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Anti shock psiof the prostacyclin analog, iloprost, in traumatic shock
Prostaglandins
Leukotrienes
and Medicine 25: 175-185,
1986
M. Andrew Levitt and Alian M. Lefer Department of Physiology Jefferson Medical College Thomas Jefferson University Philadelphia, Pennsylvania 19107
The effects of iloprost, a synthetic carbacyclin derivative of prostacyclin (PC1 1 was studied in a standardixed model of traumatic shock. Pentobarifital anesthetized rats (35 mg!kg) subjected to NobleColiip drum trauma were characterized by a 84 !: 10 minute survivai time, a K-fold increase in plasma cathepsin D activity, and a S-fold increase in plasma myocardiai depressant factor WDP) activity. Iloprost sieQificantly attenuated the accumulation of MDF activity in the plasma (69 k 14 vs. 20 + 6 U/ml) vehicle vs. drug (p
Since the discovery of PG12 by Vane and associates (I), a variety of beneficial effects of this endogenous cicosanoid product of arachidonic acid metabolism has been observed. PG12 has been found to potently prevent platelet aggregation (2), stabilize cell membranes (3, B), and dilate coronary arteries (5). However, prostacyclin (PG12)is unstable in aqueous media -in vitro and is rapidly degraded -in vivo (6). Several stable 175
prostacyclin analogs with potent PGI2-like actions have been developed recently with iloprost being the most stable and useful of those currently available. Iloprost has been studied -in vitro demonstrating a powerful antiaggregatory effect on platelets (3, 5, 6, 7, 8, 9) challenged by a variety of aggregatory agents including ADP, collagen, and thrombin. Animal studies have shown beneficial effects of iloprost in reducing the extension of ischemic damage in acute myocardial ischemia (3, 8, IO). Cellular membrane protection and preservation of ischemic tissue have been observed with iloprost in monkey, cat, and rat models of myocardial ischemia. To date, iloprost has not been evaluated in the setting of traumatic shock. The present study was conducted to see if prostacyclin would exert a beneficial effect in a standardized model of traumatic shock which is known to be responsive to drug therapy, and if so, to determine the basic general mechanisms of its protective effect.
Male Sprague-Dawley rats weighing 210 to 270 g were utilized in this study. All rats were placed in one of four experimental groups. These groups were as follows: a) sham shock rats given 0.9% NaCl as the vehicle for iloprost, b) sham shock rats given iloprost (100 ng/kg/min), c) traumatic shock rats receiving the vehicle (0.2 ml/h), and d) traumatic shock rats receiving iloprost. Prior to initiation of trauma or sham trauma, all rats (35 mg/kg) given anesthetized with sodium pentobarbital were intraperitoneally. Traumatic shock rats were subjected to a standardized degree of trauma prior to the above surgical procedures utilizing a Noble-Collip drum rats were placed in the drum for 525 apparatus (11). Anesthetized revolutions at 45 rpm. All rats underwent the following surgical procedures following trauma or sham trauma. A tracheostomy was performed utilizing a polyethylene cannula (PE 250) to maintain a patent airway during the The left carotid artery and right external course of the experiment. jugular vein were cannulated with PE-50 tubing filled with 0.9% NaCl and 50 U/ml of heparin to prevent coagulation of the blood. Within IO-15 minutes of completion of the trauma, the arterial catheter was connected to a Statham P23AC pressure transducer to allow mean arterial blood pressure (MABP) and heart rate (HR) to be continuously recorded on a Grass Model 7 osciilographic recorder. All rats were monitored for five hours after trauma or sham trauma, or until the animal declined to a MABP of 45 mm Hg. At the completion of physiologic monitoring, a 2 ml blood sample was drawn and all surviving rats were given an overdose of pentobarbital. Survival time was thus defined as that time following completion of trauma until MABP fell to 45 mm Hg. Any rats with a MABP less than 60 mm Hg within 30 min after the
176
onset of trauma were excluded from further study. All rats subjected to trauma were autopsied to confirm the presence The presence of of a significant degree of blunt traumatic injury. serosanguinous peritoneal fluid with bowel cyanosis and venous engorgement secondary to mesenteric strangulation were d6cwncntod. If these sequelae were not present to a significant degree, that animal was not included in the study. During the course of this study, 15% of the traumatized rats were excluded on this basis. At the termination of each experiment, 2 ml of blood was drawn into a heparinized (50 U/ml) tube at 4’C. The blood was centrifuged at 5000 x g for 15 minutes at 4’C. The plasma was decanted, and the samples were assayed for cathepsin D and myocardial depressant factor (MDF) activities. Plasma cathepsin D activity was determined by the method of Anson (12) and was used as an index of lysosomal disruption. Plasma MDF was measured according to the method of Barenholz et al. (13) with the chromatographic modifications of Yamada and Pettit (14) followed by its MDF activity is expressed throughout in spectrophotometric analysis. chromatographic units (MDF Units). In Vitro Methods Cats were anesthetized with pentobarbital sodiunl (30 mg/kg, i.v.1 removed and placed in ice-cold Krebs-Henseleit solution. Tissue was minced and then homogenized in Krebs-Henseleit solution (lr10, w:v) according to the method of Bridenbaugh et al. (15). Aliquots of homogenates were incubated at 37’C for 30 minutes in the presemz of 100 ng/ml iloprost and its vehicle (i.e., 0.9% NaCI). Incubates were then deproteinized and assayed for free amino-nitrogen according to the method of Kabat (16). and the pancreas
Statistics All values presented in this study are means 2 standard errors of the mean. Statistical anaiysis of survival time was performed utilizing C&an’s generalized Wilcoxon test as modified for shack studies (17). A one-ta&d Student’s V-test was used for statistical analysis of other variables. RESULTS Iloprost at the infusion rate selected (i.e., 100 ng/kg/min) did not exert any significant Cffect on MABP, HR, survival time, plasma cat%epsin D, or MDF activities in non-shocked control rats. Higher infusion re&ced arterial blood pressure significantly. The initial MABP of 126 2 7 mm Hg did not significantly differ from a final MA&P of ll5 f 3 in this group (see Fig. I). More importantly the MABP after 30 minutes (Le., 123 f 8 mm Hg) did not show any immediate effect soon after the onset of ilciprost infusion in the sham shock rats. Furthermore, the plasma cathepsin D for this group was 0.72 f 0.28 U/l and the plasma MDF for this group was 12 f 1 MDF
177
units. All animals in this group survived the entire five hour observation period, and were within normal limits at the end of the 300 minutes.
120 to 4)
(6)
100 (6
11
(6)
4) 03)
(5)
4)
OH (4)
(4)
(4)
O-OTrauma
+ lloprost
Time course of the changes in mean arterial biood pressure (MAW) over the five hour experimental period. Traumatic shock was induced 10-U minutes prior to time 0 in the two trauma entheses Indicate the number of rats surviving groups. Numbers in at that time. Syrnbo far s represent mean 2 SEM.
-
In the two trauma groups, initial W#P U-20 min after the onet of trauma was in significantly reduced (i.e., 73 to 93 mm lie;) as shown in Fig. Therefore, drum trauma significantly depressed initial MABP by &proximately 33%. This h on fNIcrJenrIy aggravated the condition of the traumatized rats and led to enhanced martaMy. Thus, at 90 minutes, there was a marked difference in survival between trauma + vehicle vs. trauma and iloprost treated rats (i.e., 16% vs. 100%). At 180 minutes, 64% of the iloproet treated trauma group survived as compared with 0% in the trauma + vehicle group. Even more impresstve was the fact that 44% of the iloprost tritatda trauma group survived 300 minutes and therefore may have been candidates for long term survivors.
178
Figure 2 depicts a summary of survival times for rats utilized in this study. A significant improvement in svvival time (p(O.805) was observed for the iloprost treated trauma group (213 f 36 min) compared to the trauma and vehicle group (84 + 18 min). Ail sham trauma rats survived the entire five hour observation period.
-P
l-_-1
T
+
lloprost
+
+
Vehicle
lloprost
Mean survival time in hours following drum trauma. ptvrc Iloprost significantly improved survival time when compared to administration of only the vehicle (pCO.005). Bar heights represent mean values, brackets are f SEM. Numbers within the bars represent the number of animals in each group. Figure 3 summarizes the plasma cathepsin D data. Only very modest elevations were observed in plasma cathepsin D activities in the sham trauma rats. In contrast, the trauma + vehicle,rats exhibited a large increase in plasma cathepsin D activity. However, an appreciable difference in total plasma cathepsin D activities was observed between the two trauma groups. In the trauma + vehicle group, plasma cathepsin D activity was much higher than the trauma and iloprost group (12 + 1.8 vs. 6.2
179
k 2.1). Both groups demonstrated an 8- to 16-fold increase in plasma cathepsin D compared to the sham trauma group. The difference in plasma cathepsin D activities in the two trauma groups may represent a lysosomal membrane stabilizing effect of iloprost.
(-P
1
1-_1~P<0.02-_1
T
I
T
IW Sham Trauma +
lloprost
,,_.
Trauma + Vehicle
+ lloprost
Pilum? 3. Final plasma cathepsin D activities for the three experimental groups. Trauma increased plasma cathepsin D activity 16-f old. Iloprost did not significantly alter the accumulation of cathepsin D in the plasma after trauma. Bar heights indicate mean, brackets are 2 SEM and the numbers in the bars indicate the number of samples measured. Figure 4 demonstrates plasma MDF activities in the three groups of rats stuidied. Sham trauma rats developed only modest plasma MDF In contrast, a !Lfold increase in plasma MM: activity in rats activities. subjected to traumatic shock. Iloprost significantly blunted MDF production from 69 f 14 in the trauma and vehicle group to 20 ?: 6 (pCO.00. This may represent an important protective effect of iloprost in that it retards the formation of the cardiotoxic peptide, MDF, during traumatic shock.
180
Iloprost also exerted direct & vitro anti-proteolytic effects which are relevant to the effects of this a@+%?%shod<. At 100 r&ml, &post tes reduced the rate of pancreatic proteoiysis in five pancreatic by 0.325 2 0.094 rpnoles of serine compared to its vehicle @(0.02). This inhibition of pancreatic proteolysis would significantly contribute to inhibition of MDF generation.
IP
1.1
-P
1,
60 -
40 -
20 -
i! 1
O-
+
h
Vehicle
+
lloprost
Plasma myocardial depressant factor (MDF) activity in E!FTrauma results in a 5-fold increase in plasma MDF activity whiih is significantly blunted by iloprost (flO.01). Bas heights represent the mean value, brackets are + SEM and the numbers in the bars indicate the number of sampies assayed. DISGUSSION Prostacyclin is produced primarily from the vascular endothelium and exhibits a major platelet antiaggregatory and vasodilator effect which should be useful in the treatment of circulatory shock states (2). However, several difficulties were encountered tiith the widespread use of PGl2 as a therapeutic agent. Prostacyclin is unstable at physiologic pH in aqueous
181
solutions and also is rapidly metabolized & + (3). More stable carbacyclin analogs have been developed, the most promising being iloprost. Carbacyclin and its analogs are chemically stable at physiologic pH (3), and have the added advantage of being much less vasoactive than prostacyclin, thus avoiding the hypotensive effect usually encountered with infusion of PGI2. Iloprost has been studied in myocardial ischemia and it is clearly effective in reducing the extension of ischemic damage in this situation (4, 5, 6). In several -in vitro studies iloprost has been shown to exert a potent antiaggregatory effect on platelets. Thus, Darius and associates (3) showed ADP induced iloprost to be equally effective to PC1 in attenuating platelet aggregation. This work was furt b er supported by Wadenvik and fashion was found to strongly Kutti (7). Iloprost in a dose-dependent antagonize the primary and the secondary wave of ADP induced platelet aggregation -in vitro human plasma (7). In this regard, Smith et al. (8) demonstrated a platelet antiaggregatory effect of iloprost in rhesus monkeys with a minimal hypotensive effect. Moreover, other investigators have shown iloprost to be even more potent than PC+ in attenuating platelet aggregation (5, 6, 9). Darius (3) further demonstrated in collagen and thrombin induced platelet aggregation that iloprost was superior to PGI2 in its antiaggregatory effect. Additional studies showed that platelet formation of TxA was not attenuated by iloprost or PC12 when arachidonic However, thrombin induced thromboxane A acid was intro d2uced. formation at the platelet membrane level was attenuated by iloprost an 2 PC12 (3). membrane stabilizing effect of iloprost was A significant demonstrated in acute myocardial ischemia in rats. In addition, myocardial membrane phospholipid loss was attenuated by iloprost infusion following induced myocardial ischemia and may serve as the basis of the membrane stabilizing effect of iloprost (4). This is consistent with the study of Smith et al. (8) who found beneficial effects with early and late administration of iloprost (0.5 - 4.0 hours) in a feline myocardial ischemic model. These authors concluded that iloprost exerted a a direct cellular protective effect as opposed to increased myocardial perfusion (8). Langen et al. (10) showed a protective effect of iloprost following acute myocardial ischemia. In a closed chest porcine model of ischemia, pathological studies one week after induced MI demonstrated a reduced extent of infarcted myocardium compared to the non-treated group. Schriir et al. (5) found iloprost to have a vasodilator effect on isolated bovine coronary arteries, an effect which may also play an important role in preserving ischemic myocardial tissue. The effect of iloprost in traumatic induced shock has not been previously evaluated. In the present study, no significant changes in MABP were seen in non-shock control rats. This finding of a minimal depressor effect of iloprost on MABP is supported by other in vivo animal experiments (4, 6) and in clinical trials (18). Survival time G hamatically
182
improved
by iloprost from a mean of ,g4 ?: lg minutes in the trauma and vehicle group to a mean of 243 t_ 36 minutes &HUH)in the ibprwt treated trauma group. This significant increase in survival time could be due to several mechanisms. The primary protective mechanism seems to be a Plasma cathepsin D activities were cytoprotective effect of iloprost. significantly lower in the trauma rats treated with iloprost. This is supported by previous studies where preservation of bound cathepsin D was shown in iloprost treated ischemic myocardial tissue (6, 8). A second related effect is a marked reduction in MDF formation. MDF was significantly lower in the trauma and iloprost group. This may be secondary to improvement of splanchnic circulation or to a lysosomal stabilizing or anti-proteolytic effect with subsequently less MDF release due to reduced pancreatic ischemia or to reduced pancreatic proteolysis (191. The well demonstrated platelet antiaggregatory effect of iloprost may also have an important role in the protection observed here in This may be due to limitation of some of the protraumatic shock. inflammatory cyclooxygenase metabolites of arachidonic acid from platelets (e.g., thromboxane A21 (20). This effect would prevent endogenous eicosanoid mediators of shock from being produced and would limit the progression of shock to an irreversible state. In summary, iloprost demonstrated a marked beneficial effect in traumatic shock in rats. If these results apply to human trauma, iloprost may prove to be an important therapeutic agent in the management of traumatic shock states. In this regard, PC1 has been found previously to protect in this same model of traumatic s%oc k in rats (21) and is more potent than PGEl in this regard (22). The present finding of iloprost being effective reduces the potential hazards of the use of PG12 (e.g., hypotension) and makes its use more practical in shock. ACKNOWLEDGEMENTS
We gratefully acknowledge the expert technical assistance of Margaret Groh during the course of these studies. We also thank Dr. E. Schillinger of Schering AC, Berlin, West Germany for the generous supply of iloprost. This work was supported by Research Grant No. HL-25575 from the National Heart Lung and Blood Institute of the NIH. MA. Levitrs present address is Division of Emergency Medicine, Department of Surgery, Thomas Jefferson University Hospital, Philadelphia, PA.
1.
Moncada S, Gryglewski RJ, Bunting S, Vane JR. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263:663-665, 1976.
183
Johnson RA, Morton DR, Kinner JH, Gorman RR, McCuire JC, Sun FF, Whittaker N, Bunting S, Salmon JA, Moncada S, Vane JR. The chemical structure of prostaglandin X (prostacyclin). Prostaglandins 12:915-928, 1976. 3.
Darius H. Platelet and vascular effects of iloprost (ZK 36,374) and its stereoisomere ZK 36,375 - two chemically stable prostacyclin analogues. (Schriir, ed.) Prostaglandins and Other Eicosanoids in the Cardiovascular System. Proc. 2nd ht. Symp., Ntirnberg-Fiirth, 1984, pp. 454-464 (Karger, Basel, 1985).
4.
Darius H, Osborne JA, Reibel 03, Lefer AM. Protective actions of iloprost against ischemia induced membrane damage in rat myocardium. 6th Int. Conf. on Prostaglandins and Related Compounds, Florence, Italy, June 3-6, 1986 (abstract).
5.
Schro”r K, Darius H, Matzky R, Ohlendorf R. The anti-platelet and cardiovascular actions of a new carbacyclin derivative (ZK 36,374) vitro. equipotent to PC1 Naunyn-Schmiedeberg’s Arch. Pharmacol. 316:252-$56,i~981b.
6.
Schriir K, Ohlendorf R, Darius H. Beneficial effects of a new carbacyclin derivative, ZK 36,374, in acute myocardial ischemia. J. Pharm. Exp. Therap. 219:243-249,1981.
7.
Wadenvik H, Kutti J. Effect of iloprost (ZK 36,374), a novel prostacyclin analogue, on ADP-induced platelet aggregation. Acta Haemat. 73:224-227, 1985.
8.
Smith EF, Gallenkamper G, Beckmann R, Thomsen T, Mannesmann G, Schror K. Early and late administration of a PG12-anaiogue, ZK 36,374 (Iloprost): effects on myocardial preservation, collateral blood flow and infarct size. Cardiovasc. Res. 18:163-173, 1984.
9.
Casals-Stenzel J, Buse M, Losert W. Comparison of the vasodepressor action of ZK 36,374, a stable prostacyclin derivative. PG12 and PGEl regation and bleeding time in rats. with their effect on platelet Prostagl. Leuko. Med. 10:197-21 a?g,1983.
10.
Lan en CDJ, Weik H, Wesseling H. Sustained protection by iloprost of t!l e porcine heart in the acute and chronic phases of myocardial infarction. J. Cardiovasc. Pharmacol. 7:924-928,1985.
II.
Noble
12.
Anson ML. Estimation of cathepsin with hemoglobin and the partial purification of cathepsin. J. Gen. Physiol. 20:656-674,1936.
RL, Collip JB. A quantitative method for the production of experimental shock without hemorrhage in unanesthetized animals. Quart. J. Exptl. Physiol. 31:187-199,1942.
184
13.
Barenholz Y, Leffler JN, Lefer AM. Detection by chemical methods of a myocardiai depressant factor in plasma of animals in circdatory shock. Israel J. Med. Sci. 9:640-647,1973.
14:
Yamada T, Pettit GW. Modified paper chromatographic method for assay of myocardial depressant factor. Circ. Shock 4:379-386,1977.
15.
Bridenbaugh GA, Flynn JT, Lefer AM. Role of arachidonic acid in splanchnic artery occlusion shock. Am. J. Physiol. 231:112-119,1976.
16.
Kabat EA. Estimation of protein with the biuret and ninhydrin In: Experimental Immunochemistry (2nd Edition), C.C. reactions. Thomas, Springfield, 1961, pp. 559-565.
17.
Knapp RD, Wise WC. A more appropriate statistical method for analyzing mortality data in shock research. Circ. Shock 16:375-381, “’ 1985.
18.
Darius H, Hossmann V, Auel H, Schr& K. Hemodynamic and platelet effects of iloprost (ZK 36,374) in patients with peripheral arterial disease. In: (S&r&, ed.) Prostaglandins and Other Eicosanoids in the Cardiovascular System. Proc. 2nd Int. Symp., Niirnberg-FUrth, 1984, pp. 392-398 (Karger, Basel, 19851.
19.
Lefer AM. Production of a myocardial depressant factor circulatory shock. Curr. Top. Crit. Care Med. 2:80-93,.1977.
20.
Lefer AM, Ogletree ML, Smith 38, Silver Ml, Nicolaou KC, Barnette Prostacyclin: A potentially valuable agent for WE, Gasic GP. preserving myocardial tissue in acute myocardial ischemia. Science I 200:52-54, 1978.
21.
Lefer AM, Sollott SJ, Galvin MJ. Beneficial actions of prostacyclin traumatic shock. Prostaglandins 17:761-767,1979.
22.
Lefer AM, Araki H. Analysis of potential beneficial actions of prostaglandins in traumatic shock. In: Molecular and Cellular Aspects of Shock and Trauma, (AM Lefer, W Schumer eds.) Alan R. Liss, Inc., New York, NY, 1983, pp. 199-210.
185
in
in
Leukotrienes
and Medicine 25: 175-185,
1986
M. Andrew Levitt and Alian M. Lefer Department of Physiology Jefferson Medical College Thomas Jefferson University Philadelphia, Pennsylvania 19107
The effects of iloprost, a synthetic carbacyclin derivative of prostacyclin (PC1 1 was studied in a standardixed model of traumatic shock. Pentobarifital anesthetized rats (35 mg!kg) subjected to NobleColiip drum trauma were characterized by a 84 !: 10 minute survivai time, a K-fold increase in plasma cathepsin D activity, and a S-fold increase in plasma myocardiai depressant factor WDP) activity. Iloprost sieQificantly attenuated the accumulation of MDF activity in the plasma (69 k 14 vs. 20 + 6 U/ml) vehicle vs. drug (p
Since the discovery of PG12 by Vane and associates (I), a variety of beneficial effects of this endogenous cicosanoid product of arachidonic acid metabolism has been observed. PG12 has been found to potently prevent platelet aggregation (2), stabilize cell membranes (3, B), and dilate coronary arteries (5). However, prostacyclin (PG12)is unstable in aqueous media -in vitro and is rapidly degraded -in vivo (6). Several stable 175
prostacyclin analogs with potent PGI2-like actions have been developed recently with iloprost being the most stable and useful of those currently available. Iloprost has been studied -in vitro demonstrating a powerful antiaggregatory effect on platelets (3, 5, 6, 7, 8, 9) challenged by a variety of aggregatory agents including ADP, collagen, and thrombin. Animal studies have shown beneficial effects of iloprost in reducing the extension of ischemic damage in acute myocardial ischemia (3, 8, IO). Cellular membrane protection and preservation of ischemic tissue have been observed with iloprost in monkey, cat, and rat models of myocardial ischemia. To date, iloprost has not been evaluated in the setting of traumatic shock. The present study was conducted to see if prostacyclin would exert a beneficial effect in a standardized model of traumatic shock which is known to be responsive to drug therapy, and if so, to determine the basic general mechanisms of its protective effect.
Male Sprague-Dawley rats weighing 210 to 270 g were utilized in this study. All rats were placed in one of four experimental groups. These groups were as follows: a) sham shock rats given 0.9% NaCl as the vehicle for iloprost, b) sham shock rats given iloprost (100 ng/kg/min), c) traumatic shock rats receiving the vehicle (0.2 ml/h), and d) traumatic shock rats receiving iloprost. Prior to initiation of trauma or sham trauma, all rats (35 mg/kg) given anesthetized with sodium pentobarbital were intraperitoneally. Traumatic shock rats were subjected to a standardized degree of trauma prior to the above surgical procedures utilizing a Noble-Collip drum rats were placed in the drum for 525 apparatus (11). Anesthetized revolutions at 45 rpm. All rats underwent the following surgical procedures following trauma or sham trauma. A tracheostomy was performed utilizing a polyethylene cannula (PE 250) to maintain a patent airway during the The left carotid artery and right external course of the experiment. jugular vein were cannulated with PE-50 tubing filled with 0.9% NaCl and 50 U/ml of heparin to prevent coagulation of the blood. Within IO-15 minutes of completion of the trauma, the arterial catheter was connected to a Statham P23AC pressure transducer to allow mean arterial blood pressure (MABP) and heart rate (HR) to be continuously recorded on a Grass Model 7 osciilographic recorder. All rats were monitored for five hours after trauma or sham trauma, or until the animal declined to a MABP of 45 mm Hg. At the completion of physiologic monitoring, a 2 ml blood sample was drawn and all surviving rats were given an overdose of pentobarbital. Survival time was thus defined as that time following completion of trauma until MABP fell to 45 mm Hg. Any rats with a MABP less than 60 mm Hg within 30 min after the
176
onset of trauma were excluded from further study. All rats subjected to trauma were autopsied to confirm the presence The presence of of a significant degree of blunt traumatic injury. serosanguinous peritoneal fluid with bowel cyanosis and venous engorgement secondary to mesenteric strangulation were d6cwncntod. If these sequelae were not present to a significant degree, that animal was not included in the study. During the course of this study, 15% of the traumatized rats were excluded on this basis. At the termination of each experiment, 2 ml of blood was drawn into a heparinized (50 U/ml) tube at 4’C. The blood was centrifuged at 5000 x g for 15 minutes at 4’C. The plasma was decanted, and the samples were assayed for cathepsin D and myocardial depressant factor (MDF) activities. Plasma cathepsin D activity was determined by the method of Anson (12) and was used as an index of lysosomal disruption. Plasma MDF was measured according to the method of Barenholz et al. (13) with the chromatographic modifications of Yamada and Pettit (14) followed by its MDF activity is expressed throughout in spectrophotometric analysis. chromatographic units (MDF Units). In Vitro Methods Cats were anesthetized with pentobarbital sodiunl (30 mg/kg, i.v.1 removed and placed in ice-cold Krebs-Henseleit solution. Tissue was minced and then homogenized in Krebs-Henseleit solution (lr10, w:v) according to the method of Bridenbaugh et al. (15). Aliquots of homogenates were incubated at 37’C for 30 minutes in the presemz of 100 ng/ml iloprost and its vehicle (i.e., 0.9% NaCI). Incubates were then deproteinized and assayed for free amino-nitrogen according to the method of Kabat (16). and the pancreas
Statistics All values presented in this study are means 2 standard errors of the mean. Statistical anaiysis of survival time was performed utilizing C&an’s generalized Wilcoxon test as modified for shack studies (17). A one-ta&d Student’s V-test was used for statistical analysis of other variables. RESULTS Iloprost at the infusion rate selected (i.e., 100 ng/kg/min) did not exert any significant Cffect on MABP, HR, survival time, plasma cat%epsin D, or MDF activities in non-shocked control rats. Higher infusion re&ced arterial blood pressure significantly. The initial MABP of 126 2 7 mm Hg did not significantly differ from a final MA&P of ll5 f 3 in this group (see Fig. I). More importantly the MABP after 30 minutes (Le., 123 f 8 mm Hg) did not show any immediate effect soon after the onset of ilciprost infusion in the sham shock rats. Furthermore, the plasma cathepsin D for this group was 0.72 f 0.28 U/l and the plasma MDF for this group was 12 f 1 MDF
177
units. All animals in this group survived the entire five hour observation period, and were within normal limits at the end of the 300 minutes.
120 to 4)
(6)
100 (6
11
(6)
4) 03)
(5)
4)
OH (4)
(4)
(4)
O-OTrauma
+ lloprost
Time course of the changes in mean arterial biood pressure (MAW) over the five hour experimental period. Traumatic shock was induced 10-U minutes prior to time 0 in the two trauma entheses Indicate the number of rats surviving groups. Numbers in at that time. Syrnbo far s represent mean 2 SEM.
-
In the two trauma groups, initial W#P U-20 min after the onet of trauma was in significantly reduced (i.e., 73 to 93 mm lie;) as shown in Fig. Therefore, drum trauma significantly depressed initial MABP by &proximately 33%. This h on fNIcrJenrIy aggravated the condition of the traumatized rats and led to enhanced martaMy. Thus, at 90 minutes, there was a marked difference in survival between trauma + vehicle vs. trauma and iloprost treated rats (i.e., 16% vs. 100%). At 180 minutes, 64% of the iloproet treated trauma group survived as compared with 0% in the trauma + vehicle group. Even more impresstve was the fact that 44% of the iloprost tritatda trauma group survived 300 minutes and therefore may have been candidates for long term survivors.
178
Figure 2 depicts a summary of survival times for rats utilized in this study. A significant improvement in svvival time (p(O.805) was observed for the iloprost treated trauma group (213 f 36 min) compared to the trauma and vehicle group (84 + 18 min). Ail sham trauma rats survived the entire five hour observation period.
-P
l-_-1
T
+
lloprost
+
+
Vehicle
lloprost
Mean survival time in hours following drum trauma. ptvrc Iloprost significantly improved survival time when compared to administration of only the vehicle (pCO.005). Bar heights represent mean values, brackets are f SEM. Numbers within the bars represent the number of animals in each group. Figure 3 summarizes the plasma cathepsin D data. Only very modest elevations were observed in plasma cathepsin D activities in the sham trauma rats. In contrast, the trauma + vehicle,rats exhibited a large increase in plasma cathepsin D activity. However, an appreciable difference in total plasma cathepsin D activities was observed between the two trauma groups. In the trauma + vehicle group, plasma cathepsin D activity was much higher than the trauma and iloprost group (12 + 1.8 vs. 6.2
179
k 2.1). Both groups demonstrated an 8- to 16-fold increase in plasma cathepsin D compared to the sham trauma group. The difference in plasma cathepsin D activities in the two trauma groups may represent a lysosomal membrane stabilizing effect of iloprost.
(-P
1
1-_1~P<0.02-_1
T
I
T
IW Sham Trauma +
lloprost
,,_.
Trauma + Vehicle
+ lloprost
Pilum? 3. Final plasma cathepsin D activities for the three experimental groups. Trauma increased plasma cathepsin D activity 16-f old. Iloprost did not significantly alter the accumulation of cathepsin D in the plasma after trauma. Bar heights indicate mean, brackets are 2 SEM and the numbers in the bars indicate the number of samples measured. Figure 4 demonstrates plasma MDF activities in the three groups of rats stuidied. Sham trauma rats developed only modest plasma MDF In contrast, a !Lfold increase in plasma MM: activity in rats activities. subjected to traumatic shock. Iloprost significantly blunted MDF production from 69 f 14 in the trauma and vehicle group to 20 ?: 6 (pCO.00. This may represent an important protective effect of iloprost in that it retards the formation of the cardiotoxic peptide, MDF, during traumatic shock.
180
Iloprost also exerted direct & vitro anti-proteolytic effects which are relevant to the effects of this a@+%?%shod<. At 100 r&ml, &post tes reduced the rate of pancreatic proteoiysis in five pancreatic by 0.325 2 0.094 rpnoles of serine compared to its vehicle @(0.02). This inhibition of pancreatic proteolysis would significantly contribute to inhibition of MDF generation.
IP
1.1
-P
1,
60 -
40 -
20 -
i! 1
O-
+
h
Vehicle
+
lloprost
Plasma myocardial depressant factor (MDF) activity in E!FTrauma results in a 5-fold increase in plasma MDF activity whiih is significantly blunted by iloprost (flO.01). Bas heights represent the mean value, brackets are + SEM and the numbers in the bars indicate the number of sampies assayed. DISGUSSION Prostacyclin is produced primarily from the vascular endothelium and exhibits a major platelet antiaggregatory and vasodilator effect which should be useful in the treatment of circulatory shock states (2). However, several difficulties were encountered tiith the widespread use of PGl2 as a therapeutic agent. Prostacyclin is unstable at physiologic pH in aqueous
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solutions and also is rapidly metabolized & + (3). More stable carbacyclin analogs have been developed, the most promising being iloprost. Carbacyclin and its analogs are chemically stable at physiologic pH (3), and have the added advantage of being much less vasoactive than prostacyclin, thus avoiding the hypotensive effect usually encountered with infusion of PGI2. Iloprost has been studied in myocardial ischemia and it is clearly effective in reducing the extension of ischemic damage in this situation (4, 5, 6). In several -in vitro studies iloprost has been shown to exert a potent antiaggregatory effect on platelets. Thus, Darius and associates (3) showed ADP induced iloprost to be equally effective to PC1 in attenuating platelet aggregation. This work was furt b er supported by Wadenvik and fashion was found to strongly Kutti (7). Iloprost in a dose-dependent antagonize the primary and the secondary wave of ADP induced platelet aggregation -in vitro human plasma (7). In this regard, Smith et al. (8) demonstrated a platelet antiaggregatory effect of iloprost in rhesus monkeys with a minimal hypotensive effect. Moreover, other investigators have shown iloprost to be even more potent than PC+ in attenuating platelet aggregation (5, 6, 9). Darius (3) further demonstrated in collagen and thrombin induced platelet aggregation that iloprost was superior to PGI2 in its antiaggregatory effect. Additional studies showed that platelet formation of TxA was not attenuated by iloprost or PC12 when arachidonic However, thrombin induced thromboxane A acid was intro d2uced. formation at the platelet membrane level was attenuated by iloprost an 2 PC12 (3). membrane stabilizing effect of iloprost was A significant demonstrated in acute myocardial ischemia in rats. In addition, myocardial membrane phospholipid loss was attenuated by iloprost infusion following induced myocardial ischemia and may serve as the basis of the membrane stabilizing effect of iloprost (4). This is consistent with the study of Smith et al. (8) who found beneficial effects with early and late administration of iloprost (0.5 - 4.0 hours) in a feline myocardial ischemic model. These authors concluded that iloprost exerted a a direct cellular protective effect as opposed to increased myocardial perfusion (8). Langen et al. (10) showed a protective effect of iloprost following acute myocardial ischemia. In a closed chest porcine model of ischemia, pathological studies one week after induced MI demonstrated a reduced extent of infarcted myocardium compared to the non-treated group. Schriir et al. (5) found iloprost to have a vasodilator effect on isolated bovine coronary arteries, an effect which may also play an important role in preserving ischemic myocardial tissue. The effect of iloprost in traumatic induced shock has not been previously evaluated. In the present study, no significant changes in MABP were seen in non-shock control rats. This finding of a minimal depressor effect of iloprost on MABP is supported by other in vivo animal experiments (4, 6) and in clinical trials (18). Survival time G hamatically
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improved
by iloprost from a mean of ,g4 ?: lg minutes in the trauma and vehicle group to a mean of 243 t_ 36 minutes &HUH)in the ibprwt treated trauma group. This significant increase in survival time could be due to several mechanisms. The primary protective mechanism seems to be a Plasma cathepsin D activities were cytoprotective effect of iloprost. significantly lower in the trauma rats treated with iloprost. This is supported by previous studies where preservation of bound cathepsin D was shown in iloprost treated ischemic myocardial tissue (6, 8). A second related effect is a marked reduction in MDF formation. MDF was significantly lower in the trauma and iloprost group. This may be secondary to improvement of splanchnic circulation or to a lysosomal stabilizing or anti-proteolytic effect with subsequently less MDF release due to reduced pancreatic ischemia or to reduced pancreatic proteolysis (191. The well demonstrated platelet antiaggregatory effect of iloprost may also have an important role in the protection observed here in This may be due to limitation of some of the protraumatic shock. inflammatory cyclooxygenase metabolites of arachidonic acid from platelets (e.g., thromboxane A21 (20). This effect would prevent endogenous eicosanoid mediators of shock from being produced and would limit the progression of shock to an irreversible state. In summary, iloprost demonstrated a marked beneficial effect in traumatic shock in rats. If these results apply to human trauma, iloprost may prove to be an important therapeutic agent in the management of traumatic shock states. In this regard, PC1 has been found previously to protect in this same model of traumatic s%oc k in rats (21) and is more potent than PGEl in this regard (22). The present finding of iloprost being effective reduces the potential hazards of the use of PG12 (e.g., hypotension) and makes its use more practical in shock. ACKNOWLEDGEMENTS
We gratefully acknowledge the expert technical assistance of Margaret Groh during the course of these studies. We also thank Dr. E. Schillinger of Schering AC, Berlin, West Germany for the generous supply of iloprost. This work was supported by Research Grant No. HL-25575 from the National Heart Lung and Blood Institute of the NIH. MA. Levitrs present address is Division of Emergency Medicine, Department of Surgery, Thomas Jefferson University Hospital, Philadelphia, PA.
1.
Moncada S, Gryglewski RJ, Bunting S, Vane JR. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263:663-665, 1976.
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Johnson RA, Morton DR, Kinner JH, Gorman RR, McCuire JC, Sun FF, Whittaker N, Bunting S, Salmon JA, Moncada S, Vane JR. The chemical structure of prostaglandin X (prostacyclin). Prostaglandins 12:915-928, 1976. 3.
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