Note: Descriptions are shown in the official language in which they were submitted.
This invention relates -to composi-tions of matter that
are useful in the -treatment oE schizophrenia. More particularly,
the invention concerns the treatment of schizophrenia by the
use of polypeptides, omega-N-acylated and/or hydrocarbon-
containing esters of polypep-tides. The polypeptides have from
three to about eight alpha-aminoacid moieties and a characteristic
O O O
Il 11 li
-NE-I-CH-C-~II-CH-C-NH-CM-C- I.
CHO- CH
CH3 CH3 CH3 2H3) ~C 3
(T) (V) (L)
structure, especially such N-acylated T-V-L-containing poly-
peptides, and the corresponding amides such as, for instance,
the N-acyl-threonylvalylleucinamides. ~ -
Schizophrenia is the most serious of all the mental
illnesses with which the psychiatrist must deal. The incidence
rate is approximately one in a thousand, and the prevalence
rate approximately one in a hundred. It is responsible for from
25 to 50 percent of all the admissions to state hospitals for
the mentally ill and psychiatric services in general hospitals
(Kolb, Modern Clinical Psychiatry, pp. 311-312, ]973).
The majority of patients who develop this illness
have an onset that is slow and insidious, although a small
percentage of patients may have an acute onset. The
j illness is characterized by a disturbance in emotional
responses with dulling or inappropriate expressions, by a
disturbance in thinking characterized by inability to
reach a goal idea, by fragmentation, by a disturbance in
associations, blocking, neologisms, etc.; by a withdrawal
from the environment and a preoccupation with internalized
~.
' -2-
; : '
thougllts; by a loss o~ the ability to experience pleasure;
and most ~requently by disturbances in perception a~
mani~ested grossly in hallucinations and delusions.
Unfortunately, schizophrenia most frequently
begins in late adolescence or early adulthood and, ~n
spite of the use of anti-psychotic drugs, persists by
incapacitating the individual more or less for the duration
of the individual's life.
There is considerable evidence now that this
illness is a genetically determined condition although,
undoubtedly, environmental factors, particularly early
life experiences, most probably are important too. There
seems to be no doubt that there is a basic metabolic
disturbance which will be described in detail herein.
Unfortunately, it cannot be absolutely proven
that there is an animal model for schizophrenic studies
since one can not converse with the animal to establish
that it is a schizophrenic. The maternally and sensory
deprived Macaque monkeys at the Wisconsin Primate Laboratory
are disturbed in behavior (being fearful, aggressive
towards themselves, and unable to function sexually),
thereby, coming closest in behavior to patients with schizo-
phrenia ~Harlow, "The Development of Patterns of A~fection",
Thomas William Salmon Lectures, New York Academy of Medicine,
New York, Dec., 1960). The most severely deprived monkeys
have been studied and show a disturbance in an alpha-2-
globulin, herein described, of their blood similar to the
disturbance that characterizes patients (Beckett, Frohman
et al., "Schizophrenic-like Mechanisms in Monkeys",
The Amerlcan Journal of Psychiatry, 119:835-842, 1963).
Piloreover, these monkeys also demonstrated electro-encephalo-
graphic disturbances, particularly of the septum of the brain,
--3--
similar to wha-t have been previously described as characteristic
o~ patients with schi%ophrenia (Heath, "Electroencephalographic
Studies in Isolation-raised Monkeys with Behavioral Impair-
ment", Diseases o~ the Nervous System, 33:157-163, 1972).
The use of animals, there~ore, has been primarily to
demonstrate the presence of abnormal metabolic substances
in the blood o~ patients with schizophrenia. When an
abnormal blood protein is given to rats in a ~ood reward,
rope climbing situation, the rats are slowed down considerably
10 in their performance (Bergen et al., "Further Experiments ~ -
with Plasma Proteins from Schizophrenics". In: Serological
Fractions in Schizophrenia, edited by R. E. Heath, p. 67,
. .
Paul B. Hoeber, Inc., Medical Book Department of Harper & ~-~
Row, New York, 1963).
There is some evidence that extracts of the limbic
system o~ the brain of patients who have died~ when injected
into normal volunteers produces a simulated schizophrenic
reaction (Garey, "Focal Electroencephalographic Changes
Induced by Anti-septal Antibodies", Biological Psychiatry,
20 8:75-88, 1974). The most consistent studies involve the
injection of the isolated alpha-2-globulin from the blood
o~ schizophrenic patients in microliter amounts, lnto
ventricles of rats who have implanted electrodes in the
median forebrain bundle. Rats so implanted will reward
themselves by pressing a bar as often as they can at the
expense o~ all other activity. When microscopic amounts
of the isolated alpha-2-globulin are injected into the
ventricles, there is a reduction in the bar-pressing which
does not occur when the comparable substance from a control
subject is injected. Some of the rats injected with the
schizophrenic isolated blood protein may hover over the
bar poised as i~ to press the bar, weaving and unable to
--4--
... .. ... .
,
V '~
continue, simulating a schi.zophrenic disturbed motor
state. The interpretat~on of the reduction in bar-pressing
is that the anlmal has lost its ability to experience
pleasure much as that which characterizes schizophrenic
patients (Caldwell, et al., "The Effects of the S-protein
on Intracranial Self-Stimulation in the Rat'l~ Biologlcal
Psychiatry, 8:235-244, 1974~.
I have been intimately involved for many years
in considerable research efforts devoted to determining
the cause of schizophrenia in terms of biochemiskry, and
to determine psychopharmalogical modes for its treatment.
In this work it was found that a high molecular weight
protein, estimated as having a molecular weighc about
263,000 and being about 80 percent lipid, is more active
in schizophrenics than normal lndividuals, and the more
rapidly a schizophrenic deteriorates, the more active
is the protein. This protein has been classified as an
alpha-2-globulin, and has been referred to as the S-
protein; Frohman, C.E.; Latham, L. K.; Beckett, P. G. S.;
and Gottlieb, J. S.; "Biochemical Studies of a Serum
Factor In Schizophrenia", Molecular Basis of Some Aspects
of Mental Activity, Vol. 2, Walaas, 0., ~d., pp. 241-255,
Academic Press, New York (1967); and Frohman, C. E.,
'iStudies on the Plasma Factors in Schizophrenia", Mind as
a Tissue, Rupp, C., Ed.~ pp. 181-195, Hoeber Med. Div.,
Harper & Row, New York. The S-protein when administered
to, for instance, rats, blocks the en;oyment of pleasure
st.imulations in the rats, but does not apparently alter
their avoidance response.
3o Endeavors to reduce the activity of the S-
protein in schizophrenics have included obtaining ex~racts
from brains, for example~ cattle brain, beef pineal glands,
... ~ ~ . , , . . ), .
,' : , ' ' ' ~ ~ '
6~X,~
etc., in the search for materlals whlch control the level
or activity of the S~protein. Frohman, et al., in "Control
of the Plasma ~'actor in Schizophrenia", ~ecent Ad ances in
Blological Psychiatry, Volume 7, pp. 45 to 51, 1964,
described that an isolated protein from animal tissue
counterac-ted the activity of the S-protein. This protein
has been named the anti-S-protein and ls found in both
human and animal tissue. In schizophrenics, S-protein in
an alpha~helical conformation is found, whereas, in normal
-10 persons the S-protein predominates as a random chain or beta-
helical con~ormation. Production o~ therapeutic quantities of
the anti-S-protein involves great difficulties and high expense,
and its extraction for general medical use is impractlcal.
Moreover, the production of a few milligrams of khe anti-S-
protein from cattle would require the sacriflce o~, say, 100,000
cattle. Thus, the natural supply of anti-S-protein is
- clearly insufficient for treatment of other than a few
inàividuals and would entail an unbearable expense. Other
publications relating to these prior studies include the
following and the citations therein: Frohman, C. E.;
Arthur, R. E.; Yoon, H. S. and Gottlieb, J. S.; "Distribution
and Mechanism of Action of the Anti-S-Protein in Human
Brainl', Biological Psychiatry, Vol. 7, No. 1, pp. 53 to
61, 1973; and Harmison, C. R. and Frohman, C. E., "Con~orma-
tional Variation in a Human Plasma Lipoprotein", Biochemistry,
Vol. 11, No. 26, pp. 4485-4493, 1972.
It has also been noted in studies in ~hich I
have been involved that the extracted anti-S-protein is an
antigen. While schizophrenia may be dramatically abated
3 upon the initial administration of the anti-S~protein to a
patient, the anti-S-protein is rapidly inactivated through
production of antibodies, and subsequently administered
.
.
,
anti-S-proteln can be so rapidly inactivated that lt
provides no discernible effect in trea~ing schlzophrenia.
I have been involve~ ~n attempts to break doT~n the antl~S-
protein into ~ragments which may have an acceptable activity
in treating schizophrenia wlthout exhibltin~ the unde-
sirable antigen activity.
In accordance with the present invention~ the
digestion of the anti-S-protein ~itl~, for example, pepsin
and trypsin, to provide small peptides of less than about
1000 molecular weight, yields a tripeptide-containing
fraction which has been found to be active in reducing the
undesired activity of the S-protein. The tripeptide
exhibits activity for causing the conversion of alpha-
helical S-prokein to its random chain conformation. This
tripeptide has been characterized as threonyl-valyl-
leucine. Work has been undertaken to synthesize this
- tripeptide, and a substantially pure, i.e., crystalline
form, of the tripeptide has been obtained. This tripeptide
exhibits activity against the S-protein of schizophrenics,
i.e., the alpha-helical conformation of the S-protein
is converted to the random form. When administered in
tests to counteract the effect of the S-protein in rats
the period of effectiveness of the tripeptide is, however,
of short duration, therefore frequent periodic doses of
the tripeptide would be required in any effective treatment
of schizophrenia.
By the present invention, it has also been found that
T-V-L peptides and their derivatives can be employed to
alleviate the schizophrenic symptoms in mammals a~flicted
3o with such symptoms. These polypeptide materials contain
the residues of at least three alpha-aminoacids and have
the T-V-L structure hereinabove defined. The polypeptide
--7--
- ~
: :
may be an aminoacid, i.e., having one terminal aminoacid moiety
in acid form and the other terminal aminoacid moiety in alpha-
amino (-NH2) form. It is preferred, however, that the poly-
peptide materials employed to alleviate the symptoms of schizo-
phrenia contain at least one of its end aminoacid moieties in
deriva-tive form, especially the terminal alpha-amino group being
acyl-substituted or the terminal carboxylic acid group being in
amide or ester form. Preferably both of these terminal moieties
are substituted in such manner, and particularly preferred are
the acylated amides. These polypeptide materials can also be
further substituted, and the pharmaceutically-acceptable, non-
toxic derivatives, e.g., acid or base salts, of the polypeptides
or their derivative forms can be employed to alleviate the
symptoms of schizophrenia in accordance with this invention.
The T-V-L-containing polypeptide materials of the
present invention are apparently effective in the treatment of
schizophrenic symptoms by causing the alpha-helical configuration
of S-protein present in the internal system, e.g., the brain, of
the schizophrenic, to assume its randomly-structured form. The
polypeptides or acylated polypeptides, esters or amides thereof
have the structure:
O O O O
H~R -Ctp(~)t-NH-CH-C-NH-CH-C-NH-CH-C-(B)X-R II.
CHOH CH
CH3 CH3 CH3 (( 2H3t~CH
wherein Ra is a saturated aliphatic hydrocarbon group, e.g.,
alkylene, of 1 to about 20 carbon atoms, preferably lower
alkylene, say of 1 to about 4 carbon atoms; Rb is -O(Re) wherein
Re is hydrogen or a saturated aliphatic hydrocarbon group, e.g.,
alkyl, of 1 to about 20 carbon atoms, preferably lower alkyl,
say of 1 to about 4 carbon atoms, or aryl or aralkyl of 1 to
about ~ rings, preferably of 6 to about 24 carbon atoms, or Rb
is -NRCRd wherein Rc and Rd may be the same or different and may
,
be hydrogen or lower alkyl, e.g., 1 to ahout 6 carbon atoms or
Rc and Rd may be lower alkylene and ~oin -to form a cyclic
structure includin~ the nitrogen atom to which they are bonded;
A and s is each a monoiminoacyl radical, preferably an ~-
monoiminoacyl radical, containing 2 to about 10 carbon atoms,
and B is hydrolyzable from the (L) component of the essential
T-V-L structure such that the carboxyl function of the (L)
component is made available in the host; p is 0 to 2; t is from
0 to about 5; x is from 0 to about 5; and t plus x is from 0 to
about 5. When t is 2 or more, each A may be the same or dif-
ferent, thus (A)t may be, for instance, phenylalanylprolyl.
Similarly, when x is 2 or more, each B may be the same or dif-
ferent. In a preferred group of compounds, -Rb is -NR R or at
least one of p, t or x is other -than zero. The (T) component
shown in structural formulas above and below may be in either
diastereoisomeric form, e.g., as threonyl or allothreonyl where-
in the stereochemistry at the ~ carbon is reversed. Preferably,
the (T) component is threonyl. The (L) component in the struct-
ural formulas above and below may be
~NH- IH_C_ (commonly referred
C~H2
CH (CH3 ) 2
to as leucyl), which is preferred, or in another form such as
-NH-fH-~- as in isoleucyl.
fH-CH3
CH2-CH3
The compounds of this invention include compounds
wherein any substituent, e.~., monoiminoacyl radical herein
designated by B, on the carboxylic group of the (L) moiety in the
30 essential T-~-L sequence is hydrolyzable. Advantageously, in
order to provide the desired activity the substituent is hydro-
lyzable under the conditions present in the intended environment
E~ g
"
in a host. Suitable hydrolyzable substituents are those which
hydrolyze in the presence oE red blood cells at an incubation
temperature of about the body temperature of a host, e.y., about
35 to 40C. One convenient procedure for determining whether
or not a substituent is hydrolyzable invol~es incubating the
compound containing the essential T-V-L structure and a sub-
stituent on the (L) moiety in a liver medium at about 37C.
The peptide moieties in the compounds of this invention
include peptide moieties which possess optical activity. Various
peptide moieties can have configurations designated as L- or D-.
The monoiminoacyl radicals comprising A of formula II and the
essential T-V-L portion of the compounds may be in the L- or D-
form or mixtures thereof such as racemic mixtures. Due to the
frequent difficulty in the hydrolysis of D- form peptides which
are substituents on carboxyl functions of peptides to preserve
the carboxylic function, advantageously the monoiminoacyl
radicals comprising B of formula II can have no optical activity
or, when optically active, can be in the L-configuration. Among
the preferred compounds are those in which (L) of the essential
T-V-L sequence is of the D-configuration.
The tripeptide, threonyl-valyl-leucine, or its deriv-
atives described herein, e.g., the corresponding amides, may be
obtained in relatively high purity, and may be crystalline,
e.g., for instance, at least about 90 or 95 percent pure,
preferably su~stantially 100% pure.
Various compounds of formula II may be intermediates
for materials of the formula which are active in the treatment
of schizophrenia. For example, when p is zero and Re is hydrogen, -
the compound may be an intermediate for the corresponding
material in which p is 1 and/or Re is other than hydrogen. The
compounds which are more desired for the treatment of schizo-
phrenia are polypeptides in which p is 0 to 2, and polypeptides
-10-
-
~ .
containing at least ~hree pep-tide or aminoacid moieties and t is
O or more, whether in aminoacid or a derivative form. Polypep-
tide materials wherein at leas-t one of p, t or x is other than
zero may exhibit a longer period of activity than provided by
the tripeptide T-V-L in aminoacid form.
Preferred acylated polypeptides for use as active
agents or intermediates therefor have the structure:
O O O O O O
a ~ b
H~R -ctp-~NH-cH-c~tNH-cH-c-NH-cH-c-NH-cH-ctNH-fH-ctx-R III.
R CHOH C~l CH R
~ 2
CH3 CH3 CH3 CH
CH3 CH3
(T) (V) (L)
wherein the group ~NH-CH-C~ is a monoiminoacyl radical and
Rl
is, for instance, hydrogen or a lower alkyl group, e.g., of 1 to
about 4 carbon atoms, or a -CH-OR group in which R and R3 are
hydrogen or lower alkyl, say of 1 to about 4 carbon atoms, or
aryl or aralkyl of 1 to about 4 rings, preferably having 6 to
about 24 carbon atoms. Such groups may be substituted as in the
case of, for example, tyrosyl. The letters R , R , p, t, and x
have the same meaning as in formula II.
In the compounds illustrated by formula III, R is
part of an alpha-aminoacid or peptide moiety or residue as in ~ -
the case of, for instance, threonyl or allothreonyl in which
is an alpha-hydroxyethyl group, leucyl in which Rl is a 2- -
methylpropyl group, valyl in which Rl is an isopropyl group,
seryl in which Rl is a hydroxymethyl group, isoleucyl in which
Rl is a l-methylpropyl group, glycyl in which Rl is hydrogen, or
alpha-alanyl in which Rl is methyl, arginyl in which Rl is
--11--
.:
,~C - NH(CH2)3 ~, -tyrosyl in which R is HO - ~ ~ 2 '
and the like. When the group including R has an alpha-hydroxy
group it may be converted to a lower alkoxy or aryloxy group,
preferably benzyloxy or t-butyloxy. This may be done during
synthesis to protect the hydroxyl function. The peptide
moieties, i.e., monoiminoacyl radicals, attached to the essential
T-V-L structure may also be prolyl, arginyl, and the like. Also
these moieties may be a dicarboxylic acid moiety such as a
residue from glutamic or aspartic acid.
The Ra group in -the compounds described herein may
have up to about 20 carbon atoms, although it is desirable that
it be alkylene of up to about 4 carbon atoms. Thus, the N-acyl
group H~R -~, may be, for example, acetyl, propanoyl, butanoyl,
pentanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl,
undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, penta-
decanoyl, palmitoyl, heptadecanoyl, stearyl, and the like. The
ester group, Re, may be methyl, ethyl, propyl, butyl, pentyl,
hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,
tetradecyl, pentadecyl, heptadecyl, octadecyl, phenyl, benzyl,
tertiary butoxycarbonyl, tertiary butyl, dihydro-epi-hydroxy-
androsteronyl, and the like. Preferably, the N-acyl group and
the ester group are normal and thus have straight carbon to
carbon chains. The compounds of the formulae or their pre-
cursor intermediates may have substituent groups which do not
interfere with the desired chemical or pharmacological activity
of the materials.
Intermecliates for preparing active compounds of
this invention inc-lude compounds of the formula
-12-
G- (A) t-N~-CH-I~-NH-~H-~-NlI-C~ - (B) X-E IV .
~HOR 5~I ¦ ~E13
CH3 CH3 \CH3 (C2H3t~
wherein t, x, A and s are as defined above; G is ~I or an ~-
amino protecting group; E is -OH; a terminal carboxylic pro-
tecting group; a complex of metal, :Eor instance, zinc, copper,
nickel, cobalt, iron, magnesium, or aluminum, the complex
preferably being a metal hydroxide complex; or an acid or base
addition salt; R4 is H, aliphatic hydrocarbon, preEerably
saturated, e.g., alkyl of 1 to about 20 carbon atoms such as
lower alkyl of say 1 to 4 carbon atoms, or araliphatic or aryl
of 1 to about 4 rings, preferably of 6 to about 24 carbon
atoms; acyl including alkanoyl or aralkanoyl of 1 to about 20
carbon atoms, preferably lower acyclic acyl of 1 to about 4
carbon atoms; tetrahydropyranyl; a monovalent metal such as an
alkali metal, e.g., sodium; and the like. A metal such as
zinc, copper, nickel, cobalt, iron, magnesium or aluminum may
form a complex with functions of the polypeptide such as a
carbonylic function.
The ~-amino protecting groups may be (1) acyl
or thioacyl type, preferably of 2 to about 21 carbons, for
example, lower aliphatic acyl of 2 to about 7 carbons, e.g.,
acetyl, etc.; lower araliphatic acyl of 8 to about 15
carbon atoms such as phthalyl, naphthoyl, benzoyl, etc.;
trifluoroacetyl; chloroacetyl; ~ -chlorobutyryl; toluenesul-
fonyl; benzenesulfonyl; nitrophenylsulfenyl; tritylsulfenyl;
o-nitrophenoxyacetyl; and the like; (2) aromatic urethane
type protecting groups illustrated by benzyloxycarbonyl
and substituted benzyloxycarbonyl such as p-chlorobenzyloxy-
-13-
.'',~,`,
3~
carbonyl, p-nitrober.zyloxycarbonyl, p-bromobenzyloxycarbonyl,
p-methoxybenzyloxycarbonyl; (3) aliphatic urethane protecting
groups illustrated by tert-butyloxycarbonyl, diisopropyl-
methoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,
allyloxycarbonyl; (4) cycloalkyl urethane-type protecting
groups illustrated by cyclopentyloxycarbonyl, cyclohexyloxy-
carbonyl; (5) thio urethane type protecting groups such as
phenylthiocarbonyl; (6) alkyl and aralkyl type protecting
groups as illustrated by triphenylmethyl (trityl) and
benzyl; (7) trialkylsilane groups such as trimethylsilane.
The terminal carboxylic protecting groups may be
(l) -OG wherein G is, for instance, an aliphatic moiety
preferably having 1 to about 20 carbon atoms, or araliphatic
moiety of l to about 4 rings, preferably having 6 to about
24 carbons, and providing an ester terminated with a carbon-
containing radical; or acyl, e.g., lower acyclic acyl preferably -~
having 1 to about 6 carbons; (2) an amide providing function,
e.g., -NH2, aminoaliphatic or aminoaraliphatic (e.g.,
monocyclic araliphatic) of 1 to about 10 carbon atoms,
thus, the amine may be primary, secondary or tertiary,
e.g., -NRCRd wherein Rc and Rd are as defined above; or
(3) an anchoring agent such as - NH - fH - C6H4 - resin
C6H5
support; - O - CH2 - C6H4 - resin support, chloromethylated
resin, hydroxymethyl resin, Merrifield resin, and the like.
Preferably, the foregoing aliphatic or araliphatic groups are
hydrocarbyl.
The polypeptide materials of this invention, e.g.,
compounds of formula II, can be employed to relieve the
symptoms of schizophrenia in humans and other mammals, and it
is believed this is accomplished by reducing the activity of
-14-
s~
the S-protein, i.e., by causiny a reduction in the degree of
alpha-helical conformation of -the S-protein in the warm-blooded
animal. These active compounds may be effective in counter-
acting -the symptoms of schizophrenia and maniEest forms thereof
such as anxiety tension states, manic depressive psyehosis,
etc., when pharmacologically-administered internally to a
living animal. The administration of an active compound of
this invention, for instance, mixed ~ith a pharmaceutieal
carrier may be internal, e.g., may be within the aigestive
tract and parenteral administration is preferred. The active
eompound may be in solid or liquid form and may be eombined
with a pharmaeeutieally-aeeeptable earrier as a solid, solution,
suspension, emulsion or other form. Paren-teral administration
may be by, for instanee, subeutaneous, intraperitoneal,
intravenous, intraarterial, intramuseular, or like, routes.
For oral administration, an aetive eompound and a pharmaeeuti-
eally-aeeeptable earrier may, for example, take the form of a
pill, lozenge, tablet, eapsule or a liquid suspension. Exemplary
earriers are solids such as lactose, magnesium stearate,
calcium stearate, starch, terra alba, diealeium phosphate,
suerose, talc, stearic acid, gelatin, agar, pectin, or acacia,
and liquids such as sesame oil, olive oil, water and the like.
An enteric eoating, sueh as cellulose aeetate phthalate, may
also be used. The compositions of this invention may also
inelude preserving agents, stabilizing agents, wetting agents,
emulsifyiny agents, buffers or salts and the like. The aetive
eompounds used in the method of the invention, or compositions
containing the same, may be either administered together with
or inelude other physiologically-active materials and/or
medieaments, e.g., buffering agents, antacids, sedatives,
-15-
tranquiliæers, analgesics, hormones or the like.
Various dosages of active compounds may be employed
depending on the particular active compound employed, the
severity of the condition being treated, the mode of admini-
stration, -the extent oE desired effect on the host, e.g.,
mammal, and the like Thus, the amount of active compound
administered may vary, but is sufficient to relieve schizophrenic
symptoms in the mammal to a significant extent. The dosages
may generally range from at least about 0.01 mg/kg/day (milli-
grams per kilogram of body weight of mammal per day), say, upto about 2 or more mg/kg/day, more preferably about 0.1 to 1
mg/kg/day. The treatment may consist of a single daily dose,
or the abové dosages can be given fractionally at periodic
intervals, for example, about 2 to 4 doses of about 0.05 to 0.2
mg/kg may be administered per day. The active compound may be
included in time-release formulations whereby the above amounts
are made available to the body over an extended time period.
The period of activity may be prolonged by incorporating the
polypeptides into organic, mostly polymeric compounds, such
as gelatin, polyphloretinphosphate, or polyglutamic acid.
The compounds of the invention including those
active in the treatment of schizophrenic symptoms or their
precursor intermediates, may be synthetically-prepared from
individual amino acids or from polypeptides to provide the
desired T-V-L peptide series. In one mode of synthesis, amino
acid components are reacted in the desired order to obtain the
peptide structure, e.g., the amine function of a first amino
acid may be condensed with the carboxylic acid function of a
second amino acid to provide a dipeptide structure which can be
further reacted to obtain the desired structure having 3 to
about 8 amino acid units or moieties. To obtain the desired
combination of amino acid moieties, it is usually expedient to
B -16-
.
block the sltes on the amino acids which mlght lead to an
undesirable si.de reaction, ~or instance, the carboxylic f~nction
o~ a first amlno acld reactant with an amine ~lmction of a
first amino acid or a second amlno acid molecule. Bloc~ing can
be effected by conventional means. Particularly advantageous
blocking groups ~or the amlno ~unc-tion of amino acids are
the ~-amino protecting groups, for instance, phthalyl,
carbobenzyloxy, and t-butyloxycarbonyl groups in that they can
be easily attached to the amlno group without causing
racemlzation of the amino acid, may be relatively inert to
reaction conditions, and may be easily removed ~rom the
amino group without adverse effect on the amino acid.
These protecting groups may be reacted as the acid halide,
e.g., chloride, ester, or acid anhydride, e.g., mixed acid
anhydride with alkyl formate, e.g., a lowe~ alkyl formate,
of the blocking group. The reaction may proceed at ambient
temperatures in an inert, organic liquid solvent such as
benzene. Higher or lower temperatures, e.g., about 0 to
50C. may, however, be employed if desired. The carboxylic
function of the amino acid may be blocked by reaction with
the amino group of the preceding amino acid in the peptide
series or by another susceptible blocking group when the
acid is the initial acid in the series.
For purposes of ease of recovery of the amino acid
synthesis product after each stage of the reaction, providing
the initial or terminal amin~ acid moiety as a resin ester
or resin amide can be quite advantageous. Particularly
useful resins are, for instance, benzyhydrilamine resin,
chloromethylated resin, Merrifield resin, and hydroxylmethyl
resin. The preparation of a benzhydrilamine resin is
described by Rivallile~ et al., ~Ielv. 51~, 2772 ( 1971), and
the preparation of a hydroxymethyl resin is described by
17
Bodanszlcy et al., Chem. :[nd. (London~ 38, 159r/-98 (196~).
The blocking groups should be stable in the
reagent and under the reac-tion conditions for preparing
the peptide. Blocking groups for the termlnal carboxylic
acld group should be stable under the conditions used for
removing the alpha-amino protecting group at each stage of
the synthesis. The blocklng group should retain its
protecting propertiesg i.e., not be spllt O~fg under
synthesis conditions, and when removed upon completion of
the synthesis the reaction conditions employed should not
alter the peptide chain in an undesired manner. The
blocking groups may be removed by various conventional
methods, depending on the nature of the group to be removed,
for example, in the presence of trifluoroacetic acid, or by
hydrogenolysis with, for instance, hydrogen and a catalyst such
as platinum, palladium or the like; or with hydrogen bromide in
glacial acetic acid or trifluoracetic acid or with anhydrous
hydrofluoric acid. The hydrogenolysis medium is preferably
essentially anhydrous.
The react~on between an existing peptide unit and a
subsequent amino acid can be accomplished by solid phase
reaction. In preparing resin esters or amides, the resin
support and amino acid may be intimately admixed in an inert
organic menstruum, for instance, tetrahydrofuran, dioxane,
dimethyl formamide, benzene; ethanol, and the like. The reaction
proceeds at ambient temperature, although higher and lower
temperatures, e.g., about 0~ to 80C., may be employed if
desired. The reaction is usually permitted to go essentially
to completion to maximize the yield of peptide product having
the desired order. The use of substantial excesses of amino
acid reactant, e.g., at least about 1.5 to about 200 or more
tlmes the amount required for reaction on a stoichiometric
3~
basis, and subs-tantlal reactio~ times, e.g.~ at least about 1
hour, preferably at least about 5 hours to 500 or more hours,
enhance the comp:Letlon of the reaction. The degree of com-
pletlon o~ the reaction rnay be determined by the Kaiser color
reaction. A pos:~tive reaction to ~aiser color reaction
indicates unsubstituted sites on the amino acid. A Dorman
titration may also be employed to determine completion o~ the
reaction.
Suitable linking methods to provide the polypeptide
chain of the compounds of this invention are described in the
literature. In general, the amino acid and/or peptide fragments
are linked so that, ~or example, an amino acld or peptide
containing a protected alpha-amino group and a terminal carboxyl
group is reacted with an amino acid or peptide containing a
~ree alpha-amino group and a protected terminal carboxyl group,
or an amino acid or peptide containing an active alpha-amino
group and a protected terminal carboxylic group is reacted with
an amino acid or a peptide containing a ~ree terminal carboxylic
acid and a protected alpha-amino group. The carboxylic group
can be activated for instance by conversion into an acid azide,
anhydride or imidazolide or into an act~ated ester such as
cyanoethyl ester, thiophenyl ester, p-nitrothiophenyl ester,
thiocresyl, p-methanesul~onylphenyl, p-nitrophenyl, 2,~-
dinitrophenyl, 2,1l,5- or 2,1~,6-trichlorophenyl, pentachlorop-
henyl, N-hydroxysuccinimide, N-hydroxyphthalimide, 8-hydroxy-
quinoline, N-hydroxypiperidine ester, or by reaction with a
carbodiimide (optionally with addition of N-hydroxysuccinimide)
or N,N'-carbonyldiimidazole or an isoxazolium salt, for
example, Woodward's reagent, the amino group for instance by
3o reaction with a phosphite. Frequently used methods include the
carbodiimide method, the Weygand-Wuensch method (carbodiimide
--19--
~3~ 3
in the presence of N-hydroxysuccinlmide), the azlde method, the
method o~ the activated esters and the anhydride method, also
the Merrifield method and the Method o~ the N-carboxyanhydrides
or N-thiocarboxyanhydrides.
Conve~iently, the carbodilmid~ method may be
employed, the carbodiimlde coupling agent may be, for
instance, N-ethyl-N'-( r -dimethylaminopropyl carbodiimide)
or a dihydrocarbyl-carbodiimide, e.g., dialkyl o~ 1 to
about 20 carbons, for instance, dicyclohexyl carbodiimide.
The carbodiimide may be provided in an inert solvent, for
instance, methylene chloride~ to assist in handling.
Frequently, the mole ratio o~ carbodiimide to moles of the
least abundant amino acid reactant is about 0.9:1 to 10:1.
The linking reaction may be conducted in an iner~ medium, e.g.,
dichloromethane, in solvent-providing quantiti~s, ~or
instance, at least about 1 to about 100 or more milliliters per
gra~ o~ amino acid. The reaction proceds at ambient temper-
ature, although higher and lower temperatures, e.g., about -30
to 50C., may be employed i~ desired.
The unblocking of an amino acid or peptide
~ragment for ~urther reaction may be conducted in any
convenient manner, and such methods are well known.
Advantageously, blocking agents on amino groups, e.g.,
acyl blocking groups may be removed in a two stage process,
using a hydrogen halide, for instance, hydrogen chloride,
in each stage. The resultant product is the acid addition
salt, e.g., hydrogen chloride salt, which may be neutra-
li~ed with a base, ~or instance, triethylamine, pyridine,
sodium or potassium hydroxide, sodium or potassium carbonate,
or the like. The hydrogen halide may be prepared by
bubbling the hydrogen halide into a li~uid medium, ~or
instance, dioxane or methylene chloride. A resin ester
-20-
may be converted to an unblocked peptide by bubbling
hydrogen halide, e.g., hydrogen bromide~ yas through a
medium containing the peptide resin ester and trifluoroacetic
acid.
As no-ted above, it is desirable to modify -the
polypeptide containhlg the T-V-L linkage to provide a
compound which exhibits a longer effective life in treating
schizophrenic symptoms than the aminoacid tripeptide, and
thus, the latter polypeptide may be converted to an
omega-N-acylated polypeptide. Known acylation procedures
can be employed although it may be most desired to use
a procedure which does not disturb the optical activity
of the amino acid moieties. The alpha-amino group
of the polypeptide may be acylated by reaction with the
corresponding acid halide, e.g., acid halide, acid anhydride,
or the like, using conventional methods. The terminal
carboxylic acid or ester group may be esterified or
transesterified in accordance with conventional procedures
to provide a peptide which may exhibit a larger effective
dose. Esterification can be effected by reaction of the
polypeptide having an unblocked, terminal carboxylic
function with the corresponding alcohol. The amides
having the characteristic T-V-L linkage of the compositions
of this invention may be prepared in accordance with known
procedures. For instance, the corresponding acid or acid
halide, e.g., chloride, of the peptide may be reacted with
ammonia or a primary or secondary amine. The ammonia or
amine is often pxovided in at least a stoichiometric
amount for complete reaction with the peptide structure. The
reaction may be conducted in an inert solvent such as
tetrahydrofuran and may conveniently be at a temperature
of about 10 to ~0 C. or more. Generally, the higher the
-21-
'
molecular weigh-t of the acyl and ester groups on the poly-
peptide the slower and more prolonged will be its efEect in
treating schizophrenic symptoms.
The functional derivatives of the compounds and
intermediates of this invention include the pharmaceutically-
acceptable, salts, including acid and base addition salts, of
the polypeptides. The acid addition salts can be obtained by
reacting the polypeptide with an organic or inorganic acid,
such as hydrochloric acid, hydrobromic acid, sulfuric acid,
phosphoric acid, acetic acid, maleic acid, tartaric acid,
citric acid, malic acid, ascorbic acid, benzoic acid, and the
like. The compounds having the T-V-L linkage may also be in
the form of metal complexes prepared by contacting the poly-
peptides with a sparingly-soluble salt, hydroxide or oxide of
metal. Metals which may be employed include cobalt, copper,
calcium, iron, zinc, magnesium, sodium, potassium and ammonium.
Other metals which may be employed include nickel and aluminum.
Thus, for example, a metal complex can be obtained by adding
the polypeptide and a sparsely-soluble, metal salt, metal
hydroxide, or metal oxide to an aqueous medium, or by adding an
alkaline medium to an aqueous solution of the polypeptide and
an essentially insoluble metal salt to form an insoluble
polypeptide-metal hydroxide complex. An insoluble polypeptide-
metal salt complex can also be prepared in situ by adding to an
aqueous alkaline medium the polypeptide and a metal salt.
The invention will be described further by the
following examples. In the examples, the peptide moieties having
optical activity are in the L- configuration unless otherwise
stated.
EXAMPLE 1
Preparation of Leucine Benzyl Ester p-Toluenesulfonate
-22-
~ ~ .
, ' ' .
3~3
In a 200 ml 3 neck flask equipped with a ~an and
Stark trap, re~lux condenser (with dry:lng tube attached),
and mechanical stirrer, 15 grams (0.0789 mole) of p~
toluenesulfonic acid monohydrate i5 refluxed until one
equivalent o~ water is released. At this point 10 grams
(o.o7634 mole) leucine and 17.0 grarns (0.15 mole) benzyl
alcohol are added. The mixture is re~luxed for 4 hours
and allowed to cool to room temperature. The solvents are
removed in vacuo and a white, solid residue is produced.
L~ The white solid is washed with anhydrous ether and recrystallized
from ethanol/ether to yield in three crops 28.2 grams of
leucine benzyl ester p-toluene sulfonate (~1~% yield)
having a melting point of 157-158C. (with decomposition).
EXAMPLE 2
Preparation of Benzyl N-~-t-Butyloxycarbonylvalylleucinate
Approximately 13.2 grams (o.o336 mole) of benzyl
leucinate p-toluenesulfonate is treated with sodium bicarbo-
nate to provide benzyl leucinate, which is recovered by
methylene chloride extraction. In a 200 ml 3 neck flask equipped
with a nitrogen gas inlet and outlet adaptors and a magnetic
stirrer, 6.25 grams (0.34 mole) of dicyclohexylcarbodiimide is
dissolved in 30 milliliters of freshly distilled dry
CH2C12, and the recovered benzyl leucinate is added thereto.
The mixture is maintained under a nitrogen atmosphere and
is cooled to approximately -30C. using dry ice and carbon
tetrachloride. To the mixture 7.1~5 grams ~0.0336 mole) of
N ~-t-butyloxycarbonylvaline dissolved in 50 ml of dry
CH2C12 is added dropwise. The resulting mixture is
stored at -10C. for 2 days. After removing a resultant
white precipitate by filtration, the methylene chloride
layer is washed successively with 25 percent aqueous
-23-
:: ' . ' , ' ' ' :
- . : .: .
acetic acld, aq~leous sodium bicarbonate solution, water,
and saturated aqueous saline solution. The organic layer
is then dried over anhydrous MgS0ll and the solvent ls
removed in vacuo. The residue is taken up in ethyl acetate,
filtered, and a~ain concentrated in vacuo. The resulting
white solid is recrystallized from ethanol/hexane to yield in
two crops 9.17 grams of benzyl N ~-t-butyloxycarbonylvalyl-
leucinate (65~ yield) having a melting point of 90-91C.
EXAMPLE 3
Preparation of Benzyl N ~-t-Butyloxycarbonyl-0-benzylthreonyl
valylleucinate.
In a 100 ml 3 neck ~lask equipped with gas inlet and
outlet adaptors and magnetic stirrer, 4.o8 grams (0.00972
mole) o~ benzyl N-OC-t-butyloxycarbonylvalylleuclnate is dis-
solved in 50 milliliters of glacial acetic acid. The mixture
is cooled to 5C. and hydrogen chloride gas is bubbled through
the mixture for 30 minutes. The mixture is then taken to dry-
ness in vacuo. The resulting white powder, benzyl valylleucine-
hydrochloride, is repeatedly washed with anhydrous ether. Thepowder is suspended in 30 milliliters of dry CH2C12, cooled
to -30C., and treated with 1.38 millillters (0.010 mole) of
triethylamine and stirred ~or 30 minutes to provide a solution.
The resulting triethylamlne solution is then added to
1.94 grams (0.010 mole) o~ dicyclohexylcarbodiimide dissolved
in 30 milliliters of freshly distilled~ dry CH2C12. This
solution is maintained under a nitrogen atmosphere and 3.0
grams (0.00972 mole) o~ N ~-t-butyloxycarbonyl-0-benzylthreo-
nine dissolved in 30 milliliters dry CH2C12 is added dropwise.
The reaction mixture is kept at -10C. for two days. The
resulting white precipitate is removed by filtration, and the
methylene chloride layer is successively washed with water, 25%
-24-
3~
aqueous acetic acid, aqueous sodlum bicarbonate solution,
water, and saturated aqueous saline solution. The orga~ic
layer is dried over anhydrous MgSO~ and the solvent ls removed
in vacuo. The resldue :Ls taken up in ethyl acetate, ~iltered,
and concentrated in vacuo to yield 3.56 grams of a white powder
which is benzyl N ~-t-butyloxycarbonyl-O-benzylthreonyl-
valylleucinate (60% yield, m.p. 115 to 119C.).
EXAMPLE 4
Preparation of ~enzyl O-Benzylthreonylvalylleucinate Hydrochlo-
ride.
Hydrogen chloride gas is slowly bubbled ~or 50
minutes through a cold (5C.) and stirred solutlon of 4.1
grams (6.71 m mole) of benzyl N ~-t-butyloxycarbonyl-O-
benzyl-threonylvalylleucinate in 50 milliliters of glacial
acetic acid. The solvent is removed in vacuo. The residue
is ~ashed with ether and recrystallized from ethanol/ether
to yield 3.14 grams of benzyl O-benzylthreonylvalylleucinate
hydrochloride (86% yield) having a melting point o~ 200-202.5C.
EXAMPLE 5
Preparation of Threonyl-Valyl-Leucine
One gram of benzyl O-benzylthreonylvalylleucinate
hydrochloride is neutralized to provide benzyl O-benzylthreonyl-
valylleucinate. A hydrogenolysis mixture is prepared containing
the benzyl O~benzylthreonylvalylleucinate, 0.5 grams of 10
percent palladium on charcoal per 0.01 mole o~ the material
to be hydrogenated~ and absolute e~hanol. To the mixture is added ~-
1.1 mole equivalents of acetic acid based on the benzyl ester.
-25-
.
The mixture is hydrogenated ror 4 hours. The catalyst is
removed by ~iltration and the solvents are removed in ~acuo.
The residue ls washed with anhydrous ether and recrystalllzed
from an ethanol/ether mixture to provide 544 milligrams of the
free peptide, threonylvalylleucine (85% yield).
EXAMPLE 6
Preparation of Benzyl N- ~-Acetyl-O-benzylthreonylvaly1leucinate.
Benzyl O-benzylthreonylvalylleucinate is prepared
from 1 gram of benzyl O-benzylthreonylvalylleucinate hydro-
chloride as in Example 5 and is dissolved in acetic acid, and
2 equivalents acetic anhydride based on the benzyl ester are
added. The mixture is stirred overnight. The solvents are
evacuated in vacuo. The residue is recrystallized from
methanol/ ether, to yield 806 milligrams of benzyl N- ~-
acetyl-O-benzylthreonylvalylleucinate (85% yield).
EXAMPLE 7
Preparation of N-~-acetylthreonylvalylleucinamide
~20 One gram of benzyl N-~-acetyl-O-benzylthreonylvalyl~
leucinate is dissolved in 95% ethanol. Ammonia is bubbled
through the solution for 8 hours. After stirring at room
temperature for 48 hours, the solvents are removed in vacuo.
The residue is washed with ether to yield 795 milligrams (99%)
of N~X-acetyl-O-benzylthreonylvalylleucinamide. One gram of
the N-~-ace~yl-O-benzylthreonylvalylleucinamide is subjected
to hydrogenolysis in absolute ethanol to yield 705 milligrams
(90% yield) of N-a~-acetylthreonylvalylleucinamide.
-26-
3~3
E~AMPLE 8
Merrif-ield Synthesis Or Threonyl-valyl-leucirle
A solution of 5.3 grams of t-butyloxycarbonyl-L-
leucine (21.2 mmole), 2.5 mill-Lliters of triethylamine (18
mmole) in a 2-rnethyltetrahydrofuran solvent ls preQared and 20
grams o~ Merrifield resin, which is polystyrene cross-linked
with 2% divinylbenzen~e, providing 21.2 mmole of chlorine is
added there to. The Merrifleld resin is analyzed to contain
about l. o6 mmole of chlorine per gram of resin, and prior to
use, the resin is washed with methanol, distilled water,
ethanol, and methylene chloride and then dried in vacuo at
100C. Prior to use the 2~methyltetrahydrofuran is distilled
over a sodium dispersion, and the triethylamine is distilled
over phenylisocyanate and then redistilled over a sodium
dispersion. The mixture is refluxed for approximately 70 hours
to effect esterification of the blocked aminoacids to the
resin. After the esterification reaction, the resin is washed
with tetrahydrofuran, ethanol, glacial acetic acid, ethanol,
distilled water, ethanol, and methylene chloride, and then
dried in vacuo at 100C. for three hours. Approximately 21.5-
22 grams of t-butyloxycarbonyl-L-leucine resin ester is
obtained.
The t-butyloxycarbonyl-L-leucine resin ester is sus-
pended in 200 milliliters of methylene chloride and stirred by
bubbling high purity nitrogen through the suspension. The t-
butyloxycarbonyl protecting group is removed by adding 400
milliliters of an equal volume mixture of trifluoroacetic
acid and methylene chloride. The resin is washed and then
neutralized using 400 milliliters of 10% by volume triethylamine
in chloroform. The resin is washed with chloroform and
mekhylene chloride. About 4.82 grams of t-butyloxycarbonyl-
L-valine is added to the resin followed by an equi~alent
-27-
amount, i.e., about 11.57 grc~ms, o~ d1cyc~ohexylcarbodilmide,
(24 mmole), and the coupling reaction ls allowed to proceed for
a-t least 16 hour~. The resultant t-butyloxycarbonyl-L-valyl-L~
leucyl resin ester is then washed with methylene chlorlde,
essentially an~y~rous ethanol, glacial acetic acid, ethanol,
and methylene chloride to remove the dicyclohexylurea by-
product. The completeness of the reaction is checked by Kaiser
color reaction.
Essentially the same procedure as described above is
employed to remove the t-butyloxycarbonyl (boc) protecting
group. To the unblocked resin ester is added 5.00 grams of t-
boc-0-benzyl-L-threonine then an equivalent amount (i.e., 3.6
grams) of dicyclohexylcarbodiimide t18.~ mmole) is added, and
the coupling reaction is allowed to proceed for four hours.
The resultant resin ester is washed in essentially the same
manner as described above and then dried overnight at room
temperature in vacuo over phosphorus pentoxide. Approximately
22-23 grams of t-butyloxycarbonyl-0-ben~yl-L-threonyl-L-valyl-
L-leucyl resin ester is obtained.
Cleavage of the tripeptide from the resin is ef~ected
by suspendlng the dried t-butyloxycarbonyl-0-benzylthreonyl-
valylleucine resin ester in 100 milliliters o~ 100% trifluoro-
acetic acid through which anhydrous hydrogen bromide is bubbled.
During this process the resin ester linkage and simultaneously
the 0-benzyl protecting group on threonine are cleaved, yielding
the soluble hydrobromide and trifluoroacetate salts of threonyl-
valylleucine in trifluoroacetic acid.
The trifluoroacetic acid solution is then evaporated
to dryness, and the residue is dissolved in 100 milliliters of
an equal volume mixture of methanol-distilled water. The mix-
ture is taken to dryness under reduced pressure, and the residue
is dissolved in 100 milliliters of ethanol, and again recovered
by drying under reduced pressure. The dried material is then
-28-
r ~
dissolved in 10 to 20 millilite.rs of a minimum of 30% glacial
acetic acid and filtered. Solid sodium carbonate or sodium
bicarbonate is slowly added until precipitation occurs. The
final tripeptide product is recrystallized from ethanol/water
or by dissolving the product in a large excess of distilled
water, concentrating the solution by distilling off some of the
water, and allowing crystal formation to occur at refrigerator
temperature, about 5C. About 657 milligrams of L threonyl-L-
valyl-L-leucine (75% yield) having a melting point range of
240-242C. (with decomposition) is obtained.
EXAMPLE 9
Preparation of L-threonyl-L-valyl-D-leucine
The procedure of Example 8 is essentially repeated
except that D-leucine is employed instead o~ L leucine and L-
threonyl-L-valyl-D-leucine is prepared. The product is a
white crystalline mixture which is observed to have a
decomposition point of about 240C. with charring.
EXAMPLE 10
Preparation of N~ ~-Acetyl-threonyvalylleucine
t-Butyloxycarbonyl-O-benzylthreonylvalylleucine resin
ester is prepared in essentially the same manner as disclosed
in Example 8. The t-butyloxycarbonyl protecting group is .
removed in essentially the same manner the groups on the leucyl ~
resin ester and valylleucyl resin ester are removed in Example ~.
8, to provide O-benzylthreonylvalylleucine resin ester. About
5.0 grams of dried O-benzylthreonylvalylleucine resin ester is
acetylated by first washing the resin ester with dimethyl-
formamide, and then reacting the resin with the acetylation
reagent comprising 100 milliliters of 44 parts by volume
--2g--
dimethylformamide, 5 parts by volume acetic anhydride, and 1
part by volume triethylamine for 20-50 m:inutes and -the resin is
washed with dimethylformamide, followed by methylene chloride,
and then dried under vacuum at room temperature overnight.
The N-acetyl peptide is cleaved from the resin in a
manner essentially the same as descr:ibed in Example 8. The
trifluoroacetic acid solution is then evaporated to dryness under
reduced pressure. The residue is washed with an equal volume
mixture of methanol and water, recovered by evaporation to dryness
under reduced pressure, washed with ethanol, and again recovered
by evaporating to dryness under reduced pressure. The final
product is then recrystallized from ethanol-ether, providing
400 milligrams of N-~-acetyl-threonyl-valylleucine (55% yield)
having a melting point range of 210~214C. (with decomposition).
EXAMPLE 11
Merrifield Synthesis of the Hydrochloride Salt
of Glycylthreonylvalylleucine
Starting with 10 grams of O-benzylthreonylvalylleucine
resin ester as prepared in a manner essentially the same as that
disclosed in Example 10, the coupling of t-butyl oxycarbonyl-
glycine is accomplished by suspending the resin ester in 100
milliliters of methylene chloride, adding 1.94 grams to t-butyl-
oxycarbonylglycine (10 mmole), followed by an equimolar amount,
i.e., 2.06 grams, of dicyclohexylcarbodiimide(10 mmole) and
allowing the condensation reaction to proceed for four hours.
The t-butyloxycarbonylglycyl-O-benzyl-threonyl valylleucine resin
ester is cleaved from the resin in essentially the same manner
as previously described in Example 8. After appropriate washings
with methanol, distilled water,
-30-
, 3,ilf 9~
ethanol, and addition oE sodium carbonate -to attain a pH o~
4.5, the product is taken down to dr~ness under reduced
pressure.
The glycylthreonylvalylleucine product is converted
to its hydrochloride salt by dissolving i-t in lO0 mil]iliters
of a mixture o~ 3 parts by volume o~ ethanol to one part by
volume ether and bubbling anhydrous hydrogen chloride gas
through the solution for approximately 10 minutes. The product
is filtered and dried overnight at room temperature in a vacuum
dessicator to provide 600 milligrams of the hydrochloride
salt of glycylthreonylvalyllleucine ~606 yield) having a
melting point range of 225~226C. (with decomposition).
EXAMPLE 12
Merrifield Synthesis of N- ~-Acetyl-tyrosyl-
threonylvalylleucine
O-Benzyl threonylvalylleucine resin ester (prepared
essentially as described in Example 10) in an amount of 7.4
grams is added to a reaction flask and suspended in 75 milli-
liters of methylene chloride. To the suspension is then added
3.735 grams of t-butyloxycarbonyl-O-benzyltyrosine (lO mmole), -
followed by an equivalent amount of dicyclohexylcarbodiimide
(2.06 grams, 10 mmole), and the coupling reaction is allowed to
proceed for four hours. Stirring is accomplished by bubbling
high purity nitrogen through the suspension. The resin is then
washed with methylene chloride, ethanol, acetic acid, ethanol
and methylene chloride to remove the dicyclohexylurea by-
product, and then dried overnight at room temperature in vacuo
over phosphorous pentoxide. The dried product, O-benzyltyrosyl-
O-benzylthreonylvalylleucine resin ester, typically increases
in weight to 7.50-7.55 grams.
The N-ace-tylation is carried ou-t essentially by -the
process described in Example 10 for the synthesis of N~ -
acetyl-threonylvalylleucine except that 7.5 milliliters of
acetic anhydride, 2.5 mil]iliters of triethylamine and 50
milliliters of dimethylformamide are employed. After the N
acetylation, the resin is washed wit:h dimethylformamide,
followed by methylene chloride and clried under vacuum at room
temperature overnight.
The cleavage of the pepticle from the Merrifield resin
is conducted in essentially the same manner as disclosed in
Example 8 except to avoid possible electrophilic aromatic
substitution on the tyrosine ring by bromine, the hydrogen
bromide is first passed through a resorcinol-trifluoroacetic
acid mixture in a ratio of 2 grams resorcinol per 100 milli-
liters of trifluoroacetic acid to remove traces of Br2. As an
added precaution against electrophilic substitution, 20 milli-
liters of anisole are added directly to the cleavage vessel.
The final product, N ~ -acetyl-tyrosylthreonylvalyllleucine, is
recrystallized from ethanol-ether providing 900 milligrams of
the tetrapeptide ~59% yield) having a melting point range of
234.5-235C. (with decomposition). Amino acid analysis of the
product provides 0.93 tyrosine, 1.00 threonine, 1.00 valine,
and 1.00 leucine.
EXAMPLE 13
Merrifield Synthesis of Threonylvalylisoleucine
A solution of 5.3 grams of t-butyloxycarbonyl-L-
isoleucine (21.2 mmole~, 2.5 milliliters of triethylamine (18
mmole) in a 2-methyltetrahydrofuran solvent is prepared and 20
grams of Merrifield resin is added thereto. Prior to use, the
resin is washed with methanol, distilled water, ethanol, and
-32-
. 3 ~
methylene chloride and then dried in vacuo at 100C. Prior to
use, the 2-methyltetrahydrofuran is distilled over a sod:ium
dispersion, and the triethylamine is distilled over phenyliso-
cyanate and then redistilled over a sodium dispersion. The
mixture is refluxed for approximately 72 hours to effect
esterification of the blocked amino acids to the resin. After
the esterification reaction, the resin is washed with tetra-
hydrofuran, ethanol, glacial acetic acid, ethanol, distilled
water, ethanol, and methylene chloride, and then dried in vacuo
at 100C. for three hours. ~pproximately 21.5-22 grams of t-
butyloxycarbonyl-L-isoleucine resin ester is obtained. This
coupling of the t-butyloxycarbonylvaline to the t-butyloxy-
carbonylisoleucine resin ester is performed in essentially the
same manner as the coupling of t-butyloxycarbonyl-L-valine to
the t-butyloxycarbonyl-L-isoleucine resin ester disclosed in
Example 8. However, because of the even greater steric
hindrance, the reaction is allowed to proceed for 2~ hours.
The resin is then washed as usual to remove by-products and
completeness of the reaction is checked by the Kaiser color
reaction. The addition of t-butyloxycarbonyl-threonine to
obtain t-butyloxycarbonyl-threonylvalylisoleucine resin ester,
the cleavage of the peptide from the resin to obtain t-butyl-
oxycarbonyl-threonylvalylisoleucine, and its subsequent
isolation and purification are performed in essentially the
same manner as in the preparation of threonylvalylleucine
disclosed in Example 8. The tripeptide, threonylvalyliso-
leucine, is obtained in an amount of about 600 milligrams (68
yield).
-33-
.
EX MPLE 14
Merrifield Synthesis of Threonyl-
valylleucylarginine
Essentially the same procedure as in Example 8 is
followed except employing 6.78 grams of t-butyloxycarbonyl-
nitro(guanidinyl)-L-arginine (21.2 nu,lole) as the amino acid
esterified to 20 grams of the Merrifield resin. The esterifi-
cation is conducted by refluxing the mixture for about 72 hours
and the resin ester is washed and dried as in Example 8 to
provide about 22 or 23 grams of t-butyloxycarbonyl-nitroarginine
resin ester. About S grams of t-butyloxycarbonyl-L-leucine
(24 mmole) is coupled to the nitroarginine resin ester by the
addition of an equivalent amount of dicyclohexylcarbodiimide
(4.6 grams, 24 mmole), allowing the reaction to proceed for 16
hours. After the resin ester is washed, the coupling of
t-butyloxycarbonyl-L-valine and t-butyloxycarbonyl-O-benzyl-L~
threonine to the resin ester is conducted by essentially the
same procedure as previously described in Example 8. Approxi-
mately 24 grams of t-butyloxycarbonyl-O-benzoyl-L-threonyl-L-
valyl-L-leucyl-arginine resin ester is obtained.
Because of the stability of the nitro group to
hydrogen bromide, the protected peptide resin ester is cleaved
with anhydrous hydrogen fluoride in order to also effect
removal of the nitro group. After passage through an appro-
priate ion-exchange resin, the eluant is concentrated down to
an oil and then isoelectrically precipitated by the addition of
solid sodium carbc)nate. The product, L-threonyl-L-valyl-L-
leucyl-L-arginine, is recrystallized from an ethanol-water
solution yielding approximately 900 milligrams of the
tetrapeptide.
-34-
-`' J ~i
Pep-tides having the characteristic T-V-L structure
may also be prepared using a solid phase reaction with
chloromethyla-ted resin which is a polystyrene cross-linked
with divinylbenzene resin and is cornmercially available
from siO Rad Laboratories, Richmond" California, under the
trade name of siO seads SX~ n these processes for the
addition of peptide moieties, for instance, a t-butyloxycar-
bonylleucine resin ester is suspended in dioxane and
unblocked using 4 normal hydrochlor:ic acid. The resultant
hydrochloride is neutralized with triethylamine and washed.
The coupling of additional amino acid moieties may be
conducted using a coupling agent such as dicyclohexyl
carbodiimide. This procedure has not been found to be
economically attractive as other synthesis procedures, for
example, the Merrifield synthesis.
EXAMPLE 15
Threonylvalylleucine Methyl Ester
Crude threonylvalylleucine (0.066 g., 0.2 mmole) is
placed in a 5 milliliter reaction vial and is dried at 100C.
under vacuum overnight over phosphoric anhydride desiccant.
The dried trip~ptide is dissolved in 0.5~ milliliter of
anhydrous methanol. A previously prepared mixture of dimeth- -
oxypropane and reagent grade hydrogen chloride in a weight
ratio of 5~2:83 is added in an amount of 0.63 milliliter to the
reaction mixture. The reaction vial is tightly sealed. The
mixture is thoroughly mixed and then is allowed to stand over
night to provide the threonylvalylleucine methyl ester. The
solvents are then removed by evaporation under a stream of
nitrogen, followed by vacuum drying the ester at lOOaC. over
phosphoric anhydr:ide desiccant overnight.
-35-
~ ~ 3~,~
EXAMPLE 16
N-O-diacetyl-Threonylvalylleucine Methyl Ester
The product of Example 15 is dissolved in 0.38
milliliter of pyridine. Acetic anhydride in the amount of 0.1
milliliter (2 m. mol.) is added as the acylation ayent and the
reaction mixture is maintained at 37C. for 20 minutes in a
tightly closed vial. The solvent is removed from the acylated
product by evaporation under a stream of dry nitrogen, followed
by vacuum drying at 100C. over phosphoric anhydride desiccant
overnight.
The acylated tripeptide ester N-O-diacetyl-threonyl-
valylleucine-methyl ester can be purified by dissolving the
crude product in methanol and passing the sample through a high
pressure, liquid chromatograph operation. A Waters modular
liquid chromatograph equipped with a 10 feet by 3/8 inch
diameter preparative column packed with Phenylporasil-B~ is
employed using methanol as the eluent. The elution is moni-
tored for changes in refractive index and U~ absorbance at 225
nm. the flow rate of the methanol eluent is 1 milliliter per
minute. The major peak with a retention volume of approxi-
mately lO0 milliliters is collected as the purified fraction.
The solvent from this fraction is removed by evaporation under
a stream of dry nitrogen followed by vacuum drying at 100C.
over phosphoric anhydride desiccant overnight.
The following example illustrates the isolation and
production of the tripeptide threonylvalylleucine from animal
sources.
-36-
.
EX~MPLE 1
Ex-trac-t:ion and Isolation of Threonylvalyl-
Leucine-Containing Polypeptide From Beef Brain
The hypothalami from several beef brains are dissected
out and homogenized in buffer solution, hereinafter referred to
as "tris citrate buffer", which has been prepared as follows:
Tris Citrate suffer
A 0.498 molar aqueous solution of tris citrate ~-
huffer is prepared as follows. To 1 liter of
distilled water is added 9.58 grams of the
citric acid and 49.~ grams tris(hydroxymethyl)-
aminomethane. The pH of the resulting solution
is adjusted to 8.65 by the addition of 0.1
molar aqueous solution of sodium hydroxide.
Homogenization of the hypothalami is conducted in
four parts by volume (milliliters) of the tris citrate buffer
per each part by weight (grams) of hypothalami. The homogenized
mixture is centrifuged at 15,000 rpm for 20 minutes at 4C. and
the supernatant liquid, which contains extracted anti-S
protein factor together with numerous impurities, is retained.
Four kilograms of starch hydrolysate is washed once
with distilled water, then twice with the tris citrate buffer~
The wet starch is formed into a block measuring 123 centimeters
long by 60 centimeters wide by 5 centimeters thick. The two
ends of the block are held in place by filter paper to permit
excess buffer to drain out of the block. After such drainage
the block has a thickness of only about 1.5 centimeters.
Acr~ss the Width of the starch block, beginning at a
point 43.5 centimeters from one end thereof, is excavated
a 1 inch wide section of the block. The temperature of the
block is adjusted to 4C. The anti-S protein factor-contain-
-37-
33
ing supernatant liquid Erom the foregoing centrifuyation
(measuring about 35-~0 ml.) is mixed with 10 drops of brom-
phenol blue. The resultant solution is mixed with the
excavated starch and -that mixture is poured into the 1-
inch wide trough in the block.
The block is then subjected to an electrophoretic
current of 750 volts and 50 milliamperes, the cathode being
connected to that end of the block which is 43.5 centimeters
distant from the trough, and anode t:o the other end. The cur-
rent is continuously passed through the block until the blueline across the width of the block (provided by the presence
of the bromphenol blue) moves toward the anode a distance of
42 centimeters. This requires about 18 hours. During this
time a bright red line (provided by the hemoglobin that was
present in the extract from the hypothalami) moves toward
the cathode a distance of about 5 centimeters from the trough.
The area between the blue and red lines after elec-
trophoresis is complete is divided into 10 strips, each strip
extending across the width of the block and being about 2
inches wide. Each strip is separately and completely exca-
vated from the block and is slurried in 12 ml. of the tris
citrate buffer for about lO minutes. The slurry is then
vacuum filtered through a Buchner funnel. Each of the result-
ant 10 fractions is assayed according to the tryptophan up-
take method described in Biological Psychiatry, VolO 7, No. 1,
p. 53, (1973) as an indication of the amount of anti-S protein
factor therein. I'he two or three of the active fractions are
retained. Numbering the strips on the starch block from l to
10 beginning with that ad~acent the blue line, the fractions
containing the greatest anti-S protein factor activity are
usually those obtained from strips Nos. 2, 9, and 10.
The foregoing procedure is repeated a sufficient
-38-
~, . ~ . .
.f~3~
number of times to provide 20 fractions having relatively
high anti-S protein factor activity. These 20 fractions are
then combined and subjected to evaporation through di~lysis
tubing to reduce the volume from an initial 100-160 ml. to
about 40-60 ml.
A packed column for use in chromatographing the
concentrated solution of crude anti-S protein factor ls pre-
pared as follows:
DEAE Cellulose Column
800 Grams of "Selectacel" brand of DEAE-type
cellulose (a diethylaminoethyl cellulose sold
by Brown Company of Berlin, New Hampshire) is
washed twice with 5 gallon portions of a 0.19
normal aqueous solution of sodium hydroxide.
Eollowing that, the packing material is washed
with 5 gallon portions of 0.19 normal hydro-
chloric acid. The material is then washed with
0.005 molar phosphate buffer until chloride-free
(usually 25 washes). The packing material is
then placed in a 110 centimeter high glass column
having an internal diameter of 2.5 centimeters.
The concentrated, crude solution of anti-S protein
factor is placed on the DEAE column and the column is then
eluted using a 2 flask gradient elution system in which flask
A contains 1,000 ml. of 0.005 molar aqueous cation phosphate
buffer solution (pH 7.4) and flask B contains 1,000 ml. of
0.04 molar aqueous cation phosphate buffer solution (pH 4.3)
which has been augmented with sodium chloride in a ratio of
405.5 grams of sodium chloride per 50 liters of buffer solution.
The eluate is collected in 15 ml. fractions, numbering about
120 in all. Each fraction is assayed for anti-S protein factor
-39-
r, ~
activity by -the tryp-tophan uptake method and the 10 fractions
having the greatest anti-S fac-tor activity are combined and
concentrated by lyophilization to about 10 percent of -their
original volume.
The entire foxegoiny procedure is repeated a suffi-
cient number of times to provide enough anti-S protein Eactor-
containing concentrate to show a total protein content of about
200 milligrams as determined by the Lowry method. This will
require about 300-400 ml. of concentrate, in turn requiring
10 about 400-1,000 grams of beef hypothalamus, in turn requiring
about 160 head of cattle.
The concentrate containing about 200 milligrams of
protein is dialyzed against distilled water for 24 hours.
For every 17 ml. of the concentrate, 4.25 ml. of 0.1 molar
trypsin and 2.1 ml. of 1 molar CaC12 is added. The pH of the
entire mixture is adjusted to 7~8 with 1 molar NaOH and
then incubated for two hours in a waterbath shaker at 37C.
After incubation, the solution is filtered through a 10,000
molecular weight Diaflo membrane (Amicon UM-10). After fil-
20 tration, 4.25 ml. of 0.1 molar pepsin solution is added per
17 ml. of the filtrate mixture. The pH is adjusted to 1.5
with 1 molar HCl and the mixture is incubated again for -two
hours at 37C. After incubationr the p~I is raised to 7.8
with 1 molar NaOH and the solution is filtered through a
1,000 molecular weight Diaflo membrane (Amicon UM-2).
The filtrate is subjected to flash evaporation to
reduce its volume to about 50 ml. The resulting mixture is
centrifuged, and the supernatant liquid retained. The pH of
the supernatant liquid is adjusted to below 2.2 by the addition
of concentrated hydrochloric acid. The acidified liquid is
then diluted with distilled water to a volume of 75 ml.
This anti-S protein factor-containing solution is then sub-
-40-
: ~,, . -
jected to column chromatography on a column prepared as follows:
Flrs-t Sulphonated ~esin Column
__ _
The packing material employ~d is "Aminex 50-WX2"
brand by sio Rad Company, which is a hydroyen ion
form of "Dowex" cation exchange resin having a
particle size of 200-325 mesh. The resin is a
sulphonated polystyrene having 2 percent nominal
cross-linking by divinylbenzene. The packing
material is washed in a Buchner funnel with 0.1
normal hydrochloric acid until the wash is
colorless. Next the material is washed with
distilled water until the wash exhibits a pH
of 5, following which the material is washed with
approximately 3 volumes of 2.0 normal aqueous
solution of sodium hydroxide. Again the material
is washed with distilled water until the wash
has a pH of 5. The packing material is then trans-
ferred to a beaker and slurried in two volumes of
1 normal aqueous solution of sodium hydroxide at
35C. for 2-3 hours. The resultant slurry is then
filtered through a Buchner funnel and the filter
cake washed with distilled water until the wash
water has a pH of 5. The packing material is
then slurried in two volumes of 0.2 molar aqueous
solution of sodium citrate (pH 3.1) and the slurry
is poured into a glass column 150 centimeters high
and 2 centimeters in internal diameter.
The acidic, aqueous solution of crude anti-S protein
factor is placed on the Aminex column and the column is then
eluted using a 2 flask gradient elution system in which flask
A contains 2,000 ml. of 0.2 molar aqueous solution of sodium
-41-
citrate (pM 3.1) and flask B contains 2,000 ml. of an ace-tate
citrate buffer (pll 9.1) prepared from 315 yram citric acid and
402 gram sodium acetate in 4 liters of deionized wa-ter. I'he
column is maintained under 10 psi of- nitrogen. The elua-te is
collected in 10 milliliter fractions, numbering about 120 in
all.
The fractions are numberecl in the order in which
they are taken from the column. Each of the thus-obtained
fractions is assayed by the tryptophan uptake method Eor
anti-S protein factor activity. A graph is prepared plotting
the numbers of the fraction along the x-axis and the activi-
ties of the fractions along the y-axis. At least one peak of
activity will be noticed in the resulting graph, usually in
the region between the 20th fraction and the 30th fraction,
and often other peaks will be observed, e.g., one in the 40's,
one in the 70's, one in the 90ls, and one in the region
between 110 and 120. The fractions which make up each peak,
e.g., about 2 or 3 fractions in the 20's, are pooled toge-ther
for further treatment. Each such pool is thereafter treated
separately, in the following manner.
PARTITION CHROMATOGRAPHY
METHOD A
The pool of fractions exhibiting high anti-S pro-
tein factor activity is flash evaporated on a rotary evapora-
tor to about 5-10 ml. The resultant concentrate is then sub-
jected to curtain electrophoresis using a Beckman Spinco Model
CP, curtain electrophoresis apparatus~ 20 Liters of an
electrolyte solution is prepared by mixing 22.4 ml. of 0.5
molar aqueous solution of KH2PO4 with 259.2 ml. of 0.5 molar
aqueous solution of Na2HPO4, diluting the mixture to 20
-~2-
~ ~L $ ~
liters with distilled wa-ter, and adjus-ting the pH of the
resulting solution -to 8.0 by the addition of either phosphoric
acid or sodium hydroxide, whichever is required. 50 Milli-
amperes of 950 volt direct current :is employed. Only about
5 liters of the electrolyte solution is used for each run.
The electrophoretically fractionated solution of anti-S
protein factor plus impurities is collected from the electro-
phoresis apparatus in 32 fractions, each having a volume of
about 15 ml. Each of these fractions is assayed for anti-S
protein factor activity by the tryptophan uptake method. The
fractions having the greatest activity are retained and pooled
together. This is often the 8th through the 12th fractions ,,
taken from the apparatus.
The pool of the most active fractions is subjected
to flash evaporation on a rotary evaporator to reduce its
volume to about 1-5 ml. The concentrated solution, which now
contains as little as about 5 milligrams of polypeptide mater-
ial, is then subjected to separation using columns of a
molecular sieve prepared as follows:
Molecular Sieve Columns
Two glass columns are used in series, each column
being 20Q centimeters high and 0.81 centimeter in
internal diameter. ~oth columns are packed with
"Sephadex G-15 fine" brand of molecular sieve
material supplied by Pharmacia Fine Chemicals
of Piscataway, New Jersey. This material is a
particulate dextran having a particle size range
of 4Q-120 microns.
The anti-S protein factor-containing solution is
passed through the two columns of "Sephadex" using 0.02
-43-
molar acetic acid as the eluant. The eluate is collec-ted in
two-milliliter fractions, numbering about 120 in all. F,ach
of these fractions is assayed by the tryptophan uptake method
for anti-S protein factor activity and is also analyzed for
total polypeptide content by UV absorption, using a wavelength
of 220 nanometers. Enough of the most active fractions (in
-terms of anti-S protein factor activity) are pooled toyether
to provide a total polypeptide eontent in the range of about
60 micrograms to 1.5 milligrams. The resulting solution is
then subjected to flash evaporation on a rotary evaporator to
reduce its volume to about 1-3 ml.
The eoncentrated solution of anti-S protein factor
plus impurities is then subjected to ion exchange ehroma-
tography using the following eolumn:
Second Sulphonated Resin Column
The glass column is 200 centimeters high and has
an internal diameter of 0.81 eentimeter. The
paeking is the "Aminex 50-WX2" brand of eation
exehange resin hereinbefore described. The packing
is plaeed in the column and equilibrated with 0.2
normal sodium citrate buffer solution (pH 3.0)
and the system is then brought to a temperature of
35C.
The solution to be chromatographed is placed on the
column and the column is then eluted using a 2 flask gradient
elution system in which flask A contains 1 liter of 0.2 normal
sodium citrate buffer solution (pH 3.0) and Elask B eontains
250 ml. of a buffer solution that has been prepared as follows:
A solution is made of 14.6 grams eitric aeid, 20.6 grams sodium
30 acetate, 3.1 ml. of glacial acetic acid and 800 ml. of distilled
--D,a~--
2.~
water; the pH of the resulting solu-tion is adjusted to 4.0 by
the addition of concentrated hydrochloric acid; and the volume
of the solution is then brought to 1 liter by the addition of
distilled water.
The eluate from the column is collected in 5 ml.
fractions at a rate of about 4 to 5 fractions per hour. About
130 to 150 such fractions are collected in all. Each fraction
is assayed for anti S protein factor activity by the trypto-
phan uptake method. The fraction or fract:ions having the
1~ highest activity constitute solutions of anti-S protein factor
that are relatively pure, i.e. are devoid of most of the other
peptide material, cellular material, etc. tha-t accompanied the
factor when it was extracted from the beef hypothalamus in the
tris citrate buffer solution. The fractions having the great-
est concentration of anti-S protein factor are often found in
the range of fraction numbers 80 to 86. Amino analysis of
these fractions shows the factor to be a polypeptide consti-
tuting some combination of some of the following amino acids:
glutamic acid, threonine, valine, leucine, phenylalanine,
tyrosine, aspartic acid, serine, and glycine. Statistical
evaluation of the amino acid analysis data, coupled with enzyme
studies, indicates that the anti-S protein factor contains the
tripeptide L-threonyl-L-valyl-L-leucine.
MET~OD B
Alternatively, each pool from the foregoing described
fractionation and assay by the tryptophan uptake method, may be
treated separatel~v and purified by reverse phase partition ~ ;
chromatography. The reverse phase partition chromatography is
conducted using a Waters Associates Model 660 Solvent Pro-
grammer and a Waters Associates Model 6000 Solvent Delivery
-45-
System in conjunction with a 6 or 10 foot by 3/8 inch Phenyl
Porasil B Column obtainable from Waters Associates. The column
is housed in a cons-tant tempera-ture compartment made of styro-
foam blocks surrounding a Haake Type FE constan-t temperature
bath. Introduction of samples to the column is effected by a
Waters Associates Model U6K Universal Injector (1 microli-ter to
10 ml.), and the effluent from the column is collected with a
Scientific Manufacturing Industries 1205 Fraction Collector.
The effluent from column is monitored with a Beckman Model 25
Ultraviolet Spectrophotometer equipped with a Waters Assoeiates
LC-25 mieroeell assembly and with a Waters Assoeiates ~-401
Differential Refraetometer.
For each pool from the sulphonated resin eolumn, the
fraetion is dried and then dissolved in 5-10 ml. of solvent and
injeeted on the eolumn. The elution from the eolumn is monitored
for refraetive index as well as absorbanee at 225 nanometers.
As peaks are eluted, the flow from the eolumn is stopped and a
sample is seanned for UV absorbanee from 350 to 210 nanometers.
Fraetions of 5 milliliters eaeh are eolleeted and homogeneous
peaks are pooled. The fraetion, or pools, are taken to dryness
and submitted for amino acid analysis and for tryptophan uptake
analysis to determine the peaks having inhibition aetivity.
The fraetion eontaining the L~threonyl L-valyl-L-
leueine may be N-aeylated and/or esterified in accordance by
substantially the same procedures set forth in Examples 10, 15
and 16~
As indicated above, research concerning schizophrenia
has led to -the isolation of an ~ 2-globulin (alpha-helical S-
protein? from plasma of sehizophrenic patients which has
aetivities different than similar ~-2-globulin (random S-
protein) obtained from plasma of normal individuals. See
Frohman, et al., Recent Advanees in Biological Psyehiatry,
-46-
supra. The alpha-helical S-protein isolated from schiæophrenic
pa-tients, when, for instance, administered to rats provides
observable psychological responses in the rats. Caldwell, et
al., in siological Psychiatry, s_prcl, indicate that administra-
tion of the alpha-helical S~protein to rats reduces self-
stimulation for pleasure of the rats. Employing the procedures
established by Caldwell, et al., various peptides of this
invention are evaluated to determine their ability to reverse
the effect of alpha-helical S-protejn. In this manner the
activity of components of this invention for alleviating the
symptoms of schizophrenia in humans is illustrated. It has
also been found that peptides which counteract the activity of
the alpha-helical S-protein exhibit tryptophan uptake inhi-
bition, and, as noted in Example 17, the tryptophan uptake
analysis can be employed to assist in isolating peptide
fractions which are active against the alpha-helical S-protein.
E~AMPLE 18
The procedure for intracranial self-stimulation
in rats described at pages 237 to 239 of Caldwell, et al.~
Biological Psychiatry, supra, is essentially repeated except as
indicated helow. The rats having the electrodes for stimulation
planted in the median forebrain bundle are tested to determine
the number of bar presses to be expected from the animals in a
given period of time. The alpha-helical S-protein is inter-
cisternally administered, and the number of bar presses is
observed to drop to about 77 percent of the previous amount.
Various polypeptides are intramuscularly administered to the
rats in an amount of 0.2 mgfkg of body weight to determine
whether the polypeptides reverse the effect of the alpha-
helical S-protein. Tryptophan uptakes are also conducted for
-47-
~
Qi~5~
the tes-ted polypeptides. The tryptophan uptake inhibition is
determined at -lO minutes. The results of the tests are provided
in the followin~ table.
Peptide Tryptophan Uptake~ar Presses
Administered Inhibition %Restoration %
Threonylvalylleucine 19.8 85
N-Acetyl-threonylvalylleucine 20.2 100
Glycylthreonylvalylleucine 24.3 100
Phenylalanylprolylthreonyl-
valylleucylprolyl phenyla-
lanine 39.5 100
10 N-Acetyl-threonylvalylleucin-
amide 56.5 100
Threonylvalylleucinamide 21.6 90
Threonylvalylisoleucine 5.6 41
The following polypeptides are tested for tryptophan
uptake inhibition. The tryptophan uptake inhibition is
determined at lO minutes.
Peptide Tryptophan Uptake ~-
Administered Inhibition %
N- ~-Acetyl-Threonylvaly-
leucine, methyl ester 24.2
N- ~-Acetyl-tyrosylthreonyl-
valylleucine 21.3
20 Prolylthreonylvalylleucyl-
prolylgylcine 44.2
The Eollowing polypeptides are tested for tryptophan
uptake inhibition at times of 10, 15 and 30 minutes.
Tryptophan Uptake Inhibition %
Peptide
Administered Time: lO min.15 min. 30 min.
D-Alanyl-L-threonyl-
L-valyl-L-leucine 15 16 0
L-Threonyl-L-valyl-D-
leucine 12 18 21
The foregoing data illustrate the use of D configuration-
-48-
- . ' ~ '
,: ,, . . ~
containing aminoacid moieties to inhibit tryptophan uptake. As
demonstrated with D-alanyl-l-threonyl-L-valyl-l,-leucine, af-ter a
period of time tl~e inhibition of tryp-tophan uptake may be
diminished. However, the essential polypeptide having the T-V-L
structure exhibited an enhanced duration of activity when the
leucine moiety was in the D configuration.
The following polypeptides are similarly tested but
for comparison purposes. The tryptophan uptake inhibition is
determined at 10 minutes.
10 Peptide Tryptophan Uptake Bar Presses
AdministeredInhibition ~ _ Restoration ~6_
Glycylglycylglycine 0 3
O-Benzyl-threonylvalylleucin-
amide 0 toxic
N- ~-Acetyl-O-benzyl-threonyl-
valylleucinamide 0 toxic
Benzyl-O~benzyl-threonylvalyl-
leucate hydrochloride 0 toxic
L-Threonyl-L-valyl-L-leucyl-
D-alanine 2 not tested
As illustrated in the foregoing data relating to the
testing of L-threonyl-L-valyl-L-leucyl-D-alanine, the presence
on the (L) moiety of the essential T-V-L structure of a sub-
stituent which is relatively difficult to hydrolyze from a
carboxylic function of an aminoacid, renders the peptide without
significant tryptophan uptake activity.
Following essentially the same procedures as described
above, N-acetyl-tyrosylthreonylvalylleucine, prolylthreonyl-
valylleucylprolylglycine are tested to determine their effect
on rats which have been administered the alpha-helical S-
protein. It is found that each of these polypeptide-containing
materials restores the bar pressing activity of the rats. Other
polypeptides exhibiting activity useful for alleviating the
symptoms of schizophrenia include threonylvalylleucylarginine.
_~9_
