Note: Descriptions are shown in the official language in which they were submitted.
11~6~
1 BACKGROUND OF THE INVENTION
The Government has rights in this invention pursuant
to Grant No. AM-14228 awarded by the National Ins-titute of Health.
The invention relates to a method and composition for
treating depression in ~umans by increasing the level of nore
pinephrine in neuronal synapses.
It is well known that the neurotransmitters dopamine
and norepinephrine are derived from dihydroxyphenylalanine (DOPA).
DOPA is, in turn, produced in neurons b~ the enzymatic hydroxy
lation of the amino acid tyrosine. This process is catalyzed ~y
the enzyme tyrosine hydroxylase. The DOPA is decarboxylated to
dopamine by the enzyme aromatic L~amino acid decarboxylase (AAAD)
and norepinephrine is produced from dopamine in neurons that also
contain the enæyme dopamine betahydroxylase. It is also knoh7n
that within this reaction chain, the rate-limiting step is the
conversion of tyrosine to DOPA. For this reason, DOPA has been
administered to patients who suffer medical disability resulting
from dopamine deficiency in diseases such as Parkinson's Disease.
Unfortunately, DOPA, when adminstered, is taken up by cells
throughout the body and converted to dopamine and this interferes
with the normal metabolic processes in these other cells. In
addition, DOPA interferes with the body's normal storage of the
neuro transmitter serotonin, and lowers brain levels of the com-
pound S-adenosylmethionine. It is believed that these effects
contri~ute to such unwanted side~effects as the "On~Off Phen-
omenon" and, in some patients, psychotic symptons. Other types
of drugs that act by increasing dopamine and norepinephrine levels
in synapses include the Monoamine Oxidase Inhi~itors ~which slow
the destruction of these neurotransmitters~ and the tricyclic
antidepressants; these compounds, which are used in treating
-1~ ~
1 diseases like depression, also relatively non-specific - pro-
ducing many chemical effects besides increasing synaptic dop-
amine and norepinephrine levels and thus have a range of unwanted
side-effects such as the dangerous increases in blood pressure
that occur when people receiving monoamine oxidase inhibitors
eat certian foods.
Other diseases appear to be cuased by the presence
of excessive quantities of dopamine or norepinephrine within
synapses including psychosis (too much dopamine~, and hyperten
sion and cardiac arrhythmias (too much norepinephrine released
from synpathetic neurons~. These diseases now usually are
treated by drugs that block the interactions of dopamine or nore-
pinephrine with their post-synaptic receptors, such as pheno-
thiazines or butyrophenones. However, these agents all exhibit
some non-specific actions as well, and thus cause side-effects.
Prior attempts to increase or decrease the levels of
dopamine or norepinephrine ~y modifying neuronal tyrosine levels
had been deemed unsuccessful because the total amounts of these
compounds in brains and tissues were not noted to change. It
~O was first observed in ~urtman et al, CScience 185:183-184, July
12, 1974) that increases in brain DOPA concentrations, which,
under the conditions of the experiments, varied in proportion to
the rates at which dopamine and norepinephrine were being syn-
thesized could be obtained by increasing brain tyrosine concen-
trations, and that decreases in brain DOPA concentrations could
be produced by giving rats treatments that decreased brain
tyrosine. An example of a treatment that increased ~rain tyro-
sine was the administration of tyrosine itself; an example of
a treatment that decreased brain tyrosine was the administration
of one of the other neutral amino acids, e.g., leucine, that
1 competes with plasma tyrosine for uptake into the brain. Prior
to that disclosure, it had been belleved that the rate-limiting
enzyme, tyrosine hydroxylase, was so saturated with tyrosine,
that increases or decreased in brain tyrosine levels would not
affect tyrosine's conversion to DOPA, In neither the above
Wurtman et al. article nor a su~sequent paper by Gibson and
Wurtman (~iochem, Pharmacology, 26:1137-1142, June 1977~ was it
actually shown that such changes in DOPA accumulation were
accompanied by changes in brain dopamine or norewinephrine
levels. Furthermore, in neither was it shown that changing
brain tyrosinelevels had any effect on the amounts of dopamine
or norepinephrine released into synapses.
It would be highly desirable to provide a means for
increasing the amounts of norepinephrine that actually are pre-
sent with synapses. Such changes in synaptic transmitter levels
need not be associatesd with changes in the total amount of
norepinephrine present in the brain or other tissues, inasmuch
as it is now well known that not all of the molecules of the
transmitters that are stored in neurons are equally accessible
for release into synapses. Furthermore, it would be desirable
to provide such a means which is biochemically specific and
which lacks the undesirable side effects associated with admin-
istration of DOPA, the MAO inhibitors~ the phenothiazines, and
the other drugs described above~ Such a means might be itself
be therapeutic in the treatment of depression, Alternativeiy,
it could be used in combination with drugs now used to treat
depression to amplify their therapeutic effects.
SUMMARY OF THE INVENTION
The present invention provides a method for treating
depression associated with a deficiency of norepinephrine in
1 ds 8
synapses. This invention is ~ased upon the discovery that treat-
ments that increase neuronal tyrosine levels can also cause
corresponding increases in the amounts of norepinephrine released
into synapses, The tyrosine, and its precursor, phenylalanine,
can be administered alone or in admixture, with other neutral
amino acids with or without drugs, in order to raise or lower
brain tyrosine ~and phenylalanine~ levels, and thereby to treat
depression associated with deficiency of norepinephrine in
synapses. sy varying the proportion of tryptophan, another amino
acid, in the mixture, the synthesis and synaptic release of
sero-tonin, another brain neurotransmitter, can similarly be
controlled. Increased synaptic norepinephrine levels are obtain-
ed ~y giving tyrosine regardless of whether the norepinephrine-
releasing neurons are or are not especially active. Decreases
in norepinephrine release into synapses can be obtained by lower-
ing brain tyrosine levels by administering neutral amino acid
compositions low in tyrosine levels~ Decreases in serotonin
release can similarly be obtained by lowering brain tryptophan
levels. By regulating the proportion of tyrosine in ~iven mix-
ture of neutral amino acids, it can be caused to increase or
decrease norepinephrine release, Phenylalanine can, in lowdoses,be used in place of tyrosine. Tryptophan's proportion in
the neutral amino acid mixture can be used to regulate serotonin's
release into synapses while regulating norepinephrine release
as descri~ed herein~
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
In accordance with this invention, tyrosine and/or
phenylalanine and/or other neutral amino acids is administered
to a patient either alone or in combination with one of more
drugs there~y to increase the level of norepinephrine which is
1 released into synapses. Serotonin release also can be controlled
at the same time by varying the proportion of tryptophan present
in the amino acid mixture. Release of norepinephrine or sero-
tonin into synapses can be varied using amino acid mixtures
whether or not the norepinephrine releasing or serotonin-releas-
ing neurons are especially active. Similarly, decrease in nore-
pinephrine release can be produced by administering amino acid
mixtures that compete with tyrosine for uptake for the brain
thereby decreasing brain tyrosine levels~
The composition of the amino acid mixture that i5
utilized depends upon the nature of the illness in the atient
that is to be treated. When there is need to increase nore-
pinephrine release without increasing that of serotonin, tyrosine
(and/or phenylalanine) is administered, with or without other
amino acids not including serotonin's precursor, tryptophan, in
doses ranging between 5 mg/kg and 200 mg/kg. ~his therapy is
useful, alone or as an adjunct to drug therapies, in treating
certian types of depression. In some situations, phenylalanine
can be used as a substitute for tyrosine, inasmuch as much of
this amino acid is converted to tyrosine in the liver, and re-
leased into the blood stream for uptake into the brain. However,
plasma phenylalanine levels should be less than ahout double
those of tyrosine, since at the higher levels, phenylalanine
competes with tyrosine for uptake into the brain, and can inhib-
it the enzyme tyrosine hydroxylase.
In some instances, it may be desirable to treat de-
pression by also increasing brain serotonin levels while increas-
ing norepinephrine release since it appears that increasing brain
serotonin levels tend to reduce depression. In these instances,
the compositions administered also contain tryptophan in addition
1 to tyrosine and/orphenylalanine and other neutral amino acids.
Other neutral amino acids than these compositions can contain
include the branched-chain amino acids ~leucine, isoleucine,
valine), as well as methionine, threonine, and histidine. The
amino acids can be supplied as monomers or as natural or syn-
thetic polymers, e.g., peptides. The phenylalanine, tryptophan
`and tyrosine will be referred to collectively as "the useful
amino acids",
The ratios of the plasma concentrations of tyrosine,
phenylalanine and tryptophan to the sum of the other neutral
amino acids are normally about Q.Q8-Q.12, 0~Q7~0.12 and 0,06-0.14
respectively, depending on the composition of the diet. In some
diseases, e.g., cirrhosis of the liver leading to coma; diabetes;
hyperinsulinism; such catabolic states as starvation, cachexia,
disseminated cancer, or severe burns or trauma~ these ratios
are abnormal, causing changes in brain dopamine, norepinephrine
and serotonin release. The particular compositions used in
these situations are desinged to restore the plasma ratios to
normal. In the primarily neurologic or psychiatric diseases
listed above~ the goal of amino acid therapy is to raise or lower
these ratios above or below their normal ranges, in order to in
crease or decrease the release of norepinephrine ~or serotonin)
into synapses.
The tyrosine, phenylalanine and other neutral amino
acids can be administered as free amino acids, esters, salts,
natural or synthetic polymers, or as constituents of foods. The
route of administration can be oral or parenteral, e,g~, intra
venous.
The following examples illustrate the present invention
and are not intended to limit the same.
lL ~
1 X~IPLE
This example illus-trates that brain norepinephrine
can be synthesized by increasing brain tyrosine levels~
This example shows that the rate at which 3-methoxy-
4-hydroxy-phenylethyleneglycol~sulfate (MOPEG-S04), the major
brain metabolite of norepinephrine, accumulates in rat brain
also varies as a function of brain tyrosine levels. This shows
that brain tyrosine levels affect not only the synthesis, but
also the turnover and release of brain norepinephrine.
Male Sprague-Dawley rats ~Charles River Breeding Lab-
oratories, Wilmington, MAl weighing 150 g were housed in hanging
cages (6-8 per cage), given ad libitum access to tap water and a
26% protein diet (Charles River Rat-Mouse ~Hamster Maintenance
Formula 24RF), and maintained under light (300 microwatts/cm2;
Vita-Lite, Duro-Test Corp., North Bergen, M,J,) between 8 AM and
8 PM daily. Rats used for diet experiments were fasted over~
nigh-t and then allowed to consume the experimental diet starting
at 10 AM. Diets of different compositions were prepared in agar
gel ~35 g/lQO ml of water) as described by Gibson et al., Bio-
chem. Pharmacol., 26, 1137-1142 (1977). All amino acids and
drug~ were injected intraperitoneally.
Norepinephrine synthesis and turnover in brain neurons
were estimated by measuring the rate of accumulation of MOPEG-S04
after probenecid administration of exposure to a cold environ-
ment. The MOPEG-S04 in brain homogenates was isolated using an
anion exchange column (A-25 DEAE Sephadex*; Pharmacia, Piscataway,
N.J.); the method used was basically that of Meek and Neff~ Br,
J. Pharmacol., 45, 435-441 (1972), but modified to allow both
tyrosine and MOPEG-S04 to be measured in the same sample, An
aliquot of each homogenate ~in 0.15 M ZnS04) was first assayed
*Trade Mark _7~
~"
l~S~48
1 for tyrosine by the method of h~aalkes and Uden-friend, J Lab.
~lin, Med., 50, 733-736 ~1957). An equal volume of 0 15 M bar-
ium hydroxide was then added to the remaining homogenate, which
was rehomogenized (Polytron, Brinkman Instruments, N. Y ),
centrifuged and assayed for MOPEG-S04 by the method of Meek and
Neff above. Recoveries of MOPEG-S04 and tyrosine from whole
brain homogenates were 70-75% and 85~95%, respectively
Tyrosine (Grand Island Biological Co , Long Island,
N. Y.~ and probenecid (Sigma Chemical Co,, St. Louis, MO~, w~ich
are poorly soluble in water, were dissolved in dilute NaOH; the
solutions were then buffered to pH 7.4 with hydrochloric acid
and brought to a known volume with saline~ This yielded a fine
suspension that was suitable for injection.
In experiments onstress produced by exposure to cold,
animals received the more soluble ethyl-ester form of tyrosine
(J.T. Baker, Phillipsburg, N.J.), instead of tyrosine itself,
to raise brain tyrosine levels. Data were analyzed ~y one-way
or two-way analysis of variance
Probenecid treatment significantly raised the MOPEG-S04
level in brain from 123 ng/g indiluent-injected controls to 175
ng/g in probenecid-treated animals (P ~ 0.0011 ~Table 1). Tyro-
sine administration alone had no effect-on brain MOPEG-S04; how-
ever, pretreatment with this amino acid significantly enhanced
the probenecid-induced rise in MOPEG-S04 Cto 203 ng/g, as com-
pared ~ith 175 ng/kg in rats receiving probenecid alone ( P C
0.01; Table 1),
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1 TABLE
Accumulation of MOPEG-SC4 after Probenecid Administration and
Pretreatment with tyrosine
-
Brain Tyrosine Level Brain ~IOPEG-S04 Level
(~g/g) (ng/g)
Pretreatment Diluent Probenecid Diluent Probenecid
~ .
Diluent 13.2 + 0.5 15.7 + 0.7 123 + 6 175 + 6
Tyrosine 23.3 + 1.5 24.7 ~ 1.3 127 + 2 203 + 8
Note: In each of 3 experiments, groups of 4-6 rats
were injected with either a dose of tyrosine ~lOQ mg/kg, i.p 3
known to accelerate brain dopa synthesis or its diluent and,
30 min. later, with probenecid ~400 mg/kg, i p.) or its diluent.
Animals were }iilled 6~ min. after the second injection, and
their whole brains were analyzed for tyrosine and MOPEG~S04
Tyrosine administration significantly raised brain tyrosine levels
~P + 0.001), whereas probenecid failed to modify brain tyrosine
or its response to exogenous tyrosine. Probenecid significantly
raised brain MOPEG-S04 ~P + 0 . 0011, and tyrosine pretreatment
significantly enhanced this response (P + 0.01). Data were
analyzed by two-way analyis of variance, Values are expressed as
means + SEM.
Placing the rats in a cold environment (.4CL increases
norepinephrine turnover; this accelerates the formation of bo~h
nore inephrine itself and its metabolite, MOPEG~S04, in brain
neurons. The rats were exposed to cold to determine whether
treatments that changed brain tyrosine levels could influence the
rate at which the brain accumulates MOPEG~S04 in rats exposed to
cold stress and not given probenecid ~Fig. 1~,
_g_
~S~4~
1 Exposure to cold for 1 hour increased brain MOPEG-SO4
levels by about 40% (from 80 ng/g to 114 ng/g; P < 0.012. In
animals treated with either of the amino acids or with saline,
brain tyrosine levels parallel, and were significantly corre-
lated with, those of MOPEG-SO4 ~r = .77, P < 005; Fig. 1). Pre-
treatment with tyrosine raised brain tyrosine levels by about
80% (from 13.3 ~g/g, in saline-injected animals, to 24.6 ~g/g;
P ~ 0.012 and those MOPEG-S04 by 70~ ~from 114 ng/g to 193~ng/g;
P < 0.012. Pretreatment with valine failed, in this study, to
cause significant alterations in brain tyrosine or MOPEG-SO4
levels (14.3 ~g/g and 117 ng/g, respectively2; however, brain
tyrosine and MOPEG-SO4 levels were also significantly correlated
in these animals, as in ot~er experimental groups (Fig, 1)~
The relationship sllown in Fig. 1 was obtained as
follows: Groups of rats were injected intraperitoneally with
valine C200 mg/};g~, an amino acid that competes with tyrosine for
uptake into the ~rain (81, or with tyrosine (125 mg/kg of the
ethyl esterl or saline; 3Q min. later they were placed in single
cages in a cold (4C) environment. After 1 hour, all animals
were killed, and their whole brains were analyzed for tyrosine
and MOPEG-SO4. Control animals were injected with saline and
left at room temperature C22C2, also in single cages, for 90
min. Each point represents the tyrosine and MOPEG-SO4 levels
present in a single brain. Data were pooled from several exper-
iments. Brain tyrosine and MOPEG-SO4 levels in animals kept at
room temperature were 14.6 ~g/g and 80 ng/g, respectively. In
Fig. 1, these symbols are as follows: closed circles, animals
pretreated with valine; open circles, animals pretreated with
saline; closed squares, animals pretreated with tyrosine.
To determine whether physiologic variations in ~rain
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1 tyrosine level might also influence brain norepinephrine syn-
thesis and turnover (as estimated by measuring ~IOPEG-S04 levels),
the accumultion of this metabolite in animals exposed to a cold
environment was examined after being allowed to consume a single
meal that would be likely to elevate tyrosine levels
Animals that had been fasted overnight were given
access to either a protein-free (0% caseinl or a 40% casein meal
between 10 AM and 11 ~; they were then placed in the cold (4C)
for 1 hour, after which they were killed, and their brains
analyzed for tyrosine and MOPEG-S04. Fasted control animals
remained at room temperature ~22C~ during this 2 hour period.
Exposure to cold accelerated the accumulation of MOPE~-
S04 in brains of fasted rats, from 123 ng/g ~in fasted control
animals kept at 22C~ to 163 ng/g (P ~ 0 05~; this treatment had
no effect on brain tyrosine levels (10.1 ~g/g vs, 10.5 g/g).
Among animals placed in the cold, consumption of either a 0% or
a 40% casein meal enhanced brain MOPEG-S04 accumulates by 40-50%
~Table II; P ~ 0 01). The 0% casein meal increased brain tyro-
sine by about 40% (P < Q.Ql) r ~hereas the 4Q% casein meal increas--
ed brain tyrosine by 77% (P < 0.01~.
~hen the consumption of a protein-free meal failed to
elevate brain tyrosine levels, brain MOPEG-S04 levels also failed
to rise (Table II~. Among protein-fed animals in this study,
the brain tyrosine level increased by about 50% (from 13.4 to
19.5 ~g/g, P ~ 0.01), and brain MOPEG-S04 rose in parallel.
These data's show that treatments that increased brain
tyrosine levels can accelerate the accumultion of the norepine-
phrine metabolite MOPEG-S04 in the brains of rats pretreated
with probenecid or exposed to a cold environment. Such treatments
can be pharmacologic (i.e. intraperitoneal injection of tyrosine)
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11~6t4~
1 or physiologic ~i.e., consumption of a high-protein meal). They
are compatible with the high Km of tyrosine hydoxylase for its
substrate, relative to brain tyrosine concentrations. The enzyme
is especially vulnerable to substrate limitation when it has
been activated, inasmuch as activation selectively enhances its
affinity for its cofactor.
MOPEG-S04 is the major metabolite of norepinep~rine
formed inrat brain and it is transported out of the ~rain by a
probenecid-sensitive mechanism. After probenecid administration,
MOPEG-S04 accumulates at a linear rate in rat brain for at least
60 min. Since brain norepinephrine levels remain constant during
this interval, the rate of MOPEG-S04 accumulation provides a
useful index of the rate of norepinephrine synthesis. This rate
apparentl~ is lower in unstressed, prohenecid-treated rats than
in animals placed in the cold (Tables I and II~, however, in
both circumstances, it is dependent on brain tyrosine levels.
TABLE II
Brain MOPEG-S04 Accumulation after Ingestion of a Single Protein-
free or 40% Protein Diet among Rats Placed in a Cold Environment
Treatment Tyrosine MOPEG-S04
(~g/g) (ng/g~
- ,
EXPERIMENT I
Fasted 10.5 + 0.55 163 + 9
Protein-free
(0% Casein) 14.4 + 0.24* 239 + 17*
40% Cas~in 18.1 + 0,85* 228 + 9*
EXPERIMENT II
Fasted 13,4 + 0.67 195 + 9
Protein-free
(0% Casein) 13.3 + Q.81 182 + 18
40% Casein 19.5 f 1.03* 264 + 20*
1 T~LE II cont.
* Values are significantly different from correspond-
ing fasted group (P ~ 0.01),
Values are significantly differellt from corresponding
protein-fee group (P < 0.01).
Note: Groups of 4-6 rats were fasted overnight and
then allowed access to one of the tests diets at lQ AM. At
11 AM r animals were placed in an environmental chamber at 4C
for 1 hour. They were killed at noon, and their whole brains
were analyzed for tyrosine and MOPEG-S04. Animals given protein-
free and 40% protein diets consumed 9,7 and lQ,5 g~ respectively,
in Experiment I, and 6.2 and 8,0 g in Experiment II. Data -
presented as means + SEM.
EXAMPLE II
This example illustrates that the administration of
tyrosine to a patient suffering from depression significantly
alleviates the depression~
A female middle-agedpatient who has histor~ of depres-
sion was alternately administered a placebo for three consecu-
tive weeks ~ollowed by the administration of tyrosine ~or threeconsecutive wee~s at a daily dosage of 3.6 grams per day. This
schedule was repeated three times, The patient was tested for
depression levels periodically during each three week period by
~amilton Depression Scores ~the higher the score the greater the
depression), The results are shown in Table III.
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1 TABLE III
Range of Hamilton
Comoosition Score
Placeho 25
Tyrosine C3-6 g/day~ 1-9
Place~o 24
Tyrosine (3-6 g/day~ 13
Place~o 28
10 Tyrosine C3-6 g/day~ 1-5
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