U.S. patent application number 13/068439 was filed with the patent office on 2011-09-08 for therapeutic composition comprising a botulinum neurotoxin.
This patent application is currently assigned to MERZ PHARMA GmbH & CO. KGaA. Invention is credited to Hans Bigalke, Jurgen Frevert.
Application Number | 20110217287 13/068439 |
Document ID | / |
Family ID | 7910318 |
Filed Date | 2011-09-08 |
United States Patent
Application |
20110217287 |
Kind Code |
A1 |
Bigalke; Hans ; et
al. |
September 8, 2011 |
Therapeutic composition comprising a botulinum neurotoxin
Abstract
A pharmaceutical preparation comprising one of the botulinum
neurotoxins from Clostridium botulinum of types A, B, C, D, E, F or
G or a mixture of two or more of these neurotoxins, wherein the
neurotoxin or the mixture of neurotoxins is free of the complexing
proteins which naturally form the botulinum neurotoxin complexes
together with the neurotoxins.
Inventors: |
Bigalke; Hans; (Hannover,
DE) ; Frevert; Jurgen; (Berlin, DE) |
Assignee: |
MERZ PHARMA GmbH & CO.
KGaA
Frankfurt am Main
DE
|
Family ID: |
7910318 |
Appl. No.: |
13/068439 |
Filed: |
May 11, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10018373 |
Dec 6, 2001 |
7964199 |
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PCT/DE00/01777 |
May 26, 2000 |
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13068439 |
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Current U.S.
Class: |
424/94.67 ;
435/220 |
Current CPC
Class: |
A61P 17/16 20180101;
A61P 17/00 20180101; A61P 25/08 20180101; A61P 21/00 20180101; A61P
25/04 20180101; A61P 25/14 20180101; A61P 27/02 20180101; A61P
43/00 20180101; A61K 38/4893 20130101; A61P 25/02 20180101; Y02A
50/469 20180101; C07K 16/1282 20130101; Y02A 50/30 20180101; A61P
21/02 20180101; A61P 29/00 20180101; A61P 25/00 20180101; A61P
25/06 20180101 |
Class at
Publication: |
424/94.67 ;
435/220 |
International
Class: |
A61K 38/48 20060101
A61K038/48; A61P 25/00 20060101 A61P025/00; C12N 9/52 20060101
C12N009/52 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 7, 1999 |
DE |
19925739.6-41 |
Claims
1. A pharmaceutical preparation comprising a Clostridium botulinum
neurotoxin selected from types A, B, C, D, E, F or G or a mixture
of two or more of these neurotoxins, wherein the neurotoxin or the
mixture of the neurotoxins is free of complexing proteins which
naturally form complexes with botulinum neurotoxin.
2. The pharmaceutical preparation of claim 1, wherein the
neurotoxin or mixture of neurotoxins exhibits reduced antigenicity
compared to botulinum toxin complexes.
3. The pharmaceutical preparation of claim 1 which is
non-antigenic.
4. The pharmaceutical preparation of claim 1, wherein the
neurotoxin is a natural neurotoxin or the mixture of neurotoxins
comprises a natural neurotoxin.
5. The pharmaceutical preparation of claim 1, wherein the
neurotoxin is a recombinant neurotoxin or the mixture of
neurotoxins comprises a recombinant neurotoxin.
6. The pharmaceutical preparation of claim 1, wherein the
neurotoxin is from Clostridium botulinum type A or B.
7. The pharmaceutical preparation of claim 1, wherein the mixture
of the neurotoxins comprises a neurotoxin from Clostridium
botulinum type A or B.
8. The pharmaceutical preparation of claim 1, wherein the mixture
of the neurotoxins consists essentially of a neurotoxin from
Clostridium botulinum type A or B.
9. The pharmaceutical preparation of claim 1, wherein the
neurotoxin does not comprise hemagglutinins.
10. The pharmaceutical preparation of claim 1, which is in the form
of an injectable aqueous solution.
11. The pharmaceutical preparation of claim 1, which is
lyophilized.
Description
FIELD OF THE INVENTION
[0001] A pharmaceutical preparation comprising one of the botulinum
neurotoxins from Clostridium botulinum of types A, B, C, D, E, F or
G or a mixture of two or more of these neurotoxins, wherein the
neurotoxin or the mixture of neurotoxins is free of the complexing
proteins which naturally form the botulinum neurotoxin complexes
together with the neurotoxins.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to pharmaceutical preparations
which comprise a botulinum neurotoxin from Clostridium botulinum,
the neurotoxin being free of the complexing proteins naturally
present in the complex. The direct consequence thereof is the
realization, on which the present invention is based, that with the
free neurotoxin, in contrast to the complex, there is only a
distinctly reduced, or no, induction of neutralizing antibodies in
the patient. The present invention further relates to the use of
botulinum neurotoxins from Clostridium botulinum for producing a
medicine for treating disorders of the nervous system. Another
aspect of the present invention relates to the use of the botulinum
neurotoxins from Clostridium botulinum for cosmetic treatment.
[0003] Clostridium botulinum toxin complex type A (M.sub.r 900,000)
has been employed for several years for the therapy of various
dystonias. At present two different products comprising this
complex are approved for the treatment of blepharospasm, hemifacial
spasms and spasmodic torticollis: BOTOX.RTM. and DYSPORT.RTM..
Clinical trials of the therapy of other disorders of the nervous
system (e.g. spasticities, migraine, low back pain, cervical spine
disorders, hypersalivation) are currently in progress. The products
are also employed for cosmetic indications such as hyperhidrosis
and pronounced wrinkling. The other Clostridium botulinum toxin
complexes (of types B, C, D, E, F, G) are also suitable for these
therapies. However, at present there is no approved product
comprising one of the type B-G toxins on the market.
[0004] Botulinum toxin complexes are composed of a mixture of
clostridial proteins. These are hemagglutinins with different
molecular masses, a nontoxic, non-hemagglutinating protein (M.sub.r
about 120,000) and a neurotoxin (M.sub.r about 150,000). They form
an acid-stable complex which is responsible for the oral toxicity
in cases of food poisoning. In contrast to the pure neurotoxin, the
complex resists the aggressive environment in the gastrointestinal
tract and makes enteral absorption of the neurotoxin possible, and
this reaches the target cells via the bloodstream or the lymphatic
system and there induces blockade of transmitter release. This is
followed by a paralysis of striped and smooth muscles and cessation
of various autonomic functions. Poisoned patients die of
respiratory muscle failure. Since the pure neurotoxin is degraded
in the gastrointestinal tract and thus does not undergo enteral
absorption, it is not toxic after ingestion. On parenteral
administration, the therapeutic effects of the neurotoxin and of
the complex do not differ since the complex decomposes into its
constituents in tissue, and only the neurotoxin is taken up by the
target cells.
[0005] For therapeutic use, the complex is in the current state of
the art injected directly into dystonic or spastic muscles, where
the neurotoxin is released at physiological pH from the complex and
elicits the desired pharmacological effect. Although the complex is
administered only in extremely low doses (1-25 ng, depending on
indication and size of the affected muscle), repeated injection is
followed in a considerable number of patients by formation of
specific neutralizing antibodies which are also directed against
the neurotoxin. The direct consequence is that antibody-positive
patients no longer respond to the complex. However, they might be
treated with other toxin types, although none of them is approved,
for therapy. When the patient has been tested with all the toxin
types and has formed antibodies against them, further
administration of a botulinum toxin complex (irrespective of the
type) no longer provides a remedy. It must be taken into account in
this connection that each dose of complex contributes to increasing
the antibody titer until further administration of the complex no
longer makes sense because no effect is now achieved. It often
takes years for the antibody titer to fall significantly, so that
these patients are not (cannot be) treated (with botulinum
neurotoxin) for long periods.
[0006] The formation of specific antibodies is favored by two
factors. On the one hand, the neurotoxin, fixed in the complex,
remains in the tissue for a long period and may activate immune
cells which migrate into the tissue to form antibodies. The long
residence time does not result in increased uptake by the target
cells, however, since poisoned target cells are no longer able to
take up toxin. The neurotoxin which slowly dissociates out of the
complex thus now has only immunological activity. On the other
hand, the proteins present in the complex intensify an immune
response. Hemagglutinins are lectins, that is to say proteins which
are distinguished by a high affinity for certain sugars. Because of
their binding to sugar structures, lectins have immunostimulating
effects. Thus, it has been possible to show that the lectins
concanavalin A, phytohemagglutinin and pokeweed mitogen activate T
and B lymphocytes. The hemagglutinins of the botulinum toxin
complexes, which likewise bind to membrane-associated sugars, are
thus able in a similar way to act as immunoadjuvants and contribute
to antibody formation and thus to failure of the therapy.
OBJECTS OF THE INVENTION
[0007] The object of the inventors of the present invention was
therefore to develop an alternative mode of treatment of the
above-mentioned disorders and disturbances. In particular, the
inventors wish to propose a suitable active ingredient with which
patients who have already formed neutralizing antibodies can be
treated.
[0008] To achieve the object stated above, as alternative to the
two commercial type A botulinum toxin complex products, BOTOX.RTM.
and DYSPORT.RTM., and also as alternative to the complexes
described in the prior art of the other types (B, C, D, E, F, G), a
novel pharmaceutical has been developed which comprises only pure
neurotoxin (type A or B, C, D, E, F, G) and is free of
hemagglutinins and other exogenous proteins. Because of its lower
molecular mass, it diffuses more quickly to the target cells in
which it is taken up, before immune cells, attracted by
hemagglutinins, are activated. We found in antigenicity studies
that the pure neurotoxin of all types--in distinction from
commercial products of type A and the complexes of types B to
G--induces no, or at the most very little, formation of antibodies.
On therapeutic use of this newly developed pharmaceutical (pure
neurotoxin of types A, B, C, D, E, F, G) there is no failure of
therapy due to antibodies even after repeated administration. It
has also been possible to show that the pure neurotoxins are,
because of their immediate bioavailability, still suitable for the
therapy of patients who have developed, after administration of a
botulinum toxin complex, e.g. after treatment with BOTOX.RTM. or
DYSPORT.RTM., an antibody titer against the appropriate type
(so-called secondary non-responders), that is to say are no longer
amenable to further treatment with BOTOX.RTM. or DYSPORT.RTM.,
because administration of the commercial toxins no longer
alleviates the symptoms.
[0009] The pharmaceutical provided according to the invention is
suitable as therapeutic composition in particular for patients who
exhibit an antibody titer against a botulinum toxin, in particular
against that of type A. The novel pharmaceutical (pure neurotoxin
or mixture of a plurality of pure neurotoxins) is particularly
suitable for patients who exhibit an antibody titer not exceeding
50, preferably not exceeding 30, more preferably not exceeding 20,
particularly preferably not exceeding 10, and very particularly
preferably not exceeding 5, mU/ml. In this connection, 1 mU of
antibody is the amount of antibody which neutralizes 10 U of
toxin.
[0010] On the other hand, the novel pharmaceutical can be employed
with particular advantage for patients who have never, or not for
many years, been treated with botulinum neurotoxin, because their
antibody titer is low or zero from the outset. The advantage of the
present invention is then that the increase in the titer in these
patients due to the treatment with the pure toxin according to the
present invention is zero, or at the most very insignificant. In
other words, the novel therapeutic composition can be administered
over long periods without losing its effect.
[0011] The induction of antibodies during therapy with a
Clostridium botulinum toxin is thus prevented by administering a
pure neurotoxin in place of the high molecular weight toxic
complexes. The neurotoxin which has been completely separated from
the complex proteins is immediately bioavailable and can bind
directly to the nerve endings of the motor endplates.
SUMMARY OF THE INVENTION
[0012] What we therefore believe to be comprised by our invention
may be summarized inter alia in the following words:
[0013] One aspect of the present invention thus relates to a
pharmaceutical preparation which comprises at least one of the
botulinum neurotoxins from Clostridium botulinum of types A, B, C,
D, E, F or G (or a mixture of two or more of these neurotoxins),
all the neurotoxins being free of the complexing proteins naturally
present in the complex.
[0014] In a preferred embodiment, the pharmaceutical preparation is
such that the induction of neutralizing antibodies in the patient
by the neurotoxin or the mixture of neurotoxins is reduced by
comparison with the complexes or is zero.
[0015] A further preferred embodiment provides a pharmaceutical
preparation which comprises as neurotoxin or as mixture of
neurotoxins a natural neurotoxin or a mixture of natural
neurotoxins.
[0016] A further preferred embodiment provides a pharmaceutical
preparation which comprises as neurotoxin or as mixture of
neurotoxins a recombinant neurotoxin or a mixture of recombinant
neurotoxins.
[0017] Another preferred embodiment of the novel pharmaceutical
preparation provides a preparation which comprises as neurotoxin
the neurotoxin from Clostridium botulinum type A or B or as mixture
of neurotoxins a mixture of the neurotoxins from Clostridium
botulinum type A and B.
[0018] A further aspect of the present invention relates to the use
of the botulinum neurotoxins from Clostridium botulinum of types A,
B, C, D, E, F or G or of a mixture of two or more of these
neurotoxins for producing a medicine for treating disorders of the
nervous system and dystonias. The disorders of the nervous system
and the dystonias are in a preferred embodiment spasmodic
torticollis and blepharospasm, spasticities such as footdrop,
hemifacial spasms, migraine, low back pain, cervical spine
disorders or hypersalivation.
[0019] Another aspect of the present invention in turn relates to
the use of the botulinum neurotoxins from Clostridium botulinum of
types A, B, C, D, E, F or G or of a mixture of two or more of these
neurotoxins for cosmetic treatment, particular preference being
given to a cosmetic treatment for treating hyperhidrosis and
wrinkling, especially in the facial region.
[0020] Very particularly preferred for the purpose of the present
invention is the use of one of the neurotoxins alone or in a
mixture for producing a medicine for treating the above-mentioned
nervous disorders in persons (preferably humans, but also animals)
who already exhibit neutralizing antibodies against a botulinum
neurotoxin complex, in particular against the complex of
Clostridium botulinum type A or B, or against a plurality of
complexes, in particular against the complexes of Clostridium
botulinum type A and B (so-called secondary non-responders).
[0021] The neurotoxins, mixtures thereof and the novel
pharmaceutical preparations can be in the form of an aqueous
solution, in particular as aqueous solution for injection, but also
as lyophilized products.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The pure neurotoxins of types A-G, which are known per se,
were produced by the protocols present in the publications detailed
in the list of references. The purification of two neurotoxins
(type A and B) is described by way of example in the following
examples.
EXAMPLE 1
Isolation of the Pure Neurotoxin
[0023] The pure neurotoxin from Clostridium botulinum type A is
obtained by a process based on the process of DasGupta &
Sathyamoorthy. Clostridium botulinum type A is cultivated in a 20 I
fermenter in a medium consisting of 2% proteose peptone, 1% yeast
extract, 1% glucose and 0.05% sodium thioglycolate. After growth
for 72 hours, the toxin is precipitated by adding 3 N
H.sub.2SO.sub.4 (final pH=3.5). The precipitated and centrifuged
biomass is extracted with 0.2 M sodium phosphate buffer pH 6.0.
[0024] After removal of the nucleic acids by precipitation with
protamine sulfate, the toxin is precipitated by adding ammonium
sulfate. The precipitate which has been solubilized and dialyzed
against 50 mM sodium phosphate pH 6.0 is bound to a DEAE-Sephadex
column at the same pH and detached with 150 mM NaCl. This is
followed by a chromatography on a QAE-Sephadex column which has
been equilibrated with a 50 mM tris/HCl buffer pH 7.9. The toxin is
eluted via an NaCl gradient. In the last step, the toxin is
chromatographed on SP-Sephadex at pH 7.0. In this case, the bound
toxin is detached from the column using an NaCl gradient (0-300
mM). The purified toxin is analyzed in an SDS polyacrylamide gel
electrophoresis (SDS-PAGE) and exhibits a purity of 95.+-.5%. The
biological activity is determined in the mouse LD.sub.50 assay: one
LD.sub.50 unit corresponds to 4.8 pb of protein.
EXAMPLE 2
Production of a Finished Pharmaceutical Containing Botulinum
Neurotoxin
[0025] The purified neurotoxin from Example 1 is used to prepare a
solution which comprises 200 mouse LD.sub.50 units, 10 mg of
sucrose and 2 mg of human serum albumin per ml. The solution (0.5
ml) is dispensed into vials and freeze-dried. The lyophilizates are
reconstituted with physiological saline, and the biological
activity is determined. The vials comprise 100.+-.30 LD.sub.50
units.
EXAMPLE 3
Isolation of Pure Neurotoxin B
[0026] Clostridium botulinum type B is cultivated in the same
medium and under the same conditions as type A and is processed as
far as the ammonium sulfate precipitation. This is again followed
by a DEAE-Sephadex chromatography at pH 6.0. The fractions eluted
from the column with 150 mM NaCl are combined and dialyzed against
sodium phosphate pH 7.0, followed by a chromatography on
QAE-Sephadex. The toxin-containing fractions are chromatographed
further on a DEAE-Sephadex column at pH 8.5 (50 mM tris/HCl pH
8.5).
[0027] Finally, the high-purity botulinum toxin type B is obtained
by a chromatography on hydroxyapatite equilibrated with 10 mM Na
phosphate pH 8.0. The bound homogeneous toxin is eluted with 80 mM
Na phosphate pH 8.0 and subsequently the biological activity is
determined in the LD.sub.50 assay (2-4.times.10.sup.7 LD.sub.50
units/mg of protein).
EXAMPLE 4
Detection of Antibodies
[0028] 20 rabbits received intracutaneous injections of 25 U of
BOTOX.RTM. at intervals of 14 days over a period of 12 weeks (5
injections). Serum was obtained after 3 weeks and then at intervals
of 14 days.
[0029] Antibodies against Clostridium botulinum neurotoxin A were
detected in an enzyme immunoassay by immobilizing the homogeneous
neurotoxin on a microtiter plate. Antibodies binding to the
neurotoxin were quantified using a second, enzyme-labeled
antibody.
[0030] The result is shown in Table 1. Antibodies were detected in
5 rabbits as little as 5 weeks after the first administration.
After 11 weeks, sera from 17 rabbits, that is to say 85% of the
animals employed, contained antibodies against the neurotoxin. It
was shown in the biological activity assay that 12 of the 17 sera
contained neutralizing antibodies (Table 2).
TABLE-US-00001 TABLE 1 Determination of serum samples (diluted
1:100) from rabbits treated with BOTOX .RTM. using an enzyme
immunoassay. OD.sub.490 nm > 0.1 are indicated. All OD values
are corrected for the OD values of the preimmune sera (OD about
0.150). Rabbit No. 3rd week 5th week 7th week 9th week 11th week 1
-- -- -- 0.11 0.36 2 -- -- -- 2.36 2.23 3 -- -- 0.57 1.43 1.44 4 --
-- 0.68 1.68 0.93 5 -- 0.97 3.52 3.49 3.44 6 -- -- 1.34 2.32 2.70 7
-- -- 2.13 3.09 3.00 8* -- 0.53 1.47 2.75 2.75 9 -- -- 0.43 2.44
2.85 10 -- -- 2.99 3.15 2.73 11 -- 0.10 2.42 2.45 1.93 12 -- -- --
1.13 1.95 13 -- -- -- -- 1.89 14 -- -- -- -- -- 15 -- -- -- -- --
16 -- -- -- -- -- 17 -- 2.93 3.62 3.72 3.44 18 -- -- 1.18 2.28 2.62
19 -- -- 0.43 0.43 0.81 20 -- 1.65 3.20 2.97 2.88 *The values were
not corrected because no preimmune serum was available "--" means
optical density (OD.sub.490nm) < 0.1
TABLE-US-00002 TABLE 2 Neutralization by sera from rabbits treated
with BOTOX .RTM. (week 11 after the first immunization) in the
mouse hemidiaphragm assay (detection limit: 0.35 mU/ml antibodies)
Neutralization Rabbits mU/ml 1 2.0 2 n.d. 3 n.d. 4 >10 5 >100
6 n.d. 7 >10 8 >10 9 n.d. 10 n.d. 11 n.d. 12 >10 13 n.d.
14 n.d. 15 <0.35 16 0.4 17 >10 18 >10 19 2.0 20 >10
n.d. = not determined
EXAMPLE 5
Antigenicity Assay with Market Product and Pure Neurotoxin
[0031] After it had been shown that the complex of neurotoxin and
hemagglutinins and the nontoxic, non-hemagglutinating protein
induces the formation of neutralizing antibodies, the immunogenic
effect of the pure neurotoxin (type A) was tested. For this
purpose, 8 rabbits were treated with the toxin complex and 12
rabbits were treated with the pure toxin. 25 U of the respective
product were administered intracutaneously by the method described
above (see Example 1). The amount of neurotoxin, measured by
weight, was the same in both products (200 pg/dose), as was
demonstrated in an ELISA. BOTOX.RTM. additionally contained complex
proteins (about 800 pg/dose).
[0032] Four of the eight animals treated with BOTOX.RTM. showed an
antibody titer in the ELISA, whereas no antibodies against the pure
neurotoxin were detectable in the 12 animals treated with pure
neurotoxin. The result was confirmed in the biological activity
assay. All four rabbit sera contained neutralizing antibody titers
preventing an effect of the toxin (Table 3).
TABLE-US-00003 TABLE 3 Neutralization by sera (diluted 1:3) from
rabbits treated with BOTOX .RTM. (week 11 after the first
immunization) in the mouse hemidiaphragm assay (detection limit: 1
mU/ml antibodies) Neutralization Rabbits mU/ml 1 12 mU 2 >30 mU
3 4.5 mU 8 >30 mU
EXAMPLE 6
Comparative Example
[0033] This experiment compared the antibody formation due to
BOTOX.RTM. with that due to DYSPORT.RTM.. For this purpose, groups
of ten rabbits were treated either with BOTOX.RTM. (group 1), with
DYSPORT.RTM. (group 2) or with the pure neurotoxin (group 3) in
accordance with the scheme described.
[0034] Whereas more than 50% of the animals formed a neutralizing
antibody titer in group 1 and 2, the sera from the animals in group
3 were free of antibodies.
EXAMPLE 7
Clinical Test
[0035] A patient (45 years of age) who had been treated for a
period of 5 years with BOTOX.RTM. for spasmodic torticollis had
developed an antibody titer of 3 mU/mI of serum. Neither BOTOX.RTM.
nor DYSPORT.RTM. was therapeutically effective for this patient. An
attempt at therapy with the pure botulinum neurotoxin in a dose of
145 U, which was equivalent to the last BOTOX.RTM. dose injected,
resulted within 72 hours in loosening of the muscle, normalization
of the posture of the head and disappearance of the muscle pain. No
adverse effects occurred.
EXAMPLE 8
Clinical Test
[0036] A patient (52 years of age) had been treated with BOTOX.RTM.
for 3 years for cerebral palsy. He had developed an antibody titer
of 1 mU/ml of serum and it was thus necessary to discontinue the
therapy. Injection of 200 U of pure neurotoxin made successful
therapy possible.
[0037] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description.
[0038] All patents, applications, publications, test methods,
literature, and other materials cited herein are hereby
incorporated by reference.
REFERENCES
[0039] DasGupta, B. R. & Sathyamoorthy, V. (1984), Purification
and Amino Acid Composition of Type A Botulinum Neurotoxin; Toxicon
22(3), p. 415-424
[0040] De Jongh, K. S., Schwartzkoff, C. L. & Howden, M. E. H.
(1989), Clostridium botulinum Type D Neurotoxin Purification and
Detection; Toxicon 27(2), p. 221-228
[0041] Schmidt, J. J. & Siegel, L. S. (1986), Purification of
Type E Botulinum Neurotoxin by High-Performance Ion Exchange
Chromatography; Analyt. Biochemistry 156, p. 213-219
[0042] Nukina, M., Mochida, Y., Sakaguchi, S. & Sakaguchi, G.
(1988), Purification of Clostridium botulinum Type G Progenitor
Toxin; ZbL Bakt. Hyg. A 268, p. 220-227
[0043] Terajima, J., Syuto, B., Ochandra, J. O. & Kubo, S.
(1985), Purification and Characterization of Neurotoxin Produced by
Clostridium botulinum Type C 6813; Infection and Immunity 48(2), p.
312-317
[0044] Wadsworth, J. D. F., Desai, M., Tranter, H. S. et al (1990),
Botulinum type F neurotoxin: Large-scale Purification and
Characterization of its Binding to Rat Cerebrocortical
Synaptosomes; Biochem. J. 268, p. 123-128
* * * * *