Antibodies to Arvin following prolonged intravenous therapy

vol.

THROkBOSIS RESEARCH Printed in the United

ANTIBODIES

TO

ARVIN

FOLLOWING

R.P.SAPRU, Departments Institute

A.K.

of Cardiology,

of Medical

ABSTRACT

PROLONGED

MOZA,

INTRAVENOUS

M.KUMAR

Experimental

Education

(Received

Medicine

and Research,

Intravenous

administration antibodies

rise

in fibrinogen

dies

could

be detected

to be a mixture

of

indicate

that

possible

because

of blood

and IgM class

prolonged

Arvin

a period

of two months

of its action

to control weeks

values,

of therapy

in clinical

response

and a

The

antibo-

and were

of immunoglobulins.

therapy

of the antibody

over

neutralisation

by the end of four

IgG

(India).

form 28.8.1975. Copley)

in rabbits

consequent

concentration

THERAPY

and Microbiology,Post-Graduate

Chandigarh-160011

of Arvin

with

1975 Inc.

and N.K.GANGULY

21.4.1975; in revised Accepted by Editor A.L.

produced

7, PP~ 635-641, Pergamon Press,

States

The

disorders

found

results

may not be

to the drug.

INTRODUCTION * Arvin

(Ancrod)

(Agkistrodon been

investigated

the ability virtue

with

of this

protein

of the study invariably showing

used this

fibrinogen became failure

been reported

*Arvin

was kindly

lower

thus

to the action

therapy

of this

Arvin

of this

antibody

by M/S

have

drug.

Although

immune

immune

out.

is reported.

Twyford 635

Labs.

Ltd.,

U.K.

has in

in blood from

the effect

in rabbits. that

isolated

of The

plan

rabbits case reports

of the drug

In the present to therapeutic

by

the

(7,8).

inactivation

reaction

Viper

resides

are-removed

and it was noticed

not been worked

mediated

action

to study

of atherogenesis

due to presumed

intravenously

supplied

with

formed

designed

Pit

practice

concentration

phagocytosis

in an investigation therapy

Malayan

in clinical

the fibrinogen

on the evolution

prolonged

from

Th e anticoagulant

(l-6).

the microclots

(9) the details

administered

obtained

and reticula-endothelial

resistant

the characterisation of Arvin

activity,

product

of Arvin

results

to drastically

depletion

required

derivative

Its use as an anticoagulant

encouraging

by fibrinolysis

We have selective

protein

venom.

of its thrombin-like

circulation

have

is a purified

rhodostoma)

study doses

ANTIBODIES TO ARVIN

636

MATERIALS Animals Institute,

Female albino Bombay.

AND

Vol.7,No.4

METHODS

I .5 to 2 Kg were obtained

rabbits weighing

Seven rabbits received

a high cholesterol

from the Haffkine

diet and three rabbits were

fed on rabbit chow. Arvin

treatment

Arvin diluted

I2 hours into the marginal

in normal saline,

various time periods in different least two months. fibrinogen

twice daily

The Arvin

treatment

at intervals

units/l<9

body weight)

of

was started at

batches of animals but once started was continued

The dosage of Arvin(2

for at

was worked out to achieve

levels of less than 100 mg per 100 ml.

The blood fibrinogen

concentration

of the animals was determined

for the first week after the start of Arvin ghout the remaining freely

was iniected

ear vein of the animals.

flowing

period of the study.

blood which was collected

content was estimated

on alternate

days

therapy and later at intervals

of one week throu-

A sharp nick on the marginal

ear vein provided

in 3.8 percent

sodium citrate.

The fibrinogen

by the method of Ratnoff and Menzie(l0).

When the blood fibrinogen

levels started to rise inspite of continued

Arvin

therapy an

attempt was made to achieve

better control by increasing the dose of Arvin two to four much success. The potency of Arvin was frequently ch$cked by compar-

folds but without

ing the simultaneously Detection

determined

of antibodies

Arvin

against Arvin

clotting

time with the thrombin

The various tests for Arvin

clotting

antibodies

time(il).

were perfor-

med on the separately pooled sera of cholesterol fed and normal rabbits treated with Arvin. Blood samples’were

obtained

at the end of 2 months of treatment

just before the animals

were sacrificed. l)

Clottin

time

0.2X&z--

The serum from Arvin-treated

in for I minute at 37oC.

rabbits was then added and the clotting from normal (untreated) 2)

Passive cutaneous anaphylaxis

fixation

time at 37°C

(PCA)

determined.

test

was investigated

Sera obtained

against sera from normal untreated Complement

with 0.1 or

A control

serum sample

rabbits was run concurrently.

ina to the method of Ovary. (12). -

3)

rabbits was first incubated

An equal volume of plasma from normal (untreated)

from Arvin

in three guinea pigs accordtreated

rabbits were tested

rabbits and normal saline.

was performed

according to the method of Lennette(l3). showing 100 percent hoemolysis. Arvin

The end point was taken as the maximum dilution in a concentration

of 2 unidml

was used as antigen.

4) Indirect haemagglutination test was performedusing tanned sheep erythrocyks accodinotoStavit$kyk (l4) modtffeatien of Boyden’s (15) method. Arvin in a concentration of 2 ;nitJml wasVused as the antigen to coat tanned sheep erthrocytes. The maximum dilution showing distinct one plus hoemagglutination was taken as the end point.

* Bovine thrombin was kindly supplied by A@

Parke Davis 8 Co.,

U.S.A.

ANTIBODIES

Vol.?,No.4

TO ARVIN

637

Roth complement fixation and indirect haemagglutination tests were repeated with aliquots of antisera diluted to I :2. These were then treated with 0.1 vol. of IM betamercaptoethanol and incubated for I hour at room temperature, the final dilutions were adjusted to 114. RESULTS Following the first injection of Arvin the fibrinogen level in the blood promptly fell from a control level of 230 (+ 14.30) mg/lOO ml to 79 (t I4 .OO) mg/lOO ml and then ranged between 75 and 120 mg/lOO ml upto the end of four weeks. Subsequently the fibrinogen content started to rise progressively inspite of a two fold increase in the dose of Arvin. When the dose of Arvin was increased four folds at six weeks there was a slight reduction in fibrinogen concentration to 126 (A 16.90) mg/lOO ml but subsequently this progressively increased to 238 (+ 12.13) m&00 ml at the end of eight weeks (Fig.1). The possible presence of antibodies to Arvin was indicated by the marked prolongation of clotting time when normal rabbit plasma was clotted with Arvin previously incubated with sera from Arvin-resistant rabbits (Table I).

FISRINOSEN

CONCENTRATION

WITH

ALVIN

(MEAN

IN RASSITS VALUES

TREATED

+- s. E.)

320 ARVIN 280

E” . ,>;,. :::I,: . _...., t

1..

.=Kg.:”

012S4s6?*1

DOSE

‘,..‘,.. :.::.,.,,:... ..‘..,.... “.:‘.

.‘-I

2

3

BAYS

4

5 WEERS

6

7

s

(Fig.1)

fibrinogen concentration in blood of rabbits treated with Arvin over a period of 8 weeks. The mean values (t S.E.) are shown.

ANTIBODIES

638

TABLE

S.No.

Vo1.'7,No.4

TO ARVIN

I 0.2

’Arvin

I.

Undiluted

>

30 min

2.

>

30 min

units

Norma I

Arvin

control

treated

44 sec.

>

30 min

7

30 min

3.

30 min

61 sec.

4.

I :4

32lsec.

37 set D

5.

I:8

II6 sec.

31 sec.

6.

I:16

106 set a

30 sec.

Results of Clotting In the PCA test, treated of I:6

Time in the Pooled Sera of Arvin

normal rabbit

animals showed distinct

Normal

I

III 1:2

7

of Arvin control

il 31 sec.

Treated Rabbits.

sera did not show any reaction

blue patches with induration

while

set-a of Arvin

upto an antisera dilution

(Table II) o TABLE II

S.No.

Dilution

of sera

obtained

from

Diameter

of the reacted tmerrment

Arvin resistant

I.

patch in ems.

II

Ill

1.3

I.8

1.9

rabbits

I

Undiluted

2.

1:2

0.8

0.8

3.

I :4

0.7

0.7

0.7 0.7

4.

0.4

0.4

0.5

5.

I:5 I:6

0.3

6.

Normal

0.3 0

0.5 0

0

rabbit

serum Results of Passive Cutaneous Anaphylaxis Tables III and IV give the results of complement nation tests,

A significant

treated animals. reduced

titre of antibodies

Furthermore

pretreatment

the titre by I to 4 dilutions

of antisera

in either

and indirect

haemaggluti-

with beta-mercaptoethanol

test.

TABLE S.No.

fixation

was present in both batches of Arvin

Ill

Antisera

, Antisera

treated

with

beta-rnercaptoethanol I 2 3 Complement

1164 I:32 I:32 Fixation

Titre in Sera from Arvin

I :I6 I:8 I:16 Resistant Animals.

4

ANTIBODIES

VoL.7,No.4

TABLE

s.No.

TO ARVIN

639

IV

Antisera

Antisera

treated

with

beta mercaptoethanol

I.

I :320

I:80

2.

l-:&lo

I :80

3.

I :80

4.

11640

I :40 I :40

5.

I :40

I :20

Indirect

Haemngglutination

Titre in Sera from Arvin

Immune-electrophoresis of Arvin gave a single distinct sera from Arvin-treated animals (Fig.2).

Resistant Animals

sharp precipitating

arc against

(Fig .2) Immuno-electrophoresis contained

anti-serum

side wells contained

of Arvin. concentrated Arvin

(.I2

The central ten times. unit).

slit The

DISCUSSION In the present study Arvin was used to produce selective rabbits but the fibrinogen spite of continued

concentration

Arvin therapy even when the dose was

sera from these animals

fibrinogen

started to rise after an interval

taken at the time the fibrinogen

considerably

concentration

depletion

in

of four weeks inincreased.

The

had increased

again to control levels showed prolongation of the in-vitro Arvin clotting time. A significant antibody titre was demonstrated by both complement fixation and indirect also showed a clear precipitating haemagglutination tests. Immuno-electro-phoresis

arc.

640

ANTIBODIES

The partial antibodies Arvin

reduction

in antibody

are a mixture of IgG is a protein

TO ARVIN

Vo1.7,No.4

titre with beta - merceptoethanol

suggests that the

and IgM class of immunoglabulins.

which exists in both monomer and dimer forms and the molecular

weight of monomer is about 30,000 (II). The drug is therefore potentially antigenic and in fact recently Lewis et al (16) and Barlow et al (17) have raised specific antibodies against Arvin

in goats.

In the report of Pitney et al (9), only one out of I2 patients venously developed

resistance

receiving

to the drug whereas 3 out of 4 patients

Arvin

intra-

receiving

the

drug by the intramuscular route showed this phenomenon. The higher incidence following intramuscular injection is understandable since this would facilitate the immunological response as has been shown in guinea pigs (18). consistent antibody

The present study has shown a

response i n all animals after what appears to be a critical

about 4 weeks of continuous

i ntravenous administration

time of

of the drug in the recommended

therapeutic dose. In view of these findings the long term use of Arvin in the treatment of clinical disorders is likely to be seriously limited. Although we did not encounter an anaphylactic given repeated

reaction

the possibility

short intravenous

of such a reaction

in patients

who may be

courses of the drug remains a cause for concern.

_ACKNOWLEDGEMENTS The authors wish to acknowledge of Miss Uzwal

the skillful

technical

and secretarial

assistance

Kaur and Iv+ G .P.Gera. REFERENCES

I.

BELL, W.R.,

PITNEY,

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KAKKAR,

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DAVIES, J.A., MERRIC K, M.V., SHARP, A.A. and HOLT, J.M. Contra Iled trial of ancrod and heparin in treatment of deep-vein thrombosis of lower limb. LancetIl, 113, 1972.

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fibrinogen ESNOU

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360,

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on er ythrocyctes

acid and subsequen t ha emag glutination Med: L.J

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1951. D .L. , BUCKN

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FI NLEY,

R.,

LAZER, L. and FE D OR , E .J . Studies of type specific immunity to the whole v enom and a fr action of Agkis trodon rhodostoma. Res.Comm.Clin. Path and Pharm. 2,649 , 1971. 17.

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1