US2002012708

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Page 1

as) United States

a2) Patent Application Publication co) Pub. No.: US 2002/0012708 Al

RUEPP

(43) Pub. Date: Jan. 31, 2002

(54) USE OF ARTICHOKE (CYNARA) EXTRACTS

(75) Inventor: MICHEL O. RUEPP, REMSCHEID

(DE)

Correspondence Address:

BROBECK, PHLEGER & HARRISON, LLP
ATTN: INTELLECTUAL PROPERTY
DEPARTMENT

1333 H STREET, N.W. SUITE 800
WASHINGTON, DC 20005 (US)

(73) Assignee: arr Pharma

(*) Notice: — This is a publication of a continued pros-
ecution application (CPA) filed under 37
CER 1.53(d).

(21) Appl. No: — 09/225,573

(22) Filed: Jan. 5, 1999

Related U.S. Application Data

(63) Continuation-in-part of application No. PCT/EP97/
03561, filed on Jul. 5, 1997.

(30) Foreign Application Priority Data
Jul. 6, 1996 (DE). 196 27 376.5
Publication Classification
(51) Int. Cl’ .... . AGLK 35/78

(52) US. Ch 424/725

(57) ABSTRACT

The present invention relates to a novel use of artichoke
(Cynara) extracts, especially dry extracts, optionally in
combination with Echinacea extracts and/or nettle (Urtica)
extracts, for the preparation of medicaments, and to orally
applicable medicaments.

In particular, the invention relates to the use of artichoke
(Cynara) dry extracts, also in combination with Echinacea
and/or Urtica extracts, for the preparation of medicaments
for the treatment of diseases of the small intestine (damages
from medicaments or infections), of the bone marrow (apla-
sia and insufficiency, for example, as a consequence of
agranulocytosis caused by medicaments or radiation), thy-
mus (dysfunction, aplasia or hypoplasia), spleen (dysfunc-
tion), lymph nodes (aplasia or hypoplasia due to damage
from medicaments or radiation), for the adjuvant treatment,
also in combination with chemopharmaceuticals, of analge-
sia, liver, pancreas and kidney diseases, of hypertension, of
malignant tumors, especially of carcinomas of the mamma,
cervix, colon or prostate gland. Cynara dry extracts are
further suitable for cellular immunostimulation, for the
therapy of leucocytopenia, granulocytopenia, lymphocy-
topenia, erythrocytopenia, and immunoglobulin deficien-
cies; in addition, it is suitable for bacterially or virally
induced diseases, such as inflammatory diseases of the small
intestine, pancreas and kidneys, hepatitis A, B and C, skin
lesions (Uleus cruris), Herpes simplex I and II, as well as
Herpes zoster.

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US 2002/0012708 A1

USE OF ARTICHOKE (CYNARA) EXTRACTS

[0001] This application is a continuation-in-part of Inter-
national Application PCT/EP97/03561, filed Jun. 6, 1997,
designating the U.S., the text of which is incorporated herein
by reference in its entirety.

[0002] The present invention relates to a novel oral use of
artichoke (Cynara) extracts, optionally in combination with
Echinacea extracts and/or nettle (Urtica) extracts, especially
dry extracts.

[0003] Since about the 16th century, the wide-spread use
of herbal books has provided a general knowledge of the
utility of artichoke as a medicament.

[0004] In folk medicine, it is recommended for treating
indigestions if caused by an underproductivity of bile, and it
is a liver remedy and a cholagogue. The drug has been
employed in the form of its aqueous or alcoholic extracts.

[0005] As a second field of action, a significant diuretic
effect had been indicated, which laid the foundations of the
plant’s use as a dehydration agent as well. The associated
increased elimination of uric acid salts by the increased
diuresis is the reason for the drug’s use as an antirheumatic
agent. It has also been proposed as an antidiabetic in singular
cases. As early as 1934, a compound, at the time chemically
undefined, was isolated from artichoke leaves, and it was
already referred to as the therapy-relevant component. This
compound was identified as cynarin only in 1954. The whole
extract evidently contains supplementary or synergistic sub-
stances. The whole extract may also be replaced by the
uptake of the fresh plant. The active substance cynarin is not
genuinely contained, but is produced in the course of the
processing when the plant is boiled in aqueous media. As
important components, there may be mentioned the three
classes of substances, caffeoylquinic acids, bitter principles
and flavonoids.

[0006] For pharmaceutical purposes, the large basal leaves
are employed, possibly from the annual basal leaf culture.
These provide the highest content of choleretically active
components. The value of the drug is determined by the total
of the caffeoylquinic acids, because the therapeutic principle
of action of artichoke extracts is based on the total of the
caffeoylquinic acids rather than on the isolated Cynara
component cynarin alone. Good drugs contain at least 0.2%
caffeoylquinic acids.

[0007] It has been the object of the present invention to
provide novel medicaments and thus novel possible thera-
pies by using artichoke extracts.

[0008] Accordingly, a first embodiment of the present
invention is the use of artichoke (Cynara) extracts, espe-
cially dry extracts, for decreasing the serum levels of blood
glucose, creatinin and/or bilirubin, for cellular immuno-
stimulation, for the therapy of leucocytopenia, granulocy-
topenia, lymphocytopenia and of organ and tissue damages
caused by infections and chemically, especially of the
0-MALT system of the small intestine, of the bone marrow,
thymus, spleen, lymph nodes, liver, pancreas and kidneys.

[0009] Preferred embodiments can be seen from the
dependent claims. According to the invention, defined
parameters of the circulation and immune system and of
defined functions of liver, pancreas and kidneys could be

Jan. 31, 2002

influenced in in-vivo studies with rats with a dry extract, in
a galenic formulation as granules, from the fresh plant of
artichoke leaves.

[0010] The test substance which was employed in an
amount of 100 mg per kg of body weight induced:

[0011] a slightly negative deviating effect, as com-
pared to the control group, on the average develop-
ment of the body weight which is attributed to a
diuretic effect;

[0012] no utilizable changes in the average organ
weights of spleen and thymus;

[0013] an increase of the cell counts of leucocytes,
polymorphonuclear granulocytes and lymphocytes,
T, B, helper, suppressor and NK (natural killer) cells
as a manifestation of cellular immunostimulation,
wherein the dominant specific increase in B lympho-
cytes may be particularly pointed out;

[0014] a decrease of the levels of creatinin, bilirubin,
urea, cholesterol, triglycerides, glucose and GPT.

[0015] The following are considered therapeutically desir-
able effects:

[0016] a decrease of the cholesterol and triglyceride
levels in terms of a positive influence on pathological
disorders of lipid metabolism and thus reduction of
atherogenic risk factors;

[0017] a general increase of defined immunocompe-
tent cells as a possible inspecific treatment of inflam-
matory processes of different causal pathogeneses;

[0018] a dominant specific increase of the cell counts
of B lymphocytes as an adjuvant in the therapy of
toxically initiated B cell suppression;

[0019] a reduction of the bilirubin level and of the
liver-specific activity value of GPT under the aspect
of a hepatocurative effect;

[0020] a decrease of the serum glucose level as a
functional equivalent of a hypoglycemic effect;

[0021] a decrease of the serum creatinin and urea
levels as an indication of an improvement of renal
function and detoxification of the ammonia produced
in protein metabolism, and in connection with this
reaction cycle, an enhanced mitochondrial perfor-
mance of the hepatocytes;

[0022]

[0023] The recorded changes of defined hematological
and clinical-chemical analytical values are to be considered
under two aspects:

a lack of indications of undesirable effects.

[0024] The results observed in the parameters used
according to the invention speak in favor, on one hand, of the
hepatocurative, hepatoprotective and hypolipidemic effects,
described in the literature, of the artichoke extracts
employed, and on the other hand, of novel, as yet unknown,
biological activities which could be utilized under therapeu-
tic aspects in disorders of carbohydrate metabolism, liver
and kidney functions, and deficiencies of the cellular
immune state.

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US 2002/0012708 A1

[0025] The orally administered artichoke extracts of vari-
ous origins and concentrations exhibited bioequivalent
potentials with respect to the increase of cell counts and the
changes of the relative cell count values of all lymphatic
cells. Bioequivalence was also found in the clinical-chemi-
cal parameters bilirubin, creatinin, GPT, cholesterol, cal-
cium, potassium and protein.

[0026] According to the invention, it has been found that
the artichoke (Cynara) extracts employed are particularly
suitable for cellular immunostimulation. The use of the
extracts caused an increase of the cell counts of leucocytes,
polymorphonuclear granulocytes and lymphocytes, T, B,
helper, suppressor and NK cells, especially B lymphocytes

[0027] The artichoke (Cynara) extracts are particularly
useful for the treatment of disorders of carbohydrate
metabolism, especially for improving the prediabetic meta-
bolic condition, and for the dosage reduction of chemically
defined antidiabetics including insulin, and for the simulta-
neous administration of the extracts when there is some
residual function of the endocrinic pancreas. In addition to
a treatment of liver and kidney functional disorders, the
artichoke (Cynara) extracts are also useful for the adjuvant
therapy of a disordered immune system as a consequence of
endogenous and exogenous factors, for example, in chemo-
or radiotherapy.

[0028] In addition, the artichoke (Cynara) extracts are also
useful for the treatment of prediabetic, for example, senile
forms, for the treatment of a latent diabetic metabolic
condition, for example, due to pregnancy, infection, stress,
obesity, and as an adjuvant therapy of Diabetes mellitus,
when there is still some residual pancreatic function, in the
form of a combination therapy.

[0029] In animal experiments, the Cynara preparation
improves the morpho-functional substrate of the Langerhans
islets in the pancreas in terms of an increased endocrinic
pancreatic function. Both in animal experiments and clini-
cally, an unambiguous hypoglycemic effect could be
detected.

[0030] From a therapeutic point of view, this means, when
there is still some residual pancreatic function:

[0031] reduction of the insulin dose (when 3x600-
900 mg of the Cynara preparation/day is simulta-
neously administered, an individual decrease of the
insulin quantity is required)

[0032] reduction of the chemically defined antidia-
betics (individual dosage required); effects thereof:
protective effect against the hepato-, nephro- and
cardiotoxic potential of a chronical administration of
chemically defined oral antidiabetics.

[0033] In order to further clarify the mechanism of action
of the extract employed on the central metabolic organ, the
liver, partial toxic alterations of the liver parenchyma were
induced in vivo by a simultaneous administration of carbon
tetrachloride. The liver necrosis caused by carbon tetrachlo-
ride was not prevented by the use of the artichoke (Cynara)
extract according to the invention, but its extension was
clearly limited. Perifocal, less damaged or undamaged
parenchyma areas impressed due to a functional swelling of
the nuclei, characterized by particularly large, bright nuclei
with a loose chromatin structure and large, highly basophilic
nucleoli.

Jan. 31, 2002

[0034] These micromorphological indications of an
increased protein biosynthesis of the intact liver cell areas
between the nucleic necroses were coupled with an
increased occurrence of liver cell mitoses and dinuclear or
polynuclear hepatocytes. These findings can be interpreted
in terms of a mobilization of the liver cell metabolism and
a beginning liver cell regeneration, induced by the simulta-
neous treatment with the artichoke (Cynara) extracts.

[0035] This evaluation was supported by the detection of
the qualitative and quantitative enzymatic activity of succi-
nate dehydrogenase. Under the influence of the use of the
artichoke extracts, a clear reduction of the succinate dehy-
drogenasenegative areas took place. The intact perifocal
areas were characterized by a succinate dehydrogenase
hyperactivity. The latter can be considered the manifestation
of a compensatory hyperintensive reaction of the intact
parenchyma.

[0036] In a further in-vivo study, the effect of artichoke
extracts, also in combination with Echinacea extracts (E.
pallida and E. angustifolia), on toxically induced alterations
of the liver and lymphatic organs by cytostatics, especially
cyclophosphamide, was examined. In rats, the latter sub-
stance induces atrophy of the red bone marrow, the lym-
phatic organs, Peyer’s plaques, the thymus, the spleen and
the lymph nodes. By a simultaneous treatment with Cynara
extracts, the damaging effect of cyclophosphamide was
reduced. By a further simultaneous treatment with Cynara
and Echinacea extracts, the cell-damaging effects were even
clearly reduced (synergistic effect of Cynara and Echinacea).
After the administration of the alkylating cytostatic, a partial
accidental regeneration can be histologically detected in the
bone marrow, thymus, spleen, mesenteric and cervical
lymph nodes and Peyer’s plaques after simultaneous treat-
ment with the Cynara preparation, which gets a synergistic
positive influence from the simultaneous administration of
Echinacea extracts. As to micromorphology, the correspond-
ing organs show clear indications of a restoration of the
organ-specific architecture and a clear repopulation of the
reticular stroma with intact cells of the myeloid and espe-
cially the lymphatic group.

[0037] This accidental regeneration of the blood-forming
and lymphoreticular tissues does not reveal any signs of an
increased mitotic activity in terms of a pathologically
increased proliferation. Cyclophosphamide induced an atro-
phy of the thymus in the form of an elimination of lympho-
cytes by an antiproliferative effect which was generally
more pronounced in B cells than it was in T cells. These
different effects of the lymphocytotoxic influence of cyclo-
phosphamide is due to different metabolic performances of
these cell qualities.

[0038] After simultaneous peroral administration of the
artichoke extracts and cyclophosphamide to rats over a
period of 14 days, a complete elimination of the lympho-
cytes from the cortex and marrow of the thymus could be
prevented by the antitoxic potential of the artichoke extracts.
This effect could even be increased by the additional admin-
istration of Echinacea extracts.

[0039] Already under the therapeutic influence of the
artichoke extracts alone, a clear repopulation of the thymus.
with lymphocytes took place which was about 40% as
compared to the controls.

[0040] In the case of the lymph node, for example, a
repopulation of the reticular tissue with cells of the lym-

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US 2002/0012708 A1

phatic group can be seen histologically. In the paracortical
zone, densely packed intact lymphatic cells prevail, and in
the cortical zone, there are loosely arranged intact lympho-
cytes with a clear tendency to form subcapsular follicles.
With the restoration of the characteristic microstructure of
the lymphoreticular tissue in the lymph node, its function
evidently also returns.

[0041] Recently, the different response forms of the lymph
node have gained practical importance: if a stimulation of
the T cells (paracortical zone) is observed in the drainage
area of carcinomas (carcinoma of the mamma, carcinoma of
the cervix), there is a better prognosis for the patients than
in the case where the B cell region or no region of the lymph
node is activated.

[0042] A successful defense against living exogenous
pathogens by the endogenous immune system is quite criti-
cally dependent on the strength and quality of the host’s
immune response. An intact cellular and humoral defense by
the host organism can control the formation and spread of a
tumor. However, the most important source of immunode-
ficiency today is the cytostatic corticoid therapy for cancer
patients.

[0043] Ultimately, the conclusion can be drawn that the
Cynara preparation, also in combination with Echinacea
formulations, in oral administration is suitable as a thera-
peutic adjuvant for the treatment of malignant tumors as
well as chemocytostatic and radiologic tumor treatment.

[0044] Although various secondary surrogates describe
the pharmacodynamic activity profile in an extract-specific
way, a “basic scheme of general reactions” can be seen in the
activity.

[0045] Depending on the active substance, dosage and
duration of the administration, target organs were influenced
due to interactions between the endocrine, nervous and
immune systems.

[0046] Similar effects on the O-MALT system of the small
intestine, of the bone marrow, thymus, spleen, lymph nodes,
liver, pancreas and kidneys as described above for Cynara
and Echinacea were also experimentally observed in a rat
animal model after the administration of Urtica extracts and
Cynara extracts from the roots, the leaves and the herbage.

[0047] In the same study, artichoke extracts exhibited an
identical therapeutic action on the mesenteric lymph node.
In this organ as well, the complete mobilization under
cyclophosphamide was compensated by a repopulation with
lymphocytes under a treatment with artichoke extracts. This
protective effect primarily concerned the lymphoblasts in the
T regions, and in a less pronounced way the lymphocytes
from the B regions and the mature microcellular lympho-
cytes from the T regions.

[0048] In a clinical outpatient study, the effect of the
artichoke extracts was examined over a period of 14 weeks
in patients suffering from hyperlipidemia and accompanying,
hepatopathy. The extracts had a positive influence on the
pathological metabolic situation of the patient suffering from
hypercholesterolemia and/or hypertriglyceridemia. At the
same time, a reduction of increased liver-specific enzymatic
activity values occurred.

EXAMPLES

[0049] A clinical study on the effect of artichoke extracts
in patients suffering from hyperlipidemia and hepatopathy

Jan. 31, 2002

was performed with 40 patients of the age of 50-80 years
who were treated with the following composition for an
average of 19 days:

[0050] dry extract from artichoke leaves (25-35:1)
60.00% by weight

[0051]
[0052] (DAB=German pharmacopoeia)
[0053] further ingredients:

extractant: purified water DAB 10

glucose 12.75% by weight
highly disperse silica 7.15% by weight
talcum 2.8% by weight

magnesium stearate
calcium carbonate
saccharose

potato starch

0.8% by weight
11.4% by weight
0.6% by weight
3.9% by weight

[0054] The patients were treated for existing disorders of
lipid metabolism and hepatopathies while a medicamental
permanent treatment of other basic diseases (diabetes,
hypertension, cardiopathy, hyperuricemia, asthma, epilepsy,
diseases of the thyroid gland and osteoporosis) which were
simultaneously present was maintained unchanged

[0055] Of this collective of patients, 28% had liver dis-
eases, 12% were diabetics who obtained chemically defined
antidiabetics, and 47% showed increased lipid and y-GT
values, in part with cardiac diseases, as well as hypertension.
The patients suffering from cardiovascular diseases simul-
taneously continued to be treated with ACE inhibitors
(45%), a-2 receptor antagonists (5%), Ca antagonists (40%),
diuretics (45%) or with a corresponding combined prepara-
tion of these classes of substances (15%) during the phase of
treatment with the Cynara preparation (300 mg).

[0056] It was remarkable that under the simultaneous
intake of the Cynara preparation together with ACE inhibi-
tors, @-2 receptor antagonists, Ca antagonists, diuretics or
with a corresponding combined preparation of these classes
of substances, the y-GT values fell by 8.7%, the GPT values
fell by 11.6%, and the GOT values fell by 10.0% after an
average of 19 days.

[0057] In 21 patients with hyperlipidemic and hyperten-
sive values, not only a reduction of the increased cholesterol
and triglyceride values, but also a decrease of the systolic
blood pressure values by an average of 5% and of the
diastolic ones by 6.8% could be observed due to a lipid level
reducing Cynara therapy. This allows to conclude that the
Cynara therapy also has an antihypertensive effect in a limit
value hypertensive condition, and in addition a synergistic
adjuvant hypotensive effect after simultaneous administra-
tion with chemically defined antihypertensive agents for
increased blood pressure values. Ultimately, this has the
consequence that the necessary dosage of chemically
defined antihypertensive agents can be reduced, and thus the
risk of undesired side-effects can be limited.

[0058] It is known that, for example, the synthesis of the
y-GT in the liver can be induced by cholestase, chronical
alcohol consumption and pharmaceuticals in therapeutic
dosage. Both an increase in activity of the soluble y-GT in
the hepatocytes and a spread of the membrane-bound form

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US 2002/0012708 A1

occur. The increased y-GT level to be measured in the serum
after an induction of the synthesis depends on the kind and
extent of the noxa.

[0059] These relationships, especially the increase of the
y-GT activity values, for example, due to a chronical admin-
istration of chemically defined pharmaceuticals, and, on the
other hand, the reduction of increased y-GT activity values
due to the simultaneous administration of the Cynara prepa-
rations allow to conclude that the artichoke dry extracts, due
to their simultaneous administration, do not enhance, but
rather significantly reduce the hepatotoxic potential, pro-
nounced in part, of the ACE inhibitors, a-2 receptor antago-
nists, Ca antagonists, diuretics or corresponding combined
preparations.

[0060] These hepatoprotective and -curative effects of the
Cynara preparation have been supported by results found in
animal experiments in a simultaneous administration with
clofibrate of the cyclophosphamides.

[0061] The clinical-chemical parameters used in the
evaluation were essentially identical with those used in the
present in-vivo studies. The results obtained in this patient
study showed a remarkable similarity in a number of the
parameters:

total cholesterol -12.0% (patients) versus -30% (in vivo)
Ger versus 11.6%

bilirubin versus -5.1%

glucose versus -8.5%

[0062] It may be concluded therefrom that the reproduc-

ible data, collected in a methodologically precise manner, of
the present in-vivo studies can altogether be transferred to
the conditions in humans by way of an argument by analogy.

[0063] Male Wistar rats with an average body weight of
310 g were kept under conventional conditions at a room
temperature of 21° C. +/-1° C., a relative humidity of about
60% and a 12 hour day/night rhythm. Before the experiment
was begun, they were subjected to an acclimatization to the
keeping conditions for 12 days.

[0064] The rats were fed with a pelletized standard diet,
Altromin C1000.

[0065] The animals were given tap water ad libitum as the
drinking water.

[0066] The artichoke extract and dosage employed were as
follows:

[0067] 1. Calcium carbonate and saccharide contain-
ing granules with a standardized dry extract from the
fresh plant of artichoke leaves (25-23:1) and the
above minor components;

[0068]

0069] starting drug: fresh artichoke
is 8:
(Cynarae folium), Hagers Handbuch/PFX;

extractant: purified water DAB 10;

leaves

[0070] dosage form: granules 1.67 mg (#1 mg of
native Cynara dry extract, FC, suspended in 0.02
mil of dist. water), 0.5 ml=25 mg/250 mg of body
weight.

Jan. 31, 2002

[0071] Ten male Wistar rats were divided into the follow-
ing treatment groups for the examination:

Dosage
group (mg/kg of body weight) number of animals

I control group — 5

I FC 100 5

[0072] The test substance FC according to the invention
was administered once a day to the non-sedated animals
intragastrically by means of a rigid bulbous probe for a
period of 7 days. The suspensions were freshly prepared
immediately before the administration and administered in a
homogeneous form. The control animals were given physi-
ological saline in an equivalent volume.

[0073] The present studies aimed at simultaneously estab-
lishing, by means of a defined in-vivo model, specific
markers which indicated early the therapeutic effect as a
synergistic effect of differently influenced physiological
feedback mechanisms.

[0074] In detail, there was detected in the peripheral
blood:
[0075] combinedly:

erythrocytes , leucocytes >

0076 hi RBC), 1 WBC)
platelets (PLT), hemoglobin (HGB), hematocrit
(HCT), erythrocyte indices:

[0077] mean corpuscular volume (MCV), mean corpuscu-
lar hemoglobin (MCH), mean corpuscular hemoglobin con-
centration (MCHC), and red cell distribution width (RDW)

[0078] by flow cytometry:

[0079] leucocyte differentiation: — granulocytes,
monocytes, lymphocytes, B cells, T cells, helper
and suppressor cells

[0080] combinedly:
[0081] GPT, GOT;
[0082] glucose,
[0083] cholesterol, triglycerides
[0084] Na, Cl;
[0085] creatinin, urea.

[0086] Using the Sysmex K-1000 fully automated hema-
tology analytic device, the following could be determined:
WBC, RBC, PLT, HGB, HCT, MCV, MCH, MCHC. The
main unit of this device essentially consisted of a hydraulic
system (HS) and an electronic system (ES). The HS served
to aspirate, pipette, dilute, mix and lyse. The ES analyzed
and converted the signals of the HS and transmitted the
results to a printer. Using microprocessors, the ES also
monitored the test procedures and the test station and
performed the quality control.

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US 2002/0012708 A1

[0087] The hematocrit value indicated the percent volume
fraction of erythrocytes in the blood. Hemoglobin, which is
the chromoprotein contained in the erythrocytes, was an
important cirterion for the diagnosis of anemias in addition
to the erythrocyte count and the hematocrit value. Classifi-
cation was performed through the erythrocyte indices.
Erythrocyte size and hemoglobin content were characterized
by the erythrocyte volume (MCV =mean corpuscular vol-
ume), the hemoglobin content of the erythrocytes (MCH
=mean corpuscular hemoglobin), and the mean corpuscular
hemoglobin concentration (MCHC). The red cell distribu-
tion width (RDW) was a measure of anisocytosis.

[0088] The parameters, such as enzymes, glucose, lipids,
electrolytes, creatinin, urea and protein, were determined
using the analytical device Cobas Mira in a selective,
method-oriented, photometric or ion-selective way. The
supplementary report provided data of quality control and
statictics in a analysis-specific way.

[0089] The leucocyte differentiation was performed using,
the flow cytometer FACScan after appropriately lysing a
whole blood sample (scattered light measurement).

[0090] The lymphocyte differentiation was performed
after a specific monoclonal incubation by means of fluores-
cence-activated cell sorting (fluorescence measurement)

[0091] The following were quantitatively analyzed on the
7th treatment day:

[0092]
[0093]
[0094]

leucocytes (total), lymphocytes (total),
T lymphocytes (CD2+/CD45 RA-),

B lymphocytes (CD2-/CD45 RA+),
[0095] helper lymphocytes (CD4-/CD8b+), and
[0096] NK cells (CD8a+/CD8b-).

[0097] For determining the phenotypes of the lympho-
cytes, they were incubated with the following antibodies
supplied by Pharmingen, San Diego, U.S.A., to which a
fluorochromeis coupled:

[0098] fluorescein isothiocyanate (FITC) conjugated
mouse anti-rat CD2 monoclonal antibody,

[0099] R-phycoerythrin (R-PE) conjugated mouse
anti-rat CD45RA OR A/B monoclonal antibody,

[0100] fluorescein isothiocyanate (FITC) mouse anti-
rat CD4 monoclonal antibody,

[0101] R-phycoerythrin (R-PE) conjugated mouse
anti-rat CD8 (BB chain) monoclonal antibody,

[0102] fluorescein isothiocyanate (FITC) conjugated
mouse anti-rat CD8a monoclonal antibody.

[0103] Approach:
[0104] a) CD2/CD45RA=T and B cells

Jan. 31, 2002

[0105] b) CD4/CD8b=T, and TS cells
[0106] c) CD8a/CD8b=NK cells

[0107] 5 4d each of the antibodies was incubated with 50
Hof Na EDTA blood at room temperature in the dark for 20
min. The suspension was agitated with 2 ml of Lyses
Reagent of Becton-Dickinson and incubated for 10 min as
described. This was followed by a centrifugation at 400xg
for 6 min, and the supernatant was poured off. The sediment
was washed with 3 ml of Cell Wash and centrifuged at
400xg for 6 min. The pellet was taken up in 100 ul of Cell
‘Wash. The suspension was analyzed using the flow cytom-
eter.

[0108] During the acclimatization prior to the start of the
experiment and throughout the duration of the experiment,
the general condition of the animals was monitored. In
addition, their body weights were determined daily.

[0109] The animals of all groups were sacrificed pain-
lessly at the end of the experiment and dissected. The organs
of the abdominal, pelvic and thoracic cavities were inspected
macroscopically, and the spleen and thymus were weighed.

[0110] All animals had withstood the intragastric admin-
istration without impairment. The animals which had been
treated with the test substance did not show any disadvan-
tageous behavior clinically until the end of the experiment.
During the treatment period, the average body weight
increased in the groups as follows:

[0111]
[0112] I FC by 8.1%.

[0113] The average body weight of the treatment group FC
increased by a lesser percentage as compared to that of the
control group. This was predominantly due to the aquaretic
effect of the test substance.

I control group by 12.6%

[0114] The potential of the test substance FC was deter-
mined indirectly through its stimulating effect on the leu-
cocyte (WBC), erythrocyte (RBC) and platelet (PLT)
counts.

[0115] Table 1 contains the individual measured values of
these cell counts after the end of the treatment, and Table 2
shows the corresponding average cell counts per group and
the corresponding percent change of these values as com-
pared to those of the control group.

[0116] The following was measured for the test substance
FC according to the invention:

[0117] a clear increase in leucocyte count by 35%”;

[0118] the other parameters, such as RBC and PLT,
varied within a physiological range of from -1% to
-2%.

D corresponding values of the control group=100%)
¢ ponding group

TABLE 1

WBC RBC HGB HCT MCV MCH MCHC PLT

group T

Average

Standard deviation

$820 5.954 7.74 0.3712 62.38 1301.2 20.86 1049.8
617.74 0.20 0.19 0.01 1.40 4794 0.43 41.37

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US 2002/0012708 A1

TABLE 1-continued

Jan. 31, 2002

WBC RBC HGB HCT MCV MCH MCHC PLT

group IL

11.920 5.834 7.86 0.3696 63.34 1347.6
1410.53 0.09 0.14 0.01 1.59 24.79

Average
Standard deviation

21.26

1038.25
87.01

[0119] Erythrocyte count (RBC) in 1017/1, leucocyte
(WBC) and platelet (PLT) counts in 10°/1. Molar concen-
tration of hemoglobin (HGB) in mmol/l, hematocrit (HCT)
in 1/1, MCV in fl, MCHC in mmol/l, MCH in amol.

[0120] (conversion:mmol/l g/d1x0.6206;
[0121] 1/1%x100; fl=um*; fmol=pgx0.062;
[0122] amol=62xpg)

group mg/kg of body weight/day
I control —
IL FC 100
[0123]
TABLE 2
WBC RBC HGB HcT MCV MCH
group I 8820 5.954 7.74 0.3712) 62.38 1301.2
group IT 11,920 5.834 7.86 0.3696 63.34 1347.6
change with respect to group I (%)
WBC RBC HGB PLT HCT MCV
group IT 35.15 -2.02 «1.58 140 -0.43 1.54

[0124] Average erythrocyte cell counts in 10 /1, leucocyte
and platelet counts in 10 /1, Molar concentration of hemo-
globin in mmol/l, hematocrit in 1/1, MCV in fl, MCH in
amol, MCHC in mmol/l.

[0125] Percent changes of the stated values as compared to
the corresponding values of the control group.

group mg/kg of body weight/day
T control —
1 FC 100

[0126] The test substance FC does not show any physi-
ologically important influence on HGB, HCT, MCV and
MCHC.

[0127] The following clinical-chemical metabolic measur-
ing, quantities were analyzed in a combined and method-
specific manner:

[0128] bilirubin, creatinin, urea, glutamate oxalacetate
transaminase (GOT), glutamate pyruvate transaminase
(GPT), y-glutamyl transferase (GGT), glucose, cholesterol,
triglycerides, calcium and sodium.

[0129] For the test substance FC, there was measured:

[0130] a clear reduction of:
[0131] creatinin by 16.7%
[0132] GPT by 11.6%
[0133] cholesterol by 30.0%
[0134] glucose by 8.5%

[0135] a slight reduction of:
[0136] _ bilirubin by 5.1%
[0137] urea by 4.6%
[0138] GLDH by 3.2%
[0139] triglycerides by 1.0%

[0140] For the test substance FC, bioequivalent changes of
bilirubin, creatinin, GPT, cholesterol, calcium, potassium
and protein were measured which were comparable in nature
and intensity.

[0141] In the treatment group II, the cell counts of the
polymorphonuclear granulocytes, monocytes, T and B lym-
phocytes, helper, suppressor and NK cells were represented
as compared with the control group.

[0142] For the test substance FC, there was measured an
increase of:

[0143]
[0144]
[0145]
[0146]
[0147]
[0148]
[0149] monocytes by 24.5%

( corresponding values of the control group=100%)

leucocytes by 35.2%”
lymphocytes by 31.1%
T lymphocytes by 15.3%
B lymphocytes by 58.5%
helper cells by 14.0%

suppressor cells by 20.3%

[0150] The present invention provides improvements in
therapeutic effectiveness for various established therapies,
the improvement consisting of co-administration of arti-
choke extracts (extracts of Cynara sp.) as an adjunct to the
established therapy. Optionally, combination adjunctive
therapy, in which extracts of artichoke are administered
along with extracts of Echinacea or nettles (Urtica), is also
provided. Adjunctive therapy according to the present inven-
tion is of particular value for the treatment of organ and
tissue damages caused chemically or by radiation or infec-
tions, especially of the 0-MALT system of the small intestine
(inflammatory diseases, Enteritis regionalis Crohn), of the
bone marrow (bone marrow aplasia), thymus (dysfunction,

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US 2002/0012708 A1

aplasia or hypoplasia), spleen (dysfunction) and the lymph
nodes (aplasia or hypoplasia due to damage from medica-
ments or radiation).

[0151] Adjunctive therapy according to the present inven-
tion is also of particular value for the treatment of organ and
tissue damages of the liver (atrophy, necrosis, hepatitis A, B
and C), pancreas (insufficiency of the exocrinic secretory
function of proteases, esterases, carbohydrases and
nucleases, and the endocrinic functional disorder of carbo-
hydrate metabolism [Langerhans islets]) and the kidneys
(insufficiency) . Adjunctive therapy according to the present
invention is likewise of particular value for the treatment of
general immunesuppressed conditions, for cellular immu-
nostimulation, for the therapy of leucocytopenia, granulo-
cytopenia, lymphocytopenia, and in immunoglobulin defi-
ciencies.

[0152] In one mode, the adjunctive therapy of the present
invention provides the cellular immunostimulation which
causes an increase of the cell counts of leucocytes, poly-
morphonuclear granulocytes and lymphocytes, T, B, helper,
suppressor and NK cells, especially B lymphocytes, in the
terminal vascular system. Thus, upon therapy according to
the present invention, an increase in vital lymphocytes can
be seen in the cortical zone and follicles in Peyer’s plaques
(small intestine) which is associated with a secretory induc-
tion of sIGA into the small intestine, an increase of all blood
forming elements can be seen in the bone marrow, a cellular
stimulation of immunocompetent cells can be detected in the
thymus, spleen and mesenteric lymph nodes, and/or the
metabolic functions of the liver, pancreas and kidneys
(reduction of the creatinin serum levels) are clearly acti-
vated.

[0153] In another mode, the present invention provides
improvement, by administration of artichoke extracts, to
methods for the treatment of disorders of carbohydrate
metabolism and of liver and kidney functions, for the
treatment of prediabetes, especially senile forms, for the
treatment of infections, stress and/or obesity, or for the
treatment of blood hypertension in a limit value hyperten-
sive condition, or by use of artichoke extracts as synergistic
adjuvants after simultaneous administration with other
chemically defined antihypertensive agents.

[0154] In yet another mode, the present invention provides
for the use of artichoke extracts as oral adjuvants for the
treatment of malignant tumors, especially for the treatment
of carcinomas of the mamma, cervix, colon or prostate
gland, also in combination with cytostatics, especially cyclo-
phosphamide, or radiation therapy. In particular, adminis-
tration of artichoke extracts according to this invention can
increase the tolerance of chemotherapy, cytostatic or radio-
logic therapies, especially in combination with chemically
defined substances selected from clofibrate, ACE inhibitors,
a-2 receptor antagonists, Ca antagonists and/or diuretics.
The artichoke extract of this invention is thus effective as an
analgetic adjuvant, especially as a pharmacological compo-
nent having a positive influence on the interactions between
the endocrine, nervous and immune systems.

[0155] In another mode, this invention provides for use of
artichoke as an adjuvant for the treatment of bacterially or
virally induced diseases, especially inflammatory diseases of
the small intestine (Enteritis regionalis Crohn), pancreas and

Jan. 31, 2002

kidneys, as well as liver atrophy, hepatitis A, B and C, skin
lesions (Ulcus cruris), and also Herpes simplex I and I, and
Herpes zoster.

[0156] The present invention provides for improved thera-
peutic efficacy based on administration of artichoke
(Cynara) extracts. The artichoke extract may be obtained a
fresh plant, especially extracted juice as extracted using
aqueous, organic and/or supercritical solvents, especially
carbon dioxide, in a dried form, especially powder as dry
extracts from the fresh plant or drug (dried plant), as
granules, especially capsule, and/or as a tablet, especially
coated tablet, or pastille. Typically, Cynara extracts with a
drug-to-extract ratio (DEV native, i-e., the weight ratio of
plant matter to extractant) of from 25:1 to 35:1 are
employed. In a preferred mode, artichoke (Cynara) dry
extracts are administered as an orally applicable medicament
in the form of calcium carbonate and saccharide containing
granules. Optionally, artichoke (Cynara) extracts are
employed in combination with Echinacea extracts, espe-
cially dry extracts (E. pallida and E. angustifolia), and/or
with nettle (Urtica) extracts, especially dry extracts from the
roots, leaves or herbage. Typically Echinacea extracts with
a drug-to-extract ratio of from 4:1 to 8:1 are employed
and/nettle (Urtica) extracts with a drug-to-extract ratio of
from 6:1 to 15:1 are employed. The dose of artichoke extract
may be from 1 to 10,000 mg per kg body weight. As an
initial dose, the extract may be administered to provide the
biological equivalent of 100 mg (based on dry matter in the
native extract) per kg of body weight. In clinical trials, the
following doses of dry extract (native dry extract, excluding
any other ingredients) have been administered:

[0157] preclinical:

[0158] 100 mg dry extract/kg body weight per day
(experimental data with rats)

[0159]

[0160] 900 mg/day/patient 15 mg/kg body weight
per day

[0161] 1800 mg/day/patient 30 mg/kg body weight
per day

[0162] 2700mg/day/patient 45 mg/kg body weight
per day

[0163] 3600mg/day/patient 60 mg/kg body weight
per day

[0164] Based these doses, the skilled clinician will, as a
matter of routine, optimize the dose for the individual patient
by monitoring the effects described herein for the particular
therapeutic application, which may include increasing the
dose in certain cases.

clinical:

1. Use of artichoke (Cynara) extracts, especially dry
extracts from the fresh plant, for the preparation of medi-
caments for the treatment of organ and tissue damages
caused by radiation, infections and chemically, especially of
the O-MALT system of the small intestine (inflammatory
diseases, Enteritis regionalis Crohn), of the bone marrow
(bone marrow aplasia), thymus (dysfunction, aplasia or
hypoplasia), spleen (dysfunction) and the lymph nodes
(aplasia or hypoplasia due to damage from medicaments or
radiation), liver (atrophy, necrosis), hepatitis A, B and C,
pancreas (insufficiency of the exocrinic secretory function of

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US 2002/0012708 A1

proteases, esterases, carbohydrases and nucleases, and the
endocrinic functional disorder of carbohydrate metabolism
[Langerhans islets]) and the kidneys (insufficiency), and of
general immunesuppressed conditions, for cellular immu-
nostimulation, for the therapy of leucocytopenia, granulo-
cytopenia, lymphocytopenia, and in immunoglobulin defi-
ciencies.

2. The use according to claim 1, characterized in that the
cellular immunostimulation causes an increase of the cell
counts of leucocytes, polymorphonuclear granulocytes and
lymphocytes, T, B, helper, suppressor and NK cells, espe-
cially B lymphocytes, in the terminal vascular system, that
an increase in vital lymphocytes can be seen in the cortical
zone and follicles in Peyer’s plaques (small intestine) which
is associated with a secretory induction of sIGA into the
small intestine, that an increase of all blood forming ele-
ments can be seen in the bone marrow, that a cellular
stimulation of immunocompetent cells can be detected in the
thymus, spleen and mesenteric lymph nodes, and/or that the
metabolic functions of the liver, pancreas and kidneys
(seduction of the creatinin serum levels) are clearly acti-
vated.

3. The use according to claim 1 or 2 for the treatment of
disorders of carbohydrate metabolism and of liver and
kidney functions, for the treatment of prediabetic, especially
senile forms, for the treatment of infections, stress and/or
obesity, for the treatment of blood hypertension in a limit
value hypertensive condition, and as a synergistic adjuvant
after simultaneous administration with chemically defined
antihypertensive agents.

4. The use according to claim 1 or 2 as oral adjuvants for
the treatment of malignant tumors, especially for the treat-
ment of carcinomas of the mamma, cervix, colon or prostate
gland, also in combination with cytostatics, especially cyclo-
phosphamide, or radiation therapy.

5. The use according to claim 1 for increasing the toler-
ance of chemotherapy, cytostatic or radiologic therapies,
especially in combination with chemically defined sub-
stances selected from clofibrate, ACE inhibitors, a-2 recep-

Jan. 31, 2002

tor antagonists, Ca antagonists and/or diuretics, as an anal-
getic adjuvant, especially as a pharmacological component
having a positive influence on the interactions between the
endocrine, nervous and immune systems.

6. The use according to claim 1 or 2 as an adjuvant for the
treatment of bacterially or virally induced diseases, espe-
cially inflammatory diseases of the small intestine (Enteritis
regionalis Crohn), pancreas and kidneys, liver atrophy,
hepatitis A, B and C, skin lesions (Ulcus cruris), and also
Herpes simplex I and II, and Herpes zoster.

7. The use according to one or more of claims 1 to 6 as
a fresh plant, especially extracted juice as extracted using
aqueous, organic and/or supercritical solvents, especially
carbon dioxide, in a dried form, especially powder, dry
extracts from the fresh plant or drug (dried plant), granules,
especially capsule, and as a tablet, especially coated tablet,
or pastille.

8. The use according to one or more of claims 1 to 7,
characterized in that Cynara extracts with a drug-to-extract
ratio (DEV native) of from 25:1 to 35:1 are employed.

9. The use according to one or more of claims 1 to 8,
characterized in that said artichoke (Cynara) extracts are
employed in combination with Echinacea extracts, espe-
cially dry extracts (E. pallida and E. angustifolia), and/or
with nettle (Urtica) extracts, especially dry extracts from the
roots, leaves or herbage.

10. The use according to claim 9, characterized in that
Echinacea extracts with a drug-to-extract ratio of from 4:1 to
8:1 are employed.

11. The use according to claim 9, characterized in that
nettle (Urtica) extracts with a drug-to-extract ratio of from
6:1 to 15:1 are employed.

12. An orally applicable medicament containing artichoke
(Cynara) dry extracts as defined in one or more of claims 1
to 11 in the form of calcium carbonate and saccharide
containing granules.