Synthesis and use of L-.gamma.-glutamyl-DOPA

The synthesis of L-.gamma.-glutamyl-DOPA (L-.gamma.-glutamyl-L-3,4-dihydroxy-phenylalanine) and its use selectively to increase renal blood flow in mammals.

BACKGROUND OF THE INVENTION 
At the present time, there are drugs available which affect the metabolism 
of kidney cells, influence renal blood flow, alter water and electrolyte 
excretion and other functions of the kidney. A serious drawback of some of 
these drugs is that they exert their pharmacological activity on parts of 
the system other than the kidney and thereby cause toxicities and 
undesirable systemic side effects such as acceleration in heart rate, and 
elevation of blood pressure. 
Such problematic systemic effects are often reported in the pharmacology of 
dopamine (Formula I) and its metabolic precursor L-DOPA (Formula II). 
##STR1## 
L-DOPA is a compound that is metabolized in the body of mammals, including 
man, by aromatic amino acid decarboxylase to dopamine (Christenson et al., 
Arch. Biochem. Biophys. 141: 356, 1970). Dopamine is a naturally occuring 
catecholamine considered to be a precursor of norepinephrine. Dopamine 
plays an important role as a neurotransmitter in the central nervous 
system and as an inhibitory transmitter in autonomic ganglia. The ability 
of dopamine to interact with .alpha. and .beta. receptors in the body 
often leads to undesirable side effects such as changes in systemic 
hemodynamics and increases in cardiac output and gangrene. U.S. Pat. No. 
3,903,147 and U.S Pat. No. 3,947,590 disclose that this problem is 
encountered when dopamine is used to increase renal blood flow. U.S. Pat. 
No. 3,769,424 discloses similar systemic problems when L-DOPA is used as 
an anti-Parkinson drug to treat disturbances in the brain. 
U.S. Pat. No. 3,903,147 and U.S. Pat. No. 3,947,590 disclose the use of a 
dopamine derivative, .gamma.-glutamyl-dopamine to increase renal blood 
flow in the kidney without desirable systemic hemodynamic changes. 
Although these patents disclose the fact that .gamma.-glutamyl-dopamine is 
a specific renal vasodilator it does not disclose the mechanism by which 
that effect was achieved. 
According to the method of this invention, the .gamma.-glutamyl derivative 
of DOPA 
##STR2## 
is used as a specific renal vasodilator.

DETAILED DESCRIPTION OF THE INVENTION 
Due to the high concentration of the enzyme .gamma.-glutamyl transpeptidase 
in the kidney, .gamma.-glutamyl-DOPA is kidney specific, and the compound 
is preferentially concentrated in the kidney by the action of the enzyme. 
When .gamma.-glutamyl-DOPA is administered to experimental mice, DOPA is 
released from its glutamyl linkage in the kidney by the action of 
.gamma.-glutamyl transpeptidase and immediately undergoes enzymatic 
degradation to dopamine by the action of aromatic amino acid 
decarboxylase. Since the dopamine is released by enzyme action in the 
kidney where the pharmacological action is needed, the systemic side 
effects are minimized. 
.gamma.-Glutamyl transpeptidase is an enzyme which is capable of 
hydrolysing the .gamma.-glutamyl bond of .gamma.-glutamyl peptides as 
follows: 
.gamma.-glutamyl-DOPA + H.sub.2 O .fwdarw. glutamate + DOPA 
the high concentration of .gamma.-glutamyl transpeptidase in the kidney is 
shown as follows. If the activity of the enzyme in human kidney is taken 
as 100, the relative activities in other tissues may be expressed as 8.3 
for the pancreas, 3.9 for the liver, 1.5 for the spleen, 0.95 for 
intestine, 0.5 for the brain, 0.31 for the lung, 0.045 for the heart 
muscle and 0.067 for skeletal muscle (Orlowski and Szewczuk, Acta Biochem. 
Polon. 8:189, 1961; Orlowski, Arch. Immun. Therap. Exptl. 11:1, 1963). 
Experiments were conducted in which mice were tested to measure the 
distribution of dopamine in the kidney after administration of 
.gamma.-glutamyl-DOPA to verify that the L-DOPA that was being released in 
the kidney was in fact remaining in the kidney and being converted to 
active dopamine. The following procedure was used to determine the 
dopamine content of the mouse tissue: Male Swiss-Albino mice weighing 
20-25 grams received an intraperitoneal injection of .gamma.-glutamyl-DOPA 
(0.5 micromole/g) dissolved in 0.15 M NaCl. Twenty minutes after the 
administration of the drug the animals were decapitated and the kidney, 
liver, heart, brain, lung, duodenum-pancreas, spleen, and muscle were 
removed for analysis of dopamine. The tissues were homogenized in five 
volumes of cold 1 N HCl, cnetrifuged and an aliquot removed and analysed 
for dopamine by gas chromatography. 
It was determined that the dopamine formed from .gamma.-glutamyl-DOPA 
reached the kidney in approximately the same time as when L-DOPA was 
administered. A peak level of dopamine was observed 10 minutes after 
intraperitoneal administration (0.5 .mu.mole/g) of either drug (Table 1). 
TABLE 1 
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Dopamine (.mu.g/g) .+-. Standard Error 
After After 
Time (min) .gamma.-glutamyl-DOPA 
L-DOPA 
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10 66.9 .+-. 14.9 23.2 .+-. 1.9 
20 64.5 .+-. 7.4 13.9 .+-. 1.6 
60 9.9 .+-. 2.3 2.6 .+-. 0.6 
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Tissue levels of dopamine after intraperitoneal administration of 
.gamma.-glutamyl-DOPA were compared to those found 20 minutes after 
intraperitoneal administration of 0.5 .mu.mole/g L-DOPA. The data in Table 
2 show that the level of dopamine in the kidneys following 
.gamma.-glutamyl DOPA was considerably higher than when the compound was 
administered as L-DOPA. In addition, it can be seen, with the exception of 
the spleen, that when .gamma.-glutamyl DOPA is administered less dopamine 
is found in organs other than the kidney. This illustrates that the 
.gamma.-glutamyl-DOPA is being selectively concentrated in the kidney. 
TABLE 2 
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Dopamine distribution in tissues 20 minutes after 
intraperitoneal administration of .gamma.-glutamyl DOPA or L-DOPA 
(0.5 .mu.mole/g). 
Dopamine* (.mu.g/g) .+-. Standard Error 
After After 
Tissue .gamma.-glutamyl-DOPA 
L-DOPA 
______________________________________ 
Kidney 64.5 .+-. 7.4 13.9 .+-. 1.6 
Heart 2.5 .+-. 0.5 3.2 .+-. 0.1 
Brain 2.8 .+-. 0.5 2.9 .+-. 0.3 
Liver 0.6 .+-. 0.4 1.4 .+-. 0.2 
Lung 0.6 .+-. 0.2 0.9 .+-. 0.2 
Duodenum-Pancreas 
6.1 .+-. 1.7 12.3 .+-. 2.2 
Spleen 2.3 .+-. 0.8 1.7 .+-. 0.4 
Muscle 0.2 .+-. 0.1 1.0 .+-. 0.2 
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*Each value represents the mean .+-. Standard Error of four 
determinations. 
The ratio of the concentrations of dopamine in the kidney to that of the 
heart can be used as an index of organ specificity. The higher the ratio, 
the more kidney specific the compounds. The kidney/heart ratio was 25.8 
after administration of .gamma.-glutamyl-DOPA, 4.3 after L-DOPA (Table 1), 
and 2.5 after dopamine (in the guinea pig, reported by Halushka and 
Hoffman, J. Pharm. Pharmac. 20:943, 1968). The accumulation of dopamine in 
the kidney was greater when .gamma.-glutamyl-DOPA was administered. This 
results in .gamma.-glutamyl-DOPA's being a more efficient and effective 
form of administering dopamine. 
Dopamine is useful as a vasodilator and because of the kidney specificity 
of .gamma.-glutamyl-DOPA it is useful as a specific renal vasodilator for 
the treatment of congestive heart failure, shock, hypertension, cirrhosis, 
acute renal failure, drug intoxication and edema. It has been found that 
.gamma.-glutamyl-DOPA significantly increases renal blood flow in test 
rats without the unwanted side effects encountered when DOPA and dopamine 
are administered in the free form. In experiments, .gamma.-glutamyl-DOPA 
significantly increased blood flow at doses 40 times lower than the doses 
of .gamma.-glutamyl-DOPA that were found to cause significant increases in 
blood pressure. 
The tests to measure the increase in renal blood flow after administration 
of .gamma.-glutamyl-DOPA were carried out in female Sprague-Dawley rats 
weighing 275-325 grams by determining the clearance of .sup.14 C- p-amino 
hippurate according to the method of Dick and Davies, J. Clin. Path. 2:67, 
1949. Rats were anesthetized with ether and the femoral artery and vein 
cannulated. The bladder was exposed and catheterized with a #8 
polyethylene catheter. The urethra was ligated. An infusing solution of 
0.45% saline-p-amino hippurate (PAH) was prepared by diluting normal 
saline with distilled water and adding 0.025.mu.Ci glycyl-1-.sup.14 
C-PAH/ml infusing solution (specific activity 43 mCi/mmole). The animals 
were infused at a rate of 10 ml/hr using a Sage model 355 syringe pump. 
After a 1 hour equilibration period, urine was collected continuously over 
30 minute intervals. Blood was withdrawn from the femoral artery at the 
midpoint of each 30 minute collection period. A total of 5 collections 
were made. The effect of .gamma.-glutamyl-DOPA on PAH clearance was 
studied by adding known concentrations of the drug to the infusing 
solution. 
The mean plasma flow of 2.54 ml/min/100 g was found in control rats. A dose 
of 5 nmoles/g/30 min .gamma.-glutamyl-DOPA increased the mean plasma flow 
to 4.8 ml/min/100 g. 
In order to determine the side effect, if any, of .gamma.-glutamyl-DOPA on 
mean arterial blood pressure, animal tests were conducted using 40 times 
the amount of .gamma.-glutamyl-DOPA that was needed to achieve 
satisfactory increase in renal blood flow. 
Female Sprague-Dawley rats weighing 275-325 g were anesthetized with sodium 
pentobarbital (50 mg/kg intraperitoneal). The femoral vein and artery were 
cannulated. The arterial catheter was connected to the blood pressure 
inducer and blood pressure was recorded with Grass model 7 polygraph. Upon 
stabilization of arterial blood pressure, the effect of 
.gamma.-glutamyl-DOPA was evaluated by infusing the drug dissolved in 
saline into the femoral vein at a constant rate of 10 ml/hr. Increases in 
systolic pressure of up to 20 mm Hg were observed at a dose of 200 
nmole/g/30 min. The blood pressure elevation was gradual. A peak response 
was obtained 5-17 minutes after initiation of the infusion. These results 
indicate that the dosage needed to increase renal blood flow (5 nmole/gr 
30 min) would not significantly affect arterial blood pressure. 
The compound of this invention can be administered as a solution in 0.15 N 
sodium chloride as an intravenous infusion. The dose can be varied from 5 
.mu.mole (1.7 mg) per kg per 30 min up to 150 .mu.mole (52 mg) per kg per 
30 min. Sterile aqueous or saline solutions of the compound can be 
prepared. 
The compound of this invention may be prepared by the following methods. 
The following examples illustrate one novel method of synthesis which is a 
departure from the general procedure of King & Kidd (J. Chem. Soc. 3315, 
1949) and one typical enzymatic synthesis: 
EXAMPLE 1 
1.97 g of L-DOPA (0.01 mole) was dissolved in 50 ml of 0.5 M Na.sub.2 
CO.sub.3 under nitrogen. The flask was cooled in an ice bath to 
0.degree.-5.degree. C. and 5.2 g (0.02 mole) of phthaloyl-L-glutamic 
anhydride, dissolved in 30 ml of dry dioxane was added dropwise with 
stirring. The mixture was stirred for an additional 20 minutes and then 
acidfied to approximately pH 1.0 by the addition of 6 M HCl. The mixture 
was extracted with several portions of ethyl acetate and the pooled 
extracts were dried with anhydrous, solid sulfate. Ethyl acetate was 
removed from the extract by flash evaporation and the residue was 
dissolved in 50 ml methanol. 3 ml of hydrazine hydrate (99%) were added to 
the methanol solution and the mixture was left for 2 days at 26.degree. C. 
Methanol was then removed by flash evaporation and the residue was 
suspended in 50 ml of water. The suspension was acidified to pH 3.0 by the 
dropwise addition of 1 M HCl. The precipitated white solid (phthaloyl 
hydrazide) was removed by filtration and the filtrate was adjusted back to 
pH 5.0. The solution was then applied at 4.degree. C. to the top of a 
Dowex-1 (acetate) column (2.5 .times. 45 cm). The column was washed with 
100 ml of 0.01 M acetic acid and then eluted with a linear gradient 
established between 2 liters of 0.01 M acetic acid and 2 liters of 2 M 
acetic acid. Fractions of approximately 20 ml were collected. The presence 
in the eluate of ninhydrin-positive material is determined by a spot test 
on Whatman No. 1 filter paper. The product of the reaction emerged from 
the column when approximately 2 liters of the eluent passed through the 
column. The fractions containing .gamma.-glutamyl-DOPA were pooled and 
acetic acid was completely removed by flash evaporation under reduced 
pressure at 37.degree. C. An amorphous white solid was obtained. The yield 
was 1.4 g (43%) L-.gamma.-glutamyl-L-3,4-dihydroxyphenylalanine 
(.gamma.-glutamyl-DOPA). 
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Microanalysis for C.sub.14 O.sub.7 N.sub.2 H.sub.18 . H.sub.2 O 
Calculated % Found % 
______________________________________ 
C 48.84 49.39 
H 5.85 6.02 
N 8.14 7.42 
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EXAMPLE 2 
Enzymatic synthesis of .gamma.-glutamyl-DOPA 
Glutathione and L-DOPA were incubated with a partially purified preparation 
of sheep kidney .gamma.-glutamyl transpeptidase. .gamma.-Glutamyl-DOPA was 
isolated from the incubation mixture by preparative ion exchange 
chromatography on a Dowex-1 (acetate) column. 6.14 of glutathione (0.02 
mole) and 1.97 g of L-DOPA (0.01 mole) were dissolved in 200 ml of 1 M 
Tris (base) solution. 2.5 ml of .gamma.-glutamyl transpeptidase solution 
(100 units/ml) were added and the flask was flushed with nitrogen to 
protect DOPA from oxidation and the mixture was incubated at 37.degree. 
for 6 hours. L-.gamma.-glutamyl-L-3,4-dihydroxyphenylalanine 
(.gamma.-glutamyl-DOPA) was isolated by ion exchange chromatography as in 
Example 1. It emerges from the column when approximately 2 liters of the 
eluent passed through the column. Unreacted. glutathione, free glutamate 
and cysteinylglycine emerge from the column in earlier fractions. 
Both the chemically and enzymatically prepared compounds were identical. A 
singly ninhydrin positive spot with an Rf of 0.32 was obtained on paper 
chromatography (Whatman No. 1. paper) in a solvent system consisting of 
1-butanol-pyridine-water (1:1:1). A single peak was also observed on amino 
acid analysis using the Technicon TSM amino acid auto-analyzer and a 
lithium citrate buffer system. 
Although the compound has been isolated in the form of the free acid, 
addition salts with organic bases can be prepared by conventional 
procedures. For example, an addition salt with the basic amino acid 
arginine has been prepared by reacting equivalent amounts of 
.gamma.-glutamyl-DOPA and arginine base in methyl alcohol and isolating 
the precipitated salt.