Note: Descriptions are shown in the official language in which they were submitted.
2&'~3
The present invention relates to aminopeptidase
inhibitor.
Hypertension is a major factor leading to heart
disease and stroke. Extensive efforts have been made to
understand the etiology of this condition to develop drugs
capable of treating it. The physiological regulation of
blood pressure is in part medicated by the renin-angioten-
sin system. See Freeman, R.H., Davis, J.O., Lohusen, T.E.
and Spielman, W.S., Fed. Proc. 36, 1766 (1977).
.
Among the compounds believed to affect blood pres-
sure in the renin-angiotensin system are three related pep-
tides designated angiotensin I, II and III. Abbreviations
used in describing the amino acid sequences of peptides
are shown in Table I.
TABLE I
Arg = L-arginine
Asp = L-aspartic acid
Gly = glycine
His = L-histidine
Ile = L-isoleucine
Leu = L=leucine
Phe = L-phenylalanine
Pro = L-proline
Tyr = L-tyrosine
Val = L-valine
EDTA = Ethylene diamine tetraacetic acid
Tris = 2-Amino-2-hydroxyethyl-1,3-propanediol
Angiotensin I, hereinafter "I", is a decapeptide
having the sequence AspArgValTyrIleHisProPheHisLeu. By
convention, the reading order of the sequence is from the
N-terminal amino acid on the left to the C-terminal amino
acid on the right. Angiotensin II, hereinafter "II", is
the same structure as above,
.. . ~
~L4~
except lacking the C-terminal HisLeu. Angiotensin III,
hereinafter "III", is des-Aspl angiotensin II, that is,
angiotensin II lacking the N-terminal Asp.
I is converted to II by the action of angiotensin
converting enzyme~ which removes the C-terminal dipeptide by
catalyzing the hydrolysis of the peptide bond between Phe8 and
His of I. III may be formed from I or II by the selective
removal of the N-terminal Asp to form des-Asp angiotensin I or
III. The des-Asp1-angiotensin I may be converted to III by
angiotensin converting enzymeO
Angiotensin I appears to have no effect on blood
pressure. However, angiotensin II is known to have a powerful
blood-pressure elevating effect. Compounds found to be
inhibitors of angiotensin converting enzyme are effective in
lowering blood pressure and thus alleviating hypertension. See
Ondetti, et al., 4,0~6,889, issued September 6, 1977, Cushman et
al., 4,~52,511, issued October 4, 1977, and Ondetti, et al.,
4,053,651, issued October 11, 1977.
The physiological role of angiotensin III is less well
characterized. However, it is a normal constituent of blood,
and it has demonstrable effects of significance in elevating
blood pressure. Very little is known of how III might be formed
_ vivo. In either of the two routes of synthesis from
angiotensin I, described supra, a specific cleavage of the
N-terminal Asp residue is required, either from I or II.
Therefore, an aminopeptidase enzyme specific for an N-terminal
aspartyl peptide (any peptide having an N-terminal aspartic acid
residue) is likely involved in the production of angiotensin
III. Suitable inhibitors of such an en~yme are expected to have
30 anti-hypertensive properties. The inhibitors are also expected
to be useful in the treatment of non-hypertensive edematous
states in which the renin-angiotensin system plays a role.
~3--
~. .
.
11~ .
1 ~ 4 ~3$~3
Aminopeptidases are hydrolytic enzymes which remove an N-terminal
amino acid from a peptide or protein. The substrate specificity is
variable from enzyme to enzyme, for example, the well-known leucîne !
aminopeptidase is relatively non-specific, being capable of sequentially ¦
hydrolyzing an entire peptide. Two aminopeptidases specific for N-terminal
aspartylpeptides have been described. Aminopeptidase A, first isolated by
Glenner, G.G., McMillan, P.J. and Folk, J.E., Nature 194, 867 (1962),
preferentially removes N-terminal dicarboxylic acids. The enzyme is
activated by Ca~+ and inhibited by EDTA. A second enzyme, called aspartate
aminopeptidase~ has been described by Cheung, H.S. and Cushman, D.W.,
Biochim. _iophys. Acta 242, 190 (1971). Aspartate aminopeptidase is not
_ .
affected by Ca+~ or EDTA, but is activated by MnC12. Both enzymes
hydrolyze cc-L-aspartyl-~ -naphthylam;de, which reaction may be measured
fluorimetrically to provide a convenient assay. The two enzymes are
further distinguished by their ability to hydrolyze c~-L-glutamyl-,67 -
naphthylamide. Aminopeptidase A hydrolyzes the glutamyl substrate and
the aspartyl substrate equally well. The aspartate aminopeptidase
hydrolyzes the aspartyl substrate three times faster than the glutamyl
substrate.
lt remains to be determined whether one of the known aminopeptidases
or some other is responsible for the in vivo formation of angiotensin III.
In the meantime, a specific inhibitor of aminopeptidases acting only on
N-terminal aspartylpeptides is useful for differential analysis of s~ch
enzymes in biological materials, for in vitro and in vivo research on
angiotensin III-mediated hypertension and for conversion of angiotensin III
from purified angiotensins I or II.
A reversible inhibitor is useful in research on the kinetics
of enzyme activity and on relative abundance of the enzyme
compared to less specific aminopeptidases. Additionallyg a
. .
-4-
& ~
reversible inhibitor will be useful as a probe to distinguish
between different N-terminal aspartylpeptide amiropeptidases
coexisting in the same biological sample.
An irreversible inhibitor is useful for labeling the active-
S site on the enzyme, for tissue localization of the enzyme, both
grossly and microscopically~ and for molecular ablation studies.
Ultimately, it is expected that such inhibitors will prove
useful as antihypertensive drugs.
Specific or at leas~ highly selective enzyme inhibitors
have been developed for certain enzymes, based upon detailed
- background knowledge of the nature of the active site, including
details of the ionic and hydrophobic binding sites and their
steric relationship, whether a metal atom is involved in binding
or catalysis, and the location o~ the metal atom relative to
the bound substrate. Such detailed knowledge has been obtained
from extensive enzymological studies, including X-ray
diffraction, and kinetic data obtained with a variety of sub-
strate analogs. Exploiting such inormation, Byersr L.D. and
Wolfe~den, R., Biol.Chem. 247, 606 (1972~ and Byers, L.D. and
Wolfenden, R., J.Biochemistry 12, 2070 C1973? have developed an
unusually potent competitive inhibitor of carboxypep~idase A,
D-2-benzylsuccinic acid. Ondetti, M.A., Rubin, B. and Cushman,
D.W., Science 196, 441 (1977) and Cushman, D.W., Cheung, H.S.,
Sabo, E.F., and Ondetti, M.A., Biochemistry 16, 5484 (1977~
2~ have extended this approach to include the development of highly
selective inhibitors o~ angiotensin converting enzyme. The
most potent of these inhibitors is ~-3-mercapto-2-methylpropanoyl-
L-proline. Certain chloromethyl ketones have been observed to
inhibit certain proteolytic enzymes. Trypsin was s~own to be
irreversibly inhibited by N-tosyl-L-lysine chloromethyl ketone
~142~
by Shaw, E. and Glover, G., Arch.Biochem.Biophys. 139, 298
(1970). The inhibitor acted as an alkylating agent of a
histidine moiety at or near the active site of the enzyme.
However 7 chloromethylketone analogs of DL-leucine and DL-
alanine were observed to he reversible inhibitors of leucine
aminopeptidase. See Birch, P.L., El-Obied, H.A. and Akhtar~
M., Arch.Biochem.Biophys. 148, 447 (1972).
Relatively little is known about the substrate
binding sites of the aminopeptidases specifically active
on N-terminal aspartyl peptides. It remains to be seen
whether the two enzymes that have been isolated, amino-
peptidase A and aspartate aminopeptidase, are the major
enzymes responsible for ln vivo removal of N-terminal aspar-
tyl groups, or for the d-aspartyl-~-naphthylamide hydro-
lytic activity of biological materials such as rat serum.
Although there is suggestive evidence implicating one or
both of the two enzymes in the in vivo formation of angio-
tensin III, neither has been clearly shown to be involved.
Additional unknown factors include the range of substrate
specificity, whether a metal atom such as Zn++ is present
at the active site, and what amino acid side chains con-
tribute to catalysis.
In accordance with the present invention, there
is provided an inhibitor of aminopeptidase capable of cataly-
zing the removal of an N-terminal aspartyl residue from
a peptide, the essential active ingredient of which has
the chemical formulao
Z-(CH ) ~-C*H-C-CH X
Y O
wherein Z is an anionic group in a~ueous solution at phys-
iological pH; Y is a cationic group in aqueous solution
-
2~
at physiological pH; n is 1 or 2, and X is hydrogen or
halogen,and C* is an asymmetric center in the L-configura-
tion.
Preferably, the essential active ingredient has
the formula:
~ o
Il
~ OOC- ~CH2 ) n-c*H-c-cH2x
: NH 2
where X is Cl or Br and n is 1 or 2. The asterisk denotes
10 an asymmetric centre in the L-configuration.
Aminopeptidases specific for N-terminal aspartyl
peptides are inhibited by compounds of the present inven-
` tion. The inhibiting compounds have the general formula:
Y O
11
Z-(CH2) -C*H-C-CH -X
where Z is an anionic group in aqueous solution at phys-
iological pH, Y is a cationic group in aqueous solution
at physiological pH, n is 1 or 2, X is either hydrogen or
halogen, and the asymmetric carbon denoted by an asterisk
20 is in the L-form. In preferred compounds, Z is COO , Y
is NH3+, n is 1 and Z is eithér Cl or Br. The most pre-
ferred compound is L-aspartyl bromomethane,
O.
HOOC-CH2-CH-C-CH2Br.
NH2
In aqueous solution at physiological pH (approximately pH
6.0 - pH 8.0) the ionic structure is
1 3 11
OOC - CH2 - CH - C - CH2Br
The inhibitor may be used _ vitro or in vivo to
inhibit the aminopeptidase-catalyzed removal of an N-
terminal aspartyl residue of a peptide, or to inhibit the
hydrolysis of ~ -L-aspartyl- ~ -naphtylamide, or to inhibit
the hydrolysis of ~ -L-aspartyl-3[H]benzylamideO The inhi-
bitor is introduced by appropriate means, such as pipetting,
perfusion, injection and the like, into reactive proximity
with the enzyme. By reactive proximity it will be under-
stood that
- 7a -
the mode of introduction is suitable to provide an adequate
concentration of inhibitor capable of diffusing to, and
interacting with, the active site of ~he enzyme. The
interaction may be a binding or a formation of a coordinate or
covalent chemical bond or the disruption of such a bondO For
example, in an n vitro reaction, merely pipetting an amount of
inhibitor sufficient to provide a predetermined final inhibitor
concentration will ordinarily suffice. For ln vivo inhibition,
such factors as the route of administration, the approximate
dilution factor to be expected, rates of metabolism and
excretion of the inhibitor and the like, must be taken into
account, as is well understood in pharmacology, in order to
introduce the inhibitor in an appropriate amount into reactive
proximity with the enzyme.
Synthesis of the inhibitor is carried out starting
with the appropriate dicarboxylic amino acid, having protecting
groups attached to the amino group and to the ~- or ~ - carboxyl
group, an expedient well-known in the art of peptide chemistry.
Suitable protecting groups include, for example tert-butyl,
benzyl, or 2-methoxybenzyl groups for the carboxyl group, and
tert-butoxycarbonyl (also known as tert-butyloxycarbonyl),
carbobenzoxy (also known as benzyloxycarbonyl) or
tert-amyloxycarbonyl groups for the amino group. Choice of
protecting group will be based upon considerations of
convenience, ease of deprotection, availability and the like.
The key intermediate for the synthesis of all inhibitors of the
present invention is the diazomethane derivative. The structure
of the key intermediate is given by the formula r
NH-B
A-OOC-(CH2)n-CH-C-CHN2
o
where A is a carboxyl protecting group, B is an amino protecting
group, and n is 1 or 2.
--8--
~r`
.
~42~
Example 1
~-L-Aspartyl chloromet_ane.
A solution of ~-carbobenzoxy-L-aspartic acid-
~-benzvl ester (ha~ing a free ~ -carboxyl group) in
tetrahydrofuran was cooled to ~10C prior to addition of
N-ethylmorpholine and reaction with isobutyl chloroformate to
form a mixed anhydride. The reaction mixture was stirred at
-10C for 12 minutes, then warmed to 0C and allowed to react
for an additional 10 minutes. A pre-cooled excess of ethyl
ether at -10C was added to the reaction mixture, which was then
filtered.
The resulting mixed anhydride solution was added
dropwise into a solution of diazomethane in ether at 4C and
allowed to stand for 2 hours after which the mixture was slowly
warmed to room temperature and washed sequentially with acetic
acid, sodium bicarbonate solution, saturated sodium chloride and
dried over anhydrous magnesium sulfate. The mixture was then
filtered and excess solvent and fre~ diazomethane removed by
rotary evaporation. The resulting c:Lear oil, ~ -benzyl ester of
~-carbobenzoxy- ~ -L aspartyl diazomethane, was analyzed by
infrared spectrophotometry, in chloroform. The compound had a
sharp absorption band at 2117 cm corresponding to CH=N =N ,
and lacked any bands in the region 2300-2800 cm , correspondin~
to a free carboxyl group.
The ~-benzyl ester of ~-carbobenzoxy- ~-L-aspartyl
~ diazomethane was decomposed by dropwise addition of 3.5 ~ HCl in
ethyl ether at -10C. The mixture was stirred at -10C until
there was no further evolution of nitrogen gas. A yellow, oily
product was obtained in 82~ yield, having an infrared spectrum
(in chloroform) almost identical with ~-carbobenzoxy~L-aspartic
acid- ~-benzyl ester except that no absorption was observed in
the 2300-2800 cm 1 region of free carboxyl absorption. The
product, ~-benzyl ester of ~ -carbobenzoxy- ~ -L-aspartyl
chloromethane, was pure as judged by migration as a single
_g _
~42E3~i~
discrete component on thin layer chromatography in five separate
solvent systems.
The product was deprotected by txeatment with
anhydrous HF in the presence of anisole to yield ~ -L-aspartyl
chloromethane.
Example 2
~-L-aspartyl bromomethane.
The ~-benzyl ester of ~ -carbobenzoxy -~ -L-aspartyl
diazomethane, prepared as described in Example 1, was decomposed
by dropwise addition of HBr in ether, under conditions
essentially identical with those described in Example 1 for
decomposition with HCl. The resulting product was the ~-benzyl
ester o ~-carbobenzoxy - a-L-aspartyl bromomethane, which was
deprotected to yield a-L-aspartyl bromomethane.
Following the procedure of Examples 8 and 9 using
~ -L-aspartyl-3[H]benzylamide and ~ -L-lysyl- [H]
benzylamide as the substrates, the I50 was determined to be 2 x
10 6M for rat lung or serum aminopeptidase-catalyzed
a -L-aspartyl-3[H]benzylamide hydrolysis and 8 x 10 3~ for
a -L-lysyl- ~H]benzylamide hydrolysis.
Example 3
-L-a_ ~ ethane.
3 -benzyl ester of a -carbobenzoxy- ~ -L-aspartyl
chloromethane, prepared as described in Example 1, was dissolved
in methanol and subjected to catalytic hydrogenolysis in the
presence of 10% palladium on barium sulfate.
Example 4
-L-aspartyl aminomethane.
The ~-benzyl ester of ~-benzyloxycarbonyl-L-aspartyl
bromomethane, as prepared in Example 2 r is mixed with potassium
phthalimide in redistilled dimethylformamide and stirred at room
temperature overnight. Ethylacetate was added to the reaction
mixture. The organic phase was washed three times with water,
-1~
- ~ ~
four times with lN NaHCO3 and finally three times with saturated
NaCl solution. The organic layer was dried over anhydrous
MgSO4, filtered, and the solvent was removed with a rotary
evaporator. The residue was crystallized from isopropanol to
yield a semi-solid material. The product was identified by
infrared spectroscopy and behaved as a pure substance on thin
layer chromatography using silica gel and three solvent systems.
The phthaloyl group may be removed by hydrazinolysis or acid
hydrolysis. The resulting compound may be deprotected by (a)
anhydrous HF in the presence of anisole, (b) acid hydrolysis
using HCl, (c) HBx in glacial acetic acid, or (d) hydrogenation
(10~ Pd/C) in a solution of acetic acid-H2O-methanol to yield
a -L-aspartyl aminomethane.
Example 5
L-3-amino-5-benzoylthio-4-oxo~ _anoic acid
.
The ~-t-butyl ester of ~-t-butyloxycarbonyl-L-aspartyl
chloromethane is prepared as in Example 1 using the ~ -t-butyl
ester of ~ -t-butyloxycarbonyl-L-aspartic acid as the starting
matexial.- A mixture of thiobenzoic acid, anhydrous potassium
carbonate and the ~-t-butyl ester of a-t-butyloxycarbonyl-
L-aspartyl chloromethane in redistilled dimethylformamide was
stirred overnight at room temperature. Ethyl acetate was added
to the reaction mixture and the organic solvent was washed
thoroughly with water. The organic phase was dried over
anhydrous MgSO4, filtered and the solvent removed with a rotary
evaporator. The crude material was purified by LH-20 column
chromatography, eluted with isopropanol. This product was
identified by infrared spectroscopy and behaved as a pure
substance on thin layer chromatography using silica gel in four
solvent systems. The ~ t-butyloxycarbonyl and t-butyl ester
protecting aroups were removed by arhydrous trifluoroacetic acid
in the presence of anisole. The resulting compound was purified
by column chromatography and identified as L-3-amino-5~
--11--
'~
~ ~2~613
benzoylthio-4-oxo-pentanoic acid. The I50 for this compound is
approximately 1 x 10 5M when assayed follo~ing the procedure of
Example 8 using ~ -L-aspartyl-3[H]benzylamide as the substrate.
Example 6
-L-aspartyl mercaptom thane.
Alkaline hydrolysis with NH3 in methanol of L-3-amino-
5-benzoylthio-4-oxo-pentanoic acid gives a -L-aspartyl
mercaptomethane. The I50 for this compound is approximately 1 x
10 6M when assayed following the procedure of Example 8 using
0 a -L-aspartyl-3[H]benzylamide as the substrate.
Example 7
Glutamic acid analogs.
Analogs of the compounds synthesized according to
Examples 1-5 are readily prepared starting from ~-carbobenzoxy-
I.-glutamic acid- y -benzyl ester or preferably y -t-butyl ester
of a-t-butoxycarbonyl-L-glutamic acid thaving a free a-carboxyl
group~. The reactions of Example 1 yield y-benzyl ester of
~ -carbobenzoxy-~ -L-glutamyl diazomethane or preferably y -t-
butyl ester of 6-diazo-4-N-t-butoxycarbonyl-5-oxo-L-hexanoate~
i.e. y-t-butyl ester of a -t-butoxycarbonyl- a -L-glutamyl
diazomethane. The corresponding chloromethane and bromomethane
derivatives are prepared as described in Examples 1 and 2,
respectively. The compound ~-L-glutamyl methane is prepared
from the ~ -benzyl ester of a -carbobenzoxy- ~ -L-glutamyl
bromomethane or preferably the y -t butyl ester of
a-t-butoxycarbonyl- ~ ~L-glutamyl bromomethane as described in
Example 3. The aminomethane and mercaptomethane derivatives are
prepared as described in Examples 4 and 5 respectively.
Example 8
Ami o ~ e inhibition by a-L-aspartyl_chloromethane.
An aminopeptidase assay was employed, using ~-
aspartyl- ~-naphthylamide as substrate for N-terminal aspartic-
-12-
!
~4;~6~
specific aminopeptidase activity and ~-arginyl- ~ ~naphthyl-
amlde as substrate for nonspecific aminopeptidase activity.
Hydrolysis of the substrate amide bond releases the fluorophor,
-naphthyl-amine.
Rat serum, 25 ul, was added to an assay mixture
containing 90 ~moles of Trls-HCl buffer, pH 7.0, 0.45 ~moles
of substrate, 0.9 ~moles calcium chloride and sufficient water
to give 0.9 ml total reaction volume. The concentration of
inhibitor was varied from 10 1OM to 10 3M, and a control
reaction was assayed in the absence of added inhibitor. The
percent of inhibition was determined as a function of inhibitor
concentration in order to establish I50, the concentration
required to inhibit the reaction rate 50%, under the reaction
conditions used. The I50 of a-L-aspartyl chloromethane was 3 x
10 M for rat serum aminopeptidase-catalyzed ~ -L-aspartyl-
-naphthylamide hydrolysis, and 6 x ]0 3M for ~ -L-arginyl-
-naphthylamide hydrolysis.
Example 9
Aminope~tidase inhibition by a-L-aspartylmethane.
The inhibitor, prepared as described in Example 3, was
tested for activity in the assay system described in Example 8.
The I50 for a-L-aspartyl- ~ ~naphthylamide hydrolysis was
-13-
8 ~
6 x 10-5 M, indicating the methane derivative was only about 1~20th as
effective a$ the chloromethane derivative.
Example 10
Inhibition of Rat Lung Aminopeptidase.
A homogenate of rat lung tissue, pumped free of blood, was fractio~ated
by low speed centrifugation at 1600 x 9 for 10 minutes at 4C. The
supernatant was further fractionated by high speed centrifugation in
0.1 M Tris-HCl, pH 7.4, and MgC12, 10 mM, at 30,000 x 9 for 30 minutes
at 4C. The majority of the aminopeptidase activity was calcium dependent,
requiring 5 mM to 10 mM Ca++ for optimal activity, resembling~ in this
regard, aminopeptidase A. The Iso f c~-L aspartyl chloromethane for
the rat lung enzyme was substantially the same as that of the rat serum
activity described in Example 8, using the same assay system.
~ Example 11 I.
i5 In vivo aminopeptidase inhib;tion by c{ -L-aspartyl bromomethane.
It is genèrally agreed that the effects of angiotensin II on
systemic arterial blood pressure are two to Four times as great as those
of angiotensin TII. Further, it is often assumed that angiotensin III
is among the first degradation products~of angiotensin II. Thus, after
a dose of angiotensin II is given to an animal, the blood pressure should
rise and then drop as the angiotensin II is degraded or eliminated.
I~ an inhibitor against the aminopeptidase which degrades angiotensin
II to III is administered, angiotensin II will be degraded less rapidly
resulting in a higher blood pressure response at a given time interval.
In this example the in vivo effect of GC-L-aspartyl bromomethane was
examined by measuring the difference in the blood pressure response
after administration of angiotensin II.
~ C 6'~
Sprague-Dawley rats were anesthetized with pentobarbital. A common
carotid artery and a femoral vein were exposed. The vein was cannulated
and heparin was injected. The artery was cannulated and connected to a
pressure transducer. Angiotensin II, 40 ng/kg, was injected intravenously
at 5 minute intervals until a reproducible blood pressure response was
obtained (usually 3-4 injections). An arterial blood sample was collected
at this time. Four doses of angiotensin II - 40, 80, 160 and 320 ng/kg -
were injected at 5 minute intervals in order to construct a log dose-
response curve. The effect of these doses is shown in Table I. The
blood pressure (BP~ response is shown by the systolic (Sys.), diastolic
(Dias.) and mean arterial pressure (MAP).
A second blood sample was collected. ~ ~L-aspartyl bromomethane
~8 mg/kg) - ;ndicated infra as either inhibitor or drug - was injected
and followed by ;njections of 160 ng/kg of anglotensin II at 2~ 7, 17 and
28 minutes following the inhibitor administration. Blood samples were
also collected at timed intervals. The bloocl pressure was measured after
each inject;on and the results are shown in Table II. Table III shows the
percent change in blood pressure response when compared to a control.
Table IV shows the blood pressure response in terms of equivalents of
angiotensin II as interpolated from the log dose-response curve.
The blood samples which were collected were centrifuged to obtain
the plasma. The plasmas were then assayed for residual aminopeptidase A-like
activity using cC L-aspartyl~[3H~benzylamide as substrate. To test for
specificity of the oC-L-aspartyl bromomethane, the plasmas were also
assayed for lysine aminopeptidase using cC-L-lysyl-C3H~benzylamide.
As shown in Table III, at 2 and 7 minutes after the injection of
O~ -L-aspartyl bromomethane~ the mean arterial blood pressure showed an
increase of 17% over the control in response to the injection of
angiotensin II. The systolic blood pressure showed an even more marked
_15_ ~ ~ b
.~ ~``
~14
TABLE I
.
Before Drug
Dose of BP response (m~ Hg)
an~iotensin II (ng/kg) MAP _ Sys. Dias.
5 . 40 2~ 32 22
56 28
160 48 60 32
. 320 61 76 40
TABLE II
.
Af~er Drug
Dose of BP response (m~ Hg)
. Time (min)angiotensin II ~ng/kg) MAP Sys. Dias.
2 160 56 75 34
7 160 56 76 40
17 160 50 6~ 34 .
28 160 48 60 40
.
_ ,
TAB~E III
; `
. After Drug
Dose of BP response, as % of control
20 Time ~min)angiotensin II, ng/kg MAP Sys. _Dias.
2 160 117 125 106
: 7 160 117 127 125
17 160 10~ 113 106
28 160 100 100 125
.
TABLE IV .
ter Drug
BP response, in terms of angio-
tensin II equivalents
Time (min) a~ n ~ (estimated from ~MAP) - _
2 160 248 ng/kg
7 160 248
17 160 172
2~ 160 160
_ , . . __. _._ .
increase. Ta~le IV indicates that the 17~ increase in the
response of the MAP to the angiotensin ~I at a dose of 160 ng/kg
is the pressure response expected of a dose of 24~ ng/kg of
angiotensin II without administration of the inhibitor. This is
a 60~ increase in apparent potency. In four similar
experiments, the increase in apparent potency ranged from
48%~135%.
~ -L-aspartyl bromomethane had the expe~ted result on
residual plasma aminopeptidase A-like activity. One minute
after inhibitor injection, enzyme activity fell by 48%. At 5
minutes, enzyme activity was at 64% of control, and at 15
minutes it was at 68% of control. The activity of plasma lysine
aminopeptidase was not changed when compared to the control.
In an additional experiment, norepinephrine or
angiotensin III was examined in place of angiotensin II.
~ -L aspartyl bromomethane showed no effects on the blood
pressure response to these two compounds.
While tlle invention has been described in connection
with specific embodiments thereof, it will be understood that it
is capable o~ further modifications and this application is
intended to cover any variations, uses, or adaptations of the
invention following, in general, the principles of the invention
and including such departures from the present disclosure as
, come within known or customary practice within the art to which
the-invention pertains and as may be applied to the essential
features hereinbefore set forth, and as follows in the scope of
the appended claims.
-17-
