DE102004036079A1 - Preparation of cell culture based reaction pattern in vertebrate cell (RiV)-specific medicament, for e.g. treating tumor, HIV-infection, psoriasis and rheumatic diseases, comprises strongly heating RiV-preparation for ten minutes - Google Patents

Abstract

Preparation of cell culture based reaction pattern in vertebrate cell (RiV)-specific medicament with high effect, controls against foreign virus, comprises strongly heating a RiV-preparation preferably for 10 minutes at 100[deg]C. ACTIVITY : Cytostatic; Virucide; Immunostimulant; Anti-HIV; Antirheumatic; Antiallergic; Antipsoriatic. No suitable data given. MECHANISM OF ACTION : None given.

Description

The This invention relates to drugs produced from stressed cell cultures for the therapy and prophylaxis of cancer and viral diseases and Process for their preparation.

The state of the art corresponds to medicaments for the treatment of cancerous, viral and other diseases based on biocomplex (BK) reaction patterns in vertebrate cells (RiV) or preparations containing RiV-specific components (US Pat. DD 228739 . DD 283330 , Utility model G9218127.9 . EP 0702955 . DE 19728323 A1 ). The term "reaction patterns in vertebrate cells" was coined in 1986 after extensive electron microscopic examinations (Solisch, P. 1986, Zentralbl.Allg.Pathol.Pathol, Anat., 131, 49-57) .The morphologically characteristic components are approximately 50 nm RiV particles in addition to smaller RiV-specific components / subunits, a reaction product of the cell on stress.

The may be mentioned et al for prophylaxis and therapy of tumors, HIV infections, rheumatic diseases, allergies, psoriasis and in combination be used with chemo- and radiotherapy. Draw the preparations This is because they have no or very minor side effects have them in almost all ultima ratio cases (about 90%) to a clear improved quality of life cancer patients not only through pain reduction or ablation (even in morphine-resistant pain conditions), but also life-prolonging and mentally lightening. In animal experiments, the tumor growth inhibiting Effect can be detected (Solisch et al., (1987) Zentralbl. Allg. Pathol. Pathol. Anat. 133; 293-298 and illustration, see below).

In the BK-RiV preparations, the proteins Annexin I and V, 14-3-3ζ, 14-3-3ε, galectin 3, S100A2, S100A4, glutathione-S transferase, Nm23, aldolase, gCAP39, myosin (light Chain), actin, vimentin, alpha-actinin, histone 2B, histone 2A, ribosomal protein L7, nucleolin, HSP27, HSP70, proteasome C6, proteasome (6), proteasome (I), ezrin, ER60, PDI, enolase, natural killer enhancing factor, and stratin-homologous protein (about 50 kD) and an annexin I isoform (H156Y). In addition, it has been shown that non-viral RNA is an essential component of BK-RiV preparations ( DE 197 28 323 A1 ).

The preparation of previous drugs based on BK-RiV is described essentially by the following procedure:
Cultivation of primary, diploid or indefinitely growing vertebrate cells, stressing the cells to enrich for BK-RiV-specific components, in particular 50nm RiV particles, preferably by starvation stress, after cell disruption separation of larger cell debris, ultracentrifugation and short-term solvent treatment of the resuspended sediment, recovery of aqueous phase after low-speed centrifugation, if necessary, expulsion of dissolved solvent by means of inert gas and subsequent testing for identity and quantity, sterility, harmlessness, etc. according to the European Pharmacopoeia. In contrast to the above variant of the workup, the ultracentrifugation and solvent treatment can also be dispensed with, depending on the application or intended indication, ie, after cell disruption, only a removal of larger cell debris takes place. Optionally, an ultrafiltration can be carried out.

Unwanted side effects So far also came after several months and several years of application of 5ml each in the. in 14 days intervals not for observation (Solisch, P., Liebermann, H., Wazel, W., Poster CLI 09, Annual Meeting of Ges. F. Virology, Berlin, March 2003).

A disadvantage of the above method is the relatively low effectiveness. The hitherto known work-up processes for the production of BK-RiV drugs (inter alia utility model G 92 18 127.9 ; DE 197 28 323 A1 ) also have the disadvantage that potential foreign viruses are not depleted, but are enriched more or less quantitatively depending on the size or sedimentation by the ultracentrifugation or the respective concentration method. Only the solvent treatment (usually short chloroform treatment) leads to inactivation of enveloped viruses. "Naked" viruses are usually not or little inactivated EP 0702955A1 Although it is stated by Solisch and Bergmann (medicines for the treatment of AIDS) that the BK-RiV preparations can be inactivated "by methods known per se" (eg thermally in a water bath, chemically with formalin) ", but not only no concrete A common means of viral inactivation is the heating to 56 ° C for a period of 30 min, see, inter alia, Virological Methods Bd.2, Mayr Bachmann Bibrack Wittmann However, depending on the virus still residual infectivity may occur, especially the inactivation temperature or the thermal inactivation point for a virus, ie the temperature which is necessary to inactivate the virions under predetermined conditions and within a given time, for certain Viruses and virus groups is charactristic and therefore with their Cha is used (Schuster, In: BI Encyclopedia Virology, Ed. E. Geissler, Bibliographic Institute Leipzig 1986).

The The present invention aims at the efficacy of BK-RiV and analog Preparations essential to increase and the risk of contamination with viruses or other pathogens in the To avoid or significantly reduce pharmaceuticals.

Farther The purpose is the shelf life / shelf life of the drug, especially in liquid form.

It has been found, quite surprisingly, that the therapeutic efficacy of BK-RiV preparations significantly increases when the drug prepared according to already proposed methods (Utility Model G9218 127.9 ) is briefly subjected to heat treatment for 10 minutes at 100 ° C.

• 1. Durch die kurzzeitige Hitzebehandlung erhöht sich die Wirksamkeit des Arzneimittels bei Krebsbehandlung signifikant. Das BK-RiV-spezifische Arzneimittel, das hergestellt wurde nach dem im Gbm G 92 18 127.9 angegebenen Verfahren, beinhaltend im wesentlichen die Schritte Kultivierung von primären Vertebratenzellen, Stressung der Zellen zwecks BK-RiV-Partikeln, Abtrennung größerer ZeAnreicherung von lltrümmer, Ultrazentrifugation und kurzzeitige Lösungsmittelbehandlung ( Chloroformbehandlung) des resuspendierten Sediments, zeigte, nachdem es erst 10 min. gekocht wurde, überraschendennreise einen signifikant besseren tumorwachstumshemmenden Effekt als die nichtgekochte Vergleichsprobe. So betrug die mittlere Tumormasse im Versuch mit Balb c-Mäusen und transplantierten J 558L (mouse balb/c myeloma) Zellen nur 54% im Vergleich zur Kontrolle mit physiologischer Kochsalzlösung. Die Tumormasse nach der Behandlung mit unbehandeltem BK-RiV-Präparat betrug hingegen 72%, siehe 1 und Legende dazu. Der Unterschied zwischen den 3 Mittelwerten der Tumormassen ist mit 5% (p≤0,05) und auch noch mit 1 % Irrtumswahrscheinlichkeit signifikant. Es wurden bei beiden Gruppen keinerlei Nebenwirkungen festgestellt. Offenbar führt diese Hitzebehandlung zur Inaktivierung von Substanzen, die entweder inhibitorisch auf die das Tumorwachstum hemmenden Proteine oder Komplexe wirken oder selbst einen schwach tumorwachstumsfördernden Effekt haben.
• 2. Durch die kurzzeitige Hitzebehandlung (vorzugsweise 10 min. kochen) werden eventuelle Fremdviren mit sehr großer Sicherheit inaktiviert. So ist bekannt, dass eine Wärmeeinwirkung von 80°C direkt am Viruspartikel kein Virus im natürlichen Milieu länger als einige Minuten übersteht, ausgenommen die Parvo- und Papovaviren. Kochen zerstört die Infektiosität aller Viren (Büttner, M. Allgemeine Virologie, S. 84. In: Medizinische Mikrobiologie, Infektions- und Seuchenlehre, Hrsg. Anton Mayr. Enke Verlag Stuttgart, 7. Auflage 2002). So ist Maul- und Klauenseuchevirus bei 80 bis 100°C-Behandlung sofort inaktiviert (Handbuch der Virusinfektionen bei Tieren; Hrsg. H. Röhre; Fischerverlag Jena 1967, S. 84 ff.) Hinzu kommt die Inaktivierung von vegetativen Formen von Bakterien und Pilzen, die normalerweise in unserer Umwelt vorkommen. Eine chemische Inaktivierung hätte den Nachteil, dass z. B. bei Einsatz von Formaldehyd die Inaktivierungskurve keine Reaktion 1. Ordnung sein kann, wie dies unter anderem vom MKS-Virus bekannt ist, bzw. bei anderen Inaktivierungsmitteln die Handhabung problematischer ist und außerdem für jedes Virus erst die optimalen Inaktivierungsbedingungen gefunden werden müssen. Weiterhin ist durch die chemische Inaktivierung eine Reduktion der Wirksamkeit des Arzneimittels wahrscheinlich.
• 3. Durch die Hitzebehandlung kommt es auch zu Inaktivierungsprozessen von (noch) vorhandenen Proteasen. Die Reduzierung möglicher Proteaseaktivitäten wiederum führt zu einem längeren Erhalt der Wirksamkeit des Arzneimittels insbesondere dann, wenn es in flüssiger Form bei Kühlschranktemperatur aufbewahrt werden soll.

The medicament according to the invention has the following advantages:

• 1. Short-term heat treatment significantly increases the efficacy of the drug in cancer treatment. The BK-RiV-specific drug that was prepared according to the in Gbm G 92 18 127.9 The following methods, comprising essentially the steps of culturing primary vertebrate cells, stressing the cells for BK-RiV particles, separating larger Ze enrichment of lltrümmer, ultracentrifugation and short-term solvent treatment (chloroform treatment) of the resuspended sediment, showed, after only 10 min. was cooked surprisingly, a significantly better tumor growth inhibitory effect than the non-cooked control sample. Thus, the mean tumor mass in the experiment with Balb c mice and transplanted J 558L (mouse balb / c myeloma) cells was only 54% compared to the control with physiological saline. The tumor mass after treatment with untreated BK-RiV preparation was 72%, see 1 and legend about it. The difference between the 3 means of tumor mass is significant at 5% (p≤0.05) and also at 1% probability of error. No side effects were noted in either group. Apparently, this heat treatment leads to the inactivation of substances that either inhibit the tumor growth-inhibiting proteins or complexes or even have a weak tumor growth-promoting effect.
• 2. Due to the short-term heat treatment (preferably boil for 10 min) any foreign viruses are inactivated with great certainty. It is known, for example, that a heat exposure of 80 ° C directly to the virus particle does not survive a virus in the natural environment for more than a few minutes, with the exception of the parvoviruses and papovaviruses. Cooking destroys the infectivity of all viruses (Büttner, M. General Virology, p. 84. In: Medical Microbiology, Infection and Epidemiology, ed. Anton Mayr Enke Verlag Stuttgart, 7th Edition 2002). So foot-and-mouth disease virus is inactivated immediately at 80 to 100 ° C treatment (Handbook of virus infections in animals, edited by H. Röhre, Fischer Verlag Jena 1967, p 84 ff.) In addition, the inactivation of vegetative forms of bacteria and fungi that normally occur in our environment. A chemical inactivation would have the disadvantage that z. B. in the use of formaldehyde, the inactivation curve can not be a 1st order reaction, as is known, inter alia, the FMD virus, or in other inactivating the handling is more problematic and also for each virus first optimal inactivation conditions must be found. Furthermore, chemical inactivation is likely to reduce the effectiveness of the drug.
• 3. The heat treatment also leads to inactivation processes of (still) existing proteases. Reduction of potential protease activities, in turn, results in prolonged maintenance of the drug's efficacy, particularly when it is to be stored in liquid form at refrigerator temperature.

embodiments

• 1. calf kidney cells will be in MEM with 10% FCS (from BSE-free land) to confluency cultivated in roll culture bottles, then there is a change of medium and the cells are maintained in maintenance medium (EM) (MEM) without serum addition held. Then the cells are supernatant with or without cell culture harvested, the cells are disrupted (sonication, repeated freezing or osmotic shock) and after a low-speed centrifugation the supernatant so clarified a high-speed centrifugation (44000 g 3h at 4 ° C) subjected. The thoroughly resuspended in 1/30 volume of PBS (phosphate buffered saline) Sediment is shaken with 5% by volume of chloroform for 1 min, low-speed centrifuged and the aqueous Phase carefully removed and cooked for 10 min. The whole process takes place under sterile conditions.
• 2. In contrast to Example 1 takes place in the Stress phase additionally a heat stress (43 ° C for 30 min) followed by several hours of incubation at 37 ° C.
• 3. Instead of the chloroform treatment in Example 1 is a Arctone or an ether treatment performed.
• 4. Instead of the solvent treatment of the resuspended sediment after ultracentrifugation the sonicated resuspended sediment was only clarified.
• 5. In contrast to Example 1 takes place after cell destruction Cook for 10 minutes and then one Clarification process (membrane filtration through a 0.45 μm filter or a low-speed centrifugation). The order (cooking and Klärprozeß) can also be reversed. This BK-RiV-specific preparation can still be about 10-fold concentrated by ultrafiltration (cutoff limit, e.g., 100 kD) become.

The in the examples 1 and 5 indicated heat treatment can under Such temperature and time conditions occur, both for Inactivation of concomitant substances affecting the effectiveness of the drug reduce, as well as for safe inactivation ev. Existing Viruses (foreign viruses) leads.

The exams on sterility, Toxicity, pyrogenicity is done according to European Pharmacopoeia.

Treatment of patients with the cell culture-based drug "BK-RiV heat-treated" (BK-RiVh):
The term "BK-RiV heat-treated" is to be understood as meaning the preparation which has been incubated preferably at 100 ° C. for 10 minutes and which is used as medicament.

BK-RiVh can be used intramuscularly according to the following schedule in cancer patients (adults) in monotherapy:
On the first day 1-2 ml; on the 2nd day 2-3ml; on the 3rd day 2ml; after a week's repetition, then every week 2-3ml or 14-day 5ml.

All 2 days 3ml were also well tolerated by a patient.

An overdose has not been determined yet. If the therapy is in combination with a chemo- or radiotherapy especially for reduction the side effects of these therapies, treatment with BK-RiVh should preferably offset in time, i. from the 3rd day after the chemo- or Radiotherapy begin or take place in the breaks of these therapies.

Claims (4)

A method for producing a cell culture-based RiV-specific drug with high efficacy and safety against foreign viruses, characterized in that the RiV preparation is heated strongly, preferably for 10 min at 100 ° C. Process for the preparation of a medicament according to Claim 1, characterized in that the strong heating for 1 to 50 min. at 80 to 100 ° C or under such conditions before or after any step the workup after cell disruption takes place (heat and time) that possible foreign viruses and / or vegetative forms of normally occurring in the environment Bacteria and fungi are safely inactivated and the therapeutic Effectiveness greater or equal to the effectiveness of the untreated sample. Use of the medicament prepared according to claim 1, for the treatment, metaphylaxis and prophylaxis of tumor and viral diseases. Use of the medicament prepared according to claim 1, to improve the body's own Defense of injurious influences in humans and animals.