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;,. :::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,
in the treatment 2.
1,493,
J .F.
disease.
: 1,490,
Lancet
Therapeutic
defibrination
1968.
KAKKAR,
V.V
Bri t. Med. --_ REID, Lancet: --
JII,
. , FLANC 806,
, C.,
HOWE, C.T., O’SHEA, M. and FLUTE, P.T. A trial of Heparin, Streptokinase and Arvin.
th rombosis. 1969.
H .A . and C HAN, 1,485,
M. J . venom.
1968.
Treatmen t of deep vein
4.
and GOODWIN,
SHARP, A .A., WARREN, B.A., PAXTON, A.M. and ALLINGTON, Anticoagulant therapy w ith a purified fraction of Molayan Pit Viper Lancet:
3.
W.R.
of thrombotic
K .E.
The paradox
in therapeutic
defibrination.
1968.
5.
and McCARTHY, C.F. Treatment BOWELL, R.E ., MARMION, V.J. central retinal vein thrombosis with Ancrod. Lancet: 1,173, 1970.
of
6.
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.
ANIXBODIEZ
Vol.7,No.4
7.
REGOECZ
I, E.,
GERGELY,
TO AIWIN
641
J. and McFARLANE,
A.S.
In vivo effects of
A gkis tr odon rhodostom a venom 1 Studies with Fibrinogen J.Clin. 8.
Invest 145,
PITNEY,
W .R.,
1202,
9.
PITNEY,
W.R.,
resistan IO.
I I.
0.D
in small
I3,5 12.
FF, M.P.
OVARY
C . A new method for the determination
and TUN NAH,
G .W.
J.Lab.Clin.Med:
37,316,1951.
The isolation
and properties
ty fr om Ancist rodon rhodostoma veno m.
,Z . Immediate
of
of the
Brit. J .Haematol
reaction
- anti gen reaction.
in the ski n of experimentaf’animals Prog. Allergy: ---
5,459,
provoked
1958.
York 4th ed . 1969 p .I-65.
Proce dure and gene r al applic ati ons of haem agglutination
reaction
with
tannic acid
J.Immunol: --_-
72,
BOY DEN,
S.V .
tannic J.Exptl. 16.
Acquired
STAV ITSK Y, A. B. Mic rome th ad for the study of proteins and antibodies, I.
15.
G.
1,79,1969.
LENNETTE , E .H . Dia no s ti c pr aced ures for Vira I and Rickettsial Infections. Lenne tt e, E .H . and +l SC midt , N .JmAmerican Public Health Association, New
14.
Lancet:
activity
1969.
BRAY, C. and BOLTON,
A rvin.
samples of plasma.
activi
Blood fibrinolytic
16,165,
81,1967
by antibody 13.
with
. and MENZIE,
RATNOFF,
G.
J. Haematol: --
HO LT, P.J .L.,
fibrinogen ESNOU
Brit.
ce to treatment
thr om bin- like
1966.
BELL, W .R. and BOLTON,
during A r vin therapy .
- l3l- I.
LEWIS,
360,
and protein
- treated
1954.
The absorption
of proteins
on er ythrocyctes
acid and subsequen t ha emag glutination Med: L.J
.,
93,1 07, MART IN,
- inhibition
red blood cells.
by anti-protein
treated
with
serum.
1951. D .L. , BUCKN
ER , S.,
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.
LEWIS, L.J .,FlN LEY, R., MARTIN, D. and STOCKER,K. BARLOW, G.H., lmmunochemi cal identif i c at ion of A NC ROD (A 38414) and Reptilase (D efibrase)
18.
Thrombasis
ROSS , J .W., BU N N, La n cet: 1,310, 1969.
Research: D .R.G
2,17,1973.
. and ASHFORD,
A. Antigenicity
of Arvin.
1
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;,. :::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,
in the treatment 2.
1,493,
J .F.
disease.
: 1,490,
Lancet
Therapeutic
defibrination
1968.
KAKKAR,
V.V
Bri t. Med. --_ REID, Lancet: --
JII,
. , FLANC 806,
, C.,
HOWE, C.T., O’SHEA, M. and FLUTE, P.T. A trial of Heparin, Streptokinase and Arvin.
th rombosis. 1969.
H .A . and C HAN, 1,485,
M. J . venom.
1968.
Treatmen t of deep vein
4.
and GOODWIN,
SHARP, A .A., WARREN, B.A., PAXTON, A.M. and ALLINGTON, Anticoagulant therapy w ith a purified fraction of Molayan Pit Viper Lancet:
3.
W.R.
of thrombotic
K .E.
The paradox
in therapeutic
defibrination.
1968.
5.
and McCARTHY, C.F. Treatment BOWELL, R.E ., MARMION, V.J. central retinal vein thrombosis with Ancrod. Lancet: 1,173, 1970.
of
6.
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.
ANIXBODIEZ
Vol.7,No.4
7.
REGOECZ
I, E.,
GERGELY,
TO AIWIN
641
J. and McFARLANE,
A.S.
In vivo effects of
A gkis tr odon rhodostom a venom 1 Studies with Fibrinogen J.Clin. 8.
Invest 145,
PITNEY,
W .R.,
1202,
9.
PITNEY,
W.R.,
resistan IO.
I I.
0.D
in small
I3,5 12.
FF, M.P.
OVARY
C . A new method for the determination
and TUN NAH,
G .W.
J.Lab.Clin.Med:
37,316,1951.
The isolation
and properties
ty fr om Ancist rodon rhodostoma veno m.
,Z . Immediate
of
of the
Brit. J .Haematol
reaction
- anti gen reaction.
in the ski n of experimentaf’animals Prog. Allergy: ---
5,459,
provoked
1958.
York 4th ed . 1969 p .I-65.
Proce dure and gene r al applic ati ons of haem agglutination
reaction
with
tannic acid
J.Immunol: --_-
72,
BOY DEN,
S.V .
tannic J.Exptl. 16.
Acquired
STAV ITSK Y, A. B. Mic rome th ad for the study of proteins and antibodies, I.
15.
G.
1,79,1969.
LENNETTE , E .H . Dia no s ti c pr aced ures for Vira I and Rickettsial Infections. Lenne tt e, E .H . and +l SC midt , N .JmAmerican Public Health Association, New
14.
Lancet:
activity
1969.
BRAY, C. and BOLTON,
A rvin.
samples of plasma.
activi
Blood fibrinolytic
16,165,
81,1967
by antibody 13.
with
. and MENZIE,
RATNOFF,
G.
J. Haematol: --
HO LT, P.J .L.,
fibrinogen ESNOU
Brit.
ce to treatment
thr om bin- like
1966.
BELL, W .R. and BOLTON,
during A r vin therapy .
- l3l- I.
LEWIS,
360,
and protein
- treated
1954.
The absorption
of proteins
on er ythrocyctes
acid and subsequen t ha emag glutination Med: L.J
.,
93,1 07, MART IN,
- inhibition
red blood cells.
by anti-protein
treated
with
serum.
1951. D .L. , BUCKN
ER , S.,
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.
LEWIS, L.J .,FlN LEY, R., MARTIN, D. and STOCKER,K. BARLOW, G.H., lmmunochemi cal identif i c at ion of A NC ROD (A 38414) and Reptilase (D efibrase)
18.
Thrombasis
ROSS , J .W., BU N N, La n cet: 1,310, 1969.
Research: D .R.G
2,17,1973.
. and ASHFORD,
A. Antigenicity
of Arvin.
1
