Note: Descriptions are shown in the official language in which they were submitted.
WO92/2069s PCT/EPg1/00918
- 20871~8 ~- ~
METHOD FOR T~E PREPARATION AND PURIFICATION
OF A MIXTURE OF GLYCOSPHINGOLIPIDS FREE FROM
CONTAMINATION BY NON-CONVENTIONAL VIRUSES
.
Field of the Invention
The present invention relates to a process for
the preparation of a specific mixture of ~
gangliosides, and the product produced by such -
process, obtained by a process which selectively
eliminates contaminants associated with non-
conventional, life-threatening viruses, without
altering the biological and pharmacological
characteristics o~ the mixture with regard to its
ef~ects on the central and peripheral nervous
systems.
Backaround of the Inventio~
Gangliosides, glycosphingolipids containing
sialic acid, are normal constituents of all cell
me~branes in mammals and are abundant in the nerve
tissue (Ando S.: ~Neurochem. Int. 5:507, }983).
~20 - ~ Four gangliosides, GMl, GDl., GD1b~and GT~
(nomenclature~according to Svennerholm L., J.
N~urochem, 10:613, 1963), constitute 80-90% of the
; total ganglioside content of the mammal brain.
Gangliosides are specifically localized in the outer
2s~ ~ layer of the plasma membrane, suggesting that they
play an important~role in many biological
.
W092/2~95 PCT/EP91/00918
2 087 1~8 2
activities, for instance as a "sensor" and/or
receptor for various molecules, and in the transfer
of information through the cell membranes (Fishman
et al.: Science 194:906, 1976). They therefore
play a key role in the regulation of ne~ronal
development and repair in the central and peripheral
nervous systems.
There is indeed ample documentation that
gangliosides are able to favorably influence
functional recovery following lesion in the
peripheral nervous system ~PNS) and central nervous
system (CNS), by the involvement of specific
membrane mechanisms and by interaction with
neurotrophic factors as revealed by in vitro studies ;
on neuronal cultures (Doherty P. et al., J.
Neurochem. 44:1259, 1985; Skaper S. et al., ~ -
Molecular Neurobiology, 3:173, 1989). ;
In particular, it has been reported that the
administration of gangliosides in vivo facilitates
nerve regeneration and functional recovery in the
PNS under pathological conditions: positive effects
have been described in models of traumatic
neuropathies (Ceccarelli B. et al., Adv. Exp. Med.
Biol. 71:275, Plenum Press, New York, 1976; Gorio A.
et al., Brain Res. 7:236, 1980; Gario A. et al.,
Neuroscience 8:417, 1983), metabolic neuropathies
(Norido F. et al., Exp. Neurol. 83:221, 1984) and
toxic neuropathies (Di Gregorio F. et al., Cancer
Chemother., Pharmacol. 26:31, 1990).
With regard to the CNS, positive effects have
been widely reported of recovery induced by
monosialoganglioside GMl in models of ischemia
(Cuello A.C. et al., Brain Res. 376:373, 1986;
Karpiak S.E. et al., CRC Critical Rev. in
Neurobiology, Vol. 5, Issue 3, 1990), traumatic
lesion (Toffano G. et al., Brain Res. 296:233, 1984)
and neuronotoxic lesion (Johnsson J., Dev. Brain
, . ,. . , . ,.,. .. , . .. . . ~ . .. , . ,., - ~ - - -, . . .
W092/20695 PCT/EPg1/~918
2~71~8
Res., 16:171, 1984) in various neuronal systems of
different animal species. It has recently been
discovered that gangliosides can inhibit the
translocation and activation of protein kinase C
S induced by glutamate (Vaccarino F. et al., Proc.
Nat. Acad. Sci. USA, 84:3707, 1987). This action is
very important in conditions of ischemic damage,
where there have been reports of a crucial role
played by excitatory amino acids, such as glutamate,
which trigger a cascade of events leading to
neuronal death. This mechanism could favor the
survival of neurons in the area around the lesion,
prevent retrograde degeneration, and accelerate the
reparative growth response to local trophic factors.
The results of experimental research have been
amply confirmed by those from the clinical use of
gangliosides. For over ten years gangliosides have
been used as therapeutic agents in almost all forms
of peripheral neuropathy, from those forms resulting
from mechanical damage to those caused by toxic
factors or deficiencies, from infectious and
inflammatory disorders to metabolic dysfunctions.
These drugs have proved to be equally efficacious in
mono and polyneuropathies, in sensory-motor
disorders and in pathologies affecting the autonomic
nervous system, such as in many neuropathies
affecting the cranial nerves, for instance Bell's
palsy, trigeminal neuralgia, and neuralgia caused by
herpes zoster. Gangliosides, and in particular the
monosialoganglioside, can be widely used in all ~ -
pathologies connected with acute lesions in the CNS
of a vascular or traumatic type and in the sequelae
of such pathologies (cerebral ischemia, cranial and
spinal trauma).
Their proven reparative activity in the CNS
also supports their use in chronic neurodegenerative
. .... .. , ., .. , ...... ., .. , . . . , . . - . - - . . ., , ,, . . .. . -
.. ~ . .. .. , . . : , .................. ... . . . . ..
~ .' " .: ' .: : . ~ , '
W092/20695 PCT/EP91/~9l8
20 87 1~8 4
pathologies, such as Parkinson's disease and
Alzheimer's disease. The fact that they are
"endocoids" (endogenous drugs) by nature, being
natural components of the neuronal membranes,
explains their excellent tolerability and the
absence, even in prolonged treatments with high
doses, of side effects which are so frequent in some
conventional therapies for peripheral neuropathies.
In general, suitable ganglioside mixtures, for
example a formulation of the following kind: GM~ ;
from 18% to 24%, GD~. from 36% to 44%, GDlb from 12%
to 18%, GT1b from 16% to 22%, or the single
ganglioside fractions, particularly the
monosialoganglioside GMl, present biological
activities such as those described. These
gangliosides, as suitable mixtures or single
fractions, in particular the monosialoganglioside
GM~, are extracted from mammal brains and it is
therefore necessary, given their particular
biological function and their therapeutic
application previously described with regard to the
peripheral and central nervous systems, to utilize
purification methods which guarantee a final product
which is absolutely pure and free from biological
and chemical contaminants.
It has long been known that it is possible to
extract, on a research level, mixtures of
gangliosides (Tettamanti et al., Biochim. e 8iophys. -
Acta, 296:160, 1973; Trams et al., Biochim. e
Biophys. Acta, 60:350, 1962: Bogoch et al., British
J. Pharm., 18:625, 1962; Wiegandt et al., Angew
Chem. 80:89, 1968; U.S. Patent No. 3,436,413; and
C.A. 61, 9851C, 9895d), but none of the aforesaid
methods was developed with a view to demonstrating
the elimination and destruction of components
- associated with non-conventional viruses. One
reason for this is that, at the time, such diseases,
. . . .
. : :
W092/2~95 2 0 8 715 8 PCT/EP91/00918
affecting the mammalian spe~ies to which the brains
used for extraction belonged, were as yet unknown.
Another reason is that no reagents were available
for the specific identification of potentially
dangerous components, whereas today such reagents
have been made available by specific methodologies
developed on the basis of newly-acquired knowledge
gleaned from the scientific evolution of molecular
biology techniques.
Sometimes situations of a pathological type can
arise wherein the pathogenic agent or agents cannot
be identified. One such pathological situation is
called bovine spongiform encephalopathy (BSE), first
reported in England in 1986 (Wells G. et al., Vet.
Record, 419, 1986). This name derives from the
spongy appearance of the brain tissue from afflicted ~-
animals. When sections of tissue are analyzed by
microscope, the main lesions are comprised by
extensive neuronal vacuoles.
All available evidence points to the fact that
BSE belongs to a group of degenerative
encephalopathies of the central nervous system which
are invariably fatal in outcome and are caused by a
group of non-conventional, infectious agents (Fraser
et al., Vet. Record 123:472, 1988: Hope et al.,
Nature 336:390, 1988). This group also includes
scrapie of sheep and goats, the chronic emaciating
disease which afflicts captive deer, infectious
encephalopathy of mink on mink farms, and two human
diseases; kuru and Creutzfeldt-Jacob disease. ~he
histopa-~ological lesions caused in the brain by
these diseases are similar in all cases and are
comparable to those caused by BSE. Many theories
have been put forward on the nature of these
etiological agents, which are neither bacteria nor
virus, are unlike any other kn~wn organism and are
therefore known as unconvention~l viruses. On -
"
.., ,.: . .. .. ....
.' ' . : . ., ,, ': , , : ' . ! . . . ~ . ' . . . : . . ' ' '
W092/20695 PCT/EP91/~918
2087i~8 6
account of their long incubation periods, running
from the moment of infection to the onset of
symptoms, these viruses are also known as "slow -I -
viruses".
Since the few cases observed in 1986, the
disease spread and has reached epidemic proportions
in Britain, affecting some 14,000 cattle and
increasing steadily by about 250-300 cases each
week. The infected cattle show no signs of disease
for several years ~the incubation period being 4-5
years), but once symptoms have appeared the animals
rapidly deteriorate and die.
An epidemiological study by the Central
Veterinary Laboratory-of the British Ministry of
Agriculture (Wilesmith et al., Vet. Record. 123:638,
1988) showed the source of infection to be animal
fodder made with the processed carcasses of other
ruminants, sold in the form of powdered meat or
bone. Since the encephalopathy can be transmitted
to a wide range of animal species, it seems
reasonable to assume that BSE is the result of
inSection by the etiological agent responsible for
6crapie, transmitted from sheep by means of these
contaminated foodstuffs (Norgan KL, Vet. Record
122:445, 1988).
on the basis o~ the results of this study, the
British government banned/ by an order which came
into forca on 18th July 1988, the sale and supply of
animal foodstuffs containing animal proteins derived
from ruminants.
The general opinion is that many factors have
contributed together to the sudden appearance of BSE
in Britain (Cherfas J., Science, Feb. 1990, 523).
Firstly, the number of sheep in Great 8ritain
increased rapidly in the late 70's and early 80's,
and with this the incidence of scrapie, an endemic
disease of sheep in Europe for over 250 years
. . .- .. ~ ., . ,, . ..................... : , . ..
. ~
~, . ~. ;. ' ' ~ ' ''':' . ,. ' ,
W092/20695 2 0 8 71~ 8 PCT/EP9t/~918
.
(Pattison et al., Vet. Record 90:465, 1972). At the
same time, in the wake of the petrol crisis,
factories producing animal fodder changed their
methods of processing carcasses to a lower-
temperature system which was probably less efficientin destroying the highly resistant scrapie agent.
All except one of the producers of these foodstuffs
abandoned the use of solvents such as benzene,
hexane and trichloroethylene, to remove excess fats
from soybean and bone meal. Perhaps most
significant of all was that the final stage of
heating of the products to remove the solvents was
coneequently left out: indeed this phase required
very high temperatures.
Moreover, government policy encouraged breeders
to produce more milk, and wean calves early by
feeding them protein-rich diets. These were often
of poor quality, since meal made from meat and bone
was cheaper than products made with soybean and fish
are surer sources of protein. Studies to find how
the disease is transmitted are fundamental to BSE
research. The most important aspect of these
experiments is that, by identifying the limits of
the inter-species barriers to transmission of the
pathogenic agent, it is possible to assess the ris~
of BSE infection to any one specie9. Fraser et al.
(Vet. Record, 123:472, 1988) demonstrated that the
disease could be passed from cattle to mice. They
inoculated extracts from the brains of cattle which
had died from BSE into the brains of mice which
subsequently developed the disease. Later, Barlow
et al. (Vet. Record, 3 Feb. 1990) transmitted the -
disease to mice by feeding them infected brains. It
was the first proof that BSE could be contracted by
3S eating infected material. No other tissue from
afflicted animals (spleen, spinal cord, lymphatic
W092/2069~o 8 7 ~ 5 8 PCT/EP91/00918
tissues, milk etc.) was able to produce the disease
in mice.
There is proof that scrapie can be transmitted
to lambs by their mothers, but so far no evidence
has come to light of possible vertical or horizontal
transmission of the etiological agent of BSE in
cattle. -
The agents which cause subacute infectious
encephalopathies are extremely resistant to standard
decontamination processes. Available data on this
aspect mostly originate from studies on the
inactivation of agents of scrapie and Creutzfeldt-
Jacob disease. The etiological agent of scrapie is
highly resistant to temperature change. When
exposed to temperatures of up to 80-C their
infectiousness is only slightly reduced; higher
temperatures however markedly reduce infectiousness
(Hunter et al., J. Gen. Nicrobiol. 37:251, 1964). A
small quantity of infectious "virus" sometimes
persists when suspensions of infected material are
heated to lOO-C for 1 hour or to 118-C for 10
minutes.
Recently, the need was felt to renew standards
of sterilizing these infectious agents under high
steam pressure in autoclaves. The current standaras
govern~ng autoclaving in the United States for the
decontamination of Creutzfeldt-Jacob disease involve
treatment at 132-C for 1 hour (Rosenberg et al.,
Annals of Neurology 19:75, 1986), and is based on
studies carried out on brain homogenates containing
scrapie or Creutzfeldt-Jacob agents (Brown et al.,
J. of Infectious Diseases 153:1145, 1986). In
Bxitain the current standard of autoclaving for
decontamination from Creutzfeldt-Jacob disease
involves treatment in an autoclave at 134-138-C for
18 minutes, on the basis of some studies including
one by Ximberlin (Ximberlin et al., Journal of ~-
.
.
W092/2~95 2 ~ 8 71~ 8 PCT/EP91/00918
9 ,.
Neurological Sciences 59:3S5, 1983). Unfortunately,
the bovine spongiform encephalopathy agents are very
resistant even to common chemical treatments, as
well as physical ones. Solvents such as benzene,
hexane, petrol and trichloroethylene have been used
as extraction solvents, but little is known of their
effects on infectivity. Only a small quantity of
data is available on the chemical inactivation of
infective agents, mainly because studies require
large numbers of animals and long observation times.
Concentrations of 0.3% - 2.5% of sodium hypochlorite
greatly reduced infectivity in the biological assays
used, but often did not completely eliminate it
(Walker et al., Am. J. Publ. Health 73:661, 1983).
Data regarding treatment with up to 0.2S N sodium
hydroxide are very variable; at concentrations of
over 1 N it appears however to be the most
efficacious chemical agent of all those studied.
Treatment with 6M-8N urea was also reported to be
highly variable.
The results of the studies on decontamination
thus show that, although most of the infectivity is
quickly destroyed by many of the different physical
and chemical processes, the existence of small
subpopulations of resistant infectious agents makes
sterilization of contaminated materials extremely
dif~icult in practice.
Once BSE had been identified as a "scrapie-
like" disease, important questions began to be asked --
on epidemiological and analytical levels, the latter ~ -
in particular being aimed at identifying the agent
associable with infectivity. However, all efforts
so far made to identify nucleic acids associated
with the etiological agent have been unfruitful.
The only component isolated, which is associated
unequivocally with the infective action, is a
W 0 92/20695 ~8 lo P~r/EP91/00918
sialoglycoprotein called scrapie prion protein
prpsc)
Genetic studies conducted on this protein
subses~uently provided some surprising information.
Some DNA probes synthesized according to the N
terminal seS~uence of the protein have made it
possible to show the presence of a chromosome gene -
in individual copy that exhibits the same
restriction pattern both in the brains of healthy
animals as in the brains of infected animals. This
gene, which is conserved even in very different
species, codes a protein called cellular prion
protein ~PrPC) with an apparent molecular weight of
33-35.0 kilodaltons (kd), which shows particularly
evident differences with respect to the PrPsG:
1) pr~C is susceptible to protease, while
prpSC is resistant. In particular, while prpC is
degraded completely by the enzyme proteinase K, prpSC
is hydrolyzed at the level of the N terminal for a
fragment of about 5 kd and gives rise to a protein
called PrP2,30. This form copurifies with the
$nfectivity and is the most abundant component that
is obtained in the preparations of infective
material.
2) Both prpC and PrPs are membrane proteins,
but while the first i5 solubilized by treatment with
detergents, the second tends to polymerize into 7 '' '
amyloid fibrous structures. Similar structures
(scrapie-associated fibrils, SAF) have been found in
in~ected brains and are peculiar of this type of
infection. The resistance of this infective protein
to inactivation is unusual: it is sensitive, for
example, to treatments with concentrated alkaline
solutions or to exposure to temperatures above 120C
and to their combinations or combinations with
different denaturing agents. Conse~uently, the only
diagnostic methods available for unes~uivocal
.
, . :. .. . . . . .. .. ... . . .. . ...
W092/20695 2 0 8 71~ 8 PCT/EP91/0~918
11
identificatio~ of these spongiform encephalopathies
are the verification of the presence of the SAF in
the infected cerebral tissues, extraction and
immunochemical identification of the protein PrP2~30,
methodologies applicable only during pathological ~ -
anatomy.
The SAF have been identified on infected bovine
brains, then the homolog of the PrPs' was isolated
and showed reactivity with a serum obtained against
mouse SAF. Further, the N terminal sequence of the
first 12 amino acids showed 100% homology with the
prpSC of sheep and a difference from that of the
mouse, hamster and man by a single insertion of
glycine. As soon as it was established that BSE is
a "scrapie-like" disease, some important questions
arose at the epide~iological and analytic level, the
latter particularly devoted to identifying the
protein associable with infectivity.
The unexpected cropping up of BSE and all the
aspects still to be explained on these neurological
disorders have caused a necessary consideration to
be given to the problem, especially by those ~ -
involved in the preparation of products that derive
from bovine material.
It could, in fact, not be enough to use, for
obtaining compounds or their mixtures oS
pharmaceutical interest, raw material certified for
food use. Consequently, it is necessary to develop
the process of production of the products in -
question by using extraction methodologies that
guarantee the elimination of the protein associated
with infectivity and the infectivity itself. It is
obvious that the process of extraction of the
infectively active fraction should, at the same
time, preserve the biological activity of the active -
principles desired as final product.
.
' .
..
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W092/20695 PCT/EP91/00918
2087 ~8 12
The other potentially dangerous protein at the
level of these preparations for man is the myelinic
basic protein (MBP).
It is a protein that in man and most
vertebrates has a molecular wei~ht (MW) of about
19.5 kd. It is present in three molecular forms in
human myelin and in six in that of the mouse, coding
by a single gene located on chromosome 18 and it
constitutes about 30% of the total myelinic protein.
Its exact topographical location is not yet certain.
It has been observed in the cytoplasm of
oligodendrocytes only at the moment of
myelinization. A protein considered identical is
present in the peripheral nervous system (protein
Pl), and the ability of peripheral myelin to induce
experimental allergic encephalomyelitis (EAE) in
laboratory animals is due to it. - '
Not all the molecule of MBP is encephalitogenic
but only a portion that varies from species to - ,
species: in the rabbit the encephalitogenic portion , ,
is amino acid fragment 64-73, in the Lewis rat 71-
85, in the guinea pig 113-121, in the SJL/J mouse ~- '
89-169. The EAE is a typical cell-dependent , ,
autoimmune disea9e; indeed, it is transferable from
one animal to another by infusion of the sensitized
T lymphocytes and not by infusion of serum. In this
case, the disease is supported by transfused
lymphocytes and not tho,se of the recipient. Another
cell-dependent autoimmune form is allergic
experimental neuritis (EAN). It too can be induced
in all species of higher vertebrates by inoculation '''
of crude homogenate of peripheral myelin in complete
Freund's adjuvant. It is considered that the
antigen mainly responsible for this autoimmunization
is protein P2, with an MW of 12.0 kd, present in the
peripheral nervous system.
. . .
W092/2~95 2 0 8 715 8 PCT/EP91/~918
13
Another contaminant to be considered in these
preparations is bovine genomic DNA. The possibility
of being able to produce, by recombinant DNA
technology, biologically active proteins, which can
be used as pharmaceutical agents, has made it
necessary ~o analyze the final products for the
presence of DNA residues, belonging to the cell -
where the desired protein is expressed. The
presence of DNA fragments in pharmaceutical
preparations to be used in man poses the problem of
the danger of an incorporation in the genome of
these fragments with a possible uncontrolled
transfer of genetic type information. Even though
it is not yet possible to obtain gan-gliosides by
recombinant DNA technology, it is necessary to apply
this type of control analytical technology to
extraction products that use raw material of animal
origin.
Finally, the gangliosides to be used in vitro ; ~;
and in vivo studies should be free from other
compounds such as asialogangliosides and
glycocerebrosides. These substances, if present in
high concentrations, can have important
immunological implications and can also lead to
erroneous experimental considerations.
The sudden onset of BSE and all the other
aspects still to be clarified on these neurological
disorders have caused necessary consideration to be
given to the probIem, especially by those involved
in the preparation of products deriving from bovine
material.
Earlier processes for preparation of
gangliosides, such as cited above, required the
product to be pharmaceutically acceptable, free from
those biological contaminants which were known at
the time to be potentially damaging to the health.
But clearly, the subsequent onset of the aforesaid
.:,
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W092/2~9~ PCT/EP91/00918
2 ~87 ~8 14
pathology in adult cattle has made it necessary to
obtain an active principle which, without losing the
aforesaid therapeutic properties, is characterized
by the assured absence of non-con~entional viral
agents, to be achieved by the use of specific
processes to guarantee the inactivation of these
non-conventional viral agents and the complete
elimination of infectivity, and to use specific
methodologies by which to identify such agents.
Indeed, it may not be enough to use raw material
which has been certified as suitable for
consumption, to obtain compounds or mixtures of the
same for pharmaceutical purposes. Obviously, the
onset of BSE must be assessed by taking into account
lS its biological action in vivo, which must be
considered as an example of verification of the
various phases of the process, but not as a summary
of the same. This analysis of the biological action
in vL~o is necessary since scientists are not yet in
agreement over associating the infection with
certain proteins such as PrP2730. Clearly, the
extraction process which eliminates infectious
activity must at the same time leave the biological
activity of the active principle intact, since this
i8 essential for its therapeutic use. (Ad hoc
working party on biotechnology/pharmacy: Validation
of virus removal and inactivation process.
Commission of the European Communities, March 1990).
Scientific research has produced, on the one hand,
methods which guarantee suitable mixtures of
gangliosides or their single fractions to be
obtained in forms free from protein, chemical and
biological contaminants, and on the other hand,
methods with demonstrated efficacy in destroying
infectivity associated with slow viruses, but no
method is known by which it is possible to obtain,
also on an industrial scale, the similarly unknown
,
'~`~ ~ : ' ' ' ' ' ' ' ' ' . . ' ' ' '
W092/2~95 2 0 8 71 ~ 8 PCT/EP91/00918
result of a product, as desired, pure,
pharmacologically active product, free from
infectivity associable with pathogenic agents
definable as slow viruses.
Brief Description of the Drawings
Figure l is a schematic diagram of the process
of the invention.
Figure 2 is a photograph showing results
obtained by the use of anti-Prp2,30 antibodies by the
Western Blot technique to analyze some samples taken
from intermediate passages of the process of the !
invention.
Figure 3 is a photograph showing results of the
analysis of bovine genomic DNA on some samples taken ~ -
from various passages of the process of the
invention.
Figure 4 is a photograph of the results
obtained by the use of anti-NBP antibodies in the
Western Blot technique to analyze some samples taken
from intermediate passages of the process of the
invention.
Figure S is a photograph of the results of -
silica gel chromatography analysis of the
ganglioaide mlxture prepared according to the
process of the invention.
Figure 6 is a photograph showing the biological
activity of the ganglioside mixture prepared
according to the invention.
Figure 7 is a photograph showing the results of
immunochemical analysis with anti-PrP2,30 antibodies
of samples taken from intermediate passages of the
process of the invention.
Detailed ~escri~tion of the Invention -
The aim of the present invention is to s~pply a
product characterized by the absence of slow viruses
. . .
W092/2~95 PCT/EP91/~918
20~7 ~8 16
obtained by an advantageous process which can be
applied to industrial production, and a process, the
innovativeness of which is founded on the suitable
sequencing of its various extraction phases. This
process which eliminates during its various phases
infectious contaminants associable with slow viruses
such as bovine spongiform encephalopathy, allows the
activity of the mixture, which represents the
therapeutic activity of the product itself, to
remain unaltered. The product deriving from this
process is constituted by a definite mixture of
gangliosides or single fractions obtained from
bovine brain or parts of the same.
The process according to the present invention
is composed, for the above reasons, of the following --
main phases:
a) subjecting bovine brain tissue to lipid
elimination in acetone;
b) suspension of an acetone precipitate in an
mixture of methylene chloride/methanol/sodium
hydroxide at a temperature of between 30-C and 35-C
for at least 3 hours to partition hydrophobic and
hydrophilic substances;
c) solubilization of the precipitate with
water/chloroform/methanol and a strong base, such as
an hydroxide of an alkali metal, pre~erably sodium
hydroxide (pH 12) by heating to between 38-C and
43-C for 4 to 8 hours;
- d) solubilization of the precipitate with a
strong base, such as an hydroxide of an alkali
metal, preferably sodium hydroxide lN, at room --
temperature for at least one hour; and
e) neutralization and dialysis of the
solution containing the ganglioside mixture through
a membrane with a MW cutoff of at least 10 kd.
Hereafter, for the purpose of illustration and
not limitation, examples are described of
W092/2~95 2 ~ ~ 7 t S ~ PCT/EP91/00918
17
preparations made from infected bovine brains where
the spongiform encephalopathy form was encountered
or from protein raw materials obtained from
uninfected bovine brains to which are added constant
amounts of infected material from the 263X scrapie
strain.
Materials and Methods
The bovine brains used in the process for
extraction of the ganglioside mixture showed, on
histological analysis, fibrils typical to tissues
belonging to materials from animals with the
infection.
' -
Pre~aration ExamDles
Exam~le l ~-
A diagram of the preparation process is shown
in Figure l.
lO00 grams of infected bovine brain, ground and
suspended in distilled water, were left in contact
with 3-lO liters of acetone (ratio 1:5 ~-
weight/volume) for about 3 hours at room temperature
under stirring. The solution was then centrifuged
at 6000 x g at a temperature of between 7-C and 4-C
until precipitation was complete. The solvent was
then eliminated and at least lO volumes of a mixture
of methylene chloride/methanol and a solution of
sodium hydro~ide (40:20:1.5) was added to the wet
powder placed in a suitable glass container and was
left again under magnetic stirring for at least
three hours at a temperature of between 30-C and
35-C. It was finally left to cool and then was
centrifuged ~or 20 minutes at 6000 x g at +lO-C.
The liquid phase was filtered through a filtering
funnel at a temperature of +4-C. Acetone (0.3-0.6
v/v) in the presence of a solution of calcium -
ehloride was added to the liquid, it was left under
. ~ . . ... . . . . .
W092/2~95 PCT/EP91/~918
08~ ~8 18
stirring for about 30 minutes and centrifuged at
6000 x g at +10-C. The precipitate (raw material 1)
was finally allowed to dry overnight and then for 5
hours in a high vacuum. Recovered raw material 1
was resuspended in a mixture of chloroform/methanol/
water in volume at a concentration of not less than
20 mg/ml. The pH was adjusted to around 12 with 5N
NaOH. The whole was heated to b,etween 38- and 43-C
from 4 to 8 hours and left under stirring. At the --
end, after being allowed to cool, it was neutralized
with 6N HC1 and at least one volume of a mixture of
chlorofor~/n-butanol/water (75:25:7) in volume was
added. It was stirred for 15 to 30 minutes and left
to stand for between 2 and 4 hours. Finally, the
lower organic phase was discarded, and after the
addition of sodium chloride, the product was
precipitated from the remaining aqueous phases with
acetone (2-5 v/v), they were stirred for about 30 ,
minutes and centrifuged for 20 minutes at 6000 x g
at +15-C (raw material 2).
The product was dried in a high vacuum,
resuspended in absolute methanol at a concentration
of between 10 and 50 mg/ml and then kept hot for
about 2 hours while stirring the solution from time
to time. The suspension was then qu~ckly
centrifuged at 6000 x g and the supernatant was
placed in a freezer for about 2 hours. The
opalescent white solution was then centrifuged at
O-C at 600 x g and the precipitate was dried in a
high vacuum. The product was gathered in lN sodium
hydroxide at a concentration between 50 and 300
mg/ml and left in contact with the solution for at
least 1 hour at room temperature. Finally, the pH
of the suspension was brought to an approximate pH
value of 9 and dialysed with a membrane having a MM
cutoff of at least 10 kd against a suitable volume
of distilled water. An amount of 2.5-3 gr/liter of
, - :
. . ,.. :: , : : .. , - .
W092/20695 2 0 8 71~ 8 PCT/EP91/~918
19
sodium chloride was added to the suspension which
was then precipitated with at least 9 volumes of
acetone. The suspension was centrifuged at +5 J C at
6000 x g, and then dried in a high vacuum (finished
product). The sample was taken up in 10 mM of
phosphate buffer pH 7.2 and sterilized at +121-C for
30 minutes (finished, sterilized product).
. .
Evaluation of Process:
As explained above, an important aspect of the
process of the invention is the provision of a
ganglioside product which is free of undesirable
contaminants, particularly free of non-conventional
viruses. To evaluate the process, samples at
various stages of the procedure were tested for
possible contamination.
The procedure and results for
biological/clinical testing are as follows: ~ -
:
~ioloaical test for scraDie:
The animals used in these experiments were
Golden Syrian hamsters (LVG/Lak). Tests for
infection were carried out on groups of four weaned,
female animals which had received intracerebral -
~i.c.) inoculation with 0.05 ml of the samples
diluted ten times in sterile PBS. The intracerebral
inoculations were effected by trained staff using
disposable glass syringes with 26G, 3/8-inch sterile
needles. -
The final, sterilized product, concentrated 20
times, was used entirely as follows:
4.0 ml injected intracerebrally in 40
animals. -:
3.0 ml diluted 1:20 and injected,
undiluted, intracerebrally in 50 animals and
i.p. in 22 animals. The volume injected i.p.
was 2.5 ml. ,
.
':
.. . . . . .
~. . ,, , . .. ,,, ; . . .
W092~20695 PCT/EP91/00918
2087 ~5~ 20 -
The animals were examined twice a week or more,
for a period of 12 months, for the onset of the
characteristic neurological, clinical symptoms. The
onset of early symptoms in each animal was recorded,
and the animals were sacrificed when the disease was
well established. ~heir brains were divided in two
halves, one ~ixed in 10% formalin and the other
preserved at -70-C. Pathological diagnosis was made
in all animals which died of suspect causes and
those which had shown signs of neurological
disorders. At the end of the observation time, all
surviving animals were sacrificed and pathological
assessment was made of their brains.
The infective titer was calculated at the
"final end point" according to the method of Reed
and Munch, and is expressed as log LDsO/ml.
The samples tested were the following,
utilizing names for the products as noted in Figure
1:
All samples were resuspended in sterile PBS in
the following volumes, so calculated as to ensure a
homogeneous titer per volume compared to the 16.7%
w/v homogenate as the starting material:
Powdered brain ml 2.0 brain homogenate
16.7% w/v,
undiluted
Raw material l ml 5.4 undiluted
Raw material 2 ml 9.7 undiluted
Finished product ml 14.4 undiluted
Finished, sterile ml 7.0 concsntrated 20
product times ~ -
Results of the biological/clinical tests are
set forth in Table l. -~
, ' . ' ' - . '
WO 92/20695 2 0 8 715 8 PCI/EP91/00918
21 : .
~ _ ~ N ~ I o-
S ai N N . . . O ~ ~
~ ~ ~ ' ~ ~ 1~ - ,'' ,''''' , . .'
1 0 ~,~_ N N N O . _ _ ~
I5 59~ I1~ ~r ~
2 0 v ¦ ~ 2 0 j r N
C~ ~ ~ IS l~ lr ~
2 6 ~ e o o N ~ ~N N
~ ~ . . ~0 c~O~ ~ ~0 r~ r~ ~ ' .
~ o . ~r ~ ~ q~ ~r ~ ,'~
_ I N ~ O ~ C ~ O ~ ~ ~
. .
SUBSTITUTE SHEET - ~
:
W092/2069~ PCT/EP91/~918
20 87 ~58 2~ ~
All animals which had shown clinical signs of scrapie and
all animals still surviving at the end of the one-year
monitoring time were included into the study. Animals which
had been put down because of accidents and/or poor health,
and those which had died of causes unrelated to scrapie or
which had been killed for meat, not having shown any
clinical signs of the disease, were not included.
The "sample" column reports the number of animals injected
at the beginning of the experiment and the cage number,
which distinguishes the different samples and dilutions. The
"sick" column reports the number of animals which showed
clinical signs of scrapie/number of animals injected minus
number of animals which died of causes unrelated to scrapie.
~. . -
.,' ' .
. :
SUBSTITUTE SHEET
.: . '
.. . . .
: ; :
W092/20695 2 0 8 71 5 8 PCT/EP91/~918
23 -
Additional Evaluations:
Determination of scrapie protein PrP is made by
polyclonal antibodies specific for the purified
protein analog from murine brain and which cross-
react with the scrapie PrP of bovine origin. The
Western Blot method was used for the determination.
The presence of the scrapie PrP was evaluated by
comparison with the standards for proteins with
various molecular weights.
Determination of the NBP protein was made by
polyclonal antibodies specific for the purified
protein analog from bovine cerebral tissue. The
method used for the determination is that of the
~estern Blot. The presence of the MBP was evaluated
by comparison with the standard of proteins with
various molecular weights.
Determination of the bovine genomic DNA was
performed by the DOT BLOT technique on samples taken
- at different phases of the processing according to :
the method known to those s~illed in the art. To
consider the sample valid, the presence of spots
should not be noted in the depositions of the -
heterologous DNA. The absence of spots in the - -
samples examined in the radioautography shows the
absence of bovine genomic DNA.
Figure 2 shows the resuits determined by ;
immunochemlcal analysis with anti-PrP2730 antibodies
of samples coming from some intermediate stages of
the ganglioside preparation and purification '~
process. The num~er codes of the analyzed samples
corresponds to the numbers in parentheses given in
the purification diagram of Figure 1.
Lane 1: code 1 sample
Lane 2: code 2 sample
Lane 3: code 3 sample
Lane 4: code 4 sample -
Lane 5: code 5 sample
'
~i;UBST~TUTE SHEET
W092t20695 PCT/~Pg1/~918
2087 ~ 8 2
Lane 6: finished product
Lane 7: protein with standard MW (downward the
kd's are as follows: 97~4, 66.2, 4~.0,
31.0, 21.5)
Bands with asterisk refer to nonspecific cross-
reactions attributable to the system of
amplification of the assay.
Figure 3 shows the analysis of bovine genomic
DNA by the DOT BLOT technique on samples taken from
various passages of the process.
Intersection of letters with numbers indicates:
Standard curves
lA-7A bovine DNA (l mg)
lB-7B bovine DNA (l ng)-
lC-7C bovine DNA (l00 pg)
lD-7D bovine DNA (l0 pg)
lE-7E bovine DNA (l pg)
lF-7F bovine DNA (0 pg)
Samples examined
20 2A-2F code l sample ~ -
2B code 2 sample
2C code 3 sample '
2D code 5 sample
2E code 6 sample
25 3A crude 3 sample
3B finished product
3C finished, sterilized product
The points of the standard curve and the
analyzed samples are given in duplicate.
Figure 4 reports results of immunochemical
analysis with anti-MBP antibodies of samples coming
from some intermediate passages of the preparation
and purification process. The code of the samples
corresponds to the numerals indicated in the
purification diagram of Figure l.
Lane l: code l sample
Lane 2: code 2 sample
SUBSTITUTE S~EET
.i ,. . ` `, .. ; .,.`, ..... ... . . ,. . . . . .. - . . .. ' . . ` . ... ` -
W092/20695 2 0 8 71 5 ~ PCT/EP91/~918
Lane 3: code 3 sample
Lane 4: code 4 sample -
Lane 5: code 5 sample (raw material 1)
Lane 6: crude 2 sample
Lane 7: code 6 sample
Lane 8: crude 3 code sample
Lane 9: finished product
Lane 10: finished, sterilized product
The various forms of MBP recognized by the
polyclonal antibodies used are in parentheses.
Figure 5 shows the results of chromatography on
silica gel of the following samples (also according
to the process outlined in Figure 1):
Lane 1: finished, sterilized product
Lane 2: finished product ,
Lane 3: standard of trisialoganglioside GTlb
Lane 4: standard of disialoganglioside GDlb
Lane 5: standard of disialoganglioside GDl,
Lane 6: standard of monosialoganglioside GMl
Figure 6 is an example photograph to show the
biological activity of the ganglioside mixture
prepared according to the inven~ions (the arrows
indicate the sprouting) on a peripheral nerve.
Figure 7 shows results of immunochemical
analysis w~th anti-PrP2730 antibodies of samples -
taXen from some intermediate passages of ganglioside -
purification and preparation processes. The code of
the analyzed samples corresponds to the numerals
indicated in the purification diagram of Figure 1.
Lane 1: solution of raw material 1 to which has
been added 1.5 micrograms/ml of PrP
Lane 2: raw material 2 code sample
Lane 3: code 6 sample
La~e 4: raw material 3 code sample
Lane 5: finished product
Lane 6: finished, sterilized product
.
~;UBSTITUTE SHFET
W092/2~95 PCT/EP91/~918
20~ 8 2~
Example 2
Preparation of a clarified homo~enate from infected
brains
Four hamster brains, infected with the 263 X
scrapie strain, corresponding to a net weight of 3.9
g, were homogenized in 10 ml of distilled water.
The volume was then brought to 15.5 ml in order to
obtain a homogenate of 25% w/v. The suspension was
centrifuged for 40 minutes at 1800 x g at 4-C: 7.5
ml of supernatant were recovered, divided into
aliquots and kept at -70-C until use.
pre~aration of the samDles
5 ml of the infected homogenate, 120 ml of
methylene chloride/methanol 2:1 in volume and 0.71
ml of 5.7 N sodium hydroxide were added to 10 g of ~ -
acetone powder of bovine brains. This addition was
made in order to keep the total volume of the
aqueous phase in the solvent constant and to obtain
the most suitable operative conditions. The final ~ -
titer of the starting solution was 1% w/v. The
suspension was magnetically.stirred for 3 hours at a
temperature of 33-C. It was then cooled and
centrifuged for 20 minutes at 6000 x g at 10-C. The
liquid phase was filtered through a Gooch funnel
(pore size No. 3) at a temperature of +4'C to avoid
evaporation of the solvents; at the end of this
stage 78 ml of liquid phase were recovered. A 2 ml
aliquot was retained for biological assays. To both -
aliquots acetone was added in the presence of a
solution of calcium chloride, and after being
magneticalIy stirred for about 30 minutes at room
temper~ture, the samples were centrifuged for 10
minutesiat 6000 x g at 10-C. The precipitate (raw
material 1) was dried in a hood overnight and then
for 2 hours in high vacuum. One aliquot was kept at
-70- for the purposes of biological assay.
~' ' ' -
SUBSTITUTE SHEET :: -
:
W092/2069~ 2 0 8 71~ 8 PCT/EP91/~918
2~
The 925 mg of raw material 1 recovered from the
reaction container were resuspended in 18.5 ml of a
mixture of chloroform/methanol/water and 0.74 ml of
the homogenate to obtain a titer of 1% w/v. The pH
was adjusted to an approximate value of 12 (assessed
with a litmus paper) adding 5N NaOH, and the mixture
was magnetically stirred at 40 C for 6 hours. After
cooling to room temperature, the solution was ;
neutralized with 6N HC1 and an aliquot of 250 ~l was -
retained for the purposes of biological assay.
Both aliquots were treated with at least 1
volume of a mixture of chloroform/n-butanol/water
(75:25:7) in volume, and after stirring for 15
minutes they were left to stand for 4 hours.
Finally, the lower organic phases were discarded.
Sodium chloride is added and then the product is
precipitated from the remaining a~ueous phases with
acetone (2-5 v/v) and, after magnetic stirring at
room temperature for about 30 minutes, they were
centrifuged for 10 minutes at 6000 x g at 15-C ~raw ;
material 2).
This product was dried in high vacuum and the
aliguot set aside for biological assay was preserved
at -70-C. The remaining material was resuspended in
6.5 ml of absolute methanol and left at 50'C for 2
hours and stirred from time to time. The suspension
was quickly centrifuged at 6000 x g and the
supernatant placed in a freezer at -20-C for 2
hours. The cold, white, opalescent solution was
then centrifuged at 0-C at 6000 x g for 5 minutes
and the precipitate was dried in high vacuum. The
yield at this point was 6 mg of product. This was
then resuspended in 500 ~l of sodium hydroxide, 460
~l of distilled water and 40 ~l of homogenate and
left in contact with this solution for 1 hour at
room temperature. Finally, the pH of the suspension
was adjusted to an approximate value of 9 (assessed
:
SUE~STITUTE SHEET
. ..... ... ... .. , .. . . .. . . . . ... .. ,, , ............ . . ~ ~ .. . ~ .
. ~ . . . . . . . .
W092/20695 PCT/EP91/00918
~ 0 8~ 2s
with litmus paper) and dialysed with a membrane
having a MW cut off of at least 10 kd for 4 hours
against 20000 volumes of distilled water. The final
volume after dialysis was 980 ~l. It was divided
into two aliquots of 500 ~l and 480 ~l respectively;
to the first was added 2.5-3 gr/l of sodium chloride
and it was then precipitated with at least 9 volumes
of acetone and then centrifuged for 10 minutes at
6000 x g at 5-C. This sample was dried in high
vacuum (final product).
To the second aliguot was added 20 ~1 of
homogenate and 50 ~l of PBS lOx and it was
sterilized at 121-C for 30 minutes (final,
sterilized product).
Evaluation of the Process:
As described above for Example 1, samples taken
from the various stages of the process were tested
for potential presence of contaminants.
The samples tested and results are as follows:
A11 of the samples were gathered in sterile PBS
in the following volumes, so calculated as to ensure
a homogeneous titer per volume compared to the 1%
homogenate w/v as the starting material:
SUBSTI~U~ $HEI~r : ,:
.
W092/20695 2 0 8 71 ~ 8 PCT/EPgl/00918 ~ .
Brain homogenate undiluted
susp .
25% w/v diluted to 1~
Raw material 1 ml 3.2 undiluted
Raw material 2 ml 1.0 undiluted
Finished product ml 0.5 undiluted
Finished, sterilized ml 0.5 undiluted
product
.
Table 2 reports the results,obtained by
biological assay.
". , ' .
.: . . .
.
SuB~ JTE ~;tlEEl~
, . ~ . , . . - ..... - .
. . , . . . ~ . . . ..
. . ... , ~ . ., ~ . . . ..
WO 92/20695 PCI'/EP91/00918
2087 ~5~
~,. ~ ~ ~ ~ ~ ~ o. ,
5 ~_
' 1~' . . ~
1 O _ Y O O O O O N
IG U ~ ~'' lo 1 ~ ~ ~
_" _ ~ o ~ ~ ~ ~ , ~ '
2 0 ~ x ~ Nl N N l l l ~
Q~ ~ ~ ~ ~ ~ ~
. ~ o~ ~ ~ ~ ~ ~,.
as ' ' Jc . .
o ~R ~ o o or o
: ~ ~ ;' ~ . ~ ~: . ~ ~ .
: ~ ~ : ~ i S~ ~ ~o ~
~ :~ : . ~: I , ,~.. '
;; ~ ~ ~ o o 1~ ~ o o
3= ~ t~ o b
;SUBSTITUTE SHEET ~ ~
:, .` ', .;' ! ' ", " ; '
W092/2~95 2 0 8 71~ ~ PCT/EP91/~918
All animals which had shown clinical signs of scrapie and
all animals still surviving at the end of the one year
monitoring time were included into the study. Animals which
had been put down because of accidents and/or poor health,
and those which had died of causes unrelated to scrapie or
which had been killed for meat, not having shown any
clinical signs of the disease, were not included.
The "sample" column reports the number of animals injected
at the beginning of the experiment and the cage number,
which distinguishes the different samples and dilutions. The
"sick" column reports the number of animals which showed
clinical signs of scrapie/number of animals injected minus
number of animals which died of causes unrelated to scrapie.
SUBSTITUTE SHEFT
- . . ` . . . ~ ~. . -
.. . . . .
W092/20695 PCT/EP91/00918
2087 ~8 3~
Example 3
Biolo~ical activity of ~anglioside mixture
A series of experiments was carried out in
vitro in order to verify whether the ganglioside
mixture (obtained according to the aforesaid
process) possessed any biological activity
predictive of therapeutic application to treat
pathologies of the peripheral nervous system (PNS)
and of the central nervous system (CNS). In
particular, the activity of gangliosides was tested
in vitro to assess neurite formation in cultures of
neuroblastoma cells (N2A). These cells, as described
in literature tDenis - Donini et al., Neuronal
Development, part II, 323:348 - Academic Press, NY
1980; Leon et al., Dev. Neurosci. 5:108, 1982) may
induce, in certain conditions, the expression of
various functions characteristic of mature neurons,
thus allowing a qualitative and quantitative
analysis of biochemical parameters correlated with
each stage of development.
Therefore, the assessments made in this model
(% of cells with neurite formation, neurite length ~
and relative branching) are valid instruments when --
investigating the possible therapeutic application
of a drug in functional recovery of the nervous
system.
; .
Materials and Methods
Cell çultures
Mouse C 1300 cells, clone N2A, supplied by
America Ce}l Type Collection (Bethesda, Maryland),
were plated at a concentration of lO,000 cells per
well (24-castor) in the presence of Dulbecco's
modified Eagle medium (DMEM) containing P/G (lO0
U.penicillin/ml) and 10% fetal calf serum (FCS from
Seromed, batch 4-C04).
SUBSTITUTE SHEET
.-
, . .. . ....... ., . . .. . . , . ~ . . . . . . . . . . . . . . .
~ .
. . . ~ ~ . - .
W092/2~95 2 V ~ 715 8 PCT/EP91/~918
33
The next day the culture medium was changed
with the same volume of fresh medium containing
gangliosides (see further). The cultures were kept
at 37C in 5% Co2 in a humidified atmosphere (Haerus
incubator). The cultures were then fixed with 2%
- glutaraldehyde at the appointed time (24 hours
later).
Pre~aration of the test solutions of the product
The ganglioside mixture (3 different batches,
Nos. 1-2-3) was dissolved in chloroform/methanol
2:1, dried in a stream of nitrogen, resuspended in
DMEM + P/G + 10% FCS until the desired
concentrations were reached.
Concentrations examined: l x lO-~M; 5 x 10-~
and 1 x 10-5M.
. Four different experiments were carried out as
follows:
- 3 experiments to assess the effect of
ganglioside mixture (batch Nos. 1 and 2)
at a concentration of 1 x 10-~M
- 1 experiment to assess the dose-response
effect of different concentrations (1 x 10-
', 5 x 10-5 and 1 x 10-5M3 of the
ganglioside mixture under examination ~;
(batch No. 3).
Process
The medium was drawn off the wells and
substituted with 350 ~l of D~EM ~ P/G I 10% FCS _
product under examination (at the aforesaid
concentrations).
The control cultures were treated in the same
way, without the addition of the ganglioside
mixture. The cultures were then kept in an ~
; incubator for 24 hours, after which the cells were -
-
SUBSTITUTE SI~EET
~: . ", . : .
:.' ' ~' . ', ,' . ~, ' ~ , . '':
W092/2~95 PCT/EPgl/~918
~o~7~ 34
fixed with 2% glutaraldehyde and observed under a
microscope.
Parameters
Morphological examination set out to assess:
- the percentage (%) of ce}ls with neurites
- length of neurites and relative branching.
Results
As reported in Table 3, it is clear that the
products under examination are efficacious (1 x lO-
~M) in inducing the formation of neurites in N2A
cells.
The effect of the ganglioside mixture is dose-
dependent with maximum efficacy at the dose of
1 x 10-~M (Tab. 4).
Tab~e 3
Effect of ganglioside mixture (batches 1, 2) on : i .
neurite formation in mouse neuroblastoma N2A cells.
All products were added to the cells at a final
concentration of 1 x 10-'M, morpho}ogical evaluations
were made 24 hours later.
.. _.. _ , .
Product _ % of cells with neuri~es
_ 1st exD. 2nd exp. 3rd exD.
Control 2 + 1 1 + 1 2 + 2 ~
GA (1) 29 + 6 2 7 + 6 2 4 + 5 .
GA ( 2 ) 2 5 + 4 2 5 + 2 2 5 + 5
. ,: ,
(batch No in brackets)
. .
. ' - '
' .
. ' ' '
~asrlT~TE ~;HE~
` ' ' '' ,''','"' .
W092/20695 2 0 8 7 1 ~ 8 PCT/EP91/00918
3S
Table 4
Effect of different concentrations of
ganglioside mixture (batch 3) on neurite formation
in mouse neuroblastoma N2A cells; dose-effect
response.
Product concentration % cells with
neurites
.. . _ .. ..
Control 3 + 2
GA (3) 1 x 10-4 35 + 6
GA (3) 5 x 105 17 + 5
GA (3) 1 x 10-~ 4 + 2 -:~
(batch No. in brackets)
.:
Statistical assessment of data on the
biological activity in vitro indicates that there is . .
no significant difference between the various
batohec o~ ganglioside nixture (Table 5).
.
s
. ~ .
:
'
:~ 8~BS'flTlJ~ SHI~ET
W092/20695 PCT/EP91J00918
2087~8 3~
'
50,000
u~ . . ..
.~ 40,000
~ 30,000
~20,000 _
o10,000, '
OIP , ,:
0000 _ .:
'~. ~ ",.
C 1 2
Sample ~-
.~" ~-.,
--
Table 5 :~
Overall statistical assessment of data obtained
with batches 1 and 2. The values reported are the
means of 9 independent tests ~ S.D. ::
Moreover, morphological assessment of the
neurites shows that the cells treated with .-
ganglioside ~ixture present long, notably branched
neurites (i.e. marked~branchingj.
, ~ ~ .. -
- ......
. ..
: , ~ : .:
~ : ...
UBSriTU rE SHEi~
W092/20695 2 0 8 71~ 8 PCT/EPg1/~918
Conclusions
The aforesaid observations, therefore, affirm
that the ganglioside mixture under examination has a
biological activity, indeed, the product obtained,
~y a process which guarantees its particular
- characteristics, can induce neurite formation in N2A
cells. This fact indicates that the product is
efficacious in repair phenomena of the peripheral
and central nervous systems. The mixture of
gangliosides, obtained as described and free from
contaminants associated with potentially dangerous
non-conventional viruses, can also be used for the
preparation of individual components or the
ganglioside mixture, such as monosialoganglioside
GM1.
In view of the pharmacological properties
described above, the ganglioside mixture can be
generally used as a drug in numerous pathologies
(with various etiopathogenic causes) in both the
peripheral and central nervous systems. Specific
conditions which can be trea*ed are: retrobulbar
optic neuritis, paralysis of the oculomotor nerves,
trigeminal neuralgia, paralysis of the facial nerve
and Bell's palsy, Garcin's syndrome, radiculitis,
traumatic lesions of the peripheral nerves, diabetic
and alcoholic polyneuritis, obstetrical paralysis,
paralytic sciatica, motor neuron diseases,
amyotrophic lateral sclerosis, myelopathic muscular
atrophy, progressive bulbar paralysis, myasthenia
gravis and Lambert Eaton's syndrome, muscular
dystrophy, impairments in synaptic nerve
transmission in the CNS and PNS, consciousness
de~iciencies such as confusion, concussion,
thrombosis, cerebral embolism, cerebral and spinal
trauma.
Administration is usually by injection,
intramuscular, subcutaneous or intravenous, or by
~l~B~TITWTE ~
. ... .. ., . , . . ~ .. - .~ . . . . - , ~ ....... .. .. .. . . .
.~ . ~... , ... .. .. , .. " ,., . .. .- . . .. ..
W092/20695 PCT/EP91/~918
2o87~58 3~ ;
transdermal, pulmonary or oral routes, preferably in
suitably buffered a~ueous solutions. Safe storage
of the pharmaceutical can be ensured by preparing it
in the form of vials containing solutions of the
product, possibly together with other auxillary
ingredients, as indicated in the examples of
pharmaceutical preparations reported hereafter. For
the therapeutic, or possibly also preventive
application by the aforesaid parenteral route, the
dosage varies preferably between 10 mg and lOO
mg/day of active substance.
For purely descriptive and not limitative
purposes, the following are examples of
pharmaceutical compositions made according to the
present invention.
.
Exam~le 1
One vial is composed as follows: ;
Active ComDonent
- Gangliosides as sodium saltsl0.0 mg
_ monosialotetrahexosylganglioside (GMl)
- disialotetrahexosylganglioside (GDl.) - -
- disialotetrahexosylganglioside (GDlb)
- trisialotetrahexosylganglioside (G~lb)
Other com~onents
- dibasic sodium phosphate 12 H2O 6.0 mg
- monobasic sodium phosphate 2 H20 0.5 mg
- sodium chloride 16.0 mg
- water for injection to 2.0 ml
.. ,~ ~ .. . .
Exam~le 2
One vial is composed as follows:
Active com~onent
- Gangliiosides as sodium salts20.0 mg
- monosialotetrahexosylganglioside (GMl)
- disialotetrahexosylganglioside (GDl,) -~ -
- disialotetrahexosylganglioside (GD~b)
':
$UBSTIT~JTE SHEET -
;-- . . . . .. ':
. ., . . ..... ~ ,. . ...... . - . . . . ... ..
... : .. . ,: . . ` , . .. . , ,, . ,., . ~. , - . . . .
.... . . . ... . .. ... . . . . ... .
W092/20695 2 0 ~ 715 8 PCTtEP91/00918
39
- trisialotetrahexosylganglioside (GTlb)
Other comDonents
- dibasic sodium phosphate 12 H2O 6.0 mg
- monobasic sodium phosphate 2 ~2 0.5 mg
- sodium chloride 16.0 mg
- water for injection to 2.0 ml
Exam~le 3 , : -
One vial is composed as follows:
Active com~onent
- Gangliosides as sodium salts100.0 mg
- monosialotetrahexosylganglioside (GM1) .
- disialotetrahexosylganglioside (GDl,) , . '
- disialotetrahexosylganglioside (GD~b)
- trisialotetrahexosylganglioside (GT~b) . :
15 Other comDonents
- dibasic sodium phosphate 12 H2O 12.0 mg
- monobasic sodium phosphate 2 H2O 1.0 mg
- sodium chloride 32.0 mg
- water for injection to 4.0 ml
~ . .
The invention being thus described, it,will be
obvious that the same may be varied in many ways. ,'
Such variations are not to be regarded as a
departure ~rom the spixit and scope o~ the '
invention, and all such modi~ications,as would be
obvious to,one skilled in the art are intended to be
included within th- sFope oi' the ~ollowLng claias.
':
':
` ' ' ~ .
.
:: BUBSTITU~E SHEE~
