Note: Descriptions are shown in the official language in which they were submitted.
CA 02311546 2000-OS-19
h
SMB
Use of Putamen Ovi
The present invention pertains to the use of processed putamen ovi for the
preparation of orally applicable medicaments and locally applicable bone
replacement.
Egg shell as a medicament has been used since 1930. Formulations made
s.
of egg shells are still being used as a mineral and trace element supplying
agent for the substitution of, in particular, calcium.
WO 96/00579 relates to a process for the preparation of putamen ovi,
putamen ovi having a defined grain size, and the use of putamen ovi for the
treatment of calcium deficiency conditions.
The prior art shows that the egg shell is formulated in the form of standard-
ized, orally applicable preparations and is used for calcium substitution.
In contrast, it has been the object of the present invention to provide the
use of an especially processed orally applicable medicament and locally
applicable bone replacement.
According to the invention, the above object has been achieved by the use
of processed putamen ovi (egg shell of Gailus gallus domesticus), in
particular saccharide-containing granules of micronized egg shells (PO), egg
shell components of the central palisade zone (POM), shell matrix with
organically bound minerals (MPM), for the preparation of orally applicable
medicaments for the treatment and prevention (protection) of organ and
tissue damage caused by radiation (radioprevention, radioprotection),
infections (infection prophylaxis) and chemically (chemoprevention, chemo-
protection), especially of the O-MALT system of the small intestine (inflam-
CA 02311546 2000-OS-19
_2_
matory diseases, Enteritis regionalis Crohn), the bone marrow (bone
marrow aplasia), of bone and cartilage genesis disorders, of diseases of the
locomotor system, of diseases of the thymus (dysfunction, aplasia or
hypoplasia), the spleen (dysfunction) and the lymph nodes (aplasia or
hypoplasia due to damage caused by medicaments or radiation), the liver
(atrophy, necrosis), the pancreas (insufficiency of the exocrine, secretory
function of proteases, esterases, carbohydrases and nucleases, as well as
insufficiency of the endocrine function of the islets of Langerhans and the
carbohydrate metabolism) and the kidneys (insufficiency), and in general
immunosuppressed conditions, for cellular immunostimulation, for the
therapy of leucocytopenia, granulocytopenia, lymphocytopenia, thrombocy-
topenia, erythrocytopenia and in immunoglobulin deficiency conditions, also
due to AIDS and tumors, as well as for the therapy of hyperlipoproteine-
mias and hyperlipidemia; further, for the treatment of primary or secondary
disorders of or damage to chondral or desmal ossification, also in combina-
tion with sodium fluoride and hormones, especially estrogens (new genera-
tion estrogens, e.g., estrogen sulfamate), calcitonin, pyrophosphates
(biphosphonates) and vitamins, especially vitamin D (D3 and dihydroxy-
cholecalciferol), of the bony substance with external substantia corticalis
(lamellar bones) and internal substantia spongiosa (framework of minute
trabeculae) as well as bone marrow due to a reduction of all hematopoietic
cell forms (bone marrow aplasia, depression or bone marrow metastases)
due to a cytostatic or radiologic therapy or after radiation accidents, of the
skeleton including osteocytes, intercellular substance with collagenous
fibrils
and calcified cement, of bone metabolism including the function of osteo-
clasts and osteoblasts, the balance between bone absorption and formation
(bone tissue remodelling); for local and oral application for the reconstruc-
tion of bone deficiencies, in fracture healing, in the filling of damaged
bones
after tumor operations, and for the removal of bone damage in oral surgery
and plastic surgery of the face; further, for the treatment of bone necrosis
due to irradiation and prolonged corticoid medication.
CA 02311546 2000-OS-19
-3-
It is known that the bone structure is subject to a variety of regulation
mechanisms. It is generally known that the minerals calcium, phosphorus
and magnesium essentially participate in the bone metabolism. In addition,
a number of particular trace elements, such as manganese, copper and
zinc, are also required as cofactors of collagen and mucopolysaccharide
synthesis for the maintenance of metabolic equilibrium.
Deficiency in such essential trace elements leads to a pathophysiological
condition with interference of enzymatic reactions in the cells of the hard
tissue. These adverse effects on the bone metabolism can also consecu-
tively spread to the immune system.
If human and animal hard tissues are compared under these aspects, a
strikingly similar spectrum of minerals and trace elements can be detected
in bones, teeth, and also in putamen ovi (egg shell, Gallus gallus domesti-
cus) which is formed outside the bones by membrane passage. In a com-
parison between bones and putamen ovi (PO), this correspondence does
not only concern the inorganic, but also certain organic compounds, such as
mucopolysaccharides based on chondroitin sulfate and hyaluronic acid.
In the case of Gallus species, minerals and trace elements from the PO
matrix membrane are presented to the skeleton system of the developing
organism in a bioavailable manner during the embryonic phase, which
process can be compared to the osteoneogenesis of the mammal organism
including humans.
In the putamen ovi preparations according to the invention, the biologically
active organic and the mainly complex-bound inorganic components, such
as calcium, iron, fluorine, potassium, silicic acid, magnesium, copper,
molybdenum, selenium, zinc etc., which are necessary for bone metabo-
lism, are contained in a mostly bioavailable state for possible therapeutic
applications.
CA 02311546 2000-OS-19
-4-
Since reduced serum concentrations of manganese (Mn), copper (Cu) and
zinc (Zn) are associated with poorly healing bone fractures, and since
manganese deficiency, if present for extended periods of time, induces
osteoporosis, and since the strontium concentration is negatively correlated
with bone density (g of calcium hydroxyapatite/cm3), putamen ovi, due to
its physiological content of trace and ultratrace elements, is a therapeuti-
cally active substitute product for these elements (especially for manga-
nese, copper, zinc, strontium, molybdenum, vanadium, cobalt, fluorine,
iron, magnesium, and others) and an effective natural substance therapeu-
tic agent for improving the bone metabolism including the function of
osteoblasts (bone tissue remodelling).
In the preparation of PO, especially validated requirements for the active
component with respect to selection (quality, purity), processing (purity,
sterility) and galenics (standardization, bioavailability) are observed.
Under such conditions, it has been shown in a patient study that it is
possible to inhibit the progression of a bone density decrease dose-
dependently by a PO treatment and even to reverse the effect, i.e., to
induce osteoneogenesis.
The active components of egg shells can be classified as follows according
to the invention:
1. egg shell components of the central palisade zone, freed from inner
and outer shell membranes (PO);
The central palisade zone consists, in particular, of the egg shell
which has been freed from the shell membrane or from both the shell
membrane and the shell matrix by proteases or alkaline denaturing
followed by purification, or by the use of the palisade matrix which is
CA 02311546 2000-OS-19
-5-
obtained either by decalcification or by aqueous and/or organic sol-
vents, or using supercritical fluids (COZ).
2. egg shell components of the central palisade zone, freed from inner
and outer shell membranes and from the shell matrix of the palisade
zone (POM);
3. shell matrix of the palisade zone with organically bound minerals
(MPM).
A preferred embodiment of the invention pertains to the use of putamen ovi
for the oral and local application of P0, POM and MPM as powders, granules
or pastes as bone cement, for bone remodelling and implantation as well as
for bone replacement with an osteoneogenesis-active potential for the well-
aimed local bone formation.
Further preferred embodiments of the invention can be seen from the
dependent claims.
In the studies made for the present invention, defined functions of the
bones, bone marrow, circulatory and immune systems, and of the liver,
pancreas and kidneys were influenced in a study with rats (treatment
period 7 days) with putamen ovi, micronized (PO), in a galenic formulation
as coated tablet core granules according to WO 96/00579, also with the
simultaneous administration of cyclophosphamide (CyS) and furosemide
(FUR).
The influence of PO on clinically healthy rats (group II) can be summarized
as follows:
CA 02311546 2000-OS-19
-6-
The PO test substance employed
- had no influence on the average increase of body weights and of the
weights of the spleen and thymus organs of the test animals;
- induced, in the peripheral blood:
a slight increase of the cell counts of polymorphonuclear granulo-
cytes, B and suppressor cells, and a slight decrease in helper cells;
- resulted, in the bone marrow,
in an increased tendency to formation of cell-rich nests of granulo-,
erythro-, lympho- and thrombocytopoiesis with functional nuclear
swelling and, in addition, in the presence of activated osteocytes (no
resting nuclei) and activated osteoblasts, deposited on the trabeculae
of the spongiosa;
- caused, in the thymus,
an increase in lymphoblastic cellular elements in the cortex and
medulla; it was striking that, due to the increase of the cell number, a
corresponding light-optical zone separation into cortex and medulla
could no longer be seen;
- resulted, in the spleen,
in a slight broadening of the marginal zones, especially in the region
of the periarterial sheathes (PALS);
- induced, in the mesenterial lymph node,
a considerable increase in lymphocytic cellular elements in the B and
T lymphocyte areas and in the medullary strands, so that the corre-
sponding areas did not appear differentiated in light-optical examina-
tion in this case either;
CA 02311546 2000-OS-19
_7_
- clearly stimulated the performance of liver metabolism:
in morphologic terms, in the form of a functional nuclear swelling with
the picture of uniformly sized nuclei (coordination);
- caused, in the peripheral blood,
a decrease in creatinin, bilirubin, urea, GLDH, AP, SP, cholesterol and
triglyceride contents; the calcium level remained unaffected.
The simultaneous administration of CyS and PO (group VII)
- showed a percental decrease of the average body weight develop-
ment and of the weights of the spleen and thymus organs of the test
animals;
- induced, in the peripheral blood:
a) as compared with the control values (group I),
a decrease in leucocytes (-37.4%) and their subsets (e.g., T
lymphocytes: -20.7%) which had a lesser value than that of
group V (CyS: WBC -51.3%, e.g., T lymphocytes: -51%) and
group VIII (CyS + CC: WBC -51.8%, e.g., T lymphocytes: -
46.5%) and group IX (CyS + NaF: WBC -39%, e.g., T lympho-
cytes: -23.4%);
b) as compared with CyS (group V),
an increase in the cell counts of leucocytes (+28.4%), poly-
morphonuclear granulocytes (+4.3%) and of lymphocytes
(+35%), T (+64.8%), B (+69.2%), helper (+44.4%) and sup-
pressor cells (+23.2%), as the manifestation of a cellular che-
moprotective effect;
CA 02311546 2000-OS-19
- $ _
c) as compared with CyS + calcium carbonate CC (group VIII),
an increase in the cell counts which correlates with that deter-
mined with group V (CyS), i.e., a chemoprotective effect is not
present with pure calcium carbonate;
d) as compared with CyS + sodium fluoride (NaF) (group IX),
an increase in the cell counts which is smaller than with CyS +
PO (group VII);
- resulted, in the bone marrow,
as compared with CyS (group V),
in a clearly increased tendency to formation of cell-rich nests (envi-
ronments) of granulo-, erythro-, lympho- and thrombocytopoiesis
(comparable with the cell picture of the control): no remarkable cell
count decrease, no indications of a degeneration of nucleated cells,
unchanged erythrocytes in the sinuses, i.e., erythrocytes in thorn-
apple form (acanthocytes), as encountered under the influence of
CyS even in combination with CC and NaF, are not present;
also in group VII (CyS + PO) - as compared with group II (PO) -
there are encountered activated osteocytes and osteoblasts as well as
active cell division;
- caused, in the thymus,
as compared with CyS (group V), CyS + CC (group VIII) and CyS +
NaF (group IX),
CA 02311546 2000-OS-19
_g_
a clear increase in lymphoblastic cellular elements in the cortex and
medulla;
- resulted, in the spleen,
as compared with CyS (group V), CyS + CC (group VIII) and CyS +
NaF (group IX),
in a broadening of the marginal zones, especially in the region of the
periarterial sheathes (PALS);
- stimulated, in the liver cells,
as compared with CyS (group V),
the metabolic performance, i.e., partial functional nuclear swelling in
hepatocytes;
- induced, in the mesenterial lymph node,
as compared with CyS (group V), CyS + CC (group VIII) and CyS +
NaF (group IX),
a considerable increase in lymphocytic cellular elements, in particular
in the T lymphocyte areas, and in the medullary strands;
- caused, in the peripheral blood, as compared with CyS (group V),
an increase in potassium (+15.6%) and acid phosphatase (+5.8%),
so that the latter parameter reached the content level found in the
serum of the control group.
CA 02311546 2000-OS-19
- 1~ -
Calcium carbonate (CC) showed neither positive effects in clinically healthy
rats nor chemoprotective effects after a simultaneous administration of
cyclophosphamide.
The present invention confirms the clinically obtained findings from a two-
year study with 361 healthy females in post-menopause in which the effect
of calcium carbonate and an organic calcium compound on the osteopenic
progression of bone density decrease was examined. In contrast to the
"organic" calcium compound, the treatment with pure calcium carbonate
showed only a slightly pronounced inhibitory influence on the bone mass
losses.
Sodium fluoride (NaF) did not induce an effect with respect to an increase in
cell counts of T, helper or suppressor cells in the terminal vascular system;
in contrast, however, the counts of B lymphocytes and polymorphonuclear
granulocytes were increased (selective induction).
A chemoprotective effect was seen with NaF after a simultaneous admini-
stration of CyS in leucocytes in the peripheral blood, but not in the bone
ma rrow.
In healthy animals, the number of erythrocytes was not increased by CC,
slightly increased by PO and clearly increased by CyS, but decreased by
NaF.
After the simultaneous administration of CyS and P0, the erythrocyte count
was normalized as compared with the increased values of CyS, i.e., lowered
(substance specific release effect of CyS was inhibited).
The decreasing effect on the erythrocyte count of NaF and the release effect
of CyS were counterbalanced after simultaneous administration; in this
CA 02311546 2000-OS-19
-11-
case, the value correlated with that of the control group, but not with
respect to the bone marrow.
In the administration of putamen ovi, there are considered desirable from a
therapeutic point of view:
- the general increase in defined immunocompetent cells in the bone
marrow, in primary and secondary lymphatic organs and in the ter-
minal vascular system, especially under the simultaneous influence of
cytotoxic substances (cytostatic agents, corticoids, diuretics etc.) as
the manifestation of a chemoprotective (chemopreventive) effect.
This could also be confirmed clinically by an inhibition of the decrease
in immunocompetent cells and a demineralization of the hard tissue;
- the increased tendency of granulo-, erythro-, lympho- and thrombo-
cytopoiesis as well as activation of osteoblasts and osteocytes as an
indication of an increased hemato-, angio- and osteogenesis. Indi-
rectly, this could also be confirmed clinically by the increase of bone
density with a concomitant reduction of the loading pain of the loco-
motor system;
- the stimulation of the metabolic performance of hepatocytes as a
functional equivalent of a hepatocurative and hepatoprotective activ-
ity;
- the decrease of the serum levels of bilirubin, creatinin and urea as an
indication of an improvement of renal function and thus of detoxifica-
tion of the ammonia generated in protein metabolism and, in connec-
tion with this reaction cycle, an increased mitochondrial performance
of hepatocytes;
CA 02311546 2000-OS-19
-12-
- the decrease of cholesterol and triglyceride values in terms of a
positive influence on pathological lipid metabolism disorders and thus
reduction of atherogenic risk factors;
- the reduction of alkaline and acid phosphatases in animal experi-
ments and clinically in terms of a positive influence on pathological
disorders of bones, liver, kidneys, intestine or on leucemia or carci-
nomas;
- lack of indications of micromorphological structural interferences or
pathologically altered organic function in terms of undesired, in par-
ticular cancerogenic or mutagenic, effects, as seen from the results of
the evaluated hematological, clinical-chemical and histological exami-
nations.
From these experimental and clinical results, it can be concluded that the
recorded changes of defined hematological and clinical-chemical analytical
values as well as of morphofunctional clinical pictures of bones and bone
marrow, of primary and secondary lymphatic organs, and of liver, pancreas
and kidneys, have to be considered under two aspects: the present results
speak in favor, on one hand, of the presence of osteopathy-inhibiting
effects of the test substance P0, and on the other hand, of novel biological
effects which could be used under therapeutic aspects in deficiencies of the
cellular immune condition and in disorders of bone metabolism (osteo-
neogenesis) and of hepatic and renal functions.
In a pilot study, it could be proven that the addition of PO to the medium in
a culture of human osteoblasts could induce the latter to increased forma-
tion of type I collagen as well as osteopontin and osteocalcin.
In a pilot study in rabbits, the local application of extracorporal shock
waves
to the perineal bone induces a restricted neogenesis of bone tissue in the
CA 02311546 2000-OS-19
-13-
spongy substance. When PO is additionally administered orally, the extent
of neogenesis was increased, and the additional course of remodelling
accelerated.
In the PO therapy according to the invention, the focus is on:
- the prevention of certain spontaneous fractures in typical positions in
static insufficiency of the skeleton by an improvement of the microar-
chitecture due to induction and stimulation of local microcallus forma-
tion;
- the stimulation of osteoneogenesis and hematopoiesis;
- a chemoprevention in an accompanying therapy with chemically
defined diuretics, cytostatic agents and corticoids (inhibition of sub-
stance-specific osteoporosis/bone marrow aplasia effects), and im-
provement of the tolerance for an accompanying hormone treatment;
- reduction of the loading pains of the cerebral column and the joints,
which often set in early.
The process for the preparation of putamen ovi (an organotherapeutical
agent, from a medical point of view), in particular with a grain size of less
than 0.1 mm, according to WO 96/00579 is preferably realized by using the
egg shells of fresh eggs (not from long-term storage) from controlled
keeping in accordance with EG or US law.
The selection is performed after individual transillumination of each egg for
examining its quality state (integrity and degree of freshness).
After breaking the egg by hand or with technical means (use of egg-white
and egg-yolk unchanged, cooled for direct use by bakeries and confection-
CA 02311546 2000-OS-19
- 14-
eries (as an additional quality criterion)), collecting the egg shells and
deep-
freezing (-25 °C), it is recommended to complete the further processing
within 4 days after an extensive analytical examination of the egg shell
quality by a visual and olfactory test (fresh egg odor must be retained).
The egg shells, especially from Gallus gallus domesticus, are washed at
room temperature or elevated temperature with purified water with stirring,
and the egg shells, purified from contaminants, are subjected to autoclave
treatment.
The autoclave treatment is preferably performed discontinuously; thus, air
and water vapor are completely removed at given intervals and replaced by
freshly prepared steam. The function of the autoclave treatment is not only
to inactivate microorganisms and to eliminate contaminants and storage
protecting agents, but also to inactivate a potential of toxins and allergens
which is associated with the presence of defined, and partly also denatured,
ovoproteins. By the use and the relatively long time of action of the live
steam, membrane and palisade proteins and glucoproteins are dissolved as
a gel and essentially eliminated, in particular, by the discontinuous opera-
tion of the process.
By means of the time control, 2 different products are produced:
1. autoclave treatment for up to about 3 hours:
egg shell components of the central palisade zone, freed from the
inner and outer shell membranes (PO);
2. autoclave treatment for about 6 to 10 hours:
egg shell components of the central palisade zone, freed from the
inner and outer shell membranes and from the shell matrix of the
palisade zone (POM).
CA 02311546 2000-OS-19
-15-
While both PO and POM have osteoneogenetic effects when orally adminis-
tered, POM is also suitable as a starting material for the local application
as
a bone substitute.
The egg shells are subsequently dried and crushed to the desired grain size
following or during the drying.
The active ingredients of the egg shells can be classified as follows:
The POM egg shell components can be prepared not only by the process
according to WO 96/00579 (POM1), but also by
- enzymatic treatment (POM2)
The starting material is PO which is incubated with a 1 to 10%
solution of proteases (proteolytic enzymes), buffered at pH 7 to 8, at
30 to 40 °C for a period of 24 to 72 hours and then again subjected
to autoclave treatment for purification (1 to 3 hours)
- alkaline treatment (POM3)
with boiling aqueous 1-5% NaOH solution, followed by purification by
means of autoclave treatment (1 to 3 hours).
The shell matrix of the palisade zone, MPM, with organically bound mineral
substances can be isolated by one of three routes:
- The mineral-containing glucoproteins (MGP1) are dissolved as gels by
autoclave treatment; MGP1 is isolated from the exchanged aqueous
phase by thermal, vacuum and/or freeze drying;
CA 02311546 2000-OS-19
- 16-
- the mineral-containing glucoproteins (MGP2) are dissolved as gels by
means of aqueous and/or organic solvents or with supercritical fluids
(COZ extraction) and then isolated by thermal, vacuum and/or freeze
drying;
- MGP3 (denatured) is obtained in the preparation of POM3. After
alkaline treatment of POM with boiling 1-5% NaOH solution, the solu-
tion is filtered off from the residual shell skeleton and neutralized.
Then, MGP3 is isolated by vacuum and/or freeze drying.
Examples la to c/Comparative Examples is to 1h:
Animals and animal keeping
Male Wistar rats having an average body weight of 322.5 g were kept under
conventional conditions at a room temperature of 21 ~ 1 °C, a relative
atmospheric humidity of about 60% and a 12 hour day/night cycle. Prior to
the start of the experiment, they were subjected to an acclimatization to
those keeping conditions for 12 days.
Feeding was performed with the pelletized standard diet Altromin C1000,
Misch. Nr. 100 (Altrogge, Lage). The animals were given tap water ad
libitum as drinking water.
Testing substances and dosage
Example 1a:
Coated tablet core granules, with putamen ovi, micronized; prepared by
autoclave treatment by analogy with Example 1 of WO 96/00579 with a
CA 02311546 2000-OS-19
- 17-
weight of 640 mg containing 440 mg of putamen ovi, micronized, corre-
sponding to 160 mg of calcium.
Dosage form
Putamen ovi, coated tablet core granules, 146 mg (c 100 mg of putamen
ovi, micronized ~ 36.5 mg of Ca) is suspended in 2 ml of dist. water (POS),
0.5 ml (POS) = 9.125 mg of Ca (PO)/250 mg of body weight.
Comparative Example la:
Calcium carbonate (CC)
91.18 mg of CC ~ 36.5 mg of Ca
0.5 ml of CCS ~ 9.125 mg of Ca (CC)/250 mg of body weight
Comparative Example 1b:
Sodium fluoride (NaF)
Comparative Example ic:
Cyclophosphamide (CyS)
Dosage form: 1 mg of cyclophosphamide/0.05 ml of dist. water
Comparative Example id:
Furosemide (FUR)
Example 1b:
Cyclophosphamide (CyS) + putamen ovi (PO)
Comparative Example le:
Cyclophosphamide (CyS) + calcium carbonate (CC)
Comparative Example lf:
Cyclophosphamide (CyS) + sodium fluoride (NaF)
CA 02311546 2000-OS-19
- 18-
Example 1c:
Furosemide (FUR) + putamen ovi (PO)
Comparative Example 1g:
Furosemide (FUR) + calcium carbonate (CC)
Comparative Example 1h:
Furosemide (FUR) + sodium fluoride (NaF)
Treatment groups and application
Thirty-one male Wistar rats were grouped into the following treatment
groups for the examination:
CA 02311546 2000-OS-19
-19-
Table 1
group dose (mg/kg body number of
weight) animals
I control (compari- --- 5
son)
II PO (invention) 36.5 5
III CC 36.5 2
IV NaF 20 2
V CyS 5 2
VI FU R 50 2
VII CyS + PO (inven- 5 + 36.5 3
tion)
VIII CyS + CC 5 + 36.5 2
IX CyS + NaF 5 + 20 2
X FUR + PO (inven- 50 + 36.5 2
tion)
XI FUR + CC 50 + 36.5 2
XII FUR + NaF 50 + 20 2
The test substances, PO, CC, NaF, CyS, FUR, CyS + PO, CyS + CC, CyS +
NaF, and FUR + PO, FUR + CC and FUR + NaF, were administered to the
untranquilized animals intragastrally by means of a rigid button probe once
daily for 7 days. The suspensions were freshly prepared immediately before
the administration and homogeneously administered.
The control animals were given physiological saline in an equivalent vol-
ume.
CA 02311546 2000-OS-19
-20-
Measurement of hematological and clinical-chemical parameters
In detail, there were detected in the peripheral blood:
- combinedly:
erythrocytes (RBC), leucocytes (WBC), platelets (PLT), hemoglobin
(HGB), hematocrit (HCT)
erythrocyte indices: '~
mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean
corpuscular hemoglobin concentration (MCHC), and erythrocyte dis-
tribution width (RDW)
- by flow cytometry:
leucocyte differentiation: granulocytes, monocytes, lymphocytes, B
and T cells, helper and suppressor cells
- combinedly:
GPT, GOT,
glucose,
cholesterol, triglycerides,
Na, K, CI, Ca,
creatinin, urea, total protein.
By means of the fully automated hematology analyzer Sysmex K-1000,
there could be determined: WBC, RBC, PLT as well as HGB and HCT, further
MCV, MCH and MCHC. The main unit of this device essentially consisted of a
hydraulic (HS) and an electronic (ES) system. The HS was used for sucking,
pipetting, diluting, mixing and lysing. The ES analyzed and converted the
CA 02311546 2000-OS-19
-21-
signals from the HS and submitted the results to the printer. With the aid of
microprocessors, the ES also monitored the test runs, the testing station,
and performed a quality control.
The hematocrit value corresponded to the percent volume fraction of
erythrocytes in blood. In addition to the erythrocyte count and the hema-
tocrit value, the level of hemoglobin, the chromoprotein contained in the
erythrocytes, was an important criterion for the diagnostics of anemias.
Classification was performed by the erythrocyte indices. Erythrocyte size
and hemoglobin content were characterized by the erythrocyte volume
(MCV = mean corpuscular volume), the hemoglobin content of the erythro-
cytes (MCH = mean corpuscular hemoglobin), and the mean corpuscular
hemoglobin concentration (MCHC). The erythrocyte distribution width (RDW
= red cell distribution width) is a measure of anisocytosis.
Parameters such as enzymes, glucose, lipids, electrolytes, creatinin, urea
and protein were detected in a selective, method-oriented, photometric or
ion-selective way using the analyzer Cobas Mira. The supplemental report
furnished analysis-specific data of quality control and statistics.
The leucocyte differentiation was performed using the flow cytometer
FACScan following appropriate lysing of the whole blood sample (scattered
light measurement).
The lymphocyte differentiation was performed after specific monoclonal
incubation by means of fluorescence-activated cell sorting (fluorescence
measurement).
On the 7th day of treatment, there were analyzed quantitatively:
- leucocytes (total), lymphocytes (total),
- T lymphocytes (CD2+/CD45 RA-),
CA 02311546 2000-OS-19
-22-
- B lymphocytes (CD2-/CD45 RA+),
- helper lymphocytes (CD4-/CDBb+),
- NK cells (CDBa+/CDBb-).
For determining the phenotypes of the lymphocytes, they were incubated
with the following antibodies of Pharmingen, San Diego (USA), to which a
fluorochrome was coupled:
Fluorescein isothiocyanate (FITC) conjugated mouse anti-rat CD2 mono-
clonal antibody, R-phycoerythrin (R-PE) conj. mouse anti-rat CD45RA or
A/B monoclonal antibody, fluorescein isothiocyanate (FITC) conj. mouse
anti-rat CD4 monoclonal antibody, R-phycoerythrin (R-PE) conj. mouse
anti-rat CD8 (~~3 chain) monoclonal antibody, fluorescein isothiocyanate
(FITC) conj. mouse anti-rat CDBa monoclonal antibody.
Approach: a) CD2/CD45RA = T and B cells
b) CD4/CDBb = T4 and T8 cells
c) CDBa/CDBb = NK cells
pl each of the antibodies is incubated with 50 pl of Na-EDTA-blood at
room temperature in the dark for 20 min. The suspension is agitated with
2 ml of Lysis Reagent of Becton-Dickinson and incubated for 10 min as
described. This is followed by centrifuging at 400 x g for 6 min, and the
supernatant is poured off. The pellet is washed with 3 ml of Cell Wash and
centrifuged at 400 x g for 6 min. The pellet is taken up in 100 pl of Cell
Wash. The suspension is analyzed by means of the flow cytometer.
Clinical observations:
During the acclimatization prior to the start of the experiment and in the
course of the entire experimental period, the general condition of the
CA 02311546 2000-OS-19
-23-
animals was examined. In addition, their body weights were determined
daily.
Histological examinations
The histological examinations were performed after formaldehyde fixation
of the organ samples with paraffin slices (Medim-Plast~) and hematoxylin-
eosin staining (H.E.).
The following selected organs were subjected to a light-microscopic exami-
nation: Peyer's plaques, bone marrow (sternal), thymus, spleen, lymph
nodes (mesenterial catchment area) from the defense system, and the
liver, pancreas and kidney parenchymas.
Results:
Erythrocytes and leucocytes
The potentials of the test substances, P0, CC, NaF, CyS, FUR, CyS + P0,
CyS + CC, CyS + NaF, and FUR + PO, FUR + CC and FUR + NaF, were
determined indirectly via their stimulating effect on the number of leuco-
cytes (WBC) and erythrocytes (RBC).
Measurements were performed with the following test substances (corre-
sponding values for the control = 100%):
CA 02311546 2000-OS-19
-24-
Table 2
W BC RBC
PO changes only within the changes only within the
physiologically normal physiologically normal range
range
CC changes only within the changes only within the
physiologically normal physiologically normal range
range
NaF +13.8% ~ -5.8%
CyS -51.3% +12.0%
FUR +61.0% unchanged
CyS + PO -37.4% unchanged
CyS + CC -51.8% unchanged
CyS + NaF -39% +3.5%
FUR + PO n.d. n.d.
FUR + CC +48.7% unchanged
FUR + NaF unchanged -5.0%
n.d. = not determined
Bilirubin, creatinin, urea, glucose, lipids, electrolytes and protein
The following clinical-chemical metabolic measurement values were ana-
lyzed in a combined method-specific manner:
Bilirubin, creatinin, urea, glutamate oxalacetate transaminase (GOT),
glutamate pyruvate transaminase (GPT), gamma-glutamyl transferase
(GGT), glucose, cholesterol, triglycerides, calcium, sodium, potassium,
chloride and total protein.
CA 02311546 2000-OS-19
-25-
Measurements were performed with the following test substances:
Table 3
bilirubincreatininurea glucose cholesteroltri-
glycerides
PO -27.6% -21.9% -5.4% unch. -9.3% -11.1%
CC -17.3% -19.7% -13.9% +6.7% unCh. -7.8%
NaF +13.8% -20.y% -13.2% +15.7% -8.7% -4.2%
CyS +58.6% -28.3% unch. +5.2% -25% +10.1%
FUR +20.7% -49.7% -7.3% unCh. -24.9% -29.6%
CyS + PO -12.6% -12.2% unCh. -10.1% -25.5% -3.1%
CyS + CC +27.6% -38.7% -5.1% -4.9% -7.5% ---
CyS + NaF +24.1% -41.1% -23.4% +3.4% -28.5% ---
FUR + PO n.d. n.d. n.d. n.d. n.d. n.d.
FUR + CC +27.6% -21.3% -27.7% unch. -34.9% -31.7%
FUR + NaF +10.3% -23.7% -6.6% -7.4% -21.5% -18.0%
n.d. = not determined
unch. = unchanged
CA 02311546 2000-OS-19
-26-
Measurements were performed with the following test substances:
Table 4
GLDH GOT GPT AP SP Ca
PO -11.8% unch. unch. -17.7% -4.4% unch.
CC +6.7% -7.1% unch. +5.5% unch. unch.
NaF -17.3% -9.8% -9.7% unch. -7.5% unch.
CyS -21.3% -24.6% -30.8% -14.7% -9.1% -23.2%
FUR -31.3% unch. -24.4% -13.4% -9.1% -28.9%
CyS + PO -27.6% -13.0% -24.4% -13.0% -4.7% -33.6%
CyS + CC -35.8% -3.2% -23.9% -24.8% -6.5% -36.0%
CyS + NaF -25.2% unch. -33.1% -19.6% -21.5% -33.6%
FUR + PO unch. unch. unch. unch. unch. unch.
FUR + CC -6.4% -20.4% -7.3% -13.2% -9.1% -14.1%
FUR + NaF unch. -39.4% -23.8% unch. -13.1% -17.5%
n.d. = not determined
unch. = unchanged
CA 02311546 2000-OS-19
Lymphocyte differentiation
Measurements were performed with the following test substances:
Table 5
lympho- lympho- lympho- helper supp. poly-
cytes cytes cytes lymphocyteslymphocytesmorphs
PO unch. uncli: +6.9% -5.4% +10.9% +7.1%
CC unch. unch. +7.6% -4.3% +12.4% unch.
NaF unch. unch. +16.5% unch. unch. +61.8%
CyS -55.0% -51.9% -79.9% -45.9% -30.9% -28.1%
FUR +60.5% +46.2% n.d. +41.5% unch. +65.7%
CyS + PO -39.3% -20.7% -64.0% -21.8% -14.9% -24.9%
CyS + CC -58.8% -46.5% -79.8% -47.9% -46.6% unch.
CyS + NaF -41.9% -23.4% -72.4% -25.4% -22.3% -28.3%
FUR + PO +82.6% +54.9% n.d. +47.0% +50.9% +62.0%
FUR + CC +44.6% +46.7% +47.0% +52.3% +41.9% +93.4%
FUR + NaF unch. -12.1% +20.0% -8.3% -12.6% n.d.
n.d. = not determined
unch. = unchanged
Example 2
Clinical study on the induction of osteoneogenesis and on the reduction of
the loading pain of the locomotor system
Patient study
The clinical examinations on the effect of PO according to Example 1 on
osteoneogenesis in 41 female patients with decreased bone density in post-
CA 02311546 2000-OS-19
_2$_
menopause and on the decrease of an existing loading pain (pain reduction)
of the vertebral column and joints.
The results of the study have been obtained from repeated osteodensi-
tometric determinations.
Osteodensitometry
The bone mineral density measurements were performed using quantitative
digital radiography (Hologic QDR-1000 TM bone densitometer) on the
lumbar vertebrae (LWK 1-4), the result of the bone mineral density calcula-
tion being expressed as density in grams of calcium hydroxyapatite per
cm3.
In this osteodensitometric method, the measured value is corrected for
absorption in the soft-tissue coat; thus, the bone mineral content can be
determined without soft-tissue errors.
Initial examination of the lumbar vertebral column (LWK 1-4) in
female patients in post-menopause
The results of the initial densitometric examination of a larger sample of
patients, after at least half a year from the last menstruation, were com-
pared age-specifically on the basis of reference data (nearly 1000 lumbar
vertebral column measurements). The measured bone density (g/cm3) was
also given as a percentage of the corresponding age-specific reference
value. Subsequently, 41 female patients in post-menopause having a
determined bone density of between 61 and 92% were included in the
study.
CA 02311546 2000-OS-19
-29-
Comparison of two vertebral column scans for follow-up
During the phase of therapy with PO, no additional hormone, vitamin D3,
calcitonin or other calcium preparations were administered.
For the follow-up of the therapeutic effect of PO in the 41 selected patients
with decreased bone density in post-menopause, the respective value
measured after the end of the therapy was compared with that of therapy
start. This absolutely required that the measuring fields of the vertebral
column scans in the two analyses be identically adjusted with respect to
size, shape and position. This is possible when a Holgic-specific computer-
controlled comparing device is employed in which the measuring field of the
repeated examination is set in an optically identical manner with that of the
initial examination by matching it to the stored scan. Thus, the reproducibil-
ity of the accuracy of measurement is optimized. This method thus ensures
that changes in the measured values at different times must be predomi-
nantly caused by changes in the bone metabolism.
Results
The overall balance of the osteodensitometric follow-up values on the basis
of the 41 follow-up examinations on the effectiveness of PO shows an
average increase of bone mineral density by +9.4% (from 78.1% to
85.5%).
In group A, which was administered PO for less than 200 days, the bone
density already increased by an average of 5.5%. In group B, which was
administered PO for less than 300 days, the measured value increased by
7.3%, and in group C (more than 300 days), the measured value even
increased by 9.4%.
CA 02311546 2000-OS-19
-30-
The increases of the measured values obtained after the end of the therapy
in patient group A (bone density prior to start of therapy > 80%) and group
B (< 80%) are clearly different. The results show a clear dose-action
relationship: in the first group (A), the value increased by 6.9% with
administration of 3 x 1 coated tablet of PO per day, and in the second group
(B), it increased by 10.9% with 3 x 2 coated tablets of PO per day.
The efficiency of PO therapy in decreased bone mineral density in post-
menopause (responder/non-responder ratio) is as follows:
With 12 of the patients examined, the increase of bone density was less
than 5%, with 18 patients, it was between 5 and 10%, and with 11 pa-
tients, the value was more than 10% (+15.4%). The responder rate can
thus be assumed to be about 70% (B + C).
In none of the patients, the bone density after the end of the therapy was
inferior to that of the initial examination.
By a therapy with P0, it is evidently possible to generally counteract a bone
density decrease in the vertebral column of female patients in post-
menopause.
In addition, the study showed that the extent of the increase of bone
mineral density is dependent on both the level of daily dose and the
duration of the PO therapy.
