 
may be used - for example the Merrifield solid phase peptide synthesis or 
the so - called FMOC procedure (see "Solid Phase Peptide Synthesis - A 
Reassessment" by R. C. Sheppard - from Molecular Endocrinology eds. 
MacIntyre and Szelke, Elsevier (1977) P 43-56; E. Atherton et al J. C. S. 
Chem. Comm (1981) p 1151-1152; and G. Barany and R. B. Merrified in "The 
Peptides" eds E. Gross and J. Jeienhofer, Academic Press, New York (1980) 
p 3. 
Cardiovascular actions of human CGRP 
Using the amino acid sequence predicted by nucleotide sequence analysis of 
phT-B58, amidated human CGRP has been chemically synthesised and then 
purified using a combination of ion and reverse phase chromatography. The 
final human CGRP preparation was pure as judged by mass spectrometry where 
a single ion was observed (Mr 3786). We have compared the cardiovascular 
effects of this preparation with a synthetic rat CGRP preparation of 
similar purity. 
Groups of 4 to 6 male Sprague-Dawley rats (285-315 g) were anaesthetised 
with sodium pentobarbitone (60 mg Kg.sup.-1 i.p.). The trachea, left 
carotid artery and left jugular vein were cannulated. Blood pressure was 
recorded from the carotid artery on a Grass polygraph via a Statham P23 ID 
pressure transducer and mean arterial pressure derived from the trace. 
Heart rate was measured by a tachograph (Grass model 7P4) triggered by the 
blood pressure signal. Human CGRP was obtained either crude off the resin, 
then purified, or latterly in a purified form from Peninsula Laboratories. 
Purified rat CGRP was from the same source. All synthetic preparations 
were subject to mass spectrometry (M-Scan Ltd.) to confirm structure and 
purity before use. Human CGRP, rat CGRP, propranolol 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 for mepyramine (s.c.). 
Saline (), human CGRP (o) and rat CGRP (.DELTA.) were administered 
cumulatively in volumes of 0.1 ml per rat at 2 min intervals. The peak 
fall in mean arterial pressure (MAP) and heart rate (HR) after 2 min was 
measured. Saline administration did not alter MAP or HR, and both human 
CGRP and rat CGRP evoked a dose-dependent fall in MAP and an increase in 
HR. Propranolol, 3.4 .mu.mol Kg.sup.-1 (.quadrature.), 5 min before human 
CGRP did not prevent the increase in heart rate. Mepyramine (12.4 .mu.mol 
Kg.sup.-1) and cimetidine (59.5 .mu.mol Kg.sup.-1, as an infusion over 30 
min) () significantly reduced the hypotensive response to histamine 
(10.sup.-8 -10.sup.-5 mol Kg.sup.-1) but did not significantly alter the 
hypotensive effect of human CGRP. Seven injections of saline over the 
duration of the experiment did not significantly alter either MAP or HR in 
the presence of propranolol or mepyramine and cimetidine. 
In the pentobarbitone anaesthetised rat, intravenous human CGRP evoked a 
rapid dose-related fall in blood pressure, maximal within 1 min, and 
representing a 50% decrease in blood pressure at a dose of 0.28 nmol 
Kg.sup.-1. This was not significantly different (p 0.05; t-test) from 
results obtained with rat CGRP(0.23 nmol. Kg.sup.-1) FIG. 5. The 
hypotension was associated with small but significant increases in heart 
rate. Since a fall in blood pressure may be evoked indirectly by basic 
peptides through the release of histamine (Goth, A. (1973) In Histamine 
and antihistamines (Ed. Schachter, M.) Vol. I. Int. Encyclopedia of 
Pharmacology and Therapeutics, Section 74, 25-43 Pergamon Press, Oxford), 
and human CGRP is a relatively basic peptide, we have examined the effect 
of pretreatment with the histamine H.sub.1 -receptor antagonist mepyramine 
and the histamine H.sub.2 -receptor antagonist cimetidine prior to human 
CGRP administration. Neither antagonist had a significant effect on the 
hypotensive response to human CGRP (FIG. 6) or the associated tachycardia 
(data not shown). It has also been suggested that rat CGRP may increase 
heart rate through increased sympathetic drive (Fisher, L. A., Kikkawa, D. 
O., Rivier, J. E., Amara, S. G., Evans, R. M., Rosenfeld, M. G., Vale, W. 
W. & Brown, M. R. (1983) Nature, 305, 534-536). We have investigated this 
possibility by i.v. administration of human CGRP after pretreatment with 
the .beta.-adrenoceptor antagonist propranolol (in a dose sufficient to 
shift an isoprenaline dose-response curve to the right by 2 log units). 
Following propranolol the basal heart rate was significantly reduced but 
human CGRP continued to evoke a tachycardia, and furthermore the threshold 
dose for this effect remained unchanged, compared with saline treated 
controls (FIG. 6). From these results we conclude that the fall in blood 
pressure and increase in heart rate evoked by human CGRP are not mediated 
indirectly via the release of either histamine or catecholamines. The 
duration of the response to human CGRP was measured following 
administration of a single dose. Human CGRP 1 nmol. Kg.sup.-1 lowered 
arterial pressure from 125.0.+-.7.6 mm Hg to 68.3.+-.9.3 mm Hg and the 
time taken for the blood pressure to recover by 50% was 3.8.+-.0.5 min. 
Examination of the cardiovascular action of i.v. PDA-4 in phenobarbitone 
anaesthetised rats as described above showed a small but significant 
effect. As can be seen in FIG. 7 PDA-4 was 50-100 fold less potent than 
human CGRP. Thus 1 ug/kilo human CGRP resulted in an immediate 50 mmHg 
drop in mean arterial pressure and an increase in heart beat rate of 25-30 
b.p.m. Whilst 10 ug/kilo PDA-4 caused a drop in mean arterial pressure of 
15-20 mm Hg and an increase in heart beat rate of 15-20 bpm. The mechanism 
of action of PDA-4 was not examined. 
The cardiac effects of human CGRP were also studied in vitro in order to 
eliminate the possible influence of reflex or other factors resulting from 
the fall in blood pressure in vivo. 
Male Dunkin Hartley guinea-pigs (280-400 g) were killed by cervical 
dislocation and exsanguinated. The heart was removed, the right atrium 
dissected out and mounted under 0.5 g tension in Krebs solution (mM): NaCl 
118, KCl 4.7, CaCl.sub.2 2.5, KH.sub.2 PO.sub.4 1.18, NaHCO.sub.3 25, 
MgSO.sub.4 1.18 and glucose 11.1, at 34.degree. C. bubbled with 95:5 
0.sub.2 :CO.sub.2. Atrial force and rate were measured isometrically by a 
Grass FT.03 transducer and recorded on a Grass polygraph. Single doses of 
either human () or rat () CGRP were given in a random order. Preliminary 
experiments showed that two consecutive dose-response curves to CGRP gave 
reproduceable results. 
Propranolol (o) (300 nM) or cimetidine (100 .mu.M), equilibrated for 30 
min, antagonised the effects of isoprenaline (3-30 nM) or histamine (500 
nM) respectively in the same experiments where these antagonists did not 
significantly reduce the increased rate and force evoked by CGRP. The 
antagonists alone did not significantly alter the basal rate (187.+-.9 
b.min.sup.-1) or force (264.+-.34 mg) of the isolated atria. 
In the guinea-pig right atrial preparation, human CGRP evoked concentration 
dependent increases in the rate and force of contraction (FIG. 8). In 
agreement with results obtained in vivo, effective .beta.-adrenoceptor 
blocking concentrations or propranolol or of the histamine H.sub.2 
-receptor antagonist cimetidine did not alter the responses to human CGRP. 
Interestingly, although rat CGRP was equipotent with human CGRP in evoking 
increases in force, it was approximately 10 times more potent at 
increasing rate (FIG. 8). Neither the increase in rate or force produced 
by rat CGRP was blocked by propranolol. Thus rat and human CGRP appear to 
act directly on the isolated atria, and their actions are independent of 
catecholamine and histamine receptor mechanisms. 
Our studies demonstrate that the peripheral cardiovascular effects of human 
and rat CGRP in the anaesthetised rat are similar to those reported for 
peripherally administered rat CGRP in conscious animals, though rat CGRP 
evoked a greater increase in heart rate in the conscious preparation 
possibly due to the blunting of cardiovascular reflexes by anaesthesia in 
our experiments (Morrison, J. L. Walker, H. A. & Richardson, A. P. (1950) 
Arch. Int. Pharmacodyn. 82, 53-62). Studies with antagonists demonstrate 
that the vasodepressor effect of CGRP is not mediated via histamine or 
catecholamines. Therefore CGRP may act on the cardiovascular system 
directly via a novel receptor mechanism and not through the release of 
other mediators. Similarly the activity of CGRP on isolated atria which 
was unaffected by propranolol and cimetidine supports the concept of a 
CGRP receptor mechanism in cardiac tissue in addition to the vasculature. 
The similar potency of human and rat CGRP at lowering blood pressure in 
vivo and increasing the force of contraction of the atria in vitro 
contrasts with their differing potency at increasing the rate of 
contraction of the atria. The results obtained in vitro suggest that rat 
CGRP has a preferential chronotropic (compared with inotropic) effect, a 
property which was not evident in vivo, possibly due to the different 
species employed or to the presence of the anaesthetic. We also 
demonstrate that PDA-4 when administered i.v. has a hypotensive effect 
with parallel tachycardia. 
Diagnostic applications 
Using the amino acid sequence predicted for human CGRP in FIG. 2, we have 
raised antibodies in rabbits against amidated CGRP, and also the 
tyrosinated analogue; Tyr-(CGRP-amino acids 25-37)-amide conjugated to 
ovalbumin (Reichlin, M. (1980), Meth. Enzym. 70 159-165). Both proved 
antigenic as determined by their ability to bind .sup.125 I Tyr(CGRP amino 
acids 25-37)-amide. We have used antibody raised against Tyr-(CGRP amino 
acids 25-37)-amide to set up a radioimmunoassay sensitive to lng 
CGRP/tube, using a 1:12500 dilution of serum and 1800 cpm .sup.125 
I-Tyr-(CGRP amino acids 25-37) -amide tracer in a 400 .mu.l assay as 
described above. Using this assay (FIG. 9), we demonstrate that the 
antibody does not cross-react with human calcitonin, tyrosinated PDA-4, 
katacalcin, Tyr-CGRP (amino acids 1-8) or salmon calcitonin, but that the 
antibody recognises antigenic determinants in rat CGRP, and shows the 
expected displacement curve when titred with increasing amounts of human 
CGRP. Using this assay we have identified the presence of human CGRP in 
extracts from human medullary thyroid carcinoma tissue (Bennett, H. P. et 
al 1978 Biochem J. 175, 1139-1141) and in tissue culture medium removed 
from a human small cell carcinoma cell-line (DMS 153), a cell-line derived 
from a liver metastasis (lung primary) at autopsy (Pettengill et al (1980) 
Cancer 45, 906-918) and known to produce low levels of calcitonin. Tissue 
culture medium alone did not react with the antiserum, whilst medium 
removed 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 humanCGRP. Parallel displacement curves were 
observed for MCT extract and DMS 153 medium. Normal human plasma did not 
contain detectable levels of CGRP within the range of the assay, whilst 
plasma from some MCT patients, contained measurable amounts. 
We have also localised human CGRP producing cells by immunocytochemical 
means at the light level in a paraffin wax embedded human medullary 
thyroid carcinoma tissue, using immunostaining by the 
peroxidase-antiperoxidase method (Sternberger, L. A. 1979 
Immunocytochemistry 2nd Edition, J. Wiley & Son, N. Y.), demonstrating the 
diagnostic application of these antibodies in the classification of human 
tumour pathology. 
We have also demonstrated the diagnostic value of human CGRP gene probes, 
to investigate tissue and cell-specific calcitonin CGRP gene expression, 
and also to investigate CGRP gene rearrangement in tumour tissues. Total 
poly (A)-containing RNA was isolated, from two different medullary thyroid 
carcinoma tumours and from the DMS 53 and 153 cell-lines (see 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 
FIG. 1) was investigated by RNA blotting involving separation by 
electrophoresis on 1.1% (w/v) agarose gels followed by transfer to Biodyne 
membranes (Taylor, J. B. et al, 1984 Biochem J. 219, 223-231). In separate 
experiments the membranes were probed with Bg1II/PstI .sup.32 P-labelled 
calcitonin specific cDNA fragments (Sp. Ac 3.2.times.10.sup.8 cpm/ug), a 
Bg1II/PstI CGRP specific cDNA fragment (Sp. Ac. 7.9.times.10.sup.8 
cpm/ug), and a Bg1II/Bg1II `intron` specific cDNA fragment (Sp. Ac. 
2.8.times.10.sup.8 cpm/ug)-see FIG. 1. The filters were washed, then 
autoradiographed. This demonstrated (FIG. 10), in agreement with RLA data, 
that calcitonin and CGRP mRNA species were expressed at different levels 
in the cell-lines and tumours, and that calcitonin and CGRP mRNA were 
probably processed from a common transcript via different processing 
pathways. Also that the intron specific sequence was not present in the 
mature processed calcitonin or CGRP mRNA species. 
Investigation of genomic organisation of the human CGRP gene sequence after 
restriction endonuclease digestion of normal placental DNA and also MTC 
tissue DNA and blood lymphocyte DNA from a patient (FIG. 11) also produced 
differences in gene organisation. Thus 20 .mu.g of each DNA sample was 
restricted with BamHI, and the fragments separated on the basis of size by 
electrophoresis on a 1% (w/v) agarose gel, blotted onto Gene Screen Plus 
membrane (NEN), then probed using a Bg1II/PstI CGRP specific cDNA probe 
labelled to a specific activity of 10.sup.8 cpm/ug. Hybridisation was 
performed overnight in 50% (v/v) formamide, 1% (w/v) SDS, 10% (w/v) 
dextran sulphate in 50 mm Tris-HCl pH 7.5. Filters were then washed 
successively in 2.times.SSC at RTP; 2.times.SSC, 1% (w/v) SDS at 
65.degree. C. for 30 min., and 0.1.times.SSC at 65.degree. C. for 15 min 
then autoradiographed for 48 h. This demonstrated (FIG. 11) a single major 
band of hybridisation (2.8 kb) and a minor band (2.6 kb) in all DNA 
samples, but an additional minor band (3.0 kb) in the placental DNA. Thus 
using a CGRP specific cDNA probe we have identified gene rearrangements, 
in this instance in a gene which shows homology with the probe, as opposed 
to being homologous. Such observations point to the diagnostic value of 
sequence specific probes in the investigation of gene rearrangements in 
tumour tissue, and in this instance the possibility of using a CGRP gene 
probe to investigate linked restriction enzyme polymorphisms in the 
familial form of medullary thyroid carcinoma. 
Summary 
Our studies on human calcitonin gene expression at the molecular level, and 
investigation of the cardiovascular activity of synthetic human CGRP 
corroborates 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., Mermod, 
J--J., Amara, S. G., Swanson, L. W., Sawchenko, P. E., Rivier, J., Vale, 
W. W. & Evans, R. M. (1983) Nature, 304, 129-135) (Fisher, L. A., Kikkawa, 
D. O., Rivier, J. E., Amara, S. G., Evans, R. M., Rosenfeld, M. G., Vale, 
W. W. & Brown, M. R. (1983) Nature, 305, 534-536). We have identified 
calcitonin and CGRP mRNA sequences in human thyroid and lung carcinoma, 
observations supported by the identification using antibodies raised 
against human CGRP or fragments thereof, of CGRP in lung carcinoma cell 
lines and in medullary thyroid carcinoma tissue and plasma. Thus 
measurement of plasma CGRP levels, the histological examination of tissues 
using in situ hybridisation or immunocytochemical techniques, or the 
examination of gene structure and expression, using DNA and RNA blotting 
may be of value in the management of medullary thyroid carcinoma (Hill, C. 
S., Ibanez, M. L., Samaan, N. A., Ahearn, M. J. & Clark, R. L. (1973) 
Medicine, 52, 141-171) lung carcinoma and diseases known to be associated 
with abnormal calcitonin gene expression for example osteoporosis. 
Our observations that amidated human CGRP peptide PDA-4 have an effect on 
the cardiovascular system causing an increase in rate and force of 
contraction of the heart, and a hypotensive effect, suggests a role for 
the peptides in the clinical management of hypertension. Our studies also 
demonstrate that the peripheral action of CGRP is independent of 
catecholamine .beta.-receptors and histamine receptors. In the light of 
the rapid onset of the hypotensive response following the peptide, the 
present results are consistent with the view that CGRP may act directly on 
the cardiovascular system through a novel receptor mechanism independent 
of other mediators.