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Absorption, metabolism, and excretion studies of carbon 14- and tritium-labeled derivatives of a ketomethylene containing tripeptide
Life Sciences, Vol. 37, pp. 299-305 Printed in the U.S.A.
Pergamon Press
ABSORPTION, METABOLISM, AND EXCRETION STUDIES OF CARBON 14- AND TRITIUM-LABELED DERIVATIVES OF A KETOMETHYLENE CONTAINING TRIPEPTIDE Ronald G. Almquist,
Thomas Steeger,
Stewart Jackson, and Chozo Mitoma
Bio-Organic Chemistry Laboratory and Biomedical Research Laboratory, SRI International, Menlo Park, California 94025 (Received in final form May 13, 1985)
Summary Tritium and Carbon 14 analogs of the angiotensin converting enzyme inhibitor ketoACE were synthesized and their oral absorption, metabolism and excretion in rats were investigated. KetoACE, a ketomethylene analog of the tripeptide Bz-Phe-Gly-Pro, was slowly absorbed at a 35% level upon oral administration. It is rapidly eliminated from the blood with a half-life of about I0 minutes. Its excretion is primarily via the bile duct and it is excreted as 80% unchanged drug. The only identified metabolite consisting of 5-10% of the excreted radioactivity was determined to be the reduced ketoACE in which the ketone group was reduced to a hydroxyl.
Previous studies of the ketomethylene trlpeptide analogue, 5(S)benzamido-4-oxo-6-phenylhexanoyl-L-proline (ketoACE) have shown it to be a potent angiotensin converting enzyme (ACE) inhibitor (Isu = 70 nM) (i) with poor antihypertensive activity in the renal hypertensive rat (2,3). The insertion of a ketomethylene linkage in place of the normal amide linkage connecting Phe and Gly in the tripeptide Bz-Phe-Gly-Pro was expected to yield a tripeptide analogue with good metabolic stability.
COOH
~_
~>_~,~-~-~~..~ o
~ o
o
ketoACE
TO investigate the reason for the poor in vivo activity of ketoACE, two radiolabeled derivatives of it were synthesized and used for studies in the rat. The first derivative H3-ketoACE contained tritium in the 3 and 4 positions of the proline ring. The second derivative contained carbon 14 at the carbonyl carbon of the benzamido group. Using these two radiolabeled derivatives, the stabilities of the benzoyl amide linkage and the proline amide linkage following oral or intravenous administration in a rat were inves tiga ted.
0024-3205/85 $3.00 + o00 Copyright (c) 1985 Pergamon Press Ltd.
300
Metabolism Studies of KetoACE
0 o
cooH 0 ~.~ a H
CH2 0
Vo[. 37, No. 4, 1985
0 ~
0
3H_ketoACE
cooH 0 ~.~
CH2 0
1"C-ketoACE
Chemical Methods Figure I shows the chemical pathway used to prepare the tritium labeled ketoACE analogue. Tritiated L-proline from Amersham was adjusted to 5 mCi/mmole by addition of dissolved unlabeled L-proline and evaporating to dryness.
cooH
CH20H
+
o
COCH2"
HN
~
HCI'H
3H
0 CH2 0 0 , i , N CNH--CH--CCH2CH2COH
~,**~I'-.3H
~ aH HCI.HN + k~3,
+
Et3N
+
DCC
1
© o
CH20
0 }~
_3H
H2[I0% Pd/C
©
o C0H
~-~-~N" Figure
-]/"
I
A standard m e t h o d was u s e d to p r e p a r e the L-proliae benzyl ester i n 70% y i e l d . This benzyl ester was u s e d a s d e s c r i b e d previously the desired 3H-ketoACE. C r u d e 3 H - k e t o A C E was c o m b i n e d w i t h c o l d
crystallized to yield pure 311-ketoACE with a specific activity of 3.04 mCi/mmole.
hydrochloride (1) to yield ketoACE and
Vol.
37, No. 4, 1985
Metabolism
Studies
of KetoACE
0
301
©
CN2 0 0 HCI.NH2__CH__CCH2CH2COCH3 +
0 1. Cl
0 CH~ 0 0 I. N'H-- -- CH2CH20CH~
) Na2C03
I NaOH
© ~
o
©
~=0
o
O
~
COCH=
ok_
" I II n f ~ ="CNH--CH--CCH=CHmCN~
o
'
~H~o
o
~--I~CNH--¢H--CCH2CH2COH Et3N 'I
+ DCC
COOH
Figure
II
Figure II outlines t h e s y n t h e t i c p a t h w a y u s e d to p r e p a r e 14 C-ke toACE. The p r e p a r a t i o n of t h e s t a r t i n g a m i n o k e t o n e was d e s c r i b e d p r e v i o u s l y ( 2 ) . C a r b o n 14 l a b e l e d b e n z o y l c h l o r i d e ( 5 . 4 4 mCi/mmole) was o b t a i n e d from o u r radiosynthesis laboratory and used in the preparation of 14C-ketoACE u s i n g m e t h o d s d e s c r i b e d i n r e f e r e n c e 2. The c o r r e c t i s o m e r o f 14C-ketoACE was obtained by three recrystallizations of the mixture of two diastereomers obtained and seeding with cold ketoACE to yield a product with specific activity of 4.54 mCi/mmole. Both 3H- and 14C-ketoACE were shown to be homogeneous and greater than 95% pure by TLC autoradiography with Rf identical to ketoACE. Melting point, HPLC, and optical rotation were used as criteria of purity of the 3H- and i~C-ketoACE samples obtained; specifically for 3H-ketoACE: [a] 22= -88.2 ° (c 0.55, CHCI3) , Lit. (I) [a]~l= _83.2 ° (c 0.98, CHCI3) ; and for l~C-ketoACE: mp 154-155°C, Lit. (i) mp 151~-153°C. Metabolic
Disposition
Methods
Adult Sprague-Dawley rats were purchased locally from Simonsen Laboratories, Inc., Gilroy, California. New Zealand white rabbits were obtained from Elkhorn Rabbitry, Watsonville, California.
302
Metabolism
Studies
of KetoACE
Vol.
37, No.
4, 1985
The animals were given Purina Laboratory Chow and water ad libitum except that the food was withdrawn from them the night before the experiment was to be conducted. After dosing the animals with radiolabeled compounds, serial blood samples were taken from the ear vein of rabbits and by the orbital bleeding technique from the rats. The blood half-time for decline of the radioactivity level was calculated by subjecting the blood data to linear regression analysis using a Hewlett-Packard 9815A desktop computer. For biliary cannulation, rats were anesthetized with Nembutal and the common bile duct was cannulated with Silastic medical-grade tubing after a midline abdominal incision was made. The rats were kept in modified Bollman cages for the collection of bile. For radioactivity assays, aliquots of urine and bile were counted directly in a Searle Analytic Mark III liquid scintillation spectrometer. Correction for quenching was made with automatic external standardization and the use of a previously determined quench curve. Aliquots of whole blood and homogenates of feces were combusted in a Packard Tri-Carb Sample Oxidizer Model B306 before assaying for radioactivity. Results KetoACE exhibited a short biologic half-llfe and was poorly absorbed after oral administration. The blood level of ketoACE after intravenous a d m i n i s t r a t i o n to the rat is shown in Table I. Linear regression analysis of the blood levels of ketoACE indicated the blood half-life to be 9 minutes based on 14C measurement and ii minutes based on 3H measurement. The tritium level in the blood was detectable beyond one hour whereas the 14C was not, indicating that some tritium on the ketoACE may have exchanged with the hydrogen atom in the body water. The average blood level of ketoACE peaked at 0.03 ~g equivalent per ml blood 45 minutes after oral administration of 2 mg of ketoACE to each of three rats averaging 151 g in weight. TABLE
Blood Half-Life
I
of Radiolabeled Blood
Time (min) 5 I0 20 30 60 t i/2 Three male Sprague-Dawley rats 2 mg of dually labeled ketoACE are expressed as avg. ± S.D.
KetoACE
levels
in the Rat
(~g equivalents/ml)
14C 4.91 2.68 1.43 0.70 0.08
± ± ± ± ±
3}I 0.57 0.22 0.42 0.29 0.03
9 min
5.12 2.90 1.64 0.83 0.17
~ ~ ± i •
0.42 0.22 0.47 0.29 0.05
Ii min
(avg. wt. 269 g, range 251-294 g) received iv (2.69 ~Ci 14C and 3.6 ~Ci 3H per rat). Data
K e t o A C E was excreted predominantly in feces. As shown in Table II, approximately 4% of the administered dose appeared in the 24-hour urine after intravenous administration and less than i% was excreted in urine after oral administration. The data on urinary excretion indicate that absorption of the oral dose is at best 20% of the administered dose over 24 hours.
Vol. 37, No. 4, 1985
Metabolism Studies of KetoACE
303
TABLE II Excretion Pattern of Radiolabeled ketoACE Intravenous 14 C % in Urine % in Feces
3.50 ±
Oral 3H
0.41
87.77 ± 27.98
4.18 ± 0.20 67.94 i 39.53
14 C
3H
0.60 ± 0.45 92.13 ± 6.23
0.37 ± 0.14 89.75 ± 5.16
Three male rats (avg. wt. 181 g, range 180-184 g) were intravenously administered with 2 mg each of dually labeled ketoACE (5.04 ~Ci 14C and 7.41 ~Ci 3H per rat). Four male rats (avg. wt. 212 $, range 209-222 ~H) were orally administered the labeled ketoACE (3.38 ~Ci ~4C and 6.70 ~Ci per rat). Urine and feces were collected for 24 hours. Data are expressed as average percentages of dose administered ± S.D.
Because the molecular weight of ketoACE is 422.5, the compound is expected to be excreted in the bile (4,5). When two bile duct cannulated rats were intravenously dosed with i0 mg of 14C-ketoACE, 85.5% and 84.9% of the doses appeared in the bile in the first four hours. Additional 0.3% and 0.48% of the dose appeared in the bile collected between 4 to 24 hours after the dose. The percentages of the dose appearing in 24-hour urines were 6.54% and 6.33%. KetoACE was apparently metabolized without cleaving the molecule to a smaller fragment. After administration of dually labeled ketoACE, the percentages of 14C and 3H appearing in blood~ urine, and bile were comparable. Furthermore, when the feces from rats intravenously administered with the dually labeled ketoACE was extracted with ethyl acetate, over 90% of both 14C and 3H was extracted. Ethyl acetate extracts of bile, urine, and feces were chromatographed on silica gel G plates using chloroform/ethyl acetate/acetic acid 4:5:1 (v/v) as the developing solvent. After autoradiography three major spots were observed. The major spot in all cases had an Rf identical to that of ketoACE and accounted for greater than 80% of the radioactivity appearing on the silica gel G plate. Two metabolite spots consistently appeared on these plates. One metabolite or mixture of metabolites remained at the origin and a m o u n t e d to 2 to 10% of the radioactivity. The second metabolite traveled just behind ketoACE and amounted to 5 to 10% of the radioactivity. This second metabolite had an Rf identical to that of the reduced derivative of ketoACE in which the ketone group has been reduced to a hydroxyl. Mass spectral and HPLC examination of this metabolite confirmed that it was the reduced derivative of ketoACE. Table III shows the biliary and urinary excretion of 3H-ketoACE following oral administration. Very little radioactivity was excreted in the urine (1.3%) in this experiment. A total of 34% of the administered radioactivity was excreted in the bile over a 48 hour period. Thin layer chromatography of ethyl acetate extracts of 24 and 48 hour bile samples indicates that 80% of the radioactivity present has the same Rf as ketoACE. This experiment demonstrates that at least 35% of an oral dose of ketoACE is absorbed from the gastrointestinal tract of the rat.
304
Metabolism
Studies
of KetoACE
TABLE Biliary
and Urinary
Excretion
Vol.
4, 1985
III
After Oral Administration 9-24 h
% in Bile (Ave ± S.D.) % in Urine (Ave ± S.D.)
37, No.
of k e t o A C E
24-48 h
21.5 i 3.0 0.37 ± 0.20
0-48 h
12.7 ~ 6.4 0.93 ± 0.94
34.2 ± 6.7 1.3 ± 1.1
Three male rats (avg. wt. 294 g, range 290-297 g) with bile ducts cannulated were orally administered 2 mg (14.4 ~Ci) of 3H-labeled ketoACE and bile and urine were collected for 48 hours. Some preliminary studies using radiolabeled ketoACE were also performed on two New Zealand white rabbits. Since there was a limited supply of radiolabeled compound, additional rabbits could not be included in these studies. Therefore, the results from the rabbits tested are not statistically significant, but they suggest that the rabbit handles k e t o A C E in a similar manner to the rat. As shown in Table IV, the peak level of ketoACE was 0.68 ~g equivalent per ml blood after the rabbit received 20 mg/kg of ketoACE orally. After intravenous dose, the blood level of ketoACE in the rabbit declined rapidly with a half-life of 9 minutes in the first hour. TABLE Blood
Half-Life
IV
of Radiolabeled Blood
levels
KetoACE
in the Rabbit
(~g equivalents/ml)
Time (hr)
Ora I
In travenou s
0.08 0.16 0.25 0.33 0.50 0.75 1 2 3 4 6 8 24
0.45 0.52 0.68 0.60 0.17 0.19 0.18 0.15 0.i0
35.71 12.87 7.08 1.55 2.13 0.38 0.23 0.23 0.13 0.07
t.1;2= 9 min
One male New Zealand white rabbit (1.65 kg) was orally dosed with 42.74 ~Ci of 14C-ketoACE and another rabbit (2.13 kg) was intravenously dosed with 15.53 ~Ci of I~C-ketoACE. The total dose of ketoACE given to both rabbits was 20 mg/kg. Discussion These studies demonstrate that insertion of a ketomethylene group in place of the normal am±de linkage in a trlpeptide does stabilize the resulting tripeptide analog to peptldase cleavage. KetoACE was excreted predominately in its unmetabolized form, and the one metabolite that could be identified was the reduced analog of ketoACE rather than an am±de cleavage product. The stability of the proline and benzamido am±de linkages in ketoACE to peptidase
Vol. 37, No. 4, 1985
Metabolism Studies of KetoACE
305
cleavage is probably the result of two factors. First, few enzymes in the gastrointestinal tract and serum will efficiently cleave these two types of amide linkages. Secondly, the enzymes that will cleave them may not bind well with ketoACE because it contains a ketomethylene group rather than an amide group adjacent to the amide linkages which must be cleaved. The low antihypertensive activity of ketoACE in hypertensive rats can be explained in two ways. First and most importantly, the rapid excretion of ketoACE into the bile does not allow the blood level of ketoACE to reach and maintain a high enough level to get the desired antihypertensive effect. Secondly, on oral administration ketoACE is slowly absorbed which further reduces the ability to obtain the blood levels needed for achieving good antihypertensive activity. Studies of factors effecting the billary excretion of compounds indicate that with compounds that are organic anions such as ketoACE there is a molecular weight threshold above which extensive biliary excretion would be expected (4,5). For rats this threshold is about 325. The molecular weight of ketoACE at 422 is well above this threshold, therefore it is not surprising that it is excreted so rapidly in the bile. The molecular weight threshold for excretion of organic anions in the bile is reported to be higher in other species (4,5). In man it is 500 and in rabbits 475. Our preliminary results in one rabbit (Table IV) indicate that ketoACE has a very short blood half-life (9 minutes). This indicates that either ketoACE is still rapidly excreted in the bile in rabbits even though its molecular weight is below the reported threshold for this species or that ketoACE is rapidly eliminated from the blood by some other mechanism. What the fate of ketoACE would be in man, remains to be investigated. Acknowledgement This work was supported by NIH Grant HL19538. References i. 2.
3.
4.
5.
R. G. ALMQUIST, W.-R. CHAO, M. E. ELLIS and H. L. JOHNSON, J. Med. Chem. 23 1392 (1980). R . G . ALMQUIST, J. CRASE, C. JENNINGS-WHITE, R. F. MEYER, M. L. HOEFLE, R. D. SMITH, A. D. ESSENBURG and H. R. KAPLAN, J. Med. Chem. 25 1292 (1982). R. F. MEYER, E. D. NICOLAIDES, F. J. TINNEY, E. A. LUNNEY, A. HOLMES, M. L. HOEFLE, R. D. SMITH, A. D. ESSENBURG, H. R. KAPLAN and R. G. ALMQUIST, J. Med. Chem. 24 964 (1981). C . D . KLAASSEN, D. L. EATON and S.-Z. CAGEN, Progress in Drug Metabolism, Vol. 6, Eds. J. W. Bridges and L. F. Chasseaud, p. i, John Wiley and Sons Ltd., New York (1981). C . D . KLAASSEN and J. B. WATKINS, Pharmacol. Rev. 36 1 (1984).
Pergamon Press
ABSORPTION, METABOLISM, AND EXCRETION STUDIES OF CARBON 14- AND TRITIUM-LABELED DERIVATIVES OF A KETOMETHYLENE CONTAINING TRIPEPTIDE Ronald G. Almquist,
Thomas Steeger,
Stewart Jackson, and Chozo Mitoma
Bio-Organic Chemistry Laboratory and Biomedical Research Laboratory, SRI International, Menlo Park, California 94025 (Received in final form May 13, 1985)
Summary Tritium and Carbon 14 analogs of the angiotensin converting enzyme inhibitor ketoACE were synthesized and their oral absorption, metabolism and excretion in rats were investigated. KetoACE, a ketomethylene analog of the tripeptide Bz-Phe-Gly-Pro, was slowly absorbed at a 35% level upon oral administration. It is rapidly eliminated from the blood with a half-life of about I0 minutes. Its excretion is primarily via the bile duct and it is excreted as 80% unchanged drug. The only identified metabolite consisting of 5-10% of the excreted radioactivity was determined to be the reduced ketoACE in which the ketone group was reduced to a hydroxyl.
Previous studies of the ketomethylene trlpeptide analogue, 5(S)benzamido-4-oxo-6-phenylhexanoyl-L-proline (ketoACE) have shown it to be a potent angiotensin converting enzyme (ACE) inhibitor (Isu = 70 nM) (i) with poor antihypertensive activity in the renal hypertensive rat (2,3). The insertion of a ketomethylene linkage in place of the normal amide linkage connecting Phe and Gly in the tripeptide Bz-Phe-Gly-Pro was expected to yield a tripeptide analogue with good metabolic stability.
COOH
~_
~>_~,~-~-~~..~ o
~ o
o
ketoACE
TO investigate the reason for the poor in vivo activity of ketoACE, two radiolabeled derivatives of it were synthesized and used for studies in the rat. The first derivative H3-ketoACE contained tritium in the 3 and 4 positions of the proline ring. The second derivative contained carbon 14 at the carbonyl carbon of the benzamido group. Using these two radiolabeled derivatives, the stabilities of the benzoyl amide linkage and the proline amide linkage following oral or intravenous administration in a rat were inves tiga ted.
0024-3205/85 $3.00 + o00 Copyright (c) 1985 Pergamon Press Ltd.
300
Metabolism Studies of KetoACE
0 o
cooH 0 ~.~ a H
CH2 0
Vo[. 37, No. 4, 1985
0 ~
0
3H_ketoACE
cooH 0 ~.~
CH2 0
1"C-ketoACE
Chemical Methods Figure I shows the chemical pathway used to prepare the tritium labeled ketoACE analogue. Tritiated L-proline from Amersham was adjusted to 5 mCi/mmole by addition of dissolved unlabeled L-proline and evaporating to dryness.
cooH
CH20H
+
o
COCH2"
HN
~
HCI'H
3H
0 CH2 0 0 , i , N CNH--CH--CCH2CH2COH
~,**~I'-.3H
~ aH HCI.HN + k~3,
+
Et3N
+
DCC
1
© o
CH20
0 }~
_3H
H2[I0% Pd/C
©
o C0H
~-~-~N" Figure
-]/"
I
A standard m e t h o d was u s e d to p r e p a r e the L-proliae benzyl ester i n 70% y i e l d . This benzyl ester was u s e d a s d e s c r i b e d previously the desired 3H-ketoACE. C r u d e 3 H - k e t o A C E was c o m b i n e d w i t h c o l d
crystallized to yield pure 311-ketoACE with a specific activity of 3.04 mCi/mmole.
hydrochloride (1) to yield ketoACE and
Vol.
37, No. 4, 1985
Metabolism
Studies
of KetoACE
0
301
©
CN2 0 0 HCI.NH2__CH__CCH2CH2COCH3 +
0 1. Cl
0 CH~ 0 0 I. N'H-- -- CH2CH20CH~
) Na2C03
I NaOH
© ~
o
©
~=0
o
O
~
COCH=
ok_
" I II n f ~ ="CNH--CH--CCH=CHmCN~
o
'
~H~o
o
~--I~CNH--¢H--CCH2CH2COH Et3N 'I
+ DCC
COOH
Figure
II
Figure II outlines t h e s y n t h e t i c p a t h w a y u s e d to p r e p a r e 14 C-ke toACE. The p r e p a r a t i o n of t h e s t a r t i n g a m i n o k e t o n e was d e s c r i b e d p r e v i o u s l y ( 2 ) . C a r b o n 14 l a b e l e d b e n z o y l c h l o r i d e ( 5 . 4 4 mCi/mmole) was o b t a i n e d from o u r radiosynthesis laboratory and used in the preparation of 14C-ketoACE u s i n g m e t h o d s d e s c r i b e d i n r e f e r e n c e 2. The c o r r e c t i s o m e r o f 14C-ketoACE was obtained by three recrystallizations of the mixture of two diastereomers obtained and seeding with cold ketoACE to yield a product with specific activity of 4.54 mCi/mmole. Both 3H- and 14C-ketoACE were shown to be homogeneous and greater than 95% pure by TLC autoradiography with Rf identical to ketoACE. Melting point, HPLC, and optical rotation were used as criteria of purity of the 3H- and i~C-ketoACE samples obtained; specifically for 3H-ketoACE: [a] 22= -88.2 ° (c 0.55, CHCI3) , Lit. (I) [a]~l= _83.2 ° (c 0.98, CHCI3) ; and for l~C-ketoACE: mp 154-155°C, Lit. (i) mp 151~-153°C. Metabolic
Disposition
Methods
Adult Sprague-Dawley rats were purchased locally from Simonsen Laboratories, Inc., Gilroy, California. New Zealand white rabbits were obtained from Elkhorn Rabbitry, Watsonville, California.
302
Metabolism
Studies
of KetoACE
Vol.
37, No.
4, 1985
The animals were given Purina Laboratory Chow and water ad libitum except that the food was withdrawn from them the night before the experiment was to be conducted. After dosing the animals with radiolabeled compounds, serial blood samples were taken from the ear vein of rabbits and by the orbital bleeding technique from the rats. The blood half-time for decline of the radioactivity level was calculated by subjecting the blood data to linear regression analysis using a Hewlett-Packard 9815A desktop computer. For biliary cannulation, rats were anesthetized with Nembutal and the common bile duct was cannulated with Silastic medical-grade tubing after a midline abdominal incision was made. The rats were kept in modified Bollman cages for the collection of bile. For radioactivity assays, aliquots of urine and bile were counted directly in a Searle Analytic Mark III liquid scintillation spectrometer. Correction for quenching was made with automatic external standardization and the use of a previously determined quench curve. Aliquots of whole blood and homogenates of feces were combusted in a Packard Tri-Carb Sample Oxidizer Model B306 before assaying for radioactivity. Results KetoACE exhibited a short biologic half-llfe and was poorly absorbed after oral administration. The blood level of ketoACE after intravenous a d m i n i s t r a t i o n to the rat is shown in Table I. Linear regression analysis of the blood levels of ketoACE indicated the blood half-life to be 9 minutes based on 14C measurement and ii minutes based on 3H measurement. The tritium level in the blood was detectable beyond one hour whereas the 14C was not, indicating that some tritium on the ketoACE may have exchanged with the hydrogen atom in the body water. The average blood level of ketoACE peaked at 0.03 ~g equivalent per ml blood 45 minutes after oral administration of 2 mg of ketoACE to each of three rats averaging 151 g in weight. TABLE
Blood Half-Life
I
of Radiolabeled Blood
Time (min) 5 I0 20 30 60 t i/2 Three male Sprague-Dawley rats 2 mg of dually labeled ketoACE are expressed as avg. ± S.D.
KetoACE
levels
in the Rat
(~g equivalents/ml)
14C 4.91 2.68 1.43 0.70 0.08
± ± ± ± ±
3}I 0.57 0.22 0.42 0.29 0.03
9 min
5.12 2.90 1.64 0.83 0.17
~ ~ ± i •
0.42 0.22 0.47 0.29 0.05
Ii min
(avg. wt. 269 g, range 251-294 g) received iv (2.69 ~Ci 14C and 3.6 ~Ci 3H per rat). Data
K e t o A C E was excreted predominantly in feces. As shown in Table II, approximately 4% of the administered dose appeared in the 24-hour urine after intravenous administration and less than i% was excreted in urine after oral administration. The data on urinary excretion indicate that absorption of the oral dose is at best 20% of the administered dose over 24 hours.
Vol. 37, No. 4, 1985
Metabolism Studies of KetoACE
303
TABLE II Excretion Pattern of Radiolabeled ketoACE Intravenous 14 C % in Urine % in Feces
3.50 ±
Oral 3H
0.41
87.77 ± 27.98
4.18 ± 0.20 67.94 i 39.53
14 C
3H
0.60 ± 0.45 92.13 ± 6.23
0.37 ± 0.14 89.75 ± 5.16
Three male rats (avg. wt. 181 g, range 180-184 g) were intravenously administered with 2 mg each of dually labeled ketoACE (5.04 ~Ci 14C and 7.41 ~Ci 3H per rat). Four male rats (avg. wt. 212 $, range 209-222 ~H) were orally administered the labeled ketoACE (3.38 ~Ci ~4C and 6.70 ~Ci per rat). Urine and feces were collected for 24 hours. Data are expressed as average percentages of dose administered ± S.D.
Because the molecular weight of ketoACE is 422.5, the compound is expected to be excreted in the bile (4,5). When two bile duct cannulated rats were intravenously dosed with i0 mg of 14C-ketoACE, 85.5% and 84.9% of the doses appeared in the bile in the first four hours. Additional 0.3% and 0.48% of the dose appeared in the bile collected between 4 to 24 hours after the dose. The percentages of the dose appearing in 24-hour urines were 6.54% and 6.33%. KetoACE was apparently metabolized without cleaving the molecule to a smaller fragment. After administration of dually labeled ketoACE, the percentages of 14C and 3H appearing in blood~ urine, and bile were comparable. Furthermore, when the feces from rats intravenously administered with the dually labeled ketoACE was extracted with ethyl acetate, over 90% of both 14C and 3H was extracted. Ethyl acetate extracts of bile, urine, and feces were chromatographed on silica gel G plates using chloroform/ethyl acetate/acetic acid 4:5:1 (v/v) as the developing solvent. After autoradiography three major spots were observed. The major spot in all cases had an Rf identical to that of ketoACE and accounted for greater than 80% of the radioactivity appearing on the silica gel G plate. Two metabolite spots consistently appeared on these plates. One metabolite or mixture of metabolites remained at the origin and a m o u n t e d to 2 to 10% of the radioactivity. The second metabolite traveled just behind ketoACE and amounted to 5 to 10% of the radioactivity. This second metabolite had an Rf identical to that of the reduced derivative of ketoACE in which the ketone group has been reduced to a hydroxyl. Mass spectral and HPLC examination of this metabolite confirmed that it was the reduced derivative of ketoACE. Table III shows the biliary and urinary excretion of 3H-ketoACE following oral administration. Very little radioactivity was excreted in the urine (1.3%) in this experiment. A total of 34% of the administered radioactivity was excreted in the bile over a 48 hour period. Thin layer chromatography of ethyl acetate extracts of 24 and 48 hour bile samples indicates that 80% of the radioactivity present has the same Rf as ketoACE. This experiment demonstrates that at least 35% of an oral dose of ketoACE is absorbed from the gastrointestinal tract of the rat.
304
Metabolism
Studies
of KetoACE
TABLE Biliary
and Urinary
Excretion
Vol.
4, 1985
III
After Oral Administration 9-24 h
% in Bile (Ave ± S.D.) % in Urine (Ave ± S.D.)
37, No.
of k e t o A C E
24-48 h
21.5 i 3.0 0.37 ± 0.20
0-48 h
12.7 ~ 6.4 0.93 ± 0.94
34.2 ± 6.7 1.3 ± 1.1
Three male rats (avg. wt. 294 g, range 290-297 g) with bile ducts cannulated were orally administered 2 mg (14.4 ~Ci) of 3H-labeled ketoACE and bile and urine were collected for 48 hours. Some preliminary studies using radiolabeled ketoACE were also performed on two New Zealand white rabbits. Since there was a limited supply of radiolabeled compound, additional rabbits could not be included in these studies. Therefore, the results from the rabbits tested are not statistically significant, but they suggest that the rabbit handles k e t o A C E in a similar manner to the rat. As shown in Table IV, the peak level of ketoACE was 0.68 ~g equivalent per ml blood after the rabbit received 20 mg/kg of ketoACE orally. After intravenous dose, the blood level of ketoACE in the rabbit declined rapidly with a half-life of 9 minutes in the first hour. TABLE Blood
Half-Life
IV
of Radiolabeled Blood
levels
KetoACE
in the Rabbit
(~g equivalents/ml)
Time (hr)
Ora I
In travenou s
0.08 0.16 0.25 0.33 0.50 0.75 1 2 3 4 6 8 24
0.45 0.52 0.68 0.60 0.17 0.19 0.18 0.15 0.i0
35.71 12.87 7.08 1.55 2.13 0.38 0.23 0.23 0.13 0.07
t.1;2= 9 min
One male New Zealand white rabbit (1.65 kg) was orally dosed with 42.74 ~Ci of 14C-ketoACE and another rabbit (2.13 kg) was intravenously dosed with 15.53 ~Ci of I~C-ketoACE. The total dose of ketoACE given to both rabbits was 20 mg/kg. Discussion These studies demonstrate that insertion of a ketomethylene group in place of the normal am±de linkage in a trlpeptide does stabilize the resulting tripeptide analog to peptldase cleavage. KetoACE was excreted predominately in its unmetabolized form, and the one metabolite that could be identified was the reduced analog of ketoACE rather than an am±de cleavage product. The stability of the proline and benzamido am±de linkages in ketoACE to peptidase
Vol. 37, No. 4, 1985
Metabolism Studies of KetoACE
305
cleavage is probably the result of two factors. First, few enzymes in the gastrointestinal tract and serum will efficiently cleave these two types of amide linkages. Secondly, the enzymes that will cleave them may not bind well with ketoACE because it contains a ketomethylene group rather than an amide group adjacent to the amide linkages which must be cleaved. The low antihypertensive activity of ketoACE in hypertensive rats can be explained in two ways. First and most importantly, the rapid excretion of ketoACE into the bile does not allow the blood level of ketoACE to reach and maintain a high enough level to get the desired antihypertensive effect. Secondly, on oral administration ketoACE is slowly absorbed which further reduces the ability to obtain the blood levels needed for achieving good antihypertensive activity. Studies of factors effecting the billary excretion of compounds indicate that with compounds that are organic anions such as ketoACE there is a molecular weight threshold above which extensive biliary excretion would be expected (4,5). For rats this threshold is about 325. The molecular weight of ketoACE at 422 is well above this threshold, therefore it is not surprising that it is excreted so rapidly in the bile. The molecular weight threshold for excretion of organic anions in the bile is reported to be higher in other species (4,5). In man it is 500 and in rabbits 475. Our preliminary results in one rabbit (Table IV) indicate that ketoACE has a very short blood half-life (9 minutes). This indicates that either ketoACE is still rapidly excreted in the bile in rabbits even though its molecular weight is below the reported threshold for this species or that ketoACE is rapidly eliminated from the blood by some other mechanism. What the fate of ketoACE would be in man, remains to be investigated. Acknowledgement This work was supported by NIH Grant HL19538. References i. 2.
3.
4.
5.
R. G. ALMQUIST, W.-R. CHAO, M. E. ELLIS and H. L. JOHNSON, J. Med. Chem. 23 1392 (1980). R . G . ALMQUIST, J. CRASE, C. JENNINGS-WHITE, R. F. MEYER, M. L. HOEFLE, R. D. SMITH, A. D. ESSENBURG and H. R. KAPLAN, J. Med. Chem. 25 1292 (1982). R. F. MEYER, E. D. NICOLAIDES, F. J. TINNEY, E. A. LUNNEY, A. HOLMES, M. L. HOEFLE, R. D. SMITH, A. D. ESSENBURG, H. R. KAPLAN and R. G. ALMQUIST, J. Med. Chem. 24 964 (1981). C . D . KLAASSEN, D. L. EATON and S.-Z. CAGEN, Progress in Drug Metabolism, Vol. 6, Eds. J. W. Bridges and L. F. Chasseaud, p. i, John Wiley and Sons Ltd., New York (1981). C . D . KLAASSEN and J. B. WATKINS, Pharmacol. Rev. 36 1 (1984).