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Fenoprofen: Inhibitor of prostaglandin synthesis
FENOPROFEN:
INHIBITOR OF PROSTAGLANDIN SYNTHESIS
Peter P. K. Ho and Michail A. Esterman
The L i l l y Research Laboratories Eli L i l l y and Company Indianapolis, Indiana 46206
ABSTRACT Fenoprofen, an anti-inflammatory agent, is a potent i n h i b i t o r of prostaglandin synthesis at physiological substrate concentration. 150's of l ~M to lO0 ~M were obtained when the concentration of arachidonate was increased from l uM to 82 uM. In c o n t r a s t , a small change in 150 was observed with indomethacin. Kinetic studies indicated that i n h i b i t i o n by fenoprofen was competitive while that of indomethacin was noncompetitive.
Accepted March 15
PROSTAGLANDINS APRIL 25, 1974
VOL. 6 NO. 2
107
PROSTAGLANDINS
INTRODUCTION Prostaglandins have been shown to induce fever and inflammation; and the inhibition of prostaglandin synthesis by aspirin-like drugs has been suggested as the explanation for their anti-inflammatory and antipyretic activity (1). In vitro synthesis of prostaglandin E2 (PGE2) from sachidonic acid by bovine seminal vesicle microsomes, measured by a radioactive assay, was inhibited by several anti-inflammatory agents, such as indomethacin and aspirin with relative potency of 2000 to 1 (2). However, in a spectrophotometric assay (3), indomethacin has been shown to be approximately 400 times more active than aspirin but only 47 times in the guinea pig lung homogenate system (1) and 100 times using the oxygen electrode assay (4). These quantitative discrepancies have partially been attributed to the method of assay. By the spectrophotometric method, 2-phenoxy-phenylpropionic acid (fenoprofen Lilly) was shown to be less active than indomethacin with potency ratio of 1 to 200 Nevertheless, fenoprofen is a potent anti-inflam(3). matory agent as demonstrated by animal activity and clinical efficacy (5,6). Since the prostaglandin synthetase system is present in a wide variety of tissues and organs, one important limiting factor in endogenous biosynthesis of the prostaglandin is the concentration of free essential fatty acid precursors which is generally very low in tissue (7). In view of the clinical efficacy of fenoprofen, we have studied its in vitro inhibition of prostaglandin synthesis at substrateconcentrations of 0.1 to 10 uM which are probably in the physiological range, and may resemble the conditions Included in these of prostaglandin biosynthesis in situ. experiments, high substrate concentration (82 pM) was also performed to verify the reported results (3). MATERIALS
AND
METHODS
A microsomal prostaglandin synthetase preparation was made from bovine seminal vesicles. Frozen bull seminal vesicles were obtained from Pel Freeze Biologicals, Inc., The thawed vesicles were first trimmed Rogers, Arkansas. of connective tissue, fat, and muscle, and then cut into
108
APRIL
25, 1974
VOL. 6 NO. 2
PROSTAGLANDINS
small pieces. The minced tissue was homogenized in a Waring Blendor with the addition of two volumes of 0.1 M All operations were potassium phosphate buffer, pH 8.0. performed at 4'C and the microsomal pellet was suspended in 0.01 M potassium phosphate buffer, pH 8.0, and lyophilized (3). Prostaglandin synthesis was measured by determining the amount of radioactive PGE2 formed from [3H]-arachidonate. Reduced glutathione was the only cofactor and hydroquinone or its equivalent was not added. Reaction tubes contained unlabeled arachidonate acid (NuChek Prep., Elysian, Minnesota) at the concentration indicated, 0.2 pCi of [5,6,8,9,11,12,14,15-3H] arachidonate acid i;;eai,fic activity 80 Ci/mmole; New England Nuclear Corp., Mass.) 3 mM glutathione (GSH), enzyme (0.5 mg proteiA/assay) in 0.05 M Tris buffer, pH 8.3. The reaction was started by the addition of 1.0 ml of enzyme (freeze-dried powder reconstituted in buffer plus GSH) to tubes containing 20 pl of tritiated substrate and inhibitor. The reactions were conducted for 15 min at room temperature (22-24°C). Three tenth ml of 0.2 M citric acid to stop the reaction and 10 pg of PGE and PGF2 (Fuji Chemical Industries, Ltd., Tokyo, Japan) in et 6 anol (0.2 ml) were added. The mixture was diluted to 5 ml with water and extracted twice with 5 ml of ethyl acetate. The ethyl acetate was evaporated and the residue was dissolved in 0.2 ml ethanol. A 50 pl sample was spotted on silica gel F for TLC separation in chloroform-methanolacetic acid (18:l:l v/v). Prostaglandins were located by visualization in an iodine chamber. Zones corresponding to prostaglandins E and F were scraped from the thin-layer plate into scintillation vials with an Auto-Zonal Plate The radioactivity was Scraper (Isolab, Akron, Ohio). determined by scintillation counting in Triton-toluene (1:l) scintillation solution using a Packard Scintillation Counter. Counting efficiency was 30% and quench corrections were made by using the sample channels-ratio method. RESULTS
AND
DISCUSSION
Fig. 1 shows the results obtained when indomethacin and fenoprofen were evaluated as inhibitors of PGE2 synthesis in the bovine seminal vesicle microsome system. At low substrate concentration (0.1 to 1 PM), both compounds As the concentration inhibited PGE2 synthesis at 1 to 2 pM. of arachidonate increased, the degree of inhibition by both at substrate concentration compounds decreased. However,
APRIL
25, 1974
VOL. 6 NO. 2
109
PROSTAGLANDINS
of 82 pM, indomethacin showed only a slight increase of 150 to 10 pM, while fenoprofen exhibited a drastic change of 150 to 100 pM. These values of 150, obtained at high substrate concentration, were consistent with the reported data (2,3). At levels of 0.1 to 1 pM of arachidonate, which are considered to be in the physiological range, fenoprofen is a potent inhibitor of prostaglandin synthesis. Kinetic studies indicated that inhibition by fenoprofen was apparently competitive, while that of indomethacin was noncompetitive (Fig. 2). A Km value of 1.65 x 10-5 M for arachidonic acid was obtained from the Linew aver-Burk plot, and KI values of 5 x 10-7 M and 5.2 x lo- 7 M were observed for indomethacin and fenoprofen, respectively. These results show that fenoprofen is a potent inhibitor of prostagl;;i;n synthesis and is more active than previously reported. caution should be used when comparing inhibitors of piostaglandin synthetase only at high substrate concentrations, since improper relative potencies may be obtained at high substrate concentrations. REFERENCES
1.
Vane, J. R. a Mechanism New Biology
2.
Tomlinson R. V., H. J. Ringold, M. C. Qureshi and Forchielli. Relationship Between Inhibition of Support Prostaglandin Synthesis and Drug Efficacy: the Current Theory of Mode of Action of Aspirin-like Drugs, Biochem. Biophys. Res. Comm. 46:552, 1972. -
Inhibition of Prostaglandin of Action for Aspirin-like 231:232, 1971.
Synthesis as Drugs, Nature-
E. for
3.
Takeguchi, C., and C. 3. Sih. A Rapid Spectrophotometric Assay for Prostaglandin Synthetase: Application to the Study of Nonsteroidal Anti-inflammatory Agents, Prostaglandins 2:169, 1972.
4.
Smith, W. L., and W. E. Blockade of Prostaglandin 246:6700, 1971.
5.
Nickander, R. C., R. J. Kraay and W. S. Marshall. Anti-inflammatory and Analgesic Effects of Fenoprofen, Abstract 2059, Fed. Proc. -30:563, 1971.
110
M.
Lands. Stimulation and Biosynthesis, J. Biol. Chem.
APRIL
25, 1974
VOL. 6 NO. 2
PROSTAGLANDINS
6.
Gruber, C. M., A. S. Ridolfo and R. C. Nickander. Delay of the Erythemic Response of Human Skin to Ultraviolet Irradiation by Anti-inflammatory Drugs, Therap. -12:292, 1971. Clin. Pharmacol.
7.
Hinman, J. W. 161, 1972.
Prostaglandins,
APRIL 25, 1974 VOL. 6 NO. 2
Ann.
Rev.
Biochem.
41:
111
PROSTAGLANDINS
Figure
1.
Inhibition of Prostaglandin E2 Formation by Anti-inflammatory Drugs: Incubation and assay conditions are described in the text. Values for percent conversion of arachidonic acid to PGE2 at various substrate concentrations are as follows: uM
arachidonate
% Conversion
82 10
2.4 :; 44
i.1 The concentration reaction mixture of the symbol. loo-
I DONATE,
FENOPROFEN
ARACHIDONATE,
112
to the top
1NDOMETHAClN
ARACH lT
of drug (pM) added is indicated on the
MM ’
MM
APRIL
25, 1974
VOL. 6 NO. 2
PROSTAGLANDINS
Fig. 2.
APRIL
25, 1974
Double reciprocal plot of the amount of PGE2 formation versus the arachidonic acid concentration in the presence and absence of inhibitors. Determinations were made at room temperature for five minutes.
0-0
= absence
•-_~
= add
1 x 10S6
M of
fenoprofen
A-A
= add
4 x low6
M of
indomethacin
VOL. 6 NO. 2
of
inhibitors
113
INHIBITOR OF PROSTAGLANDIN SYNTHESIS
Peter P. K. Ho and Michail A. Esterman
The L i l l y Research Laboratories Eli L i l l y and Company Indianapolis, Indiana 46206
ABSTRACT Fenoprofen, an anti-inflammatory agent, is a potent i n h i b i t o r of prostaglandin synthesis at physiological substrate concentration. 150's of l ~M to lO0 ~M were obtained when the concentration of arachidonate was increased from l uM to 82 uM. In c o n t r a s t , a small change in 150 was observed with indomethacin. Kinetic studies indicated that i n h i b i t i o n by fenoprofen was competitive while that of indomethacin was noncompetitive.
Accepted March 15
PROSTAGLANDINS APRIL 25, 1974
VOL. 6 NO. 2
107
PROSTAGLANDINS
INTRODUCTION Prostaglandins have been shown to induce fever and inflammation; and the inhibition of prostaglandin synthesis by aspirin-like drugs has been suggested as the explanation for their anti-inflammatory and antipyretic activity (1). In vitro synthesis of prostaglandin E2 (PGE2) from sachidonic acid by bovine seminal vesicle microsomes, measured by a radioactive assay, was inhibited by several anti-inflammatory agents, such as indomethacin and aspirin with relative potency of 2000 to 1 (2). However, in a spectrophotometric assay (3), indomethacin has been shown to be approximately 400 times more active than aspirin but only 47 times in the guinea pig lung homogenate system (1) and 100 times using the oxygen electrode assay (4). These quantitative discrepancies have partially been attributed to the method of assay. By the spectrophotometric method, 2-phenoxy-phenylpropionic acid (fenoprofen Lilly) was shown to be less active than indomethacin with potency ratio of 1 to 200 Nevertheless, fenoprofen is a potent anti-inflam(3). matory agent as demonstrated by animal activity and clinical efficacy (5,6). Since the prostaglandin synthetase system is present in a wide variety of tissues and organs, one important limiting factor in endogenous biosynthesis of the prostaglandin is the concentration of free essential fatty acid precursors which is generally very low in tissue (7). In view of the clinical efficacy of fenoprofen, we have studied its in vitro inhibition of prostaglandin synthesis at substrateconcentrations of 0.1 to 10 uM which are probably in the physiological range, and may resemble the conditions Included in these of prostaglandin biosynthesis in situ. experiments, high substrate concentration (82 pM) was also performed to verify the reported results (3). MATERIALS
AND
METHODS
A microsomal prostaglandin synthetase preparation was made from bovine seminal vesicles. Frozen bull seminal vesicles were obtained from Pel Freeze Biologicals, Inc., The thawed vesicles were first trimmed Rogers, Arkansas. of connective tissue, fat, and muscle, and then cut into
108
APRIL
25, 1974
VOL. 6 NO. 2
PROSTAGLANDINS
small pieces. The minced tissue was homogenized in a Waring Blendor with the addition of two volumes of 0.1 M All operations were potassium phosphate buffer, pH 8.0. performed at 4'C and the microsomal pellet was suspended in 0.01 M potassium phosphate buffer, pH 8.0, and lyophilized (3). Prostaglandin synthesis was measured by determining the amount of radioactive PGE2 formed from [3H]-arachidonate. Reduced glutathione was the only cofactor and hydroquinone or its equivalent was not added. Reaction tubes contained unlabeled arachidonate acid (NuChek Prep., Elysian, Minnesota) at the concentration indicated, 0.2 pCi of [5,6,8,9,11,12,14,15-3H] arachidonate acid i;;eai,fic activity 80 Ci/mmole; New England Nuclear Corp., Mass.) 3 mM glutathione (GSH), enzyme (0.5 mg proteiA/assay) in 0.05 M Tris buffer, pH 8.3. The reaction was started by the addition of 1.0 ml of enzyme (freeze-dried powder reconstituted in buffer plus GSH) to tubes containing 20 pl of tritiated substrate and inhibitor. The reactions were conducted for 15 min at room temperature (22-24°C). Three tenth ml of 0.2 M citric acid to stop the reaction and 10 pg of PGE and PGF2 (Fuji Chemical Industries, Ltd., Tokyo, Japan) in et 6 anol (0.2 ml) were added. The mixture was diluted to 5 ml with water and extracted twice with 5 ml of ethyl acetate. The ethyl acetate was evaporated and the residue was dissolved in 0.2 ml ethanol. A 50 pl sample was spotted on silica gel F for TLC separation in chloroform-methanolacetic acid (18:l:l v/v). Prostaglandins were located by visualization in an iodine chamber. Zones corresponding to prostaglandins E and F were scraped from the thin-layer plate into scintillation vials with an Auto-Zonal Plate The radioactivity was Scraper (Isolab, Akron, Ohio). determined by scintillation counting in Triton-toluene (1:l) scintillation solution using a Packard Scintillation Counter. Counting efficiency was 30% and quench corrections were made by using the sample channels-ratio method. RESULTS
AND
DISCUSSION
Fig. 1 shows the results obtained when indomethacin and fenoprofen were evaluated as inhibitors of PGE2 synthesis in the bovine seminal vesicle microsome system. At low substrate concentration (0.1 to 1 PM), both compounds As the concentration inhibited PGE2 synthesis at 1 to 2 pM. of arachidonate increased, the degree of inhibition by both at substrate concentration compounds decreased. However,
APRIL
25, 1974
VOL. 6 NO. 2
109
PROSTAGLANDINS
of 82 pM, indomethacin showed only a slight increase of 150 to 10 pM, while fenoprofen exhibited a drastic change of 150 to 100 pM. These values of 150, obtained at high substrate concentration, were consistent with the reported data (2,3). At levels of 0.1 to 1 pM of arachidonate, which are considered to be in the physiological range, fenoprofen is a potent inhibitor of prostaglandin synthesis. Kinetic studies indicated that inhibition by fenoprofen was apparently competitive, while that of indomethacin was noncompetitive (Fig. 2). A Km value of 1.65 x 10-5 M for arachidonic acid was obtained from the Linew aver-Burk plot, and KI values of 5 x 10-7 M and 5.2 x lo- 7 M were observed for indomethacin and fenoprofen, respectively. These results show that fenoprofen is a potent inhibitor of prostagl;;i;n synthesis and is more active than previously reported. caution should be used when comparing inhibitors of piostaglandin synthetase only at high substrate concentrations, since improper relative potencies may be obtained at high substrate concentrations. REFERENCES
1.
Vane, J. R. a Mechanism New Biology
2.
Tomlinson R. V., H. J. Ringold, M. C. Qureshi and Forchielli. Relationship Between Inhibition of Support Prostaglandin Synthesis and Drug Efficacy: the Current Theory of Mode of Action of Aspirin-like Drugs, Biochem. Biophys. Res. Comm. 46:552, 1972. -
Inhibition of Prostaglandin of Action for Aspirin-like 231:232, 1971.
Synthesis as Drugs, Nature-
E. for
3.
Takeguchi, C., and C. 3. Sih. A Rapid Spectrophotometric Assay for Prostaglandin Synthetase: Application to the Study of Nonsteroidal Anti-inflammatory Agents, Prostaglandins 2:169, 1972.
4.
Smith, W. L., and W. E. Blockade of Prostaglandin 246:6700, 1971.
5.
Nickander, R. C., R. J. Kraay and W. S. Marshall. Anti-inflammatory and Analgesic Effects of Fenoprofen, Abstract 2059, Fed. Proc. -30:563, 1971.
110
M.
Lands. Stimulation and Biosynthesis, J. Biol. Chem.
APRIL
25, 1974
VOL. 6 NO. 2
PROSTAGLANDINS
6.
Gruber, C. M., A. S. Ridolfo and R. C. Nickander. Delay of the Erythemic Response of Human Skin to Ultraviolet Irradiation by Anti-inflammatory Drugs, Therap. -12:292, 1971. Clin. Pharmacol.
7.
Hinman, J. W. 161, 1972.
Prostaglandins,
APRIL 25, 1974 VOL. 6 NO. 2
Ann.
Rev.
Biochem.
41:
111
PROSTAGLANDINS
Figure
1.
Inhibition of Prostaglandin E2 Formation by Anti-inflammatory Drugs: Incubation and assay conditions are described in the text. Values for percent conversion of arachidonic acid to PGE2 at various substrate concentrations are as follows: uM
arachidonate
% Conversion
82 10
2.4 :; 44
i.1 The concentration reaction mixture of the symbol. loo-
I DONATE,
FENOPROFEN
ARACHIDONATE,
112
to the top
1NDOMETHAClN
ARACH lT
of drug (pM) added is indicated on the
MM ’
MM
APRIL
25, 1974
VOL. 6 NO. 2
PROSTAGLANDINS
Fig. 2.
APRIL
25, 1974
Double reciprocal plot of the amount of PGE2 formation versus the arachidonic acid concentration in the presence and absence of inhibitors. Determinations were made at room temperature for five minutes.
0-0
= absence
•-_~
= add
1 x 10S6
M of
fenoprofen
A-A
= add
4 x low6
M of
indomethacin
VOL. 6 NO. 2
of
inhibitors
113