Human intestinal hormone and its use

A human intestinal hormone having the following peptide structure: His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Glu-Gly-Ala-Arg-Le u-Gln-Arg-Leu-Gln-Gly-Leu-Val-NH.sub. 2 ; compositions containing such hormone; and a method of stimulating pancreatic secretion.

The present invention relates to human intestinal hormone, namely the 
hormone secretin which stimulates pancreatic secrection. 
Secretin is an intestinal hormone formed by the mucosa of the upper portion 
of the small intestine, which stimulates the secretion of water and 
bicarbonate from the pancreas. The structure of porcine secretin has been 
known for some time and it has been isolated from porcine intestine and 
has been found to be constituted by a peptide composed of 27 amino acid 
residues (Mutt, V., Jorpes, J. E. and Magnusson, S. (1970) Eur. J. 
Biochem., 15, 513-519). Moreover, it has been found that bovine and 
porcine secretins are identical but that they are markedly different from 
chicken secretin (Carlquist, M., Jornvall, H. and Mutt, V. (1981) FEBS 
Lett., 127, 71-74). 
Although bovine and porcine secretins behave identically with human 
secretin in some respects they are not structurally identical. In 
accordance with the instant invention it has now been found that amino 
acids number 15 and 16 differ in that the human secretin at said positions 
contains the residues of glutamic acid (Glu) and glycine (Gly), 
respectively. Thus, the human intestinal hormone of this invention has the 
peptide structure: 
His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Glu-Gly-Ala-Arg-Le 
u-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH.sub.2. 
In the instant disclosure the abbreviations used for characterizing the 
amino acids and their residues are the traditional ones as found for 
example in the textbook Organic Chemistry, second edition, Ralph J. 
Fessenden & Joan S. Fessenden, Willard Grant Press, Boston, Mass., pages 
852 and 853. 
In the same way as the known secretins find diagnostic uses the human 
secretin according to this invention is highly useful in determining 
pancreatic and gallbladder functions. According to this aspect of the 
invention a composition for diagnostic use in this respect comprises an 
effective diagnostic amount of the secretin of this invention in 
combination with a carrier which does not interfere with the diagnostic 
procedure used. 
The human secretin of this invention is in addition therapeutically useful 
in that it stimulates pancreatic secretion in man if administered in a 
suitable manner. According to this aspect of the invention a composition 
for such use is provided comprising an effective therapeutic amount of the 
human secretin of the invention in combination with a non-toxic, 
pharmaceutically acceptable carrier. In this context the invention also 
covers a method of treating gastro-intestinal disorders comprising 
administering a therapeutically effective amount of the hormone of this 
invention or a composition of this invention on a patient to be treated. 
The present invention thus includes within its scope pharmaceutical 
compositions, which comprise the human intestinal hormone according to 
this invention in association with a pharmaceutically acceptable carrier. 
In clinical practice the compositions of the present invention will 
normally be administered parenterally due to the fact that being a peptide 
the hormone is sensitive to biologically active environments. Oral or 
rectal administration may, however, be conceivable using compositions of 
the slow release type making it possible for the active ingredient to 
reach the site of primary interest, namely the small intestine. 
Preparations according to the invention for the preferred parenteral 
administration includes sterile aqueous or non-aqueous solutions, 
suspensions or emulsions. Examples of non-aqueous solvents or suspending 
media are propylene glycol, vegetable oils, such as olive oil, and 
injectible organic esters, such as ethyl oleate. These compositions may 
also contain adjuvants, such as preserving, wetting, emulsifying and 
dispersing agents. They may be sterilized, for example, by filtration 
through a bacteria-retaining filter, by incorporation of sterilizing 
agents in the composition, by irradiation or by heating. They may be also 
be manufactured in the form of sterile solid compositions, which can be 
dissolved in a sterile injectible medium immediately before use. As well 
as the more customary intravenous and intramuscular routes the 
compositions may also be administered by intraarticular injection. 
The percentages of active ingredient in the compositions of the invention 
may be varied as long as they constitute a proportion such that a suitable 
dosage for the desired stimulatory effect on the pancreas is obtained. 
Obviously several unit dosage forms may be administered at about the same 
time. Generally, the compositions should contain from about 0.1% to about 
80% by weight of active ingredient. 
The dose employed depends upon the desired stimulatory effect, the route of 
administration and the duration of the treatment. The hormone of this 
invention may be administered each day or, according to the wishes of the 
medical practitioner, less often, e.g. weekly.

The invention will be further illustrated below in an example describing 
the isolation and characterization of the human secretin of this 
invention. 
EXAMPLE 
Pieces of human duodeni were obtained from patients undergoing surgery. The 
tissue was immediately rinsed and stored at -20.degree. C. The combined 
frozen material (181 g wet weight) was immersed into boiling water for 10 
min., cooled on ice, minced and extracted with 0.5M acetic acid (400 ml) 
for 16 h at 5.degree. C. The suspension was filtered through Whatman 541 
with the aid of 10 g Hyflo Super Cel. The filtrate was adjusted to pH 2.7 
with 0.2M HCl and peptides were absorbed to alginic acid (40 g wet weight) 
during stirring for 1 h. The alginic acid was collected on a filter and 
successivley washed with ice-cold 0.005M Hcl, ethanol and again with the 
acid, whereafter peptides were eluted with ice-cold 0.2M HCl (190 ml). 
Sodium acetate was added to the eluate to pH 3.8 and peptides were 
precipitated with NaCl at saturation. 
The precipitate obtained (460 mg) was dissolved in 4.6 ml water, diluted 
with two volumes of ethanol, brought to pH 7.2 with 0.3M NaOH and 
centrifuged. To the supernatant, two volumes of cold ethanol was added and 
the suspension was allowed to sediment at -20.degree. C. for 24 h. The 
precipitate was removed by filtration and the filtrate was adjusted to pH 
3.0 with 0.1M HCl. Peptides were recovered after addition of 100 ml 
methanol followed by three volumes of ether (Carlquist, M., Kaiser, R., 
Tatemoto, K., Jornvall, H. and Mutt, V. (1984) Eur. J. Biochem., 144, 
243-247.) This precipitate was dried under vacuum, dissolved in 1 ml 0.2M 
acetic acid and chromatographed on a Sephadex G-25 (fine) column 
(0.6.times.95 cm) in 0.2M acetic acid. Fractions of 1 ml were collected 
and tested in the secretin bioassay (Mutt, V. and Soderberg, U. (1959) 
Arkiv. f. Kemi, 15, 63-68). The fractions containing the bulk of the 
secretin activity were combined and submitted to ion-exchange HPLC on an 
LKB TSK 535 CM column (7.5.times.150 mm) in a Waters instrument. Elution 
was performed with a gradient of sodium chloride (0.075-0.3M, 75 min) in a 
0.02M sodium phosphate buffer, pH 6.4, at 1 ml/min and fractions of 1 ml 
were collected. Final purification was carried out by reverse-phase HPLC 
using an LKB TSK ODS-120T column (4.6.times.250 mm). Elution was performed 
with a gradient of acetonitrile (25-50%, 50 min) in 0.1% trifluoroacetic 
acid at 1 ml/min (Carlquist, M. and Rokaeus, .ANG.. (1984) J. Chromatogr. 
296, 143-151). 
Hydrolysis was carried out for 24 h at 110.degree. C. in evacuated tubes 
with 6M HCl containing 0.5% phenol. Amino acides were analyzed by 
reverse-phase HPLC after precolumn derivatization with 
phenylisothiocyanate (Koop, D. R., Morgan, E. T., Tarr, G. E. and Coon, M. 
J. (1982) J. Biol. Chem. 257, 8472-8480). 
Edman degradation of the peptide was performed with an Applied Biosystems 
Model 470A gas-phase sequence and amino acid derivatives were analyzed by 
HPLC (Zimmerman, C. L., Apella, E. and Pisano, J. J. (1977) Anal. Biochem. 
77, 569-573). 
RESULTS 
The isolation procedure described above, starting with 181 g tissue, 
resulted in 500 pmol secretin. Through all steps up to and including the 
ion-exchange step, the material behaved identically to porcine/bovine 
secretin. However, on the C.sub.18 column, the elution time for the human 
hormone was not identical to that of the porcine. These findings indicate 
that human and porcine secretins are not identical, although they have the 
same net charge. 
The result of the amino acid analysis, performed on 20 pmol, is shown in 
Table 1 as enclosed hereto. Like porcine secretin, human secretin is, 
composed of 27 amino acid residues, but with a different composition. 
Differences are seen for position 15, Asp (-1), Glu (+1) and position 16, 
Ser (-1) and Gly (+1). 
The amino acid sequence of the human secretin of this invention is shown in 
appended FIG. 1. The figure shows gas-phase sequence degradation of 450 
pmoles of the intact peptide. The values shown constitute pmoles 
recovered. Residues within parenthesis are not fully identified. 
Repetitive yield calculated on Leu.sub.10-22 is 96%. 
The structure shown in FIG. 1 differs from that of porcine/bovine secretin 
at positions 15 and 16, by having Glu-15 and Gly-16 instead of Asp-15 and 
Ser-16. This is in full agreement with the amino acid analysis and 
explains the observations during the isolation. 
##STR1## 
TABLE 1 
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Amino acid composition of human secretin 
Amino acid Human secretin 
Porcine/bovine secretin 
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Asp 1.0 (1) 2 
Glu 4.0 (4) 3 
Ser 2.7 (3) 4 
Gly 3.1 (3) 2 
His 1.1 (1) 1 
Thr 2.0 (2) 2 
Ala 1.2 (1) 1 
Arg 3.7 (4) 4 
Val 1.3 (1) 1 
Leu 5.7 (6) 6 
Phe 1.0 (1) 1 
Total 27 27 
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