DE1617886A1 - Process for the preparation of drugs with selective tropism against cancer and certain organs - Google Patents

Description

Process for the preparation of drugs with selective tropism against cancer and certain organ.

In the case of spontaneous tumors, the body's own immunological Tuxor defense is sufficient in terms of quantity due to antibody globulins. The antibodies can be selective Have tropism to the tumor cells without damaging them. The immunological Cancer therapy must therefore include both tumor-specific antibody formation and increase the cell-damaging effect of the antibodies on tumor cells.

The active immunization of the tumor patient with tumor antigens, especially from the body's own tumor, can only slightly influence the formation of antibodies, because an immunological. Tolerance to this Forms tumor antigens. If organ-specific antigens are also used with this type of treatment there is a risk of auto-sensitization to the organ tissue of the topsoil of the tumor.

According to the invention, the formation of antibodies against tumors can now be initiated or activated when sensitizing messenger ribonucleic acids from lymphatic cell elements of the blood and body fluids, such as macrophages, , monosytoid elements, small lymphocytes and spindle-shaped cells (histiocytes) instead of the antigen transfers to the patient. According to this method is also the Initiation of antibody formation in intermediate animals as well as in tissue cultures of lymphatic tissues and in the cell-free synthesis systems produced from such tissues possible in vitro.

This method of inducing antibody formation by messenger.

RNA and the generation of antibodies in vitro was carried out by JACHERTS Described in Z. med. Microbiol. and Immunol. 152, 112-133 (1966), as in the further publications specified there under nos. 3, 11, 12 and 13. However, this method has not heretofore been produced in the present manner used by messenger RNA and antibodies against tumors and normal organs. That This is because the process can also be based on organ extracts or organ antigens as starting materials transfer.

In the present procedure, the particular method is active immunization by means of RNA and the production of antibodies, a component that is considered a process step A is designated.

(A) Obtaining tumor antigens and suitable tumor extracts as well as organ antigens and organ extracts are carried out according to known methods, e.g. after DBP announcement T 33 o36 IVa / 3oh; the DBP 1 090 821i to POTTER, APPELLA and GEISSER: j. Mol. Biol.

(1963) et al. To generate the M-RNA in cells, the leukocyte blood cells, in particular the antibody-forming cells, are isolated in the present method in known ways, for example by gravity separation, spontaneous sedimentation, electrophoresis and sieving from blood preservation and in the natural milieu of the blood plasma, optionally with the addition of antibiotics and or . or chemotherapeutics or in a suitable tissue culture medium, e.g. Hanck's solution + 10 * calf serum as well as the addition of antibiotics and / or chemotherapeutics (see D. JACHERTS: Z. Med. Mikrobiol. o. Immunol, 152, 1 - 19 (1966 ), if necessary cultured with mechanical movement and aeration. The tumor extract or the tumor antigens are added accordingly in dilutions, eg from 10-4 to 10-12 g of the tumor or organ dry substances or the corresponding @@ / concentrations of fresh extracts per ml of tissue culture medium. After an incubation time of several hours to days, the leukocytes are irradiated with UV light (cf.

JACHERTS: Z. med. Microbiol. and Immunol. 152, 262-272 (1966), or ab.r by other physical methods, e.g. by brief heating to 40 ° C, Irradiation with ionizing rays, or by chemical action of radiomimetic acting substances are damaged.

After the premorbid stage has occurred, the egg begins to die can be recognized by cells of the culture, the cells are carefully thrown off isolated and washed several times in physiological Koc salt solution and freeze-dried. It is also possible, nec KIRBY: Progress in nucleic acid research and molecular biology, ed. by DAVIDSON and COHN. New York and London: "Acadeiie Press," the RNA to be prepared and then preserved by freeze-drying.

The lymphatic cells or RNA obtained in this way are now in concentrations from 10-3 to 10-9 g of dry matter per ml of physiological saline solution as a suspension or solution, if necessary (53 injected repeatedly in operation B medium animals or in tissue cultures using other lymphatic cells or the like. tor artificially assembled cell-free synthesis systems for the generation of antibodies used.

The stimulation of antibody formation and the production of antibodies (C) from cancer patients corresponds to operation C of the present method. Here, too, the injections are made at intervals of hours to a few days.

The antibody globulins obtained according to operations B) and C) can now in step D individually or as a mixture according to known processes organ extracts from the tissues of the normal topsoil of the tumor, or the corresponding homologous or heterologous organ extracts or homogenates are saturated will.

The process of adsorption using column chromatography is suitable for this purpose, like you are familiar with immunological methods of precipitation. This step is particularly important if no isolated antibodies or M-RNAs are to be obtained Tumor antl genes, but total tumor extracts were used, which are still organ-specific Contain normal tissue factors. The unprecipitated part of the antibody is predominantly tumor-specific, (E) A further narrowing can be done in step E) by saturation on the corresponding tumor extract. That through antigen-antibody formation coming about Precipitate is isolated and washed according to known methods and into both of them Components dissociated, whereby the antibodies and antigens or haptens are recovered (cf. e.g. GRAMLICH: Naturwissenschaften 49 (1962), 451.) The antibody globulins can e.g. 3. but also after dissociation from the antigen due to half saturation with ammonium sulfate precipitated, separated from the supernatant antigen solution and treated with physiological saline solution getting washed. From the supernatant antigen solution, z.3. possibly according to known methods, by gel filtration or dialysis, the antigens and hapters isolated. The isolated antigens can then be used in process step A) and B) or but also used directly for active immunization and new antibody production will.

The isolated tumor-specific antibodies are now in the working :) step F) according to known methods by mild reduction, z.3. with 3-mercaptoethanol, split into fragments by bursting the disulfide bridges (cf. POTTER, APPELLA and GEISSER, j. Mole.

Biol. (1965), 14, 361-372, Dreesman: Proc. nat. Acad. Sci. 54 (1965) 822-830) Fracturing is also possible by other chemical methods, e.g. using saline solutions and heating, as well as chemical hydrolysis (see DBP) and by fermentative cleavage, z.3. with papain (see KRONVALL : Vox. Sang.

10: 303-313 (1965) possible. The tropism to the antigen must be for some of the fragments will be preserved. Use known chemical methods to be cancer-effective drugs are now bound to these fragments. These may have to be chemically activated prior to coupling. For example, the free sulfhydryl groups of the antibody fragments sulfur mustard derivatives, which were previously reduced at the sulfur atom have been coupled by mild oxidizing agents. However, others can also do so known chemical methods of coupling, depending on the use of a particular Drug, such as diazotization, alkylation, the oxazolone process, u.e .. If necessary, the antibody globulins not used for the reaction are now again precipitated by salting out and the coupled conjugated present in the supernatant Molecules e.g. isolated by dialysis or gel filtration and preserved by known methods.

The isolated tumor antigens can also have a tropism to the same Own tumor. They are then also suitable as a vehicle for drugs which they can be coupled together using known chemical methods. One prior separation and isolation of the chemically reactive group of the molecule, which is responsible for the tropism can also be done here before the chemical Connection with the tumor-active drug takes place The advantages and possible applications Tumor-specific drugs lie in the selective enrichment of the drugs at the desired place of effect. For example, the well-known cytotoxic ones are used against cancer or static substances, or else cytolytic substances such as histamine bacteria, puryl histamine, epsilon puryl lysine, or dipuryl ethylenediamine used, as well as a transfer of nucleic acids or internal repressors, as well as protein bodies possible. The targeted transport becomes a premature Inactivation of the active substances avoided. The enlargement of the drug molecule coupling it to an antibody or antigen fragment reduces the possibility absorption by other cells in the body that do not have receptors for the antibody vehicle own. There are no dangerous side effects.

Because of the individual and tumor specificity and the multitude of possible Medicinal and chemical coupling methods are suitable for the present process extensive application possibilities.

Separate application of individual process steps is also possible, e.g. from process step A; A and B; A and C; D and Eg D, E and F, as well as such E and F. As a starting product or antigen for the manufacture of preparations Any type of neoplasm are used, including the so-called blood clot.

The inventive demonstration can be applied to the production of organotropic Transfer drugs. Been there instead of the Tumor extracts or antigenic organ extracts, or isolated organ antigens are used. In the process step D) the saturation of the antibody globulins takes place on one extract from another Organ type, e.g. 3. in the isolation of antibodies against cerebrum on the extract lcleinbrain, diencephalon, or spinal cord; in those against the diencephalon to the spinal cord or cerebrum; for antibodies against kidneys on the liver or heart; with such against Heart to kidneys liver or spleen; in those from lymph glands on the thymus; at them against the thymus, on the spleen, etc. Obtaining organ antibodies according to the invention The process of step A) is also #### as well as the combination of the others Processing into organotropic drugs, novel. There are particularly many here Possible applications through the coupling of the most varied of drugs and chemical molecules. These can be both direct and indirect biologically act F by, for example, electromagnetic rays emitted by a particular Transmitters are emitted, are stimulated. This is how wireless delivery works from electrical impulses, e.g. on the heart or certain parts of the brain, likewise the amplification of local bioelectrical processes and theirs specific derivation. At the place of action can not be directly biologically active Molecules are activated by other molecules.

Be-sniel 1 @ Tumor-specific mustards for the treatment of a Bronchial carcinoma are produced. The production is divided into 6 work steps. In work step A), the tumor antigens and large-molecule RNA are used to generate antibodies won. To do this, the tumor is broken into smaller particles after surgical removal cut up. These are rinsed in physiological saline solution, because in verdiüssia tem nitrogen suddenly frozen and pulverized in deep-frozen mills, because lyophilized, then by anhydrous acid vapor hydrolysis in a vacuum unlocked according to DBP 1 090 821 and sterile under the exclusion of air as a durable permanent preparation kept.

Now fresh blood supplies of human blood or search from fresh animal blood by sedimentation and / or others acquaintance Procedure to separate the leukocytes from the red blood cells and platelets and in the corresponding blood serum according to known methods, if necessary with an addition of antibiotics and / or chemotherapeutic agents, grown in the manner of a tissue culture. Other suitable nutrient media can also be used for this purpose, such as Hanck's solution with the addition of lo% calf serum and a combination of antibiotics. Within one hour after the creation of the leukocyte culture, it is diluted with an aqueous solution Extract from the prepared preparation of the tumor in a concentration of 10-6 g dry substance added per ml of the nutrient medium. U @ the lipids of the total tumor extract in solution To bring, 0.01 mg of lauryl sodium sulfate per ml are also used. It can too fresh tumor extracts or Homog.nate can be used.

After an incubation period of 2 days at 37 ° C, the cells of the culture for 10 seconds with UV light from a low-pressure mercury lamp with 8 Watts and. UG filter irradiated in, distance of 13 cm.

After about 15 minutes, the cells are removed from the culture, then Washed several times in PBS solution and freeze-dried.

B) In operation B), antibody globulins were now obtained using Medium animals or from in vitro cultures of antibody-producing cells or of suitable ones duty-free systems. The method corresponds to an active immunization in which instead of the antigen, the preparations obtained according to step A) in a dilution of 10-6 g Trookensubstanz per ml of physiological saline solution can be used In the case of the macroorganism, the injection is carried out parenterally, three times at intervals of 1, 2 and 3 days. The antibody production can be done using the tumor antigen be monitored by known methods of gel precipitation, so that after reaching A sufficient concentration of the antibody globules according to known methods obtained from blood or body fluids or the nutrient media and e.g. by gel filtration on Sephadex G 200 or by half saturation with ammonium sulfate be enriched. The precipitation product of the ammonium sulfate solution is centrifuged and, after the supernatant has been eliminated, with physiol. NaCl washed C) In step C), the antibody formation is stimulated and intensified in the tumor patient. In the same way as with Mittlertisern, those obtained from operation A) are obtained Park preparations terally injected. The antibody globulins are obtained as in step B).

In operation D), those obtained after operation 13) and C) are now obtained Antibody globulins mixed together in a ratio of 1s1 and made from organ extracts healthy tissues of the normal topsoil of the tumor are saturated. Unless there is it is not possible to preserve healthy tissue from the surgical specimen Animal organs can also be used for satiety. That after got Methods obtained precipitate then contains the organ and gag also type and individual-specific factors of the antibody globulins.

The antibodies not used for the reaction are both against the Tumor antigens, as well as directed against exogenous antigens. To get rid of the latter, takes place in step E) the precipitation of the so far not exhausted gas and itself Antibodies in solution on the tumor extract obtained in step A). Here, too, the permanently soluble components of this extract are used by this by adding lauryl sodium sulfate of the above

Concentration to be emulsified. The precipitate is now more common Way isolated, washed and dissociated into its components.

The antibody globulins are made by half saturation with ammonium sulfate precipitated and centrifuged. The remaining tumor antigen can then optionally used later when repeating operation A) or used as an active one Immunization of middle animals or for the production of organotropic drugs to be used.

@) In operation F) the isolated antibodies are now in # -mercaptomethanol carefully reduced. A solution of 0.3 # mercaptosethanol, 7 M guanidine HC1 ; 0.5 M TRIS-HCl buffer (Ph 8.2) is used at a content of 1 to 2 * der Antibody globulins.

After an exposure time of 1/2 hour at 37 ° C, 1 M S-mustard was added to the solution and this was kept at 30 ° C. for a further hour. so is cooled to 10 ° C. and 0.7 M methylene blue is added as a weak oxidizing agent. After an exposure time of 1/2 hour, the solution becomes against physiological for 24 hours Dialyzed saline and then lyophilized. Before that, those who cannot be divided Antibody globulins precipitated by saturation with ammonium sulfate, centrifuged off The supernatant is then reassigned against physiological saline dielysed. The finished preparation can be used therapeutically in the fresh state or it is lyophilized and dissolved in a solvent before use.

Example 2s Bs are intended to be selective drugs against muscle sarcoma getting produced. The work steps A and C correspond to example 1.

Step B is omitted. In step D, the antibodies obtained from the patient are precipitated with a fresh cell-free extract from beef muscle tissue that was produced immediately after slaughter. The precipitate is centrifuged and the supernatant is incubated with the tumor extract. The precipitate that forms is washed and washed at 37 ° C. for 16 hours in a ratio of 100: 1 with merouripapain as enzyme in a solution of 0.01 M cysteine, 0.002 M EDTA, 0.19 M NaCl, 0.1 M phosphate buffer pH 7.0% The digested precipitate is digested on Sephadex-G-100 in a column of 4.4 x 38 cm after equilibration of the columns with 0.05 M phosphate buffer of pH 8, o separated from the papain used at a rate of 20 ml per hour.

The fragments of antibodies and antigens or haptens are Now coupled to tri #ethylene melamine (TEM) by diazotization. The unbound TEM is removed by gel filtration.

Example 3r A drug is said to act selectively on the cerebrum For this purpose, in step A, a surgical preparation is used human brain obtained and used to generate antibody-forming RNA used, in step B antibodies are obtained from the horse with it. These are saturated in operation D on an extract from beef spinal cord. in the Operation E is the precipitation of the organ-specific that did not react Antibodies with the extract from human brain. The precipitate is through 2-5 days Glycol solution dissociated according to OLITZKI and FRANKEL (see NATURE: 126, 723 (1930) and proc. Social exp. Biol. A. Med. 28, 492 (1931)) and Globulin. through half saturation precipitated with ammonium sulfate and centrifuged. The antigens are obtained from the supernatant obtained and for diagnostics of the tropism of the preparations obtained later as well of the patient's degree of senibilization against the brain. The Antibody are broken down into fragments by fermentation and by ion exchange chromatography in DEAE cellulose (cf. FRANKLI @ J. clin. Invest. 39t 1933 (1960)). By Gel-PrSzlpitation + with the organ antigen, the organotropic part is determined and this coupled to the drug by diazotization.

Claims (3)

Claims 1.) Process for the production of organ-specific and Anti-cancer drugs, characterized in that A.) the in known way from blood reserves or fresh human or animal blood isolated leukocytes, especially lymphocytes, plasma cells and macrophages rit Tumor antigens are incubated and known physical and / or chemical Methods placed in a premorbid state, preserved or removed from the cells after known methods that isolate and conserve ribonuclic acids, these preparations according to known methods via medium-sized animals or in cultures of antibody-forming Cells or the cell-free systems produced from them or artificially produced, used to obtain antibody globulins and / or corresponding antibodies from the blood or body fluids of the corresponding cancer patient after pretreatment rit wins the preparations made in step A, then (D) these antibody globulins obtained in steps B and C by known methods individually or as a mixture of organ extracts from the tissues of normal topsoil the tumor is satiated and E! the non-binding antibody globules with Tumor extracts precipitated, this precipitate isolated by known methods and dissociated into their two components, the antibodies and antigens, or Haptens, chemically or fermentatively in fragments with retained tropis and on these fragments are carcinogenic or cytolytic by known chemical processes Coupling drugs, the process steps Ag A and B; A and C; D and E, as well as D, E and F and E and F can be carried out on their own 2.) Procedure according to claim 1, characterized in that instead of the tumor extracts, organ extracts or organ antigens are used as starting materials and others in step D) Organ species than those of the starting product are used. 3.) Method according to claim 1 and 2, characterized in that both health-promoting, as well as harmful, as well as directly and indirectly pharmacologically active chemical molecules to the organotropic or tumor tropic Tractor substances are coupled 4.) The method according to claim 3, characterized in that that molecule can be used by electromagnetic rays or by others Substances become biologically active.