Note: Descriptions are shown in the official language in which they were submitted.
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PCT996
Use of aQ expressed juice, digestive juice or extract from flesh-eating plants
for the inhibition of protein kinases
The invention relates to the inhibition of protein kinases through the use of
an expressed juice,
digestive juice or extract from flesh-eating plants
Recent research in the field of drug active substances has been concentrated
on the field of
drugs produced on a natural basis. Besides the surprisingly great variety of
active substances
that can be found in plants and naturally occurring microorganisms, drugs
produced on a
natural basis often have the surprising advantage of better tolerance compared
to synthetically
produced active substances, which often provoke severe, undesired side-
effects. The flesh-
eating plants are an interesting example of a source for natural active
substances. Thus for
example the expressed juice of certain flesh-eating plants is used for the
control of malignant
diseases such as cancer, Crohn's disease, ulcerative colitis, herpes and aids.
This is for
example stated in EP-A-249465, EP-A-219295 and DE-A-2920631.
At the same time, there is a whole range of diseases in which the conventional
medication with
synthetic active substances shows practically no result. Gastric ulcers are an
example of such
a disease, for the control of which costly chemotherapeutic treatments are now
given. The
therapies currently most commonly used at present are the modified triplet
therapies. These
therapies are for example described in the technical journal "hunch. med.
WSCHR., 140
(1998), No.7, pp 90 to 93" and in "Deutsches Arztemagazin No.51/52, December
1997, der
Kassenarzt" on pages 31 to 34. These therapies have however the disadvantage
that, like
comparable chemotherapies in cancerous diseases, they are accompanied with
severe side-
effects.
Hence the purpose of the present invention is to provide an active substance
or an active
substance combination with which it is possible to control diseases which
respond to the
inhibition of protein kinases, with as far as possible no occun-ence of side-
effects.
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This purpose is achieved through the use of an expressed juice, digestive
juice or extract of
flesh-eating plants for the inhibition of protein kinases. These are also
referred to below
collectively as juices.
Protein kinases play a central role in the growth, division and
differentiation of cells. Growth
signals, which in the form of growth factors bind to specific recognition
structures (receptors)
on the outer side of the cell membrane, are transmitted into the cell nucleus
via various kinase
cascades, and there lead to the modification of transcription signals. As a
consequence of this,
specific genes become activated or the transcription of other genes is
stopped.
Surprisingly, it has now been found that the expressed juice, an extract or
the digestive juice
from flesh-eating plants makes inhibition of protein kinases possible,
simultaneously making it
possible to control various diseases, previously treatable only with very
great difficulty.
In particular, it has been found that the juices of flesh-eating plants show
effects which
resemble those of conventional antibiotics, but with the occurrence of
markedly fewer side-
effects. The juices of flesh-eating plants show action against gram-positive
and gram-negative
bacteria, fungi and other germs.
In particular, the juices used according to the invention are suitable for the
control of
plasmodia, chlamydia, trypansomes, phototropic bacteria, staphylococci,
streptococci,
pneumococci, klebsiella, candida, mucor, aspergillus and bacterium pyocyaneus.
Presumably
the aforesaid germs, bacteria and fungi are inactivated by inhibition of the
protein kinases. In
particular, the expressed juice, an extract or the digestive juice of flesh-
eating plants are
suitable for the control of helicobacter pylori, one of the conunonest causes
of gastric ulcers.
By series of experiments, it was further confirmed that inhibition of the
following germs, fungi
and bacteria can be achieved by means of the juice of flesh-eating plants:
Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus aureus,
Streptococcus pneum-
oniae, Escherichia coli, Staphylococcus epidermis, group B Streptococci
(haemolysing),
Streptococcus pyogenes (A), Candida albicans, Aspergillus fumigatus,
Pneumococcus
mucosus, Bact. pyocyaneus, Bact. Proteus vulgaris, Klebsiella oraeanae,
Candida mycod.,
Candida tropicaus and Mucor racemosus.
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The obtention of an expressed juice, digestive juice or extract of flesh-
eating plants, and also
preferred presentations, are discussed below.
The flesh-eating plant can be any flesh-eating plant. Preferably, it belongs
one of the genera
Heliamphora, Sarracenia, Darlingtonia, Cephalotus or Nephentes. Also preferred
are the
flesh-eating plants of the Drosera family, especially Dionaea muscipula, the
Venus flytrap.
The expressed juice, an extract or the digestive juice of flesh-eating plants
can be used.
The use of the digestive juice has the advantage that the plants do not have
to be destroyed for
its obtention, since the digestive juice can be tapped off. Here a significant
increase in the
yield can be achieved by constant feeding of the plants. The flesh-eating
plants are usually fed
with meat fragments of the same tissue over a long period. This also has the
result that the
composition of the digestive juice is stabilised, i. e. that the individual
components of the
digestive juice in the same type of plants are present in similar proportions.
Without feeding,
this is not the case, so that the active substances are present in different
concentrations from
plant to plant, and hence the therapeutic effect only leads to a therapeutic
result by mixing the
digestive juices of several plants. In order to increase the quantity of
digestive secretions
which are supplied by the plants, a stimulation of the secretion can be
performed. For this the
spraying of the plants with uric acid has been found to be particularly
suitable, the remaining
residues of uric acid showing no adverse effects. Further suitable agents for
stimulation of the
secretion are bactopeptones, kalifora, glutamic acid, yeast extract, peptones,
taurine, proline
and asparagine. The removal of the digestive juice is conveniently effected by
pipetting off or
by making small puncture wounds on the inner side of folds, and subsequent
collection of the
secretion in micropipettes.
However, according to the invention the expressed juice can also be used. The
expressed
juice is advantageously produced using fresh plants, i.e. plants in which no
enzymatically
triggered wilting process has yet begun. Usually, the harvested plants are
first washed, which
removes extraneous components. The cleaned plants are then chopped, grated or
mashed, and
then pressed in the wet state. Advantageously, they can be treated for a few
seconds with
pressurised steam before pressing, in order to inactivate enzymes, effect a
disintegration by
partial destruction of the cell walls and, through albumin precipitation, to
facilitate a later
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clarification process. Similariy, shock freezing can be performed.
For the pressing, both continuously operating fly-presses, hydraulic filter
presses and also
continuous screw presses are commonly used. The parameters to be set for this
and other
process operations are well known to the specialist. The expressed juice can
then be treated
by normal processes, such as filtration, centrifugation and separation. If
necessary, it is next
ultrapasteurised and then filled into sterile bottles. Next it is preferably
cooled as quickly as
possible to temperatures below 70°C.
Also usable are extracts from flesh-eating plants. Here, the following
extractants and
processes can be used:
Eatractants:
cold water, hot water, dilute acetic acid, sweet wine, ethanol-water mixtures,
ethanol
and fatty oils or neutral oils and supercritical gases, for example
supercritical CO2.
Extraction Processes:
static maceration, stirred maceration, turbulent extraction, digestion,
percolation,
repercolation, evacolation, diacolation, combinations of maceration and
percolation,
ultrasonic extraction, countercurrent extraction and extraction with
separators,
centrifuges and decanters.
The equipment to be used for this and the process parameters normally used are
familiar to the
specialist.
The products obtained by tapping off the digestive juice, by pressing and by
extraction can be
further processed in normal ways. In particular, these juices can be freeze-
dried, especially
lyophilised. For this, the usual additives, such as mannitol or glycerol can
be used during the
lyophilisation. The equipment needed for this and the process parameters are
well known to
the specialist. Further, various additives which have a favourable influence
on the therapeutic
action on administration, such as for example formic acid, benzoic acid,
citric acid, malic acid,
cyanidine-3-glucoside, sodium, magnesium, potassium, calcium, chlorine,
naphthoquin-ones,
L-arginine, L-asparagine, L-serine, L-glutamic acid, L-alanine, L-threonine, L-
lysine, glycine,
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L-tyrosine, L-phenylalanine, L-valine, L-leucine and L-isoleucine, can be
added to these juices.
The juices of flesh-eating plants can be administered in various ways.
The juices can be used directly orally or topically; alternatively, they can
previously be diluted
with a harmless solvent. Examples of suitable solvents are water, sweet wine,
ethanol-water
mixtures and salt solutions.
Usually, they are diluted to a dry residue of 0.1 to 10 wt %, preferably 0.5
to 5 wt %, and
especially preferably 0.8 to 2.5 wt %.
Further, the juice of the flesh-eating plant can be administered orally in the
form of tablets and
capsules. The equipment needed for this and the processes for formulation are
well known to
the specialist. Further, topical applications in the form of the juices
themselves or in the form
of ointments are also possible. The equipment and the materials and processes
needed for the
production of ointments are well known to the specialist.
Further, it is also possible to produce parenteral formulations from the
juices ofthe flesh-
eating drugs. These can be administered intramuscularly, subcutaneously or
intravenously.
The equipment, additives and processes needed for the production of such
parenteral formul-
ations are well known to the specialist.
The following experiments illustrate the invention:
1. Inhibition of Protein Kinases
Sample material Carnivora expressed juice dilute and also C:arnivora~
expressed juice neat,
produced by Carnivora Research GmbH.
Until the start of the experiment, both samples were stored unopened in the
refrigerator. On
the day of the first experiment, both samples were each prediluted 1:10 and
1:100 with water
and DMSO, so that a 1:10 and a 1:100 initial solution of both samples in 10 %
DMSO was to
hand. On addition to the test, a further 1:10 dilution step was performed on
each occasion.
Both samples were tested at 1:100 and 1:1000 dilutions. The DMSO concentration
in the
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assay was 1 %. The prediluted initial solutions were kept at 4°C and
used for all measure-
ments over a period of 10 days.
Protein Kinases Used:
Cyclin-dependent kinase 4 (CDK 4) and cyclin-dependent kinase 2 (CDK 2)
Tyrosine kinase of the (i receptor of platelet-derived growth factor (PDGF-R(3-
TK)
Tyrosine kinase of the receptor for epidermal growth factor (EGF-R-TK)
Tyrosine kinase of the ERBB2 receptor ERBB2-TK
Tyrosine kinase of the receptor-1 of fibroblast growth factar (FGF-R1-TK)
Tyrosine kinase of insulin-like growth factor receptor, type 1 (IGF-1-R-TK)
Tyrosine kinase of the insulin receptor Ins-R-TK
Protein kinase C, subtype y (PKC-y)
Protein kinase C, subtype E (PKC-e)
Janus kinase 2 (JAK 2).
The protein kinases were cloned from the RNA of suitable cells by means of RT-
PCR. The
isolated DNA sections were then subcloned in baculovirus transfer vectors and
recombinant
baculoviruses were then produced. Using the recombinant baculoviruses, the
proteins were
expressed in Sf~ insect cells. The kinases were purified from the insect cell
lysates using
glutathione agarose gel.
All kinase assays were performed in 96-well microtitre plates in a reaction
volume of 100 ~l.
For the measurement of PKC activity, the test (mixture] contained 50 mM HEPES-
NaOH, pH
7.5, 1 mM EDTA, 1.25 mM EGTA, 5 mM MgCl2, 1.32 rr>Nf CaCl2, 5 pg/ml
phosphatidyl-
serine, 1 ~g/ml 1,2-diolein, 1 mM DTT, 0.1 uM [33P]-ATP, 50 ng recombinant
kinase and
0.5 ~g histone Hl as substrate. For all other kinases, the test mixture
contained 50 mM
HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnClz, 3 u.M Na orthovanadate, 1 mM DTT,
0.1 I,WI ATP, 100 ng recombinant kinase and a suitable substrate.
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The reaction mixture was incubated for 80 mins at 30°C. The reaction
was stopped by
addition of 100 ul H3P0~ (2 %). Next the plates were washed three times with
HZO. The
incorporation of [33P)-phosphate into the relevant substrate was determined
using a scintill-
ation counter for microtitre plates. The results are summarised in the
following table.
Table 1
Percentage Action
(Inhibition)
Kinase dilute expressed
juice neat
expressed juice
1:100 1:100
1:1000
CDK4/cyclin D - 66 % - 109 % - 41
1
CDK2/cyclin E _ gg %
PDGF-R(3-TK -78 % - 98 % - 64
EGF-R-TK -78 % - 96 % - 68
ERbB2-TK - 62 % - 88 % - 82
FGF-RI-TK - 70 % - 100 % - 67
IGF-1-R-TK - 59 % - 9Ei % - 66
Ins-R-TK - 68 % - 99 % - 7~
PKC-y -57% _34%
'' PKC-E - 6~ % -. _ 37
J~ ~ -81% ~% -79%
The results clearly show that both the neat expressed juice from Carnivora and
also a diluted
expressed juice cause inhibition of protein kinases.
II. Control of Helicobacter pylori
Studies relating to the eradication of HeIicobacter pylori were carried out on
the duodenal
ulcer model in the rat. In this ulcer model, rats which are predominantly
colonised with
Helicobacter pylori were used. After oral administration of t:he ulcerogen
cysteamine
hydrochloride, duodenal ulcers and gastric ulcers were 100 °,%
detectable macroscopically and
microscopically 48 hrs after administration.
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Carnivora~' expressed juice (2 % dry residue) was administered orally in the 3
dosages 0.06,
0.6 and 2.83 ml/kg body weight (BW) 3 x daily over a period of 20 days after
ulcer induction.
The same administration regime was used for the reference group (combination
of tetracycline
(6 mg/kg BW), metronidazole (15 mg/kg BW) and basic bismuth nitrate
(30 mg/kg BW)) and for the controls (isotonic common salt solution, 0.9 %
NaCI).
2, 5, 8, 14 and 21 days after the ulcer induction, the respective groups were
examined for
ulcer healing by macroscopic assessment, and in addition a determination of
Helicobacter
pylori was performed.
The following parameters were covered:
mean assessment score (mAS) based on the degree of severity of the duodenal
and gastric
ulcers, percentage frequency of the ulcers (% U), ulcer index (UI) (mAS x %
U), area of the
ulcers, body weight and mortality.
In this model, the combination of the three reference substances showed the
expected action.
The ulcer index was lowered, the lesion areas of the duodenal or gastric
ulcers were decreased
and the incidence of Helicobacter pylori was lowered.
The three dosages of Carnivora expressed juice tested showed very good dose-
dependent
effects. The ulcer index was lowered, and the lesion areas of the duodenal or
gastric ulcers
were decreased, or the ulcers completely eliminated. The incidence of
Helicobacter pylori
could be lowered, and at the highest dosage the eradication of this germ was
effected. Further
beneficial effects of the test substance manifested themselves in a decrease
in the spontaneous
deaths occurring during the first 3 days after administration of the ulcerogen
and a greater
weight gain compared to the controls over the test period from 0-21 days.
The demonstrated effects of Carnivora expressed juice confirm its outstanding
action on this
model.
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Table 2
Group 2 5 8 14 21
days
post
ulcerogen
DU GU DU GU DU GU DU GU DU GU
controlmAS 2.2 1.9 2.6 18 2.7 1.6 2.2 0.6 2.1 1.2
sd 0.-10.5 0.4 0.9 0.5 1.5 0.3 0.8 0.4 0.7
U 100 100 100 100 100 60 100 40 100 80
UI 219.0191.0258.0 204.0268.096.0 220.024.8 206.096.0
referencemAS -- -- 2.1 2.0 1.3 l.3 0.3 0.2 0.5 0.3
sd -- -- 0.5 0.5 1.3 1.3 0.7 0.5 0.8 0.6
U -- -- 100 100 60 60 20 20 40 20
UI -- -- 206.0 204.076.8 76.8 6.0* 4.4 21.6*5.2
dose m.~.S-- -- 2.:1 1.8 2.2 1:.3 1.8 0.8 1.4 0.6
1
sd -- -- 0.6 0.9 0.5 1..2 1.1 1.3 0.9 0.9
U -- -- 100 100 100 ti0 80 40 80 40
UI -- -- 238.0 178.0218.078.0 145.633.6 115.225.6
dose mAS -- -- 2.1 1.6 1.1 1.1 0.6 0.7 1.2 1.0
2
sd -- -- 0.7 1.1 1.0 I.0 0.8 0.9 1.1 1.0
U -- -- 100 80 60 fi0 40 40 60 60
UI -- -- 214.0 131.267.2*6:3.624.8*26.4 69.6 62.4
dose mAS -- -- 2.1 1.3 1.2 0.9 0.7 0.9 1.1 0.7
3
sd -- -- 0.2 0.9 1.2 0.9 1.0 0.9 1.0 0.7
U - -- 100 80 60 60 40 60 60 60
UI -- -- 208.0*107.274.4*5fi.428.8*55.2 66.0 43.2
AS assessment score
mAS mean assessment score
U percentage of animals with ulcers of all degrees of severity
UI ulcer index
significance calculation, t-test, p<0.05, versus control
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Table 3
Area
of
duodenal
ulcer
[mmZ]
days post 2 5 8 1.1 21
ulcerogen
Group r/n A r/n A r/n A r/n A r/n A
Control 8110 .10.95/5 25.1 4/5 17.5 4/5 13.0 315 14.9
Reference -- -- 3/5 20.9 2/5 21.2 0/5 0 O/S 0
Dose 1 -- -- 4/5 23.4 4/5 12.6 3/5 11.3 2/5 5.9
Dose 2 -- -- 3/5 39.8 2/5 4.7 0/5 0 1/5 3.1
Dose 3 -- -- 4/5 5.7 2/5 8.3 1/5 3.1 0/5 0
Table 4
Area
of
gastric
ulcer
[mm2]
days post 2 5 8 14 21
ulcerogen
Group r/n A r/n A r/n A r/n A r/n A
Control 5/10 207.33/5 130.93/5 110.00/5 0 1/6 78.5
Reference -- -- 4/5 90.4 2/5 53.4 O/S 0 O/S 0
Dose 1 -- -- 3/5 238.83/5 93.2 1/5 113.11/5 28.3
Dose 2 -- -- 215 64.4 1/5 28.3 0/5 0 115 12.6
Dose 3 -- -- 2/5 28.3 1/5 12.6 0/5 0 O/S 0
r/n number of animals with ulcers of severity degree 2-3/number of animals
studied
A area (determined for ulcers of severity degree 2-3).
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Table 5
_ . Helicobacter
pylori
(Hp+)
[%]
days post 2 5 8 14 21
ulcerogen
Group
Control 80 100 100 80 80
Reference -- 60 40 20 20
Dose 1 -- 100 60 60 60
Dose 2 -- 60 :l0 40 40
Dose 3 -- 60 20* 0* 0*
* significance calculation, exact Fisher Test, p < 0.05, one-sided, versus
control
Table 6
Mortality
days post 1 2 3 Total
uicerogen e/n e/n e/n e/n
Group
Control 1/30 3/30 x/30 9130
Reference 1/20 0/20 0/20 1/20
Dose 1 0/20 1/20 1/20 2120
Dose 2 1/20 0120 0/20 1120
Dose 3 0/20 0/20 0/20 0/20
e/n number of animals died/total number of animals per group
