CA2311546A1 - Use of putamen ovi - Google Patents

Abstract

The invention relates to the use of treated putamen ovi in the production of medicaments which can be administered orally or locally for treatment and prevention.

Description

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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-_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.

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.

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 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:

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;

_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;

- $ _ 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), _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.

- 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 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;

- 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 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-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).

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;

- 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 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) 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:

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 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.

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 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-), - 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 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%):

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.

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 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 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-_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.

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%.

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.

Claims (13)

1. Use of putamen ovi for the preparation of orally applicable medicaments for the treatment and prevention of organ and tissue damage caused by radiation, infections and chemically, of the O-MALT system of the small intestine, the bone marrow (bone marrow aplasia), bone and cartilage genesis disorders, of diseases of the locomotor system, dysfunction, aplasia or hypoplasia of the thymus, dysfunction of the spleen and aplasia or hypoplasia due to damage to the lymph nodes caused by medicaments or radiation, atrophy or necrosis of the liver, insufficiency of the exocrine, secretory function of proteases, esterases, carbohydrases and nucleases of the pancreas, insufficiency of the endocrine function of the islets of Langerhans, the carbohydrate metabolism and kidney insufficiency, and in immunosuppressed conditions, for cellular immunostimulation, for the therapy of leucocytopenia, granulocytopenia, lymphocytopenia, thrombocytopenia, erythrocytopenia and in immunoglobulin deficiency conditions, also due to AIDS and tumors, and for the therapy of hyperlipoproteinemias and hyperlipidemia.

2. The use according to claim 1, characterized by a cellular immunostimulation, especially following immunosuppression after radiation or chemotherapeutic treatment to increase the cell counts of leucocytes, thrombocytes, erythrocytes, polymorphonuclear granulocytes and lymphocytes, T, B, helper, suppressor and NK cells, especially of B lymphocytes, in the terminal vascular system, wherein an increase of vital lymphocytes is seen in the cortex zone and the follicles of Peyer's plaques which is accompanied by a secretory induction of sIGA in the small intestine, a cellular stimulation of immunocompetent cells in the thymus, spleen and mesenterial lymph nodes, activation of the metabolic functions of liver, pancreas and kidneys through a lowering of the serum levels of creatinin, uric acid, glucose, bilirubin, cholesterol and triglycerides as well as GOT, GPT and GGT.

3. The use according to claim 1 or 2 for the treatment of primary or secondary disorders of or damage to chondral or desmal ossification, osteoneogenesis and hematogenesis, optionally in combination with sodium fluoride and hormones, especially estrogens, calcitonin, pyrophosphates, especially biphosphonates, and vitamins, especially vitamin D, especially D3 and dihydroxycholecalciferol, the bony substance with external substantia corticalis and internal substantia spongiosa the skeleton including osteocytes, intercellular substance with collagenous fibrils and calcified cement, bone metabolism including the function of osteoclasts and osteoblasts, and the balance between bone absorption and formation.

4. The use according to any of claims 1 to 3 for the stimulation of the metabolic performance of osteoblast-specific transcription factors, of the differentiation of precursor cells to osteoblasts by a regulation of osteoblast differentiation, and stimulation of the provision of bone-specific alkaline phosphatase, collagens, especially type I collagen, and of osteopontin and osteocalcin.

5. The use according to any of claims 1 to 3 in oral and bone surgery after oral and local application for the stimulation of osteoneogenesis and for early callus formation.

6. The use according to claim 1 or 2 in combination with cytostatic agents, especially cyclophosphamide, or radiation therapy, as oral adjuvants of malignant tumor treatment, especially the treatment of primary and secondary bone tumors with osteoclastic or osteolytic metastases, tumor-like bone defects with intra-ossal space occupation, for the treatment of bone cysts.

7. The use according to claim 1 for increasing the tolerance for chemotherapy, cytostatic and radiologic therapies, especially in combination with chemically defined substances selected from cytostatic agents, especially cisplatin, cyclophosphamide, methotrexate, fluorouracil, bleomycin, and/or diuretics, especially acetazolamide, hydrochlorothiazide, chlorothalidone, furosemide, amiloride, inhibiting the bone substance destruction due to diuresis and saluresis, as an adjuvant of analgesia, and as a pharmacological component of the positive influencing of interactions between the endocrine, nervous and immune systems.

8. The use according to any of claims 1 to 4 for the inhibition of bone decalcification upon daily cigarette consumption.

9. The use according to claim 1 for the additive treatment of hyperlipoproteinemias, especially hypercholesterolemia and/or hypertriglyceridemia.

10. The use according to claim 1 or 2 for the treatment of anemia, especially in tumor patients.

11. The use according to any of claims 1 to 10 in the form of putamen ovi pulvis and in a form extracted with aqueous and/or organic and/or supercritical solvents, especially carbon dioxide, as powder, granules, capsule, tablet, coated tablet, pastille or paste.

12. The use according to claim 11 for oral and local application on the basis of - egg shell components of the central palisade zone, freed from inner and outer shell membranes;
- egg shell components of the central palisade zone, freed from inner and outer shell membranes and from the shell matrix of the palisade zone; and/or - shell matrix of the palisade zone with organically bound minerals;

for the preparation of an agent for bone remodelling and implantation, for use a bone cement and bone replacement with an osteoneogenesis-active potential for the well-aimed local bone formation, reconstruction of bony deficiencies, in the filling of bone damage after tumor operations, and for the removal of bone damage in oral surgery and plastic surgery of the face, and for the local induction of osteoneogenesis by the application of extracorporal shock waves in combination with oral administration of putamen ovi.

13. The use according to any of claims 1 to 12, characterized in that putamen ovi preparations are employed in combination with Viscum extracts, Echinacea extracts, Cynara extracts, Urtica extracts, Betula extracts, Equisetum extracts and/or Taraxacum extracts, optionally in combination with chemically defined diuretics/cytostatic agents, for the inhibition of bone demineralization and/or depression.