Effects of nicardipine in rats subjected to endotoxic shock

Gen. Pharmac.Vol. 23, No. 1, pp. 71-74, 1992 Printed in Great Britain. All fights reserved

0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press pie

EFFECTS OF NICARDIPINE IN RATS SUBJECTED TO ENDOTOXIC SHOCK HING=CHuNG LEE,* JOHN. M. HARDMAN and BERT. K. B. LUM'~ Departments of Pharmacology and of Pathology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96822, U.S.A. [Tel. (808) 956-8936]

(Received 3 June 1991) Abstract--l. The calcium channel blocker, nicardipine, produced a dose-dependent reduction in the mortality caused by endotoxin in rats. 2. The drug also reduced most of the hematological and gross pathological manifestations of disseminated intravascular coagulation (DIC) caused by endotoxin. 3. The endotoxin-induced monocytopenia but not the granulocytopenia, lymphocytopenia or thrombocytopenia was inhibited by the drug. 4. The results suggest that the protective action of nicardipine is causally related to prevention of the endotoxin-induced DIC and that an effect of the drug on monocytes may be of importance.

INTRODUCTION

calculated from the plasma platelet count and the hematocrit. Total white blood cells (WBC) were counted with a Coulter Counter after the red cells were lysed with Zap-Oglobin II (Coulter). Blood smears were stained with DiffQuick Differential stain. The differential WBC count was obtained by counting at least 100 cells on the peripheral blood smear and neutrophil, lymphocyte and monocyte counts were calculated from the differential and total WBC counts.

Calcium channel blockers (CCBs) have been reported to have a protective action against shock produced by hemorrhage, trauma and endotoxin (ETX) (Hackel et al., 1979, 1981; Lefer and Carrow, 1981; Hock et al., 1984; Bossun et al., 1985). We previously reported that the CCBs, nilvadipine, verapamil and nitrendipine were highly effective in reducing mortality caused by ETX in experimental animals (Lee and Lum, 1986). In the present investigation, we studied the effects of the CCB, nicarpidine, on the hematological changes and mortality produced by ETX in rats.

Drugs Drugs and chemicals which were utilized included E. coli endotoxin (Lipopolysaccharide 0127:B8, Difco Laboratories) and nicardipine hydrochloride (1,2-dihydro-2, 6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid methyl 2-[methyl-(phenylmethyl) amino] ethyl ester hydrochloride).

MATERIALS AND METHODS

Mortality studies Male albino Wistar rats weighing 220-260g, fasted overnight, were used. E. coil endotoxin (Lipopolysaccharide 0127: B8, Difco Laboratories) was injected i.v. as a bolus in awake rats via a tail vein. Nicardipine was dissolved in a 50% aqueous-ethanol solution and was given as a bolus via a tail vein 15 min before ETX. Control animals were injected with an equivalent amount of the vehicle (1 ml/kg) instead of the CCB. Mortality was followed for 48 hr after ETX.

Statistics The chi-square test with Yate's correction was used to analyze mortality data. Mean values + SE were compared using ANOVA with the Newman-Keuls Studentized range test or using the paired t-test, as appropriate (Snedecor and Cochran, 1967). P values of <0.05 were considered significant.

Hematological measurements An indwelling carotid catheter was surgically implanted and the animals were studied while fully awake a day later. Arterial blood samples were taken before and at 15 min and 3 hr after ETX. Serum fibrin(ogen) degradation products (serum FDP) were measured by means of a latex agglutination method using a Dade Diagnostics commercial kit. Serial dilutions of the serum allowed quantification of the FDP. Plasma fibrinogen was determined by a modified thrombin titration method using a Dade Diagnostics kit. Platelet counts were made on plasma obtained after sedimentation of the red blood cells using a Scientific Products kit and a Coulter Counter. Blood platelet counts were

Effect o f nicardipine on endotoxin-induced mortality The i.v. administration of E T X (10 mg/kg) in control rats produced an 84% mortality, 48 hr after the lipopolysaccharide was given (Fig. 1.). Nicardipine, administered i.v. 15min before ETX, produced a dose-dependent reduction in the mortality caused by the toxin (Fig. 1). Pretreatment with 1.0 mg/kg of the CCB produced optimum protection; all 6 animals in this series survived the E T X challenge. Less protection was obtained with a higher (2.0 mg/kg) and a lower (0.5 mg/kg) dose of the drug (Fig. 1).

RESULTS

Studies on serum FDP and plasma fibrinogen Arterial blood samples obtained prior to and 15 min and 3 hr after E T X (10 mg/kg) showed that the lipopolysaccharide produced an increase in serum

*Present address: Department of Medicine, Creighton Medical Center, Omaha, NB 68131, U.S.A. 1"To whom all correspondence should be addressed. 71

72

HING-CHUNG LEE et al.

100 ~-. 7s

5O

o

O' I

~

post-ETX platelet and neutrophil counts were somewhat higher in the pretreated series than in the controls, the baseline (pre-ETX) values were also higher in the former than in the latter series; thus, the ETX-induced changes in platelet and neutrophil counts were not significantly different in the 2 groups of animals (Figs 3 and 4).

CONTROl.

/

NIc 0'5 "O/kg

/ / ~ . ..... 7"..... o ................. []

25

:

Morphological findings =

i m NIC 1 0 mg/kg

0 ~

'i

i

i

i

i

12

24

36

48

HOURS AFTER ETX ( I 0 MG/K0) Fig. I. Effect of nicardipine on ETX-induced mortality.

CONTROL = animals pretreated with vehicle. NIC = animals pretreated with nicardipine. Number of animals=32, 6, 6 and 14 in the CONTROL, NIC 0.5 mg/kg, NIC 1.0mg/kg and NIC 2.0mg/kg series, respectively. Pretreatment with nicardipine, 15rain before ETX, produced a dose-dependent reduction in mortality. fibrin(ogen) degradation products (serum FDP) (Fig. 2) and a decrease in plasma fibrinogen (Fig. 2). Pretreatment with nicardipine (1.0mg/kg) reduced or abolished the increase in serum F D P and the decrease in plasma fibrinogen caused by ETX (Fig. 2).

Studies on formed blood elements ETX (10 mg/kg) caused a marked decrease in the counts of all of the blood elements which were measured, including total white cells (WBC), monocytes, neutrophils, lymphocytes and blood platelets (Figs 3 and 4). Pretreatment with nicardipine (1.0 mg/kg) significantly reduced the monocytopenia caused by ETX (Fig. 4) but had little or no effect on the ETX-induced decrease in total WBC, neutrophil, lymphocyte and blood platelet counts (Figs 3 and 4). Although the IBE~E

Five of the controls were given an overdose of pentobarbital 3 hr after ETX (10 mg/kg) and examined for gross and microscopic pathology. Gross pathological findings which were observed included pronounced and diffuse hemorrhagic congestion of the stomach and small intestine, and small discrete hemorrhagic areas in various visceral organs including the heart, lungs, spleen, liver, pancreas and kidneys. Under light microscopy, examinations of tissue sections stained with hematoxylin-eosin, PTAH and Masson-trichrome revealed numerous fibrin microthrombi as well as leucocytic and erythrocytic aggregates in the small vessels of the various visceral organs. Sections through small intestine showed prominent mucosal and submucosal hemorrhages. Four animals pretreated with nicardipine (I.0 mg/kg) were examined for gross pathology 3 hr post-ETX. All 4 animals showed little or no evidence of the gross pathology typically seen in the controls; thus, little or no gastrointestinal hemorrhage was observed and the discrete hemorrhages in other organs seen in the controls were largely absent.

DISCUSSION

Disseminated intravascular coagulation (DIC) is considered to be an important contributory factor in the circulatory shock and death caused by ETX (Hardaway and McKay, 1959; McGovern, 1971,

ETX ~JlS .m AF....TER ETX 1~3 HRS AFTER ETX 400

350

(23} []3} (t5) {13Xt3} {Jt) '=

(231 (t3)(t61 (t31 1t31 lit)

300

250

..

LIJ m

=.

,.H

t50

m ,,~

= m,m

I00

..~

50

iO(

0

300

Z LU C.~ Z

200

CONTROL

NICkI~IPIM

200

tJ~

100

CONTROL NICAROIPINE

Fig. 2. Effect of nicardipine on ETX-induced changes in serum FDP and plasma fibrinogen. Left graph = serum FDP. Right graph = plasma fibrinogen. CONTROL = animals pretreated with vehicle. NICARDIPINE =animals pretreated with the calcium channel blocker (1.0mg/kg). *Significantly different from before ETX (baseline) value. **Significantly different from corresponding value in the CONTROL series. Number in parenthesis = number of animals. Nicardipine pretreatment antagonized the elevation in serum FDP and the reduction in plasma fibrinogen induced by ETX.

Nicardipine and endotoxic shock NBEFORE ETX

m

N

o

N I 5 MINAFTER ETX

650

m ~

550

450

HRSAFTER ETX

(t21 (it)191

1i31 1121(t01

CUNIMUL

~t~ANUIW~C

i I--"--i

z

350

o

~LtJ

250

~=

--J LlJ I---

<

N3

o

E I,--

73

._J ,,~ I--

150

o

o_

uJ

50

CONTROL

"' NICARDIPINE ,5-

Fig. 3. Effect of nicardipine (1.0 mg/kg) on ETX-induced changes in blood platelet and total white blood cell counts. Left graph = blood platelet count. Right graph = total WBC count. Other explanatory notations same as in Fig. 2. Pretreatment with the calcium channel blocker had little or no effect on the thrombocytopenia and the leukopenia produced by ETX. 1972; Hinshaw et aL, 1983). The syndrome is characterized by thrombi formation and internal bleeding stemming from a massive activation of both the blood coagulation and fibrinolytic pathways. In our study, an approximate LD-80 dose of ETX was found to produce full-blown manifestations of the coagulopathy including elevated serum FDP, decreased plasma fibrinogen, leukopenia, thrombocytopenia, fibrin microthrombi in blood vessels of visceral organs and gross and microscopic pathological findings of diffuse hemorrhage in the gastrointestinal tract and more discrete areas of hemorrhage in other visceral organs. We previously reported that the CCBs, nilvadipine, nitrendipine and verapamil, were highly effective in reducing mortality caused by ETX (Lee and Lum, 1986). In those experiments, ETX did not increase

,,,

total tissue calcium so that the protective action of the CCBs could not be related to prevention of gross tissue calcium overload. In the present study, we found the CCB, nicardipine, to be similarly effective in protecting rats against death caused by the lipopolysaccharide. We also observed that nicardipine prevented or reduced the major manifestations of DIC including the elevation in serum FDP, reduction in plasma fibrinogen and the gross pathological findings of diffuse hemorrhage in the gastrointestinal tract and more discrete hemorrhage in other organs. These findings suggest that the protective action of nicardipine against death caused by the lipopolysaccharide is causally related to prevention of the ETXinduced DIC by the CCB. The mechanism by which ETX causes DIC has been a topic of considerable controversy. However,

600[

750

*ImLLl

c.r)

"~ 600

HO)(iO)(6)(to)(to) (6)

-H

500

(to) (io)(7)

"' c~ "44

(to)(1o} (s)

'

x

400

450

300

g

•

200 ~00

0

CONTROL

NICAROIPINE

"'

CONTROL

NIC~DIPINE

".,r'

~

CONTROL

NIC/~DIPINE

Fig. 4. Effect of nicardipine (1.0 mg/kg) on ETX-induced changes in monocyte, lymphocyte and neutrophil counts. Left graph = monocyte count. Middle graph = lymphocyte count. Right graph = neutrophil count. Other explanatory notations same as in Fig. 2. Pretreatment with the calcium channel blocker antagonized the ETX-induced monocytopcnia but not the lymphocytopenia or neutropcnia. GP 23/I--F

74

H1NG-CHUNGLEE et al.

many investigators believe that the procoagulant effect of ETX on blood monocytes is crucial to the development of the DIC. ETX is known to increase tissue factor (thromboplastin) formation in monocytes and macrophages and this action is thought to trigger the coagulopathy (Rivers et al., 1975; Osterud and Bjrklid, 1982; Muller-Berghaus and Lohman, 1983; and others). It is perhaps significant that in our investigation, nicardipine was found to inhibit the monocytopenia caused by ETX but had little or no effect on the thrombocytopenia, lymphocytopenia or neutropenia. The results suggest that inhibition of the known ability of ETX to stimulate platelet aggregation is not the mechanism by which nicardipine prevented the ETX-induced DIC and further suggest the possibility that an action of the drug on monocytes may be of importance.

SUMMARY

We previously reported that the calcium channel blockers (CCBs), verapamil, nitrenidipine, and nilvadipine, exerted a protective action against endotoxin shock (Lee and Lum, 1986). In the present study, nicardipine was found to produce a dose-dependent reduction in the mortality induced by E. coli endotoxin in male Wistar rats. Endotoxin produced changes indicative of disseminated intravascular coagulation (DIC) in control animals. These included gross pathological findings of diffuse hemorrhagic congestion in the stomach and small intestine, clinical laboratory findings of elevated serum fibrin(ogen) degradation products (FDP), reduction in plasma fibrinogen, thrombocytopenia and leukopenia as well as microscopic findings of fibrin thrombi in small blood vessels of visceral organs. Nicardipine prevented the major manifestations of ETX-induced DIC. Pretreatment with the CCB reduced or prevented the gross gastrointestinal hemorrhagic congestion, the elevation in serum FDP and the hypofibrinogenemia. Nicardipine also inhibited the monocytopenia but not the granulocytopenia, lymphocytopenia or thrombocytopenia caused by ETX. The results suggest that the protective action of nicardipine is causally related to prevention of the DIC induced by ETX and that an effect of the drug on monocytes may be of importance.

Acknowledgements--This research was supported in part by

grants-in-aid from the Hawaii Heart Association, the Leahi Trust and Syntex Research. The authors wish to thank Dr A. P. Roszkowski of Syntex Research for generouslyproviding us with nicardipine. REFERENCES

Bossun S., Kuenzig M. and Schwartz S. I. (1985) Verapamil improves cardiac function and increases survival in canine E. coli endotoxin shock. Circ. Shock 16, 307-316. Hackel D. B., Mikat E. M. and Whalen G. (1981) Effects of verapamil on heart and circulation in hemorrhagic shock in dogs. Am. J. Physiol. 241 (Heart Circ. Physiol.) 10, H12-HI7. Hackel D. B., Mikat E. M., Whalen G., Reimer K. and Rochlani S. P. (1979) Treatment of hemorrhagic shock in dogs with verapamil. Lab. Invest. 41, 356-359. Hardaway R. M. and McKay D. G. (1959) Disseminated intravascular coagulation: A cause of shock. Ann. Surg. 149, 462-470. Hinshaw L. B., Belier-Todd B. K., Archer L. T. and Dexton M. D. (1983) Septic endotoxemia. In Handbook o f Shock and Trauma (Edited by Altura B. M., Lefer A. M. and Schumer M. D.), Vol. 1, pp. 425-435. Raven Press, New York, U.S.A. Hock C. E., Su J. Y. and Lefer A. M. (1984) Salutary effects of nitrendipine, a new calcium entry blocker, in hemorrhagic shock. Eur. J. Pharmac. 97, 37-46. Lee H.-C. and Lum B. K. B. (1986) Protective action of calcium entry blockers in endotoxin shock. Circ. Shock 18, 193-201. Lefer A. M. and Carrow A. B. (1981) Salutary actions of nimodipine in traumatic shock. Life Sci. 29, 1347-1353. McGovern V. J. (1971) Shock. In Pathology Annual (Edited by Sommers S.), Vol. 6, pp. 279-298. Appleton Century Crofts, New York. McGovern V. J. (1972) The pathophysiology of gram-negative septicaemia. Pathology 4, 265-271. Miiller-Berghaus G. and Lohmann E. (1983) Pathogenesis of disseminated intravascular coagulation. In Disseminated Intravascular Coagulation (Edited by Abe T. and Yamanaka M.), pp. 3-14. University of Tokyo Press, Tokyo. Osterud B. and Bjrklid E. (1982) The production and availability of tissue thromboplastin in cellular populations of whole blood exposed to various concentrations of endotoxin. An assay for detection of endotoxin. Scand. J. Haematol. 29, 175-184. Rivers R. P. A., Hathaway W. E. and Weston W. L. (1975) The endotoxin induced coagulant activity of human monocytes. Br. J. Haemat. 30, 311-316. Snedecor G. W. and Cochran W. G. (1967) Statistical Methods, 6th edn. The Iowa State University Press, Ames.