Aminopeptidase P inhibitors and uses thereof

The present invention is directed to a compound of the formula: ##STR1## or a pharmaceutically acceptable addition salt thereof, wherein C.sup.3 and C.sup.2 in combination have the configuration S,R or R,S; PA1 wherein Y is straight or branched chain lower alkyl having 1 to 6 carbon atoms, straight or branched chain lower alkenyl or alkynyl having 2-6 carbon atoms, cyclic alkyl or alkenyl having 5 or 6 carbon atoms, or benzyl; and PA1 wherein X is an amino acid or an oligopeptide having from 1 to 8 amino acid residues, the first amino acid residue at the N-terminus of X being a natural or a synthetic L-amino acid having a radius of gyration of less than 1.54 .ANG., X also having a carboxyl or a carboxyamide moiety at its carboxy terminus. The present invention is further directed to a pharmaceutical composition and to a method of inhibiting bradykinin degradation in a patient using the above-described compound.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention is directed to a compound that is capable of 
inhibiting the enzyme, aminopeptidase P, whose natural substrate is 
bradykinin. The compound of the present invention is useful as a 
pharmaceutical agent because by inhibiting bradykinin degradation, the 
compound of the present invention allows bradykinin to exert its 
beneficial effects on the cardiovascular system (including decreasing 
blood pressure, dilating the coronary arteries, and providing protective 
effects on the heart during myocardial ischemia reperfusion injuries), to 
improve renal function, and to improve glucose tolerance and 
insulin-sensitivity. The present invention is also directed to a method 
for inhibiting bradykinin degradation in a mammalian patient. 
2. Background of the Invention 
Bradykinin (Bk) is a nine-amino acid peptide hormone which has recently 
been shown to have numerous beneficial effects on the cardiovascular 
system. These include decreased blood pressure, dilation of coronary 
arteries leading to increased blood flow to heart muscle, and direct 
protective effects on the heart during myocardial ischemia-reperfusion 
injuries. Bradykinin can also enhance renal function and improve glucose 
tolerance and insulin-sensitivity (2). See references as disclosed at the 
end of the Detailed Description (1-7). However, Bk is rapidly degraded in 
vivo. Almost complete inactivation of Bk occurs during a single 
circulation through the lung by peptidases located on the plasma membrane 
of vascular endothelial cells (8-10). One of the enzymes responsible for 
inactivation is angiotensin converting enzyme (ACE) (11). 
Aminopeptidase P is known to cleave the N-terminal amino acid from peptides 
that have a prolyl residue in the second position (12, 14, 19). It has 
been suggested that membrane-bound aminopeptidase P may also have an 
important role in vivo in the pulmonary degradation of Bk (10-18) by 
cleaving its Arg.sup.1 -Pro.sup.2 bond. It has also been suggested that 
other peptidases may also play a role in Bk degradation (13). To date, 
studies to determine the role of aminopeptidase P in Bk metabolism in vivo 
have been hampered by the lack of a potent and specific inhibitor of 
aminopeptidase P. Accordingly, it is an object of the present invention to 
determine which enzymes, other than ACE, are involved in Bk degradation. 
It is a further object of this invention to discover and provide an 
inhibitor of aminopeptidase P. It is also an object of the present 
invention to provide a method for inhibiting bradykinin degradation in 
vivo. 
SUMMARY OF THE INVENTION 
It was discovered that the degradation of the nonapeptide hormone, Bk, by 
aminopeptidase P is capable of being inhibited by a compound of the 
formula: 
##STR2## 
or a pharmaceutically acceptable addition salt thereof, wherein C.sup.3 
and C.sup.2 in combination have the configuration S,R or R,S; 
wherein Y is straight or branched chain lower alkyl having 1 to 6 carbon 
atoms, straight or branched chain lower alkenyl or alkynyl having 2-6 
carbon atoms, cyclic alkyl or alkenyl having 5 or 6 carbon atoms, or 
benzyl; and 
wherein X is an amino acid or an oligopeptide having from 1 to 8 amino acid 
residues, the first amino acid residue at the N-terminus of X being a 
natural or a synthetic L-amino acid having a radius of gyration of less 
than 1.54 .ANG., X optionally having a carboxyamide moiety replacing the 
carboxyl moiety of its carboxy terminus (i.e., C-terminus). Accordingly, 
in its first aspect, the present invention is directed to this compound. 
In another aspect, the present invention is directed to a pharmaceutical 
composition comprising: 
(a) a therapeutically effective amount of a compound of the formula: 
##STR3## 
or a pharmaceutically acceptable addition salt thereof, wherein C.sup.3 
and C.sup.2 in combination have the configuration S,R or R,S; 
wherein Y is straight or branched chain lower alkyl having 1 to 6 carbon 
atoms, straight or branched chain lower alkenyl or alkynyl having 2-6 
carbon atoms, cyclic alkyl or alkenyl having 5 or 6 carbon atoms, or 
benzyl; and 
wherein X is an amino acid or an oligopeptide having from 1 to 8 amino acid 
residues, the first amino acid residue at the N-terminus of X being a 
natural or a synthetic L-amino acid having a radius of gyration of less 
than 1.54 .ANG., X also having a carboxyl or a carboxyamide moiety at its 
carboxy terminus; and 
(b) a pharmaceutically acceptable carrier. 
In yet another aspect, the present invention is directed to a method for 
inhibiting bradykinin degradation in a patient comprising: administering 
to a patient in need of inhibition of bradykinin degradation, 
(a) a therapeutically effective amount of a compound of the formula: 
##STR4## 
or a pharmaceutically acceptable addition salt thereof, wherein C.sup.3 
and C.sup.2 in combination have the configuration S,R or R,S; 
wherein Y is straight or branched chain lower alkyl having 1 to 6 carbon 
atoms, straight or branched chain lower alkenyl or alkynyl having 2-6 
carbon atoms, cyclic alkyl or alkenyl having 5 or 6 carbon atoms, or 
benzyl; and 
wherein X is an amino acid or an oligopeptide having from 1 to 8 amino acid 
residues, the first amino acid residue at the N-terminus of X being a 
natural or a synthetic L-amino acid having a radius of gyration of less 
than 1.54 .ANG., X also having a carboxyl or a carboxyamide moiety at its 
carboxy terminus; and 
(b) a therapeutically effective amount of an inhibitor to angiotensin 
converting enzyme. 
In the compound, the pharmaceutical composition and the method of the 
present invention, the first amino acid residue at the N-terminus of X is 
a natural or a synthetic L-amino acid having a radius of gyration less 
than 1.54 .ANG., preferably said first amino acid is Pro, Ala, Ser, Thr, 
Gly, Vat, Cys or hydroxyproline. In a particularly preferred embodiment, X 
has 2 amino acid residues. More preferably, X has two amino acid residues 
and their sequence is -Pro-Ala-NH.sub.2, wherein the NH.sub.2 at the 
C-terminus reflects that this oligopeptide has a carboxamide moiety 
instead of the typical carboxyl moiety at its C-terminus.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention has multiple aspects. In its first aspect, it is 
directed to a compound of the formula: 
##STR5## 
or a pharmaceutically acceptable addition salt thereof, wherein C.sup.3 
and C.sup.2 in combination have the configuration S,R or R,S; 
wherein Y is straight or branched chain lower alkyl having 1 to 6 carbon 
atoms, straight or branched chain lower alkenyl or alkynyl having 2-6 
carbon atoms, cyclic alkyl or alkenyl having 5 or 6 carbon atoms, or 
benzyl; and 
wherein X is an amino acid or an oligopeptide having from 1 to 8 amino acid 
residues, the first amino acid residue at the N-terminus of X being a 
natural or a synthetic L-amino acid with a radius of gyration less than 
1.54 Angstroms (i.e., .ANG.), X further having a carboxyl or a 
carboxyamide moiety at its carboxy terminus. 
In a particularly preferred embodiment of the compound of the present 
invention, Y is benzyl or isobutyl; preferably Y is isobutyl. 
In the compound of the present invention, the oligopeptide X may include 
naturally occurring or synthetic amino acid residues. Consistent with 
convention, the Applicants have utilized herein the three letter 
abbreviations for the various amino acids and have capitalized the first 
letter of the amino acid in those instances wherein the amino acid has the 
"L" configuration, e.g., Pro=L-proline. X also includes amino acids that 
have the D-configuration. 
Any amino acid residues that are utilized in the compound of the present 
invention as the first amino acid at the N-terminus of X should have the 
L-configuration and should not be so large as to preclude the compound of 
the present invention from binding to the active site of aminopeptidase P. 
A convenient measure for determining whether the first amino acid at the 
N-terminus of X will be sufficiently small to allow the inhibitor of the 
present invention to fit within the active site of the enzyme is the 
radius of gyration such as reported by Levitt et al., (1976) J. Mol. 
Biol., 104, 59-107 or Sherman et al., (1985) BioEssays, 3: 27-31. 
Applicant has determined that when the first amino acid in X is 
hydroxy-proline, which has a radius of gyration slightly greater than 1.25 
.ANG., or is a smaller amino acid, the potential inhibitor fits into the 
active site of aminopeptidase P, whereas when the first amino acid in X is 
Leu, which has a radius of gyration of 1.54 .ANG., the potential inhibitor 
does not fit into the active site. Accordingly, the first amino acid in X 
is an amino acid (natural or synthetic) with a radius of gyration that is 
less than 1.54 .ANG.. 
By way of example, the radius of gyration in ascending size (.ANG.) for the 
various naturally occurring amino acids, are as follows: Gly(-); Ala 
(0.77); Set (1.08); Cys (1.22); Thr (1.24); Pro (1.25); Val (1.29); Asp 
(1.43); Asn (1.45); Leu (1.54); Ile (1.56); Gln (1.75); Gh (1.77); His 
(1.78); Met (1.80); Phe (1.90); Lys (2.08); Tyr (2.13); Trp (2.21); and 
Arg (2.38). Thus, the first amino acid at the N-terminus of X is 
preferably a member of the group consisting of Gly, Ala, Ser, Cys, Thr, 
Pro, Vat and hydroxyproline. 
Although X may have from 1 to 8 amino acid residues, the preferred number 
of amino acid residues in X is 1 to 3, more preferably 2. 
The compound of the present invention also includes a pharmaceutically 
acceptable acid addition salt such as the hydrochloride, hydrobromide, 
hydroiodide, sulfate, phosphate, acetate, propionate, lactate, maleate, 
realate, succinate, tartrate and the like. Additionally, the compound of 
this invention may be administered in a suitable hydrated form. 
The compound of the present invention may be prepared by any number of 
methods known to those skilled in the art. For example, the particular 
sequence of reactions by which a oligopeptide is joined to N-t-BOC-(2S, 
3R)-3-amino-2-hydroxy-4-phenylbutyric acid (i.e., Y=benzyl) to form a 
compound of the present invention is generally not of critical importance, 
the sequence being chosen principally for convenience or for maximum 
yields. Moreover, the choice of activating reagents and conditions for 
joining amino acids or small peptides is not limited to those specifically 
described herein. The oligopeptide intermediates that are used in this 
invention are easily synthesized using techniques well known in the art, 
including full automated peptide synthesizers, such as disclosed in the 
Examples. Several procedures are available utilizing different resins, 
different protected amino acids, and different peptide bond-forming 
strategies. A typical synthesis begins with the attachment to a solid 
polymeric support of the N-t-butyloxycarbonyl (BOC)-derivative of the 
amino acid which is to be the C-terminal residue of the peptide. The 
support may be, as an example, a 4-chloromethyl resin (if the final 
peptide is to have a C-terminal carboxyl group) or a 
4-methylbenzhydrylamine resin (if the C-terminus is to be a carboxamide). 
The BOC group is then removed by treatment of the resin with 40% (V/V) 
trifluoroacetic acid (TFA)/dichloromethane (DCM). Following neutralization 
of the resin with 10% N,N-diisopropylethylamine (in DCM), the 
BOC-derivative of the next amino acid is coupled to the .alpha.-amino 
group of the C-terminal amino acid in the form of an activated ester 
preformed with hydroxybenzotriazole (HOBt) and 
N,N'-dicyclohexylcarbodiimide (DCC) in N-methylpyrrolidone (NMP). 
Following washing of the resin with NMP, any uncoupled amino groups are 
capped with 10% acetic anhydride (in DCM). Additional amino acids are then 
added sequentially in the C-to N-terminal direction by repeated cycles of 
BOC-group removal, neutralization, coupling of the next BOC-amino acid as 
the activated ester, and capping. Any trifunctional amino acids are 
side-chain protected as required. The resulting oligopeptide is then 
capable of being coupled to a protected intermediate, such as 
t-BOC-3-amino-3-Y-2-hydroxypropanoic acid, after activation of the latter 
compounds carboxyl group, such as with 
benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium 
hexafluorophosphate. The completed peptide is simultaneously removed from 
the resin and deprotected by treatment of the resin with anhydrous HF 
containing anisole. The HF is removed in vacuo and the crude peptide is 
purified by HPLC. The oligopeptides and compounds of this invention are 
typically purified by crystallization or by column chromatography. 
The addition salts that are within the scope of the compound of the present 
invention are prepared by reacting a neutral compound of the present 
invention with an excess of the acid form of the addition salt under 
precipitating conditions. By way of example, the acetate salt of a 
compound of the present invention is prepared by reacting a compound of 
the present invention with a molar excess of acetic acid in a polar 
solvent, such as methylene chloride, and evaporating the solvent until 
salt formation occurs. 
The compound of the present invention was found to have the ability to 
inhibit the activity of the enzyme, aminopeptidase P, which is found in 
the mammalian lung. 
The kinetics of inhibition of one compound of the present invention wherein 
Y is a benzyl and X is -Pro-Ala-NH.sub.2 (hereinafter "apstatin") was 
determined as described in the Experimental Procedures using Arg-Pro-Pro 
as the substrate, and purified bovine lung membrane-bound aminopeptidase P 
as the enzyme. Apstatin exhibited mixed-type kinetics (FIG. 1A). Slope and 
1/v-axis intercept replots (FIG. 1B) showed that inhibition was a linear 
mixed-type with .alpha.&gt;0 and .beta.=0. Cf. (28). 
The inhibitory constant K.sub.i was determined, as described in the 
Experimental Procedure herein, from plots of 1/v versus i, wherein i is 
the concentration (.mu.M) of inhibitor, while .alpha.Ki was determined 
from s/v versus i, wherein i is the concentration (.beta.M) of inhibitor. 
Table 1 compares the K.sub.i and .alpha. values for purified bovine and 
rat lung membrane-bound aminopeptidase P, partially purified human lung 
membrane-bound aminopeptidase P, and recombinant E. coli aminopeptidase P, 
using apstatin as the inhibitor. All enzymes showed a linear mixed-type 
inhibition with K.sub.i values in the micromolar range. Table 1 reflects 
that human aminopeptidase P had the highest affinity for apstatin, having 
a K.sub.i =0.64 .mu.M, compared to 2.6 .mu.M for aminopeptidase P of rat 
origin and 7.8 .mu.M for aminopeptidase P of bovine origin. 
TABLE 1 
______________________________________ 
Inhibition of Various Aminopeptidase P Preparations by Apstatin 
Source of Aminopeptidase P 
K.sub.i (.mu.M).sup.o 
.alpha..sup.b 
______________________________________ 
rat lung.sup.c 2.6 5.1 
bovine lung.sup.c 7.8 4.2 
human lung.sup.d 0.64 11. 
E. coli.sup.e 14. 2.5 
______________________________________ 
.sup.a Inhibition in all cases was linear mixedtype (.beta. = 0). K.sub.i 
is defined as the equilibrium constant for enzymeinhibitor binding in the 
absence of substrate. 
.sup.b .alpha.K.sub.i is the equilibrium constant for enzymeinhibitor 
binding at infinite substrate concentration. 
.sup.c Purified membranebound form. 
.sup.d Partially purified membranebound form. 
.sup.e Recombinant (22). 
Preincubation of rat aminopeptidase P with apstatin for up to 2 hr. at 
4.degree. C. before addition of substrate did not increase the degree of 
inhibition compared with no preincubation, indicating the absence of any 
time-dependent or slow-tight binding inhibition. A 100-fold dilution of a 
preincubated enzyme-apstatin mixture led to the degree of inhibition 
expected from control experiments for the residual apstatin concentration 
without preincubation, indicating reversibility. Inhibition by apstatin 
was not dependent on the presence of Mn.sup.+2. 
The IC.sub.50 of the compound of the present invention in relation to 
aminopeptidase P was also determined. The IC.sub.50 is the concentration 
causing 50% inhibition of the cleavage of 0.5 .mu.M Arg-Pro-Pro in 0.1M 
Hepes, pH 8.0 at 37.degree. C. In particular, the IC.sub.50 of apstatin 
for human lung aminopeptidase P was 2.9 .mu.M; for bovine lung 
aminopeptidase P, it was 9.4 .mu.M; and for rat lung aminopeptidase P, it 
was 4.1 .mu.M. The specificity of the compound of the present invention 
for other aminopeptidases was similarly determined as described in the 
Experimental Procedures herein. In particular, the IC.sub.50 of apstatin 
was 600 .mu.M for aminopeptidase M and 1,100 .mu.M for 
dipeptidyl-peptidase IV. The following enzymes had IC.sub.50 values&gt;800 
.mu.M: aminopeptidase A, angiotensin converting enzyme, 
dipeptidyl-peptidase I-like activity, 
bestatin-sensitive/amastatin-insensitive membrane dipeptidase (29), 
microsomal dipeptidase, endopeptidase 24.11, endopeptidase 24.15, and 
prolyl oligopeptidase. Surprisingly, prolidase, the cytosolic 
X-Pro-specific dipeptidase, had an IC.sub.50 value for apstatin of 4.9 
.mu.M. 
The IC.sub.50 was also determined for two stereoisomers of a compound of 
the present invention wherein Y is isobutyl, X is Pro-Ala-NH.sub.2 and 
C.sup.2 -C.sup.3 are 2S,3R and 2R,3S, respectively. Specifically, the 
IC.sub.50 for the two stereoisomers were determined for aminopeptidase P 
derived from three different sources: rat lung, bovine lung and human 
lung. This data is reported in Table 2 and reflects that for human lung 
aminopeptidase P, the 2S,3R stereoisomer exhibited the lowest IC.sub.50, 
i.e., 0.23 .mu.M. The IC.sub.50 of this 2S,3R stereoisomer was 
approximately ten-fold better than the IC.sub.50 apstatin (2.9 .mu.M). 
TABLE 2 
______________________________________ 
IC.sub.50 (.mu.M) 
Aminopeptidase P From 
(2S,3R).sup.a 
(2R,3S).sup.b 
______________________________________ 
rat lung 0.56 0.31 
bovine lung 4.5 2.1 
human lung 0.23 0.43 
______________________________________ 
.sup.a The (2S,3R) stereoisomer is 
N[(2S,3R)3-amino-2-hydroxy-5-methylhexanoylL-prolyl-L- 
prolylL-alaninamide. 
.sup.b The (2R,3S) stereoisomer is: 
N[(2R,3S)3-amino-2-hydroxy-5-methylhexanoylL-prolyl-L- 
prolylL-alaninamide. 
Using the procedure described herein, under Experimental Procedures it was 
determined that aminopeptidase P contributed to Bk degradation in the 
mammalian lung. In that procedure, [.sup.3 H]-Bk (labelled in Pro.sup.2 
and Pro.sup.3) was perfused through the isolated lung in the presence or 
absence of various peptidase inhibitors. The perfusate was then analyzed 
for radiolabelled products by HPLC. FIG. 2A shows that when [.sup.3 H]-Bk 
was perfused in the absence of inhibitors, no intact Bk was found in the 
perfusate. This is consistent with previous data demonstrating almost 
complete degradation of Bk during a single passage through the rat 
pulmonary circulation (8-10, 17). Most of the radioactivity (an average of 
79%) was in the form of [.sup.3 H]-Pro-Pro [Bk(2-3)] with the remainder 
being [.sup.3 H]-Bk(1-5). It was hypothesized that pulmonary vascular 
aminopeptidase P was cleaving the Arg'-Pro.sup.2 bond followed by rapid 
removal of Pro-Pro by dipeptidylpeptidase IV (DPP IV) (12). These cleavage 
sites on Bk are shown in FIG. 3. Simultaneously, angiotensin converting 
enzyme (ACE) was sequentially removing Phe-Arg and Ser-Pro from the 
C-terminus (13). To test this hypothesis, [.sup.3 H]-Bk was perfused in 
the presence of an ACE inhibitor ramiprilat (FIG. 2B), or in the presence 
of ramiprilat plus apstatin (FIG. 2C). FIG. 2B shows that ramiprilat alone 
blocked the formation of [.sup.3 H]-Bk(1-5) and gave rise to a comparably 
sized peak of intact [.sup.3 H]-Bk. However, most of the radioactivity 
remained in the Pro-Pro peak indicating substantial cleavage of Bk at the 
N-terminal end. When a DPP IV inhibitor, diprotin A, was present along 
with ramiprilat (data not shown) most of the radioactivity eluted from the 
lung in the form of [.sup.3 H]-Bk(2-9). This indicated that in the 
presence of ramiprilat, the primary site of cleavage was at the Arg.sup.1 
-Pro.sup.2 bond and that [.sup.3 H]-Pro-Pro arose by DPP IV cleavage of 
[.sup.3 H]-Bk(2-9). FIG. 2C shows that when apstatin and ramiprilat were 
present together, cleavage at both the N-terminal and C-terminal end was 
blocked, and most of the radioactivity eluted as intact [.sup.3 H]-Bk. 
Only a small peak of [.sup.3 H]-Pro-Pro remained which was presumably due 
to incomplete inhibition of aminopeptidase P at 40 .mu.M apstatin (which 
is only fifteen times the K.sub.i concentration). 
FIG. 4 shows the percentage of the radioactivity surviving as intact 
[.sup.3 H]-Bk as a function of apstatin concentration in the presence of 
ramiprilat. An average of 22.+-.6% of the radioactivity eluting from the 
lung was intact [.sup.3 H]-Bk when ramiprilat alone was present in the 
perfusion medium. When increasing concentrations of apstatin were included 
along with ramiprilat, the percentage of intact [.sup.3 H]-Bk eluting from 
the lung increased. At 40 .mu.M apstatin, 92.+-.4% of the radioactivity 
was [.sup.3 H]-Bk. 
The above results reflect that aminopeptidase P and ACE can fully account 
for the degradation of Bk in the pulmonary circulation of the rat as shown 
in FIG. 4. Other membrane-bound aminopeptidases, namely aminopeptidases A 
and M, cannot degrade intact Bk (37). In control experiments, a potent 
inhibitor of these enzymes, [(2S, 
3R)-3-amino-2-hydroxy-5-methylhexanoyl]-Val-Val-Asp, also called amastatin 
(38) had no significant effect on the pattern of metabolites when present 
at 100 .mu.M along with ramiprilat in the perfusion medium (data not 
shown). 
Earlier in vitro studies using rat lung microsoma/membranes had indicated 
that endopeptidase 24.11 was present in the lung and could make a minor 
contribution to Bk degradation by this preparation by hydrolyzing the 
Pro.sup.7 -Phe.sup.8 bond (13). However, no [.sup.3 H]-Bk(1-7) was 
observed in the perfusate when apstatin and ramiprilat were present 
suggesting that the endopeptidase 24.11 is not involved in the pulmonary 
degradation of Bk in vivo. Endopeptidase 24.11 has been found 
immunocytochemically on lung epithelial cells but not on lung vascular 
endothelial cells that would be in direct contact with circulating Bk 
(39-40). 
Overall, aminopeptidase P was quantitatively less important than ACE in 
degrading Bk in the perfused rat lung. The ACE inhibitor ramiprilat alone 
increased the amount of intact [.sup.3 H]-Bk eluting from the lung from 
unmeasurable levels to 22% (.gtoreq.22-fold). In contrast, apstatin alone 
(dam not shown) yielded mainly [.sup.3 H]-Bk(1-5), but increased intact 
[.sup.3 H]-Bk to only about 1-3% which was difficult to quantitate 
accurately. The quantitative role of aminopeptidase P in Bk degradation 
was shown more clearly when apstatin (at 40 .mu.M) was used together with 
ramiprilat. The amount of [.sup.3 H]-Bk in the perfusate increased from 
22% with ramiprilat alone to 92% with both inhibitors present, a 4.2 fold 
increase. Presumably, these values could have been increased toward 100% 
and 4.5-fold at higher apstatin concentrations. 
In order to estimate the relative contributions of aminopeptidase P and ACE 
to the pulmonary cleavage of BK, the integrated form of the two enzyme/one 
substrate model (32) under first-order conditions was used as described in 
the Experimental Procedures. Assuming an overall pulmonary inactivation of 
Bk (H.sub.APP+ACE) of 99.75% as recently determined in vivo by Ryan et al. 
(10), it was calculated that aminopeptidase P is responsible for 25% of 
the cleavage of Bk while ACE is responsible for 75%. Other assumptions for 
overall pulmonary activation from 98% (17) to 99.9% gave calculated 
contributions for aminopeptidase P ranging from 39% to 20%, respectively. 
While aminopeptidase P appears to be quantitatively less important than 
ACE, there is now evidence that aminopeptidase P is nevertheless a 
physiologically important regulator of the blood pressure response to Bk 
in the rat (15,41). Apstatin alone was shown to significantly potentiate 
the magnitude as well as the duration of the blood pressure decrease 
caused by intravenous administration of Bk. Apstatin plus lisinopril (an 
ACE inhibitor) was also more effective in this regard than lisinopril 
alone. Thus, aminopeptidase P appears to play a significant role in the 
pulmonary degradation of Bk. Further, apstatin has proven itself to be an 
effective inhibitor of aminopeptidase P and a useful pharmaceutical agent 
when used in combination with an ACE inhibitor so as to allow Bk to exert 
its well known and useful effects over a prolonged period of time. 
Thus, in its second aspect, the present invention is also directed to a 
pharmaceutical composition that is capable of inhibiting bradykinin 
degradation. The pharmaceutical composition of the present invention 
comprises: 
(a) a therapeutically effective amount of a compound of the formula: 
##STR6## 
or a pharmaceutically acceptable addition salt thereof, wherein C.sup.3 
and C.sup.2 in combination have the configuration S,R or R,S; 
wherein Y is straight or branched chain lower alkyl having 1 to 6 carbon 
atoms, straight or branched chain lower alkenyl or alkynyl having 2-6 
carbon atoms, cyclic alkyl or alkenyl having 5 or 6 carbon atoms, or 
benzyl; and 
wherein X is an amino acid or an oligopeptide having from 1 to 8 amino acid 
residues, the first amino acid residue at the N-terminus of X being a 
natural or a synthetic L-amino acid with a radius of gyration less than 
1.54 .ANG., X further having a carboxyl or a carboxyamide moiety at its 
carboxy terminus; and 
(b) a pharmaceutically acceptable carrier. 
The compound of the present invention is typically administered in a 
pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers 
are well known in the art. These carriers may be liquid or solid and 
include oils, polymers, vitamins, carbohydrates, amino acids, salts, 
buffers, albumin, surfactants, bulking agents, binding agents and fillers. 
In liquid formulation, preferred carbohydrates include sugar or sugar 
alcohols, such as mono, di, or polysaccharides, or water soluble glueans. 
The saccharides or glucans can include fructose, dextrose, lactose, 
glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, 
dextrin, alpha and beta cyclodextrin, soluble starch, hydroxethyl starch 
and carboxymethylcellulose, or mixtures thereof. Sucrose is most 
preferred. "Sugar alcohol" is defined as a C.sub.4 to C.sub.8 hydrocarbon 
having an --OH group and includes galactitol, inositol, mannitol, xylitol, 
sorbitol, glycerol, and arabitol. Mannitol is most preferred. These sugars 
or sugar alcohols mentioned above may be used individually or in 
combination. There is no fixed limit to amount used as long as the sugar 
or sugar alcohol is soluble in the aqueous preparation. Preferably, the 
sugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %, 
more preferably between 2.0 and 6.0 w/v %. Amino acids include levorotary 
(L) forms of carnitine, arginine, and betaine; however, other amino acids 
may be added. Preferred polymers include polyvinylpyrrolidone (PVP) with 
an average molecular weight between 2,000 and 3,000 daltons, or 
polyethylene glycol (PEG) with an average molecular weight between 3,000 
and 5,000 daltons. It is also preferred to use a buffer in the composition 
to minimize pH changes in the solution before lyophilization or after 
reconstitution. Most any physiological buffer may be used, but citrate, 
phosphate, succinate, and glutamate buffers or mixtures thereof are 
preferred. 
In solid formulations, the compound of the present invention is combined 
with a filler and a binding agent such as known to the art. Some agents 
may act both as a filler and a binder. Suitable fillers include one or 
more polysaccharides, such as the starches, the celluloses, and 
derivatives thereof, xanthan gum, gum arabic and the like. Suitable 
binders include any of the binders known to the art. By way of example, a 
suitable binder is one or more of the carbohydrates mentioned above. 
It is also within the scope of the present invention that the compound of 
the present invention be formulated in a slow release formulation, such as 
disclosed in U.S. Pat. No. 4,917,893, entitled "Prolonged Release 
Microcapsules" or in U.S. Pat. No. 4,359,483, entitled "Process for 
Producing A Multi-Layered Slow Release Compound," both of which are hereby 
incorporated herein by reference. 
The pharmaceutical composition of the present invention is typically 
administered in oral dosage form such as tablets, capsules, pills, 
powders, granules, suspensions, or solutions. It may also be administered 
rectally or vaginally, in such forms as suppositories or bougies. It may 
also be administered intraperitoneally, subcutaneously, intramuscularly or 
intravenously using forms known to the pharmaceutical art. In general, the 
preferred route of administration is oral or intravenous. 
In its third aspect, the present invention is directed to a method of 
inhibiting bradykinin degradation in a mammalian patient comprising: 
administering to a mammalian patient in need of inhibition of bradykinin 
degradation, 
a therapeutically effective amount of a compound of the formula: 
##STR7## 
or a pharmaceutically acceptable addition salt thereof, wherein C.sup.3 
and C.sup.2 in combination have the configuration S,R or R,S; 
wherein Y is straight or branched chain lower alkyl having 1 to 6 carbon 
atoms, straight or branched chain lower alkenyl or alkynyl having 2-6 
carbon atoms, cyclic alkyl or alkenyl having 5 or 6 carbon atoms, or 
benzyl; and 
wherein X is an amino acid or an oligopeptide having from 1 to 8 amino acid 
residues, the first amino acid residue at the N-terminus of X being a 
natural or a synthetic L-amino acid having a radius of gyration less than 
1.54 .ANG., X further having a carboxyl or a carboxyamide moiety at its 
carboxy terminus. 
Optionally, the method of the present invention further includes the step 
of administering a therapeutically effective amount of an inhibitor to 
angiotensin converting enzyme. 
The method of the present invention comprises coadministering an inhibitor 
of aminopeptidase P, preferably in combination with an inhibitor of 
angiotensin convening enzyme (ACE). By the term "coadministering" as used 
herein, is meant that the aminopeptidase P inhibitor of the present 
invention and the ACE inhibitor be administered such that both are present 
in the patient's bloodstream at the same time in therapeutically effective 
amounts. Thus, it is within the scope of the present invention that both 
compounds be administered as a single tablet, substantially simultaneously 
as two tablets, or in other instances, such as where one of the inhibitors 
has a long half-life in vivo, it may be sufficient that the two compounds 
be administered within the same forty-eight hour period. Preferably, the 
pharmaceutical composition of the present invention is administered in 
unit dosage form. However, regardless of how or when the inhibitors to 
aminopeptidase P and ACE are administered, the method of the present 
invention is limited to administering them such that the patient in need 
of treatment has a therapeutically effective amount of each member of the 
combination in their bloodstream at any particular time. 
An effective but nontoxic quantity of the compound of the present invention 
is employed in any treatment. The dosage regimen for inhibiting bradykinin 
degradation by the compound of this invention is selected in accordance 
with a variety of factors including the type, age, weight, sex and medical 
condition of the mammal, the severity of the symptoms, and the route of 
administration of the particular compound employed. A physician or 
veterinarian of ordinary skill will readily determine and prescribe the 
therapeutically effective dosage based on the route of administration of 
the Bk inhibitor to prevent or arrest the progress of the condition. In so 
proceeding, the physician or veterinarian would employ relatively low 
dosages at first, subsequently increasing the dose until a maximum 
response is obtained. Because the compounds of the present invention are 
excreted through the kidney, patients with impaired renal function would 
receive a lesser dose than patients with normal renal function. Physicians 
would assess a patient's renal function by monitoring the patient's serum 
creatinine. Serum creatinine concentrations increasing above 1.0 mg/dl 
reflect decreasing renal function. Thus, by the term "therapeutically 
effective mount" as used herein is meant the amount of the compound that 
is effective to cause substantial inhibition of its respective enzyme such 
that the combination substantially increases the half-life of any 
endogenous Bk that is formed in the patient. 
In rats, Bk potentiation by apstatin has been observed with 0.08-0.8 mg/kg 
intravenously when administered over a one hour period. More potent 
inhibitors of the present invention, wherein Y=isobutyl, and wherein X is 
Pro-Ala-NH.sub.2 should be effective at five to tenfold lower dosages. See 
e.g., Table 2. Less potent inhibitors would require a greater dosage to 
provide the same therapeutic result. A typical therapeutically effective 
dose of a compound of the present invention is from about 0.008 mg/kg to 
8.0 mg/kg, when given intravenously. 
Inhibitors of ACE are well known in the art and are used for inhibiting the 
in vivo conversion of angiotensin I to angiotensin II. Typical inhibitors 
of ACE include captopril, enalapril, enalaprilat, lisinopril, quirtapril, 
benazepril, fosinopril, ramipril and ramiprilat. The method of 
administration and dosages for each of these ACE inhibitors is well known 
in the art and are disclosed in the 1995 Physician's Desk Reference. 
Captopril, which is also known as 1-[(2S)-3-mercapto-2-methyl 
propionyl]-L-proline, is typically administered to humans as tablets at 
between 18.75 mg to 150 mg/day with a target of 150 mg/day, but never to 
exceed 450 mg/day. Enalapfil, which is also known as 
(S)-1-[N-[1-(ethoxycarbonyl)-3-phenylpropyl)]-L-alanyl]-L-proline, (Z) 
maleate (1:1), is typically administered to human patients as tablets at 
between 10 mg/day to 25 mg/day, not to exceed 50 mg/day. Enalapril is 
convened in vivo to enalaprilat, the acid form enalapril. Enalaprilat has 
the formula: (S)-1-[N-(1-carboxy-3-phenylpropyl)-L-alanyl]-L-proline 
dihydrate and is typically administered intravenously. Lisinopril, which 
is also known as (S)-1-[N.sup.2 
-(1-carboxy-3-phenylpropyl)-L-lysyl]-L-proline dihydrate, is typically 
administered to human patients as tablets at a dosage of 20 mg/day to 40 
mg/day. Ramipril, which is also known as 
(2S,3aS,6aS)-I[(S)-N-[(S)-1-carboxy-3-phenylpropyl]alanyl]octahydrocyclope 
nta[b]pyrrole-2-carboxylic acid, 1-ethylester, is converted in vivo to its 
aliacid form ramiprilat. Ramipril is administered as tablets with the 
typical dosage for human patients of 2.5 mg/day to 20 mg/day. 
In the process of the present invention, a physician or veterinarian would 
coadminister the ACE inhibitor component of the present invention at the 
above described dosages, allowing for variations due to the patient's 
weight, health, age, and renal condition. For example, serum creatinine 
concentration increasing above 1.0 mg/dl reflect decreasing renal function 
and a decreased ability to excrete the inhibitors used in the method of 
the present invention. However, in each instance, the patient is 
administered therapeutically effective amount, i.e. , an amount sufficient 
to substantially inhibit the cleavage of Bk by ACE, and to substantially 
inhibit the cleavage of Bk in vivo when administered in conjunction with 
an inhibitor of aminopeptidase P as already discussed above. 
Because Bk is known to decrease blood pressure, dilate the coronary 
arteries, protect the heart during myocardial ischemia reperfusion 
injuries, enhance renal function, and improve glucose tolerance and 
insulin sensitivity (2), the present invention is further directed to a 
method for obtaining any one of these effects comprising administering to 
a patient in need of one of the above effects, 
a therapeutically effective amount of a compound of the formula: 
##STR8## 
or a pharmaceutically acceptable addition salt thereof, wherein C.sup.3 
and C.sup.2 in combination have the configuration S,R or R,S; 
wherein Y is straight or branched chain lower alkyl having 1 to 6 carbon 
atoms, straight or branched chain lower alkenyl or alkynyl having 2-6 
carbon atoms, cyclic alkyl or alkenyl having 5 or 6 carbon atoms, or 
benzyl; and 
wherein X is an amino acid or an oligopeptide having from 1 to 8 amino acid 
residues, the first amino acid residue at the N-terminus of X being a 
natural or a synthetic L-amino acid .with a radius of gyration of less 
than 1.54 .ANG., X also having a carboxyl or a carboxyamide moiety at its 
carboxy terminus. 
The above methods of the present invention may further include the step of 
coadministering to said patient a therapeutically effective amount of an 
inhibitor to angiotensin converting enzyme, such as already discussed 
herein. 
Experimental Procedures 
Materials--[2,3-Prolyl-3,4-.sup.3 H(N)]-bradykinin (lot 3109-298) was 
obtained from Dupont NEN Research Products (Boston, Mass.). The specific 
activity was 62.0 Ci/mmol and the purity as shipped was 97.1%. Ramiprilat 
was the girl of Dr. Ronald J. Shebuski (The Upjohn Company, Kalamazoo, 
Mich.). Arg-Pro-Pro and cyclo-Pro-Pro were obtained from Bachera 
Bioscience (Philadelphia, Pa.). Ile-Pro-Ile (diprotin A), amastatin, and 
N-t-BOC-(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoic acid were obtained from 
Sigma (St. Louis, Mo.). Purified rat lung aminopeptidase P was used to 
enzymatically produce the following fragments of the bradykinin ("Bk") 
nonapeptide: Bk(2-5) from Bk(1-5), Bk(2-6) from Bk(1-6), and Bk(2-8) from 
Bk(1-8). All other Bk-fragment standards were obtained from sources 
indicated in references (12, 14, 19, 21 herein.) For convenience, any 
prior art references to procedures used in the Applicants' Experimental 
Procedures are parenthetically referred to herein by number and 
numerically listed at the end of the Detailed Description. 
Enzymes--Membrane-bound aminopeptidase P was purified to homogeneity from 
bovine lung (14) and from rat lung. Partially purified membrane-bound 
aminopeptidase P from human lung was obtained by treatment of lung 
microsomes with phosphatidylinositol-specific phospholipase C (B. 
thuringiensis) (ICN, Costa Mesa, Calif.) followed by centrifugation as 
described previously (14). Membrane dipeptidase was purified from rat lung 
microsomes (manuscript in preparation). Recombinant E. coli aminopeptidase 
P (22) and purified lamb kidney prolyl oligopeptidase (23) were generally 
supplied by Dr. Tadashi Yoshimoto (Nagasaki University). Purified porcine 
kidney prolidase and purified porcine kidney aminopeptidase M was obtained 
from Sigma (St. Louis, Mo.). 
Synthesis of N-[(2S, 
3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-prolyl-L-prolyl-Lalaninamide 
(Apstatin)--The tripeptide, Pro-Pro-Ala-NH.sub.2, was prepared on resin by 
standard solid-phase techniques (24) using an Applied Biosystems 430A 
peptide synthesizer beginning with p-methylbenzhydrylamine resin. The 
N-t-BOC-(2S,3R)-3-amino-2-hydroxy-4-phenylbutyric acid was coupled to the 
tripepride-resin in a manual apparatus using 
benzotfiazole-1-yl-oxy-tris-(dimethylamino)-phosphonium 
hexafluorophosphate (BOP) as an activating agent (25). The N-terminal BOC 
group was removed with 40% trifluoroacetic acid (TFA) in methylene 
chloride, and the peptide was cleaved from the resin by treatment at 
0.degree. C. with HF-anisole (9/1) for one hour. Crude peptide was 
extracted from the resin with 50% aqueous acetic acid and lyophilized. It 
was purified by preparative reversed-phase HPLC on a Vydac C.sub.18 column 
with a linear gradient beginning with 100% water (0.1% TFA) to 80% 
water/20 % acetonitrile (0.1% TFA). After lyophilization the purified 
peptide showed a single peak on analytical HPLC on a Vydac C.sub.18 
column. It was characterized by fast atom bombardment mass spectrometry 
and showed a [M+H].sup.+ ion at m/z 460. 
Synthesis of N-[(2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl 
]-L-prolyl-L-prolyl-L-alaninamide--This compound is prepared as described 
above for apstatin replacing 
N-t-BOC-(2S,3R)-3-amino-2-hydroxy-4-phenylbutyric acid with 
N-t-BOC-(2S-,3R)-3-amino-2-hydroxy-5-methylhexanoic acid (Sigma Chemical 
Co., St. Louis, Mo.; Cat. No. B 8020). 
Synthesis of N-[(2R,3S)-3-amino-2-hydroxy-5-methylhexanoyl 
]-L-prolyl-L-prolyl-L-alaninamide--This compound is prepared, as described 
above for apstatin, replacing 
N-t-BOC-(2S,3R)-3-amino-2-hydroxy-4-phenylbutyric acid with 
N-t-BOC-(2R,3S)-3-amino-2-hydroxy-5-methylhexanoic acid. 
Synthesis of N-t-BOC-(2S,3R)-3-amino-3-Y-2-hydroxypropanoic acid or 
N-t-BOC-(2R,3S)-3-amino-3-Y-2-hydroxypropanoic acid 
A benzyloxycarbonyl-[Z-] protected amino acid is used as the starting 
material. The amino acid is a natural or unnatural .alpha.-amino acid with 
a side-chain of Y in either the D-configuration [for inhibitors having the 
(2S ,3R)-stereochemistry] or the L-configuration [for inhibitors having 
the (2R,3S)-stereochemistry]. To the Z-amino acid in ethyl acetate is 
added one equivalent of dicyclohexylcarbodiimide. Thirty minutes after 
addition, one equivalent of pyrazole is added and the mixture stirred for 
sixteen hours at 0.degree. C. After removal of dicyclohexylurea by 
filtration, the solvent is evaporated and the pyrazolide of the Z-amino 
acid (compound "1") is crystallized from ethyl acetate. To a solution of 1 
in dry tetrahydrofuran (THF) at -20.degree. C. is added two equivalents of 
LiAlH.sub.4 in THF over a period of 45 minutes followed by stirring at the 
same temperature for one hour. Excess reagent is then decomposed by the 
slow addition of 5N HCl. The solvent is evaporated and the residue 
extracted with ethyl acetate. The extract is washed with water and then 
dried to yield the benzyloxycarbonyl-amino acid aldehyde ("2"). An aqueous 
suspension of 2 is treated with two equivalents of NaHSO.sub.3 at 
60.degree. C. for two hours to form the NaHSO.sub.3 adduct ("3") which is 
then extracted with ethyl acetate followed by removal of the solvent by 
evaporation. To an aqueous suspension of 3 is added 1 equivalent of NaCN 
over a period of 1 hour to form the cyanohydrin ("4"). Compound 4 is 
extracted with ethyl acetate and the solvent evaporated. Compound 4 is 
then hydrolyzed by refluxing in 6N HCl to give 
3-amino-3-Y-2-hydroxy-propanoic acid ("5") as a mixture of 
diastereoisomers [either (2S,3R) and (2R,3R) or (2S,3S) and (2R,3S) 
depending on whether the starting material was the D-amino acid or L-amino 
acid, respectively]. The diastereoisomers are then separated by 
chromatography on Dowex 50WX4 using 0.1M pyridine-formic acid (pH 3.1). 
The (2S,3R) or (2R,3S) isomer of compound 5 is then converted to the 
N-t-BOC derivative by reaction with 
2-t-butyloxycarbonyloximino-2-phenylacetonitrile in aqueous 1,4-dioxane 
containing triethylamine [Bodanszky, M, and Bodanszky, A. (1984), The 
Practice of Peptide Synthesis, Springer-Verlag, Berlin]. The resulting 
t-BOC-(2S,3R)-3-amino-3-Y-2-hydroxypropanoic acid or 
t-BOC-(2R,3S)-3-amino-3-Y-2-hydroxypropanoic acid is then coupled to the 
Pro-X-resin as described for apstatin. 
Enzyme Kinetics--Aminopeptidase P activity was determined by measuring the 
release of arginine from Arg-Pro-Pro by a fluorescent assay described 
previously (14). The effect of apstatin on aminopeptidase P kinetics was 
determined by incubating the enzyme with various concentrations of 
Arg-Pro-Pro (0.2-1.25 mM), each in the presence of various concentrations 
of apstatin (0-160 .mu.M), in 0.1 M Hepes, pH 8.0, at 39.degree. C. Each 
mixture was run in duplicate. Aliquots were removed from each incubation 
mixture at various times and assayed for the amount of arginine present to 
determine the reaction velocity. In general, reaction velocities were 
calculated from data representing less than 20% cleavage of substrate. 
K.sub.i and K.sub.i ' (=.alpha.K.sub.i) were determined from plots of 1/v 
versus concentration of inhibitor "i" (26) and S/v versus concentration of 
inhibitor "i" (27), respectively. These parameters were also determined 
from the slope replot and 1/v-intercept replot, respectively, of the 1/v 
versus 1/S plot (28). See e.g., FIG. 1B. The values from the two 
treatments of the data were averaged for each experiment. Experiments for 
each enzyme preparation were performed twice and the results averaged. 
Specificity of Apstatin--Apstatin was assayed for its ability to inhibit 
several other peptidases. The concentration of apstatin needed to cause 
50% inhibition of enzyme activity (IC.sub.50) was determined in each case. 
Unless otherwise indicated, the source of each enzyme activity was a 
detergent extract of bovine lung prepared as described in (12-13). 
Activities were determined by a modification of the fluorescent assay 
described above (indicated by "FLUO") or by HPLC separation and 
quantitation of reaction products using the method described in (12) 
(method "HPLC-1") or the method described below (method "HPLC-2"). The 
enzymes, substrates, assay conditions, and detection methods were as 
follows: aminopeptidase A (EC 3.4.11.7) (rat lung microsomes), 1 mM 
.alpha.-Glu-.beta.-Na, 0.1M potassium phosphate ("KPhos") containing 1.1 
mM CaCl.sub.2 (pH 6.8), HPLC-1; aminopeptidase M (EC 3.4.11.2) (purified, 
porcine kidney), 0.6 mM Ala-.beta.-NA, 0.1 M KPhos (pH 6.8), by FLUO; ACE 
(EC 3.4.15.1), 0.5 mM Hip-His-Leu, 0.1M Hepes containing 0.3M NaCl (pH 
6.5), HPLC-1; dipeptidyl-peptidase 1-like activity CEC 3.4.14:1) (13), 0.5 
mM Gly-Phe-.beta.-naphthalarnide ("Gly-Phe-.beta.-NA"), 0.1M Hepes 
(containing 10 .mu.M NaCl, 12 .mu.M 2-mercaptoethanol, 0.17 mM amastatin, 
5.2% DMSO) (pH 6.5), HPLC-2; dipeptidyl-peptidase IV (EC 3.4.14.5), 0.5 nM 
Gly-Pro-.beta.-NA, 0.1M KPhos (pH 6.8), FLUO; 
bestatin-sensitive/amastatin-insensitive membrane dipeptidase (29) (bovine 
lung microsomes), 0.5 mM Arg-Trp, 0.1M KPhos containing 0.17 mM amastatin 
(pH 6.8), FLUO; membrane dipeptidase (EC 3.4.13.19) (purified, rat lung), 
0.5 mM Gly-D-Phe, 0.1M Hepes containing 20 .mu.M ZnCl.sub.2 (pH 8.0), 
HPLC-1; endopeptidase 24.11 (EC 3.4.24.11), 1 mM 
Suc-Ala-Ala-Phe-7-amido-4-methyl-coumarin, 0.1M KPhos containing 0.17 mM 
amastatin (pH 6.8), HPLC-1; endopeptidase 24.15 (EC 3.4.24.15) (rat brain 
synaptosomes)(21), 1 mM Bk(2-9), 0.1M KPhos (containing 50 .mu.M 
phosphoramidon, 20 .mu.M captopril, and 1 mM diprotin A) (pH 6.8), HPLC-1; 
prolyl oligopeptidase (EC 3.4.21.26) (purified, lamb kidney), 0.5 mM Bk, 
0.1M KPhos (containing 1 mM EDTA and 0.5 mM 2-mercaptoethanol) (pH 6.8), 
HPLC-1; prolidase (EC 3.4.13.9) (purified, porcine kidney), 1 mM Arg-Pro, 
0.1M Hepes (pH 8.0), HPLC-2. 
Perfusion of the Isolated Rat Lung--Male Sprague Dawley rats, weighing 
300-500 grams, were anesthetized with sodium pentobarbital (50-75 mg/kg) 
given by intraperitoneal injection. The trachea was cannulated and the 
lungs ventilated with air at 70 cycles per minute (Harvard Small Animal 
Ventilator) at a pressure of 70 mm H.sub.2 O. The heart and lungs were 
exposed via midsternal incision and 1000 units of heparin were 
administered intracardially. Polyethylene cannulae were secured into the 
pulmonary artery and the left ventricle. A modified Krebs buffer (118.5 mM 
NaCl, 4.7 mM KCl, 1.1 mM KH.sub.2 PO.sub.4, 1.1 mM MgSO.sub.4, 2.5 mM 
CaCl.sub.2, 19.6 mM NaHCO.sub.3, 5.6 mM dextrose), which was kept in a 
50.degree. C. water bath and aerated with 95% O.sub.2 -5% CO.sub.2 to pH 
7.35, was perfused into the pulmonary artery at 35.degree. C. and 2.6 
ml/min (LKB Microperplex peristaltic pump). The buffer was circulated 
through the isolated lung in situ until the effluent was free of blood. 
The surgical field was maintained at 37.degree. .C with a heat lamp 
throughout the procedure. Experiments were conducted by introducing 
[.sup.3 H]-Bk in the presence or absence of test peptidase inhibitors into 
the pulmonary circulation via the pulmonary artery and then collecting 
perfusion effluent from the left ventricular cannula. Ramiprilat (0.5 
.mu.M) or ramiprilat plus apstatin (4-40 .mu.M) in the modified Krebs 
buffer was perfused through the lung for two minutes. The lung was then 
perfused with 1 .mu.Ci [.sup.3 H]-Bk in 1 ml of the inhibitor solution 
(giving 16 nM Bk) followed by inhibitor solution alone. Effluent was 
collected in tubes (0.5 min/fraction) containing 150 .mu.l of 2% TFA. For 
each lung preparation, 5-6 samples were perfused over a period of about 
one hour. Control perfusions of [.sup.3 H]-Bk alone were always carried 
out before perfusions of samples containing ramiprilat because of the slow 
reversibility of inhibition of ACE by this compound. Additional 
experiments involving perfusion of apstatin or diprotin A alone with 
[.sup.3 H]-BK were similarly run before ramiprilat-containing samples for 
the same reason. The effects of the latter two inhibitors were reversible. 
Occasionally a lung failed to completely degrade the perfused [.sup.3 
H]-Bk in the absence of inhibitors. Only lungs which showed complete 
cleavage during the control perfusion were used for quantitative analysis. 
Separation and Identification of Bradykinin Metabolites--The fractions 
which were collected from each perfusion sample run were counted for 
radioactivity, and the fraction having the highest radioactive counts was 
prepared for HPLC by filtration through a Centricon-10 microconcentrator 
(Amicon). An aliquot of the filtrate containing 25000 dpm (approx. 25 
.mu.l) was spiked with a cocktail of unlabelled Bk fragments (3 .mu.l) and 
then subjected to HPLC using a Waters Radial Compression Separation System 
with a 5.mu. NOVA PAK C.sub.18 Radial Pak Cartridge. The column was 
developed at a flow rate of 1 ml/min with a 60 min linear gradient from 
100% Solvent A (0.1% TFA in water) to 75% Solvent A: 25% Solvent B (0.08% 
TFA in acetonitrile) using a Spectra Physics 8700XR Solvent Delivery 
System. The column effluent was simultaneously monitored for absorbance at 
206 nm (LKB Uvicord S) and for radioactivity (Radiomatic Flo-One HS 
Radioactive Flow Detector using Ultima-Flo M scintillation fluid from 
Packard, Meriden, Conn.). The results were displayed on an LKB 2-channel 
chart recorder, with the radioactive counts indicated at six-second 
intervals. Radioactive peaks were identified by comparison of the 
retention times to those of the unlabelled standards. The digital output 
from the radioactive flow detector was used to calculate the percentage of 
dpm present in each Bk metabolite. The HPLC method gave the following 
retention times in minutes for the indicated Bk fragments which contain 
the position 2 and/or 3 proline residues: Pro,.about.0; Bk(1-2), 1.4; 
Bk(2-3), 4.3; Bk(2-4), 5.5; Bk(1-3), 6.4; Bk(1-4), 6.9; cyclo-Pro-Pro, 
8.2; Bk(2-6), 19.1; Bk(1-6), 20.9; Bk(2-5), 23.4; Bk(1-5), 24.8; Bk (2-7) 
27.2; Bk(1-7), 27,9; Bk(1-9), 41.1; Bk(2-9), 44.4; Bk(1-8), 45.5; and Bk 
(2-8), 49.7 minutes. 
Calculation of the Relative Rates of Bk Degradation by Aminopeptidase P and 
ACE--An estimation of the relative rates of cleavage of Bk by 
aminopeptidase P and ACE in the isolated perfused lung was calculated 
based on assumptions concerning the extent of degradation of Bk in the 
absence of inhibitors. Degradation of Bk in the perfused lung was assumed 
to follow Michaelis-Menten kinetics (30) and to be first-order since the 
concentration of [.sup.3 H]-Bk (16 nM) was very much less than the K.sub.m 
value for either aminopeptidase P or ACE (14, 31). The first-order rate 
constant for the cleavage of Bk by aminopeptidase P [(V.sub.max 
/K.sub.m).sub.APP ] is given by Equation 1: 
##EQU1## 
where t is the time of perfusion (capillary transit time) and H.sub.APP is 
the % hydrolysis of Bk at time t when ACE is completely inhibited, i.e., 
when aminopeptidase P is the only active enzyme (78%, see below). Equation 
2 is the integrated form of the equation describing two enzymes acting on 
a single substrate (32) under first-order conditions. 
##EQU2## 
where (V.sub.max /K.sub.m).sub.ACE is the first-order rate constant for 
ACE. H.sub.APP+ACE is the % hydrolysis of Bk at time t when both enzymes 
are hydrolyzing Bk simultaneously (no inhibitors present). Since 
H.sub.APP+ACE approached 100% and could not be determined accurately, 
different estimates of H.sub.APP+ACE were used based on in vivo 
experiments (see below). The relative contribution of aminopeptidase P to 
the overall cleavage of Bk in the isolated perfused lung is given by the 
ratio of (V.sub.max /K.sub.m).sub.APP to [(V.sub.max /K.sub.m).sub.APP 
+(V.sub.max /K.sub.m).sub.ACE ], Equation 1 divided by Equation 2. In this 
ratio, the unknown quantity t disappears. 
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