Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
zo~s4a . POLYSACCHARIDE MIXII)RE WIT~I IMMt~NE STlM[lIATING AND ANTI-PR(~ ME;THOD OF PRODUC~IOW AND MEDIC~IES ~C~TAINING THE SUBSTANOES The invention relates to a polysaccharide with immune stimulat~ng effect, a method for its production, as well as medicines containing this polysaccharide. Immune stimulation as a therapeutic concept has long been known in medicine. In general, the term comprehends prescription of substances, which themselves have only small if any antigenic effect, but which are capable of inducing the body's own defense mechanism in an unspecific way. It is known today that a plurality of substancès are capable of stimulating the immune r¢sponse, including especially various minerals, for example AL(OH)3, MgSO~, beryllium, plant oils with or without addition of microbacterium, as well as a number of plant hormonal groups guch as alkaloids, Sesgui- and diterpene bonds, quinones and -~ polysaccharides. The whole process of ~mmune stimulation is described in detail, for example, by Chedid, L., et al. "Immunstimulation", Publisher: Springer, Heidelberg/New York, 1980: Heidelberger, M., "Structure and Immunological Specificity o~ Polysaccharides"; Fortschritte der chem. org. Naturst. 42, 288 (1982):~Drews, J., "Immunpharamacologyn, Springer Verlag, 1986, ;~; G. Dannhardt, Therapeutikon 11, November 1988, 653; ~Immunopotentionn:~Ciba~Foundation symposium, Elsevier, Amsterdam 1973,~"Immunopharamaco}ogy of Infectious Diseasesn, Ed. J.A. Ma~de,~ Alan R. ~iss, New York 1987. Up until today, in most cases the exact operation of the immune stimulating sùbstances cannot be definitively explainedl. In general, these substances show, among others, an effect on the function~and/or proliferation of immune response capable cells, however }eaving no memory reaction. This means, that macrophages, granulocytes, as well as T and B-lymphocytes are ;~ primary targets for the immune stimulating 6ubstances. The effect can occur directly or indirectly, for example, throuqh the compIement system or lymphocytes, through production of kinin, `~ for example, interferon, interleukin, tumor-necrosi6-factor, ~ '~ colony stimulating factor and others, as well as a proliferation of macro and microphagocytes. Since non-specific and specific ~ - defense mechanisms interrelate, cascaded effects and simultaneous influencing of multiple defense mechanisms can be anticipated. In medicine, preferred uses for immune stimulation include especially the therapy of mixed infections, chronically- persisting, chemotherapeutic-resisting bacterial and viral infections, the therapy of opportunistic infections in high risk patients, the therapy of malignant illnesses and to a certain extent also the therapy of autoimmune diseases. Immune stimulation could also be used during Zytostatica-therapy to partially compensate for the associated immune suppression. . . It is known that various polysaccharides, especially mushrooms and higher plants, are capable of enhancing the activity of the immune system. Of the best known immune stimulants of this type are (alpha)-1,3/1,6-glucose Lentina and Schizophylla, compare for example "Immunpharmacology of Infectious Diseases", Publisher: Springer 1986. , , From EP-A 0 246 069, a watery extract of plants of the family Nerium with proliferation inhibiting characteristics are known. It is a goal of the present invention to make available a new ~- polysaccharide with immune stimulating effect, a method for its production, and a medicine containing this polysaccharide. Surprisingly, Applicant has found that from the leaves and branches of the Nerium oleander tree a polysaccharide with immune stimulating activity can be isolated. - - ~ ~. In contrast to previously known ~entina- and Schizophylla- polysaccharides, one of the inventive polysaccharides is composed essentially exclusively of alpha (1- >43 galacturonic acid units ; - . , . : ,. 20~948 having approximately 90% of its carboxyl groups methylated. The molecular weight of this polysaccharide was identified with 30,000 to 40,000 D, preferably with approximately 35,000 D, HPLC- gelpermeation chromatography. It was further discovered that a polysaccharide mixture, obtained as an intermediate product, has not only a general immunestimulating activity but also demonstrates a cell proliferation inhibiting activity. This polysaccharide mixture, obtained through precipitation of-the polysaccharide of the plant, contains essentially the above-described alpha (1->4) galacturonics as well as additional galacturonics of varying ~ molecular weights and oligosaccharides. ~ ~ - Additionally, a low molecular polysaccharide mixture was ;~ isolated, the molecular weight of which was determined with -~ approximately 2500 to 12000 D in HPLC-gel permeation ~hromatography. This mixture thus definitely falls below the molecular weight (approx. 20000D) of the previously known -~ polysaccharide with immune stimulating effect. This polysaccharide mixture, obtained from the watery extract of the -~ Nerium oleander plant, contains three lesser fractions of varying molecular weights and content. The immune stimulating effect of the inventive polysaccharide was ;~ tested through in vitro methods structured to measure the capability of the mononuclear system as well as the stimulation of T and B-lymphocytes. Through these examinations it was surprisingly found that the ;`~ inventive polysaccharide is specially capable of enhancing the secretion of tumor-necrosis-factor. The tumor-necrosis-factor is formed in macrophages during induction and released to the blood. It plays an important role during tumor resistance. 4 ``- - -` 20~9~8 In other tests the inventive polysaccharide as well demonstrated immune stimulating activity. Branches and leaves of the oleander tree (Nerium oleander) are suitable as starting materials for the isolation of the inventive polysaccharide. Leaves are preferably used. '~ The method of isolating the inventive polysaccharide essentially comprises cooking the crushed starting material with ;- approximately 5 times the volume of distilled water, for 2 to 4, preferably 2.5 to 3 hours, followed by filtration of the solid particles, cooling of the extract to room temperature and additional filtration. The high molecular polysaccharide can be obtained from the extract as an intermediary product through precipitation with alcohol, over complex-formation with heavy metal salts or -~ quaternary ammonia salts. Preferably, the polysaccharide is precipitated with ethanol which is added in a ratio from 3:1 to 1:3, preferably 1:1. After the solution has set for at least 12 hours, it is filtered, the precipitate is dissolved in distilled water, and the precipitation is repeated twice. The final - precipitate, which again is dissolved in water, can be lyophilized if necessary. The raw fraction can be separated into high and low molecular weight portions through a customary gelchromatography procedure, for example through use of a Biogel- P-60 column. From the high molecular weight fraction the inventive polysaccharide is isolated through ion exchange chromatography, for example through use of a DEAE-Sepharose C 6-B column, through elution with for example a 0.1 molar NaCl gradient at a NaCl concentration of approximately 0.2 molar. ~.: 201~948 The method of isolating the inventive low molecular polysaccharide, that is the mixture, consist essentially of lyophilization of the filtrate obtained according to the above- described extraction step, dissolving of the freeze dried material in distilled H20 and dialyzing against distilled H20 (exclusion point of approximately loooo D). The freeze dried dialysi~ product is then treated with alcohol and the precipitate i8 further separated by known methods of gelchromatography -~ following centrifugation. ~ ~; Exam~le 1 Isolation and characterization of the hiqh molecular ;~ polvsaccharide from Nerium oleander leaves ; The yield of polysaccharide varies according to the source and time of harvest of the starting material. All the steps described below were conducted at 4-C, unlesæ ;~ otherwise stated. ; The leaves of the Nerium oleander tree were crushed and cooked in the approximately 5-fold volume distilled water for 3 hours. The solid particles were filtered off and following cooling to room temperature the solution was again filtered. The filtrate was mixed with ethanol (96%) in a 1:1 ratio. This solution was allowed to set for 12 hours, the formed gelsuspension was filtered off and the remainder taken up in distilled water. This ~ - solution was again mixed with ethanol (96%) in a 1:1 ratio. After allowing the solution to set for at least 12 hours, the gelsuspension was filtered off, the remainder taken up in distilled water and lyophilized. In this way, a raw extract was obtained as an intermediary product. ~ 6 ~ ` . . .,: : 2~ 9~8 To produce the inventive polysaccharide, the high molecular - substances were carefully separated from the low molecular substances. In that regard the raw extract was dissolved in water, the solution centrifuged and the precipitate placed on a biogel P-60-column and eluded with distilled water whereby two fractions were obtained. The first fraction contained a polysaccharide mixture of molecular weight range of 17,000 to 120,000 D. The second fraction (MG approximately ~ 10,000 D) contained lower molecular weight poly and oligosaccharides and ~ - - other substances. The inventive polysaccharide was obtained through chromatography of the high molecular weight fraction in an ion exchangP DEAE Sepharose CL 6 B column. For this the high molecular weight fraction was dissolved in water and placed on the column filled ;- with DEE Sepharose and eluded with 0.1 molar NaCl. The inventive polysaccharide eluded at approximately 0.2 molar NaCl. It shows a positive optical rotation of 38.12- (1 mg./ml. H~0, room temperature). The following structure was determined for the inventive polysaccharide: - COOR COOR ~,O/~ O'/L~/~ OH OH , 20~948 in which the residue R demonstrates an average methylization degree of approximately 90% and otherwise indicates, independent of each other, hydrogen, alkali or earth alkalation. ~- . The structure of the inventive polysaccharide was further examined through methanol determination and '3C-NMR-spectroscopy. During these examinations it was found that the polysaccharide ~ ' contains only D-galacturonic acid units which are approximately ; 90% methylated. - The permethylization analysis demonstrated the bond relationships of this galacturonic acid ~Hakomori, S.I., J. Biochem. (Tokyo) 55, 205 (1964)). The GC-MS results of the permethylization analysis showed that the polysaccharide chain is constructed of ~ >4) bonded D- galacturonic acid units, the carboxyl groups of which are ~ ; methylated at approximately 90%. Other than ~ >4) glycolic ~ ~ bonds, no other bond relationships could be determined. That ~ - means: the polysaccharide chain is constructed linearly and ; - contains no branching. For further analysis of these polysaccharides the molecular weight was next determined. Determination of the molecular weight occurred through HPLC-gel permeation chromatography (HP-GPC). The HP-GPC-System used~ ~-Bondagel E 125 + 5 Bondagel E 500 (Fa. Waters). Buffersystem- 0.5 molar phosphate buffer, pH6 For comparative substances Dextran T10, T40, T70, TllO, T2000 and glucose were used. ~ ` : , ~ , ~; ;' :,,, ': ~ ''"'"' '" ~'" 8 ; ~ . .: . - , . . ': '` "'~ l .; ' '' : ' ' ` ' ' ' ` ' :`: ZC)1~4R Using this method the inventive polysaccharide was determined to have a molecular weight of 30,000 to 40,000 D, preferably of approximately 35,000 D. However, it must be pointed out, that the molecular weight may vary according to the methods of analysis used. The uranic acid content of the inventive polysaccharide was determined using the carbazole test (Bitter T. and Muir, H.M., Analyt. Biochem. 4, 330 (1962) Uranic Aaid Content in Percentaae SamDle , ,. ~ :: 1. Test 2. Test Polysaccharide 99,9 100 To determine the content of the inventive polysaccharide, the polysaccharide was mixed with TFA and heated for two hours in a 121-C dry oven. After removal of the TFA a thin layer chromatography W8S conducted. ~ :. Adsorbent: Kieselgel GF ~5~-ready plates for the NAN0-DC(HPTLC) 20 x 20 cm (Fa. Merck). -~ ~ ~ 'Running system: n-Butanol-Acetone-acetic acid-water ~35:35:10:20). I Detection: Anilindiphenylaminophosphoric acid. Results showed that only D-galacturonic acid can be demonstrated to exist. g ZC~ 6~4~3 The ~(1 >4) bonds of the D-galactruonic acid was additionally examined with pectinase. The polysaccharide was dissolved in distilled water and incubated with pectinase for 3 hours at room temperature. Then, the liberated galactruonic acid was demonstrated through thin layer chromatography. The pharmacological effect of the inventive polysaccharide was - examined through accepted in vitro tests. Since no specific test methods for the verification of immune stimulating effect of substances exists to date, in vitro and in ; vivo methods which measiure the effect of bonds or plant extract -~ on the function and capability of mononucleated systems as well as the ability to stimulate T- and B-lymphocytes are generally -~ used. -~ : Through these examinations it was determined that the inventive polysaccharide is especially effective in the tumor-necrosis factor liberation test. TNF (Tumor-necrosis-factor) is a protein which, in people, consist of 157 amino acids. It is synthesized in macrophages during induction and delivered to the blood. TNF stimulates the - defense system and kills tumor cells. During the TNF test the ~ -~ substance to be tested is injected into experimental animals. Then the TNF concentration through necrotic effect on the tumor cell lines is measured in the blood of the experimental animals and used as a parameter for the stimulating effect of the test substances. :, .. . .. . The tumor-necrosis-factor test (TNF-T) was conducted according to ^ Stim PL, M., Proksch, A., Wagner, H. and Lohmann-Matthes, M.L., Infection and Immunity 46, 845 (1984). The results are set forth in the following table. ,'.... ~;, ... ....... ... ':',,,'-.'. .: ` 20~69~3 TNF-Test Results Polysaccharide ConcentrationTNF-Concentration (~g)(U/ml) . . PS 50 256 PS 25 64 PS 12.S 8 PS 6.2 4 PS = Inventive polysaccharide As can be deduced from the table, the inventive polysaccharide causes a definite elevation in ~NF concentration, i.e. it induces the synthesis of TNF in macrophages. The immune stimulating effect of the raw fraction obtained as an intermediary product was as well tested using the Tumor-necrosis- factor liberation test. The results are set forth in the following table. TNF-T-Results: Polysaccharide ConcentrationTNF Concentration Raw Fraction (~g) (U/ml) PS 50 1000 ~ - 500 The immune stimulating ef~ect of the polysaccharide raw fraction was additionally examined through the granulocyte test according to Brandt ~Brandt, L., Scand. J. Haemat. (Supplement) 2 (1967). . . During the granulocyte test according to Brandt, the number of ~ ~ - phagocytic in yeast cells or bacteria are determined through a granulocyte fraction, derived from human serum, under a microscope in in vitro tests. The percent elevation of phagocytes due to the inventive polysaccharide is measured. The - results are set forth in the following table. 11 2016~4~ Phagocyte Amounts in Percent by concentration (mg/ml) Polysaccharide lo-l 10-3 10-' 10-5 1o~6 ~: Fraction Raw Fraction 95 74 70 42 32 (Polysaccharide mixture~ As the results in the table show, the polysaccharide mixture produced as an intermediary prodùct effects a definite elevation in phagocytes which correlates well with the stimulation data obtained in the TNF-liberation test. The intermediary product furthermore has a cell proliferation inhibiting effect (anti-proliferation effect). ~ ~ . . .. Example 2: Isolation and characterization of the low molecular ~olvsaccharide mixture. that is the low molecular polvsacchaFide~ from Nerium oleander leaves The yield of polysaccharide mixture varies according to the source and time of harvest of the starting material. All the steps described below were conducted at 4C, unless otherwise stated. The leaves of the Nerium oleander tree were crushed and cooked in ; -~; approximately 5-fulled volume of distilled water for 3 hours. The solid particles were then filtered off and following cooling - to room temperature the solution was again filtered. The filtrate was lyophilized, the precipitate dissolved in distilled water and dialysized in a dialysis tube (having a molecular weight upper border of approximately lO,OOOD) against water for 3 24 hour periods. Following each 24 period the dialysis product was exchanged against distilled water. - 12 ~ .~, ~,,. ~ ': ~01~948 ~ - To obtain the inventive polysaccharide mixture, the dialysis product was lyophilized, the precipitate mixed with methanol, shaken and centrifuged for 10 minutes at 3,000 rpm. These steps were repeated 3 times. In additional separation steps, the precipitate obtained from centrifugation was dissolved in distilled water, placed on a Sephadex0-LH-20-column and eluded with distilled water. The SephadexD-LH-20-column separated the precipitate into numerous fractions. The first fraction (NOAG-II) contains a mixture of polysaccharide with molecular weights between approximately 2,500 and 12,000D. To isolate the lesser fraction polysaccharide from NOAG-II, this fraction was dissolved in distilled water, if necessary following lyophilization, placed on a column filled with Sephadex~-G-50 and eluded with distilled water. Through this chromatography the NOAG II was separated into 3 polysaccharide fractions. The lesser fraction will be hence forth designated as NOAG-III, -~ NOAG-IV, NOAG-V. - ., ,. ~..... By adding a 15% solution of Trichloracetic acid to the polysaccharide fractions dissolved in water, 5-7% of the protein content could be removed. To characte~ize the inventive polysaccharide mixture the ;~ ~ollowing tests were performed. : .. ~: Chemical tests: 1. Qualitative sugar determination: The fractions were mixed with 2 N TFA for hydrolysis and were heated in a desiccation chamber for 2 hours at 120-C. Following the removal of TFA a thin layer chromatography was performed. 13 2016948 Abso-bance: Kieselgel G F254 ready plates for the Nano-DC(~PT~C) 20 X 20 cm tFa. Merck). Running system: n-Butanol-Acetone-Acetic acid water - (35:35:10:20). Detection: Anilindiphenylaminophosphoric acid. The results showed that the following sugars could be determined: Galactruonic acid Arabinose ~; Rhamnose Galactose Xylose ~; Glucose ! ~', ,' "" `' 2. Quantitative neutral sugar determination: The lesser polysaccharide fractions were hydrolyzed and then , changed to Alditolacetates (BLAKENEY A.B. et al. Carbohydrate Res. 113(1983)291) and the sugar is determined by gas - : chromatography. Instrument: Perkin-Elmer 900 : . ~ . . Column: Glass, 6 x 2 mm, GP3% SP-23-30 on 100/200 Supelcoport. -~ ''. '; ~.,,',- Temperature: 210C ~ ~ :, . ~ ';;"The following table shows the molar relationship of the neutral sugar content. `:, ' ' ' '~ .:.... ' .. ~ .- - 14 -~ - ' ~-. . '~ . , 9~ Fractions Rhamnose: Arabinose: Xylose: Galactose: Glucose: NOAG II 0.3 0.2 0.2 0.9 1.0 NOAG III 1.1 4.4 -- 3.2 1.0 NOAG IV 0.4 3.9 -- 2.0 1.0 NOAG V 0.2 0,9 0.3 0.9 1.0 The calculation of the molar relationship is determined with glucose equalling 1Ø 3. Uranic acid determination: With the help of the carbazole tests of Bitter, T. and Muir, H.M. Analytical Biochemistry 4, 330 (1962) the uranic acid content o~ the polysaccharide fractions was determined to be 40 to 70~. 4. Molecular wei~ht determination: The molecular weights of the isolated polysaccharide were determined with HPLC-Gelpermeation chromatography (HP-GPC). HP-GPC system: ~-Porasil GPC 60 A + ~-Bondagel E-500. Buffer system: ~; 0.05 M phosphate buffer, pH 6.0 with 0.15 M NaCl. ~ . . Comparison substances: Glucose, Maltotriose, Maltoheptaose, Dextransulfate 5,000, ~ Dextran T-10, T-40, T-70, T-110, T-2000. ~ ; . I I , . . Result~ ~. . Polvsaccharide Molecular Weight (D~ NOAG III 10,000 to 12,000 NOAG IV 5,000 to 6,000 NOAG V 2,500 to 3,500 _~ 201~4~ .: 5. Protein content: :. . The protein content of the separate fractions was determined accsrding to Lowry O.H., Journal of Biologlcal Chemistry 193 (1951) 265. , .. .. . Through these tests it was shown that the polysaccharide mixture has a protein content of approximately 6 to 10%. ~ ; Optical orientation~ The optical orientation of the substances was determined with a ~ ~ ; polarimeter (Perkin-Elmer Polarimeter 241) at 20-C. Wavelength: 589 nm (Na) Concentration c: 0.1% Dissolving material: distilled water. ": It was shown that the polysaccharide has a strong right ~ -~ orientation. Values between 120 and 150 were found. ~- , . .: Pharmacological tests: The pharmacological effect of the inventive polysaccharide mixture was determined using known in vitro tests, whereby it was discovered that the inventive polysaccharide mixture is particularly effective in the tumor-necrosis factor liberation ~ ; ~ test . .. .; ,: ,: I ~: ..... Lymphocyte transformation test~ During the lymphocyte transformation test, lymphocyte cultures;~ are mixed with 3H labeled Thymidine (a component of DNA). 3H- ~ , Thymidine is taken up by proliferating lymphocytes and utilized in the structure of DNA. The amount of 3H-Thymidine incorporated in the DNA is proportional to the lymphocyte proliferation and 16 ;, ~ ' " ,. 2016948 can be used as a parameter to determine the stimulating ef~ect o~ the polysaccharide. . The following table shows the average value results of 3 independent measurements and shows a very good stimulation o~ the lymphocyte proliferation through the polysaccharide mixture NOAG II. Lymphocyte stimulation (%) at Fraction Concentration (ma/ml~ 10 1o~2 1o~3 lO 10-5 NOAG II 89 55 38 29 54 Tumor-necrosis-factor-test (TNF): The ~rocedure used was as that described in Example 1. -~ The results showed that NOAG II stimulates macrophages to produce TNF very well in that 1.5 ~g NOAG II parameter median induced over 500 units per ml TNF. ~ The immune stimulating effect of the polysaccharide mixture NOAG ~- II was additionally analyzed through a granulocyte test according to Brandt (Brandt, L. Scand. I. Haemat. (Suppl.) 2, 1967). - ` : . The following table shows the results (average values from two ~ ~- independent measurements). - F~action Phaaocyte values in % at Concentration ~ma/ml) 0-2 ,~Q-3 10-~ 10-~ 6 NOAG II 70 40 130 21 99 18 As the results in the table show, the polysaccharide mixture NOAG II effects a definite increase in phagocyte values which correlates well with the stimulation data obtained in the TNF liberation test. .,: - ~. ."- .., 17 -~ - - , . ~ ~ , ~., . ,, ~ , 20~i948 Chemiluminescence-Test (CL-T): -- With this test the increase in the production of reactive oxygen bonds with macrophages in granulocytes due to the test substances is determined. The reactive oxygen bonds are brought to reaction with amplifiers like Lucigenin which then results in chemiluminescence. Through measurement of this chemiluminescence the stimulatory effect of the test substances on the immune - system can be determined. The following table shows the results (average values of two independent measurements). Chemiluminescence Increase in % at Concentration ~ma/ml) lo-l lo~2 10-3 10' 10-5 10' NOAG II 7.2 9.3 1.2 24.4 - 7.6 ~ ~; As can be seen from the table, the polysaccharide mixture NOAG II indicates activity in this test as well. The inventive polysaccharide, that is the respective polysaccharide ~ ~; mixture, can be prescribed separately or as mixtures in a pure - -~ - substance or in form of a pharmaceutic preparation, however it would generally be more effective in form of a pharmaceutic preparation. Preferably the preparation lies in the following ~ormulation: (1) At least one inventive polysaccharide, that is one polysaccharide mixture and - (2) One or more suitable binders, carriers and/or other aid materials and ~- ~3) If necessary, other therapeutic agents or adjuvants. The carriers, binders and/or aid materials must be pharmaceutically and pharmacologically compatible so that they may be combined with the other contents of the formulation and have no negative effect on the organism to be treated. The formulations include those administrable through parenteral -~ (including subcutaneous, intradermal, intramuscular and intravenous) 18 20~48 or oral dispensation, although the most suitable dispensation method depends on the condition of the patient. The production of the formulation occurs utilizing methods generally known to the art of pharmacy. All methods include the step of mixing the inventive polysaccharide that is the polysaccharide mixture (i.e. the hormone) with the carrier, binder and/or help material, whereby the latter are an additional ingredient. In general, the formulations are produced by mixing the hormone with the liquid carriers or the fine particled carriers or both and then turning the obtained product into the desired dispensation form. The inventive formulation may be appropriate for oral dispensation by being contained in discrete units such as capsules or tablets, each containing a predetermined amount of the inventive hormone. Alternatively, the formulation may be in form of powders or granules or in a suspension or dissolved state in an aqueous or non-aqueous liquid or in form of an emulsion, for example, in form of lipsomes. The hormone may also be used in form of a paste. The tablets can be produced through pressing or pouring whereby one or more of the typical supplements can be added as necessary. Formu}ations to be used for parenteral dispensation include sterile aqueous or non-aqueous injection solutions, the anti-oxygens, buffers, bacteriostatiscs and dissolved materials, which make the formulation ~ ; isotonic in view of humans. Additionally, the inventive -~ polysaccharide that is the polysaccharide mixture may be contained in form of sterile aqueous or non-aqueous medium suspensions which contain suspension solutions and thickening agents. The formulations can be submitted as single or multiple doses for example in form of ampules or tightly closed flasks and can also be stored in lyophilized form so that a fluid carrier would simply be added immediately before - use, for example, injection appropriate water. The injection solutions and suspensions to be prepared immediately before use can be ~B ''' '' :' prepared of sterile powders, granules and tablets of the above- ~- described type. ~ - The inventive preparations can include other ingredients appropriate for the formulation in addition to the above named ingredients. For example, the pharmaceutic preparations to be administered orally may contain various aroma producing ingredients. ~'',. The amounts of hormone suitable for each application varies depending -' on the therapy. Generally a single dose should contain 5% to 95% active ingredient. This translates for a one time application to for - ~''-';,"' example 50 ~g to lO0 mg per dose per person parenteral and 1 mg to 500 ~,-mg peroral. This dose, however, will vary within a large boundary depending on the dispensation method, the condition of the patient and - - the f ield of therapy. , - Set forth below are several formulations of the inventive , ,--' preparations. ~ .': .- Tablet Formulation ~ . . ~ ,Formulation A hg~ 5~ '~- ;' (a) active ingredient 250 ~' ' , (b) lactose '' 210 -, (c) PVP 15 -~ (d) sodium starch glycolate 20 (e) magnesium stearate 5 ~ , , 500 , . .~ ' Eonnulation B (capsule) m~/capsule active ingredient 250 gelatinized starch 150 , -, 400 ,, ~01~8 Formulation C mg/capsule active ingredient 250 lactose 150 microcrystalline cellulose 100 500 Formulation D ma/capsule : (a) active inqredient 250 ~- (b) lactose 143 - (c) sodium starch glycolate 25 (d) magnesium stearate 2 420 Formulation E mq/cansule (a) active ingredient 250 :- - (b) polyethylene glycol 350 -- 600 Formulation F (in~ection solution) active ingredient 0.200 g hydrochloric acid solution0.1 mol/l q.s. to pH 4.0 to 7.0 sodium hydroxide solution0.1 mol/l q.8. to pH 4.0 to 7.0 :~ sterile water q.s. to lOml ~mulation G active ingredient 0.125 g ~teriIe,~pyrogen-free phosphate buffer with pH 7, g.s. to 3.00 ml : :' ~ :~:' . .: ' ' ' ":Y~ ;:, : 21 .: :-:. ~ ;~Oi69~8 ~ ~- Formulation H -- active ingredient 0.20 g : :- benzyl alcohol o 1 g tetrahydrofurfuryl-polyethylan-glycolether 1 45 g water for injection g.s. to 3.00 ml Formulation I : active ingredient 0.2500 g sorbitol solution 1.5000 g glycerine 2.0000 g ~ -~ sodium benzoate 0.0050 g . - flavoring 0.0125 ml purified water q.s. to 5.0000ml -~ ~ . .- ,- ' .. .. . , . . '' ,: ' '', ~ ':: ~ ` ,
