DE3546701C2 - New peptido:glycan jomol - Google Patents

Abstract

Peptidoglycan jomol having the characteristics listed below is new: UV absorption maximum at 286.0 nm; apparent mol. wt. of 4500, 3000 and 900; strongly hygroscopic; depending on residual water content, ivory-coloured and amorphous-crystalline flakes; 1 mg/ml aq. soln. has pH 5.5; weakly positive, pink ninhydrin reaction; can be coloured with orcinol reagent; protein content 1.4%; Rf values of 0.20, 0.45, 0.685, 0 and 0.54 with butanol-glacial acetic -water 4:1:1, benzene-glacial acetic-water 2:1:2, methanol-glacial acetic-water 4:1:1, CHCl3-MeOH 96:4 and ChCl3-MeOH 1:1; poorly sol. in non-polar solvent, very good solubility in polar solvents; detectable components (with relative amts.) are neutral sugars (glucose, galactose, ribose) (not greater than 1), amino sugars (glucosamine, muraminic acid) (3.5), amino acids (Ala,Glu,DAP), (4) (DAP 2.5), lipids (below 1), phosphorus (below 1); peptidoglycan structure in which the glycan skeleton consists of 10-80 N-acetylglucosaminyl -N-acetylmesamyl disaccharide units.

Description

The invention relates to a diagnostic agent for Tumors containing Jomol or Jomolderivate.

Jomol® and its derivatives can be used as medicines and as diagnostic agents are used. They are in particular dere as a means of increasing cellular defense and for the diagnosis and therapy of malignant tumors as well to treat weaknesses of cellular and humoral Immune defense suitable. Jomol® can be used as a carrier for diagno Static and therapeutic substances are used.

The diagnosis of malignant tumors is despite intensive research still difficult today. It is almost impossible to detect malignant tumors at an early stage, and to date no method is available with An early diagnosis can be carried out.

U. Ehrenfeld (Krebsgeschehen 5, 132 ff. (1979)) discuss the cancertoxic effect of a mixture Acetaldehyde and ethanol. The mixture contains 3 to 10 g Acetaldehyde per 1000 g of ethanol. It turned out, however, that the effect of this mixture in the treatment of solid malignant tumors as well as metastases is sufficient.

In the German patent applications P 33 34 751.4, P 33 36 583.0 and P 34 02 312.7 (corresponding to the euro  European Patent Application 84 110 118.1 and following white patent applications in foreign countries: Canada (463 991), South Africa (84/7296), Australia (33327/84) Japan (202 859/84), USSR (3 798 784.13), Poland (P. 249 725) Romania (115 800), Yugoslavia (P-1639/84), Spain (536 085), Argentina (297 977), South Korea (84-5894) GDR (267 559), Denmark (4553/84), USA (689 728)) a means of diagnosis and therapy of malignant tumors ren and for the treatment of weaknesses of the cellular and Homoral immune defense described using an immune module in and / or on liposomes or lipidated or one with a radioactive emitter, a dye or a cytostatic labeled immunomodulator ent holds.

However, there is still a need for such mit which show as few side effects as possible and in particular, are very easy to manufacture. The well-known th means have the further disadvantage that their intravenous administration with some difficulty connected is.

The present invention is based on the object a means and a product for the diagnosis of tumors available len. The agent should be highly effective and therefore in clotting  gerer concentration than the known means administered can be. The agent of the invention should bewir that the organism of the patient is less severe is loaded, and should continue its administration easy way to be possible.

The agent should continue to be administered intravenously and as a carrier for radioactive sources, drugs and dyes are used.

Surprisingly, it has been found that a compound which one gains from the microorganism Rhodococcus rhodochrous, solves the object of the invention, when combined with a Use a mixture of an alcohol and aldehyde it becomes.

The invention relates to a diagnostic agent which characterized in that it contains a microbial Ex tract, received by

• a) Breeding of Nocardia opaca = Rhodococcus rhodo chrous (DSM 43 202 or ATCC 21 953) on one broth
• b) obtaining the cells,
• c) suspension of the cells in a buffer and 15 Minutes to a few hours leaving the Sus pension,
• d) Treatment of the suspension with a murolytic Enzyme,
• e) destruction of cells,
• f) Separation of the material obtained in sediment and supernatant,
• g) Separation of the supernatant on Sephadex columns under Use of buffer as eluent,  
• h) Obtaining the active fractions in the UV spectrum show an absorption at 214 nm,
• i) Reaction of the active fractions with acetaldehyde in a molar ratio of 1 mol of active fraction (assumed average molecular weight 4000) with 1.8 to 2.2 moles of acetaldehyde, optionally coupled with a molecule that is a radioactive Spotlight and / or can bind a dye,

contains and it together with an aldehyde of general formula I.

RCHO (I)

wherein R is a hydrogen atom and a straight or branched hydrocarbon group of 1 to 4 carbon atoms, wherein the free aldehyde also directly or indirectly metabolically released from substances, and optionally an alcohol of the general formula II

R¹CH₂OH (II)

wherein R¹ has the meaning given above for R, as well as optionally conventional pharmaceutically acceptable carrier substances and / or diluents.

The microbial extract (Jomol®) is by the following own characterized by:

• (1) absorption maximum in the UV spectrum at 286.0 nm;
• (2) apparent molecular weights at 4500, 3000 and 900;
• (3) strong hygroscopicity;
• (4) ivory, partly amorphous, partly crystalline Flakes after freeze-drying, depending on the Residual water content;  
• (5) pH 5.5 (1 ml of the compound dissolved in 1 ml of water);
• (6) weakly positive, pink ninhydrin reaction;
• (7) stainable with orcine reagent;
• (8) Protein content 1.4% (protein test after L. Thomas, J. Clin. Chem. Clin. Biochem., Vol. 19, 1981, pages 203 to 208, Coomassie Brilliant Blue G 250);
• (9) the following R _f values (solvent specification volume: volume) R _f = 0.20 (butanol: glacial acetic acid: water 4: 1: 1)
  R _f = 0.45 (benzene: glacial acetic acid: water 2: 1: 2)
  R _f = 0.685 (methanol: glacial acetic acid: water 4: 1: 1)
  no migration (chloroform: methanol 96: 4)
  R _f = 0.54 (chloroform: methanol 1: 1)
• (10) Solubility: poorly soluble in nonpolar solution average, very soluble in polar solvents;
• (11) detectable components: substances Ratio (approximation) Neutral sugar (glucose, galactose, ribose) () 1 Amino sugars (glucosamine, muramic acid) (after hydrolysis) 3.5 Amino acid (Ala, Glu, DAP) @ (Ala, Glu + DAP 2.5) 4 lipids <1 phosphorus <1
• (12) Structure:
  Peptidoglycan structure whose Glycangerüst consists of 10 to 80 disaccharides from N-acetylglucosaminyl-N-acetylmuramyl.

The microbial extract can with a radioactive emitter, a dye or a cyto be marked statical, the mark under Ver use of coupling means, such as DTPA, Cryptofix, uridine, L-thyronine, L-tyrosine. As a coupling agent can be all such Kopplungsmit tel use a radioactive emitter, a Dye or a cytostatic can wear or bind NEN.  

The applicant has surprisingly found that a certain microorganism of the genus Nocardia opaca, namely Rhodococcus rhodochrous gives a compound the self surprising after reaction with acetaldehyde as a carrier for diagnostic Has substances. The strain Rhodococcus rhodochrous was at the German Collection of Microorganisms, Gries bachstraße 8, D-3400 Göttingen, on May 16, 1972 under the number DSM 43 202 (= DSM 363 = ATCC 21 953) behind sets. The strain is freely available, and its viable It was again demonstrated on 28 February 1985.

Jomol® is used in accordance with DE 35 10 795 C2 (or DE 35 10 795 A). manufactured.

In carrying out the process for the preparation of Jomol® becomes a carbon source, a nitrogen source and minerals containing culture medium used. the This culture medium may also contain antifoams and / or others re usual components. Examples of coals Sources are carbohydrates, alcohols, hydrocarbons fabrics and bran. Examples of nitrogen sources are Corn steep liquor, yeast extracts, meat extracts, peptone, Fish meal, ammonium salts, nitrate salts and urea. The Mineral source includes inorganic salts such as phosphates, Ma magnesium salts, zinc salts, calcium salts, manganese salts, molybdenum Danish salts and copper salts.  

The composition of the culture medium can be changed as needed, and During fermentation, these can be carbon, nitrogen and mineral be added in addition sources. For making an inoculum is the microorganism for example on DST-Oxoid medium, or dextrose agar (2%) in plate technique at three to four Plates cultivated for five to eight days. This gives a product that then as an inoculum to produce the material on an industrial scale can be used.

The cultivation of the microorganism on an industrial scale, for example in Erlenmeyer flasks containing 100 ml of normal nutrient broth, respectively. Breeding takes place under aerobic conditions.

The cultivation temperature is 20 to 40 ° C, and the pH of the culture mediums is 7.2 to 7.4. The preferred temperature is 30 to 37 ° C, and the suitable culture time is usually 2 to about 30 Days and may be appropriate, depending on the selection of others Culture conditions, to be changed.

The microorganisms are then harvested, preferably by centrifugation yaw. However, the microorganisms can also be filtered off via glass filters trate. The centrifuging can, for example, 4000 revolutions per Minute during a time of 5 to 10 minutes in one Refrigerated centrifuge with a rotor of 40 cm diameter done. The microorganisms obtained are usually washed, for example with water or 0.01 M Tris-HCl buffer with a pH of, for example 7.4. The Tris-HCl buffer preferably contains EDTA-Na₂, for example 0.1 to 0.04, preferably 0.06%. The washing process can be one or more Repeated times.

The microorganisms are then preferably washed after being washed as above in Tris-HCl buffer (pH 7.4), for example 0.01 M, which is preferably 0.1 to 0.05% EDTA-Na₂ and 8 to 12%, preferably 10%, contains glucose, suspended.

The resulting suspension, which was then incubated, is then added a murolytic enzyme, preferably lysozyme and Preferably, to remove the nucleic acids and to reduce the  Viscosity deoxyribonuclease added. For example, for 5 g of bacteria, 100 ml Tris-HCl buffer and Then 10 mg of lysozyme and 3 mg of deoxyribonuclease are added to this suspension.

The treatment with glucose and the enzymes is for a period of 30 Minutes to several hours, preferably for a period of two to three hours, at a temperature of 20 to 40 ° C, preferably 30 to 37 ° C, performed.

Subsequently, the cells are destroyed. This can be done with any suitable Methods happen, for example ultrasound. Preferably, Ultra sound for one minute, used. The mechanical destruction of the cells does not have to However, it is preferably performed, because thereby the Yield of the desired product is increased.

The material obtained is then in supernatant and sediment, preferably by centrifuging, separated. The centrifuging can be done in a normal Centrifuge are carried out, but it is preferred in a Kühlzen trifuge at a temperature of 4 to 6 ° C at 4000 revolutions per minute Performed for 10 to 15 minutes. The sediment may optionally washed once or several times with water or the above-mentioned buffer be, with the resulting wash solutions combined with the supernatant become. The supernatant is optionally after concentration in a high vacuum at low temperatures on Sephadex® G-75 columns. Is omitted on a washing of the obtained sediment, so is the Supernatant directly subjected to chromatography, without being concentrated becomes. For example, from 5 g of bacteria, a supernatant is obtained preferably divided into five equal parts and split into five prepared Sephadex There are G-75 columns in each case. The columns are 80 cm long, the inside diameter is 2.5 cm, the Sephadex® filling height is 60 cm. If only one column is used, then the part that is not chromatogra is frozen at -25 ° C and stored at this temperature.

When using chromatography on one of the columns to characterize the JOMOL® substance "albumin from human serum labeling", the first peak of the crude substance with the albumin (MW 69,000) and the fraction 2b * with the same for the column calibration occurs used vitamin B₁₂ (MW 1 355.4) ( Figure 2).

The eluent is 0.01 M Tris-HCl buffer (pH 7.4) containing 0.06% EDTA-Na₂ contains, used. The flow rate is 100 μl per minute (= 2 drops / min), with the same can apply lower or higher flow rates. The active fraction, the as fraction 2b * is called, in a time of 12 to 14 hours collected after the onset of gel chromatography. The elution volume is about 900 to 980 ml, the fractionation is carried out under UV detection at 214 nm The procedure for obtaining fraction 2b * may be from harvesting the cells after culturing to column fractionation also in phosphate buffer, Ammonium acetate buffer and others. The use of ammonium acetate Buffer is particularly preferred for the preparations for iodination. The resulting active fractions are lyophilized and then incubated for one hour at 80 ° C to destroy the degradation enzymes. Unless is lyophilized, the dilute solutions at -20 ° C, preferably -40 ° C, more preferably -80 ° C, frozen and stored as a solution.

Before lyophilization or freezing, the solutions obtained are at active fractions, preferably with a 1 M acetaldehyde (pure) aqueous Solution in u.g. M ratio mixed and 10 minutes to 2 hours, preferably wise z. B. 30 minutes, allowed to stand at room temperature.

The reaction of fraction 2b * with acetaldehyde takes place in a molar ratio Fraction 2b *: acetaldehyde = 1: 1.8 to 2, preferably in the ratio of 1: 2, wherein for the fraction 2b * an average molecular weight of about 4000 assumes.

Surprisingly, it was found that in the implementation of the active Fraction 2b * with acetaldehyde a "fraction 2b" obtained as JOMOL® has, has surprising pharmacological properties and can also be used as a carrier.

The yield from about 5 g of bacteria is about 50 to 70 mg JOMOL® in pure form.

The product is very hygroscopic. Optionally, by conventional methods a water content determination of the lyophilisate be carried out.  

After drying, the substance is yellow-white and flaky, with a little water At the moment she appears "crystalline", with a little more water she is colorless, glassy, gelatinous and pulls strings like household glue. at Sterile storage without access of light under nitrogen is the substance at a temperature below -25 ° C stable. The substance is under light Stable at room temperature for a few hours.

As explained above, in the method according to the invention Nocardia Bacteria used. Nocardia are Gram-positive actinomycetes. Your more layered cell wall framework indicates protein and poly deposits saccharides on. It is superimposed on top layers, which are difficult to remove are.

The cell wall contains as outer layer lipoproteins and waxes, as the middle Layer of lipopolysaccharides and as inner layer long polysaccharide chains. The antigenicity of the coating components decreases from outside to inside.

The substrate for the production is the inner layer with the long ones Peptidoglycan. These are in Glykangerüst from Dissaccharidbausteinen educated. The disaccharide building block consists of N-acetylglucosaminyl-N-acetyl muramyl:

On the acid group of N-acetylmuramic acid (N-acetylglucosamine lactate ether) are peptide units bound via L-alanine. Contain these peptide units Sequences of L-alanine → D-isoglutamine → meso-α, ε-diaminopimelic acid (DAP), where the DAP may be amidated. At this point comes rarely Uridine diphosphate or another amino acid, more commonly lysine, which may in turn be substituted. Instead of the acetyl group may be at the Muramic acid may be present a glycolyl group. Except these mentioned Units can also be present up to 10% neutral sugar.

According to the above manufacturing process, the proportion of discarded outside wall layer of the Nocardien; Lipoproteins, lipids and  Waxes are therefore neither in the overall preparation nor in the preparation fraction 2b ent hold. The preparations prepared are cleavage products of peptydoglycans and peptides of the inner wall layer. The cleavage lysozyme hydrolyzes the bond between N-acetylglucosamine and N-acetylmuramine acid of the glycan backbone. The deoxyribonuclease cleaves nucleic acids; you reduces the viscosity of the product. The peptide substituents of Peptidoglycans go as far as theirs in the above manufacturing process loss of interpeptidic bonds that they become water soluble and only are still low in fat. At the same time lose the ends of peptido glycans their peptide content. Through the manufacturing process, the Peptidoglycans largely from natural lipids, with which they together occur in the cell walls, free. The N-acetylglucosamine groups of Peptidoglycans may be partially deacetylated.

JOMOL® has unexpected properties. JOMOL® finds itself as an immune modul lator, drug or diagnostic use. JOMOL® and its coupling Products with certain coupling agents are particularly suitable as Carrier substances for radioactive compounds, dyes and pharmaceuticals. JOMOL®, its coupling products and the labeled or loaded JOMOL® Surprisingly, derivatives accumulate on cancer cells and are released by healthy cells are hardly or only to a limited extent bound. For example JOMOL® labeled with a radioactive marker and administered, so the labeled JOMOL® accumulates on the tumor cells and they can thereby easily detected by gamma camera imaging.

Examples of JOMOL® coupling products are JOMOL® products coupled a crown ether, diethylenetriaminepentaacetic acid, fluorescein isothiocyanate or stannous chloride. Such coupling products are generally prepared by placing JOMOL® in a suitable polar Solvent or in an aqueous solution of a polar solvent solves. For example, 1 to 10 mg of JOMOL® can be added to 20 μl of a Dissolve appropriate aqueous solvent. You can also be very focused Create solutions from JOMOL®. The solution takes place at the lowest possible Temperature, but the temperature must be chosen so that the Solvent not yet crystallized. The substance to be converted with JOMOL® will couple in a ratio of 1 mole of JOMOL to 0.8 to 1.2 moles of implemented the compound, wherein for the JOMOL® a medium molecular weight of about 4000 accepts. The connection coupled to JOMOL®  is dissolved in an anhydrous medium or homogeneously suspended. in the In the case of stannous chloride, a hydrochloric acid aqueous solution is used. The reaction mixture is allowed to stand for a few minutes Allowed to stand for 60 minutes at a temperature in the range of 15 to 40 ° C. The product can be used directly. It can also be done by chromatography be cleaned and isolated.

Chromatography and purification are done in a similar manner as for JOMOL® self described. Sephadex® columns are used and as elutions medium, the above-mentioned buffers are used.

If the coupling products themselves are used directly as a diagnostic agent, is it prefers to react as pure as possible solutions and the resulting solutions directly, if necessary after sterilization to use.

The following coupling products were prepared:

1. Coupling product of JOMOL® and Diethylentriaminpentaessigsäureanhydrid

This product is referred to as "JOMOL®-DTPA" or as "JOMO®-In". It is suitable for loading with 111 indium or with ^99m technetium which has been reduced with tin (II) chloride. The products labeled with a radioactive marker are particularly suitable for diagnostics.

2. Coupling product of JOMOL® and tin (II) chloride

This product is called "JOMOL®-Sn" or "JOMO®-tech". JOMOL®-Sn is particularly suitable for loading with ^99m Technetium.
The labeled product is particularly well suited for the diagnosis of tumors.
The product is prepared by reacting a JOMOL® solution with a hydrochloric acid aqueous tin (II) solution.

3. Coupling product of JOMOL® and uridine

This product is referred to as "JOMO®-J / U" and is particularly suitable for labeling with radioactive iodine. The labeled product can be used for diagnosis or therapy.
The preparation is carried out by reacting JOMOL® and uridine-2 ', 3'-dialdehyde in an anhydrous solvent.

4. Coupling product of JOMOL® and fluorescein isothiocyanate

The product is called "JOMO®-Color". It is particularly suitable for diagnosis and / or therapy. The products 3 and 4 are special good for radioactive iodine labeling. The products are used for diagnostics.

5. Coupling product of JOMOL® and a precursor of Kryptofix 222

This product is referred to as "JOMO®-Ce" and also as "JOMO®-Ra". The Product is manufactured by JOMOL® with a preliminary stage of crypto fix 222®, the CE is implemented, is implemented. The CE molecule has the following structure:

CE contains the complete amide bond in the crown ether (in contrast to the Kryptofix 222®).
The compound is particularly suitable for radioactive labeling with radium. A longer incubation period with ²²⁴Ra is necessary for its uptake than for the Kryptofix 222®. The stability constant with respect to radium binding is the same for CE and Kryptofix 222®: IgK = 6.64 (this logarithm is higher for radium than for any other metal ions in question). The bound radium can only be detached from its binding by an acidic environment (unstable in the stomach, stable in the rest of the body). Acidification must be avoided after the binding of radium to CE.

JOMOL® and its coupling derivatives described above are used as vehicleub used for dyes, drugs and radioactive markers. There It is these substances, as mentioned above, that accumulate on cancer cells thus it is possible to specifically detect or specifically treat the cancer cells spindles. It is thereby possible, the active ingredients, such as the radio  active marker, on the one hand to detect the cancer cells and others also serve to destroy the cancer cells, targeted to the cancer cells bring to.

The above-mentioned coupling product of JOMOL® and fluorescein isothiocyanate is a particularly preferred product according to the invention, as it is suitable for both the diagnosis and therapy as well as for labeling with radioactive iodine suitable is.

The preparation of labeled JOMOL® or the labeled JOMOL® derivatives, which are marked with a drug or a dye takes place, by using a JOMOL® solution as above in the manufacture of the coupling products or by adding a solution or suspension of the coupling products in a suitable concentration with a solution or suspension of a Drug or a dye. In general, equi molar amounts of JOMOL® and the dye in a polar solvent brought together. Thereafter, an equimolar amount of a crosslinking agent (Glutaraldehyde) added in a conventional manner and to Coupling of the components used. The reaction temperature is 15 to 40 ° C. This is followed by a chromatographic purification.

The preparation of the products marked with a radioactive marker he follows by JOMOL® or a coupling product of JOMOL® or a solution with a salt solution of the radioactive marker and the radioactive product in a conventional manner, for example, by säulenchromato graphy wins. The labeling with radioactive radiators is described in the literature. Examples of radioactive emitters are the emitters commonly used in the field of therapy and diagnosis of malignant tumors, such as ^99m technetium, 111 indium, 125 iodine, 13 iodine and 222 radium. For example, the radioactivity may be 185 KBq to 7.4 GBq.

When loaded with JOMOL® a radionuclide, the peak of JOMOL® with an apparent molecular weight of 3000 in an FPLC elution pattern shows the highest binding of the radionuclide. The same applies to JOMO®-tech, JOMO®-In and JOMO®-Ra. ( Figure 3) For JOMOL®-Sn ^99c Tc, JOMOL®-DTPA-111 In and JOMOL®CE-22Ra, no autoradiolysis was observed after five days storage in aqueous solution (0.5 mg / ml at -20 ° C) after labeling detected.

According to the invention, it is also possible mixtures of JOMOL® and its with a radioactive radiator, a dye or a cytostatic  to use labeled derivatives.

According to the present invention, JOMOL®, its coupling products and the appropriately labeled derivatives, as described above, on liposomes or in lipidated form. Liposomes are spherical Structure of one or more lipid bilayers with an interior. Such bubbles can be by mechanical finest distribution of Phospholipids, such as lecithin, produce in aqueous media. According to the invention liposomes are used, the single unilamellar Bubbles (SUV) are and preferably phosphatidylcholine: phosphatidyl serine: cholesterol in the molar ratio 8: 2: 10 exist and by sonication getting produced. The lipids from which they are made are in Trade available. The lipids are in Ether dissolved, purified by column chromatography, under N₂ with JOMOL® or its derivatives, in phosphate buffered saline (PBS), for example, suspended at pH 7.4 and then for example 25 minutes Sonicated at + 2 ° C with a pulsating sonicator (sonicated). The Sonication is generally carried out under N₂. After sonication (Soni cation), the liposomes are chromatographed on a Sepharose® 4-B column and preferably using the fractions of the population with radii less than 30 nm. (C. Huang, Biochemistry 15, 2362, 1969).

These liposomes are then labeled for diagnosis in a conventional manner with preferably ^99m technetium on JOMOL®. To test the radioactive label, an aliquot of the liposomes are placed on a Sepharose® 4-B column and chromatographed. It is noted that the preparation has a specific activity of 99.2% of JOMOL® bound radioactivity and 0.8% of free pertechnetate. The liposomes may be loaded with JOMOL® or its derivatives, which are with a radioactive tracer, with a dye, a cytostatic or with mixtures of these compounds be sent. Such liposomes are particularly suitable for the diagnosis ge. According to one embodiment of the invention, JOMOL® or JOMOL derivative, optionally charged as explained above, is dispersed in a lipid. The dispersion takes place by bringing the substances together and likewise subjecting them to a sonication. As lipids one can use phosphatidylcholine alone or with phosphatidylserine and cholesterol together, for example in a molar ratio of 8: 2: 10.

As mentioned above, it is known that a mixture of acetaldehyde and ethanol  has a cancerotoxic effect. Surprisingly, it has now been found that JOMOL® its derivatives develop a particularly good effect, if together with an aid called a "cocktail" be administered. The adjuvant contains an aldehyde of general formula I.

RCHO (I)

wherein R is a hydrogen atom or a straight-chain or branched carbon hydrogen group having 1 to 4 carbon atoms, and given if an alcohol of the general formula II

R¹CH₂OH (II)

wherein R¹ has the meaning given for R. The tool contains optionally conventional carriers and / or diluents. It is particularly preferred that the auxiliary in addition to the aldehyde a Contains alcohol. Surprisingly, it was found that at the same time and with gradual use of the aid together with JOMOL® or JOMOL® derivatives the efficiency of JOMOL® or JOMOL® derivatives essential is improved. The invention thus relates to a diagnostic Agent containing the above-described diagnostic agent or drug contains medium and the auxiliary described above.

The adjuvant in the diagnostic agent of the invention can be the Aldehyde as such in conventional pharmacologically acceptable carriers and / or Contain diluents. It is particularly preferred, the aldehyde to be used in aqueous and / or alcoholic solution. According to the invention it is particularly preferred, the respective aldehyde together with his associated alcohol.

Preferred tools release, directly or indirectly, and / or contain:
Formaldehyde / methanol, acetaldehyde / ethanol, n-propionaldehyde / n-propanol, n-butyraldehyde / n-butanol, iso-butyraldehyde / iso-butanol, n-valeraldehyde / n-Pen tanol or mixtures of these compounds. The substance pair acetaldehyde / Etha nol is practically non-toxic in the required concentration and amount; you can administer it in appropriately high doses. This results in the possibility of a long-term treatment, also in combination with a radiation treatment. The immunobiological system is positively influenced, and a combination with other drugs as well as with surgical and radio logical measures is possible.

Apart from this mixture ethanol / acetaldehyde are in principle also other analogous mixtures of the above type possible. Methanol is in man body degraded much slower than ethanol, propanol around the Factor 2 faster than ethanol. The agent can only be one specific each chosen. Aldehyde as well as aldehyde mixtures. The application the aldehydes is not necessarily associated with the presence of the corresponding Coupled alcohols. Also, aqueous solutions of aldehydes can be used become. Instead of the free aldehydes according to the invention, such Aldehyde derivatives are used, in the metabolism of the inventions Pharmaceutical agents treated according to the invention form the free aldehyde. Suitable aldehyde derivatives are, for example, the acetals or hemiacetals Condensation products, also as such or in dissolved form (Water or alcohols) as well as in mixtures with the aldehydes and / or Alcohols can be used. In a preferred further invention According to the embodiment, the auxiliary contains small amounts (less than 0.05 wt .-%) of peroxides, in particular the material related here Peroxides come into consideration, in particular H₂O₂ and / or the aldehyde peroxide or hydroxyhydroperoxide and the peroxide of the associated carboxylic acid. Due to the content of peroxides, the antitumoral effect is even further improved.

The concentration of the aldehyde in the preparation according to the invention is determined on the one hand by its compatibility and on the other hand by the dose to be administered. For the pair of ethanol / acetaldehyde, an acetaldehyde concentration in the alcohol below 2 × 10 ^-4 mol / liter is often unsatisfactorily slow in effect. The effect increases with increasing concentration of aldehyde and is usually limited to the top by possibly a passing incompatibility of the acetaldehyde in each case. For example, ethanol-aldehyde solutions having 5 × 10 ^-2 mol to 1 mol of acetaldehyde per liter of ethanol have proven useful in practice, and these mixtures can be used at a dose of, for example, 10 to 150 cm 3 per day. It is preferred that the aid contains 10 to 60 g of aldehyde per 1000 g of alcohol, more preferably 40 g of aldehyde per 1000 g of alcohol. In general, the adjuvant for administration is diluted with water. The alcoholic solution can be diluted with water as desired. For example, it is possible to dilute a volume of the alcoholic solution having 1 to 10 volumes, preferably 2 to 5 volumes, of water.

The adjuvant is preferably administered orally in the form of the aqueous solution and drunk by the patient. However, the aid may also be parenteral be administered.

In the product or kit according to the invention, the two constituents be combined in different ways. The tool can be used in one for oral administration and / or for parenteral administration, For example, by infusion, suitable form. The preparation of Infusion solutions is familiar to the expert and can be known per se Done way. For example, the aid in the form of Trinkam or it may be in the form of drinking ampoules containing water be diluted present.

For diagnosis, it is preferable to first use the adjuvant as an aqueous Solution and about 20 to 30 minutes later the diagnostic agent parenteral before given intravenously, to be administered.

JOMOL® and its derivatives (under "JOMOL® derivatives" are used in the present Registration all coupling products and marked products understood) penetrate surprisingly easily through the cell walls, in particular through the walls of lung alveoli, lymphatic and blood capillaries, and accumulate on cancer cells, where they can easily be detected. This will be a new diagnosis and a new type of treatment of malignant Tumors, since the labeled with a radioactive marker JOMOL® or such JOMOL® derivatives go directly to the malignant tumor and there to detect and treat the tumor most effectively. The diagnostic agent according to the invention can be used for the diagnosis of malignant Tumors are used in vivo and vitro. The malignant tumors can be diagnosed outside the body. For example, the surgeon remove malignant tissue during surgery and then remove the living tissue Incubate outside the organism, so that in the flat section of Ge webs, for example in color coding with fluorescein isothiocyanate in UV light malignant tissue will light up and dark healthy tissue immediately can be distinguished.

If JOMOL® or one of its derivatives mar with a radioactive emitter kiert, the visualization of the radioactive distribution takes place under Using an external gamma camera. Shown with the external Gamma camera different melanosarcomas, squamous cell carcinomas and adeno Carcinomas of the female breast.  

Fluorescein isothiocyanate or ^99m technetium remain bound to JOMOL® or its derivative at the cancer cell wall, where they can be detected by fluorescence microscopy or autoradiography. When imaging with the gamma camera is in the model with the SD rat Walker Carcinosarkom 256, the attachment rate of the invention claimed substance with simultaneous use of the combination (cocktail) of ethanol / acetaldehyde / water 2.5 to 8 times higher than on healthy tissue (in comparison). In humans, the connection ratio is higher. Since JOMOL® is distributed in the bloodstream in the body and is excreted via the kidneys, liver and lungs in urine, bile and exhaled air, with appropriate labeling of JOMOL® or its derivatives with radioactive isotopes when displayed by gamma camera except cancer and its metastases in soft tissues and bones also functional and clearance studies in these passages feasible.

Pharmacological investigations 1. Toxicity

JOMOL® and its derivatives are virtually non-toxic. In the screening after Robert A. Turner found for JOMOL®, JOMO®-tech, JOMO®-In and JOMO®-Color in case of overdose of Human daily dose of 4000 to 5000 times, d. H. in an amount of 2 mg / kg Body weight, on SPF mice no deviation from normal findings. The tolerability appears excellent, unwanted side effects did not occur.

2. Binding of radioactively labeled JOMOL® derivatives malignant and benign Cells in vitro

In order to show that JOMOL® and its derivatives are particularly easily bound to carcinoma cells, comparative experiments were carried out. The binding behavior of the radioactive components JOMOL®-DTPA-1 In, JOMOL®-CE-22 Ra and JOMOL®-Sn- ^99m Tc was tested on carcinoma cells and on healthy cells in vitro. The carcinoma cells used were mouse F9 embryonic cells and mouse fibroblasts L929 as healthy cells.

In a further experiment it was shown that the connection particularly out if, prior to administration of the radioactive components he is inventive cocktail is administered.

For the evaluation of binding results, it is significant that the volume an average healthy fibroblast cell L929 about three times as  big like a malignant F9 cell, the surface is twice so big. In addition, the ratio of the cell wall and adhesion matrix to Cell is significantly larger in healthy L929 cells than in malignant ones F9 cells.

The result of the binding experiments is summarized in the following Table 1. In summary, it can be stated that the mouse embryonic carcinoma cells F9 JOMOL®-DTPA-111 In, JOMOL®-CE-222Ra and JOMOL®-Sn- ^99m Tc bind better by a factor of about 30 to 60. There is a tendency to increase the binding ratio by adding the cocktail of alcohol and acetaldehyde recognizable.

Table I

Ratio of specific cell labeling (cpm / 10⁶ seeded cells normalized) of embryonic mouse carcinoma cells F9 to mouse fibroblasts L929 (F9 / L929

3. Binding of radiolabelled JOMOLS® to malignant tumor tissue in vivo a) Binding to Walker carcinosarcoma 256 in the Sprague-Dawley rat

Four days after subcutaneous transplantation of Walker's carcinosarcoma 256, 0.8 ml of a JOMOL®-DTPA-111 In solution or a JOMOL®-Sn- ^99m Tc solution was applied to three female Sprague-Dawley rats. The first solution contained about 80 nmol / ml of JOMOL®-DTPA-111 In in a specific labeling of about 175 GBq / mmol. The second solution contained about 68 nmol / ml. JOMOL®-Sn- ^99m Tc in a specific labeling of ca. 925 GBq / mmol. All animals were orally administered 1 ml of cocktail 30 minutes before iv administration of the labeled JOMOLS® (cocktail: 22.5 ml physiological saline, 2.4 ml ethanol, 0.26 ml acetaldehyde). The animals were scanned 24 hours a day in chloral hydrate anesthesia from a dorsal point of view.

The tumor was clearly distinguishable in all animals. The tumor mark, expressed as the quotient of the specific accumulation of tumor and muscle tissue, was found to be between 4: 1 to 8: 1 for JOMOL®-DTPA-11¹In, and between 4: 1 to 6: 1 for JOMOL®-Sn- ^99m Tc In subsequent experiments binding ratios were found quite frequently at 8: 1, in individual cases up to 36: 1.

b) Distribution of JOMOL®-Sn- ^99m Tc in the tumor-bearing Sprague-Dawley rat

Five female Sprague-Dawley rats were injected with 0.5 ml of JOMOL®-Sn- ^99m Tc solution into the tail vein four days after subcutaneous transplantation of Walker carcinosarcoma 256 into the right lateral flank. The solution contained 25 nmol / ml JOMOL®-Sn- ^99m Tc in a specific labeling of 925 GBq / mmol. Thirty minutes before application, 2.5 ml of the cocktail was orally administered to the animals (cocktail: 22.5 ml physiological saline, 2.4 ml ethanol, 0.26 ml acetaldehyde). Two of the animals showed in the scintigram a connection ratio (specific accumulation in the tumor to specific accumulation in the muscle tissue) of 6: 1 with a mean tumor weight of about 1.5 g.

Scintigraphically, the tumor was already 30 minutes after application abge be bordered. Extrapolated to the human conditions can be expected become, that here a tumor scintigraphy already 3 hours after application is possible. To determine the distribution in the organs were the animals Decapitated one hour after injection and largely bled. In the The organs listed in Table II were removed and weighed; the included Radioactivity was measured in a gamma counter.

The following Table II shows the distribution of radioactivity for one hour after application.

A not inconsiderable proportion of the radioactivity was within a Hour renally excreted; the estimated clearance is 0.5 ml / min.  

Table II In vivo distribution of ^99m Tc-JOMOL® in the tumor-bearing rat one hour after intrayenous administration, (n = 5, Sprague-Dawley, Walker-Carcinosarcoma 256)

organ % d. Dose / g blood 0.46 ± 0.01 liver 0.17 ± 0.05 spleen 0.09 ± 0.01 stomach 0.14 ± 0.02 kidneys 3.34 ± 0.29 heart 0.16 ± 0.02 lung 0.24 ± 0.02 muscle 0.05 ± 0.01 thymus 0.11 ± 0.04 femur 0.09 ± 0.02 tumor 0.32 ± 0.05

c) Various malignant tumors were presented in humans. With the aid of a gamma camera, 3 hours after intravenous administration of JOMO®-tech (740 MBq ^99m Tc) or JOMO®-In (74 to 111 MBq¹¹¹¹Indium)), in most cases half an hour before a dose of cocktail - 24 ml of ethanol, 1 ml of acetaldehyde, 225 ml of water - orally administered) the tumors imaged. At the site of tumor imaging, the tumor was verified with other examinations (for example histologically). The tumor area / non-tumor area ratio was more than 10: 1 with respect to the radiation detected by the gamma camera, and 8: 1 in the liver.

4. The diagnostic effect of JOMO®-Color (JOMOL®-fluorescein isothyocyanate) (JOMOL®-FITC)

Experiments with Fitc-labeled lymphocytes are known to be in vivo in vivo phagocytosed within a few hours. Segmented nuclear leukocytes (granulocytes) be sit proteolytic enzymes and lysozyme. The lysozyme serves to cleave Murein in bacteria.

Surprisingly, it has now been found that the Murein "alienated" by Fitc Fragment JOMO® (reinforced with cocktail) bound to tumor cells in the area the protease-altered cell wall matrix triggers granulocyte invasions. If Walker Carcinosarcoma 256 ascites with JOMOL® Fitc, ethanol / acetaldehyde, and p-hydro xyphenil-ketopropionic acid antagonists (those produced by competition at their receptors or tyrosine-analogous molecules and monoamine oxidase inhibitors in Sprague-Dawley's Rats is treated, it is found after 24 hours, for example, after use of JOMOL®-Fitc, ethanol / acetaldehyde and cotrimoxazole at the point of ascites one very large number of leukocytes and a subtotal regressive change in the Ascitestumorzellen. Controls show vital tumor cells and sparse leukemia cytes. Examples of the above-mentioned tyrosine analog and tyrosine degradation product antagonists are sulfamethoxazole and isoniazid with the monoamine oxidase inhibitor Tranylcypromine. This effect is like the selectively occurring fluorescence of the Cancer cell walls (fluorescence) microscopic already 10 to 15 minutes after i.v. Appli cation of JOMO®-Color.  

embodiments

The following examples illustrate the invention.

example 1 Production of JOMOL®

Nocardia opaca (ATCC 21 953 DSM 202) are grown on DST plates or dextrose agar, 2% in plate technique cultured on three to four plates for 5 to 8 days. After harvesting will be the bacteria are inoculated to about 100 ml normal nutrient broth and overnight incubated at 30 to 37 ° C.

The harvesting of the Nocardia bacteria is preferably carried out by Abzentrifu For example, at 4000 rpm, for 5 to 10 minutes in a refrigerated centrifuge with a rotor of 40 cm diameter. The resulting sediment (about 5 g Bacteria) is dissolved in 100 ml, for example, 0.01 M Tris / HCl buffer (pH 7.4), preferably with EDTA-Na₂ (0.06%) suspended (washing), as above centrifuged and in about 100 ml 0.01M Tris / HCl buffer of pH 7.4 with preferably 0.06% EDTA-Na₂ and suspended with 10% glucose. After about 30 minutes, 10 mg lysozyme and preferably, for reducing the viscosity, 3 mg of deoxy Ribonuclease added.

This phase with glucose and ferments lasts 2 hours and takes place at 30 to 37 ° C instead. Then, as stated above, can be centrifuged. The Lump can resuspended in 100 ml of 0.01M Tris-HCl buffer containing 0.06% EDTA-Na₂ be centrifuged again and resuspended in the same medium become. This washing process can be omitted, but through him is more Wall material of bacteria and nutrient medium from the final used Supernatant removed.

After that, the cells are destroyed. This can be done with any suitable methods done, ultrasound is preferred over 1 minute.

Thereafter, it is centrifuged at 4 to 6 ° C in the refrigerated centrifuge (as described above) at 4000 rpm for 10 to 15 minutes. The UV spectrum of the supernatant is shown in Fig. 1.

The supernatant is preferably divided into five parts and preceded by five He used to place Sephadex® G-75 columns in separate sections. The pillars are 80 cm long, inner diameter 2.5 cm, the Sephadex® filling height is 60 cm (UV detection at 214 nm). When using a column, the remaining Portions frozen at -25 ° C until use. The fraction has approx. 4500 to 900 daltons. For example, it elutes with 0.01M Tris HCl buffer (pH 7.4), with 0.06% ETDA-Na₂ at about 900 to 980 ml by, at a drip rate of about 2 drops per second (overnight) about 12 hours in 1 to 2 hours. This fraction contains the active frak tion, d. h., the desired product 2b *. This is followed by lyophilization. After that is preferably incubated for 1 hour at 80 ° C.

If not lyophilized, then the diluted solutions are at -20 ° C, better kept at -40 ° C or -80 ° C, frozen. JOMOL® is going through Reaction of fraction 2b * (assumed average molecular weight 4000) with acetaldehyde in molar ratio 2b *: acetaldehyde as 1: 2. Thereafter, must be re-lyophilized, optionally (see above) to be frozen.

The yield for about 5 g of bacteria is about 50 mg of pure product, ie JOMOL®. The product is very hygroscopic. It has the following properties: absorption maximum in the UV spectrum at 282.5 nm. The substance was dissolved in water, reference water 1 cm quartz cuvettes; apparent molecular weights at 4500, 3000 and 900; depending on the residual water content, ivory-colored amorphous-crystalline fluffy; pH 5.5 (1 mg of compound dissolved in 1 ml of water); weakly positive, pink ninhydrin reaction; stainable with Orcin Ragens;
Peptide content: about 50%, based on amorphous substance (protein test according to L. Thomas, J. Clin. Chem. Biochem., 19, 1981, page 203-208, Coomassie Brilliant Blue G ₂₅₀R), the following R _f values (Solvent Specification Volume: Volume)

R _f = 0.20 (butanol: glacial acetic acid: water 4: 1: 1)
R _f = 0.45 (benzene: glacial acetic acid: water 2: 1: 2)
R _f = 0.685 (methanol: glacial acetic acid: water 4: 1: 1)
no migration (chloroform: methanol 96: 4)
R _f = 0.54 (chloroform: methanol 1: 1)

Solubility: poorly soluble in non-polar solvents, very soluble in polar solvents; detectable components:

substances Ratio (approximation) Neutral sugar (glucose, galactose, ribose) (<) 1 Amino sugars (glucosamine, muramic acid) (after hydrolysis) 3.5 Amino acids (Ala, Glu, iso-Gln, Gly, Lys, DAP) 4 lipids <1 phosphorus <1

Structure:
Peptidoglycan structure whose glycane backbone consists of 3 to 17 disaccharides of N-acetylglucosaminyl-N-acetylmuramyl.

Example 2 Production of the aid "Cocktail"

1000 ml of ethanol 96% and 40 g of acetaldehyde puriss. be mixed.

An effective dose is 25 ml of it.

Application Instructions:
25 ml of the cocktail are mixed with 225 ml of water and administered orally. The daily dose is 2 to 3 effective doses, on a doctor's order can be dosed higher. The cocktail should not be administered if brain metastases are suspected.

Example 3 Production example for JOMOL® derivatives JOMOL®-Sn (also called JOMO®-tech)

• a) General working conditions:
  Work under nitrogen at 0 to 10 ° C, purging solutions with nitrogen before use, using reaction vessels acid-washed.
• b) 20 mg of JOMOL® are dissolved in 100 μl of 1N HCl, then the solution becomes 10 to 15 Minutes left. Thereafter, 1 mg SnCl₂ × 2H₂O (solid) is added and The reaction mixture allowed to stand for about 20 minutes. There will be 2 ml of one  0.1N HCl, physiological saline (0 to 1 ° C) was added.

After vigorous shaking, each 20 ul of the solution in a Stell Area temperature from -10 to -20 ° C in pre-cooled vials filled. The vials are capped immediately and until the Use frozen (about -20 ° C) kept.

Example 4 ^99m technetium loaded JOMOL®-Sn for diagnostics

For labeling with ^99m technetium, add 740 to 3700 MBq of ^99m Tc in a volume of up to 5 ml (from a ^99m molybdenum generator) to the frozen vial (Example 3) Preferably, the second eluate of the preparation day is used after 10 to 15 minutes Room temperature (avoid full light), the IV injection preparation can be used with the gamma camera for a diagnostic test.

Example 5 Coupling product of JOMOL® and diethylenetriaminepentaacetic acid

This coupling product is suitable for loading with ¹¹¹Indium or reduced by SnCl₂ ^99m pertechnetate.

• a) General working conditions:
  The reaction vessels are used washed with acid, it is carried out at 0 to 10 ° C under nitrogen, the solutions used are nitrogen blown through
• b) 5 mg of diethylenetriaminepentaacetic acid anhydride are dissolved in 5 ml of absolute Diethyl ether suspended (this suspension is referred to as S1).
• c) Approx. 200 μg of JOMOL® are dissolved in 300 μl of bicarbonate buffer (0.05M, pH 7.0) in physiological saline. With vigorous shaking (blender, 30 to 60 seconds) 25 .mu.l of the suspension S 1 are added and then incubated at 37 ° C for 1 hour. The ether evaporates.
  This is followed by chromatography:
  FPLC chromatography:
  Column: Superose 12, HR 10/30, code no. 17-0538-01.
  Applied volume: approx. 300 μl
  Eluent: physiological saline
  Flow rate: 1 ml / min
  Detection: λ = 214 nm, HPLC pump.

In the product fraction JOMOL® is completely available as JOMOL®-DTPA. The Binding of the DTPA to the amino group of the lysine of JOMOLS® is stable, hydrolyze other connections.

Example 6 Labeling of JOMOL® diethylenediaminepentaacetic acid with 11,1'-indium

To about 220 μg of JOMOL® diethylenetriaminepentaacetic acid (hereinafter referred to as JOMOL-DTPA) in 3 ml of 5 mM bicarbonate buffer (pH 7.0) in physio  logical saline solution is about 74 MBq of 111 indium in 0.2 ml of 40 mM HCl added. After shaking and waiting 15 minutes at room temperature the solution obtained can be used for intravenous injection (i.v.). The test takes place with the gamma camera.

The ¹¹¹¹¹I-labeled JOMOL® DTPA results in the elution pattern shown in FIG. 3 in FPLC chromatography (214 nm, 0.1 M phosphate buffer). In the diagram the distribution of the radioactivity is indicated on three separable fractions.

Example 7 Marking of JOMOL® diethylenediamine pentaacetic acid with ^99m technetium

For labeling with ^99m Technetium is added to 1 ml of JOMOL-DTPA solution (about 50 micrograms, frozen) 100 .mu.l of a hydrochloric acid (0.1N HCl) 3 mM SnCl₂ × 2H₂O, physiological saline and waited 10 minutes. Thereafter, 740 to 1850 MBq of ^99m technetium in a volume of up to 5 ml are added (from the second eluate of the preparation day). After 10 to 15 minutes, the preparation for iv injection is suitable for testing on the gamma camera.

Example 8 Production of JOMOL® coupling products a) JOMOL® uridine (JOMOL®-J / U)

The preparation is analogous to the connection of diethylenetriamine  pentaacetic acid, as described in Example 5. There are 30 μg uridine-2 ', 3'-dialdehyde dissolved in 10 ul of dried diethyl ether. The product obtained is, as in Bei game 5 described, chromatographed.

b) JOMOL®-L-Tyrosine (JOMO-J / Ts)

The preparation of the coupling product is carried out in the same way as in Example 5, wherein α-amino-β- [p-hydroxyphenyl] propion aldehyde in an amount of 30 ug in 10 ul of absolute diethyl ether ver be used.

c) JOMOL®-L-Thyronine (JOMO-J / Tn)

The attachment of α-amino-β- [p-hydroxyphenyl [p-hydroxyphenyl ether]] - proprionaldehyde is carried out in the same manner as described in Example.

Example 9 Preparation of an iodine-labeled coupling product of JOMOL® uridine / JOMOL®-L-tyrosine / JOMOL®-L-thyronine / JOMO®-Color

The coupling product of JOMOL and uridine prepared according to Example 8a) is treated with a suitable radionuclide according to the procedure customary for uridine performed by appropriate laboratories.

The coupling products prepared according to Examples 8b) and 8c) and JOMO®-Color (see Example 12) are prepared in a manner known per se labeled with radioactive iodine.

Example 10 JOMOL® bound to a precursor of Kryptofix (JOMO®-Ra or JOMOL®-CE)

To 200 μl of an aqueous solution of 1 mg of JOMOL® is added 1 ml of 0.2M borate buffer (pH 8.0). A solution is separated from a preliminary stage of the Kryptofix manufactured. The precursor is called CE.

Cryptofix precursor (= CE); Kryptofix 222 = 4,7,13,16,21,24-hexaoxo-1,10- diazabicyclo [8.8.8] hexacosane.

The solution has a concentration of 1 mg per ml of ethanol. 240 μl of the ethanolic CE solution are added to the above JOMOL® solution give. To the reaction mixture is added 160 ul Cyanoborhydridlösung (1 mg / ml 0.2M borate buffer, pH 8.0). The reaction mixture is 20 hours incubated at 37 ° C and can then directly for binding of radioactive Markers are used.

Example 11 Marking of JOMOL®-CE with radioactive radium

After the above-mentioned incubation, ²²⁴Radium is added to the reaction mixture as ²²⁴RaCl₂ solution to 7.4 MBq and allowed to stand at room temperature for about 1 hour. The resulting solution is placed on a Superose ^T H FPLC column. The eluent used is 50 mM potassium phosphate buffer of pH 6.8 in physiological saline, 1 ml / min. Detection at 214 nm, and radioactivity monitoring. The reaction product and free JOMOL® have essentially the same spectrum. The fractional removal allows the separation of the low molecular weight components of the reaction mixture. The product is eluted in 2 peaks. After leaving about 80% of the applied radioactivity, a shoulder becomes visible on the 2nd peak. The fractions attributable to this shoulder and the subsequent ones are discarded. These contain, among other things, the cyanoborohydride. Since JOMOL®-CE-²²⁴Ra is not only enriched in cancer cell surfaces, but also, as the immunostimulatory pure immunomodulatory molecule, monocytes, macrophages, microphages and bone marrow stem cells, care must be taken before administering JOMOL®-CE-²²⁴Ra iv that these cells can not take up the product. Restricting the uptake of JOMOL®-CE-²²⁴Ra into immunocompetent and bone marrow stem cells is necessary with high doses of cortisol iv / im at the distance known to those skilled in the art before the administration of the activity; additional measures should be taken ad hoc by consulting an immunologist , Failure to do so threatens a leukemia. The drug should be used primarily before or after microseeding of tumor cells by surgery (not for non-Hodgkin's tumors).

Example 12 JOMOL® fluorescein isothiocyanate (JOMO®-Color) for diagnosis and / or therapy

Working conditions: room temperature
Fluorescein isothiocyanate in saturated solution in ethanol 96%
JOMOL® 10 mg dissolved in 200 ul H₂O.

JOMOL® and fluorescein isothiocyanate are combined in a molar ratio of 1: 5 given and mixed for 30 seconds in the ultrasound. Then about 10 minutes from wait and about a column 30 cm × 0.6 cm, column bed 15 cm Sephadex G-75, Eluent physiological saline 0.5 ml / min, fractionate. Fraction 2.5 to 9 ml contains JOMO®-Color. The column is previously with 2 runs to equilibrate with 5 mg JOMOL®-Fitc. The resulting fraction is ent neither immediately frozen at -20 ° C, or proportioned per 300 μg JOMOL®-Fitc and lyophilized, sealed under nitrogen and until use -20 ° C stored.

Examples of kits and their applications 1) JOMO®-tech, scintigraphic imaging kit for malignant tumors with the gamma camera

• ingredients:
• a) cocktail (ethanol 96%, 50 g / acetaldehyde puriss., 2 g) 2 × 1 dose, screw cap bottle
• b) JOMO®-tech (200 μg JOMOL-Sn 20 μg dry matter, containing JOMOL + 10 micrograms SnCl₂ × 2H₂O in 0.1N HCl, physiological Saline), vial, frozen at -20 ° C.
• Application Instructions:
  to a) Cocktail: (not for brain metastases)
  1 dose = 25 ml from the screw cap bottle
  Dilute cocktail with 225 ml of water and give orally, wait 30 minutes, then
  to b) JOMO®-tech: 740 to 1110 MBq ^99m Technetium in physiological saline (second eluate of the preparation day from a ^99m molybdenum generator), in a volume up to 5 ml, are placed in the frozen vial (b) with a syringe containing you put it on it. The vial (b) is shaken several times, allowed to stand at room temperature for 10 to 15 minutes, then injected intravenously.

2) JOMO®-tech lyo, kit for the scintigraphic display of malignant tumors with the gamma camera

• ingredients:
• a) Cocktail (see [1a])
• b) JOMO®-tech lyo (1350 μg JOMOL®-Sn 813 μg dry matter, containing JOMOL® + 30 μg SnCl₂ × 2H₂O + 8 μg Na-acetate + 45 μg
  Succinic acid + 670 μg lactose), lyophilised, vial frozen at -20 ° C
• Application Instructions:
  See (1) JOMO®-tech.

3) Smil-N / Tc, a scintigraphic imaging kit for malignant tumors the lungs with the gamma camera

• ingredients:
• a) Cocktail (see [1a])
• b) JOMO®-tech (see [1b])
• c) Liposomes in physiological saline:
  saline solution, 5 ml, vial, containing liposomes, 5 mg, consisting of phosphatidylcholine:
  Phosphatidylserine: Cholesterol in the molar ratio 8: 2: 10
• Application Instructions:
  to a) cocktail (see [1a])
  to b) JOMO®-tech (see [1b]). Instead of the intravenous injection, it is instilled into the vial (c).
  c) The contents of (b) and (c) are brought together, shaken and inhaled with a closed inhalation system.

4) JOMO®-In, scintigraphic imaging kit for malignant tumors with the gamma camera

• ingredients:
• a) Cocktail (see [1a])
• b) JOMO®-In (200 μg JOMOL®, dry, +20 μg DTPA = 220 μg JOMOL-DTPA in 3 ml of physiological saline, 5 mM Na bicarbonate buffer,  pH 7.0), vial, frozen at -20 ° C.
• Application Instructions:
  to a) cocktail (see [1a])
  to b) thaw JOMO®-In and add 74 to 111 MBq of 111 indium in 0.2 to 0.3 ml of 40 mM HCl (InCl₃). After shaking and waiting for 15 minutes at room temperature, the resulting solution for intravenous injection is suitable for testing on the gamma camera.

5) JOMO®-J labeled with 13 iodine, kit for scintigraphic display of malignant tumors with the gamma camera and for therapy

• ingredients:
• a) Cocktail (see [1a])
• b) JOMO®-J labeled with ¹³¹Jod (about 250 μg JOMOL 25 μg JOMOL Dry matter + 37 MBq ¹³¹Jod) in 1 to 2 ml PBS, 25 mM, 0.05N Acetic acid, vial, frozen at -20 ° C.
• Application Instructions:
  to a) cocktail (see [1a])
  to b) thaw JOMO®-J labeled with ¹¹³ iodine and inject intravenously, followed by testing on the gamma camera.

Blockage of the thyroid with sodium perchlorate is recommended. For therapy, if necessary, by order of the acting physician higher doses used.

6) JOMO®-Ra, marked with ²²⁴Radium, kit for local radiotherapy of Trace seed of cancer cells (JOMOL®-Ce-²²⁴Ra for i.v./i.t.)

• ingredients:
• a) Cocktail (see [1])
• b) JOMOL®-CE-²²⁴Ra (about 30 μg JOMOL® CE dry substance, labeled with 7.4 MBq²²⁴Radium) in 2 ml PBS, 25 mM, vial, frozen at -20 ° C.
• Application Instructions:
  to a) cocktail (see [1a])
  to b) JOMOL®-CE-²²⁴Ra:
  Two hours before treatment with JOMO-Ra 250 mg Cortisol iv, then orally, then thaw (b) and apply intravenously or intra tumoral.

7) JOMO®-Color, Intraoperative color diagnostic kit with ultraviolet Light in malignant tumors

• ingredients:
• a) Cocktail (see [1a])
• b) JOMOL® fluorescein isothiocyanate in a molar ratio of 1: 5) from 5 ml of physiological saline, lyophilised, piercing bottle, frozen at -20 ° C.
• Application Instructions:
  to a) cocktail when used for therapy (see [1a]); When used for intraoperative diagnosis, 5 to 10 ml of the cocktail is used to prepare an infusion solution, administered 10 to 30 minutes before iv administration of (b).

It should be noted that morphine and its derivatives through the cocktail be increased in their effect 30- to 40-fold.

to b) JOMOL® fluorescein isothiocyanate is dissolved with 5 ml H₂O (Ampuwa ^⌀ ) and administered intravenously.

For intraoperative diagnosis 10 to 20 minutes p.a. from (b) is one UV lamp required (see Ophthalmology, Dentistry)

8) JOMO®-Lab for the rapid diagnosis of malignant tumors in vitro

• ingredients:
  Laboratory grade lyophilisate, not for use in vivo. 300 μg fluorescein isothiocyanate-JOMOL®, 45 mg NaCl, screw cap bottle, frozen at -20 ° C.
• Application Instructions:
  In the screw cap bottle 5 ml H₂O are added, the tissue sample is added, 5 minutes at room temperature, the tissue sample is washed in physiological saline.

In the section of the tissue sample shows up in the ultraviolet light with the Magnifying glass or in the fluorescence microscope, the tumor tissue bright, the healthy Fabric dark. The surgical preparation margin shows by Zellzerstörungsbe protease release also gave fluorescence.

Claims (9)

1. Diagnostic agent, characterized in that it contains a microbial extract, sustain by

• a) cultivation of Nocardia opaca = Rhodococcus rhodo chrous (DSM 43 202 or ATCC 21 953) on a nutrient medium,
• b) obtaining the cells,
• c) suspending the cells in a buffer and allowing them to stand for 15 minutes to a few hours,
• d) treatment of the suspension with a murolytic enzyme,
• e) destruction of cells,
• f) separation of the material obtained in sediment and supernatant,
• g) separation of the supernatant on Sephadex columns using buffer as eluent,
• h) recovery of the active fractions showing absorption at 214 nm in the UV spectrum,
• i) reacting the active fractions with acetaldehyde in a molar ratio of 1 mol of active fraction (assumed MW 4000) with 1.8 to 2.2 moles of acetaldehyde, optionally coupled with a molecule capable of binding a radioactive source and / or dye and containing it together with an aldehyde of the general formula I RCHO (I) where R is a hydrogen atom and a straight-chain or branched hydrocarbon group having 1 to 4 carbon atoms, wherein the free aldehyde can also be metabolically released directly or indirectly from substances, and optionally an alcohol of the general formula IIR¹CH₂OH (II) wherein R has the meaning given above for R, and optionally customary pharmaceutically acceptable carriers and / or diluents is used.

2. Diagnostic agent according to claim 1, characterized ge indicates that as a molecule, the one radioactive radiator and / or can bind a dye, Diethylenetriaminepentaacetic acid or 4,7,13,16,21,24-hexa oxo-1,10-diazabicyclo [8.8.8] hexacosan (Kryptofix 222®) puts. 3. Diagnostic agent according to claim 1, characterized ge indicates that the cells are in one Buffer and a glucose containing medium suspended. 4. Diagnostic agent according to claim 1, characterized ge indicates that the suspension with a murolytic enzyme and treated with deoxyribonuclease. 5. Diagnostic agent according to claim 1, characterized ge indicates that the separation of the over on Sephadex columns using a buffer, containing 0.1 to 0.05% disodium ethylenediaminetetraacetate, performs.   6. Diagnostic agent according to claim 1, characterized ge indicates that the microbial extract according to claim 1 by culturing the Bakte for 5 to 8 days on dextrose agar (2%) plates using of nutrient broth as a nutrient medium and carrying out the breeding during a period of 10 to 20 hours at a tem temperature of 30 to 37 ° C receives. 7. A diagnostic agent according to claim 1, characterized ge indicates that the microbial extract after harvesting the cells one or more times with Buffer pH 7.4, optionally 0.1 to 0.05% EDTA-Na₂ ent can hold, washes. 8. A diagnostic agent according to claim 1, characterized in that one uses as a radioactive emitter which can be bound by the molecule, ^99m technetium, ¹¹¹Indium, ¹²⁵Iod, ¹³¹Iod or ²²⁴Radium. 9. A diagnostic agent according to claim 1, characterized in that it contains the microbial extract labeled with ^99m technetium.