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Total serum angiotensin converting enzyme activity in rats and dogs after enalapril maleate (MK-421)
Pergamon Press
Life Sciences, Vol. 30, pp. 1225-1230 Printed in the U.S.A.
TOTAL SERUM ANGIOTENSIN CONVERTING ENZYME ACTIVITY IN RATS AND DOGS AFTER ENALAPRIL MALEATE (MK-421) E. H. Ulm and T. C. Vassil Merck Institute for Therapeutic Research West Point, PA 19486 (Received in final form January 28, 1982) Summary A centrifugal gel filtration separation of serum angiotensin converting enzyme (ACE) from a potent stable inhibitor is described. This, together with a 20 hr assay incubation of very dilute enzyme, permitted the assessment of the effects of enalapril maleate treatment on total serum ACE in rats and dogs. Total serum ACE increased in both species after 1 or 2 weeks at 10 mgfkgfday. Serum ACE in rats was more than doubled; whereas the increase was modest in dogs (48: 9% minimum). The effect of the drug on serum ACE combined with inherent variability of ACE precludes use of serum ACE activity as an accurate measure of inhibitor concentration in animals receiving enalapril maleate. Prolonged captopril treatment has been reported to lead to increases in ACE in both plasma (1) and lungs (1,2) of rats. A similar effect may be evident in man (3). Measurement of total ACE in samples containing captopril has proved relatively easy due to captopril's instability (4,5) and the ease with which it can be inactivated as an inhibitor by reaction with agents such as N-ethyl maleimide (6, and Ulm, unpublished observations). A new, non-sulfhydryl converting enzyme inhibitor (MK-422, N-[l(S)-carboxy-3-phenylpropyl]-L-alanyl-~-proline (7)), which arises -in vivo from hydrolysis.of the monoethylester enalapril, (MK-421), introduces the problem of inhibitor stability into the measurement of total ACE (8,9). The procedure described for measurement of total ACE in samples containing MK-422 takes into account the biochemical properties of the enzyme inhibitor interactions, e.g., Ki 10-l"M and a very slow dissociation rate constant. Materials and Methods Partially purified human plasma angiotensin converting enzyme was supplied by H. Schwamm and S. Michelson of Merck Sharp & Dohme Research Laboratories. The enzyme activity was chloride dependent, inhibited by EDTA and MK-422 and yielded HisLeu as product when incubated with benzyloxycarbonyl-phenylalanyl-histidyl-leucine (Z-PheHisLeu from Bachem Inc., Torrence CA). Sephadex G-25-40 (Sigma Chem., St. Louis) was hydrated for at least 18 hr in 0.05M All procedures were carried out at ambient Tris HCl, 0.3M NaCl pH 7.65. temperature unless noted otherwise. Columns of the gel (2 ml bed) were prepared in Sarpettes 91.787 (Walter Sartedt, Princeton, NJ). The Sarpettes were plugged with 4 mm solid glass beads, and 4 ml aliquots of a 50% settled volume slurry of G-25 in 0.05 Tris HCl, 0.3M NaCl pH 7.65 were carefully pipetted over the beads. 0024-3205/82/141225-06$03.00/O Copyright (c) 1982 Pergamon Press Ltd.
1226
ACE After
Serum
allowed
to
drain
Vol.
MK-4:I
The columns
were
Immediately polypropylene discarded.
prior to sample application, tubes and centrifuged at
30,
No.
14,
1982
completely. they were 380 x g for
placed 5 min.
inside The
i3 x 100 mm effluent was
A 50 ~1 serum sample was dililted to 1 ml with Tris NaCl buffer and allowed to stand for 3 hr. A 500 1.11 aliquot of this was placed on a centrifuged G-25 column and this effluent was subjected IJnless otherwise no ted, the centrifugation repeated. to an additional gel filtration step. The effluent from the second column was assayed for protein by the Biorad Protein and an aliquot diluted lo-fold with Tris NaCl buffer for Assay Kit method (91, assay of converting enzyme activity by modifications of the method of Piquillaud et al. (11). treated serum and 200~1 of 0.74 mM The assay mix contained 100 ul of the diluted, The tubes were capped with Z-PheHisLeu (0.49 mM final, in buffer, ca 4 x Km). The reaction was terminated with addition Parafilm and incubated for 20 hr at 30°. of 1.65 ml OPD-NaOH reagent. To prepare this reagent, a 2% in methanol solution of OPD (orthophthaldialdehyde, Sigma Chemicals, St. Louis, MO) was filtered through a 0.2 J_rrnfilter (Nucleopore, Pleasanton, CA) and diluted with 0.28 N NaOH (20 ml OPD plus 145 ml NaOH). After an additional 20-60 min 0.2 ml of 3N HCl was added and the fluorescence at 490 nm (excitation at 365 nm) determined on an Aminco SPF 125 spectrofluorometer. The interval between addition of the hydrochloric acid and reading of fluoresence was kept constant for all samples in an assay. Activity is expressed as nmoles HisLeu hr- 1 (mg protein)-1. Histidyl-leucine used as standard was from Calbiochem (LaJolla, CA). Tritiated MK-422 was prepared by base hydrolysis (1N NaOH for 10 min) of 3H-enalapril (2,3[3H]phenylpropyl) maleate supplied by the Merck Sharp & Dohme Research Laboratories, Rahway, NJ. Radioactivity was determined by liquid scintillation counting in PCS@ (Amersham Searle, Arlington Heights, IL). in water (5 mg free base/ml) Enalapril maleate (MSDRL, Rahway, NJ) was dissolved and administered on a once-daily basis by gavage (2 ml/kg for a dose of 10 mg/kg) After 7 or 14 days of to male Sprague Dawley rats (Charles River 175-225 g). treatment, they were decapitated and blood was collected for preparation of serum. Placebo treatment was 2 ml/kg of water by gavage once a day. Enalapril maleate capsules to male serum was obtained
30 mg/kg or 10 mg/kg at either beagles (10.1-12.5 kg) once daily. by venapuncture. Results
was
administered in gelatin Blood for preparation of
and Discussion
The separation of serum ACE from any stable inhibitor is required if total ACE activity is to be assessed. The tightness of binding of MK-422 to converting enzyme, primarily due to a slow off-rate of the inhibitor from the enzyme (Bull, presented a particular problem. For this reason, a 3 hr equiunpublished), It was thought that desalting, libration was allowed after the initial dilution. the inhibitor concentration to a level less than using Sephadex G-25, would reduce A further dilution of the desalted enzyme or equal to the enzyme concentration. would then yield a sample in which inhibitor concentration was sufficiently lower than KI as to be ineffectual. To test the validity of this approach, 3H-MK-422 was added to a serum sample and the concentration of inhibitor was determined at each Table I presents the data obtained in this experiment along step of the procedure.
Serum ACE After MK-421
Vol. 30, No. 14, 1982
1227
with calculations of the theoretical concentrations based on the assumptions of a total enzyme concentration in serumof 5 x 10-9M and a complete separationof bound from free inhibitor. For the calculations, 2 x 10-lo~ was used for KI.
TABLE I Separation of MK-422 from ACE
Step
Serum
Calculated Itotal
---
Measured Itotal+
Ef"/Et
3.25 x 10-7M
20-fold dilution through 1st G-25 step
2.5 x lo-l"M
4.6
x lo-l"M
0.39
Through 2nd G-25 step
3.9 X 10-llM
7.4
x lo-"M
0.85
Assay
2.5 x 10-12;'"
---
0.99
+
Radioactivity measurement
*
Ef (free enzyme) calculated as a root of 0 = Ef2 + (It-Et-KI) Ef-KIEt where It (total inhibitor) is the measured value. Et is the assumed enzyme concentration.
*:k Based on measured Itotal of 2nd G-25 step.
Stability of ACE activity over the prolonged incubations required by the procedure was examined by incubating serum diluted l/30, l/60 and l/600 for 1 hr 2 hr and 20 hr, respectively. In one such experiment the specific activities measured were 9.3, 4.6 and 0.49 nmoles HisLeu hi--l, respectively. The direct proportionality with time indicated that the enzyme had sufficient stability to be assayed over a 20 hr incubation. Linearity with enzyme concentration was determined by addition of known aliquots of purified human ACE to a serum sample. Additionally, MK-422 was added to each of these serum samples over a range of concentrations of clinical interest. ACE activity was determined before and after a single centrifugal gel filtration (figures 1 and 2). The concentrations of MK-422 in the assay done before G-25 treatment are 11600th the values shown for the original concentrations. Linearity with enzyme concentration was seen in all cases; but, as would be expected, presence of MK-422 depressed the activity observed. After a single gel filtration, only the samples containing the highest original concentration of inhibitor remained inhibited. Since two gel filtrations could be demonstrated to remove even that concentration, all sera from experimental animals were taken through two gel filtrations. Rats which had been treated with 10 mg/kg enalapril maleate p.o. daily for lor 2 weeks were found to have sharply elevated serum ACE levels (Table II). The magnitude of the enalapril effect is similar to that reported for captopril in rats (1).
Serum ACE After MK-421
1228
Vol. 30, No. 14, 1982
b SERUH
I
I
1 VOL
2 VOL
PURIFIED
PURIFIED
ACE
ACE
FIG. 1 ACE assay of human serum containing MK-422 without G-25 treatment. The concentrations of MK-422 indicated for each line were those in the original samples prepared. Assay concentrations are 1/600th of these.
SERW
PURIFIED
2 VOL PURIFIED
ACE
ACE
1 VOL
FIG. 2 ACE assay of human Serumcontaining MK-422 after a single G-25 treatment. Only the sample containing the highest original MK-422 concentration (3 x 10e6M) demonstrated inhibition.
vol. 30, No. 14, 1982
Serum ACE After MK-421
1229
TABLE II Effect of Enalapril Maleate on Total Serum ACE in Rat
ACE nmole His-Leu hr-l (mg prot) -1
Treatment
1 week
2 week
Placebo (n = 6)
123 + 17
102 + 15
Enalapril maleate d. 10 mg/kg p.o., o.d. (n = 12)
220 + 60
245 + 23
Figure 3 presents data obtained from 3 dogs over 2 weeks of 10 mglkg per day of enalapril maleate. Analysis of the data indicates a 31: 7.5% increase in total serum ACE after 1 week and 482 9.0% increase after 2 weeks. The increases were significant at HO.025 using paired t test. The enalapril effect in dog was clearly much less than that of the rat.
FIG. 3 Effect of enalapril maleate on total serum ACE in dogs. Three dogs received 10 mg/kg enalapril maleate p.o., o.d. Data from each dog are plotted separately.
1230
Serum
ACE After
m-421
Vol.
30, No.
14, 1982
Although it may be attractive to speculate that measurement of ACE (without separation from inhibitor) can provide estimates of inhibitor concentrations in plasmas or sera of patients receiving enalapril maleate (9,121, the range of normal activity and the effects of ACE inhibitors on enzyme concentrations compromise such estimates. Attempts to show a close correlation between % predose ACE and log inhibitor concentration in human studies with enalapril have revealed considerable scatter of points (13). It may be that in addition to intersubject variation, human serum ACE may also increase in the presence of inhibitor. The fact that % predose ACE activity is related to the log of inhibitor concentration means that the error in measurement of ACE is compounded when used to deduce inhibitor concentration. Because MK-422 arising from enalapril maleate in viva isolation bytnmtion can easily be measured after radioimmunoassay (15). Where concentrations of ACE inhibitor are to be assessed, the procedure presented forward approach.
is a stable inhibitor, it of ACE (14) or by in presence of a stable here-provides a straight-
References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11.
T.E. KOKUBU, M. UEDA, M. ONO, T. KAWABE, Y. HAYASHI AND T. KAN, Eur. J. Pharm. 62: 269-275 (1980). F. FYHRQLJIST, T. FORSLUND, I. TIKKANEN and C. GRONHAGEN-RISKA, Eur. J. Pharm. 3, 473-475 (1980). P. LAROCHELLE, J. GENEST, 0. KUCHEL, R. ROUCHER, Y. GUTKOWSKA, and D. MCKINSTRY, Can. Med. Assoc. J. 121, 309-316 (1979). F. M. LAI, T. TANIKELLA, H. HERZLINGER and P. CERVONI, Eur. .J. Pharm. -67: 9799 (1980). K. .I. KRIPALANI, D. N. McKINSTRY, S. M. SINGHVI, D. A. WILLARD, R. A. VUKOVICH, and B. H. MIGDALOF, Clin. Pharmacol. Ther. 27: 636-641 (1980). F. H. M. DE@?X and M. A. D. H. SCHALEKAMP, F. BOOMSMA, J. H. B. deBRUYN, Clinical Science 60: 491-498 (1981). A. A. PATCHETT, E?lARRIS, E. W. TRISTRAM, M. J. WYRATT, M. T. WU, D. TAUB, E. R. PETERSON, T. J. IKELER, J. tenBROEKE, I,. G. PAYNE, D. L. ONDEYKA, E. D. THORSETT, W. 3. GREENLEE, N. S. LOHR, R. D. HOFFSOMMER, H. JOSHUA, W. V. RUYLE, J. W. ROTHROCK, S. D. ASTER, A. L. MAYCOCK, F. M. ROBINSON, R. HIRSCHMANN, C. S. SWEET, E. H. ULM, D. M. GROSS, T. C. VASSIL and C. A. STONE, Nature 288: 280-283 (1980). D. B. BRUNNER, G. DESPONDS, .I. BIOLLAZ, I. KELLER, F. FERBER, H. GAVRAS, H. R. 11: 461-467 (1981). BRUNNER and J. L. SCHELLING, Br. .I. Pharmac. T. ANDERSON, K. KURZ, M. L. COHEN, New Eng. J-of Med. 304: 1550 (1981). M. M. BRADFORD, Anal. Biochem. 72: 248-254 (1976). Y. PIQUILLAUD, A. REINHARZ and z ROTH, Biochim. Biophys. Acta 206: 136-142
(1970). (1980). 12. M. A. PETTY, J. L. REID, and S. H. K. MILLER, Life Sci. 26: 2045-2050 P. MOJAVERIAN, R.K. FERGUSON2.H. VLASSES AND 13. B.N. SWANSON, M. HICHENS, M. DUDASH, Res. Comm. Chem. Path. Pharm. 33: 525-536 (1981). C. VASSIL and E. H. ULM, Drug 14. D. J. TOCCO, F. A. deLUNA, A. E. W. DUNCAKT. (in press). Met. and Dispos. Ligand Quarterly 4,43 (1981). 15. M. HICHENS, E. L. HAND, M. and W. S. MULCAHY,
Life Sciences, Vol. 30, pp. 1225-1230 Printed in the U.S.A.
TOTAL SERUM ANGIOTENSIN CONVERTING ENZYME ACTIVITY IN RATS AND DOGS AFTER ENALAPRIL MALEATE (MK-421) E. H. Ulm and T. C. Vassil Merck Institute for Therapeutic Research West Point, PA 19486 (Received in final form January 28, 1982) Summary A centrifugal gel filtration separation of serum angiotensin converting enzyme (ACE) from a potent stable inhibitor is described. This, together with a 20 hr assay incubation of very dilute enzyme, permitted the assessment of the effects of enalapril maleate treatment on total serum ACE in rats and dogs. Total serum ACE increased in both species after 1 or 2 weeks at 10 mgfkgfday. Serum ACE in rats was more than doubled; whereas the increase was modest in dogs (48: 9% minimum). The effect of the drug on serum ACE combined with inherent variability of ACE precludes use of serum ACE activity as an accurate measure of inhibitor concentration in animals receiving enalapril maleate. Prolonged captopril treatment has been reported to lead to increases in ACE in both plasma (1) and lungs (1,2) of rats. A similar effect may be evident in man (3). Measurement of total ACE in samples containing captopril has proved relatively easy due to captopril's instability (4,5) and the ease with which it can be inactivated as an inhibitor by reaction with agents such as N-ethyl maleimide (6, and Ulm, unpublished observations). A new, non-sulfhydryl converting enzyme inhibitor (MK-422, N-[l(S)-carboxy-3-phenylpropyl]-L-alanyl-~-proline (7)), which arises -in vivo from hydrolysis.of the monoethylester enalapril, (MK-421), introduces the problem of inhibitor stability into the measurement of total ACE (8,9). The procedure described for measurement of total ACE in samples containing MK-422 takes into account the biochemical properties of the enzyme inhibitor interactions, e.g., Ki 10-l"M and a very slow dissociation rate constant. Materials and Methods Partially purified human plasma angiotensin converting enzyme was supplied by H. Schwamm and S. Michelson of Merck Sharp & Dohme Research Laboratories. The enzyme activity was chloride dependent, inhibited by EDTA and MK-422 and yielded HisLeu as product when incubated with benzyloxycarbonyl-phenylalanyl-histidyl-leucine (Z-PheHisLeu from Bachem Inc., Torrence CA). Sephadex G-25-40 (Sigma Chem., St. Louis) was hydrated for at least 18 hr in 0.05M All procedures were carried out at ambient Tris HCl, 0.3M NaCl pH 7.65. temperature unless noted otherwise. Columns of the gel (2 ml bed) were prepared in Sarpettes 91.787 (Walter Sartedt, Princeton, NJ). The Sarpettes were plugged with 4 mm solid glass beads, and 4 ml aliquots of a 50% settled volume slurry of G-25 in 0.05 Tris HCl, 0.3M NaCl pH 7.65 were carefully pipetted over the beads. 0024-3205/82/141225-06$03.00/O Copyright (c) 1982 Pergamon Press Ltd.
1226
ACE After
Serum
allowed
to
drain
Vol.
MK-4:I
The columns
were
Immediately polypropylene discarded.
prior to sample application, tubes and centrifuged at
30,
No.
14,
1982
completely. they were 380 x g for
placed 5 min.
inside The
i3 x 100 mm effluent was
A 50 ~1 serum sample was dililted to 1 ml with Tris NaCl buffer and allowed to stand for 3 hr. A 500 1.11 aliquot of this was placed on a centrifuged G-25 column and this effluent was subjected IJnless otherwise no ted, the centrifugation repeated. to an additional gel filtration step. The effluent from the second column was assayed for protein by the Biorad Protein and an aliquot diluted lo-fold with Tris NaCl buffer for Assay Kit method (91, assay of converting enzyme activity by modifications of the method of Piquillaud et al. (11). treated serum and 200~1 of 0.74 mM The assay mix contained 100 ul of the diluted, The tubes were capped with Z-PheHisLeu (0.49 mM final, in buffer, ca 4 x Km). The reaction was terminated with addition Parafilm and incubated for 20 hr at 30°. of 1.65 ml OPD-NaOH reagent. To prepare this reagent, a 2% in methanol solution of OPD (orthophthaldialdehyde, Sigma Chemicals, St. Louis, MO) was filtered through a 0.2 J_rrnfilter (Nucleopore, Pleasanton, CA) and diluted with 0.28 N NaOH (20 ml OPD plus 145 ml NaOH). After an additional 20-60 min 0.2 ml of 3N HCl was added and the fluorescence at 490 nm (excitation at 365 nm) determined on an Aminco SPF 125 spectrofluorometer. The interval between addition of the hydrochloric acid and reading of fluoresence was kept constant for all samples in an assay. Activity is expressed as nmoles HisLeu hr- 1 (mg protein)-1. Histidyl-leucine used as standard was from Calbiochem (LaJolla, CA). Tritiated MK-422 was prepared by base hydrolysis (1N NaOH for 10 min) of 3H-enalapril (2,3[3H]phenylpropyl) maleate supplied by the Merck Sharp & Dohme Research Laboratories, Rahway, NJ. Radioactivity was determined by liquid scintillation counting in PCS@ (Amersham Searle, Arlington Heights, IL). in water (5 mg free base/ml) Enalapril maleate (MSDRL, Rahway, NJ) was dissolved and administered on a once-daily basis by gavage (2 ml/kg for a dose of 10 mg/kg) After 7 or 14 days of to male Sprague Dawley rats (Charles River 175-225 g). treatment, they were decapitated and blood was collected for preparation of serum. Placebo treatment was 2 ml/kg of water by gavage once a day. Enalapril maleate capsules to male serum was obtained
30 mg/kg or 10 mg/kg at either beagles (10.1-12.5 kg) once daily. by venapuncture. Results
was
administered in gelatin Blood for preparation of
and Discussion
The separation of serum ACE from any stable inhibitor is required if total ACE activity is to be assessed. The tightness of binding of MK-422 to converting enzyme, primarily due to a slow off-rate of the inhibitor from the enzyme (Bull, presented a particular problem. For this reason, a 3 hr equiunpublished), It was thought that desalting, libration was allowed after the initial dilution. the inhibitor concentration to a level less than using Sephadex G-25, would reduce A further dilution of the desalted enzyme or equal to the enzyme concentration. would then yield a sample in which inhibitor concentration was sufficiently lower than KI as to be ineffectual. To test the validity of this approach, 3H-MK-422 was added to a serum sample and the concentration of inhibitor was determined at each Table I presents the data obtained in this experiment along step of the procedure.
Serum ACE After MK-421
Vol. 30, No. 14, 1982
1227
with calculations of the theoretical concentrations based on the assumptions of a total enzyme concentration in serumof 5 x 10-9M and a complete separationof bound from free inhibitor. For the calculations, 2 x 10-lo~ was used for KI.
TABLE I Separation of MK-422 from ACE
Step
Serum
Calculated Itotal
---
Measured Itotal+
Ef"/Et
3.25 x 10-7M
20-fold dilution through 1st G-25 step
2.5 x lo-l"M
4.6
x lo-l"M
0.39
Through 2nd G-25 step
3.9 X 10-llM
7.4
x lo-"M
0.85
Assay
2.5 x 10-12;'"
---
0.99
+
Radioactivity measurement
*
Ef (free enzyme) calculated as a root of 0 = Ef2 + (It-Et-KI) Ef-KIEt where It (total inhibitor) is the measured value. Et is the assumed enzyme concentration.
*:k Based on measured Itotal of 2nd G-25 step.
Stability of ACE activity over the prolonged incubations required by the procedure was examined by incubating serum diluted l/30, l/60 and l/600 for 1 hr 2 hr and 20 hr, respectively. In one such experiment the specific activities measured were 9.3, 4.6 and 0.49 nmoles HisLeu hi--l, respectively. The direct proportionality with time indicated that the enzyme had sufficient stability to be assayed over a 20 hr incubation. Linearity with enzyme concentration was determined by addition of known aliquots of purified human ACE to a serum sample. Additionally, MK-422 was added to each of these serum samples over a range of concentrations of clinical interest. ACE activity was determined before and after a single centrifugal gel filtration (figures 1 and 2). The concentrations of MK-422 in the assay done before G-25 treatment are 11600th the values shown for the original concentrations. Linearity with enzyme concentration was seen in all cases; but, as would be expected, presence of MK-422 depressed the activity observed. After a single gel filtration, only the samples containing the highest original concentration of inhibitor remained inhibited. Since two gel filtrations could be demonstrated to remove even that concentration, all sera from experimental animals were taken through two gel filtrations. Rats which had been treated with 10 mg/kg enalapril maleate p.o. daily for lor 2 weeks were found to have sharply elevated serum ACE levels (Table II). The magnitude of the enalapril effect is similar to that reported for captopril in rats (1).
Serum ACE After MK-421
1228
Vol. 30, No. 14, 1982
b SERUH
I
I
1 VOL
2 VOL
PURIFIED
PURIFIED
ACE
ACE
FIG. 1 ACE assay of human serum containing MK-422 without G-25 treatment. The concentrations of MK-422 indicated for each line were those in the original samples prepared. Assay concentrations are 1/600th of these.
SERW
PURIFIED
2 VOL PURIFIED
ACE
ACE
1 VOL
FIG. 2 ACE assay of human Serumcontaining MK-422 after a single G-25 treatment. Only the sample containing the highest original MK-422 concentration (3 x 10e6M) demonstrated inhibition.
vol. 30, No. 14, 1982
Serum ACE After MK-421
1229
TABLE II Effect of Enalapril Maleate on Total Serum ACE in Rat
ACE nmole His-Leu hr-l (mg prot) -1
Treatment
1 week
2 week
Placebo (n = 6)
123 + 17
102 + 15
Enalapril maleate d. 10 mg/kg p.o., o.d. (n = 12)
220 + 60
245 + 23
Figure 3 presents data obtained from 3 dogs over 2 weeks of 10 mglkg per day of enalapril maleate. Analysis of the data indicates a 31: 7.5% increase in total serum ACE after 1 week and 482 9.0% increase after 2 weeks. The increases were significant at HO.025 using paired t test. The enalapril effect in dog was clearly much less than that of the rat.
FIG. 3 Effect of enalapril maleate on total serum ACE in dogs. Three dogs received 10 mg/kg enalapril maleate p.o., o.d. Data from each dog are plotted separately.
1230
Serum
ACE After
m-421
Vol.
30, No.
14, 1982
Although it may be attractive to speculate that measurement of ACE (without separation from inhibitor) can provide estimates of inhibitor concentrations in plasmas or sera of patients receiving enalapril maleate (9,121, the range of normal activity and the effects of ACE inhibitors on enzyme concentrations compromise such estimates. Attempts to show a close correlation between % predose ACE and log inhibitor concentration in human studies with enalapril have revealed considerable scatter of points (13). It may be that in addition to intersubject variation, human serum ACE may also increase in the presence of inhibitor. The fact that % predose ACE activity is related to the log of inhibitor concentration means that the error in measurement of ACE is compounded when used to deduce inhibitor concentration. Because MK-422 arising from enalapril maleate in viva isolation bytnmtion can easily be measured after radioimmunoassay (15). Where concentrations of ACE inhibitor are to be assessed, the procedure presented forward approach.
is a stable inhibitor, it of ACE (14) or by in presence of a stable here-provides a straight-
References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11.
T.E. KOKUBU, M. UEDA, M. ONO, T. KAWABE, Y. HAYASHI AND T. KAN, Eur. J. Pharm. 62: 269-275 (1980). F. FYHRQLJIST, T. FORSLUND, I. TIKKANEN and C. GRONHAGEN-RISKA, Eur. J. Pharm. 3, 473-475 (1980). P. LAROCHELLE, J. GENEST, 0. KUCHEL, R. ROUCHER, Y. GUTKOWSKA, and D. MCKINSTRY, Can. Med. Assoc. J. 121, 309-316 (1979). F. M. LAI, T. TANIKELLA, H. HERZLINGER and P. CERVONI, Eur. .J. Pharm. -67: 9799 (1980). K. .I. KRIPALANI, D. N. McKINSTRY, S. M. SINGHVI, D. A. WILLARD, R. A. VUKOVICH, and B. H. MIGDALOF, Clin. Pharmacol. Ther. 27: 636-641 (1980). F. H. M. DE@?X and M. A. D. H. SCHALEKAMP, F. BOOMSMA, J. H. B. deBRUYN, Clinical Science 60: 491-498 (1981). A. A. PATCHETT, E?lARRIS, E. W. TRISTRAM, M. J. WYRATT, M. T. WU, D. TAUB, E. R. PETERSON, T. J. IKELER, J. tenBROEKE, I,. G. PAYNE, D. L. ONDEYKA, E. D. THORSETT, W. 3. GREENLEE, N. S. LOHR, R. D. HOFFSOMMER, H. JOSHUA, W. V. RUYLE, J. W. ROTHROCK, S. D. ASTER, A. L. MAYCOCK, F. M. ROBINSON, R. HIRSCHMANN, C. S. SWEET, E. H. ULM, D. M. GROSS, T. C. VASSIL and C. A. STONE, Nature 288: 280-283 (1980). D. B. BRUNNER, G. DESPONDS, .I. BIOLLAZ, I. KELLER, F. FERBER, H. GAVRAS, H. R. 11: 461-467 (1981). BRUNNER and J. L. SCHELLING, Br. .I. Pharmac. T. ANDERSON, K. KURZ, M. L. COHEN, New Eng. J-of Med. 304: 1550 (1981). M. M. BRADFORD, Anal. Biochem. 72: 248-254 (1976). Y. PIQUILLAUD, A. REINHARZ and z ROTH, Biochim. Biophys. Acta 206: 136-142
(1970). (1980). 12. M. A. PETTY, J. L. REID, and S. H. K. MILLER, Life Sci. 26: 2045-2050 P. MOJAVERIAN, R.K. FERGUSON2.H. VLASSES AND 13. B.N. SWANSON, M. HICHENS, M. DUDASH, Res. Comm. Chem. Path. Pharm. 33: 525-536 (1981). C. VASSIL and E. H. ULM, Drug 14. D. J. TOCCO, F. A. deLUNA, A. E. W. DUNCAKT. (in press). Met. and Dispos. Ligand Quarterly 4,43 (1981). 15. M. HICHENS, E. L. HAND, M. and W. S. MULCAHY,