CA1052772A

CA1052772A - Peptides for control of intestinal motility

Info

Publication number: CA1052772A
Application number: CA239,188A
Authority: CA
Inventors: Irving Goodman; Robert B. Hiatt
Original assignee: Research Corp
Current assignee: Research Corp
Priority date: 1974-11-14
Filing date: 1975-11-07
Publication date: 1979-04-17
Also published as: JPS5191311A; GB1497316A; DE2550992A1; FR2290911B1; FR2290911A1; JPS6114126B2; DE2550992C2

Abstract

ABSTRACT OF THE DISCLOSURE

Peptides isolated from the bovine posterior pituitary are useful for the control of intestinal motility.

Description

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..~, This invention is concerned with compounds isolated from the bovine posterior pituitary which possess useful activity on the smooth muscle of the gastrointestinal tract and other tissues. More particularly, it is concerned with~
certain low molecular weight peptides which, because they stab~lize intestinal motility in mammals, are useful in the treatment of ileitis, colitis, spastic constipation, spastic diarrhea, diverticulitis, peptic ulcer and other conditions where irregularity of the peristaltic function is involved.
The products of the invention are certain low molecular weight peptides which can be isolated from the ,. . .
~ bovine posterior pituitary, and with the pharmaceutically :, .
! acceptable salts of such peptides. The invention is also ;~ concerned with physiologically useful compositions containing . such compounds as the principal active ingredient together : .
with a pharmaceutically acceptable carrier. Methods for the treatment of ailments such as those mentioned above and other -~
conditions associated with arrhythmia of the intestinal tract are also within the scope of the invention. One of the peptides of this invention in addition to its utility for . the control of intestinal motility also manifests a high degree of vasoconstrictor activity. The product and its pharmaceutically acceptable salts, therefore, are useful in ~;1 treatment of shock, and other conditions where it is necessary ! or desirable to effect contraction of the capillaries and ;~ arteries.
;' For convenience, the products which are isolated by the process of this invention are designated Coherin A, B

and C. Coherin A and B are the particular subjects of this ,:i ., .~

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inventlon. Both are userul ln the control Or lntestlnal motllity. Both manl~est vasocontrlctor activlty, although the actlvity Or Coherin A is rather 10W. Coherin C presently ~ppears to ~unction as a carrier.
~ he procedure for ~he isolation o~ these products i~ as ~ollows.
In step 1, acetone dried~bovine posterior pitu-itary powder (50g), ~Miles Laboratories) was homogenized or 5 minutes in 2 x 125 ml of ammonium acetate (0.2M, pH 5;6) ln an ice bath. A~ter centrifugation for 40 minutes a~ 18,000 g and 5 C the supernatant solution was ~ilkered through 3 g of diatomaceous filter aid ~Celite, Johns Man-~ille~ Analyticalj, and trans~erred (step 2) to a GOlUmn (50 x 610 mm) of diethylaminoethyl cellulose (DEAE) (Whatman, DE 23) that had been pre-equilibrated with the O.~ M ammonium acetate. The DEAE column was eluted with 0.05 M ammonium acetate pH 5~6 and the eluate was collected in fractions which were assayed for activity as measured by in~bition o~
intestinal contraction and by coherence of contraction in ad~acent segments of intestine in the dog with Roux-en-Y
~istula.
The main biological activity was eluted from DEAE
cellulose in the fraction between 1825 and 3965 ml (Total volume, 2140 ml; ~A280/ml = 4608).
The active fraction obtained froM the DEAE cellu-lose column was lyophilized. Two grams Or the solld was dissolve~ in 10 ml ammonlum aceta$e (0.~02M, pH 5.~) and transferred to a column (50 x 610 mm) o~ cross-llnked dextran gel (Pharmac.ia Corp., Sephadex G-50~ Super~ine*).

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In ~tep 3, elutlon wa~ achleved uslng 0.02 M am-~onium acetate, pH 5.6. The active fraction wa~ eluted bet~een 1460 and 2580 ml (Ye/Vo = 3.08).
The rraction eluted from Sephadex G-50*!was lyophll-ized and the product (201 ~) was dissolved ln O.OZ l~ ammon- ..
ium acetate (5 ml); the solutlon was applied as above to t~tep 4) a column (50 x 1090 mm) o~ cross-linked dextran gel (Pharmacla, Sephadex G-25 Superf'ln~ and eluted as above.
The active component was located ln the rractlons bet~een 1460 and 2580 ml (Ve~Vo = 2.33).
.. In an analogous manner the actlve fractlon obtained rom Sephadex G-25*was lyophilized, dissolved in 4 ml Or , acetlc acld, 0.1 M, and transrerred (step 5) to a column .- (10 x 1050 mm) o~ highly croissed-linked dextran gel (Sephadex ,-~ G-10~ which had been pre-equillbrated with the same sol~ent.
Coherin isolated in step 5 appears to exist as a complex bound either by electrostatic or Van der ~Jaals forces.
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It is obtained upon elution o~ the product Or step 4 from Sephadex G-10 at a rate o~ 0.5 ml per minute in fractlons ,;.j .
- between 248 ml and 363 ml (Ve/Vo = 1.47). Purification o~
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bound Coherln was achieved by dissolvlng 78 mg Or the prod-uct of step 5 in 19 ml acetic acid, 0.2 M. Thls solution j ,, was extracted (3 x 10 ml) with butanol-acetlc acid-water (4:1:5), upper (non-aqueous) phase. The non-aqueous ex-tracts were combined and concentrated in vacuo to a white !
l solid. The solld was dlssolved in 10 ml ethanol, 95% and `b`'' upon standin~ at 5 ~or 48 hours rormed rine white crystals.
Coherln complex remains bound a~ter repeated re-cycling on Sephadex G-lO*in acetic acld, 0.1 M.
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The Coherln'compl,ex lsolated in step 5 undergoes " dissociatlon to yield Coherln A~ B and C when' subJect,ed to -, r~peated cycles Or contlnuous ~low electrophoresis at pH 2.8 and 3.5. Using the Beckman-Splnco Contlnuous Flow Electro-' phoresis apparatus9 Mo~el CP,* Coherin complex (step 5) (~A280 =
;.' 175) ls dissolved ln acetic acicl~ 0.2 M, pH 2.75 to gi~e a . concentration such that A280 = 4.0; this solution is applied . at a polnt + 75 mm rrom vertical m~dline at the rate Or O.42 ,' ml per hour (18 V/cm; 0.'83 mA/cm). The electrolyte was 0.2 M
acetic acid and elution rate was 1.2 ml per tube per hour.
Coherin A was lscated in ~ractions 13 to 19 which c~rrespond'to drip tips 95 m~ cathodal to the point of appli-", ,' cation. The solid obtained a~ter pooling and lyophilizatihn' . Or these tubes was extracted with isopropanol (3 x 3 mI) and ',. collected by centrifugation. . . .
' ' U~der these conditions (pH2.8) Coher~n B is ~so- i. ' -1 lated in fractions 5 to 9, 150 mm cathodal to the point of ;
Y applicatlon and 'Coherin C 1s eluted in ~ractions 17 to 19, 60 mm cathodal to the point o~ appl~cation).
, Tubes.containing Coherin A, B and C respectively ,j '' ' are pooled, lyophilized and individually recycled to electro-' ' `
, . phoretlc homogeneity on the continuous ~low electrophoresi,s , apparatus at pH 2.8 and 3.5. The electrolyte for pH 3.5 a was ammonium acetate, 0.02 M ln acetic acid, 0.2 M. Upon recycling at pH 3.5 Coherin A is located in fractions 19-21, B in fractions 15 to 19 and C in 23 to 25. Since the . .
' ~ractlon numbers on th,e ~eckman-Spinco Model Ci*remain statlonary they indicate the posltion relative to the anode ~nd cathode. The numbers are ~rom 1 to 3~ from cathode to anode.

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~ lOSZ7'A~;2 ' Coherin A, B and C are isolated after repeated recycling on continuous flow electrophoresis at pH 2~8 and 3.5 followed by ethanolic extraction to remove traces of other peptides and free amino aclds.
Coherin A is a peptide containing at least glycine.
Coherin B is also a peptide, but`of somewhat higher molecular we1ght. It also contains glycine and additionally contains at least glutamic acid ~ E~e. Both peptides are soluble in water, slightly soluble in absolute ethanol and insoluble in acetone and ether. The physical and chemical properties of the products are shown in Table 1.

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TAB LE
PROPERTIES OF COHERIN PEPTIDES
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, Coherin A Coherin B
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max (nm) ~a) :~~ pH 1 274 276 . pH 11 240,290 239,290 min (nm) ' pH 1 248 253 pH 11 . 233,272 236,276 ~' 1%
A280~ pH 1 12.1 18.5 Electrophoresis r (b) . .
. p~l 2.0 2.22 2.77 ,. pH 3.5 1.83 2.20 pH 8.5 .39 -1.0 Isoelectric point (c) 6.0 8.5 .. Rf (d) .t 1) .70 .58

2) . .33 .03 M.P. (e) 200~205C
-~ Mol. wt. (f) 300-600 . Dose for 5 mln Inhib (g) 0.5 2.0 ( 11 g/Kg ) ':~
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Notes for Table 1 .
a) U. V. determinations were on the Cary, Model 11 Spectro- -photometer.*
Solvents were for pH 1, 0.1 N HCl pH 11, 0.1 N NaoH
b) Electrophoresis was on Research Specialties Co. apparatus using Whatman 3MM* paper and electrolyte buffers as , follows:
- pH 2 Formic, acetic acid (14.2 V/cm .85 mA/cm) pH 3.5 pyridine, Acetic acid (14.6 V/cm .85 mA/cm) pH 8.5 Barbital buffer .05m (8.13 V/cm, 1.25 mA/cm) , r = ~Ms where ~Ms is the difference (in mm) in migration MBpB between bromphenol blue and the sample, and M
is migration of bromphenol blue. BPB
c) Isoelectric point was taken as the pH resulting is zero migration from the origin using the elctrophoresis apparatus described in (b).
d) Chromatography was on thin layers of silica gel (Merck, ;~ Silica gel G)* on glass. Solvents (1) N-Butanol~pyridine , ; 20 - acetic acid - H2O (60:40:12:48). (2) N-Butanol - acetic acid - water (4:1:5).
` e) Melting points are uncorrected and were determined on the Kofler micro melting point hot stage.
` f) Molecular weight was estimated on thin layers of dextran (Sephadex G-50)*.
,! g) Bioassays were run routinely on major fractions isolated in all st~ps described above. Dogs were surgically , preparea with Roux-en-Y fistulae allowing intestinal contractions to be monitored by pressure transducers in the lumen of a segment of ileum. Coherin activity was determined by mechanical as well as electrical means.
Activity of fractions was indicated by marked inhibition of intestinal contraction within 30 seconds after intra-venous injection of Coh,erin. Fractions for assay were routinely dissolved in~normal saline solution.
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` ~05;~72 : Slnce the compounds Or thi~ inventlon are ampho-teric, both ph2rmaceutlcally acceptable metalllc salts and . . .
acid addltlon salt~ can be prepared and are use~ul. Typically l u~e~ul salts include the salts Or alkall and alkallne earth ~etals, partlcularly calcium, ~odlum and potassium, ammonium . . and amine salts such as cyclohexylamlne and plperldlne salts, and organic an~ lnorganlc aclds such as hydrochloric sul-.,.j.
~urlc phosphorlc aceticj lactic, cltric, tartaric, oxallc, : ~uccinlc, maleic, ~luconic, and the like. The compounds may - be prepared by conventlonal procedures normally utilized for the preparatlon Or such salts, ~or example by titration Or ayueous solutlons with an acid or base and removlnF the water .~1 . by rreeze drying. Alternatively they can be taken up ln water, the appropriate acid or base added, and the salt pre-. cipitated by the addition o~ a water mlscible non-solvent such as acetone.
. The products o~ thls lnvention may be adminiskered - alone but wlll generally be administered ~ith pharmaceutic-ally acceptable, non-toxiG carriers, the proportions o~ whlch are determlned by the sultability and chemical nature of the particular carrier, the chosen route o~ adminlstration, and . standard pharmaceutical practlce. For example, they may be . . .
,. administered orally in the ~orm Or tablets or capsules con-: talnlng such excipients as starch, milk su~ar, certain types . Or clay, etc. They will normally be enterlc coated so as to be more reslstant to the acid and digestive enzymes Or the .

stomach. However, since they are Or relatively low molecu-., : . lar wel~ht they may be absorbed before they are hydrolyzed ~ by the proteolytlc enzymes o~ the stomach.. For intraveneous, - lntramuscular, or subcutaneous adminlstratlon they may be .... . . ........ .. .... _... ... ~ .
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'. used in the rorm Or sterlle solutions contalnln~ other ~olute~, for example, enough sallne or glucose to make the solutlon . isotonlc. A wlde ~ariety Or dosage unit.rorms are possible.
.` The physician or veterinarian in attendance wlll determlne the dosage regimen wh'Lch will be erfectlve. Thls ., :
.. will depend upon such ~actors as the age and welght o~ the .i patient, the condltion being treated and the dosage unit orm selected. Because o~ the hlgh order Or actlvlty o~
the compounds, dosage as low as 005 to 10 yg are userul.
Typically, a dosage reglmen o~ 1 to 3 ~g/kg Or body weight ls satisfactory.
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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process of preparing a peptide selected from the group consisting of:
(a) a peptide characterized as containing glycine, soluble in water, slightly soluble in absolute ethanol and insoluble in acetone having a molecular weight of from 300 to 600, having .lambda. maximums of 274 nm at pH 1 and 240 nm and 290 nm at pH 11 with corresponding minimums at 248 nm, 233 nm and 272 nm, respectively, in the ultraviolet region of the spectrum, and the pharmaceutically acceptable salts thereof;
and (b) a peptide characterized as containing glycine and glutamic acid, soluble in water, slightly soluble in absolute ethanol and insoluble in acetone, having .lambda. maximums of 276 nm at pH 1 and 239 nm and 240 nm at pH 11 with cor-responding minimums at 236 nm, 253 nm and 276 nm, respectively, in the ultraviolet region of the spectrum, and the pharma-ceutically acceptable salts thereof which comprises isolating peptides identified by (a) or (b) above according to well-known techniques involving elution with suitable eluates, lyophilization, highly cross-linked dextran gel columns and electrophoresis, from a bovine-posterior pituitary, and when the pharmaceutically acceptable salts thereof are required, reacting with a suitable base.

2. A process of preparing a peptide characterized as containing glycine, soluble in water, slightly soluble in absolute ethanol and insoluble in acetone having a molecular weight of from 300 to 600, having .lambda. maximums of 274 nm at pH 1 and 240 nm and 290 nm at pH 11 with corresponding minimums at 248 nm, 233 nm and 272 nm, respectively, in the ultraviolet region of the spectrum, and the pharmaceutically acceptable salts thereof, which comprises isolating the peptide according to well-known techniques involving elution with suitable eluates, lyophilization, highly cross-linked dextran gel columns and electrophoresis, from a bovine-posterior pituitary, and when the pharmaceutically acceptable salts thereof are required, reacting with a suitable base.

3. A process of preparing a peptide characterized as containing glycine and glutamic acid, soluble in water, slightly soluble in absolute ethanol and insoluble in acetone, having .lambda. maximums of 276 nm at pH 1 and 239 nm and 240 nm at pH 11 with corresponding minimums at 236 nm, 253 nm and 276 nm, respectively, in the ultraviolet region of the spectrum, and the pharmaceutically acceptable salts thereof, which comprises isolating the peptide according to well-known techniques involving elution with suitable eluates, lyophili-zation, highly cross-linked dextran gel columns and electro-phoresis, from a bovine-posterior pituitary, and when the pharmaceutically acceptable salts thereof are required, reacting with a suitable base.

4. A peptide selected from the group consisting of:
(a) a peptide characterized as containing glycine, soluble in water, slightly soluble in absolute ethanol and insoluble in acetone having a molecular weight of from 300 to 600, having .lambda. maximums of 274 nm at pH 1 and 240 nm and 290 nm at pH 11 with corresponding minimums at 248 nm, 233 nm and 272 nm, respectively, in the ultraviolet region of the spectrum, and the pharmaceutically acceptable salts thereof;
and (b) a peptide characterized as containing glycine and glutamic acid, soluble in water, slightly soluble in absolute ethanol and insoluble in acetone, having .lambda. maximums of 276 nm at pH 1 and 239 nm and 240 nm at pH 11 with cor-responding minimums at 236 nm, 253 nm and 276 nm, respectively, in the ultraviolet region of the spectrum, and the pharma-ceutically acceptable salts thereof, whenever prepared by the process of claim 1.

5. A peptide characterized as containing glycine, soluble in water, slightly soluble in absolute ethanol and insoluble in acetone having a molecular weight of from 300 to 600, having .lambda. maximums of 274 nm at pH 1 and 240 nm and 290 nm at pH 11 with corresponding minimums at 248 nm, 233 nm and 272 nm, respectively, in the ultraviolet region of the spectrum, and the pharmaceutically acceptable salts thereof, whenever prepared by the process of claim 2.

6. A peptide characterized as containing glycine.
and glutamic acid, soluble in water, slightly soluble in absolute ethanol and insoluble in acetone having .lambda. maximums of 276 nm at pH 1 and 239 nm and 240 nm at pH 11 with corresponding minimums at 236 nm, 253 nm and 276 nm, respect-ively, in the ultraviolet region of the spectrum, and the pharmaceutically acceptable salts thereof, whenever prepared by the process of claim 3.