Journal of the Neurological Sciences, 109 (1992) 107-110 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00
107
JNS 03748
Inhibition of stimulated human leukocyte hydrogen peroxide generation by a novel antioxidant, OPC- 14117 M a r c F i s h e r a n d M a r g a r e t M. A r p a n o Department of Neurology and Medicine and Blood Research Laboratory, The Medical Center of Central Massachusetts, and Department of Neurology, University of Massachusetts Medical School, Worcester,MA, USA (Received 3 September, 1991) (Revised, received 19 November, 1991) (Accepted 21 November, 1991)
Key words: Leukocytes; Oxygen free radicals; Antioxidant Summary Oxygen free radicals generated by leukoeytes may contribute to tissue injury after central nervous system (CNS) focal isehemia or trauma. Inhibiting oxygen free radicals has improved outcome in experimental models of these conditions and antioxidant therapy appears promising. We evaluated the ability of a novel antioxidant, OPC-14117, to reduce hydrogen peroxide (H20 2) production by stimulated human polymorphonuclear (PMN) leukocytes and monocytes. Stimulated PMN and monocytes were incubated with several concentrations of OPC-14117 for 20 rain and H20 2 production, nmol/1 x 10~ cells/30 rain, was measured. OPC-14117 significantly reduced PMN H20 2 production (P < 0.001) and monoeyte H20 2 production (P < 0.05). A dose response relationship was observed for both leukocytes, as the 100/~M drug concentration was significantly (P < 0.05) more effective than the 10/~M concentration. These results demonstrate that OPC-14117 inhibits H202 generation by stimulated human leukoeytes and support further studies of its effects in disorders such as CNS focal ischemia and trauma, conditions where antioxidant therapy may be beneficial. Introduction Oxygen free radicals are highly toxic products of molecular oxygen or hydrogen peroxide (H 202 ), which contain an unpaired electron in their outer ring (Southern and Powis 1988). These compounds can induce cellular injury within the central nervous system (CNS) by oxidizing lipids, proteins or nucleic acids. Lipid membranes are especially vulnerable and this may be an important mediator of oxygen free radical induced CNS injury (Kontos 1987). Because of these hazardous effects of oxygen free radicals, neurons and other CNS cells are equipped with enzymes such as superoxide dismutase, catalase and glutathione peroxidose, for protection. These enzymes and others protective mechanisms, such as the free radical scavenger, vitamin E, can be easily overwhelmed when large quantities of oxygen free radicals are produced, as may occur during reperfusion after CNS ischemic or traumatic injury. Oxygen free radical mediated CNS injury has been implicated in both of these conditions (Chan and Fishman 1985). Correspondence to: Marc Fisher, M.D., Medical Center of Central Massachusetts, 119 Belmont Street, Worcester, MA 01605, U.S.A. Tel.: 508-793-6641; Fax: 508-793-6412.
Oxygen free radicals can be produced by a variety of biochemical and cellular mechanisms. Inflammatory white blood cells, polymorphonuclear leukocytes (PMN) and monocytes produce large quantities of oxygen free radicals and are recruited to areas of CNS injury acutely and subacutely (Pozzilli et al. 1985). Inhibiting PMN and monocyte oxygen free radical generation might offer a novel mechanism to inhibit CNS damage after CNS ischemia or trauma. Therefore, we evaluated the effects of a novel antioxidant, OPC14117, 7-hydroxy-l-[4-(3-methoxyphenyl)- 1-piperazinyl]acetyl]amino]-2,2,4,6-tetramethylindane, on stimulated PMN and monocyte H 2 0 2 generation. OPC14117 contains a phenolic hydroxyl group as does vitamin E. Preliminary studies have demonstrated that OPC-14117 can inhibit lipid peroxidation of rat brain homogenates, improve survival in hypoxic conditions and enhance recovery after cerebral concussion in mice (Oshiro et al. 1991).
Materials and methods
White blood cell separation Blood samples were drawn from 12 unmedicated normal volunteers, who signed a consent form ap-
108 proved by our institutional review board and were randomly assigned to one of 2 groups. One group of 6 volunteers were used for PMN experiments and a different group of 6 volunteers was used for the monocyte experiments. Two separate groups were used because of the large volume of blood required for each study. For the PMN experiment, there were 5 male and 1 female volunteers with a mean age of 34 years. One was a smoker. For the monocyte experiment, 3 male and 3 female volunteers were employed with a mean age of 26 years. Three volunteers were smokers. Monocytes were obtained by collecting blood into eighteen 10 ml EDTA vacutainer tubes (K 3, 10.5 mg/ml; Becton Dickinson, Rutherford, N J). Forty ml of whole blood was mixed with 4 ml of 6% Dextran 500 (Sigma Chemical company, St. Louis, MO) in normal saline. Blood and Dextran were gently mixed and left to sediment at room temperature for 60 min. After sedimentation, the leukocyte rich plasma (LRP) supernatant was collected and pooled. SIX ml of LRP was carefully layered over 3 ml of Nycodenz-Monocytes (Robbins Scientific, Mountain View, CA), a non-ionic derivative of tri-iodobenzoic acid which separates monocytes based on density and osmolality. L R P / Nycodenz was then spun at room temperature for 20 rain at 500 x g. After spinning, the monocyte/platelet interface was collected, pooled and mixed with an equal volume of phosphate buffered saline (PBS, Sigma). The cells were spun at room temperature for 7 min at 600 X g. To remove the platelets, the monocyte/platelet pellet was resuspended in 7.5 ml PBS and layered over a gradient containing 24 mi of 12% sucrose layered over 6 ml of 16% r lcrose and spun at room temperature for 12 min at 200 x g. The platelets were trapped in the supernatant and discarded. The monocyte pellet was washed once in phenol red working solution containing 140 mM NaCI; 10 mM potassium phosphate buffer, pH 7.0, 5.5 mM dextrose; 0.28 mM phenol red, and 8.5 U / m l horseradish peroxidase (Sigma). Cells were resuspended in the phenol red buffer at a concentration of 1.77 x 106 ceUs/ml; of which 89% were monocytes, 1% polys, and 10% iymphocytes (by a-naphthylbutyrate staining). Cell viability was determined by mixing 0.1 ml of monocytes with 0.1 ml of 0.2% trypan blue in PBS. Cell viability was 94% pre- and postincubation with OPC-14117 at the concentrations employed, as determined by the percentage of cells excluding the dye. Polymorphonuclear leukocytes were prepared by dextran sedimentation as previously described (Levine et al. 1981). The PMN cell pellet was resuspended in the phenol red buffer at a concentration of 4.06 x 106 cells/ml; of which 72% were polys, 20% lymphs, and 8% monocytes. Cell viability was 93% pre- and postincubation with OPC-14117 at the concentrations employed.
Drug preparation The drug was made up prior to use in the assay. OPC-14117, 0.04376g (Otsuka Pharmaceutical Co., Ltd., Tokyo 101, Japan) was dissolved in 1.0 ml dimethylformamide (Sigma) to a concentration of 100000 /~M. The drug was further diluted with 3% bovine serum albumin (BSA, Sigma) in PBS to 1000/~M and 100 IzM. Since the ratio of dimethyiformamide and BSA differed for each drug concentration, corresponding vehicles were prepared in the same manner as the drug. Both monocytes and PMN were incubated with various concentrations of the drug or vehicle for 20 min prior to stimulation and assay. The final concentrations of drug were, 10, 100 or 300/zM.
Hydrogen peroxide assay The H202 assay was performed using a modification of the colorimetric method described by Pick and Keisari (Pick and Keisari 1980). Samples of 0.9 ml of the monocyte preparation (1.77x 106 cells/ml) or PMN preparation (4.06 × 106 cells/ml) were preincubated with 0.1 ml of various concentrations of OPC14117 or the respective vehicle for 20 rain and then stimulated by adding 0.1 ml of 0.22/tM phorbol myrisrate acetate (PMA, Sigma). Cells were allowed to react for 30 min in a 37°C shaking water bath before the reaction was stopped by centrifuging at 4°C for 10 min at 2000 X g. The supernatant was decanted and brought to room temperature by incubating it in a 22°C water bath for 30 min. The pH of the supernatant was adjusted to 12.5 by adding 10 /tl of 1 N NaOH. Standards were run under identical conditions. Samples were analyzed on a Shimadzu 160A Spectrophotometer (Shimadzu Co., Kyoto, Japan) at an absorption wavelength of 610 nm to measure horseradish-peroxidase-mediated oxidation of phenol red by H202. Results were expressed as nmol/30 m i n / l x 106 cells. All samples were run in duplicate, and the values reported are the average of the 2 results for 6 subjects evaluated.
Statistical analysis A one-factor repeated measure, analysis of variance and paired t-test were performed in multiple and two group comparisons for data of the PMN H202 study. When a significant difference was found, specific linear comparisons were made using Scheffe's F-test. Wilcoxon signed-rank test was used for the monocyte H202 study, because of the non-normative distribution of the data. A two-tailed probability less than 0.05 was considered significant. Results Production of H 202 by stimulated human PMN was significantly inhibited by OPC-14117 ( P < 0.001). Each
109 TABLE 1 THE EFFECTS OF OPC-14117 ON H202 PRODUCTION BY STIMULATED PMN, nmol/l x 106 PMN/30 MIN Mean _+SEM. OPC-14117 (n = 6) Control (phenol red solution) 10/tM 100/tM 300/zM
Drug
Vehicle
% Suppression
40.4-+4.7 * 10.0+ 1.8 ** 7.9_+0.8 **
40.2 + 3.0 46.4-+5.3 43.2_+5.4 38.7+5.1
12.8-+1.7 71.7_+2.2 78.7_+1.7
* P < 0.0002 OPC-14117 as compared to vehicle (paired t-test). ** P < 0.001 OPC-14117 as compared to vehicle (paired t-test).
concentration of the drug in comparison to its vehicle was found to effectively inhibit PMN H 2 0 _, generation (Table 1). A dose response relationship was observed as the 100 /zM and 300 /zM concentration of OPC14117 lowered PMN H202 generation significantly more ( P < 0 . 0 5 ) than the 1 0 / z M concentration, although the 100 /tM and 300 /zM concentrations did not differ. Stimulated human monocyte H 202 production was also significantly reduced by OPC-14117 at both the 10/zM and 100/zM concentrations (Table 2). The 100/zM concentration significantly (P < 0.05) inhibited H 2 0 2 generation by stimulated monocytes when compared to the 10/zM concentration. To determine whether OPC-14117 had a scavenging effect o n H 2 0 2 , rather than inhibition of its production, 10 ttM and 100 /zM drug and their respective vehicles were incubated with known concentrations of H 2 0 2 (10 and 50/zM) and buffer for 20 minutes in the absence of monocytes. This mixture yielded the same quantity of measurable H 2 0 2 in our assay system as that concentration of H 2 0 2 and buffer without OPC14117, indicating that the drug was not scavenging the H202.
Discussion
These results demonstrate that OPC-14117 reduces generation by stimulated human PMN and
H20 2
TABLE 2 THE EFFECTS OF OPC-14117 ON H202 PRODUCTION BY STIMULATED MONOCYTES, n m o l / l × 106 MONO/30 MIN Mean + SEM.
OPC-i4117 (/zM) Drug (n 6)
Vehicle
% Suppression
30.4_+ 7.7 22.6_+4.0 18.0_+3.7
60.2±8.3 91.7_+2.8
=
Control (phenol red solution) 10/zM 100/tM
10.2_+3.6 * 1.54-0.6 *
* P < 0.05 0PC-14117 as compared to vehicle (Wilcoxon ranked sign test).
monocytes. A dose response relationship for the reduction of H20 2 measurements was observed for both cell types, as the 100/.tM concentration of OPC-14117 was significantly more effective than the 10/~M concentration. The reduction in H 202 measurements associated with stimulated monocytes and PMN primarily represents inhibition of production and not a scavenging effect. This is apparent from the observations that 10 /~M and 100/tM concentrations of OPC-14117 incubated with known concentrations of H 2 0 2 did not reduce the amount of H 2 0 z which could be measured in our assay system. OPC-14117 effectively inhibits H202 generation by stimulated PMN and monocytes, although the drug may be more effective in monocytes, because the percentage reduction of H 2 0 2 generation was greater at the 10 ~M and 100/.tM drug concentrations in these cells. Hydrogen. peroxide is formed by the conversions of superoxide to H 2 0 2 by superoxide dismutase and is not itself very toxic (Klebanoff 1980). It does diffuse readily in tissues and can be converted to the highly toxic free radical, the hydroxyl radical, by the "Fenton" or "Haber-Weiss" reaction. The hydroxyl radical is highly destructive to cellular constituents such as lipid membranes, because it can induce lipid peroxidation. Leukocytes, such as PMN can also produce hypochlorous acid, another highly toxic free radical, from the hydroxyl radical and H202. Therefore, the ability of OPC-14117 to inhibit PMN and monocyte H20 2 generation should reduce the capability of these cells to induce free radical mediated tissue injury. PMN are rapidly recruited to areas of focal cerebral ischemic injury and monocytes appear at a later time point (Pozzelli et al. 1985). Oxygen free radicals released by PMN may contribute to ischemic injury. Activated PMN in the cerebral microvasculature can injure endothelial cells and other CNS cells and well as impair perfusion by pluggillg small vessels (Ernst et al. 1987). Reducing the number of PMN prior to experimental ischemic stroke has been observed to improve electrophysiologicai consequences of focal ischemia (Dutka et al. 1989). Neutropenia induced by antineutrophil antiserum has been shown to reduce infarct size in a rabbit stroke model (Bednar et al. 1991). Another group of antioxidants, the 21-aminosteroids have been observed to improve survival, preserve neurons and reduce cerebral edema in an animal stroke model (Hall and Pazara 1989). The 21-aminosteroids have also been observed to inhibit PMN and monocyte H202 production (Fisher et al. 1990). These studies support the concept that PMN contribute to ischemic injury associated with stroke and suggest that interfering with PMN inflammatory potential, as exemplified by oxygen free radical generation, may be beneficial in acute ischemic stroke. Lipid membrane peroxidation also appears to be an important mechanism of tissue injury in CNS
110
trauma (Demopoulos et al. 1980). Drugs such as methylprednisolone and the 21-aminosteroids, which inhibit lipid membrane peroxidation, reduce the neurological consequences of experimental and clinical spinal cord and head trauma (Hall et al. 1988; Bracken et al. 1990). Inhibiting inflammatory leukocyte .oxygen free radical production may contribute to these beneficial effects on CNS trauma. Our observation that OPC-14117 reduces H202 generation by stimulated human PMN and monocytes suggests that this drug could be useful for ameliorating the neurologic damage associated with focal ischemia and trauma~ Previous studies have demonstrated that OPC-14117 is protective in animal models of hypoxia and CNS trauma (Oshiro et ai. 1991). The drug also appears to have minimal toxicity. These observations suggest that OPC-14117 has substantial antioxidant and CNS protective effects and that this drug should be evaluated more extensively in animal models of focal cerebral ischemia and CNS trauma. Acknowledgements We would like to thank Kazuo Minematsu, M.D., for his help with the statistical analysis and Joyce Ford for her help in preparing the manuscript. This study was supported in part by Otsuka Pharmaceutical, Co,, Ltd.
Refet'ences Bednar, M.M., Raymond, S., McAuliffe, T., Lodge, P,A. and C.E. Gross (1991) The role of neutrophils and platelets in a rabbit model of thromboembolic stroke. Stroke, 22: 44-50. Bracken, M.D., Sheppard, MJ., Collins, W,F., Holford, T.R, Young,
W., Baskin, D.S., Eisenberg, H.M., Flamm, E., Leo-Summers, L., Maroan, ,l., Marshall, L.F., Perot, P.L., Piepmeier, J., Sonntag, V.K.H., Wagner, F.C., Wilberger, J.E. and H.R. Winn (1990) A randomized controlled trial of methylprednisolone or naloxone in the trea~ent of acute spinal-cord injury. N. Engl. J. Med., 322: 1405-141 !.
Chan, P.H. and R.A. Fishman (1985) Oxygen free radical: potential mediators in brain injury. In: Inaba, Y., I. Klatzo and M. Spatz (Eds.), Brain Edema, Springer-Verlag, Berlin, pp. 317-323. Demopoulos, H.B., Flamm, E.S., Fietronigro, D.D. and M.L. Seligman (1980) The free radical pathology and the microcirculation in the major central nervous system disorders. Acta Physiol. Scand. (Suppl.). 492: 91-119. Dutka, AJ., Kochanek, P.M. and J.M. Hallenback (1989) Influence of granulocytopenia on canine cerebral ischemia induced by air embolism. Stroke, 20: 390-395. Ernst, E., Hammerschmidt, D.E., Bagge, U., Matrai, A. and J.A. Dormandy (1987) Leukocytes and the risk of ischemic diseases. JAMA, 257: 2318-2324. Fisher, M., Levine, P.H. and R.A. Cohen (1990) A 21-aminosteroid reduces hydrogen peroxide generation by and chemiluminescence of stimulated human leukocytes. Stroke, 21: 1435-1438. Hall, E.D. and K.E. Pazara (1989) Effects of 21-aminosteroid antioxidants on postischemic neuronal degeneration. In: Ginsberg, M.A. and W.D. Dietrich (Eds.), 16th Princeton Conference, Raven Press, New York, pp. 387-391. Hall, E.D., Yonkers, P.A, McCall, J.M. and M. Braughler (1988) Effects of the 21-aminosteroid U74006F on experimental head injury in mice. J. Neurosurg., 68: 456-461. Klebanoff, S. (1980) Oxygen metabolism and the toxic properties of phagocytes. Ann. Intern. Med., 93: 480-489. Kontos, M.A. (1989) Oxygen radicals in cerebral ischemia. In: Ginsberg, M.D. and W.D. Dietrich (Eds.), 16th Princeton Conference, Raven Press, New York, pp. 365-372. Levine, P.H., Harding, J.C., Scoon, K.L. and N.i. Krins~ (1981) Effects of corticosteroids on the production of superoxide and hydrogen peroxide and the appearance of chemiluminescence by phagocytosing polymorphonuclear leukocyte. Inflammation, 5: 19-26. Oshiro, Y., Sakurai, Y., Tanaka, T., Kikuchi, T., Hirose, T. and K. Trottori (1991) Novel cerebroprotcctive agents with central nervous system stimulating activity. J. Med. Chem., 34: 2004-2013; 2014-2023. PozziUi, C., Lenzi, G,L., Argentina, C., CaroUei, A., Rasura, M., Signore, A., Bezzae. and P. Pozzilli (1985) Imaging of leukocyte infiltration in human cerebral infarcts. Stroke, 16: 251-255. Pick, E. and Y. Keisari (1980) A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J. lmmunol. Methods, 38: 161-170. Southern, P.A. and G. Powis (1988) Free radicals in medicine. I. Chemical nature and biologic reactions. Mayo Clin. Prec., 63: 381-389.
107
JNS 03748
Inhibition of stimulated human leukocyte hydrogen peroxide generation by a novel antioxidant, OPC- 14117 M a r c F i s h e r a n d M a r g a r e t M. A r p a n o Department of Neurology and Medicine and Blood Research Laboratory, The Medical Center of Central Massachusetts, and Department of Neurology, University of Massachusetts Medical School, Worcester,MA, USA (Received 3 September, 1991) (Revised, received 19 November, 1991) (Accepted 21 November, 1991)
Key words: Leukocytes; Oxygen free radicals; Antioxidant Summary Oxygen free radicals generated by leukoeytes may contribute to tissue injury after central nervous system (CNS) focal isehemia or trauma. Inhibiting oxygen free radicals has improved outcome in experimental models of these conditions and antioxidant therapy appears promising. We evaluated the ability of a novel antioxidant, OPC-14117, to reduce hydrogen peroxide (H20 2) production by stimulated human polymorphonuclear (PMN) leukocytes and monocytes. Stimulated PMN and monocytes were incubated with several concentrations of OPC-14117 for 20 rain and H20 2 production, nmol/1 x 10~ cells/30 rain, was measured. OPC-14117 significantly reduced PMN H20 2 production (P < 0.001) and monoeyte H20 2 production (P < 0.05). A dose response relationship was observed for both leukocytes, as the 100/~M drug concentration was significantly (P < 0.05) more effective than the 10/~M concentration. These results demonstrate that OPC-14117 inhibits H202 generation by stimulated human leukoeytes and support further studies of its effects in disorders such as CNS focal ischemia and trauma, conditions where antioxidant therapy may be beneficial. Introduction Oxygen free radicals are highly toxic products of molecular oxygen or hydrogen peroxide (H 202 ), which contain an unpaired electron in their outer ring (Southern and Powis 1988). These compounds can induce cellular injury within the central nervous system (CNS) by oxidizing lipids, proteins or nucleic acids. Lipid membranes are especially vulnerable and this may be an important mediator of oxygen free radical induced CNS injury (Kontos 1987). Because of these hazardous effects of oxygen free radicals, neurons and other CNS cells are equipped with enzymes such as superoxide dismutase, catalase and glutathione peroxidose, for protection. These enzymes and others protective mechanisms, such as the free radical scavenger, vitamin E, can be easily overwhelmed when large quantities of oxygen free radicals are produced, as may occur during reperfusion after CNS ischemic or traumatic injury. Oxygen free radical mediated CNS injury has been implicated in both of these conditions (Chan and Fishman 1985). Correspondence to: Marc Fisher, M.D., Medical Center of Central Massachusetts, 119 Belmont Street, Worcester, MA 01605, U.S.A. Tel.: 508-793-6641; Fax: 508-793-6412.
Oxygen free radicals can be produced by a variety of biochemical and cellular mechanisms. Inflammatory white blood cells, polymorphonuclear leukocytes (PMN) and monocytes produce large quantities of oxygen free radicals and are recruited to areas of CNS injury acutely and subacutely (Pozzilli et al. 1985). Inhibiting PMN and monocyte oxygen free radical generation might offer a novel mechanism to inhibit CNS damage after CNS ischemia or trauma. Therefore, we evaluated the effects of a novel antioxidant, OPC14117, 7-hydroxy-l-[4-(3-methoxyphenyl)- 1-piperazinyl]acetyl]amino]-2,2,4,6-tetramethylindane, on stimulated PMN and monocyte H 2 0 2 generation. OPC14117 contains a phenolic hydroxyl group as does vitamin E. Preliminary studies have demonstrated that OPC-14117 can inhibit lipid peroxidation of rat brain homogenates, improve survival in hypoxic conditions and enhance recovery after cerebral concussion in mice (Oshiro et al. 1991).
Materials and methods
White blood cell separation Blood samples were drawn from 12 unmedicated normal volunteers, who signed a consent form ap-
108 proved by our institutional review board and were randomly assigned to one of 2 groups. One group of 6 volunteers were used for PMN experiments and a different group of 6 volunteers was used for the monocyte experiments. Two separate groups were used because of the large volume of blood required for each study. For the PMN experiment, there were 5 male and 1 female volunteers with a mean age of 34 years. One was a smoker. For the monocyte experiment, 3 male and 3 female volunteers were employed with a mean age of 26 years. Three volunteers were smokers. Monocytes were obtained by collecting blood into eighteen 10 ml EDTA vacutainer tubes (K 3, 10.5 mg/ml; Becton Dickinson, Rutherford, N J). Forty ml of whole blood was mixed with 4 ml of 6% Dextran 500 (Sigma Chemical company, St. Louis, MO) in normal saline. Blood and Dextran were gently mixed and left to sediment at room temperature for 60 min. After sedimentation, the leukocyte rich plasma (LRP) supernatant was collected and pooled. SIX ml of LRP was carefully layered over 3 ml of Nycodenz-Monocytes (Robbins Scientific, Mountain View, CA), a non-ionic derivative of tri-iodobenzoic acid which separates monocytes based on density and osmolality. L R P / Nycodenz was then spun at room temperature for 20 rain at 500 x g. After spinning, the monocyte/platelet interface was collected, pooled and mixed with an equal volume of phosphate buffered saline (PBS, Sigma). The cells were spun at room temperature for 7 min at 600 X g. To remove the platelets, the monocyte/platelet pellet was resuspended in 7.5 ml PBS and layered over a gradient containing 24 mi of 12% sucrose layered over 6 ml of 16% r lcrose and spun at room temperature for 12 min at 200 x g. The platelets were trapped in the supernatant and discarded. The monocyte pellet was washed once in phenol red working solution containing 140 mM NaCI; 10 mM potassium phosphate buffer, pH 7.0, 5.5 mM dextrose; 0.28 mM phenol red, and 8.5 U / m l horseradish peroxidase (Sigma). Cells were resuspended in the phenol red buffer at a concentration of 1.77 x 106 ceUs/ml; of which 89% were monocytes, 1% polys, and 10% iymphocytes (by a-naphthylbutyrate staining). Cell viability was determined by mixing 0.1 ml of monocytes with 0.1 ml of 0.2% trypan blue in PBS. Cell viability was 94% pre- and postincubation with OPC-14117 at the concentrations employed, as determined by the percentage of cells excluding the dye. Polymorphonuclear leukocytes were prepared by dextran sedimentation as previously described (Levine et al. 1981). The PMN cell pellet was resuspended in the phenol red buffer at a concentration of 4.06 x 106 cells/ml; of which 72% were polys, 20% lymphs, and 8% monocytes. Cell viability was 93% pre- and postincubation with OPC-14117 at the concentrations employed.
Drug preparation The drug was made up prior to use in the assay. OPC-14117, 0.04376g (Otsuka Pharmaceutical Co., Ltd., Tokyo 101, Japan) was dissolved in 1.0 ml dimethylformamide (Sigma) to a concentration of 100000 /~M. The drug was further diluted with 3% bovine serum albumin (BSA, Sigma) in PBS to 1000/~M and 100 IzM. Since the ratio of dimethyiformamide and BSA differed for each drug concentration, corresponding vehicles were prepared in the same manner as the drug. Both monocytes and PMN were incubated with various concentrations of the drug or vehicle for 20 min prior to stimulation and assay. The final concentrations of drug were, 10, 100 or 300/zM.
Hydrogen peroxide assay The H202 assay was performed using a modification of the colorimetric method described by Pick and Keisari (Pick and Keisari 1980). Samples of 0.9 ml of the monocyte preparation (1.77x 106 cells/ml) or PMN preparation (4.06 × 106 cells/ml) were preincubated with 0.1 ml of various concentrations of OPC14117 or the respective vehicle for 20 rain and then stimulated by adding 0.1 ml of 0.22/tM phorbol myrisrate acetate (PMA, Sigma). Cells were allowed to react for 30 min in a 37°C shaking water bath before the reaction was stopped by centrifuging at 4°C for 10 min at 2000 X g. The supernatant was decanted and brought to room temperature by incubating it in a 22°C water bath for 30 min. The pH of the supernatant was adjusted to 12.5 by adding 10 /tl of 1 N NaOH. Standards were run under identical conditions. Samples were analyzed on a Shimadzu 160A Spectrophotometer (Shimadzu Co., Kyoto, Japan) at an absorption wavelength of 610 nm to measure horseradish-peroxidase-mediated oxidation of phenol red by H202. Results were expressed as nmol/30 m i n / l x 106 cells. All samples were run in duplicate, and the values reported are the average of the 2 results for 6 subjects evaluated.
Statistical analysis A one-factor repeated measure, analysis of variance and paired t-test were performed in multiple and two group comparisons for data of the PMN H202 study. When a significant difference was found, specific linear comparisons were made using Scheffe's F-test. Wilcoxon signed-rank test was used for the monocyte H202 study, because of the non-normative distribution of the data. A two-tailed probability less than 0.05 was considered significant. Results Production of H 202 by stimulated human PMN was significantly inhibited by OPC-14117 ( P < 0.001). Each
109 TABLE 1 THE EFFECTS OF OPC-14117 ON H202 PRODUCTION BY STIMULATED PMN, nmol/l x 106 PMN/30 MIN Mean _+SEM. OPC-14117 (n = 6) Control (phenol red solution) 10/tM 100/tM 300/zM
Drug
Vehicle
% Suppression
40.4-+4.7 * 10.0+ 1.8 ** 7.9_+0.8 **
40.2 + 3.0 46.4-+5.3 43.2_+5.4 38.7+5.1
12.8-+1.7 71.7_+2.2 78.7_+1.7
* P < 0.0002 OPC-14117 as compared to vehicle (paired t-test). ** P < 0.001 OPC-14117 as compared to vehicle (paired t-test).
concentration of the drug in comparison to its vehicle was found to effectively inhibit PMN H 2 0 _, generation (Table 1). A dose response relationship was observed as the 100 /zM and 300 /zM concentration of OPC14117 lowered PMN H202 generation significantly more ( P < 0 . 0 5 ) than the 1 0 / z M concentration, although the 100 /tM and 300 /zM concentrations did not differ. Stimulated human monocyte H 202 production was also significantly reduced by OPC-14117 at both the 10/zM and 100/zM concentrations (Table 2). The 100/zM concentration significantly (P < 0.05) inhibited H 2 0 2 generation by stimulated monocytes when compared to the 10/zM concentration. To determine whether OPC-14117 had a scavenging effect o n H 2 0 2 , rather than inhibition of its production, 10 ttM and 100 /zM drug and their respective vehicles were incubated with known concentrations of H 2 0 2 (10 and 50/zM) and buffer for 20 minutes in the absence of monocytes. This mixture yielded the same quantity of measurable H 2 0 2 in our assay system as that concentration of H 2 0 2 and buffer without OPC14117, indicating that the drug was not scavenging the H202.
Discussion
These results demonstrate that OPC-14117 reduces generation by stimulated human PMN and
H20 2
TABLE 2 THE EFFECTS OF OPC-14117 ON H202 PRODUCTION BY STIMULATED MONOCYTES, n m o l / l × 106 MONO/30 MIN Mean + SEM.
OPC-i4117 (/zM) Drug (n 6)
Vehicle
% Suppression
30.4_+ 7.7 22.6_+4.0 18.0_+3.7
60.2±8.3 91.7_+2.8
=
Control (phenol red solution) 10/zM 100/tM
10.2_+3.6 * 1.54-0.6 *
* P < 0.05 0PC-14117 as compared to vehicle (Wilcoxon ranked sign test).
monocytes. A dose response relationship for the reduction of H20 2 measurements was observed for both cell types, as the 100/.tM concentration of OPC-14117 was significantly more effective than the 10/~M concentration. The reduction in H 202 measurements associated with stimulated monocytes and PMN primarily represents inhibition of production and not a scavenging effect. This is apparent from the observations that 10 /~M and 100/tM concentrations of OPC-14117 incubated with known concentrations of H 2 0 2 did not reduce the amount of H 2 0 z which could be measured in our assay system. OPC-14117 effectively inhibits H202 generation by stimulated PMN and monocytes, although the drug may be more effective in monocytes, because the percentage reduction of H 2 0 2 generation was greater at the 10 ~M and 100/.tM drug concentrations in these cells. Hydrogen. peroxide is formed by the conversions of superoxide to H 2 0 2 by superoxide dismutase and is not itself very toxic (Klebanoff 1980). It does diffuse readily in tissues and can be converted to the highly toxic free radical, the hydroxyl radical, by the "Fenton" or "Haber-Weiss" reaction. The hydroxyl radical is highly destructive to cellular constituents such as lipid membranes, because it can induce lipid peroxidation. Leukocytes, such as PMN can also produce hypochlorous acid, another highly toxic free radical, from the hydroxyl radical and H202. Therefore, the ability of OPC-14117 to inhibit PMN and monocyte H20 2 generation should reduce the capability of these cells to induce free radical mediated tissue injury. PMN are rapidly recruited to areas of focal cerebral ischemic injury and monocytes appear at a later time point (Pozzelli et al. 1985). Oxygen free radicals released by PMN may contribute to ischemic injury. Activated PMN in the cerebral microvasculature can injure endothelial cells and other CNS cells and well as impair perfusion by pluggillg small vessels (Ernst et al. 1987). Reducing the number of PMN prior to experimental ischemic stroke has been observed to improve electrophysiologicai consequences of focal ischemia (Dutka et al. 1989). Neutropenia induced by antineutrophil antiserum has been shown to reduce infarct size in a rabbit stroke model (Bednar et al. 1991). Another group of antioxidants, the 21-aminosteroids have been observed to improve survival, preserve neurons and reduce cerebral edema in an animal stroke model (Hall and Pazara 1989). The 21-aminosteroids have also been observed to inhibit PMN and monocyte H202 production (Fisher et al. 1990). These studies support the concept that PMN contribute to ischemic injury associated with stroke and suggest that interfering with PMN inflammatory potential, as exemplified by oxygen free radical generation, may be beneficial in acute ischemic stroke. Lipid membrane peroxidation also appears to be an important mechanism of tissue injury in CNS
110
trauma (Demopoulos et al. 1980). Drugs such as methylprednisolone and the 21-aminosteroids, which inhibit lipid membrane peroxidation, reduce the neurological consequences of experimental and clinical spinal cord and head trauma (Hall et al. 1988; Bracken et al. 1990). Inhibiting inflammatory leukocyte .oxygen free radical production may contribute to these beneficial effects on CNS trauma. Our observation that OPC-14117 reduces H202 generation by stimulated human PMN and monocytes suggests that this drug could be useful for ameliorating the neurologic damage associated with focal ischemia and trauma~ Previous studies have demonstrated that OPC-14117 is protective in animal models of hypoxia and CNS trauma (Oshiro et ai. 1991). The drug also appears to have minimal toxicity. These observations suggest that OPC-14117 has substantial antioxidant and CNS protective effects and that this drug should be evaluated more extensively in animal models of focal cerebral ischemia and CNS trauma. Acknowledgements We would like to thank Kazuo Minematsu, M.D., for his help with the statistical analysis and Joyce Ford for her help in preparing the manuscript. This study was supported in part by Otsuka Pharmaceutical, Co,, Ltd.
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