Early Humun Deuelopment, 14 (1986) 239-244 239

Elsevier

EHD 00790

Ethamsylate reduces immunoreactive prostacyclin

metabolite in low birthweight infants
with respiratory distress syndrome
Janet M. Rennie I, Joyce Doyle 2 and Richard W.I. Cooke 2
’Depcrrtment of Paediutrics, .!Jnioersi<y of Cumhridge, Cambridge,
and ’ Deportment of Child Health, lJniuersit.v of Liverpool, Liverpool, U.K.
Accepted for publication 7 July 1986

Summary

Measurement of 6 ketoprostaglandin Fr alpha was made by radioimmunoassay

during the first 3 days of life in 33 infants with respiratory distress syndrome who
were subjects in a double blind controlled trial of ethamsylate for the prevention of
intraventricular haemorrhage. Levels of 6-ketoprostaglandin Fr alpha were signifi-
cantly lower on the first and second days of life in babies receiving ethamsylate.
There was a reduction in the incidence of intraventricular haemorrhage in the
treated group. High levels of prostacyclin metabolite are found in babies who
develop haemorrhage, and reduction of prostacyclin synthetase activity may be the
mode of action of this drug in vivo.

ethamsylate; intraventricular haemorrhage; prostacyclin; 6-ketoprostaglandin Fi

alpha; preterm infant

Introduction

The incidence of intraventricular haemorrhage (IVH) continues to be reported in

45% of infants weighing less than 1500 g in studies using ultrasound diagnosis [18].
IVH remains a significant cause of mortality and morbidity in the low birthweight
infant [13]. Timing of IVH shows that it is largely a postnatal event [4] and
prevention of bleeding has been attempted with various prophylactic agents in the
past [1,2,5]. A previous trial using ethamsylate suggested a protective effect for IVH,

Address for correspondence: Dr. J.M. Rennie. Department of Paediatrics, Level 8. Addenbrooke’s
Hospital, Hill’s Road, Cambridge CB2 ZQQ, U.K.

037%3782/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

240

with a reduction of IVH to half that in the control group although numbers were
small [12]. The mode of action of this haemostatic drug is unknown with previous
theories involving increased platelet agglutination and alteration of thromboplastin
formation [3]. In a recent publication Ment and co-workers have suggested that
ethamsylate reduces circulating levels of 6-ketoprostaglandin Fi alpha (6-
ketoPGF,alpha) in the beagle puppy model of IVH. Although few animals were
studied, the dose was possibly inappropriate and assay details were not given [lo].
We have studied 6-ketoPGF,alpha, the stable hydrolysis breakdown product of
prostacyclin, on the first 3 days of life in babies receiving ethamsylate or placebo as
part of a multicentre double blind controlled trial of its use for the prevention of
IVH.

Methods

Patients

The data reported are from 33 infants born weighing less than 1500 g in the
Liverpool Maternity Hospital between November 1983 and September 1984 and
suffering from respiratory distress syndrome (RDS). Data from a further 31 inborn
infants entered into the ethamsylate trial during this period are not included: 19 did
not develop RDS, one exclusion occurred due to a diagnosis of diaphragmatic
hernia, and in 11 cases samples suitable for analysis could not be obtained.
Ethamsylate (125 mg/ml) or placebo were provided in identical preparations by the
manufacturer (Delandale Laboratories) who held the randomisation code until the
end of the period of study. Approval was obtained from the hospital Ethical
Committee. Infants received 0.1 ml/kg of drug or placebo intravenously within 1 h
of birth and thereafter every 6 h for 4 days. Clinical data including birthweight,
gestational age, details of delivery and resuscitation together with blood gas analysis
and ventilator settings in the therapy of RDS were recorded prospectively during
the study.

Diagnosis of intraventricular haemmorhage

Diagnosis of IVH was with real time ultrasound. Scans were made in the coronal
and parasaggital plane via the anterior fontanelle using an Advanced Technology
Laboratories 850A mechanical sector scanner fitted with a 5 MHz scan head.
Representative photographs were taken on Polaroid film from a slave monitor.
Ultrasound appearances were graded using a modification of the method, of Levene
[9]. Scans were made daily for the first 4 days and then weekly until discharge from
the Unit. The first scan was made within 2 h of birth.

Measurement of bketoprostaglandin F, alpha

Blood was taken on the first 3 days of life for estimation of 6-ketoPGF,alpha.
One ml of blood either from venepuncture or an indwelling arterial catheter was
 241

collected directly into ice-cold indomethacin-coated test tubes with ethylene di-
amene tetracetic acid anticoagulation, there being a good correlation between
arterial and venous blood (n = 16). Samples were centrifuged immediately at
3000 X g on ice for 15 min to prepare platelet-poor plasma which was separated and
stored at - 20°C until assay within 3 mth. Samples which showed evidence of
haemolysis in the form of pink supernatant were discarded. Measurement of 6-keto
PGFialpha was by radioimmunoassay. Antibody was obtained from Walker
Laboratories Ltd. The tracer was I-125 TME 6-ketoPGF,alpha, and the assay was
performed in a buffer of phosphate/gamma globulin (pH 6.5). Assays were per-
formed in duplicate on 100 ~1 of unextracted plasma with an incubation time of 40
h. Separation of bound from free was with dextran-coated charcoal. Counts were
made in a Nuclear Enterprises gamma counter. Under the conditions described the
lower limit of detection was 5 pg/tube (50 pg/ml). Intra-assay coefficient of
variation of 10% and inter-assay coefficient of variation of 13% was achieved. Water
blanks were included and gave a value of < 50 pg/ml (n = 6). Cross reactivity to
the structurally related PGE, PGFialpha and PGF,alpha was less than 0.5%.
Normal adult plasma gave results below the lower limit of detection using this assay
for all samples measured (n = 13) and the addition of authentic 6-ketoPGF,alpha
(Sigma Ltd) to normal adult plasma at a concentration of 200 pg/ml gave a result
of 207 f 27 pg/ml.

Analysis of Data

The 6-ketoPGF,alpha levels between groups were compared using Wilcoxon

Rank Sum and Mann Whitney U-test. Incidences of IVH, hypercarbia, pan-
curonium administration, mode of delivery and survival were compared using the
x2-test. AaDO 2 gradient data were analysed using Wilcoxon Rank Sum test.

Results

Of the 33 babies suffering from RDS and enrolled into the study, 17 received
ethamsylate. The groups were well matched for birthweight, gestational age and
mode of delivery (Table I). The severity of RDS assessed by lowest pH, occurrence
of hypercarbia and pneumothorax was comparable. Eleven infants in the placebo
group and 5 treated infants received pancuronium and this was significant at the 1%
level. There was a reduction in the incidence of IVH in the treated group and this
was significant at the 5% level. There was no reduction in mortality.
Levels of 6-keto-PGFialpha were higher throughout the 3 days of the study in the
placebo group, reaching statistical significance on the first 2 days. Levels are shown
in Table II and graphically in Fig. 1.
Measurement of capillary bleeding time by a capillary micromethod [15] failed to
show a significant reduction in the treated group, with a mean bleeding time of 3.05
min in the placebo and 2.75 min in the treated group.
 8 Ava L AQQ 1 AQa
13 ld 13 ld ld
4p ?? *
-5-
-L
;
0
. -.-
.
.
anp d UOXO3[!M
(as 1 F uuaur) sweju! Apws u! ajq JO shp Z wy aql JOJ swa!pm% uaMx0 p.ra~n-nqoa~~y
III 318V~
SN (SPI-ZL) L6
Zoo’0 (9PT-ZL) PZI
10’0 (6PI-PL) 011
a%uw aplmnbJa]u!
pur? ue!pam ‘1w/8d u! Sap ql!m sweju! u! ajq JO dsp 6 1s~~ aqi uo eqd[v1df)dolay09 JO s1aAa-I
II 3mv.L
SN s w=a
SO’0 8 HA1
SN 6 Lamlap uog3as ‘3
SN Z xeJoqloumaud
10’0 5 wn!uom3uvd
SN 01 Ed9 8 ‘03’?d
SN 91’0 TSO’L Hd WoM
SN Yfi 8’1 F 6Z a% Itwogwsaf)
SN SSZZT LLll ~$wqiq
ZPZ
 243

Analysis of blood gas data was performed using the alveolar air equation and
assuming end-tidal CO, equal to arterial PaCO, and r = 0.8, AaDO, gradients were
calculated for the three occasions on the first 2 days of life for all infants in the
study. There was a reduction on the mean AaDO, value in the ethamsylate treated
infants on the second day of life (Table III).

Discussion

Ethamsylate (diethylammonium 1,4, dihydroxy-3-benzene sulphonate) is a

haemostatic drug that has been claimed to reduce the incidence of IVH in prema-
ture babies. Its mode of action is uncertain: it has been shown to reduce the blood
lost during transurethral resection of the prostate [17] and intrauterine-device-in-
duced menorrhagia [6]. Studies using in vitro platelet function tests showed an
increased platelet adhesion, and the addition of ethamsylate to platelet-rich plasma
containing prostacyclin alters platelet aggregation [19]. In the beagle puppy model
of IVH ethamsylate reduced IVH occurring after insult as well as levels of
6-keto-PGFialpha and thromboxane B, 30 min after the administration of this drug.
Ethamsylate has also been demonstrated to block the synthesis of 6-ketoPGF,alpha
and thromboxane B2 in the pregnant human myometrium in vitro [8]. The pattern
of response in this study suggested an inhibition of prostaglandin and thromboxane
synthetase rather than an interaction with the prostaglandin pathway at cyclo-
oxygenase level as occurs with indomethacin.
Prostacyclin is the most potent vasodilating substance known and is able to
disaggregate platelets [ll]. It is produced via an enzymatic pathway involving
cycle-oxygenase in vascular tissue throughout the body, and unlike the primary
prostaglandins is not metabolised by the lung. It is released from damaged lung and
isolated ventilated heart lung preparations [14]. High levels of 6-ketoPGF,alpha
have been shown in infants with RDS [17] and these may be important in the
pathogenesis of cerebral vasodilatation in IVH [16]. We have shown that ethamsy-
late administration was associated with lower levels of circulating 6-ketoPGF,alpha
on the first 2 days of life in a group of low birthweight infants suffering from RDS
treated in a double blind trial compared with a well matched group of control
infants. This may support the animal and in vitro evidence for the effects of
ethamsylate on the prostaglandin pathway.
The reduction in AaDO, in the treated infants suggests that ethamsylate may
alter capillary leakage in the lung. Subsequent reduction in the requirement for
mechanical ventilation, and hence stimulus for prostacyclin release, may alter
circulating 6-ketoPGF,alpha.
The aetiology of IVH is multifactorial, and may in part be due to ischaemia.
However, with regard to the haemorrhagic component which undoubtedly exists,
prevention of excess release of prostacyclin by drugs such as ethamsylate and/or
modification of ventilatory techniques may prove useful.
244

Acknowledgements

We thank the medical and nursing staff of the N.I.C.U., Liverpool Maternity
Hospital for the clinical care of the patients, and Delandale Laboratories for
supplying trial materials. Dr. Janet Rennie was supported by a grant from Mersey
Regional Area Health Authority.

References

1 Bedard, M.P., Shankaran, S., Slovis, T.L., et al. (1984): Effect of prophylactic phenobarbital on IVH
in high risk preterm infants. Pediatrics, 73, 435-439.
2 Chiswick, M.L., Johnson, M., Woodham, C., et al. (1983): Protective effects of vitamin E against IVH
in preterm babies. Br. Med. J., 287, 81-84.
3 Deacock, A.R. de C. and Birley, D.M. (1969): The antihaemorrhagic activity of ethamsylate. Br. J.
Anaesthesia, 41, 18.
4 De Crespigny, L., McKay, R., Murton, M.J., Ray, R.N.D. and Robinson, P.H. (1982): Timing of
neonatal IVH with ultrasound. Arch. Dis. Childh., 57, 231-233.
5 Donn, S.M., Roloff, D.W. and Goldstein, G.W. (1981): Prevention of IVH in preterm infants by
phenobarbitone: a controlled trial. Lancet, i, 215-217.
6 Harrison, R.F. and Campbell, S.A. (1976): A double blind trial of ethamsylate in the treatment of
primary and intrauterine induced menorrhagia. Lancet, ii, 283.
7 Kaapa, P., Koinsto, M., Viinikka, L. and Ylikorkala, 0. (1982): Increased plasma immunoreactive 6
ketoPGF,alpha levels in newborns with IRDS. Pediatr. Res., 16, 817-819.
8 Kovacs, L. and Falkay, G. (1981): Ethamsylate as an inhibitor of prostaglandin biosynthesis in
pregnant human myometrium in vitro. Experientia, 37, 1182-1183.
9 Levene, M.I., Dubowitz, L.M.S., De Crespigny, L. (1983): Classifying intraventricular haemorrhage.
Lancet, ii, 39.
10 Ment, L.R., Stewart, W.B. and Duncan, CC. (1984): Beagle puppy model of IVH: ethamsylate
studies. Prostaglandins, 27, 245-256.
11 Moncada, S., Higgs, E. and Vane, J.R. (1977): Human arterial and venous tissues generate pros-
taglandin X: a potent inhibitor of platelet aggregation, Lancet, i, 18.
12 Morgan, M.E.I., Benson, J.W.T. and Cooke, R.W.I. (1981): Ethamsylate reduces the incidence of
periventricular haemorrhage in very low birthweight babies. Lancet, ii, 830-831.
13 Palmer, P., Dubowitz, L.M.S., Levene, MI. and Dubowitz, V. (1982): Developmental and neuro-
logical progress of preterm infants with IVH and ventricular dilation. Arch. Dis. Childh., 57,
748-753.
14 Piper, P. and Vane, J.R. (1971): Release of prostaglandins from the lung and other tissues. Ann. N.Y.
Acad. Sci., 180, 363.
15 Rennie, J.M., Gibson, T. and Cooke, R.W.I. (1985): Micromethod for bleeding time in the newborn.
Arch. Dis. Childh., 60, 51-53.
16 Rennie, J.M., Doyle, J. and Cooke, R.W.I. (1986): Elevated levels of immunoreactive prostacyclin
metabolite in babies who develop IVH. Acta Paediatr. Stand., in press.
17 Symes, J.M. (1975): The effect of dycenene on blood loss during and after TURP. Br. J. Ural., 47,
203.
18 Syzmonowicz, W., Schafler, K., Cussen, L.J. and Yu. V.H.Y. (1984): Ultrasound and necropsy study
of periventricular haemorrhage in preterm infants. Arch. Dis. Childh., 59, 637-642.
19 Vinazzer, H. (1980): Clinical and experimental studies on the action of ethamsylate on homeostatis.
Thromb. Res., 19, 783-791.