Note: Descriptions are shown in the official language in which they were submitted.
2170 200/PSP
WA/TON/UF
80~722
PANCREATIC SPASMOLYTIC POLYPEPTIDE
This invention is directed to a novel purified
polypeptide or a physiologically acceptable salt thereof,
as well as to a method for recovery and purification thereof
and to the use thereof as a spa ~ lytic agent. The purified poly-
peptide of this invention, wHich is recoverable from porcine
pancreas, has surprisingly been shown to possess smooth muscle
relaxing or spasmolytic effects. It has, therefore, been
accorded the trivial name of Pancreatic Spasmolytic Polypep-
tide, hereinafter for the sake of convenience abbreviated to
PSP. PSP shows interesting pharmacological properties.
Spasmolytic agents or antispasmodics, such as atropine,
congeners thereof and synthetic drugs having an atropine-like
effect, are widely used for the treatment of a variety of
ailments, in particular of smooth muscle spasms and hypermotility states~
However, the intended action of such drugs is usually accompanied
by a number of side effects attributable to their general
character of being anticholinergics.
As a diagnostic aid in gastrointestinal radiology,
particularly in conjunction with an X-ray visualizati`on medium
for improving visualization of the gastrointestinal, biliary and
urinary tracts, atropine-like anticholinergic drugs have also
been commonly used. Such a drug is usually administered parenteral-
ly and, owing to the size of the dose needed to induce relaxation,
the side effects classical to those agents are usually encountered.
Recently, parenteral administration of the peptide
hormone glucagon consisting of 29 amino acids was introduced as
an alternative means of reducing gastrointestinal motility in
conjunctiOn with radiographic examinations (vide U.S. Patent
No. 3,8~2,301). However, glucagon exerts a plurality of actions
in the human body incluaing a strong influence on metabolic
regulatory functions, the most conspicuous effects being the
induction of hyperglycemia and lipolysis. Thus, although the
introduction of glucagon in endoscopy accomplished certain
advantages, undesirable side effects were not completely
abolished. It is an object of this invention to provide a
spasmolytic agent which, whilst possessing antispasmodic and
smooth muscle relaxing effects comparable to those of known
agents, exhibits substantially reduced side effects.
According to one aspect of the present invention
there is provided a novel purified polypeptide exhibiting the
following amino acid composition:
Trp (2), Lys (4), His (l~, Arg (5), Asx (lO), Thr (3), Ser (9),
Glx (12), Pro (12), Gly (6), Ala (6), Cys~ tl4!) , Val (7), Met (2),
Ile (3), Leu (l), Tyr (2), Phe (7), wherein the determinations
are subjected to the usual error of + lO per cent of the indicated
figures. The partial amino acid sequence ccmprising a total of ~5 amino acids frcm
the N-terminal, is believed to be:
pyrGlu-Lys-Pro-Ala-Ala-Cys-Arg-Cys-Ser-Arg-Glx-Asx~Pro-~ys-Asx-
. ~0 15
-Arg-Val-Asx-Cys-Gly-Phe-Pro-Gly-Ile-Thr-Ser-Asx-Glx-Cy~-Phe-Thr-Ser-
-Gly-Cys-Cys-~he-Asx-Ser-Glx-Val-Pro-Gly-Val-Pro-Trp-,
wherein pyrGlu (residue l) stands for pyroglutamic acid.
The a~breviat~ons for the amino acids appear from
J.Biol.Chem. 243 (1968), 3558.
The present invention also provides a method for
preparing purified PSP, whic~ method comprises isolating PSP
from porcine pancreatic tissue prefera~ly from the insulin
salt cake by a com~ination of chromatograph~ and precipitation
processes. --
The insulin salt cake may ~e prepared as follows:whole, neatly defatted porcine pancreas glands are finely
comminuted under frozen cond~t~ons and then su~jected to the
conventional extraction process for recovery of insulin, that
is extracted with a mixture of water and an organic water-
misci~le solvent, such as a lower aliphatic alkanol, for
example ethanol or isopropanol, in an acid medium, for example,
a medium having a pH in the range of ~rom about 1.5 to 5 when
measured with a pH meter in the mixture. The acid pH is
o~tained ~y the addition of an acid. In the mixture, the
organic solvent is present in a concentration in the range of
from a~out 40 to 80% ~v/v) when all the components are mixed.
The resulting slurry is stirred at a temperature in the range
of from a~out 5C to am~ient, followed by removal of the gland
residue, for example ~y centrifugation. The extract is then
neutralized to a pH in the range of from about 5 ko 9, and
clarified, for example by centrifugation. The extract is
acidified to a pH in the range of from about 3 to 4, where-
after the extract is freed of any organic solvent, for example
by evaporation at reduced pressure, followed by removal of
lipid compounds, for example by centrifugation. Insulin
admixed with other proteins and polypeptides, such as PSP, is
salted out from the concentrated extract so o~tained, for
example ~y the addition of sodium chloride to a concentration
in the range of from a~out 10 to 30% ~w/v) r and the precipitate
formed is isolated, for example by centrifugation, thus
affording the salt cake.
The salt cake thus obtained may then be dissolved in
water and crude insulin isolated by isoelectric precipitation at
a pH in the range of from about 4.~ to 5.7, for example about
5.3, optionally in the presence of metal ions, for example zinc
ions, and recovered, usually by centrifugation. The supernatant
is given a pH in the range of from about 5.7 to 7, preferably
about 6.5. The precipitate formed, containing some insulin, i5
centrifuged off. In order to remove ancillary substances, such
as salts, excess of EDTA is added to the above second
supernatant, followed by the addition of a water-miscible
organic solvent, preferably ethanol (usually, from 5 to 20
volumes). The mixture is left to precipitate overnight at about
4C and then centrifuged. The precipitate is dried in vacuo,
yielding a dry powder, hereinafter referred to as "superna-tant
protein". By this procedure, practically all protein material
of the "supernatant protein" is recovered.
PSP can be obtained in a crude crystalline form from a
solution of "supernatant protein" in water (about 10 parts).
The solution is stirred gently while acid, for example acetic
acid, is added in the course of about 3 hours until a pH in the
range of from about 3.8 to 4.8, preferably about 4.3, is
attained. The mixture is then chilled and the stirring is
continued for 3 days, preferably at about 4C. A crop of
relatively large, bar-shaped, birefringent crystals is isolated,
for example by centrifugation, and dried in vacuo.
The material so obtained may be further purified,
preferably by applying consecutive steps of anion and cation
exchange chromatography.
I'o illustrate the procedure, anion exchange
chromatography may be performed on a column of "QAE-Sephadex
A-25" (supplied by Pharmacia AB, Sweden), using the eluent
stated on Fig 1 of the accompanying drawings (TRIS being tris
(hydroxymethyl) aminomethane).
P~ f~
The chromatogram obtained by monitoring the optical
density of Eractions at 276 nm shows one main peak. The pool
corresponding to the main peak is adjusted to p~ 7.4 and then
mixed with a water-miscible organic solvent, for example ethanol
~4 volumes). ~pon standing at ~C for 2 days a precipitate is
recovered by centrifugation and dried in vacuo.
The material so obtained can be further purified
by cation exchange chromatography, for example on a column of
"SP-Sephadex C-25`' (supplied by Pharmacia). Elution may be
effected with the eluent stated on Fig 2 of the accompanying
drawings. The chromatogram, obtained in the same manner as above,
shows a main peak. Pooled fractions corresponding thereto are
evaporated to dryness, the residue is dissolved in water at a pH
in the range of from about 6 to 8, for example about 7, mixed
with an excess (about 12 volumes) of a water miscible organic
solvent, for example ethanol, and left overnight under similar
conditions as described above. Purified PSP , which precipi-
tates from the solution, is isolated by centrifugation, washed
with ethanol, and dried in vacuo.
Alternatively , PSP containing protein may be obtained
from the mother liquor arising when isolating the salt cake
using sodium chloride in a concentration of from 10 to 20~ (w/v)- by
an additional salting out process. The
precipitate is recovered, for example by centrifugation. Purified
PSP can be obtained from the precipitate by the use of anion
and/or cation chromatography in any order.
By a further method, PSP containing protein may be
isolated from the above extract of pancreas glands obtained
using a mixture of water and an organic water-miscible
sol~ent by adsorption to a cation or anion exchanger,
for example alginic acid, sulphonated polystyren or
aminoethylcellulose. Thereafter, the ion exchanger is washed and
the protein is eluted with an aqueous medium. The isolation by the
use of an ion exchanger is performed by methods which are analogous
to known methods.
:~'
-- 6
PSP obtained ~y any of th~ a~ove methods has the
following characteristics:
Molecular weight, calculated from the amino acid
composit~on: aBout 11,700.
Molecular we~ght, determined ~y sodium dodecyl
sulp~ate ~ gel electrop~ores~s ~Nevil~e: J. ~iological
C~emistry 246 C1~711 6328~: a~out 10,700.
Electrophorectic characteristics:
~ asic DISC electrop~oresis (basic DEl in polyacryl-
amide gel as descri~ed ~y J. Schlichtkrull et al. (Horm.
Meta~ol.Researc~, Suppl. Series 5 (1974l 1341 shows essenkially
a single ~and with Rf 0.65 - O.75. A similar pattern is
obtained in analytical electro-focusing in polyacrylamide gel
~y which method the pI is determined to a~out 4.4. ~ry~5i~
Products o~tained upon treatment of PSP with~tr ~r
~-chymotrypsin, CNBr, acid, or pyroglutamate aminopeptidase as
descri~ed ~elow, have a spasmolytic activ~ty of the same order
as that of PSP.
''Tryp's'in''treatment:
Twenty mg of PSP was dissolved in 20 mQ of 0.01 M
NH4HCO3 (pH: 7.8) and preincu~ated for 5 minutes at 37C.
After addition of 100 ~1 of 0.001 M HCl containing 0.4 mg
TPCK-trypsin ~o~tained from Worthington Biochem. Corp.), the
mixture was incubated at 37C for 15 minutes and then lyophiliz-
ed.
a-Chymotrypsin trea*ment:
Twenty mg of PSP was dissol~ed in 2ao ~1 of 0.1 M
NaOH and 1800 ~1 of 0.05 M NH4HCO3 (pH: 8.0) was added. The
solution was preincubated for 5 minutes at 37C and 50 ~1 of
0.001 M HCl containing 100 ~g a-chymotrypsin (obtained from
Sigma Chemical Company) was added. The incubation was contin-
ued for one hour at 75C and the reaction was stopped by the
addition if 50 ~1 concentrated acetic acid, whereafter the
solution was lyophilized.
-- 7 --
~ 2
CNBr treatment
Twenty mg of PSP was dissolved ~n ~ ml of 70% (v/v)
formic acid containing 72 mg CNBr. T~e m~xture was stored at
room temperature for 40 hours and then lyop~ilized. The
lyophilizat~on was then repeated after add~tion of 2 ml of water.
Acid treatment
Samples of 1 mg PDP, dissolved in 100 ~Q of 0.5 N
hydrochloric acid, were incubated at 37C for 2, 10 and 21 days.
After incu~ation t~e protein of each sample ~as precipitated
quantitativel~ ~y the addition of 2 ml of acetone. The pre-
cipitate was isolated ~y centrifugation, washed with 2 ml of
acetone and dried in vacuo. The samples so obtained and a
sample of untreated PSP were analysed by basic DE, vide supra,
with the proviso t~at the time of electrophoresis was reduced to
give Rf = 0.53 for PSP. In the sample ;ncubated for 2 days a
series of ~ands were observed with Rf ranging from 0.53 to 0.86.
In the samples incu~ated for 10 and 21 days only a single band
with Rf 0.86 appeared. The results indicated that a partial
deamidation of PSP had occurred after 2 days and a complete
deamidation after 10 days of incubation.
Pyroglutamase aminopeptidase treatment
A sample of 6 mg PSP was dissolved in 2 ml of 50 mM,
sodium monohvdrogen phos~hate, 3~ ~ p-mercaptoethanol, 1 m~
EDTA buffer with a pH of 7.8. A so~ution of 2.5 mg p~roglut-
amate aminopeptidase (obtained from oehm~gcr ~lannhcl~) in
0.5 ml of the above buffer was added. The mixture was
incu~ated for 16 hours at 39C and thereby lyophilized.
(2.5 mg pyroglutamate aminopeptidase used contained about 10
mu enzymatic activity.
The purity of the final PSP product may be checke~ by
analytical isoelectric focusing (IEF) and basic DISC
electrophoresis (basic DE, ~ ). The product migrates
essentially as a single band in both systemsO IEF is performed
according to the instructions of LKB brochure I-1804-E02: "LKB
Ampholine PAG" plates for analytical electrofocusing on
polyacrylamide gels (LKB-Produkter AB, Bromma, Sweden).
Likewise, gel filtration of the polypeptide on "Bio-Gel
P-3O" (supplied by Biorad Laboratories, Richmond, California,
V.S.A.) using 1 molar acetic acid as the eluent, reveals only a
single peak.
PSP has been analysed for a number of
immunoreactivities according to methods known in the art. The
results obtained are presented in Table 1:
Table 1
Immunoreactant Contents (Ppm2
Insulin (lRl) 3 - 6
~'otal glucagon (total GLI) 0.02
Pancreatic glucagon (pancreatic GLI) 0.02
Vasoactive intentinal peptide (VIP) 0.02
Pancreatic polypeptide (porcine) 0.08
C-Peptide (porcine) 0.1
Somatostatin 0.002
The immunoreactivity of PSP is measured by a highly
specific radioimmunoassay which is developed to detect down to
250 pg per ml.
Antibodies were prepared by immunizing rabbits with
"supernatant protein" (0.5 ml of a solution contining
approximately 4 mg protein per ml) mixed with Freund's adjuvant
(0.5 ml) twice weekly for a period of 20 weeks. Beginning from
the 13th day after the first immunization, a total of 10 blood
samples (10 ml) ~rom each animal, taken at regular intervals
over a period of 172 days, were collected. The antisera
obtained were tested for affinity and capacity and a suitable
antiserum was selected for use in the radioimmunoassay.
- 9
125I-PSPw~s prepared by the lactoperoXidase method
developed by Thorell and Johansson (Biochim.Biophys.Acta 251
~1971) 363). The radioiodinated PSP ~s purified by anion
exchange chromatography as known in the art and used for
polypeptide radioimmunoassay according to the procedure developed
by L.G. Heding (Diabetologica 7 (i971), 10).
.
Furthermore, the present invention relates to salts
of PSP and, as examples of such salts, salts with cations such
as sodium, potassium, magnesium, calcium and zinc and acid
addition salts with organic or inorganic acids such as formic,
methansulfonic, hydrochloric and sulphuri.c acid, can be mentioned.
For the sake of brevity, the designation PSP Compounds is used
to cover PSP and physiologically acceptable salts thereof.
PSP Compounds and glucagon were found to be about
equipotent in their inhibition of the amplitude of the contractions
of electrically stimulated guinea pig ileum ln vitro, vide Table 2.
PSP and glucagon were dissolved in 0.9% sodium chloride with 0.1%
human serum albumin
Table 2
Inhibitory effect in per cent
Concentration in the
organ bat~, M PSP Glucagon
,-
10-5 89 89
lo~6 - 49 51
10 7 21 24
This effect of PSP Compounds was blocked by phentolamine
but not by naloxone. The spontaneous motility of the isolated
ileum from reserpine-treated guinea pigs was inhibited by PSP.
Likewise, PSP Compounds were found to be about
as potent as glucagon with respect to its inhibitin in vivo of
.the peristalsis in mice, vide Table 3, an effect which again could
-- 10 --
6~6Z~
be blocked b~ phentolamine.
Table 3
Drug Per cent of intestine traversed by
( 50 mg/kg sub- charcoal compared to a control
cutaneously)
,
PSP 78
Glucagon 66
Atropinsulphate 64
.. . . . .. . .. . . ..
PSP reduces intestinal motility in rabbits in vivo
after administration intravenously or intraluminally in the
intestine. The motility was recorded by means of a balloon
catheter in the intestine connected to a pressure transducer. In
5 out of 5 rabbits (fr~m 2.5 to 3.0 kg body weight)400 ~g PSP
administered intravenously or 5 cm ~Xom the balloon into the lumen
of the intestine caused a marked reduction of the intestinal motility,
almost to atonia. 200 ~g ~ad a clear effect in 3 out of 5 rabbits.
Glucagon had the same effect, but only when administered intravenously.
PSP was found to delay the absorption of [U-14CJ protein
hydrolysate in pigs and in pancreatectomised dogs and of [U-14C~
ovalbumin in panareatectomised doges, when the compound was
administered perorally in a capsule with 3 mg PSP. The pigs and the
dogs weighed about 30 kg. 100 ~Ci [U-14C] protein hydrolysate or 5
~Ci lU-14C] ovalbumin was mixed with a suspension of 1 g/kg Idon
and administered through stomach tubes. Maximum plasma dpm values
were reached from 30 to 40 minutes later after administration of PSP
as compared to placebo. This delay in absorption caused by about 100
~g/lg pf PSP orally probably reflects a reduced gastro-intestinal
motility.
`` PSP comp~unds were found to be devoid of an~ In vitro
~ 'ect on the release of glucagon or insulin or on lipolysis and
of any in vivo effect on blood glucose. Nor did an intravenously
injected dose of up to 1 mg/kg exert any significant efect on the
blood pressure of the anest~etized rat.
~ The above p~armacological data indicate the potential value
of PSP compounds for t~e prevention and treatment of smooth muscle
spastîc conditions, ~r example in the intestine. Due to the lack of
metabolic effects, PSP compounds may prove advantageous as a
substitute for gluaagon in endoscopy and in radiological procedures.
PSP compounds may ~e administered intravenously as a bolus
or as an infusion. When an effect of prolo~ged nature, slower in
onset, is desired, PSP compounds may be administered as a depot from
which it is slo~ly mobilized by the blood stream such as intra~
muscularly or su~cutaneously in a region of good peripheral
circulation supply. The fact that the biological activity and the
immunoreactivity is maintained after exposure of PSP to gastric
juice, trypsin, and chymotrysin and the experiments described above
showing delayed a~sorption after oral administration of PSP points to
the oral route as a possible way of administration. Therefore, PSP
may be administered through an endoscope during the endoscopy
procedures or PSP~may be mixed with the contrast media, e.g. barium
sulphate, during the radiology procedure.
The dosage rates of PSP Compounds can be adjusted
according to the magnitude of desired response and other factors
routinely taken into consideration in establishing the dosage.
As an example of a dosage ra~ge, from 10 to ~Oo ~ g per kg body
weight can be mentioned, although a lower or igher dosage may be
administered.
The present invention also relates to a pharmaceutical
composition comprising PSP Compounds and one or more
pharmaceutically acceptable carrier(s) . As examples of
such carriers,preservatives and sodium chloride can be mentioned.
- 12 -
6~
" I
In an attempt to secure that the desired result is
obtained after administration of a PSP Compound it is advisable
to use a starting material for preparing PSP preparations which
has a purity of at least 50%, preferably a purity of at least
90~ of a PSP Compound.
According to hitherto unpublished data pancreatin
pills contain PSP (for example about 1 per thousand).
Because of its content of enzymes pancreatin pills have been
used for pancreatectomized patients and patients with chronic
pancreatitis. Commercial insulin has now been found to contain
about 30 ppm PSP.
Any novel feature or combination of features described
herein is considered essential.
The following Examples, which, however, are not con-
sidered to be limiting, are presented to illustrate the
process for preparing PSP. Highly purified PSP is PSP which
essentially migrates as a single band in the above IEF
and basic DE systems.
- 13 -
Example 1.
A salt cake originating from 94 kg of porcine pancreas
glands was dissol~ed in water to a volume of 3.2 1 . The pH
of the solution was adjusted to 5.3, whereafter the precipi-
tate was removed by centrifugation. The pH of the supernatant
was adjusted to 6~5 and the suspension thus formed was centri-
fuged. The solution was mixed with 32 ml of 0.5 M Na4EDTA and
35 1 of ethanol. The mixture was left overnight at 4C and then
centrifuged. The precipitate was dried in vacuo yielding
50 g of dry supernatant protein powder.
A solution of the powder in 500 ml of water was
stirred gently while 1 M acetic acid was added slowly by means
of a peristaltic pump until a pH of 4.30 was attained (af~er
about 3 hours of pumping). Stirring was then continued for 3
days at 4C whereby crystallization occured. The crop of
crystals (bar-shaped by appearance,possibly orthorhombic and
showing birefrinye~ce) wére harvested by centrifugation,
suspended in 500 ml of water at 4C with stirring overnight,
centrifuged and dried in vacuo. The yield was 5.2 g.
4 g of this material was dissolved in 50 ml of 50
per cent (v/v) ethanol and 50 ml of eluent (vide Fig 1) at
pH 8.6. The solution was subjected to anion exchange chroma-
tography as shown in Fig 1. The pool from the main peak was
given a pH of 7.4, mixed with 4 volumes of 96 per cent (v/v)
ethanol and then stored at 4C for 2 days. The precipitate
was isolated by centrifugation, washed twice with 150 ml of
96 per cent (v/v) ethanol and dried in vacuo. The yield was 2.6 g.
2.5 g of this material was dissolved in 125 ml of
50 per cent (v/v) ethanol and 125 ml eluent (vide Fig 2) ~t
pH 4.7 and then subjected to a cation exchange chromatography
as shown in Fig 2. The pool from the main (only visible)
peak was evaporated to dryness. The residue was dissolved in
. ~
water and the pH of the solution was adjusted to 7.1 (the
final volume was about 90 ml). The solution was mixed with
1200 ml of 96 per cent (v/v) ethanol and the mixture was
stored at 4 C overnight. The precipitate was isolated by
centrifugation, washed twice with 150 ml of 96 per cent (v/v)
ethanol, and dried ln vacuo. The yield was 1.~ g of highly
purified PSP fulfilling the purity requirements stated in
Table 1. -
Example 2.
20 g of supernatant protein powder, produced asdescribed in Example 1, was dissolved in 200 ml of water.
208 ml of 96 per cent (v/v) ethanol was added, followed by
adjustment of pH to 4.6 with acetic acid. A small precipitate
was removed by centrifugation. The supernatant, which slowly
became turbid, was subjected to cation exchange chromatography
on a 2.5 x 80 cm column of "SP-Sephadex C-25", equilibrated
in Eluent 1 (0.4 M acetic acid, 0.05 M sodium acetate,
50 per cent (v/v) ethanol, pH: 4.6). Linear gradient elut:ion
was performed between 3 1 of Eluent 1 and 3 1 of Eluent 2
(0.3 M sodium acetate/50Fer cent;(v/v) ethanol, pH: 8.7). Fractions
of 10 ml were collected at an elution rate of 40 ml/h. The
fractions corresponding to the large peak appearing from
fractions 100 to 130 were pooled. The pool was given a pH of
8 and then mixed with 1.8 1 of 96 per cent (v/v) ethanol. The
mixture was stored at 4C for 24 hours. The precipitated
protein was isolated by centrifugation, washed twice with 150
ml of 96 per cent (v/v~ ethanol and dried in vacuo. Yield:
2.8 g. 2.5 g of this material was dissolved in 250 ml of a
TRIS bu~fer (0.0575 M TRIS, 0.05 N HCl, pH: 7.~ ). The solu-
tion was subjected to anion exchange chromatography on a
2.5 x 50 crn column of "QAE-Sephadex A-25", equilibrated in
a TRIS buffer (0.115-M TRIS, 0.1 N HCl, pH: 7.4). The column
was eluted with the equlibration buffer at a rate of 30 ml/h.
Fractions of 10 ml were collected. The fractions corresponding
to the central major part of the peak showing a maximum at
fraction 225 were pooled. The pool (620 ml) was mixed with 60
ml of 5 M sodium chloride an~ 12 1 of 96 per cent ~v/v) ethanol.
~ _ . _ . . . _ . _ _ . ... _ . ~ _
i - 15 -
~ 6;~1
The mixtu~-e was stored at 4~C for 24 hours. ~he precipitated
protein was isolaked by centrifugation, washed twice with
150 ml of 96 per cent (v/v) ethanol and dried in vacuo.
Yield: 1.7 y of highly purified PSP.
Example 3.
To 150 1 of an a~ueous solution which was obtained
b~,~ evaporation of an extract from 250 kg of porcine pancreas
gl.~nds and which was freed from insoluble material, 22.5 kg
of sodium chloride were added. The mixture was stirred to
dissolve the salt added and the resulting precipitate was
removed by centrifugation thus affording the salt cake. To the
mother liquor (162 1) was added 34 kg of ammonium sulphate
with continued stirring for 2 hours at room temperature
affording a precipitate which was isolated by centrifugation
223 g of the wet product were dissolved by addition of
50~ ml of a buffer (0.05 M formic acid, 0.01 M sodium
hydroxide buffer, pH: 3.2). The conductivity of the solution
was reduced to 4 mS by dialysis against water. The solution
was applied on a 5 x 50 cm column of "SP-Sephadex C-25"
equilibrated with Buffer I (0.1 M formic acid, 0.02 M sodium
hydroxide, pH: 3.2). After application of the solution, the
column was ~uted with a linear gradient of sodium chloride
from 0 to 0.27 M in Buffer I. The total volume of the eluent
was 5.5 1. The column was then further eluted with Buffer I
containing 0.27 M sodium chloride. The flow during the appli-
cation al~ elutioh was 100 ml per hour and fractions of 15 ml
were collected. The chromatogram obtained by monitoring the
optical density of the fractions at 276 nm showed one main
peak from fraction 420 to 530. The pool corresponding to the
main peak was adjusted to a pH of 7.4 and then mixed with
20 volumes of 96 per cent (v/v) ethanol. Upon standing at
4C for 48 hours, a precipitate was recovered by centrifugation
and dried in vacuo. Yield: 6 g. The material so obtained was
further purified by anion exchange chromatography on a column
of "Q~E-Sephadex A-25", as described in Example 2. Yield: 3.4 g
of highly purified PSP.
EXH I B I T A
SUPPLEMENTARY DI SCLOSURE
Among the interesting pharmacological properties of
PSP referred to herein, in addition to the spasmolytic effect,
PSP also possesses an inhibitory effect on gastric acid
secretion. Patients with duodenal ulcers benefit from
treatment with agents which inhibit gastric acid secretion.
However, the same patients suffer from an increased gastro-
intestinal motility. PSP combines two effects which are highly
desirable in the treatment of patients with duodenal ulcers, an
inhibitory effect on the gastrointestinal motility, and an
inhibitory effect on gastric acid secretion.
As an example of a known medicament which is used to
inhibit gastric acid secretion, cimetidin may be mentioned~
However, cimetidin possesses frequent adverse effects such as
diarrhoea, exanthema, elevation of liver enzymes, and
gynecomastia. As PSP is a polypeptide which is to be dosed
orally and as it is not absorbed in substantial amounts in the
gastrointestinal tract, it is not likely to have systemic
adverse effects.
PSP was found to inhibit pentagastrin stimulated
gastric acid secretion in rats and cats with chronic gastric
fistulas. 10 ~g PSP infused over 1 hour to rats was found to
be as effective in inhibiting the acid secretion after 5 ~g
pentagastrin s.c. as 1 ~g somatostatin, i.e. the peptides are
equipotent on a molar bas s. 10 ~g/kg PSP s.c. and 250 ~g PSP
orally in a capsule were effective in cats.
The data indicate the value of PSP in the treatment
of gastroduodenal ulcers. Therefore, PSP may be administered
orally in capsules to patients with gastroduodenal ulcers.
