Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PEAT-DERIVED ~IOACTI~fE PRODUCTS AND PHARMACEUTICAL AND COSMETIC
CDt~4~OSITIONS CONTAINING T(~IEM
The present invention relates primarily to novel peat-derived
bioactive products and to a process .for producing such products.
The invention also relates to pharmaceutical and cosmetic
compositions containing these products and to processes =or
preparing pharmaceutical and/or cosmetic formulations containing
said peat-derived bioactive products.
It is known to extract peat by various methods using various
extraction media and to use such extraets, containing peat-
derived bioactive ingredients, for cosmetic and pharmaceutical
purposes.
On o= these known processes is described in Polish patent
specification No. 124110 (Chemical Abstracts 101(10), 78854e).
According to this prior art process peat-derived bioactive
products are obtained by primary and secondary alkaline
hydrolysis of an air-dried raw peat material, followed by
acidification of the thus obtained hydrolysate and separation o~
insoluble solid parts with subseguent second alkali~atio.~.,
acidification of the c'_ear liguid phase and elim~.nar__on c~
ballast substances by means o~ alcohol and etner eat=act;on. ..
said process, the aqueous phase from the organic e_ct=act_on is a
liauid neat-derived bioactive product.
The known liquid product, being a solution ov peat--dewived
active ingredients in a highly concentrated, near-,~ saturatea
acrueous solution of sodium chloride, obtained according to t:~.°
above cited Polish patent specification, is unstab'~e wPen stores
for a long time and, moreover, contains - regardinc the b=o-
logical activity of the composition - a large excess one~~tra_
inorganic substances. As a bulk product, it is ha=d to handl°,
store and process.
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It is the main object of the present invention to provide a
product which is not afflicted with these disadvantages, i.e. a
product which is stable and which can easily be formulated into
pharmaceutical and veterinary products as well as be introduced,
either in solid state or in any suitable solution, into cosmetic
preparations.
In particular, with respect to the use of a peat-derived bio-
active product for pharmaceutical purposes, i.e. for the
production of pharmaceutical compositions, there was a strong
need for providing a solid form which would be well suited for
this purpose. As previaus attempts aiming at concentration o'
the known aqueous solution of peat-derived bioactive substances
and desalination of the same failed to give a positive result
due to the occlusion of active ingredients in a crystallising
solid phase, causing decrease of biological activity pa ~ze
composition, it was very hard to find a suitable process °or
converting the liquid composition into powder form.
Unexpectedly, it was found that a positive result is achievable
if, prior to concentration, the liquid composition is L-rst
diluted several times, i.e. by water volumes several times the
volume of the composition.
Accordingly, the prey ent invention provides a process by whic~ a
peat-derived bioactive product, featuring the above advantageous
characteristics, is obtainable from a highly concentrated
aqueous inorganic salt - especially sodium chloride - solutio:~
containing peat-derived bioactive ingredients, by diluting said
solution with demineralised water, e.g. distilled water, ~ollow~-
ed by reverse osmosis, concentration and clarification. A solut-
ion so obtained can easily be converted into a sterile and solid
product, well suited to the intended purposes, by stervlizatio.~.
and spray-drying. Dilution is preferably effected with quantit-
ies of water several times, preferably 5 to 8 times, the quanti-
ty of the concentrated solution to be diluted.
~~J~~TITtJT~ ~H~~T
CA 02083061 2001-03-14
3
Thus, in one aspect, the present invention provides a
process for producing a peat-derived bioactive product. A
concentrated solution of a peat extract containing inorganic
salts and peat-derived bioactive ingredients is subjected to
processing steps comprising: (a) a dilution step wherein the
solution is diluted with demineralized water, (b) a
subsequent desalination step wherein the diluted solution is
subjected to reverse osmosis in order to remove inorganic
salts from the solution, such that the desalinated solution
contains no more than 70% by weight, based on dry solids, of
the inorganic salts being removed, (c) a concentrating and
purifying step wherein the desalinated solution is
concentrated and clarified, and (d) optionally, a sterilizing
and/or drying step wherein the concentrated and clarified
solution is sterilized and/or spray-dried,
the steps yielding the peat-derived bioactive product.
In another aspect, the invention provides a process for
producing a peat-derived bioactive products from a highly
concentrated aqueous solution of inorganic salts containing
peat-derived bioactive ingredients. The solution is diluted
with demineralized water, the dilution being effected with
water volumes several times the volume of the solution to be
diluted. The diluted solution is subjected to reverse
osmosis in order to desalinate the solution. Inorganic salts
being removed, the desalinated solution is concentrated and
clarified.
The instant process, applied to the processing of a
product obtained according to Polish patent specification No.
124110, consists in expelling residual organic solvents from
a post-extraction aqueous phase, separating insoluble parts
CA 02083061 2001-03-14
4
by filtration under reduced pressure through a sintered
ceramic material, diluting the permeate with several times
the volume of distilled water and subjecting it to
desalination by reverse osmosis to separate excessive mineral
salts, mainly sodium chloride, as a permeate. Then, the
desalinated solution is concentrated, clarified by
centrifugation and sterilized by filtration through a
membrane filter, e.g. a Millipore(R) filter. The resulting
microbiologically clean solution may optionally be spray-
dried. The sterilized product (liquid or solid) may be
formulated into a cosmetic pharmaceutical or veterinary
composition. Optionally, the concentrated and clarified
solution may be used - without sterilisation and spray-drying
- in any suitable dilution as a component in numerous
cosmetic compositions.
Preferably, in a spray-drying step, the inlet
temperature is set to about 180°C, while the outflow
temperature is set to about 90°C.
While the process of the present invention is described
above in combination with the process according to Polish
patent specification 124110, its use is not restricted to
such combination, but is applicable generally in the context
of a process for obtaining a peat-derived bioactive product
from a highly concentrated aqueous solution of inorganic
salts, especially of sodium of sodium chloride, containing
peat-derived bioactive ingredients.
The peat-derived bioactive products provided by the
present invention do not contain more than 70s by weight,
preferably not more than 60% by weight of inorganic salts,
especially of sodium chloride. Since a sodium chloride
CA 02083061 2001-03-14
4a
concentration as low as possible would be desired for an
optimal product, especially for pharmaceutical applications
and such cosmetic applications where higher concentrations
are required, i.e. for face care, lower sodium chloride
concentrations, such as 55% and even lower, are most
preferred, especially when obtainable by the steps of
dilution and reverse osmosis.
Thus, in one aspect, the invention provides a water
soluble peat-derived bioactive product, containing not more
than 70% by weight sodium chloride, based on dry mass. In a
further aspect, the invention provides a peat-derived
bioactive product containing not more than 70% by weight of
inorganic salts, based on dry solids.
In another aspect, the invention provides a peat-derived
bioactive product obtainable by desalination of a
concentrated aqueous solution of peat extract, which solution
contains inorganic salts and water-soluble peat-derived
bioactive ingredients and originates from an extraction
process comprising alkaline hydrolysis of raw peat material,
the bioactive product containing not more than 70%, by weight
of inorganic salts, based on dry solids.
Where the process is terminated with the concentration
and clarification steps, the product is a concentrated (or
thickened) solution. "Concentrated peat extract", referred
to in this specification, is a dark-brown liquid of a density
of 1.02 - 1.09 g/ml and has a content of dry solids of not
less than 5% by weight. The chloride ion content in dry
solids, calculated as NaCl, is not higher than 70%,
preferably not higher than 60%, and the pH value of a 1%
aqueous solution is 5.0-6.5, generally about 6Ø The lack
CA 02083061 2001-03-14
4b
of a further sterilization step may not be detrimental in
certain cases, e.g. for certain cosmetic uses of said
concentrated peat extract.
On the other hand, the sterilization step will be
mandatory, particularly when the product is intended for the
preparation of pharmaceutical compositions. Particularly in
such a case, the further step of spray-drying is most
preferred, if not mandatory. The product resulting after
such a spray-drying step is in powder form and thus
particularly suited to the preparation of certain
pharmaceutical compositions. A most preferred product of
this type is the product commercialized under the designation
"TOLPA~R~ Torf Preparation", TOLPA~R~ being a registered
trade mark of Torf Corporation, u1. Mydlana 2, Wroclaw,
Poland. The abbreviation TTP will be used in the course of
this specification to designate said product.
The present invention also relates to pharmaceutical
compositions containing as active ingredient a peat-derived
bioactive product as hereinbefore described, particularly a
product which contains not more than 70% by weight,
preferably not more than 60% by weight, of inorganic salts,
especially sodium chloride, based on dry solids, together
with a pharmaceutically acceptable
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W~ 92/I6216 PCT"/EP92/Otk191
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carrier. The peat-derived bioactive product contained in such
pharmaceutical compositions is preferably TTP as defined above.
The pharmaceutical preparation contains the peat-derived bio-
active product and the gharmaceutically acceptable carrier
material, preferably in a weight ratio of between about 1:5 and
1:25, and most preferably between 1:9 and 1:19.
The present invention furthermore relates to a process for
preparing a pharmaceutical formulation containing a peat-derived
bioactive product, in the form of a gel. This process is cha-
racterised in that a sterile alcoholic herb extract is combined
with sterile glycerol, a sterile aqueous - preferably concen-
trated - solution of previously powdered peat-derived bioactive
product and a sterile menthol solution, and that the resulting
mixture is gradually combined with colloidal silica 'to convert
the liquid composition into gel form, the weight ratio of liauid
mixture to silica being from 90:10 to 94:6. Preferably TTP is
used as the powdered or concentrated peat-derived bioactive
product.
The present invention also relates to a process for preparing a
pharmaceutical formulation containing a peat-derv ved bioactive
product, in the form of an ointment. Th9.s process is charact-
erised in that a sterile herb extract is gradually combined with
a sterile solution of powdered peat-derived bioactive product,
that the resulting mixture is gelled with the addition of col-
loidal silica and that the gel thus obtained is triturated with
a previously sterilized mixture of fatty components, such as
eucerine and petrolatum, preferably with a weight ratio of
liquid components to silica of about 30:20 and of gel to fatty
composition of between 32:68 and 34:66. Also here, preferably
TTP is used as the powdered or concentrated peat-derived
- bioactive product.
Cosmetic preparations, which may comprise herbal extracts as
well as other auxiliary and enriching components, fragrant
compositions and carrier materials allowed for cosmetic use,
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WO 92!16216 PCT/EP92/00491
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contain the peat-derived bioactive product according to the
present invention in an amount of 0.01-10~ by weight, preferably
0.05-1.00 by weight, and most preferably 0.05-0.10 by weight.
Carrier materials may be aqueous solutions of alcohols, all
types of emulsions, gels, foaming compositions and fatty
carriers. Use of one specific carrier selected from the group of
the above mentioned substances allows formulation of various
types of cosmetic preparations according to the invention, such
as tonics, creams, balsams, cleaning milks etc. for daily body
care as well as shampoos, hair balms, foaming bath compositions,
al? with the addition of peat-derived bioactive produc:.s.
Peat-derived bioactive products (peat extracts in abbreviacec
form) stemming from original yaw peat material, such as famonc
others) therapeutic mud, contain well balanced quantities o~:
mineral and organic compounds, such as mineral salts of ~~e
following -elements: B, Si, Ab, Fe, Mg, Mn, Cu, Sn, Ni, Ca, ~c
and Na; organic compounds, such as aminoacids in free form ant
as salts; polysaccharides, partially degraded/reaeted - _.. she
course of hydrolysis - 'to desoxysaccharides and%or aminosacc'.~.-
arides. Peat, in particular therapeutic muc, is known ant =°-
cognised -as a material of biological plant and microorganism
origin; due to its contents of nourishing and stimul ar_~.ng cor,.-
aonents it has beneficial effects an humans and mammals; she==-
fore, peat-derived bioactive compositions contain the above-
mentioned substances in proportions characteristic for the '-=v-
ing organisms; this is considered to be an explanation of the
advantageous effects of cosmetic and pharmaceutical pr~parat~o:a
containing peat-derived active products and compositions.
Particularly good effects of the new cosmetic compositions are
observed when herb extracts are also present. in the =ormulation.
Selection of a suitable herb extract is based on a known typical
use of such extracts in cosmetics, modifying the activity of
preparations and thus enabling the content of cosmetic prepar-
ations to be matched with demands and needs of indivic~aals to be
treated.
~tJBSTiTIJTE SHEET
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'!W~ 92/16216 PCT/EP92/00491
The present in~rention is better characterised and explained in
the following examples.
Exa~le 1
Starting with 1000 kg of air-dried raw peat material, following
e.g. the known procedure according to Polish patent No. 124110,
a solution of peat-derived bioactive ingredients in a saturated
aqueous solution of sodium chloride was obtained in a quantity
of about 10 liters. The solution was filtered through a sintered
ceramic filter under reduced pressure in order to clarify the
solution before desalination of the same, The clear solution
thus obtained contains about 95~ of NaC'_ in a dry mass. The dr.:
mass constitutes about 32~ by weight of the solution. The volume
of this clear solution is about 7 liters.
The clear solution is diluted with 5 to 8 M mes the quantity o.
distilled water and in diluted form is subjected to a desalin-
ation step carried out by using a reverse osmosis technique
using a DDS apparatus. Desalination was carried out for 3 ~o
hours, whereby the excess of mineral salts - mainly NaC'_ - is
separated in the form of the permeate. The desalinated compos-
ition contained approx. 6~-70a.of sodium chloride in the soi'_ds.
The solution thus obtained, or 6-7 liters by volume, was con-
centrated 4-5 times in a Buechi rotating evaporator, so tat a
concentrated sol~ition containing approx. 20~ of dry mass was
obtained. The resulting concentrated solution was clarified
using a Biofuga-Heraeus centrifugal apparatus (flow separator)
and then sterilised by filtering through a Millipore~R) ~-lte=.
The resulting microbiologically pure solution was spray-dried in
an Anhydro dryer with the outflow temperature se't at 90°C and
supply inlet temperature set at 180°C. The yield of dried oow~:e~
was approx. 200 g.
Example 2:
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The product obtained in Example 1 was used for preparing gel and
ointment pharmaceutical compositions, containing also hero ex-
tracts synergistically improving the therapeutic effect with ,
respect to certain diseases. For example, a gel and ointment
against varicose ulcer of the shank was prepared as follows: ,
20 g of hippocastanaceous extract, 10 g of calendula extract, 60
g of glycerol, 0.1 g of TTP in the form of a powder obtained as
described in Example 1 above, 0.1 g of salicylic acid, 1.0 g of
distilled water and 8.8 g of Aerosil(R) (colloidal silica) were
used in order to obtain a gel form of the preparation.
hiquid (non-volatile) ingredients were sterilised before use, by
means of heating under reflux for two hours. Herb extracts were
combined with glycerol and an aqueous solution of TTP and also
with menthol, and silica was gradually added to the obtained
mixture, under continuous stirring.
Similarly, in order to obtain an ointment composition, t:~.e
following ingredients were used: 20 g of hippocastanaceous ex-
tract, 10 g of calendula extract, 0.1 g of salicylic acid, 0.1 g
of TTP in powdered form obtained as described in example 1 and
2.0 g o~ Aerosil(R) (colloidal silica).
As fatty components, a mixture of the following substances was
used: 22 g of eucers.:ne and 45.8 g of petrolatum. :lerb e~:.rac~s
were sterilised by heating under reflux for approx. ? hours.
Eucerine -and petrolatum were similarity sterilised. ~iauic in-
gredients wer a carefully combined with silica to obtain a ge'_,
which in turn was triturated with sterilised and 'arty com-
ponents cooled down to room temperature. A stable aintmen~ was
obtained which did not separate when stored.
The gel and ointment obtained above were simultaneously applied
in. the treatment of varicose ulcer of the shank. Ulcers were
treated with the gel preparation while the surrounding, non-
affected skin was treated with ointment. Addition c~ colloidal
silica is believed to be responsible for prompt dess,~cation
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while the herbal and peat-derived ingredients are believed to be
responsible for 'the curing effect of the preparation. Fatty
components helped to keep elastic the crust and the skin. The
results obtained were compared with a control group of patients
treated in a classic way. Those who received the new treatment
were selected from a group of patients suffering from the
disease for many months (sometimes years) without noticeable
positive effects. Patients treated with compositions according
to the invention showed better results - already within the
fir st few weeks - than control patients,
_Example 3:
Products obtained in Example 1 were used to pr°par~ pharmaceut-
ical formulations in the form of tablets, or granules to be
placed in capsules.
A sterile pe at-derived bioactive composition ;n pcwdered form
was combined with a carrier in a weight ratio of 1:9. As a
carrier, ~IYVATEX(P)TL (tradename of F_astman-:Codak), a .mixture o-_.
lactose and lubricating substances, was used in a weight =atio
of 44:1. Lactose of 50 mesh particle size and ivIYVAT°X(~)TT~ were
finely disintegrated so that approx. .70% o= its mass passed
through a 100 mesh screen. A part of the resulting mixture of
active composition and carrier was formulated into tablets con-
taining ~ mg of aci~ive ingredients. The total. mass oz -ac'.~.
tablet was SO mg. 'The other part of the same rni:<tare of active
camoosition -and a carrier was granulated using c.~. oz ec'nanol
(40% by volume). Granules were sieved and ground ~._ necessary
and then filled in capsules in such a quantity that each capsu~~.e
contained 5 mg o.f active ingredients by using TTP in mixture
with a carrier at a ratio of 19:1.
The tablets obtained as above were tested is order to measure
the time of their disintegration in an ar'~i~icial gastric juice
at 37°C +/- z°C using Erweka equipment. The artificial gastric
juice was prepared as follows: 2.0 g of sodium chloride and 3.~g
oz pepsin were dissolved in 7 ml of hydroc~loric acid and
SUBST1TUT~ SNEET
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WO 92116216 P~T~1;P92/00491
- 10 -
distilled water was added up to a total volume of 1000 ml. The
pH-value of the resulting solution was approx. 1.2.
l3esintegration time of a tablet, having a diameter of 5.1 mm and
a total mass of 0.0498 g, was 5 minutes.
Further examples relate to numerous cosmetic preparations
according to the present invention, having different forms
composition and being designed for different applications,
containing the beneficial addition of bioactive ingredients
derived from peat. Among others, preparations such as tonics,
balms, creams, milks, shampoos, foaming bath compositions etc.
are described.
Example 4:
A reaction vessel equipped with a stirrer was charged with 150 g
of camomile extract obtained by the extraction of camomile in-
florescence with a 1:1 ethanol:water solution, as we';l as i g o~
TTP as described above. SO g of glycerol were added :,o the
mixture obtained. The three substances were stirred to obtain a
uniform mixture. Subsequently, a second mixture as previously
formulated, was introduced into the same vessel. It comprised
340 g or a 95:5 ethanol: water solution, 1 g or salicyl'_c acid
and 0.5 g of menthol.. The two mixtures were combined b~ st;rring
to form a uniform solution: Next, 3 g of a fragrant composition
TILIANA H4308 were added. TILIANA H4308 is a product o~ ~abryka
Syntetykow Zapachowych Pollena-Aroma (Synthetic Fragrance works
Pollena-Aroma), of r,Varsaw, Poland. The solution was then brought
to a total volume of 1000 ml by adding 454.5 g or distil'_ed
water; stirring was continued until a homogeneous mixture was
obtained.
In the above procedure, 86~ glycerol, menthol and water accord-
ing to the requirements of Polish Pharmacopea FP T_V and ethanol
in a concentration of 95~ according to the Polish industrial
standard BN-75/6193-01 were used.
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~V~ 9211b216 PCT/EP92/(j0491
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The concentrated peat extract used was a dark-brown liquid or a
density 1.020-1.090 g/ml and a content of dry solids not less
than 5~; the pH value of a l~ aqueous solution was 5.0-6.5.
Camomile extxact was a red-brown liquid of a density of 0.9160-
0.9503 g/ml, and ethanol content of 52-56~ by volume.
The tonic preparation obtained above is suitable for all kinds
of skin. It is a clear liquid without any solids. Its colour is
yellow. The pH value is 4.28 and the ethanol content is 45.920
by volume. Total acidity calculated as salicylic acid content
was not less than 0.1~ by weight, namely 0.23a by weight. The
preparation being stored for 12 months did not lose any of the
above characteristic features.
E_x_ample 5:
The procedure described in Example 4 above was repeated, the
only difference being that instead of camomile extract and the
TILIAIdA H4308 fragrant composition, a marigold flowers extract
and a composition SINUS H4625 (also a product of Fabryka Syn-
tetykow Zapachowych Pollena-Aroma) were used in the same way and
the same molar and volume ratios. The resulting tonic prepar-
ation is suitable for dry and fragile skin. Similar==y '-.- was a
clear liquid without any solid particles. The pH value was 4.30,
total ethanol content was 45.82 by volume and total acidity was
0.27$ by weight. When stored, the preparation was unchainged
after 12 months like the preparation obtained acca-rding to
Example 4.
Example 6:
The procedure of Example 4 was repeated, except that an ext=acs
of sage leaves was chosen instead of camomile extract, and the
fragrance LELIA 90368 (Pollena-Aroma, Warsaw) was chosen instead
of TILIANA H4308. The extract of sage leaves was obtained by ex-
tracting dried gage leaves with ethanol at 50°C and had a brown-
ish colour, a characteristic sage odour and a density of C.916C
to 0.9503; it contained 52 to 56~ of ethanol. The resulting fac°
care agent is particularly su°itable for greasy skin. It is a
SUBSTITUTE SH~~T
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WO 92/16216 ~CT/IEP92/(30491
- '12 -
clear and homogeneous liquid having a dark yellow colour; the pH
value, the ethanol content and the acidity were comparable with
the values of the care agent according to Example 4.
Example 7:
The following composition proved to be a particularly effective
gel for avoiding or treating periodontosis:
24.0 g camomile extract
3.0 g sage leaf extract
0.3 g salicylic acid
0.2 g menthol v
C.1 g TTP
to 100.0 g commercial gel base
Example 8:
The following carefully weighed components were introduces =~ro
a reaction vessel of a volume of 2000 m1, equipped with a
mechanical stirrer:
270 g of camomile extract obtained by extraction of camomile
inflorescence with 50o ethanol; the extract was a =ed-brow~
liquid, having a density of 0.9160-0.9503 g%ml and a~
ethanol content of approx. 55o by volume
- 50 g of glycerol,'86~, according to the requirements o. °olis
Pharmacopea E'P IV
- 30 g of a saponaria officinalis extract obtained by ext=act~:=g
saponaria of.ficinalis roots with 70~ ethanol; the e:c~=acs
was a red-brown liquid, the density was 0.9630-0.9810 glm~-,
and the ethanol content approx. 75~ by volume
- 1.0 g of the inventive concentrated peat ext=act, being a da='-:
brown liquid having a density of 1.02-1.09 g/mi; not more
than 2~ chloride ions calculated as sodium chloride; the c~.~
solids content was no's less than 5~; the pH value oa 1~
water solution of the extract was 6Ø
The ingredients listed above were mixed thoroughly. ~ pr°-~=ous_y
prepared solution of 1 g of salicylic acid in 260 g of
ethanol was added thereto. To :he combined solution, 383 :~
SUSST'ITL~TE SHEET
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W~ 92116216 PtJT/EI'~2/Od~l9l
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distilled water and 5 g of fragrant composition TILIANA H3408
were added and stirred until a uniform solution was obtained.
The preparation was analysed and stored in retail size bottles
of 200 ml volume. The resulting preparation was suitable as a
hair care preparation. It was a clear, slightly opalescent
liquid, containing approx. 45~ by volume of ethanol; the pH
value was 4.5; the total acidity calculated as salicylic acid
was not less than 0.1~ by weight. The preparation is suitable
for blonde hair. During 12 months storing the preparation re-
mained unchanged in i'ts features.
Example 9:
The procedure described in Example 8 was followed except that
instead of camomile extract and TILIANA H4308 composition there
were used in the same sequence and ratio: horsetail herb extaact
and the fragrant composition FINUS H4625. Horsetail herb extract
was a green-brown l,;quid of a density oz 0.9160-0.9503 g/ml and
an ethanol ccntent or 55~ by volume.
The resulting preparation was suitable for a1'_ kinds of ;~a=. -..
was a clear and transparent liquid without any soled parts,
yellow-brown in colour. pH value, ethanol content, total acidity
as well as stability after a 12 months storing period were the
same as those of a preparation described in Example 5.
Fxam 1e 10:
The procedure as described in Example 8 was repeated with com-
parable results. The only difference was that, instead of camo-
mile extract and TILIANA H4308 composition, stinging nettle
leaves extract and fragrant composition LELIA 90368 (product o~
the same Fabryka Syntetykow Zapachowych Pollena-Aroma) in the
same sequence and ratio were used. 'The herb extract used was
olive-green in colour, had a density of 0.9160-0.9503 g/ml and
an ethanol content of approx: ~5~ by volume. The preparation was
suitable for all kinds of hair.
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Example 11:
rn general, cosmetic milks are dispersions of fatty substances
acting in both chemical and mechanical ways on the skin. In .
fact, due to a convenient way of application and better
interaction of the fluid and the skin, it is very appropriate
to use liquid, more specifically emulsion creams. They can
easily penetrate to deeper layers of the skin and thus prevent
changes of the skin due to age. Cosmetic milks are used mainly
to clean a dry and fragile skin. Accordingly, they must not
contain any aggressive volatile oils, while frequently they
contain suitable herb extracts like camomile extract or wheat
germ extract. Addition of peat-derived bioactive products to
such cosmetic milks further improves their positive effects. In
particular, the new recipe is as follows:
0.05 g
TTP
aloe extract 20.00 g
glycerol 3.00 g
eucerine 2.00 g
white paraffin oil 1.00 g
triethylamine 1.00 g
Aerosil(R) (colloidal silica)4.00 g
Example 12:
Improved regenerative results were observed when TT° and
carefully selected fatty carriers were used in a c7.assic
nourishing and regenerative cream formula. TTP is used in an
amount of 0.01 -1.00 by weight in combination with a herb
extract (selection depends on the type of skin for which the
cream is intended) in an amount of at least 0.05 - 1.00 by
weight, antibacterial preparation in an amount of 0.05 - l.OOj
by weight, synthetic fragrant composition in an amount of 0.01 -
0.05~ by weight and a fatty carrier in the form of a water
emulsion, constituting 97.00 - 99.500 by weight of the whole
composition. The fatty composition needs to be a good carrier
for the active ingredients and to be well accepted by the skin.
Preferably, it is an emulsion of. (a1.1 amounts ir_ o by weight)
35-45 eucerine, 8-14 petrolatum, 2.5-4 olive oil, 6-10 glycerol
ci iRRTfTi fTF SH~~T
-~ u, ,~- y:~ ~s r ~ ~
t .
-~ 3.~ J ~.# 'tJ t. .'
wo 9zn~zr~ ~cri~~z~o~a91
- 15 -
and 35-40 water. Preferred are marigold flower
herb extracts
extract, camomile extract, and the like.
thyme extract
Preferred recipes are lows:
as fol
39.00 partsby weight
1. Eucerine
Petrolatum 11.50 partsby weight
Olive oil 3.13 partsby weight
Glycerol 7.80 partsby weight
Water 38.00 partsby weight
NIPAGINA A (anti-
bacterial preparation) 0.40 partsby weight
TTP
0.05 partsby weight
Marigold extract 0.10 partsby weight
rance 0 02 parts~weiaht
fra
ti
th
g
c
e
Syn
total 100.00 by weight
parts
00 parts
by weight
42
2. Eucerine .
Petrolatum 8.50 carts
by weight
O1'_ve oil 3.08 parts
by weight
Glycerol 7.90 parts
by weight
Water 38.00 parts by weight
NIPAGINA A (anti-
bacterial preparation) 0.40 partsby weight
TTP 0.05 partsby weight
Camomile extract 0.02 partsby weight
Synthetic fragx'ance_ 0.05 parts5v weight
total 100.00 parts by weicJnc
Example 13:
An after-shave preparation contains TTP as a peat ext=act in an
amount. of 0 . 01 -- 1 ~ by weight, herb e:ctracts in ar. amount of 1 -
30~ by weight, glycerol in an amount of 1-8o by weight,
salicylic acid and menthol in aqueous-alcohol solution.
Preferred herb extracts are: camomile, marigold, thyme, aloe
extract and similar beneficial herb extracts. Addition of
glycerol is also beneficial due to its influence on the
elasticity of the skin. Tt speeds up the spreading of the
preparation on the face as well as the penet~ation into t:ze
. . ..., .~-....-r-a ,'~'-r= c~ ~-7 C ~T
~1 ~-. .;
~~ S_7 ! 7 rw.1 ~,~ L' .~'_
lV~. !16216 PC_'T/~Ptl2/U0491
16 _.
deeper layers o~ the skin, thus enhancing the beneficial effects
of the active peat composition and herb extracts. .
A preferred recipe is as follows:
0.10 parts byweight
Camomile extract 15.00 parts byweight
Glycerol 5.00 pats byweight
Menthol 0.10 parts byweight
Salicylic acid 0.10 parts byweight
Ethanol (conc.95~) 10.00 parts byweight
Fragrant composition 0.30 parts byweight
Distilled water ad 100.00 parts
by weight
Example 14:
A shampoo composition was prepared according to the ~oliowing
recipe:
Fuller's herb e:ctract 15.60 g 7.50% byweignt
Stinging nettle leaves extract20.00 g 10.00% byweight
GAMAL SBS-11 (detergent) 30.00 g 15.00% byweight
GAMAL NO-3 (detergent) 20.00 g 10.00% byweight
' 0.40 g 0.20 byweight
tina
Asep
ethanol 1.60 g 0.8U% byweight
BRONOPOL (preservative) 0.04 g 0.02% byweight
Sodium chloride 6.00 g 3.00% byweight
W 106.96 53.48% b~weight
t a
er
a
total 200.0 0 100.00 %
g by
weight
To 92 parts by weight of the above shampoo composition~8 parts
by weight of T'rP were added to obtain 100 parts by weight oz a
shampoo according to the invention. Other herb ertrac~s car, be
used in place of stinging-nettle leaves e:ct-ract.
Example 15:
The following shampoo composition was prepared:
Horse chestnut extract 13.00 g
Marigold extract 22.00 g
GAMAL SBS-11 30.00 g
GAMAL NO-3 20.00 g
w ~zmsz~s ~~c:rm~zivog9i
_ 17
Aseptina 0.40 g
Ethanol 1.60 g
BRONOPOL 0.04 g
Sodium chloride 6.00 g
Water 106.96
a
total 200.00
g
To 95 parts by weight of the above composition, 5 parts by
weight of TTP were added to obtain 100 parts by weight of
shampoo according to the present invention.
Example 16:
A tooth paste contains TTP as a concentrated peat extract, in an
amount of 0.01-0.10% by weight, etheral oils or their
compositions or else fruit essences in an amount of 1-10% by
weight, glycerol in an amount of S-10g~ by weight, herb extwac~s
in an amount of 0.10-10~ by weight and cleaning substances in a::
amount of 20-35% by weight dispersed in water in an amount of
45-60% by weight, and dyes and whitening components in an amount
of 1-2~ by weight.
Titanium dioxide may be used as a whitening component; TTP
itself may be used as an anti-bacterial additive; sage leaves,
camomile or marigold flowers extracts may be used as benef'_cial
preferred herb extracts.
The preferred recipe is as follows:
Precipitated calcium carbonate 150.00 g
Magnesium carbonate 60.00 g
Glycerol 70.00 g
Herb extract x.00 g
TTP 0.50 g
Titanium dioxide 10.00 g
Etheral oils (or mint, lemon, etc essence) 5.00 g
water 400.00g
Dye trace
S~BBTITL~'TE ~HBET
~ .~ J ~i K.1 ~ ~-a t
~~r ~ o i_l ~J 1~ ~i ..~..
WO 9. 0216 PC'f1E~92/1H~491
_ 1g _
E_xamnle 17:
Bath salt preparation: In the course of the process for
obtaining a peat-derived bioactive product according to the
present invention, in particular when oonverting a liquid form
into a powdered one, there is the desalination step in which
sodium chloride is separated as a by-product. In said by-
product, 95g constitutes sodium chloride, other mineral salts
separated are calcium salts, magnesium salts, mainly chlorides
and sulfates; these salty products also contain some organic
peat-derived low molecular compounds occluded within the crystal
structure of these inorganic salts. These organic compounds are
components of TTP and are - among others - polysaccharides,
aminoacids, fulvic acids and the like. The presence of these
components in the salty by-products is beneficial when sail is
used as a bath salt, because they may add additional bene=icial
effects to the standard activity of bath salt. Accordingly ~..is
by-product was tested for its chemical and physical grope=~ies
in the Balneologic Institute in Poznan, Poland, to find ouc
whether it can be used in cosmetic baths. Since the T_nsti~u~°
has found no undesired entity in the salt, it was approved =o.
cosmetic use.
The preferred recipe is as follows:
Salt (NaCl) containing occluded TTP 97.00 g
Pine etheral oil or etheral oils composition 3.00 g
Example 18:
A new hair balm contains TTP in an amount of 0.01-1% by weigh
herb extracts in an amount of 0.01-10% by weight, anti-elec~ro-
static components in an amount of 3-4% by weight, components
preventing excessive drying of hair and skin in an amount o= v,:p
to 2~ by weight, glycerol in an amount or 1-5% by weight, pre-
servative and stabilisers in an amount of 0.05-0.50% by weigi:~
and water to 100 by weight.
As an anti-electrostatic component, the present balm contains ar.
alcoholic solution of trimethylamine and ammonium chloride salt,
obtained from fatty animal-derived amines; as 'thickening agent -
~UBST1TUTB ~~E~~T
V1'~ ~2/1621G PC.T/EP92/00~91
- 19 -
acting also as stabilizing agent - as agent
cosmetic alcohol;
preventing excessive dryness of hair skin - t oils,
and plan
acting simultaneously as co-emulsifying glycerol
agents; and for
easying spreading and penetration of balm, in
the particular
of
its active ingredients TTP and herb acidic
extracts, As an en-
vironment stops multiplication of bacteriar
the balm according
to the invention contains citric acid fumaric
or acid in
an
amount of 0.1~ as well as a preservativeknown as
BRONOPOL
and
fragrant compositions.
The preferred recipe is as follows:
Alcoholic solution of trimethylamine
and ammonium Chloride salts 3-4~ by weight
Cosmetic alcohols 3-4~ by weight
T'rP 0.01-10$ weight
by
Thickened herb extracts 0.01-10~ weight
by
Glycerol 1.5a by weight
Plant oils up to 2; by weight
Citric or fumaric acid 0.1% by weight
BRONOPOL 0.1$ by weight
Fragrant composition 0.3~ by weight
Distilled Water to 100 weight
by
Fxamole 19:
Cosmetic masks are well known cosmetic preparations serving many
different purposes. As therapeutic mud has a known beneficial
effect on the skin and body, it was believed that also post-
extraction peat obtained in the process of separation of bio-
active peat-derived compositions from peat may be used in cos-
metic applications. Post-extraction peat contains a solution, o
active bodies freed in the alkaline hydrolysis process due 'to
extremely high sorptive properties of peat after neutralisation;
It was therefore found to be a valuable component of cosmetic
masks. To enrich the post-extraction peat with more of the
valuable components, natural therapeutic mud and humic acid
fractions were added which are present in natural peat and
separated in a process for obtaining peat-derived bioactive
compositions .from the alkaline hydrolysate. Such a composition
c.i ~czCTIT'i iTF fiHE~~
Zi 7~ ?j ~~ i~
we ; mz~6 ~criE~ziot~am
- 20 -
was tested in the above-mentioned ~alneologic Institute and was
found suitable for cosmetic use.
The preferred recipe is as follows:
post-extraction peat 100.0 g
Natural therapeutic mud or peat 20.0 g
Humic acid fraction 10.0 g
Magnesium carbonate 10.0 g
Zinc oxide 5.0 g
Citric acid or the like 0.1 g
Herb extract or powdered plant material 5.0 g
Distilled water q~s'
The following statements and explanations relate to the bio-
logical aspect of the products of the present invention, _.e. to
the bioactive characteristics and to the compatibility o~ these
products, particularly with a view to their usefulness as phar-
maceuticals. The following abbreviations will be used below:
TTP TOLPA(R) Torf Preparation (trademark of Torf Corporation,
Wroclaw, u1. Mydlana 2, °oland)
IFN Interferon exists as a ubiquitous cytokine (tissue hcrmone).
IFN genes are present in all cells. IFN is mainly i~.duced by
proteins or glyco-proteins. Substances stimulating the IFN
genes for the production of IFN are called inducers. The
process of the IFN induction is a highly regulates, soph-
isticated biochemical process; negative and positive regul-
atory genes controlling IFN production have been recognized.
Small amounts of IFN may be produced spontaneoulsy, without
any detectable induces. Such IFNs are sometimes named
"physiological IFNs". IFN exists in nature in three main
molecular forms:
IFN-a (or leukocyte IFN),
IFN-~i (fibroblast TFN), and
IFN-Y (immune IFN).
IFN-a and IFN-(i are type I--IFNs, IFN-Y is the type II-IFN.
~U~STITLIT~ ~H~~T
GZ a
~,a ~; ~I r~ c.3 :J _~.
~~fO 92/16216 hCT/EF'92100~b~1
- 21 -
The major biological activities of IFNs are antiviral, anti-
proliferative (anticancer), and immunomodulatory activities.
Various forms of IFN are produced commercially as the
natural and recombinant preparations and are used as drugs
for treatment of neoplastic, viral, and several other
diseases.
CTL - cytotoxic T lymphocyte.
NK cells - natural killer cell.
IL-1, IL-2 well known interleukins stimulating the prolifer-
ation of T cells and other lymphoid ce'_'_s including 3 cells;
RPMT_ 1640 tissue culture medium for the growt'n of human and
other leukocytes (abbreviation of the Roswell ?ark Memor~.a'_
T_nstitute, Buffai0);
FCS = fetal calf serum (for assays with leukocytes it has to
be pre-tested because it may contain mitogenic substances
mimicking the action of interleukins);
EMCV -= encephalomyocarditis virus, mouse oicorna virus non-
pathogenic fox humans, it is often used as a challenge virus
in the TFN bioassays;
A-X49 human adenocarcinoma cell line - used in t:ze i=N bio-
assays because of its high sensitivity fo.r. IFN-a,~, and Y.
The line is recommended for such use by the wii0 e~pe=~s on
IFN standardization;
MTT 3-(4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide. Reagent used to measure the cell kill or cell
growth in several bioassays, using the ELTSA scanners
(kiansen et al., J. Immunol. Meth. 1989, 119, 203-210);
m tnhTlTi iT~ C,''Ld~~"T
.7 r~ ~ ~~ ~ .9
~.$ U 9~ to ti
lVO . 16Z 16 PCI"/EP921(10491
_ zz
L929 the mouse cell line, commonly used for assaying mouse
ZFN and human or mouse TNF;
TNF tumor necrosis factor, cytokine, (relatively small pro-
tein, very susceptible to the proteolytic enzymes), produced
by monocytes and macrophages (TNF-a known also as cachectin
factor causing cachexia in humans and animals);
produced after stimulation with LPS (lipopolysaccharides),
viruses, bacteria, and many other agents, very toxic for
many virus-infected and neoplastic cells; may also act as a
growth factor for fibroblasts. Connected with inflammaLOry
reactions. The related form TNF-p (lymphotaxin) is produces
mainly by T cells and some other tolls.
PBL peripheral blood leukocytes, normal human ieukocy=es
from healthy blood donors, isolated from "buffy coats"
(interphase between red cells and plasma). The respons~ve-
ness of PBL from individual donors to various cytok'_ne '_..
ducers appears to depend on a genetic set-up of the donors.
The high responders and non-responders have been identifies.
This refers also to the response of PBL _to TTO,
The variation in the individual response to inducers c~ =FN
or TNF is more visible when Weak inducers a.r.~ useri than whe::
very strong induc ers, like viruses, are employed. This is
due to the fact that the reaction to weak inducers is o_ the
"all or none" type whereas viruses almost always induce de-
tectable amounts of the cytokines.
Tolerance to inducer: Called also a hyporeactivity state. Occurs
after administration of a single dose of an induces, e.g.
after 20 h of exposure of PBL to an induces (e. g. vir:a);
the cells stop further production of IFN. The hyporeactivity
state lasts usually about seven days. It may be complete or
partial. Such reactivity makes the therapeutic application
of strong inducers difficult and/or ineffective. weak T_FN
~UBSTITUTc ~H~BT
92/1b216 pf~~~~i'/1~.
inducers either do not induce the hyporeactivity state or
the tolerance is small.
Several natural drugs which are extracted from medicinal plants
possess immunomodulating properties. TTP appears to be one of
them. TTP alters many different immune functions in vitro and in
viva. It initiates balanced immunostimulation, with the capabil-
ity of non-specifically activating all effector pathways (CD4
helper, weakly CD8 suppressor, CTL, NK cell, and activated
macrophage) without cytotoxicity for norrnal tissues.
TTP has a restorative effect on normal wound healing. Low doses
of TTP weakly stimulate IL-1 and IL-2 production. High doses may
inhibit the cytokine synthesis.
The respective tests with TT° were carried out by the following
methods:
PBL from healthy blood donors were pur.='_ed 'oy amrnoniLm chlcrice
treatment. "'he culture medium was RPMI 1640 plus 1,0~ PCS.
proximately'8 x 1C6 cells/ml were cultured far 20 h at 3?°C, S~
COZ. The antiviral activity of interferon was assayed by ~MC'J
cytopathic effect inhibition in human A549 cells. The MTT method
to measure cell ki7.1 was also used.
TNF activity was measured in =929 cells. T_n order to de='_ne =='a
type, the individual IFN samples were trAated with different
anti-iFN -sera for 1 h. Their antiviral activities were compared
with non-treated preparations.
The tests indicate TTP stimulates the production of endogenous
interferons (IFNs) and tumor necrosis factor ('rNF). The response
is dose-related.
Seven assayed cocnmercial batches of TTP had comparable biolog-
ical activity as immunostimulant and the cytokine inducers.
3~~~~~~~TE ~~~CT
.. , y ,
',i ;; 3 '.? ~ 7 7 j...
~:: ~ E ~-.l P.j i~ a~,1 ~1.
w zm6zy6 ~c°ri~~zioo~~~
- 24 _
Considerable variation in the response to TTP of leukocytes of
the individual blood donors have been observed. PBL of several
donors wrere found to be unresponsive. This may reflect a genetic
background.
Potent polyclonal antisera were used, such as anti-IFN-a
(Cantell), anti-IFN-a Ly (Namalwa) from K. Fantes, and anti-IFN-
Y (Cantell) to neutralize antiviral activity in the supernatants
of PBL treated for 20 h with TTP.
The results of th.e neutralization assays were found to resemble
the finger prints of individual blood donors. In other words,
proportions of IFN types produced by the individuals varied con-
siderably. The separation of PHL into adherent and non-adherent
fractions may potentiate the induced cytokine synthesis.
The hyporeactivity (tolerance) a the induction of ~FN by "dDV
observed 20 h after the initial stimulation of PBL with TTY was
either minimal or absent.
All of the seven batches of TTP tested in human PBL as I=N and
TNF inducers were found to be active in inducing _TFN and/or TNF.
The optimal concentration of TTP for IFN induction was 30-100
p.g/ml'and for TNF induction 100-200 ~.~g/ml. The dose of 200ug/ml
may be subtoxic for PBL, but the synthesis of TNE" occurs much
faster than that of IFN and faster than the devE:i.oprnenL oz
moderate cytotoxici t:y .
The conclusion of the foregoing is the experience that the
active principle in TTP pharmaceuticals is an immunoactive
fraction of an extract from peat containing organic, primarily
bound sugars, aminoacids, uronic acids, hurnic ,=acid substances
and mineral salts including microelernents. Tire LD~O in animals
is >2400 mg/kg per os.
~.. r
hr ~ ~.t r.~ > > a 3 .~A.
V ~2/162i6 PCT/~pf2/0049i
- 25
No mutagenic, genotoxic, embryotoxic, teratogenic as carcino-
genic activity of TTP was found. TTP shows no allergenic pro-
perties and had no topically irritating activity.
The therapeutic indications include chronic and recurring
respiratory tract inflammations and lower leg varicose ulcers,
supglementing the treatment of vaginal erosions, and periodontal
diseases.
There are clinical observations suggesting that TTP may be use-
ful as an immunomodulator in the supporting therapy of several
neoplastic diseases. TTP is used r,_-ally (5 mg tablets) or
topically.
Of particular relevance and importance appears to be the fact
that PBL _treated with TTP for 20 h at 37°C do not develop the
hyporeactivity state because they retain the capacity to produce
IFN after induction with NDV (Newcastle Disease Virus), a very
potent IFN induces.
The attached Tables 1-24 relate to various biological, e.g.
toxicological, hematological and immunological tests. 'these
Tables appear to be self-explanatory and provide for the bio-
logist pertinent information relating to compatibility and bio-
logical activity of T'I'P.
It is worth noting that the concentration of active ingredients
in pharmaceuticals, as indicated in claim 13, may be higher than
in cosmetic compositions (as indicated in claim 14) due to the
following reasons:
Pharmaceuticals are prepared in unit dosages, wherein the con-
tent of active ingredients is under strict control; e.g. in
tablets, the concentration is adapted to 'the size of the tablet
containing the effective daily dose (or a part of it) of the
active ingredient.
SI~~ST1~'UT~ ~~~~ l'
~, r i ;vi ~~ ;~ ,.~ .~.
fft /16226 P~T/Ek'92IU043~
_ 2
The concentration of the active ingredient, e.g. cf 'TTP, in
granules with which capsules are filled, preferably is only 5
by weight in order to achieve a sufficient tablet size and to
allow an appropriate operation of the capsulating machine.
Another reason is that most pharmaceuticals are administered
orally, and the active ingredient is distributed within the
whole body..~Even though it reaches the skin cells in a very low
concentration, the therapeutic effect is remarkable.
Furthermore, .cosmetic compositions - contrary to pharmaceuticals
- are used in rather uncontrolled quantities, applied locally,
with a different penetration rate to different cells.
Some compositions, such as a shampoo which is nearly immediately
washed out, have short contact times with the body or hair, and
may contain relatively more of the beneficial component; others
are applied several times a day and therefore should have a
lower content of the active ingredient.
Finally, as in the case of tooth paste, being in contact with
the mucous membrane i.n the mouth, active ingredient penetration
is much easier than through the skin, and the concentration of
the active ingredient may be lower.
SUBSTITUTE SH~~T
;'.q _::.g ': .~ ~ a n a
hf ~ y v 1J ;"~' .,.
9xmszxs ~~'iE~zi~aa91
;_
TAB. ACUTE TOxI CITY.EFFECT OEZALLY ADMINISTERED
1 OF TTP
ON BIOCHEMICAL F BLOODOF
THE PARAMETERS RABBITS
O
Examined Parameters
Sex Dose Day
of
TT_? Test Creatinxne TotalProteinY-Globulin
;
~
g/kg ~mg%) lg/1) ~9~)
0.0 0 1.090.03 65.0 =3.49 0.53 Ø00
~
7 I 1.03=0.02 62.0--_0.330.50 Ø01
_ ~ 2.0 0 1.06=0.01 58.0 ~1.9b I 0.50x0.02
~
7 I 1.14=0.38 57.8 =1.54 i 0.54-_0.01
5.0 0 ~ 0.96-_0.12 70.3 .3.52 I 0.55-_0.00
~ I
7 I 0.94Ø13 60.0 _2.34 j 0.57x0.00
i 0.0 0 0.80_-0.11 73.8 -_3.330.72 =0.06
I
~ 7 , 0.82X0.13 75.3 -_1.020.79 =0.03
~
I 3.0 0 ~ 0.88-0.05 64.6 .2.36 ~, _0.'0
M . 0.52
j 7 I 1.24Ø34 64.5 =2.03 0.67 =~J.07
,
.,.0 0 0.85_-0.03 58.5 -3.19 0.57 =0.03
i
7 i 1.04-0.12 57.0 =4.00 0.50 -~J.J1
.
'.'AB. ACJTE TOXIC:':"_'. ___"ECT IST:?ED
~ OF '"'"
ORtIL:.'! C~I
ADMI'I ='='-~--
'
.~C'_'IVT_'~'_ ':'P.ANSAMINAScS =N ScRUM ?.a.33I':'S
OF 'rT.:=OF .
'T':?E
I I i
Exam=ned
~a~arneLers
Se ~ Dose i ay r
D o
; ~~mD ~ ~:esL Aianine ~spar ag~.~...
g%k~ j a11t1:1OL~3nSAe~a S2 SL_-35e
llTt;nOL~atl
t ~ ~ iU T_U
_ .-
0.0 __ 0 ; 8.07 X1.20 33.33 ~3.:i0
j
7 I 9.07 Ø33 28.57
2.0 0 ~ 10.33 =1.76t 18.33 =..09
I , I 12 33 .3.52 0.00 x...03
I i
i 5.0 ~ 0 ! 11.33 -'._56- - ''3,33~_.52
I 7 i 11.00 =3.05 20.07 =..06
0.0 0 10.33 :.-1.76 X3.00 =3.05
~ 7 ~ 12.00 =2.04; 25.67 .4.'.'i
M 2.0 I 0 9.00 -_1.00~ ;8.00 =..52
i '
7 9.00 =0.57; 20.66 . .53
i
5.0 0 8.00 -_1.00I - 00
18.00 =1
7 11.33 -1.66; 23.00 :
=
~~ c~~~~~
s; . ~ CJ s;~ ry to 'a
.,
r.r L~ ~~i m.) s1
~z«6z~6 ~~rr~z~~io~a~r
- 28 -
TAB. 3 ACUTE TOXICITY. EFFECT OF ORALLY ADMINIST~t~
TTP ON THE I~riIATOLOGIC?.L PARAMETERS OF RABBTTS
Examined I
parameters
Sex Dose Day
of
TTP Test Hemoglobin Hematooszt Leukocytes Erytzrocytesl
I ~ ~ -
-
glkg (mg%) (%) I x
x 10
10
0.0 0 12.4=0.4438<3_1.857.32 =0.435.21=0.20
I
7 1 11.8=0.6337.7.1.206.30 x0.661 -_0.11
1 6.11
2.0 0 11.5._0.3136.7Ø885.69 0.531 _0.65
I I 5.d6;
r
i 7 1 12.3_-0.4838.30.56 5.74 =0.76j --0.'.
i 5.69
j 0 112.3_0.1440.7--2.57.35 _0.991 Ø20
5.0 1 5.42
I ~ 111.6=0.2337.3Ø33, =1.791 -_0.19
! 7 8.6a 5.08
0 ~ 0 X12.0=0.8637.3X1.20111.10_0.631 =0.26
0 -~~3
, I 7 111.9=0.6135.7.1.8510.80=0.941 =0.79
' .~..i4
!
!9 0 112.3-_0.26X43.7-4.80~ =2.001 _0.65
! 9.94 ~.tn
2.0
7 11.8=0.39134.%=2.02111.06_1.081 =0.50
~.37
5.0 j 0 j12.7Ø6338.7_2.40j =1.361 :0.10
9.52 -.9
jl0.oX1.04,4Ø2.08I -_1..3! _0.6a
8.87 ..,_
'_~~B. -. C:IRONTC =~OXTCT'='<_'. ==~5C'.' OF ORALu..Y aDMTNIST°?c-0
T'.'? ON '.'HE MASd OF T4;; ORGANS OF ?ABBI'=S
Dose of TTP (mg/kg)
I
i '
I ~ 0.0 ! 60 i 150 ~ 0.0 I 60 160
;Sex ; .
(Organ ! Mass of Organ '_a ~ of Tocal Mass
j t I I i
jLung 0.72 X0.08 0.61 =0.1710.67 Ø0? 0.50 -0.05j0.54 _0.0910.1 =0.:.
I I
(Heart 0.23 Ø01 0.22 Ø02 0.25 _-0.03 0.24 -0.020.23 ~O.OtIO.?3 _'J.J2:
I I I
ISoleen 10.057=0.01 0.051=0.000.079=0.02 0.05 ~0.01~O.Oo =0.01i,0.J4 =O. J1
I i i ~ I, ;
jLiver 12.35 =0.1513.02 Ø08 3.22 -_0.37 2.32 =0.091'.77 _J.2813.12 -O.Jo.
1 ~ 1 !
1
:Cidney 0.59 =0.030.52 =0.0210.60 ~0.01I0.48 X0.0310.6_ _J.0110.49 -O. J3
.,
Adrenal
Gland 0.0160.01 0.015_0.01 0.020=0.01 0.013=0.01'0.013=0.02!0.012=0.0
Ovaries I I ~
with
Uterus-0.58 _~0.04 0.39 =0.02 0.60 Ø04 - -
TestiC
ales ~ - - - X0.30 -0.01 0.32 _0.0310.30 _0.02'
~U$s~s~U~~ ~~"~~~T
,~-.~;:
,a 3 S (.~ e. ~ ':9 ~,.t
. 92/ 1 b21 b PCT/EP92/00491
- 29 -
Tpg, 5 CHRONIC TOX%CXTY. EFFECT OF Ok~ALLY ADMTPIISTERED
TTY ON THE H;rMATOLOGICAL FARAMETERS OF RABBITS
Examined parameters
Sex Dose Week
l
TTP of Hemoglobin HematocritLeukoc yT~esrythsocytes
E -
'
g/kg Test (mgt) (~ ) x ,
I 10- x
10
0 0 12 X0.05138.2.1.71i 9.33=0.7915.10Ø56
0 2
N! . 7 . =0.1540.7=0.331- 9.39=1.1816.34-_0.23
~ 12.3
6 3.0 0.35141.7=1.30 9.38=1.1217.32=0.70
,
i 12 13.6 =0.19'40.7~0.57j,11.23 _1.1015.30Ø46
SO ~ 0 12.9 ~O.d6135.7=1.821 7.62=0.7815.14;0.47
I
1
6 13.3 -u.35d1.0=:c.10~ 0.00=1. 0.3_".;.
";
12 13.7 =0.11j39.3=0.94 9.36=1.0?I6.19=0.19
i ' 0 X14.20 =0.2?36.7=1.811 8.19=0.1515.34.'J.;6
150 '.
I 3 11 25j 39.5=1.531 3.33=0.'815.6d=0.-10
05 =0
i ~ 6 . . =1.451 3.18=0.0610.34='J.10
(12 =0.53f37.3 .
40
I ~ 12 . Ø32!39.0 =0.63 8.24Ø0-110.~4=0.54
~ 113.70 .
0 ~ 0 112.6 =0.=7160.2 :0.831 7.07=1.16i~.".,=0.17
j
0
. ~ 3 ~i2.1 ~0.~3~39.3 -0.721 3. =1.011.,.'~=J.12
i6
j
i ; 5 112.4 =0.86142_3 -1.20; 9.79=t.69~...36=0.?4
i ',~ j12.5 =0.87137.7 =2.181 10.?0=2.1614.00wJ.~9
i : 0 113.2=1.20135.0 =1.701 7.44=0.9615.47=~J.50
; ;
50
t t
I~ 6 113.0=0.55139.3 -1.201 7.25=0.96i7.08=0.?4
.
i 12 112.3=0.21/3'.. =4.331 9.31=0.35io.~'_'=0.32
y p '.'S.' _1.15138.3 =1.401 8.50=0.5416. =0.'J8
15p J9
i 3 '13.1 _1.10147.0 =2_901 3.10=0.76j5.35=~7
57
0 12 =0.92j41.0=1.15j 6.35-0.3115.61_~
5 30
1
~i I 12 . =0.701-'=1.'J=I.OOj 8.15=l.5di~.92='J.?3
13.1
~IIBSTITUTE SH~~T
;~ ,:, ,r., d~ " t' 4
i a ,,
r'-, i. C i c3 is ~. !.
1~(3 »/1~21G PC:l'/E~2/00~197
'TAB. 6 EFFECT OF TTP ON THE PHAGOCYTIC ACTIVITY OF
NEUTROPHTLS IId THE PERIPHERAL BLOOD OF RABBITS
Phagacytic
Activity
Neutrophil~s ~
Neutrophiles
Group n (1000/1 NBT+ L.H L.W
mm
)
0 3 6 0 3 6 I 3 6 3 6
I 0 I
U
Control
1 ml 6 1.9 2.3 2. 14.315.117.6154.560.659.81 7.:3'.511
?3S 7.53
v .0 1 4 y4 ~4 O~t9.4 .4.3c6.8I~0.77 -_0.86=0.4
i 9 1 6 6 -3
0
4
. . __ _ . _ .
. . . _ .
TTD I,
(Test *O *O *O * * * I
1)
5 mg/kg15 i.2 1.718.426.327.2 69.374.98.751U.39.38
2.0 59.2
_.v. I~0.9 _0.7.7.1.7.1Ø7_7.0~=0.7_-7.0=7.91-i.18-i.?S=1.0;
I
I TTP I ~ I
I ('='esta I xo *o *o *o ~c , _
z y .
15 mg/kgi 2.8 1.6 1.615.522.125.563.069.576.Si 3.5:3.:0
-.i6
ii. I =0.8_0.5'_1.0'_4.9_-7.0=9.11_7.0=7.1=3.91_0.33 -i.04.1.0
v.
.. Number of Test Animals
?9S ohosDhate bu=~er solution
0 3e.ore the administration o~ '~'='?
? 3 days a=ter the administration or TTP
6 6 days aster the administration _or TTP
* Statistically significant variation at ?<0.0S
in relation to the value before administration of ':
o Statistically signif'_cant variation at p<0.05
in comparison to the control group
~iJ~S~IT~ITE ~~I~~T
., ~~a ~.~ i i ~,:1 al ~.~ _~.
.92!16216 F~CT1~~°92JOii~191
- 31 -
'.CAB . 7 EFFECT OF TTP ON 'THE NUMBER OF LYMPFiOC~T~S ANn ON
~ PERCENTAGE OF ~ T-LYMEFiOCX'~5 (E-ROSETTES)
pip OF B-LYMPHOCXTES ( EAC-e'2USET'I'ES )
2N ~'FIE PERIPHERAL BL0017 OF RABBITS .
Lymphc~cyt~s ~
~ E-Rosettes
~~
EAC-Rosettes
x I
10
/mm
Group
n 6 6
0 0 I
0
I
6
Control
6 I
1 ml PBS .6 .0 9.0 0.1 42.1 ~ 42.3
i.v. .2.0 .1.9 _.,.7~~.~ !
=6.3 ' =2.051
TTF? (':a5tI o
1 ) ~ 6.6 5.4 19.2 27.1 43.7 4'7.7
S mg/kg 15 -1.5 =1.4 a4.1 =3.2 .2.6 ~ -4.9
,
I
TT? (best I
mg/kg 6.0 6.8 17.0
2) ~ 8 _1.0 I I ~
_.v. j =0.5 '_1.9248
I
41.5
I;
41.0
~
.2
9
;
_i.~
~,
-2.1
n Number of Test Animals
PBS ohosnhate buffer solution
0 3efore 'the administration oz TT?
6 6 says after the administration or TT?
Statistically significant variation at p<0.05
in relat'_on to the value before adminisLrarson cr 'L""'
o Statistically significant variation at ?<O.OS
in cornDaraon to the control group
T?,a. 3 NUMBER OF SPLF.:IOC'_'TES AF'='ER LONG-TER."'1 ~'vE?.A?v X11"- ~
~~~~°
ConL::ol~ TTP 1 601og~Kg."a:~
10 TT1 '
mg/kg.day
n
=
10
.rime v ?FCiI~~ SDlenOCyLeS
O
nmp_
in rla~kss4thDay 7th Day 4th Day Day 4th Day .~..Day
1 7th
3 I 367-_157 =41 1120=328* =240'! =236x'61=57
156 1 336 772
5 1 572.134 _47 11528_34'6* =62*j1239=280x232=90
138 292 I
7 I .45 133 .40 1 =136* =44 ~ =258'r141=63
518 699 159 722
9 I 1185 ;75 I .94 .36 ~ -_138167-62
466175 395 132 412 l
12 I =18 1S3 =30 1 .131 =39 ~ .130167=59
287 287 156 376
* Statistically significant variation at o<U.OS
in comparison to the control group
~t i'R~TiTI 1'T~ ~~~~~ly
r:d' ~.~ CJ C7 1~ '.~ v
~Yi' '213~Zt6 PfT/~F92/~191
- 32 -
TAB. 9 CONOENTRATION OF THE ANTI-SFxBG (19S+7S) AhTTIBODIES
AT KONG-TERrS Z'F1X WI'IH TTP
Control TTP a0
TTP pg/kg~ay
mg/kg.day
n ~ 10 n = 10
Time
~
of Hemagglutinin,
-log2
of Titer
TTP
Doses
in
Weeks)4th Day 7th Day I 4th Day 7th 4th Day 7th Day
Day I i
3 ~3.7St1.787.12~1.36I8.80t0.97* I8.20t0.40~6.90.1.37
9.2C1.46* 1
5 5.8 2 6.50ti.0017.70*0.84* 7.70v0.64*9.33~2.90T~~
1.40 9.10*2.02*
7 48 1010.83* 13.50=0.8313.>j=1.21
13 11% 4 i
0 40t1
60 74~4
20=0
6
. .
. .
= .
.
.
i
1 j3.2011.404.66~1.24I3.50=1.28 12.9011.226.33=1.52
9 4.0010.81
12 14.80 0.74 4.40~1.0115.11=1.09 6.60=1.51 15.11=1.19 5.1C=1.74
* Statistically significant variation at o<0.05
in _comparison to the cone=of group
TAB. 10 CONCENT:2?TION OF TEE ANTI-SC'2BC (7S) ANTT30DIES
AT LONG-T°R~! THERAPX WITH TTP
Cons=of mg/k T'"P ;ng%kg
TTP I 50
10
1 n = 10 n _- 10
Time
'
of Hemagglutinin, log2
- of Titer
TTP
Doses
in
Weeks)4th Pay 7th Day 4th Day 7th Day 4th Day 7th Day
I I '~
.
3 1.75=0.606.37=0.8513.200.87* 5.9U11.703.80;0.87*4.90=0.741
I
5 12.50=0.706.12~1.76I2.20=0.97 5.70*_1.1012.30=1.415.33-0.74x1
7 (1.00*0.893.801.0910.60=0.80 2.50=1.11'"~0..55~0.832.55=0.68!
9 10.40=0.483.66=1.2410.3010.64 3.25=0.9610.00 4.331.24
12 0.80Ø973.80=0.7611.11=0.994.401.3511.55-1.494.30.1.25
* Statistically significant variation at p10.05
in comparison to the control group
SUBSTITUTE ~HE~T
/aL~i.iBJ~~~,~.y.
VdCD 92/16216 P~"d'/EP92J00491
33 -
TAH . 1 1 zMP~ITJIdOM01?ULATORY EFFECT DF TTP ( ~SD }
Dase n PFC/10 ~ E-RosettesHemagglutininxHemagglutininxl
TTP n Splenocytes Type 19S Type 7S
mg/kg I 4th Day 4th Day + 7S 7th Day .
4th Day
Contzol30 469 13.6 5-4 82
_111 j d3.2 X1.1 -_1.3
x x
0.5 10 997 15.6 - 6.8 8.4
.139 .1.4 X1.0 ~1.1 j
x x x . x
i
2 10 839 20.3 6.9 I 9.7
. .177 .4. 1 =1.0 -1.4 i
5.0 10 746 23.2 7.2 9.8
.129 .4.7 I =O.o ;1.4
i
x '~ I
x I
I 18.4 ' 8.3 ! 10.-:
10.0 j ~ 795
.0
.1.1
l .729 ~ .4.0 ~ -_1.3 I
j ! . ~ ~ :<
'
. 3
'_5.0 ~ t 560 I 16.3 ~ 6 1 ;
,
i ~ ~ =145 ~ _?.5 ., i -__1.'
, '
' 50.3'; j 100 14.' i 5.3 I 5.~
10
I I 57 j _-4.0 _-1.0
=1.9
! x x ; x
! 100.010 375 11.3 ! 5.1 . .7
:
~ _ I
. 67 .2.'_ ; -0.3 .o
, _.
"
x :L ~, .'
250.0 I 30S 9.5 j 4g ''3
10 I
-?.Q I -_1.i '1.7
_ 67 -
x Statistically -significant variation at total comoar'_san O.OS
(Student t-Test)
-log2 of titer
_ . . .., ,-,-... a T, e-r 1"' C' L.1 C= ~T"
s
'i .
3_~ I_i r~
w~ ~~m6z~s ~~.-ri~~zioo~9~
- 34 -
TAg. 12 TTP AI7MTNTS'PER~I7 DAILY IN 3 DOS;rS
9th After 10th Day After
Day
Immunization Immunization
with SRBC with SRBC
I
' Hemagglutinin Hemagglu tinin
rP n 6 of titerI -log2 titer
Dose T l of
~ E- PFC/10og2
~ -
RosettesSoleno- -
7S 195 - 7S
7S~
oytes 19S
+ 7SI
'Control 15 13.7 469 5.7 0.3 ~ 9.8 3.4
I i ',
l .3 .125 .1.3 .1.0 I .1.3 .?.5
1 0 ~
0
3
m .
x
.
Control 15 I 15.3 579 6.1 0.5 ~ 9.0 3-5
I I ~ ~ '
'
1 ml 0 .143 -_1.1 ~_0.9 ; -1.3
0 I =4 I ~ -_?.7
. .
x
i 10.
..-~, mg% 1 ~ 1 ~ 364*7 . ~ I s~' . .
kg 7 9 . 4' .
4* i
I
I1 x 0.3 .3.3 I .190.1.1 ~.1.? , _1.0 .'...
ml I I
~ 15 19.0'' 769* 7.1* 0.5 ' 9.5 ='.0
/k I ~
m
;'
g ~
g
_.
j ~ ,
3 x 0.1 ~ ; _5.0 I .132=0.3 X0.3 i -1.0 __.0
ml , ~
~10 mg/kg ~ I i9.3*I 795*7.0* 0.3 ~, iU.4* ~.~*
13 I ~ !
3 ml I ~ .4 I -_102I Ø7 .i.0
I1 ? I -_1 .5 _'
0 .0
. .
x
I10 mg/kg 15 17.5 ~ 650 7.3'~ 1.3 j 8.9 3 3
~
~ I f
1 ml 7 I .1291.4 .1.. I .1.3
0 ~ .3 ~
3 ~
. ~ . _
x
,
SUSST1TUTE SE-l~~T
F:; i i ; 9j ;> ~..E .~.
9'YO 92/1~SZ16 PC~1'/EP92/00491
- 35 -
TAB. 13 EFFECT OF TTP ADMINISTERED ORALLY FOR 12 WEEKS ON THE
IMMUNOhOGICAL RESPONSE OF i'dICE IMMC3NIZED WITR SRBC
4th After 10th
Day Day
After
PFC/106 PFC/10
Hemagglutinin Hemagglutinin
I
Weeks
E- Spleno--log2 % E- Spleno-~-log2 titer)
of I of
titer
Rosettescytes Rosettestyres~-- ,
19S 7S ~ I'19S
+ 7S1 I - 7Si
7S
15.2 76~ 6.0 1.6 14.7 256 ~ 8.7 ~
7.9
'
3 -3.8 -~6 _0.9 .1.3=3.4 =57 j .1.0 ~=1.0
j
7* 1033* 7.5* 2.9*23.3* 406* ~ i0.4*I
23 9.7*
. =177 -0.3 =0.2=3.7 -88 -_1,2 I=1.0
7.8 I I
' 13 422 5.1 0 . 12.7 209 7.1 ~
8 ~ ~ ~ 6.7
. .53 .0 0 j -_2.i =41 ' _1.4 i_1.6
4 ~ 9 I ;
.2
. 933* . ~ I 20.* 430* I )).Ox*
23.0* 7.6* 2.7X: ~,'1.0
I, =5.1 X248 =1.5 ~,=i.3. ~2.7 -35 _ . .1.0
I ~
! 12.3 495 4.6 ~ . 14.0 245 . 3.9
i ~ 0.3 I
I -2.9 .85 .1.1 I=0.9' .5.1 .48 j -_1.6,=1.5
7 I j '
;
20.3x 1120" 7.n* ''.lxj 26.9"', i, . 'J.3"
~ .?;
437
I I '_214 =0.6 '=1.5; _7.7 ', ' _ =0.-1
~ X5.3 ~ -117 .0
I
' ; 15.0 . 531 I 6.2 ; ! 14.1 i 3.', ~
0.3 294 3.4
.
9 I -_2.1I -_67~ _0.8 ~~0.?I .2.3 I -1.3 ~=i.5
=53
I i8.6~'j 1049xI ,.7x I ~ 18.?x ; , .',.9xiiO.dW
1.9 id's '
.2.9 I .184~ Ø7 ~ ! -3.-1 I ; Ø3 .
-_1 -630 .1
.? .'J
I 15 ' 373 7.2 j ' 16.3 ! ~ 7.3 '
0 1.s 256
12 . X143 I _-1.0I=1.2~ 2.5 ~ j -_1.1;_0.9
.2.6 -5c3
14.1 ~ 770*I '.2 I ! 19.0 ~ .1.3" !i0.6'~~
1.- 294
-_2,7 -_132 ~ =1.4 ',-_1.".j =4.i _63 ' _~J.=!.1.0
i
JtatiStl.CallV si.gni~'_canc variation at p---0.05 in comparison ~o the
control g:oup. ~n eac:~ groua, t.~.e.e we_e ~30 an~.mais.
r.~ tr,r~~rnrt tT~ '~NF=~T
G'J . h 's~ b.i° i,a 7
~t ~~ L.'I e,~ t1 ,N ~~
W~ 'i~6216 PCT/E~2J0~0491
- 36
TAB..14 INE°LUENCE OF THE STORAGE CONDITIONS ON THE ACTTVITY OF
TTP IN VIEW OF THE ABILITY OF FORMING E-ROSETTES
$ E-Rosettes
after two months storage
Dose n Starting ;
Activity Temperature At Room ~ At Room
* 4°C Temperature Temperature!
Light Admittedlin the Dar~i
Control 8 I 13.0 .?.4 !~.o ~~.3 m .o -~.a
I
0.1 mg/kg 8 I 15.8 .1.7 16.1 _~3.04 15.1 X2.9 j 16,0 =S.5
I
1 mg/kg 8 ~ 18.0 -0.8~' ~ 19.8 =4.1* 15.3 =2.8 ~ 19.8 =2.
_ ._
mg/kg $ ~ 20.4 =4.2* i8.0 =4.5x 16.8 -2.4* 10.9 -~
* Statist'_cally significant variation at a = 0.5 in compa;_son ~:~ t.._
control group
TAB. 1.INFLUENCE OF T::E STORAG'c CJNDITT_ONS ON Tr-T.E AC'='=V='_"'_' O=
TTP IN VIEW OF THE NUMBER OF CvI.LS ?RODUC=NG
ANTIBODLES OF THE TYPE 19S
?~'C/106 SolenocyLes
I i after two months storage
Dose ~ ~ 5tar~ing
I Activity I Temperature I At Room at 300:
-4°C ~ Temperature "='e:npe=s-~=°-:
I
I i I , Light Admittedl in t.._ ~ar::.
I Control a ~ 571 X59 ~ 514 -_128 ~ 514 -_128 5i4 =i_3
0.1 mg/kg 8 i 747 =144 ~ 1039 -325* i '718 -_135 '11002 =?'0't
1 mg/kg 8 1204 =155* 1026 =314* 793 _~186F 1046 =331
10 mg/kg I 8 1075 ~232'* 1070 _249* j, 869 ~160x ~~', 848 =15
* Statistically signi'icanc variation at c = 0.5 in comparisc.~. _~ _.._
control group
~UB~TiTU~T~ ~H~~ i
L~ ~~ ~ :f v3 ~.
.1V~, ~Z/Ib~lb P~T/EP92/(~91
- 37 -
TAr3. 16 INFLUENCE OF THE STORAGE CONDITIONS ON THE ACTIVITY
OF TT° IN VIEW OF INFLUENCING THE FORMATION
OF ANTIBODIES OF THE TYPE ANfi I-SRBC 19Sø7S
-after
two
months
storage
' "''
n S ring
tar
Activity TemperatureAt Room At
Boom
Dose +4 C ~ Temperature
Temperature
LightAdmit~edin Dark
the
Day 4th 10th 4th 10th4th 10th ~ 4th 10th
I
Control 8 5.6 9.8 S.0 9.5 5.0 9.5 l 5.0 9.0
~
=0.9i0.6 1.1 .2.1~1.1 .2.1 I=?.1 .2.1
0.1 mg/kg 8 7.4 11.4 7.8 10.5~.4 10.8 j 6.5 12.6
=0.4*_1.3*~_1.0*s1.7*=0.8 =0.7* j=0.7'~_2.2*i
l 1 mg/kg ~ 6.8 12.2 I 6.7 ?3.85.? 13.2 I o.3
8 ii
.3..
;5.3*=1.1*Is0.4*_2.1'~=~J.3* i=i.3*=t.3m
=0.4*
mg/kg 8 5.4 11.0 5.2 12.04.8 10.5 ( 5.0 13.5
I
-1.2=1.4*-0.6 y2.3*l e2.a~ i~O.S -1.3m
_1.3
R StatiStical'_y signi==cant ;variation at a = 0.5 i:~ comparison to the
control group
TAB. . 7 INFLUENCE OF THE STORAGE CONDITIONS ON '='HE
~1C':'T_'~T_'~°v
OF '="='? IN VT_EW OF INFLUENCT_:1G THE F ORMATION
OF ANTI90DIES OF THE TVp~ ANTI-SRBC 7S
l l aster
~:~o
months
storage
Dose ~ n Starting
I
act_vity Temperature At Room at boom
;
-4C I Tert~oe=azure
'~emperatur
I l Light- 7arr.
l admittedlin
~
Dayi4th 1 4th 1 4th 1 Ot'.~.4tht Ot'.~.
Oth Oth ~
I ~
Control 8 0.4 9.2 0.3 8.1 0.3 8.1 0.38.?
t ~
0.8 ~ Ø7 .1.4~ .1.4 =0.'=1..1
Ø7 Ø7 l
0.1 mg/kg 8 0 10.8 ~ 0.7 9.3 1 10.0 O.i.1.3
0.8 ~i I
~1.U*=0.9 =0.9*=0.9 -O.o~' -_0.-1.4~'~
~
l
1 m /k 8 0.8 10.6 0.3 11.50.7 1U.3 0.711.0
9 g
'-0.9=0.4*."Ø7=1.8~=0.9 =0.7~ i=0.9-1.0*i
I
10 mg/kg 8 0 10.2 0.3 9.8 0.3 10.- i 10.3
I 0."
t1.3*=0.7 =1Ø0.7 =2.0~ ~=0.9=0.9~i
* Statistically significant variation at a = 0.5 in compariso:i to the
control group
~i IF~ST1TUT~ S~E~T
s;:
(.f ~i e7 t~ i.~
WO 1 b2 d 6 PCI'/~P9Z/0049 d
38 -
TAB. 18 HEMATOLOGICAL PA.RAMIr"TERS FOR HEALTHY VOLUNTEERS BEFORE ANIJ
AFTER TWO~WEEFC ADMYNTSTRATIONS OF TTP AT A I70SE OF 1 MG/DAY
Placebon=6 TTP n=5
Parameter
0 14 0 14 days
days
Eryt:~rocytes
I I 18 32;
4 0
x 10 4.44 .Ø464.32 ,0.454.32 -_0.32. .
/u1 .
Hemoglobin
I
g/dl ~ 12.4 t2.5 11.9 ~.8 i2.8 Ø8 11.8=0.6
Hematoc~it
I
38.1 .5.7 38 -6.9 38.7 _2.1 38.1=1.6
Leuk~cy~ces
'
x 10 /u1 5.25 X0.59 6.27 =0.8 6.23 -_0.54.86-0.6
i
Neut=oohils ~ ~
-
i ~12 57.6 =11 ~ 64.2-2.6 66 -3.6
62.6
Lymphocytes
26 X7.5 30.4 -8.3 34 _4.1 32.4~
.3
'='hro~booytes i ~ '
x 10~/~.i.l ~ 220 =40.4 214 .35.8 I 221 -_38 214 _39.o j
3lood Sedime.~.c'
mm/1h ~ 7.6 ~9 6.6 .1.9 ~i ,.3 -2.~ 6.9 .3.:
StISSTIT~JT~ SH~~T
4~' l l1 ~TC ch ~ i ~,! 'T
~~s 9,.y tJ C~ '~ ~,. .P
a ~z~mzi6 ~cr/~~>ziooa9~
- 39 -
TAB. 19 BIOCI3EMICAL PARAMETERS FOR HEALTHY VOZ.U~ITEERS BEFORE AND
AFTER TWO-Wi~EIC ADMIPdISTRATIONS OF TTP AT A DOSE OF 1 MG/DAY
Placebon=S p -
Parameter
0 14 ays 0 14 ays
Total 72.8 X1.76 68.42.3.2 73.8 _-1.9270 ;a.3
Protein 163.2X2.5 63.8~2.3 '63.5=1.8 54.4.2..
g/1
Albumina
3
Globulinat I r0.4 3 =0.5 I =0.5 2.3 _0.-'.'.3~
~ 3.1 2.8
Globulina2 I X1.9 6.8 ~1.5 ~ =0.9 06.9Ø?
~ 5.95 0.93
Globulinp ~ 14..1-2.2 14.2_1.5 13.3 -1.9 '3.2_1.3
- -
GlobulinY ~ 112.2.i.5 12.1=i.3 113.7-_0.9.2.3.i
_ -
-
IgG g/1 110.3.1.1 10.2~1 11', _'.o '1.'=i.-
- r -
-
-
TgA g/1 i -_0.6 1.g _~.j I .j ..9 =0.3
t.8
gM g/1 ~ Ø3 1.4 _0.2 I -__0.21.; ='J.-
1.4 i.1 .
i
I Complementg/= I Ø1 1.1 -_0.1 ! -_0.2 0.. Ø.
C3 1.1 ,..
Complement g/1 j =0.5 9.3 =0.041 Ø0 0.? _O.
C4 0.2 0.2 J3;
i -
Aianine 134.2 _0.05 0.3 X0.04130.?=9.'_ 3i.=
U/'_ =3.?
Asoaragine U/' ~32.d =4.4 40.2 =5.5 ~30.-~ .'... 39.x =5
~U~STiTUT~. S~~~T
KM::Ir'>~h~$rr_~.
F:a ~.y ~~.~ a~ E ~ wa
W( J16216 PCT/11'92141~91
40 -
TAB. 20 COMPARISON OF THE FREQUENCY OF ACUTE >:NFECTIONS OF THE
RESPIRATORY TRACTS DURING THE LAST QUARTERS OF THE YEARS
1989 AND 1990 IN RESPECT OF TWO GROUPS OF PATTENT5 '
TRF.ATEn WITH TTP AND PLACEBO RESP. DURING OCTOBER 1990 ,
TTP
applied
during
the
4th
quarter
1990
i
n 4th quarter4th quarterstatistical
1989 1990 signieicance
(no TTP) (with ~~p"
TTP)
average average
number number
o o
infectionsinfections
per patientper patient
Cold 20~4.0 -__1.31.0 -4.5 < 0.01
~
Sore 203.3 .1.3 0.8 -0.G < 0-01
throat
Fever
blisters 202.1 .2.2 0.3 -_0.4~ . 0.01
Caugh 201.4 _1.1 U.3 ~O.o j < 0.01
Bronchitis200.3 =0.5 0 ' ~i j
PneumoniaI20~ 0.2 Ø90
Placebo
applied during the 4th quarks= 1990
nl 4th quartez~ 4th quarter ~ scaL_s~ac~a~
1989 ~ 1990 evaluation o.-'.1
(no '='~'?) (with TTP) difference"c"~
average average
number o~ number o~
infections infections
per patient per patient
. _
Cold 20~ 3.9 =1.4 3.1 ~1.> 0.05
Sore 20 2.3 .2.0 2.0 .1.7 > 0.05
throat
Fever
blisters 20 2.3 ._1.8 1.7 .2.1 > 0.05
Cough 20 1.5 -1.5 1.9 =2.0 > 0.05
Bronchitis 20 0.1 Ø3 0.1 Ø3
Pneumonia 20 0.1 Ø3 0.1 X0.3
~~J~~T(TUT~ S~E~ T
y ~ z
t~ 3_~ U ~.~ i~ ;:G _~.
WO . 4J 1 b216 ~ PCd'!EF'92JOU49 ~
- 41 -
TAB. 21 EFFECT OF INTRADERMAL INJECTION OF BACTERIAL ANTIGENS OR
PHA (PHYTOHEMAGGLUTININ) IN PATIENTS TREATED WITH TTP
Tuber- Strepto- Staphylo- PHA NaCl
Patientculin lycin cocci/
Ser.(Init- RT~3 0 anatoxin
No. ials)
pre 3 3 pre 3 pre3 pre 3
(pre , I I I
I
1 I B.T. + + - ' ~ '
. ! , = I
_
2 P.E. + _
,. _ I _
_ _ _
- I
I + t + _ - -
3.1 + + + ~
M.H. ~+ -
I - I
-
I
'
4.I - + ' '
W.H. - I
I - -
-
., .. ~
! ~ ..
'~. - -
. ~
-
.
i
,
I
_
.
I
_
I
.
.
I
.
I
o.':
Z.N.
I-
-
I=
+
I
_
-
~
,
I
I
I
I
t r
7.; I r I
P.J. - .~ I
I
i
-
-
I
r
-
_
_
I
-
I
9.' S.M. ~- - !- ~Y ~~ i - j - i _ !
pre pretreatment
3 after 3 weeks
TAB, z? EFFEC'~' OF INTRADERMAL IN~"EC';'ION OP' 9AC'~'ERIAL ANT;GENS
OR PHA IN UNTREATED (PLACEBO) °ATIENTS
Tube.- Strepco- PHA I
I Staphylo- NaC=
!
Patientculin lycin ~
l cocci/ I
Ser.I(Tnit-RTZ~ 0 anacoxin
;
No. ials)
' e re I I
I ~ I pre pz'-
3 3 I .
I 3 3
~ '
pre
'
3
pr p
1 M.M. I ~ ~
. - + -
I _ T j
-
~'
-
,
I ' ,
. r _ _ I i
z. o.. ~ _ i
I I
._
_
,
,
! ~ + -
3. O.H. I+ ~...+~I + _
j + i
-
i,
-
I
4. W.J. I _ _ - - -
I - ~ - i
~ -
-
K.C. ~ + + - p ~,
_ _
- -
-
I -
b.~I M.J. + ++ ~+~. ~+ ~_ - I
I- -
- i
I
7. H.W. - + + - _ _
- + r
-
8. K.T. I
( _ _ + + + _ _ -
I , ~ I ~
+ -
i
pre pretreatment
3 after 3 weeks
~IISSTI"I~IJT~ ~#~FFT
1 ~:' s.
~r .~ G.t e.'? 1 ~ ~..~ .,.
ii~C) 5 6216 PC.'T/~~92/0~~91
- 42 -
TAB. 23 xMMUNOGLOHULIIVS IN THE SEFtt3M OF PATIENTS WTTa:
UI~CUS CRURZS TtZEATEI? WSTH TTP
n Test Group Immunoglobulin in
I ( mg
before after
Treatment Treatment
I
1 I 1732.872207.327 ZgG 1732.87.207.52
Ig 1 1
G
8 A 2 IgA 304.25 _ 47.508 IgA 283.87 . 44.10
~ I I I I
' I 3 ' 202.87 ~ 71.129 IgM ~ 189.75_- 75.15
IgM ' I
~
~ ~4 ~ 1 1924.371246.2510 IgG I 1972.00-_239.33
IgG ~ 1
8 g I I I 236.50_ 59.841 I j 285.50-_ 56.76
a IgA 11 IgA
6 ~ I 242.12. 56.95~ I I 532.7._ 65.;5
IgM i2 IgM
1 1 7 D O.OS ~ 4 i0 ? 0'05
StatlStlCS1 ~ VS > O.O~ 'IS >
Evaluac;on I 2 a D 0.05 I J 11 0.05
oz VS > VS 12 ~
3 9 p 5 ~
vs > vs
I , ~ 7 i0 ~ O.JS
Di=?ezence 1 ; 4 ~ 0.05 vs >
Vs > 0.05 I ' o.os
j 2 ; ~ 0.05- 1 a i1 ~ 0.05
VS > VS >
3 o a 9 12 ~
vs > ws >
SUSST1TUT~ S~~~T
