MAMMAL INTESTINAL HORMONE PRECURSOR AND ITS USE

Abstract: A mammal intestinal hormone precursor having the following peptide structure: His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-L-M-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-Gly-N-O, wherein L is Glu or Asp, M is gly or Ser, and N and O are selected from Lys and Arg; compositions containing such precursor and a method of stimulating pancreatic secretion.

Description: Mammal intestinal hormone precursor and its use. The present invention relates to a mammal intesti¬ nal hormone precursor related to the hormone secretin which stimulates pancreatic secretion. Secretin is an intestinal hormone formed by the muc of the upper portion of the small intestine, which stimulate the secretion of water and bicarbonate from the pancreas. Th structure of porcine secTβtin has been known for some time and it has been isolated from poτcine intestine and has been found to be constituted by a peptide composed of 27 amino a- cid residues (Mutt, V., Jorpes, J.E. and Magnusson, S. (1970 Eur.J.Bioche . , 15, 513-519). Moreover, it has been found th bovine and porcine secretins are identical but that they are marked different from chicken secretin (Carlquist, M. , Jδrn- vall, H. and Mutt, V. (1981) FEBS Lett., 127, 71-74). The C- terminal amino acid residue of secretins (valine in porcine/ bovine, methionine in chicken) is, as in many other hormonal ly active peptides, amidated. In accordance with the present invention variant fo of secTetins which are not amidated at its C-terminal residu but instead have a C-terminal residue of glycine have now been found. These variants which can be considered as pro- forms are precursors to the natural secretins possess high secretin-like bioactivity. Thus , according to the present invention there is provided a mammal intestinal hormone precursor having the following peptide structure: His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Seτ-Arg-Leu-Arg-L-M- Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-Gly-N-0, wherein L is Glu or Asp, M is Gly or Ser, and N and 0 are selected from Lys and Arg. It can be seen from this structure that it contains s - ***-- 30 amino acid residues corresponding to a natural secretin molecule extended by three amino acid residues. In the hormone precursor corresponding to the hu¬ man secTβtin L is Glu and M is Gly, whereas in the non-hu- man counterpart L is Asp and M is Ser. In regard to the C-terminal amino acid residues it is preferred that one of N and M is Lys and the other one is Arg. It has been found that the secretin precursors accor ding to this invention are at least as potent as natural sec¬ retins in stimulating the secretion of bicarbonate by the pan creas of the anesthetized cat (Mutt, V. and. Sδderberg, ϋ. On the assay of secretin. Ark.Kern. 15, 63-68, 1959). 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 Ch mistry, second edition, Ralph J.. Fessenden & Joan S. Fessende Willard Grant Press, Boston, Mass., pages 852 and 853. In the same way as the known natural secretins find diagnostic uses the secretin precursors according to this invention are 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 pre- cursor of this invention in combination with a carrier which does not interfere with the diagnostic procedure used. The secretin precursor of this invention is in addi¬ tion therapeutically useful in that it stimulates pancreatic secretion in mammals if administered in a suitable manner. Accor- ding to this aspect of the invention a composition for such use is provided comprising an effective therapeutic amount of the secretin precursor of the invention in combination with a non-toxic, pharmaceutically acceptable carrier. In this con text the invention also covers a method of treating gastro- intestinal disorders comprising administering a therapeuti¬ cally effective amount of the secretin- precursor of this invention or a composition of this invention on a patient to be treated. ' 4_*_ I__ --*-'".TJ ■ » • _*? ■ _- <-"_ • f - r ~ T- 9 —» The present invention thus includes within its scope pharmaceutical compositions, which compris-e the mammal intes¬ tinal hormone precursor according to this invention in asso¬ ciation with a pharmaceutically acceptable carrier. In cli- nical practice the compositions of the present invention will normally be administered parenterally due to the fact that be¬ ing a peptide the hormone precursor is sensitive to biologi¬ cally active environments. Oral or rectal administration may, however, be conceivable using compositions of the slow relea- se 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 pre¬ ferred 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 es¬ ters, such as ethyle oleate. These compositions may also con¬ tain adjuvants, such as preserving, wetting, emulsifying and dispersing agents. They may be sterilized, for example, by fil tration through a bac'teria-retaining filteτ, by inσorporation of sterilizing agents in the composition, by irradiation or by heating. They may also be manufactured in the form of sterile solid compositions, which can be dissolved in a sterile inject ible medium immediately before use. As well as the more custo- mary intravenious and intramuscular routes the composition may also be administered by intra-articular injection. The percentages of active ingredient in the composi¬ tions of the invention may be varied as long as they consti¬ tute a proportion such that a suitable dosage for the desired stimulatory effect on the pancreas is obtained. Obviously se¬ veral unit dosage forms may be administered at about the same time. Generally, the compositions should contain from about 0,H to about 80* by weight of active ingredient. The dose employed depends upon the desired sti ula- tory effect, the route of administration and the duration of the treatment. The hormone precursor of this invention may be administered each day or, according to the wishes of the medical practitioner, less often e.g. weekly. This invention also covers a method of stimulating pancreatic secretion in a patient suffering from gastrointes¬ tinal disorder, this method comprises administering to the patient an amount of the hormone precursor or the composition of the invention sufficient to combat such disorder. The invention will be further illustrated below in an example describing the isolation and characterization of a secretin precursor of this invention. EXAMPLE The starting material. The upper part of porcine intestine was boiled, frozen and minced. The resulting powder was extracted with 0.5 M acetic acid and the peptides were adsorbed on alginic acid. Then elution with 0.2 M HC1 and precipitation with NaCl were performed. The resulting eluate was gelfiltered on Sephadex G25 (Pharmacia Fine Chemicals) in 0.2 M acetic acid. Extraction was carried out with methanol and soluble materials were precipitated with ether. The resulting solu- tion was gelfiltered on Sephadex G25 (Pharmacia Fine ^ Chemi- , cals) in 0.2 M acetic acid. The resulting solution was sub¬ jected to ion exchange chromatography on CM-cellulose and elution was then carried out with 12.5 mM phosphate buffer at a pH of 8. With regard to details of the procedure see Said, S.I. and Mutt, V. Isolation from porcine-intestinal wall of a vasoactive octacosapeptide related to secretin and to glucagon. Eur.J.Biochem. 28, 199-204, 1972. The eluate was further subjected to two additional CM-chromatogτaphies. In the first one the elution was caT- ried out in a 0.02 M phosphate buffer at pH 6.4 with a salt gradient of 0-0.3 M NaCl. A fraction eluting at about 0.15 M NaCl was further chromatogτaphed on a second CMC column eluted with a stepwise gradient at pH 8 in NH 4 HC0 3 at dif¬ ferent concentrations. The fraction referred to here as the "starting material" was eluted with 0.04 M NH 4 HC0 3 from this column. The starting material was further purified on high performance liquid chromatography (HPLC) HPLC Reverse phase HPLC was carried out on a ,uBondapak C 18 column (7.8 x 300 mm) or a LKB Ultropac TSK ODS-120T col¬ umn (3.9 x 300 mm) with 0.12* trifluoroacetic acid (TFA) in water and 0.1. TFA in acetonitrile as mobile phases. Ion ex¬ change chromatography was performed on a LKB Ultropac TSK535 CM-column (7.5 x 150 mm) with 0.02 sodium phosphate buffer at pH 6.4 and a NaCl-gradient of from 0 to 0.3 M NaCl. Both Teverse phase and ion exchange HPLC were carried out on an instrument from Waters Associated. Biological activity The biological activity was measured by the ability to stimulate the secretion of alkali in the pancreatic juice in the anesthetized cat (Mutt, V. and Sδderberg, U. On the assay of secretin. Ark.Kern. 15, 63-68, 1959). The sample was compared to a secretin standard with an established activity of 3500 units per mg. Proteolytic fragmentation The peptide was digested with TPCK-treated trypsin for 6 h or TLCK-treated chy otrypsin for 4 h in 1* NH 4 HC0 3 , pH 8 at room temperature. The ratio of peptide to enzyme was 50:1 by weight. The enzymes were purchased from Worthington (trypsin) and Merck ( chymotrypsin) . The proteolytic frag¬ ments were separated by reverse phase HPLC on a ,uBondapak C.g column as described above. The gradients used were, for tryptic fragments, o-40\ acetonitrile in TFA for 40 minutes at a flow rate of 1 ml per minute and, for chymotrypsic frag¬ ments, 10-25* acetronitrile in TFA foτ 30 minutes at a flow rate of 1 ml per minute. Amino acid composition analysis Hydrolysis was carried out in evacuated tubes con¬ taining 6M HC1 with 0.1. β-mercaptoethanol at 110°C for 24 h, All hydrolysates were analyzed in a Beckman 121 amino acid analyzer except the tryptic digests which were analyzed in a Waters Assoc. amino acid analysis system using precolumn derivatization with o-phthalaldehyde (OPA) (Roth, N. Analy¬ tical Chemistry 43, 880- , 1971). Identification of free arginine The tryptic digestion mixture was treated with PITC (Koop, D.R. , Morgan, E.T., Tarr, G.E. and Coon, M.J. J.Biol.Chem. 257, 8472-8480, 1982) to form the phenylthio- carbamyl derivative of the fragments formed during tryptic digestion of the secretin variant. The mixture of the tryp¬ tic peptides and any fTee PTC-a ino acid was then run on a Spherisorb C_,g column (3/U) in a HPLC-system for separation of PTC-amino acids. The system used was as described by Hein- rikson and Meredith (Heinrikson, R.L. and Meredith, S.C. Analytical Bioche . 136, 65-74, 1984). RESULTS During the purification of VIP and a search for variant forms of it (Dimaline, R. and Dockray, G.J. Multiple i unoreactive forms of vaso-active intestinal peptide in hu¬ man colonic mucosa. Gastroenterologγ 75, 387-392, 1978) a certain fraction was observed to have a secretin- rather than a VIP-like bioactivity. The final purification of the secretin variant or precursor from the starting material was obtained by three HPLC chro atographies. The first HPLC-step was a re¬ verse phase chromatography on a ,uBondapak C_,g column using a gradient of 32-38* acetonitrile in 0.12* TFA, 30 minutes at a flow rate of 2 ml per minute. The chromatogra is shown in Fig 1. The active fraction was further subjected to an ion exchange HPLC-step on a LKB Ultropac CM column eluted with a gradient of 0.12 to 0.3M NaCl in a 0.02 M phosphate buffer pH 6.4 for 15 minutes and isocratic elution at 0.3 M NaCl in the same buffer for another 15 minutes. The flow rate was 1 ml per minute. Desalting by reverse phase HPLC using a LKB Ultropac ODS column also gave some purification (Fig. 2) and the isolated peptide was considered to be pure after this step Active fractions were identified with the bioassay for sec¬ retin (described under "The Starting Material") throughout the purification procedure The total amino acid composition of the intact pep¬ tide is shown in Table 1. Comparison of this amino acid com- e«- * TJ" * " ! - _r -__- —•- ——» J position to that of secretin indicates that the isolated pep¬ tide differs from porcine secretin by containing a Lys resi¬ due and one additional residue each of Arg and Gly. To check if the purified peptide is a variant form of secretin, tryp¬ tic digestion of it was carried out and the fragments obtai¬ ned were separated on reverse phase HPLC. The chromatograms of the tryptic fragments of the purified peptide and of sec¬ retin are shown in Figs. 3 and 4. Only one fragment eluted at different positions in the two chromatograms. This frag¬ ment corresponds to the C-terminal fragment T, as shown by the amino acid composition. Each tryptic fragment of the i- solated peptide was hydrolyzed and subjected to total amino acid composition analysis. The amino acid compositions of the tryptic peptides are shown in Table 3. The C-terminal fragment of the peptide, T,, contains an extra Gly and a Lys residue as compared to the corresponding fragment of secretin, while the other fragments (T.-T,) are identical with the secretin fragments. Chymotryptic degradation of secretin and of the isolated peptide was carried out in pa- rallel and led in both cases to numerous fragments. With one exception each fragment of the peptide appeared to be identical to a fragment of secretin and vice versa. The amino acid compositions of the two nonidentical fragments was determined (Table 2). That of the secretin fragment was Gly-, Glx.. , Leu.,, Val.,, unequivocally showing that it represented the C-terminal Gln-Gly-Leu-Val-N^ se¬ quence of secretin. That of the polypeptide fragment was Arg 1 , Gly 2 , Glx-. , Leu_ j , Lys- j , Val- j , suggesting the sequence Gln-Gly-Leu-Val-Gly-Lys-Arg. Consequently the findings sug- gested that the polypeptide consisted of secretin extended C-terminally with Gly-Lys-Arg. If so, free arginine should . be split from it on tryptic degradation and this was also shown to be the case; a tryptic digest was derivatized with PITC (Koop, D.R. , Morgan, E.T., Taττ » G.E. amd Coon, M.J. J.Biol.Chem. 257, 8472-8489, 1982) and applied to an HPLC- syste for the detection of PTC-amino acids. All five tTyp- tic peptide derivatives and free PTC-arginine were indeed " , de¬ tected. Slt ~ ~> ~~-.•-*• • -..--cr e- r 7 f " . τ The above data show that the isolated polypeptide of the invention is a variant form of secTetin with a C- ter inal extension by Gly-Lys-Arg. This is in good agreement with the knowledge that in peptide hormone precursor proteins a glycine residue followed by paired basic residues usually represents a idation and cleavage signals , respectively. The pure secretin precursor was tested for bioact- ivity in the secretin biόassay described above. As shown in Fig. 5, secretin-Gly-Lys-Arg is about 1.5 times more potent than secretin itself. It is to be noted that the invention is not limi¬ ted to the specific details and features of the example de- described above and that modifications can be made to the polypeptide structure without deviating from the inventive idea. Thus, the invention is not limited otherwise than according to the scope of the appended patent claims. SUBSTITUTE SHE T TABLE 1 Amino acid composition of the secretin-variant. Values are molar ratio without correction for destruction, incomplete hydrolysis or impurity. Figures within parantheses represent the predicted numbers of each amino acid in the secretin variant Amino acid Secretin variant Secretin Lys 1.1 (1) - His 1.3 (1) 1 Arg 4.9 (5) 4 Asx 2.1 (2) 2 Thr 1.8 (2) 2 SeT 3.3 (4) 4 Glx 3.0 (3) 3 , Gly 3.0 (3) 2 Ala 1.2 (1) 1 Val 1.0 (1) 1 Leu 6.0 (6) 6 Phe 1.0 (1) 1 Total 30 27 3£l . ~^ - _"~'? ~~ 'f . ~fr* _ -- ■ "-* ■ —••y _H ϋ i ϋ ϋ i __..- • * •'-;.- , TABLE 2 Amino acid -compositions of the tryptic fragments and the C-termi¬ nal chymotryptic fragment of the secretin variant. Values are mo¬ lar ratios without correction for destruction, incomplete hydro¬ lysis or impurity. Figures within parantheses are the numbers of amino acid in the corresponding secretin fragment. Amino T. Chymotryptic acid fragment Lys l.O(-) 0.9(-) His 0.8(1) Arg 0.8(1) 0.9(1) 0.8(1) 0.8(1) 0.9(0 Asx 1.3(1) 1.3(1) Thr 2.0(2) Ser 2.8(3) 0.9(1) Glx 1.4(1) 1.2(1) 1.3(1) 1.2(1) Gly 1.0(1) 1.4(1) 2.0(1) Ala 1.0(1) Val 1.3(1) 0.9(1) Leu 1.0(1) 1.1(1) 1.0(1) 3.0(3) 1.1(1) Phe 0.9(1) Total 12 SM^T

Claims: CLAIMS 1. A mammal intestinal hormone precursor having the following peptide structure: His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-L-M- Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-Gly-N-O, wheTein L is Glu or Asp, M is Gly or Ser, and N and 0 are selected from Lys and Arg. 2. The hormone precursor of claim 1, wherein L is Glu and M is Gly. 3. The hormone precursor of claim 1, wherein L is Asp and M is Ser. 4. The hormone precursor of any of claims 1 to 3, whe¬ rein N is Lys and 0 is Arg. 5. The hormone precursor of any of claims 1 to 3, whe¬ rein N is Arg and 0 is Lys. 6. The hormone precursor of any of claims 1 to 5 for diagnostic use in determining pancTeatic or gallbladder func¬ tion. 7. The hormone precursor of .any of claims 1 to 5 for therapeutic use to stimulate pancreatic secretion in mammals including man. 8. The hormone precursor of claim 4 or 5 for therapeu¬ tic use to stimulate pancreatic secretion in man. 9. A composition for use in stimulating pancreatic sec¬ retion in mammals including man, comprising an effective therapeutic amount of the hormone precursor of any of claims 1 to 5 in combination with a non-toxic carrier therefore. 10. A composition for use in stimulating pancreatic secretion.in man, comprising an effective therapeutic amount of the hormone precursor of claim 2 in combination with a non-toxic carrier therefore. •1 . A composition for diagnostic use in determining pan¬ creatic or gallbladder function, comprising an effective diag¬ nostic amount of the hormone of any of claims 1 to 5 in com¬ bination with a non-interfering, carrier therefore. 12. A method of stimulating pancreatic secretion in a patient suffering from gastrointestinal disorder, which comprises administering to the patient an amount of the hor¬ mone precursor of any of claims 1 to S or the composition of claim 9 or 10 sufficient to combat such disorder. ■ τ

Inventors: CARLQUIST MATS, GAFVELIN GURO, MUTT VIKTOR, CARLQUIST, MATS, GAFVELIN, GURO, MUTT, VIKTOR
Applicants: KABIGEN AB, SKANDIGEN AB
Application year: 1986
Main classification: C07K7
IPC codes: A61K38, C07K1, C07K14