HUMAN INTESTINAL HORMONE AND ITS USE

Abstract: 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-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH2; compositions containing such hormone; and a method of stimulating pancreatic secretion.

Description: Human intestinal hormone and its use. The present invention relates to human intestinal hormone, namely the hormone secretin which stimulates pancreatic sec- retion. 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 i has been isolated from porcine intestine and has been found to be constituted by a peptide composed of 27 amino acid residues C Mutt, V., Jorpes, J.E. and Magnusson, S. (1970) Eur.J.Biochem. 15, 513-519). Moreover, it has been found that bovine and por¬ cine secretins are identical but that they are marked different from chicken secretin (Carlquist, M. , Jδrnvall, 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 gluta- ic 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-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH 2 . In the instant disclosure the abbreviations used for characterizing the amino acids and their residues are the tra¬ ditional ones as found for example in the textbook Organic Che¬ mistry, 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 dia- gnostic 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 proce¬ dure used. The human secretin of this invention is in addition thera- peutically useful in that it stimulates pancreatic secretion in man if administered in a suitable manner. According to this as¬ pect of the invention a composition for such use is provided comprising an effective therapeutic amount of the human secre¬ tin of the invention in combination with a non-toxic, pharma- ceutically 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 phar¬ maceutical compositions, which comprise the human intestinal hormone according to this invention in association with a phar¬ maceutically acceptable carrier. In clinical practice the com¬ positions 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 rec¬ tal administration may, however, be conceivable using composi¬ tions 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 sol¬ vents or suspending media are propylene glycol, vegetable oils, such as olive oil, and injectible organic esters, such as ethyle 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-re¬ taining filter, by incorporation of sterilizing agents in the composition, by irradiation or by heating. They may also be ma¬ nufactured 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 intravenious and intramuscu¬ lar routes the compositions may also be administered by intra- articular injection. The percentages of active ingredient in the compositions of the invention may be varied as long as they constitute a pro¬ portion 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 ef¬ fect, the route of administration and the duration of the treat¬ ment. The hormone of this invention may be administered each day or, according to the wishes of the medical practitioner, less of ten, e.g. weekly. The invention will be further illustrated below in an ex¬ ample describing the isolation and characterization of the human secretin of this invention. EXAMPLE Pieces of human duodeni were obtained from patients under¬ going surgery. The tissue was immediately rinsed and stored at -20°C. The combined frozen material (181 g wet weight) was im¬ mersed into boiling water for 10 min., cooled on ice, minced and extracted with 0,5 M acetic acid (400 ml) for 16 h at 5°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 0.2 M HC1 and peptides were adsorbed to alginic acid (40 g wet weigh during stirring for 1 h. The alginic acid was collected on a filter and successivley washed with ice-cold 0.005 M HC1, e- thanol and again with the acid, whereafter peptides were eluted with ice-cold 0.2 M HC1 (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 0.3 M NaOH and centrifuged. To the supernatant, two volu¬ mes of cold ethanol was added and the suspension was allowed to sediment at -20 C for 24 h. The precipitate was removed by filtration and the filtrate was adjusted to pH 3.0 with 0.1 M HC1 Peptides were recovered after addition of 100 ml methanol follo¬ wed by three volumes of ether (Carlquist, M., Kaiser, R. , Tate- moto, K., Jδrnvall, H. and Mutt, V. (1984) Eur.J.Biochem. , 144, 243-247.) This precipitate was dried under vacuum, dissolved in 1 ml 0.2 M acetic acid and chromatographed on a Sephadex G-25 (fine) column (0.6 x 95 cm) in 0.2 M acetic acid. Fractions of 1 ml were collected and tested in the secretin bioassay (Mutt, V. and Sδderberg, U. (1959) Arkiv. f. Kemi, 15, 63-68). The frac¬ tions containing the bulk of the secretin activity were combined and submitted to ion-exchange HPLC on an LKB TSK 535 CM column (7,5 x 150 mm) in a Waters instrument. Elution was performed with a gradient of sodium chloride (0.075-0.3 M, 75 min) in a 0.02 M sodium phosphate buffer, pH 6.4, at 1 ml/min and frac¬ tions of 1 ml were collected. Final purification was carried out by reverse-phase HPLC using an LKB TSK ODS-120T column (4.6 x 250 mm) . Elution was performed with a gradient of acetonitrile (25-50 , 50 min) in 0.1 trifluoroacetic acid at 1 ml/min (Carl- quist, M. and Rδkaeus, A. (1984) J. Chromatogr. 296, 143-151). Hydrolysis was carried out for 24 h at 110 C in evacuated tubes with 6 M HC1 containing 0.5% phenol. Amino acides were a- nalyzed by reverse-phase HPLC after precolu n 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., Appella, 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. g co- lumn, 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 p ol, is shown in Table 1 as enclosed hereto. Like porcine secretin, human secretin is, composed of 27 amino acid resi¬ dues, 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 in¬ vention 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 1n _ 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 du¬ ring the isolation. Figure 1. 1 5 10 15 20 25 His-Ser-Asp-Gly-.Thr-Phe-Thr-Ser-Glu-Leυ r Ser-Arg-Leυ-(Arg)-Glu-Gly-Ala-(Arg)-Leu-Gln-(Arg)-Le -Leu-Gln-Gly-(Leu)-(Val 286 54 182 253 67 156 77 65 104 69 45 57 55 64 22 64 64 28 Table 1 . Amino acid composition or r.uman secrcm Amino acid Human secretin Porcine/bovine secretin 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

Claims: CLAIMS 1. 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-Leu-Gln-Arg-Leu-Leu-G-ln-Gly-Leu-Val-NH 2 . 2. The hormone of claim 1 for diagnostic use in deter¬ mining pancreatic or gallbladder function. 3. The hormone of claim 1 for therapeutic use to sti¬ mulate pancreatic secretion in man. 4. A composition for use in stimulating pancreatic sec¬ retion in man comprising an effective therapeutic amount of the hormone of claim 1 in combination with a non-toxic carrier therefore. 5. A composition for diagnostic use in determining pan¬ creatic or gallbladder function, comprising an effective dia¬ gnostic amount of the hormone of claim 1 in combination with a non-interfering carrier therefore. 6. A method of stimulating pancreatic secretion in a patient suffering from gastrointestinal disorder, which com¬ prises administering to the patient an amount of the hormone of any of claims 1-3 or composition of claim 4 sufficient to combat such disorder.

Inventors: CARLQUIST MATS, FORSSMANN WOLF-GEORG, JOERNVALL HANS, JOHANSSON CATJA, MUTT VIKTOR, THULIN LARS, CARLQUIST, MATS, FORSSMANN, WOLF-GEORG, JOERNVALL, HANS, JOHANSSON, CATJA, MUTT, VIKTOR, THULIN, LARS
Applicants: KABIGEN AB, SKANDIGEN AB
Application year: 1986
Main classification: C07K7
IPC codes: A61K38, C07K1, C07K14