Vol. June
177,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 814-820
14, 1991
ONO-5046,
A NOVEL
INHIBITOR
OF HUMAN
NEUTROPHIL
ELASTASE
Kazuhito Kawabata, Mayumi Suzuki, Masafumi Sugitani, Katsuhiro Imaki MasaakiToda andTsumoruMiyamoto Minase ResearchInstitute, Ono PharmaceuticalCo., Ltd., Shimamoto,Mishima, Osaka,618, Japan Received April
15, 1991
SUMMARY: ONO-5046, N-[2-[4-(2,2-Dimethylpropionyloxy)phenylsulfonylamino]benzoyl] aminoacetic acid, competitively inhibited human neutrophil elastase(ICsu= 0.044 pM, Ki = 0.2uM). It also inhibited leukocyte elastaseobtained from rabbit, rat, hamster and mouse.However, ONO-5046 did not inhibit trypsin, thrombin, plasmin, plasmakallikrein, pancreaskallikrein, chymotrypsin and cathepsinG even at 100uM. In in vivo studies,ONO5046 suppressed lung hemorrhage in hamster (ID 50 = 82 pg/kg) by intratracheal administrationand increaseof skin capillary permeability in guineapig (ID50 = 9.6 mg/kg) by intravenousadministration,both of which were inducedby humanneutrophil elastase. 0 1991 Academrc mess,Inc.
Human neutrophil elastase(HNE, EC 3.4.21.37) is a proteasecapable of hydrolyzing mostconnective tissuecomponents(reviewed in 1). It hasbeensuggestedto participate in the tissueinjury of emphysema(2), rheumatoidarthritis (3), adult respiratory distresssyndrome (4) and septic shock (5). Although plasma and interstitial fluid contain powerful antiproteinasessuch as al-proteinase inhibitor (al-PI) and a2-macroglobulin (a2-MG), their elastaseinhibitory activities have been suggestedto be circumvented becauseal-PI and cc2MG, macromolecularinhibitors, are excluded from close contacts between neutrophils and substrate(6, 7). In contrast to theseanti-proteinases,a low molecular weight HNE inhibitor might be delivered to achieve close contacts (8, 9), suggesting the advantage of a low molecularweight HNE inhibitor asa therapeuticagent. Under thesecircumstances,many low molecularweight synthetic inhibitors of HNE have beendeveloped(10 and reviewed in 11). A low molecular weight inhibitor of HNE which has broad inhibition on neutrophil elastasesderived from severalspeciesof animals,is highly specific and hassystemic activity Abbreviations: HNE, human neutrophil elastase;al-PI, alpha-1-proteinaseinhibitor; a2-MG, alpha-2-macroglobulin;sue-Ala-Pro-Ala-pNa, succinyl-Ala-Pro-Ala-p-nitroanilide. 0006-291X/91 $1.50 Copyright 0 I991 by Academic Press. Inc. All rights of reproduction in any form reserved.
814
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
is required for studying the role of neutrophil elastase on diseases such as emphysema and adult respiratory distress syndrome. However,
such a highly specific and systemically active
elastase inhibitor has not been reported as yet. We have found that ONO-5046, N-[2-[4-(2,2Dimethylpropionyloxy)phenylsulfonylamino]benzoyl] and intravenously
aminoacetic acid is a potent, specific
active neutrophil elastase inhibitor. In the present paper, we describe the
biochemical and pharmacological properties of ONO-5046. MATERIALS
AND METHODS
Enzvme and regents: Purified human neutrophil elastase (purulent human sputum as a source) was purchased from Elastin Products Co., Inc. (Missouri, U.S.A.). Bovine pancreas trypsin, human plasma thrombin, human plasma kallikrein, bovine pancreas chymotrypsin, n-N-benzoyl-DL-arginine-p-nitroanilide HCl, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and elastin-congo red were obtained from Sigma Chemicals Corp. (St. Louis, U.S.A.). Synthetic substrate, S-2238, S-2266 and S-2302 were obtained from Daiichikagaku Pharmaceutical Co., Ltd. (Tokyo, Japan). Succinyl-Ala-Pro-Ala-p-nitroanilide (sue-Ala-Pro-Ala-pNa) and succinyl-Ala-Ala-Ala-p-nitroanilide were purchased from Peptide Institute, Inc. (Osaka, Japan). Porcine pancreatic kallikrein was a product of Ono pharmaceutical Co., Ltd. (Osaka, Japan). ONO-5046 was synthesized in our laboratories. The chemical structure of ONO-5046 is shown in Fig. 1. Prenaration of neutrouhil extracts: Peritoneal neutrophils were obtained from rabbits pretreated with 0.2% glycogen (200 ml/body) five hrs before harvesting peritoneal cells by saline lavage, and from rat, hamster and mouse pretreated with 1% casein (100 ml/kg) 16 hrs before harvesting peritoneal cells by saline lavage. Then neutrophils were suspended in Tris-HCl buffer (0.1 M, pH 7.5) containing 0.1% Brij-35 and 1 M of MgC12 and lysed by sonication. The lysate was used as the source of neutrophil elastase. The hydrolytic activity of neutrophil elastase was Determination of urotease activitv: detemrined using two methods. (a) The hydrolysis of synthetic substrate, sue-Ala-Pro-AlapNa was measured by the spectrophotometric method of Levine et al (12): sue-Ala-Pro-AlapNa (0.1 mM) was incubated with the enzyme (1 x 1O-3 U) in Ttis-HCl buffer (0.1 M, pH 8.0) containing 0.2 M sodium chloride in a final volume of 1.0 ml. After incubation at 37°C for 30 min, the absorbance at 405 nm was measured. (b) The hydrolysis of elastincongo red was measured by the spectrophtometric method of Naughton and Sanger (13) with some modifications: elastin-congo red (2 mg/ml) was incubated with 0.2 U of enzyme in TrisHCl buffer (0.1 M pH SO), containing 0.2 M of sodium chloride in a final volume of 1.0 ml. After incubation at 37°C for 1 hour, the absorbance was measured at 500 nm. Porcine pancreatic elastase activity was determined by succinyl-Ala-Ala-Ala-p-nitroanilide hydrolysis by the method of Fujimoto et al (14) with some modifications: sue-Ala-Ala-Ala-pNA was incubated with 1 x 1O-3U of enzyme at 37’C in HEPES-NaOH buffer (20 mM, pH X.0) containing 10 mM CaC12 in a final volume of 1.O ml. The developed color was measured spectrophoto-metorically at 405 nm. One unit of elastase activity was defined as the quantity of enzyme that liberated 1 umol of p-nitroanilide in 1 min. Other protease activity was determined by the method of Matsuoka et al. (15).
CH3-~~+=+O*NH~H~ooH l&w.: 434.47
Fig. Chemical structure of ONO-5046 ( N-[2-[4-(2,2-Dimethylpropionyloxy)phenylsulfonylamino]benzoyl]aminoacetic acid ). 815
Vol.
177,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Lunar hemorrhage: Male Golden hamsters, weighing 90 to 110 g were used. Human neutrophil elastase (0.3 U in 1.50~1 PBS /site) was injected intratracheally via a small incision in the ventral neck region, using a small diameter (o.d. = 1.0 mm) polyethylene catheter under ketamine (1.50 mg/kg i.p.) anesthesia. One hour later, hamsters were sacrificed by bleeding via the abdominal aorta. The isolated lungs were homogenized with 5 ml of distilled water and the homogenates were centrifuged at 22600 x g for 30 min. The supernatant was diluted (x 10) with distilled water and absorbance at 412 nm (the index for hemorrhage) was measured spectrophotometorically. ONO-5046 was administered intratracheally five min before HNE injection. Male Hartley strain guinea pigs, weighing 250 to 350 g were Canillarv Dermeabilitv: injected with human neutrophil elastase (20 x 10m3U in 100 ~1 PBS / site) in the clipped-back skin. Soon after, 1.7 ml/kg of Evans blue solution (2% w/v) was injected intravenously. Thirty min after Evans blue injection, guinea pigs were sacrificed by bleeding via the carotic aorta and the blue spots in the skin were cut off and solubilized by 1 ml of 1 N KOH at 37°C overnight. The absorbance of acetone-O.6 N phosphate solution (13 : 5 v/v)- extracted dye was measured spectrophotometorically at 620 nm ( the index for permeability). ONO-5046 was administered intravenously 30 set before HNE injection. Statistics: Statistical comparisons were evaluated by using variance analysis of the software package SAS (Statistical Analysis System, cary, USA) followed by Student’s unpaired t-test. Only p values of 0.05 or less were considered statistically significant. RESULTS AND DISCUSSION The inhibitory activity of ONO-5046
was studied using a synthetic substrate, sue-Ala-
Pro-Ala-pNa and a macromolecular substrate, elastin-congo red. ONO-5046 potently inhibited HNE with an IC50 of 0.044 f 0.003 PM, Ki of 0.20 f 0.02 FM (sue-Ala-Pro-Ala-pNa substrate)
and ICs() of 7.0 f 0.1 pM ( e1,ds‘t’m - congo red as a substrate).
competitive
as determined by Lineweaver-Burk
as a
The inhibition is
plot analysis (sue-Ala-Pro-Ala-pNa
as a
substrate). The IC50 value with elastin-congo red as a substrate was higher than that with sucAla-Pro-Ala-pNa.
This may be due to the difference of enzyme concentration in the two assay
systems because the hydrolysis
of elastin-congo
concentration than that of sue-Ala-Pro-Ala-pNa. of SC-39027
red required 200 times higher enzyme
Nakao et al (16) have reported the IC50 value
against elastin is higher than that of synthetic
substrate. ONO-5046
also
inhibited leukocyte elastase obtained from rabbits, rats, hamsters or mise with IC,, values comparable to that against HNE (Table 1). These results show that the inhibition of ONOSO46 is not confined to HNE but extends to neutrophil elastases of several other animal species. Furthermore, plasma thrombin,
ONO-5046
was inactive against bovine pancreas trypsin,
human plasma plasmin, porcine pancreas kallikrein,
kallikrein, bovine pancreas chymotrypsin However, ONO-5046
human
human plasma
and human neutrophil cathepsin G even at 100 NM.
inhibited porcine pancreas elastase with an IC5o value of 5.6 + 0.2 PM
(succinyl-Ala-Ala-Ala-p-nitroanilide
as a substrate).
IC,o values of ONO-5046 on neutrophil
elastases derived from human or other animal species were about 130 times higher than that of 816
Vol.
177, No. 2, 1991
Table
1. Inhibitory Human
Human
of
AND BIOPHYSICAL
Effect of ONO-5046 and Various Animal
Rabbit
0.044 + 0.003 The hydrolysis
BIOCHEMICAL
sue-Al;~-Pro-Ala-pNa
on Neutrophil Species
Rat
0.036 + 0.001
O.OlY
was
f 0.001
measured. Each
RESEARCH COMMUNICATIONS
value
Elastase
of
Hamster
Mouse
0.037+ 0.004
0.049+ 0.005
is
mean
+
S.E.M.
of
three
separate
rxperiments.
porcine pancreaselastase,indicating that inhibition by ONO-5046 is highly specific to these neutrophil elastases.Thus, ONO-5046 can be useto study the roles of neutrophil elastasein severalanimal models. Evidence for in vivo activities of ONO-5046 was obtained by two animal models. We first examined the effect of ONO-5046 using lung hemorrhageinduced by intratracheal instillation of HNE. Intratracheal administration of ONO-5046 significantly and dosedependently suppressedthe lung hemorrhage (Fig. 2), with an ID,, value of 82 pg/kg. Hassallet al (17) have alsoevaluated the effect of a HNE inhibitor using HNE-induced lung hemorrhagein hamsters.Secondly, we examined the intravenous activity of ONO-5046 on a
I *** T
Vehrcle 0.02 I
0.21
ONO-5046
0.7
7 .I -.
(mg/kg)
Fig.. Effect of intratracheal administration of ONO-5046 on lung hemorrhage induced by human neutrophil elastase in hamsters. Human neutrophil elastase (0.3 U/site) was instillated intratracheally. ONO-5046 was administered five min prior to enzyme. Each value is mean f S.E.M. of five to six animals. **:P
177,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 814-820
14, 1991
ONO-5046,
A NOVEL
INHIBITOR
OF HUMAN
NEUTROPHIL
ELASTASE
Kazuhito Kawabata, Mayumi Suzuki, Masafumi Sugitani, Katsuhiro Imaki MasaakiToda andTsumoruMiyamoto Minase ResearchInstitute, Ono PharmaceuticalCo., Ltd., Shimamoto,Mishima, Osaka,618, Japan Received April
15, 1991
SUMMARY: ONO-5046, N-[2-[4-(2,2-Dimethylpropionyloxy)phenylsulfonylamino]benzoyl] aminoacetic acid, competitively inhibited human neutrophil elastase(ICsu= 0.044 pM, Ki = 0.2uM). It also inhibited leukocyte elastaseobtained from rabbit, rat, hamster and mouse.However, ONO-5046 did not inhibit trypsin, thrombin, plasmin, plasmakallikrein, pancreaskallikrein, chymotrypsin and cathepsinG even at 100uM. In in vivo studies,ONO5046 suppressed lung hemorrhage in hamster (ID 50 = 82 pg/kg) by intratracheal administrationand increaseof skin capillary permeability in guineapig (ID50 = 9.6 mg/kg) by intravenousadministration,both of which were inducedby humanneutrophil elastase. 0 1991 Academrc mess,Inc.
Human neutrophil elastase(HNE, EC 3.4.21.37) is a proteasecapable of hydrolyzing mostconnective tissuecomponents(reviewed in 1). It hasbeensuggestedto participate in the tissueinjury of emphysema(2), rheumatoidarthritis (3), adult respiratory distresssyndrome (4) and septic shock (5). Although plasma and interstitial fluid contain powerful antiproteinasessuch as al-proteinase inhibitor (al-PI) and a2-macroglobulin (a2-MG), their elastaseinhibitory activities have been suggestedto be circumvented becauseal-PI and cc2MG, macromolecularinhibitors, are excluded from close contacts between neutrophils and substrate(6, 7). In contrast to theseanti-proteinases,a low molecular weight HNE inhibitor might be delivered to achieve close contacts (8, 9), suggesting the advantage of a low molecularweight HNE inhibitor asa therapeuticagent. Under thesecircumstances,many low molecularweight synthetic inhibitors of HNE have beendeveloped(10 and reviewed in 11). A low molecular weight inhibitor of HNE which has broad inhibition on neutrophil elastasesderived from severalspeciesof animals,is highly specific and hassystemic activity Abbreviations: HNE, human neutrophil elastase;al-PI, alpha-1-proteinaseinhibitor; a2-MG, alpha-2-macroglobulin;sue-Ala-Pro-Ala-pNa, succinyl-Ala-Pro-Ala-p-nitroanilide. 0006-291X/91 $1.50 Copyright 0 I991 by Academic Press. Inc. All rights of reproduction in any form reserved.
814
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
is required for studying the role of neutrophil elastase on diseases such as emphysema and adult respiratory distress syndrome. However,
such a highly specific and systemically active
elastase inhibitor has not been reported as yet. We have found that ONO-5046, N-[2-[4-(2,2Dimethylpropionyloxy)phenylsulfonylamino]benzoyl] and intravenously
aminoacetic acid is a potent, specific
active neutrophil elastase inhibitor. In the present paper, we describe the
biochemical and pharmacological properties of ONO-5046. MATERIALS
AND METHODS
Enzvme and regents: Purified human neutrophil elastase (purulent human sputum as a source) was purchased from Elastin Products Co., Inc. (Missouri, U.S.A.). Bovine pancreas trypsin, human plasma thrombin, human plasma kallikrein, bovine pancreas chymotrypsin, n-N-benzoyl-DL-arginine-p-nitroanilide HCl, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and elastin-congo red were obtained from Sigma Chemicals Corp. (St. Louis, U.S.A.). Synthetic substrate, S-2238, S-2266 and S-2302 were obtained from Daiichikagaku Pharmaceutical Co., Ltd. (Tokyo, Japan). Succinyl-Ala-Pro-Ala-p-nitroanilide (sue-Ala-Pro-Ala-pNa) and succinyl-Ala-Ala-Ala-p-nitroanilide were purchased from Peptide Institute, Inc. (Osaka, Japan). Porcine pancreatic kallikrein was a product of Ono pharmaceutical Co., Ltd. (Osaka, Japan). ONO-5046 was synthesized in our laboratories. The chemical structure of ONO-5046 is shown in Fig. 1. Prenaration of neutrouhil extracts: Peritoneal neutrophils were obtained from rabbits pretreated with 0.2% glycogen (200 ml/body) five hrs before harvesting peritoneal cells by saline lavage, and from rat, hamster and mouse pretreated with 1% casein (100 ml/kg) 16 hrs before harvesting peritoneal cells by saline lavage. Then neutrophils were suspended in Tris-HCl buffer (0.1 M, pH 7.5) containing 0.1% Brij-35 and 1 M of MgC12 and lysed by sonication. The lysate was used as the source of neutrophil elastase. The hydrolytic activity of neutrophil elastase was Determination of urotease activitv: detemrined using two methods. (a) The hydrolysis of synthetic substrate, sue-Ala-Pro-AlapNa was measured by the spectrophotometric method of Levine et al (12): sue-Ala-Pro-AlapNa (0.1 mM) was incubated with the enzyme (1 x 1O-3 U) in Ttis-HCl buffer (0.1 M, pH 8.0) containing 0.2 M sodium chloride in a final volume of 1.0 ml. After incubation at 37°C for 30 min, the absorbance at 405 nm was measured. (b) The hydrolysis of elastincongo red was measured by the spectrophtometric method of Naughton and Sanger (13) with some modifications: elastin-congo red (2 mg/ml) was incubated with 0.2 U of enzyme in TrisHCl buffer (0.1 M pH SO), containing 0.2 M of sodium chloride in a final volume of 1.0 ml. After incubation at 37°C for 1 hour, the absorbance was measured at 500 nm. Porcine pancreatic elastase activity was determined by succinyl-Ala-Ala-Ala-p-nitroanilide hydrolysis by the method of Fujimoto et al (14) with some modifications: sue-Ala-Ala-Ala-pNA was incubated with 1 x 1O-3U of enzyme at 37’C in HEPES-NaOH buffer (20 mM, pH X.0) containing 10 mM CaC12 in a final volume of 1.O ml. The developed color was measured spectrophoto-metorically at 405 nm. One unit of elastase activity was defined as the quantity of enzyme that liberated 1 umol of p-nitroanilide in 1 min. Other protease activity was determined by the method of Matsuoka et al. (15).
CH3-~~+=+O*NH~H~ooH l&w.: 434.47
Fig. Chemical structure of ONO-5046 ( N-[2-[4-(2,2-Dimethylpropionyloxy)phenylsulfonylamino]benzoyl]aminoacetic acid ). 815
Vol.
177,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Lunar hemorrhage: Male Golden hamsters, weighing 90 to 110 g were used. Human neutrophil elastase (0.3 U in 1.50~1 PBS /site) was injected intratracheally via a small incision in the ventral neck region, using a small diameter (o.d. = 1.0 mm) polyethylene catheter under ketamine (1.50 mg/kg i.p.) anesthesia. One hour later, hamsters were sacrificed by bleeding via the abdominal aorta. The isolated lungs were homogenized with 5 ml of distilled water and the homogenates were centrifuged at 22600 x g for 30 min. The supernatant was diluted (x 10) with distilled water and absorbance at 412 nm (the index for hemorrhage) was measured spectrophotometorically. ONO-5046 was administered intratracheally five min before HNE injection. Male Hartley strain guinea pigs, weighing 250 to 350 g were Canillarv Dermeabilitv: injected with human neutrophil elastase (20 x 10m3U in 100 ~1 PBS / site) in the clipped-back skin. Soon after, 1.7 ml/kg of Evans blue solution (2% w/v) was injected intravenously. Thirty min after Evans blue injection, guinea pigs were sacrificed by bleeding via the carotic aorta and the blue spots in the skin were cut off and solubilized by 1 ml of 1 N KOH at 37°C overnight. The absorbance of acetone-O.6 N phosphate solution (13 : 5 v/v)- extracted dye was measured spectrophotometorically at 620 nm ( the index for permeability). ONO-5046 was administered intravenously 30 set before HNE injection. Statistics: Statistical comparisons were evaluated by using variance analysis of the software package SAS (Statistical Analysis System, cary, USA) followed by Student’s unpaired t-test. Only p values of 0.05 or less were considered statistically significant. RESULTS AND DISCUSSION The inhibitory activity of ONO-5046
was studied using a synthetic substrate, sue-Ala-
Pro-Ala-pNa and a macromolecular substrate, elastin-congo red. ONO-5046 potently inhibited HNE with an IC50 of 0.044 f 0.003 PM, Ki of 0.20 f 0.02 FM (sue-Ala-Pro-Ala-pNa substrate)
and ICs() of 7.0 f 0.1 pM ( e1,ds‘t’m - congo red as a substrate).
competitive
as determined by Lineweaver-Burk
as a
The inhibition is
plot analysis (sue-Ala-Pro-Ala-pNa
as a
substrate). The IC50 value with elastin-congo red as a substrate was higher than that with sucAla-Pro-Ala-pNa.
This may be due to the difference of enzyme concentration in the two assay
systems because the hydrolysis
of elastin-congo
concentration than that of sue-Ala-Pro-Ala-pNa. of SC-39027
red required 200 times higher enzyme
Nakao et al (16) have reported the IC50 value
against elastin is higher than that of synthetic
substrate. ONO-5046
also
inhibited leukocyte elastase obtained from rabbits, rats, hamsters or mise with IC,, values comparable to that against HNE (Table 1). These results show that the inhibition of ONOSO46 is not confined to HNE but extends to neutrophil elastases of several other animal species. Furthermore, plasma thrombin,
ONO-5046
was inactive against bovine pancreas trypsin,
human plasma plasmin, porcine pancreas kallikrein,
kallikrein, bovine pancreas chymotrypsin However, ONO-5046
human
human plasma
and human neutrophil cathepsin G even at 100 NM.
inhibited porcine pancreas elastase with an IC5o value of 5.6 + 0.2 PM
(succinyl-Ala-Ala-Ala-p-nitroanilide
as a substrate).
IC,o values of ONO-5046 on neutrophil
elastases derived from human or other animal species were about 130 times higher than that of 816
Vol.
177, No. 2, 1991
Table
1. Inhibitory Human
Human
of
AND BIOPHYSICAL
Effect of ONO-5046 and Various Animal
Rabbit
0.044 + 0.003 The hydrolysis
BIOCHEMICAL
sue-Al;~-Pro-Ala-pNa
on Neutrophil Species
Rat
0.036 + 0.001
O.OlY
was
f 0.001
measured. Each
RESEARCH COMMUNICATIONS
value
Elastase
of
Hamster
Mouse
0.037+ 0.004
0.049+ 0.005
is
mean
+
S.E.M.
of
three
separate
rxperiments.
porcine pancreaselastase,indicating that inhibition by ONO-5046 is highly specific to these neutrophil elastases.Thus, ONO-5046 can be useto study the roles of neutrophil elastasein severalanimal models. Evidence for in vivo activities of ONO-5046 was obtained by two animal models. We first examined the effect of ONO-5046 using lung hemorrhageinduced by intratracheal instillation of HNE. Intratracheal administration of ONO-5046 significantly and dosedependently suppressedthe lung hemorrhage (Fig. 2), with an ID,, value of 82 pg/kg. Hassallet al (17) have alsoevaluated the effect of a HNE inhibitor using HNE-induced lung hemorrhagein hamsters.Secondly, we examined the intravenous activity of ONO-5046 on a
I *** T
Vehrcle 0.02 I
0.21
ONO-5046
0.7
7 .I -.
(mg/kg)
Fig.. Effect of intratracheal administration of ONO-5046 on lung hemorrhage induced by human neutrophil elastase in hamsters. Human neutrophil elastase (0.3 U/site) was instillated intratracheally. ONO-5046 was administered five min prior to enzyme. Each value is mean f S.E.M. of five to six animals. **:P
