Note: Descriptions are shown in the official language in which they were submitted.
-
` ~ 1789~9
The present invention relates to a new
thiolated polypeptide compound derived from a fragment
of tetanus toxin, the process for obtaining this compound
and the applications of the latter, in particular as a
5 neuropharmacological transport agent and as a specific
- labelling agent of neuronal cells.
With regard to the tetanus toxin itself or its
anatoxin, it was already proposed to use them notably for
obtaining vaccines or assay reagents. In order to
10 illustrate this prior art following references ~ill be
cited:
- FR patent for addition No 74,16,936 published
under No 2,270,891, which relates to a process for
obtaining vaccines by treatment of a toxic product with
15 glutaraldehyde. This process consists in treating a toxic
product with glutaraldehyde in effecting the
polymerisation of a limited number of molecules of said
product and the detoxiiication of said product. In this
process tetanus toxin may be used as toxic product.
- FR patent application No 77,29,186, published
under No 2,366,569, relates to an immunochemical process
for assaying haptens, wherein are used a particle
sensitized by an antibody, prepared by sensitizing fine
particles by an antibody of the hapten to be assayed and
a hapten-carrier conjugate. The carrier of this conjugate
may be notably tetanus toxoid.
This hapten-carrier conjugate is used as reagent
in al immunochemical process and also for immunization of
,. ~ , . . . . . .
1 178949
an animal in order to obtain corresponding antibodies
/ see on page 4, lines 20 to 32_/.
The tetanus toxoid is therefore used as a carrier
of the hapten in the body of the animal for obtaining
antibodies, However, there exists no teaching in this
FR patent about a particular fragment of tetanus toxin
and its possible use as axonal transport agent for drugs.
- GB patent 2,013,690 relates to an antigen for
early pregnancy test and contraceptive vaccine. This
antigen is obtained from the ~-sublnit of human chorionic
gonadotrophin by reducing and cleaving of three, four,
five or six of intrachain disulphide bounds of said ~-
subunit, alkylating of the thus reducing intrachain
disulphide grcups and isolating oi' the produced antigen.
This antigen may be coupled with a protein or a hapten
to enhance its immunological specificity. Tetanus toxoid
ls cited as suitable protein.
- GB patent 1,492,445 relates to a composition
comprising a conjugate of a subject-compatiblç
' 20 immunogenic carrier and an immunochémically purified
hormone derivative. The tetanus toxoid is used as carrier
in this composition.
- DE-OS patent application 1,617,880 relates to a
process for obtaining bioactive organotrope substances,
particularly drugs. This process consists in making a
conjugateof abiologically active subætance with
organotrope receptive substances obtained from cellular
membranes or antibodies. The toxins may be used as
1 178949
organotrope substances.
Furthermore, it was proposed to use thiolated
proteins as drug carriers. Reference may be made to US
patent 3,171,831 which relates to thiolation of proteins
by reaction with homocysteine thiolactone in the presence
of a tertiary amine. The thiolated proteins thus obtained,
for example gelatin, may be used as carriers for drugs.
According to example 18 of this US patent 3,171,831 the
gelatin thus treated is used for encapsulating a
pharmaceutical product which is sens~tive to the acid
environment of the stomach. The pharmaceutical product
is therefore in this case not coupled with the thiolated
protein but coated with it.
On the other hand, it was disclosed in FR patent
application 76,37,367 published undsr No 2,334,954 a
reagent i'or immunoenzymatic determination. This reagent
is composed by an antigen and an enzyme coupled by
means of an ester of maleimidobenzoic acid and n-hydroxy-
succinimide.
It is known that tetanus toxin is retrogradely
transported to the central nervous system and the
peripheral nervous system. In this respect, reference may
be made to the article of BIZZINI et al, entitled : "An
antigenic polypeptide fragment isolated from tetanus
toxin : chemical characterization, binding to
gangliosides and retrograde axonal transport in various
neuron systems", which appeared in the "~ournal of
Neurochemistry", 1977, vol.28, pp 529-542, and to all
.. ~ . . .. . . . .
i ~78g49
the bibliographic references cited in this article.
Various studies have shs)wn that tetanus toxin may
be degraded or cleaved into several fractions or sub-
units. For example, COH~N et al. / The Journal of
Immunology vol. 104, no 6 June 1970_/ have shown that
the freezing-defreezing of the crude filtrate of
Clostridium tetani culture results in a degradation of the
molecule of tetanus toxin; the resulting degraded tetanus
toxin is practically devoid of toxicity and has a
flocculating power lower than the one of tetanus toxin.
BIZZINI ancl R~YNAUD have also studied the sub-units
A-I, A-II, A-III and B-I, B-II and B-III of tetanus toxin.
r C.R. Acad. Sc. Paris, t. 279, 1974 series D, pp. 1809-
1811 and Annales of Pasteur Institute Paris 126, 159-176
(1975)_/. French patent 74,36,622 (publication 2,249,679)
discloses an immunogenic atoxic product obtained from
tetanus toxin. This atoxic product is obtained by the
treatment of tetanus toxin with a proteinase.
BIZZINI et al. have also isolated from frozen crude
20 toxin a polypeptide fragment of the toxin which is
identical, from the immunological point of view, to the
above mentioned fragments A-II and B-II, but differs
therefrom by its slze and toxicit~r / see in this respect
~ournal of Neurochemistry, 1977, vol. 28, pp, 529-542_/.
25 This fragment, named B-IIb, which will be defined in more
detail hereinafter, is capable of binding to the
gangliosides and to the synaptic membranes with an
affinity which is even greater than that of tetanus toxin.
1 178949
Owing to this property, it is suggested in this
article that this fragment could be employed for
specifically transporting to the central nerYous system
chemotherapeutic agents or pharmacological agents for
determining specific effects in the central nervous
system.
However, up to the present time, it has not been
found hou to bind these chemotherapeutic or
pharmacological agents to said B-IIb fragment so as to,
on one hand preserve the full activity of the agent to
be transported to the central nervous system and, on the
other hand, to preserve the binding property of the B-IIb
fragment to the receptors for the tetanus toxin in the
central nervous system.
There has now been found a way of binding
chemotherapeutic or pharmacological agents to the B-IIb
fragment while preserving its property of binding to the
receptors for the tetanus toxin in the central nervous
system,
~oreover, to the knawledge of the applicant, it is
very difficult to separate the neuronal cells from the
glial cells. It has been found that the new thiolated
polypeptide compound according to the present invention
could be employed as a specific labelling agent for the
neuronal cells. Then, the separation of the labelled
neuronal cells becomes easier with the techniques well-
known to one skilled in the art, such as gel filtration,
affinity chromatography and the lilce.
1 178949
The thiolated polypeptide compound according to
the present invention is constituted by the B-IIb
fragment of the tetanus toxin to which is bound at
least one -SH group and has substantially the same
properties of retrograde axonal transport and binding
to the receptors of the tetanus toxin as the B-IIb
fragment itself.
In the compound of the invention, the -SH group or
groups are directly or indirectly bound to the B-IIb
fragment. Generally, in view of the process for producing
it, which implies a thiolation, the -SH groups will be
bound by means of the residue of the thiolation agent
The thiolation agent is moreover bound to the B-IIb
fragment by -NH2 groups born by it.
The B-IIb fragment, from which the new thiolated
polypeptide compound according to the present invention
is derived, is a polypeptide of the tetanus toxin whose
molecular weight is about 46,000
The process for obtaining this B-IIb fragment and
the physicochemical and immunological properties thereof
are described in detail in the aforementioned article
by BIZZINI et al. (Journal of Neurochemistry, 1977, vol.
28, pp. 529-542),
It will be briefly recalled that this fragment was
obtained by the process which comprises subjecting a
frozen filtrate of _ ostridium tetani culture (Harvard
Strain No 6037 of the National Collection of
Microorganism Cultures of Pasteur Institute) to the
.
1 17894~
following steps :
1. ultrafiltration in order to remove the
substances having a molecular weight lower than 10,000;
2. fractionating with ammonium sulphate;
3. gel filtration.
The B-IIb fragment has the following physico-
chemical properties :
- molecular weight of about 46,000;
- it has a disulphide bridge and two non reactive
free sulfhydryl groups;
- the N-terminal groups are tyrosine and lysine;
- its composition in aminoacids is given in the
folla.ving table I.
The B-IIb fragment of the tetanus toxin has the
same antigenic structure as the B-II fragment but differs
therefrom by its specific toxicity. The immunological
properties of the B-IIb fragment compared with that of
tetanus toxin and of the B-II fragment are gathered in
the following table II.
1 1~8949
,_
s~ ~
~ s o
U~
~ s.x
.,, _, o
C) ~ bll ~ N ~ d' N NC5~ C5) N O
t~l ~ tÇ~ ~ ~ ICi)~ ~ u) ~ N
c~ a~ O. . .
O ~ r~, o_, o ,, ~ c~ u~ ~ o
~: h ~ ~1 ~1
.,1 ~ ~
~ X O ~D
¢ a>
~5
~: ~ S~
~5
ea _~~ 00 ~' ~<oO O ~ u~
_I ~ ~ 15) r~ ~ o Oo o 1` N C~
,Q ~ N ~ _I I ~C`l Ocs) ~ u~
~ O O
E~ 4~
O O ~ ~q
s~ a
O Q ~ 1C~ I Nt9 0C`~
~1 1-l~3 ~.q lQ N I ~1 ~ N C~ N _I N
E-~
~ ~-1 ~
~ O a),,,,,~, ,, t;O O'~
¢ ~ ~ Od1d~ ~ N ~t~tS~O r-~( t`
~Z;S; ~D ~ o ~1 0 N~1 N O --i O O O
¢
t~ P
~J
O O ~~ tg ~1 ot~ ~ o ~ ~ ~
Ot~tY~t~O I tS: CO ~ ~Doo u~ ~1
ht,~;J ~ ~t d~ ~110 ~ ~ tY) ~
`_ C~
. .,.
U~ C~ C~
t~
~C a) tD
t~ ,~: P Ot~ P C~
t~ O tD'C1 ~r~ ~ 5 tDEl a P
O +> ~ tl) ~ O
UJ rA ~rlbD~:4h h :~ O
F~ h ~
3 E~ q ¢ ¢'~ ~( ~ UP~ U
, . .
~ 1~8949
.~
~a c o
U~
~ ~ X
.,, ,~ o
c~ ~ ~ bO
a~
~ O
O ~ ~ O ~ N u~
C h +~ ~1 C~ ~1 ~ C~l O O ~ ~ ~9
.,
~ X O ~ C~ _I ~ ~ _l
c; a~ ~ ~'
b~ a~ s~
S~ ~ ~1
~1 ::
~ C) ~ C~ O ~ o C~ oo L
R ~> a) ~ t~
~: ~ O ~ U~ O U~ C~ L~
a: ~ ~3 3 ~ U~ ~ ~ ~ ~ ~ ~ ~
W
P:
E~ 9~
~ o ~n
O ~ a
:Z h ~ :1
O
0~ tD O ~ ~ ~
E~ ~ ~1 C~ ~ O
~ Z ~ h ~
c ~
0~ 0~ ~ U~ o
h ~ ~ 1~ _I ~ ~I r-l C`l t- d'
:a ~z; ~, 14 o o o o .-~ ~1 o o tS:~
V ~D^
O-r~ ~ Ir~ NIr~ N o~Ir) 0 00
o o ~ ~ ~ c~t~ ~ ~ c~ oo co
c~l ~
t~
~1 ~ ~1
¢
a~
~ c
r1
c
~ ~ c ~ t3
O ~q ~r( ~1 a) ~1
~ ~ ~ c o ~ c t3
c o ~ o
O .~
c c ~ c) o c
,~ ~ E~ ~ ~ O
~ ,~ ~ ~ o
¢ ¢
1 178949
TABLE II
Comparison of the main immunological and toxic
characteristics of the B-IIb fragment with those
of the B-II fragment and tetanus toxin.
Compound Ouchterlony Specific Specific
test flocculating Toxicity
(antigenic activity LF/O.D. LF/Lf LD/mgN
structure) (LF/mgN)
_
toxin l(a,b) 3150 423 2,5x103 7.~x107
fragment l(b) 11000 1000 0.05 555
B-II~
fragment l(b) 8200 825 8 6.6x104
B-II
_
The thiolated polypeptide compound according to the
present invention is produced by thiolation of the B-IIb
fragment previously treated for eliminating the residual
toxicity thereof.
The thiolation of the B-IIb fragment can be carried
out by conventional means permitting/introduction of -SH
groups on a molecule comprising amino groups, but for the
purposes of the invention, the means in question must not
denature the axonal transport property and the binding
property to the specific receptor of the tetanus toxin
in the central nervous system, of the B-IIb fragment.
By way of example, it will be indicated that the
thiolation of the B-IIb fragment can be achieved with
the following thiolation agents :
1 1789~9
4-methyl-mercaptobutyrimidate : HS- (CH2)3-~0-CH3
(Biochemistry vol. 17 No 8,1978~ NH ~Cl-
2-iminothiolane (Schramm J.H. and Dolffer T. (1977)
Z. Physiol. Chem. 358 ; 137-139)
N-acetylhomocysteine thiolactone (AHT) (see J.Am.Chem.
Soc., l9GO, 82, 565-571)
/ S
H2C C=~::O
H C - CH
NHCOCH3
S-acetyl-mercaptosuccinic anhydride (AMS) (J. Am.
Chem. Soc. 1959, _ J3802-3803)
~z:O
C~3-CO-s- T ~ C O
CH - C ~
~0
On the other hand, it will be indicated that the
known processes of thiolation consisting of a
dithiopyridylation step and a reduction step are
unsuitable for the purpo~es of the invention. Indeed, the
properties of axonal transport and binding of the thus
thiolated B-IIb fragment are modified iTI the course of
the reduction step.
For example, the thiolation effected by reaction
with the N-succinimidyl-3-(2-pyridyl-dithio)-propionate
and by reduction of the dithiopyridylated compound so
obtained, for example according to the procedure
12
1 178949
described by CARLSSON et al. ~(Bioch. J. (1978) 173
723-724 / is unsuitable for the purpo~es of the invention.
In order to be more precise, it will be indicated
that the thiolated polypeptide compound according to the
invention comprises one or more Z-SH groups, in which Z
is the residue of the thiolation agent.
Thus, if one of the thiolation agents mentioned
above is employed, Z then represents :
IlH2 , Cl
-C- (CH2)3- ; - CO ~ I CH2 CH2 ; ~ CH- CO - ;
NH - CO - CH3 CH2-CO~H
Il (~I2)3
NH
The thiolation of the B-IIb fragment is achieved
on the NH2 groups thereof.
The residual toxicity oY the B-IIb fragment can
be previously removed, for example by an immuno-adsorption
step on a CNBr-activited Sepharose 4B gel column to wh~ch
the IgG ~raction of the anti-Ibc serum is covalently
bound. The B-IIb fragment thus obtained is not toxic in
the mouse at the dose of 1.9 mg.
It has been found that the thiolated polypeptide
compound according to the invention is suitable as a
neuropharmacological transport agent for transporting
pharmacological or chemotherapeutic agents to the central
nervous system.
In order to transport a medicine to the central
nervous system by means of the agent according to the
*Trademark
13
1 178949
invention, this medicine must be bound to the
thiolated polypeptide compound, employed as a transport
agent, without of course modifying the pharmacological
properties of the medicine or the binding property of the
B-IIb fragment to the specific receptors of the tetanus
toxin in the central nervous system. "Medicine" is
intended to des~gnate according to the invention any
substance having pharmacological properties, such as
pharmacological agents, chemotherapeutic agents and the
like. The medicines which may be bound according to the
- invention to the polypeptide compound employed as a
neuropharmacological transport agent must have-NH2 groups.
As examples of medicines which may be transported
to the central nervous system by means of the thiolated
polypeptide compound according to the invention~it may be
mentioned : alkaline phosphatase, the A fragment of
cholera toxin, the A i'ragment of diphtheria toxin,
dipyrido-indoles according to French patent 77,11,148
and, generally, any medicine having-NH2 groups.
It is known that the cholera toxin is bound to the
GMl gangliosides of the intestine wall and that the A
fragment is responsible for the increase in the cyclic
AMP rate (cyclic adenosine-monophosphoric acid). On the
other hand, in tetanus a decrease in the cyclic AMP
proportion in the central nervous system is found. The
conjugate according to the invention, formed by the
thiolated polypeptide compound coupled to the A fragment,
may be employed for controlling tetanus.
~ 1 ~894 9
The dipyrido-indoles according to French patent
77,11,148 are chemotherapeutics of utility in the
traitment of cancers. In th~ field, it is kno-~n that
the metastases are due to the fact that the cancerous
cells nestle in the central nervous system whence they
migrate to other regions of the body where they develop
tumors.
The development of the metastases could be avoided
or reduced as soon as the means for destroying these
cells in the central nervous system reach the central
nervous system.
In the same way, the inventlon may be applied to
the treatment of cerebral tumors.
The present invention consequently also relates to
the means for coupling the thiolated polypeptide compound
according to the invention to medicines.
Aeeordingly, the invention provides a proeess for
eoupling a medieine eontaining at least one NH2-gxoup to
the thiolated polypeptide eompound eonstituted by the
B-IIb fragment of tetanus toxin to whieh at least one -SH
group is direetly or indireetly bound, whieh eomprises the
steps of either:
_ a) introdueing at least one dithiopyridyl group
in the medieine; and
b) reaeting the dithiopyridyl group-bearing
medieine with the thiolated polypeptide
eompound; or:
~3
1 178~49
B c~ reacting the medicine to be bound with the
ester of meta-maleimidobenzoyl N-hydroxy-
succinimide; and
dl reacting the resulting compound with the
thiolated polypept;de compound;
whereby medicines bearing from l to 5 dithiopyridyl
groups are preferred,
The means for coupling the compound according to the
invention and the medicines to be transported use at least
a disulphide bridge or at least a sulfur irreversible link.
The present invention therefore also relates to B-IIb/
medicine conjugates comprising at least one disulphide
bridge or at least one sulfur irreversible link.
It is known to prepare protein conjugates by
formation of an intermolecular disulphide bridge. The
formation of such an intermolecular bridge is achieved
for examplo by reaction of a protein having thiol groups
with a protein having dithiopyridyl groups.
15a
,~ ,
1 178949
For example, according to the process described by
TE PIAO KING et al. / Biochemistry vol. 17 No 8,1978/,
two different proteins may be coupled by first binding
thiol groups to one of the proteins and 4-dithiopyridyl
groups to the other protein and by reacting the resulting
modified protein~ under suitable conditions in order to
form disulphide bridge and eliminating 4-thiopyridone.
The thiol groups may be bcund to one of the proteins by
means of 4-methyl-mercapto-butyrimidate and the 4-
dithiopyridyl groups to the other protein by means of,for example, 3-méthyl~4'-dithiopyridyl) propionimidate.
This coupling process produces a protein-protein
conjugate in which the fraction between the two proteins
is symmetrical relative to the disulphide bridge.
According to CARLSSON et al. (Bioch. J., 1978,
173, 723-724) the thiol group can be introduced in one of
the proteins by reaction of said protein with N-
succinimidyl-3-(2-pyridyl-dithio) propionate and subsequent
reduction; according to this process, the same reagent,
namely the N-succinimidyl-3-(2-pyridyl-dithio)propionate,
is used for introducing thiol and dithiopyridyl groups
in the proteins. The resulting conjugate also has a binding
fraction which is symmetrical relative to the disulphide
bridge.
4-methyl-mercapto-butyrimidate has also been used
for forming higher dimers and oligomers of proteins of
30 S ribosome of Escherichia Coli (Biochemistry, 12,
32~6-3273, ]973).
16
1 178949
The conjugates thus obtained have many applications,
for example as immunological assay reagents.
The coupling process according to the invention, of
the thiolated polypeptide compound used as a neuro-
pllarmacological transfer agent with a medicine by meanso~ disulphide bridge consists of the steps of :
1) - introducing dithiopyridyl groups in the medicine
to be bound ;
2) - reacting the medicine having the dithiopyridyl
groups with the thiolated polypeptide compound according
to the invention.
A preferred coupling process comprises
(a) reacting the B-IIb fragment with compounds chosen
from the group con6isting of 2-iminothiolane, N-acetyl-
homocysteine thiolactone, S-acetylmercapto~uacinimic acid
anhydride and 4-methylmercaptobutyrimidate to obtain a
thiolated polypeptide which contains between about 1 and
5 -SH groups;
(b) reacting the medicine with 2-iminothiolane
and 4,4'-dithiopyridine or N-succinimidyl-3-(2-pyridyl-
dithiopropionate) to obtain a medicine bearing one or
more dithiopyridyl groups; and
(c) reacting the product of step (a) with the
product of step (b).
17
r~
1 178949
The reaction diagram of this coupling process may
be represented in the following manner when the
dithiopyridylation agent used in step 1 is N-
1~ succinimidyl-3-(2-pyridyl-d1thio) propionate ;
1) lll- NH2 + ~? - s S-CEI2 - CH2 - C - O -
o
_~ M - NH - C - CH2-CH2-S - S~ ~ + H0-
2) NH2+,C1
~-II } N~ -(CH2)3_ZSIl+ M-~l-C C~12 C12 S S
0 ll~2 ,Cl
Nl~ -C-CH2-C~2-S- S -(CH2)3-C-NH/B-IIb_7 + ~ SH
17 a
~ 178g49
In this process, another dithiopyridylation agent
may be employed, such as dithiopyridine or any other
suitable agent for such a reaction.
Another way of coupling the polypeptide compound,
used as a neuropharmacological transport agent according
to the invention, consists of creating a~ irreversible
link between said agent and the medicine to be transported.
This process may be represented by the follcwing reaction
diagram :
1) M -NH2 + ~ ~ O - C ~ N~
~ ~ ~ ~ N - OH
0
2) M - NH - C ~ ~ ~ r-IIb_7-NHfC-(CHz)3~ SH
O
M - NH - C ~ ON ~ INlH2 ~ Cl
,~J s- ( CH2 ) 3- C-NH / B- I Ib_7
It consists of the steps of :
1) reacting the medicine to be bound with the ester
of metamaleimidobenzoyl-N-hydro~y-succinimide ;
2) reacting the resulting compound with the
polypeptide compound according to the inventi.on.
18
~ .
~ 17~949
The foregoing reaction diagrams and those which will
be given hereinafter are simplified and do not take into
accound the number of SH groups which may be bound to
the B-IIb fragment.
It has already been proposed to use the ester of
metamaleimidobenzoyl-N-hydrosuccinimide for forming
en~yme-antibody conjugates (FEBS Letters, vol. 95, No 2,
Nov. 1978). However, the teachings of this prior art did
not permit foreseeing that the use of the ester of
metamaleimidobenzoyl-N-hydrosuccinimide for coupling the
neuropharmacological transport agent according to the
invention to the medicines would not modify or inhibit
the pharmacological properties of said medicines and the
binding property of the B-IIb fragment to the specific
receptors of the tetanus toxin in the central nervous
system.
It will be obse~ved that the coupling according
to the invention of the medicinesto the thiolated
polypeptide compound employed as a neuropharmacological
transport agent is achieved by the known conventional
techniques of protein-protein coupling. However, it should
be noted that not all of the protein-protein coupling
processes available to one skilled in the art are suitable
for the purposes of the invention, Indeed, only the coupling
processes which achieve a disulphide bridge or a sulfur
irreversible link are suitable. In particular, it will be
indicated that the most conventional coupling process
which employs glutaraldehyde is unsuitable for the
19
1 178949
purposes of the invention, since the B-II fragment
treated with the glutaraldehyde loses its properties of
axonal transport and binding to the receptors of the
tetanus toxin in the central nervous system. ~hus, the
B-IIb fragment to which carbonyl groups would have been
bound, for example with glutaraldehyde, is unsuitable
for the purposes of the invention.
Another application of the thiolated polypeptide
compound according to the invention, is the labelling of
neuronal c211s. Thus, the compound of the invention may
be employed as a specific labeller of neuronal cells and
also or preparing immunological reagents. For example
an enzymatic reagent may be prepared from the thiolated
polypeptide compound according to the invention and
alkaline phosphatase. It was found that the resulting
conjugate had both the B-IIb fragment binding power and
the enzymatic activity of the phosphatase.
The invention will now describe in more detail
by means of the examples illustrating the preparation
cf the thiolated polypeptide compound of the invention
and of the coupling of the latter with medicines. In all
the examples, there has been employed the B-IIb fragment
as defined hereinbefore devoid of its residual toxicity.
EXAMPLE l
Thiolation of the B-IIb fragment by means of 2-
,
iminothiolane
The thiolation of the B-IIb fragment was achieved
by means of the method described by Schramm et al.
1 178949
æ. Physiol. Chem. 1977, 358; 137-139 7.
The B-IIb fragment (1 mg) in solution in 50% glyce-
rol (0.2 ml) was thiolated by iminothiolane (0.5 mg) in
solution in 750 ~1 of 0.2 M triethanola~ine HCl buffer,
pH 8.5-9.0, The reaction mixture was maintained at room
temperature for 2 hours. The excess of the reagent was
eliminated by filtration on a "SEPHADEX G-25" column.
The thiolated polypeptide product so obtained contained
1.2 -SH groups.
EXAMPLE 2
Thiolation of the B-IIb fragment by means of N-
acetylhomocysteine thiolactone
The B-IIb fragment was thiolated by the method
described by SINGER et al. / J, Am. Chem. Soc., 1960
82, 567-571 /.
To 3 mg o~ the B-IIb fragment in solution in 2 ml
of water, there was added the K2C03/NaHC03 buffer, pH
10.7 (1.6 ml). A nitrogen current was passed therethrough
in order to expel the air. 0.4 ml of an 80 mg/l solution
Of N-acetylhomocysteine thiolactone waæ added. The
reaction mixture was maintained for tvo hours at ~C under
nitrogen. The reaction was thereafter stopped by filtration
on SEPHADEX G 25. The obtained product contained 2.8 SH
groups.
EXAMPLE 3
Thiolation of the B-IIb fragment by means of S-acetyl-
.
mercaptosuccinimic acid anhydride
-
*Trademark. 21
I 1 789~ ~
The B-IIb fragment was thiolated by the method
described by Klotz et al. / J. Am. Chem. Soc., 1959,
81, 3802-3803_/.
The B-IIb fragment (3 mg) was introduced into 1 ml
of 0.1 M phosphated buffer, pH 7.4, in a 3-necked flask
and nitrogen was passed through the flask.
While stirring and maintaining a nitrogen
atmosphere, 140 ~g of S-acetyl-mercaptosuccinimic acid
anhydride was introduced in two times at 5 minutes
intervals,
The reaction was allowed to occur for a total of
20 minutes. The reaction mixture was then filtered on
SEPHADEX G 25. A solution of the modified B-IIb fragment
was collected and 1 M NH2 OH was added thereto until
a final concentration of 0.1 M was obtained. The
deacetylation was allowed to occur for 5 minutes. The
reaction mixture was then immediately filtered on
SEPHADEX G 2S equilibrated with a 0.1 M phosphate buffer,
pH 7.4. The product so obtained had 5.6- SH groups.
0 The reac~ion ~gram may be represented
D as follo~s :
CH3-C0-S-CH-C ~
¦ 0 + B-II ~ - >
CH2C
0
/ CH3-C0-S-CH-C0-~ - B-IIb
CH2-COOH
22
1 178949
/-- CH3-CO-S-CH-CO-_/- B-IIb + NH2
CH2-COOH
/ HS-I -CO- ~- B-IIb
CH2-COOH
EXAMPLE 4
Thiolation of the B-IIb fragment by means of 4-methyl-
mercaptonutyrimidate
The thiolation was carried out by the method
described by TE PIAO KING et al. / Biochemistry vol. 17
No 8, 1978 ~ , by means of 4-methyl-mercaptobutyrimidate
and a thiolated polypeptide compound was obtained
comprising 1.4 groups of following formula :
- C - (~I ) - SH
Il 2 3
NH2 Cl
EXAMPLE 5
L
Preparation of a thiolated polypeptide compound-A
fragment of diphtheria toxin conjugate
2,2 mg of fragment A of diphtheria toxin in 1 ml of
0.025 M borate buffer, pH 9.0, were employed. 7 mg of
2-iminothiolane and 77 ~g of 4,4'-dithiopyridine were
added in the form of a methanolic solution (100 ~1 of
a solution of 3.85 mg in S ml of methanol). This solution
was added in two times of 50 ~1 at 3 minutes intervals
while cooling in ice and stirring. The reaction was
allowed to occur for 2 hours. The reaction mixture was
imrnediately applied to the SEPHADEX G 25 (0.9 x 20 cm)
23
1 178949
equilibrated with a 0.1 M phosphate buffer, pH 6.9
containing 1 mM Na2 EDTA
Three moles of the polypeptide compound obtained
from example 4 were mixed with 2.8 moles of the A-
dithiopyridylated fragment according to the abovemethod. The exchange reaction was followed at 324 nm.
The mixture was filtered on SEP~AROSE 6 B equilibrated
with a buffer tris 0.05 M , 0.5 M NaCl, pH 8Ø
The conjugate obtained preserves the binding p~er
of the B-IIb fragment and the immunological activity of
the A fragment of the diphtheria toxin. Thus it is
possible to prepare a chimera.
The dithiopyridylation of the A fragment may also
be achieved by means of N-succinimidyl-3-(2-pyridyl-
dithio)propionate.EXAMPLE 6
Use of the thiolated ~olype~tide comPound according to
the inventio~ as a neuropharmacological transport agent
In order to show that the thiolated polypeptide
compound according to the invention may be employed as
a specific neuropharmacological agent, the peripheral
oculomotor system in the rat was used as an experimental
model.
The thiolaterl polypeptide compound obtained from
the above example 4 was used. This thiolated polypeptlde
compound was coupled to the IbC fragment of the tetanus
toxin.
The IbC fragment was obtained by papain digestion
24
1 178949
of the tetanus toxin.
The injection of this IbC fragment to mice
poisoning
produced symptoms of/similar to those of the B fragment
/ Helting et al. J. Biol. Chem. 253, (1978~ 125-129 ~ .
The IbC fragment coupled to the polypeptide
compound according to the invention was used in order to
show that it is the B-IIb fragment which is bound to the
specific receptors of the tetanus toxin in the central
nervous system and not another fragment of the tetanus
toxin, such as the IbC fragment. Indeed, the B-IIb/IbC
conjugate comprising disulphide bridges obtained
according to the coupling process defined above,
resembles, from the structural point of view, the entire
tetanus toxin, and the results mentioned below indicate
that lt is only the B-IIb which is bound to the specific
receptors of the tetanus toxin.
The B-IIb and IbC fragments and also the B-IIb/IbC
con~ugate were labelled by means of 1 5I radioactive iode.
Thls labelling was e~fected by the method of Greenwood
et al. (Biochem. J, 89, (1963) ~14-127).
For each substance to be labelled, there were
employed 400 ~g of said substance and 2 mCi of Na I125
(provided by the Amersham Radiochemical Center). The
specific radioactivity of each substance thus labelled
is indicated below :
1 178949
Substance Radioactivity
.
B-IIb fragment 3.4 ~Ci/~g
IbC fragment 2.6 ~Ci/~g
B-IIb/IbC conjugate ~Ci/~g
Experiment procedure :
For all the experiments, female Sprague-Dawley rats
weighing 250-27Gg were used. The rats were maintained at
a constant temperature of 23C and supplied with the usual
diet (Na~ag, Gossau) and water.
Five albino rats received an injection of one of
the above labelled substances in the medial rectus muscle
of the left eye according to the following procedure.
After having bared the muscle, the substances were
injected by a thermally controlled injection system by
means of a glass pipette (50-100 ~m outside diameter),
The injecting time was 35 to 50 minutes. One rat received
the above con~ugate and the other four rats received one
of the IbC or B-IIb fragments. A 30% horseradish peroxidase
(HRP type Sigma ~I) was used as a control. The rats were
sacri$i~d 24 hours after the in~ection. An intracardial
perfusion was carried out under general anesthesia first
of all with a plasma expander ("Macrodex"), then with
1.25% glutaraldehyde and 1% paraformaldehyde in O.lM
phosphate buffer, (pH 7.4) for 30 minutes, and lastly
with a 10% sucrose in a 0.1 M phosphate buffer (pH 7.4)
for 30 minutes. For more details of this procedure,
reference may be made to the article of M~SULAM / J.
26
1 178g49
Histochem. Cytochem., 26 (1978) 106-117~ .
The brain was removed immediately after this
perfusion and placed in a 30% sucrose solution for 44
hours before cutting. Frozen sections (30 ~m thickness)
were taken, starting from the abducens nucleus and
through the whole oculomotor nucleus. Each section
treated with the horseradish peroxidase was stained by
ths TMB method of M~SULAM and restained with neutral
red while the other sections were autoradiographed, The
latter sections were mounted and dipped into a liquid
emulsion NTB2 at 45C diluted 1:2 with distilled water.
The sections were exposed for 4 weeks at 4~C in the dark
and developed with "Koda~ Dektol" developer at 18C for
90 seconds, washed, and then fixed with 30% sodium
thiosulfate, washed for 2 hours, then stained with cresyl
violet and covered. All the sections were then examined
under the microscope (250 magnification) and the location
of the labelled cells was ascertained by microphotography.
In a fir8t series of experiments, the B-IIb/IbC
conjugate was injected simultaneously with the horseradish
peroxidase in the medial rectus muscle and a radioactive
labelling was found in the oculomotor nucleus for the
conjugate and the horseradish peroxidase.
The main difference between the horseradish
peroxidase (HRP) and the B-IIb/IbC conjugate resides
in the fact that the location of the HRP granules is
limite~to the the perikarya and dendrites of the
oculomotor nucleus, while the silver grains which
27
. . . . .. . .
1 178949
represent the 125I-labelled conjugate ~ere also found in
the pericellular spaces.
In another series of experiments, thel 5I-labelled
B-IIb fragment was injected into two rats according to
the above procedure and labelled cells were observed in
neurons of the ipsilateral oculomotor nucleus in both
the treated animals.
In a third series of experiments 5I-labelled IbC
fragment and HRP were injected simultaneously into the
eye muscle of a rat. This time, only a labelling of the
cells of the oculomotor nucleus by the horseradish
peroxidase was observed. In another animal, only the
125I-]abelled IbC fragment was injected and no labelling
of the cells of the oculomotor nucleus was observed. The
fact that the experiment carried out with the first
ani~lals (treated simultaneously Ylith HRP and 125I-labelled
IbC) gave positive results due to the HRP is important,
since it shows that the fact that the IbC fragment was
not transported in a retrograde axonal manner does not
result from a breakdown o~ the retrograde axonal flow
but rather from the specific properties of the IbC fragment
which seem to be incompatible with intraaxonal transport
functions.
Further, it was found that the IbC fragment does
not bind to the gangliosides and isolated synaptic
membranes. On the other hand, the B-IIb fragment binds
in a specific manner to the gangliosides and the synaptic
membra~les / see Journal of Neurochemistry (1977), vol. 2
28
1 178g49
p. 529-5427.
The above test shows that if there is to be an
axonal retrograde transport of a substance, the
substance must be able to bind specifically to the
gangliosides and synaptic membranes.
By means of the tests described in the Journal of
Neurochemistry (1977) vol.28, p. 529-542, it was verified
that the B-IIb/medicine conjugates obtained according
to the invention from the thiolated polypeptide compound
bound in a specific manner to the gangliosides and the
synaptic membranes; these results demonstrate that the
substances can consequently be transported in retro~rade
axonal manner.
29
~ 17894g
.. ~..... .......... .............. ......................
oP' ++ + + , +,
0
0 .... ,..... .......... .............. ......................
~ ~ lQ 0 0 0 0
~ ~1 ~ ~ ~ h h S~
~-rl C~ ~ I ~ S
~3 .
S~ ~ C~ C~ C~ C~
C .... ,..... .......... .............. ......................
S-p~ q~ t ~ ~/ t~t~
~ ~3V ~ ~ ~ ~ ~
~ o~
~C ~ ~ G~ 8 ~~
H ~ O ,5:: ~rl ~1 0~ ~i ~i 0 ~ii
H h .......... .......... ............ ,.
~ O ~ O g
~ 9~, ~ o ~ ~ ~ ~ ~
o .. . ... .... .... .. ... .. .... ........ ..
O ~1 R~ I~ m H P1 H
~2 /D H o C~ h t!l h C`J h C~ h
........ .......... .............. ......................
~ t- ~ r~ o
t_ CO
h .......... .............. ......................
'~0
~) ~ ~--I H H
.. ~. ~ .. .......... ....................................
