DD275692C2 - PROCESS FOR THE PREPARATION OF CYTOKIN (INERTFERONE) INDUCTORS AND ACTIVATORS OF NATURAL KILLER CELLS - Google Patents

Description

y = 0 to 3 and

ζ = O to 3

Unipolymerisate: y = ζ = O

Bipolymerisate: B ₁ = B ₂

Terpolymer: B ₁ Φ B ₂

to be obtained.

2. The method according to claim 1, characterized in that the uni-, bi- and terpolymers are prepared by Y-induced polymerization.

3. The method according to claim 1, characterized in that the uni-, bi- or terpolymer strand and the complementary polyribonucleotide are thermally denatured prior to hybridization.

4. The method according to claim 1, characterized in that left-modifications of the nucleobases in the uni-, bi- and terpolymers have functional groups with hydrophobic and polar interactions, ionic and coordinative bonds and hydrogen bonds to the neighboring strand.

5. The method according to claim 1 and 4, characterized in that the N ⁴ -linker of the 1-vinylcytosine monomer units have a length corresponding to the interaction with the complementary nucleobase or Ribosephosphatmustern.

6. The method according to claim 1,4 and 5, characterized in that for the left with hydrophobic and polar interactions hydroxy-substituted alkyl groups such as the 2,3-dihydroxy-1-propyl group (dhp) find use.

7. The method according to claim 1,4 and 5, characterized in that for the left with ionic and coordinative bonds preferably polyamine groups such as the diethylenediamine group (dd) find use.

For this, if pages drawings

Field of application of the invention

The invention relates to a process for the preparation of agents for cytokine (interferon) induction and activation of natural killer cells. These agents are useful in basic medical research, in human and veterinary medicine, and as crop protection agents and biological process control agents.

Characteristic of the known state of the art

Cytokines are antiviral (glyco) proteins released by a variety of low and high molecular weight inducers in cellular systems (DAStringfellow, Interferons and Interferon Inducers, Marcel Dekker Inc. of New York and Basel, 1980, S.Hoffmann, Z. Chem. 22 (1982) 357-367).

Among the inducers, the double-stranded ribonucleic acids (dsRNAs), and especially the polyinosinic acid polycytidic acid [(I) _n (C) _n ] complex, have long been known (PFTorrence, E. De Clercq, Pharmac (197711-88).

The cytokine (interferon) induction in human Lymphozytensuspensionon by complexes of homo- and copolymers of 1-vinylcytosine with complementary polyribonucleotides, called Semiplastics inducers, provide a favorable alternative to induction by enzymatically unstable as well as mitogenic (I) _n (C) "- double strands (Pitha, J. Pitha, Science 172 [1971] 1146-1148; K.Waschke, S.R.Waschke, S. Hoffmann, W. Witkowski, Acta biol. med. germ. 38 (1979) 739-744). The hybrid complexes of (I) _n with the homo- or copolymer of 1-vinyl cytosine show a good antiviral activity and inducing potency. Increases in activity of natural killer cells have hitherto only been described by dsRNAs (RH Wiltrout, RR Salup, TATwiley, JETalmadge, Journal of Biological Response Modifiers 4 (1985) 512-517) Disadvantages of the Use of Previous Low and High Molecular Weight Cytokine {Interferon) Inductors and Activators of natural killer cells are lack of thermal stability, different pathways of action, hyporeactivity, different in vivo and in vitro efficiencies, lack of species breadth, sometimes dramatic mitogenicity, phagocytosis and other toxic side effects.

Object of the invention

The object of the invention are agents that potentiate antiviral activities with reduced application rate and reduced toxicity by stimulating cellular systems to increase the production of cytokines (interferon) and to increase the activity of natural killer cells, while suppressing undesired activities.

Explanation of the essence of the invention

The object of the invention is to improve the previous stability of semiplastic Cytokindnferferlinduktoren by the incorporation of left-modified Nucelobasen with increased affinity for complementary polyribonucleotide crucial and by suitable hybridization techniques for the cytokine (interferon) inductions and the activation of natural killer cells required secondary and Tertiary structure education.

According to the invention, semiplastic cytokine (interferon) inductors with increased interferonogenic and reduced mitogenic activity are obtained, while at the same time significantly increasing the activity of natural killer cells as semiplastics of general formula T.

B. B-R

I ¹ I ² I ₁

PoIy (I) tfVf ^ 7

in which:

Bi = left-modified nucleobase B] = left-unmodified nucleobase Nucleobases = adenine, 2-aminoadenine, cytosine, guanine, iso-guanine, thymine, uracil R = pyrrolidin-2-one-i-yl, -COOH, -CONH ₂ , -COO (alkyl), -NH ₂ , -NH (alkyl), - N (alkyl) ₂ , -OH, -O (alkyl) with alkyl: methyl, Ethyl, propyl, isopropyl

x, y, ζ = monomer content of the polymer with

y = 0 to 3 and

Unipolymerisate: y = ζ = O Bipolymerisate: B | = B ₂ Terpolymer: B) Φ B ₂

The left-hand modifications of the nucleobases in the mono-, bi- and terpolymers preferably have functional groups which bring about stabilization of the secondary structure of Semiplastics due to hydrophobic and polar interactions, ionic and coordinate bonds as well as hydrogen bonds with appropriate spacer dimensioning to the neighboring strand ,

As N ⁴ -linker moieties of the 1-vinylcytosine monomeric units having hydrophobic and polar interactions, hydroxy-substituted alkyl moieties such as the 2,3-dihydroxy-1-propyl group (dhp), as opposed to co-laminative and ionic bonds, preferably find polyamine moieties such as for example, the diethylenediamine group (dd) use (Figures 1 ac).

The uni-, bi- and terpolymerizations of the monomers are prepared by γ-induced polymerization. Linker modifications and proportions in the homo- and copolymers are chosen to achieve sufficient water solubility and optimal binding to the complementary polyribonucleotide. The homo- and copolymers themselves are thermally stable up to 25O ⁰ C and orfahren until above this temperature a slow ·) decomposition. The semiplastics are obtained according to the invention by hybridization of the left-modified polymers with (I) _n after complete complete thermal denaturation. For this purpose, the corresponding equimolar amounts are reacted in a salt-free aqueous solution under sterile conditions. The concentration of the partner strands is determined by

Measurement of the wavelength of UV absorption. In the example, the solution of (I) _n after previous thermal denaturation for 15 minutes at 50 ⁰ C with the corresponding Mquimolaren solution of the uni-, bi- or tarmerizate mixed, cooled rapidly to 4 ⁰ C and 14 days at 4 ⁰ C. kept. This is followed by a filling of the sample solution to the physiological Salzkorvzentration of 0.01 M phosphate and 0.15 M NaCl (pH 7.15) using a 10-fold PBS buffer (0: M phosphate and 1.5 M NaCl) under sterile conditions.

The melting curves of Semiplastics (FIG. 2) prove the multiple-stranded character of the hybrids. The increase comparable to (I) _n * (C) _n indicates a very good cooperative. All new complexes have a melting point range above 20 ° C. Due to this low melting range, cytokine (interferon) induction or activation of natural killer cells can only take place in the nonphysiological temperature range around 20 ° C. An induction period of 2 hours at 20 ° C for the subsequent cytokine or Intzerferoninduktion and the activation of natural killer cells at 37 ⁰ C is completely sufficient. Fixations of these semiplastics to polymeric supports provide opportunities for extracorporeal cytokine (interferon) induction.

The semiplastics prepared according to the invention are characterized in mononuclear human leukocyte suspensions by an exceptionally high cytokine (interferon) induction potency t.us (Table 1). Compared to the mitogenic dsRNAs their mitogenicity is significantly reduced in presence during the entire induction period. Merely pretreating the lymphocyte suspension sufficient for interferon induction does not result in a mitogenic effect. Con A-stimulated lymphocyte transformation is also unaffected (Table 2). The activity of the natural killer cells is significantly increased after pretreatment with these complexes and even exceeds that of (I) _n (C) _n for (I) _n (a) (FIG. 3). The phagocytosis performance of monocytes, as the major cyfokin (interferon) producer, is not affected in any way (Figure 4).

Table 1: The influence of semiplastic inducers on interferon production in human mononuclear leukocyte suspensions

inductor Concentration ImM) Interferon titer (IU / ml) (D _n - (C) _n (D _n - (A) n (L) n- (b) "(Dn (O _n 0.15 0.27 0.33 0.10 3400 1250 250 4600 Table 2: The Influence of semiplastic en inducers to the lymph inductor Concentration ImM) Stimulation rate I Il III

without - - - 25 (D _n - (C) _n 0.15 1.0 2.8 32 (Dn-Ia) _n 0.27 1.0 4.1 34 (D _n - (b) " 0.33 1.0 2.4 32 (Dn- (O _n 0 "0 1.0 1.4 27

I: Pretreatment with inducer II: Permanent Inductor III: Stimulation by Con-Α after pretreatment with the inducer

Exemplary embodiments Preparation of N 1 -langer-modified 1-vinylcytosine

1. 1 -Vinyl ^ - ^. S-dihydroxy-i-propyl] amino-2-pyrimidinone (vC ^hp )

0.50 g (2.98 mmol of 1-vinyl-4-methylmercapto-2-pyrimidinone are dissolved with 0.55 g (6.00 mmol) of 1-aminopropanediol (2.3) in 10 ml of absolute ethanol, refluxed with stirring for 5 hours , then cooled and left overnight in the refrigerator.The solid which is concentrated by evaporation and concentrated by ether is filtered off with suction and washed several times with small amounts of cold absolute ethanol and ether

Yield = 0.53 g (83.5%) m.p. = 163 ⁰ C (decomposition)

2. 1-Vinyl-4- (3,6-diaza-hexyl] -amino-2-pyrimidinone (vC * ¹ )

0.50 g (2.98 mmol) of 1-vinyl-4-methylmercapto-2-pyrimidinone are dissolved with 0.62 g (6.00 mmol) of diethylenetriamine in 10 ml of absolute ethanol, refluxed for 5 hours with stirring, then cooled and overnight in

Leave refrigerator. The solid which has separated out after concentration and ether is filtered off with suction and washed with water for a period of time

washed small amounts of cold absolute ethanol and ether.

Yield = 0.31 g (46.7%)

Melting point = 79-80 ° C The compounds are highly hygroscopic and should be stored under vacuum in Nj atmosphere. polymerization

1. Unipolymerization of vC ^{ie |>} to [VC ¹¹¹ P) _n :

0.21 g (1.00 mmol) of vC ^dhp is dissolved in 2.5 ml of DMSO and 0.5 ml of 1 η HCl and melted in an ampoule under argon. The

Polymerization takes place at 16'C for 8 hours with a radiation dose of 90KR / h, which corresponds to a total dose rate

of 0.72 MR. After irradiation, the solvent is removed, the polymer is boiled several times with small amounts of acetone and connected ^ dried in the drying gun.

Yield = 0.15g

Melting point greater than 250 ° C (decomposition)

2. Bipolymer Analysis of vC ^dhp with 1-Vinylpyrrolidin-2-one:

0.21 g d.OOmmol) vC ^dhp and 0.11 g (1.00 mmol) of 1-vinylpyrrolidin-2-one (vPyrro) are dissolved in 5 ml of methanol and 1 ml of 1 η HCl and melted into an ampoule under argon. The polymerization is carried out at 15 ⁰ C for 8 hours with a Schrahlendosis of 90KR / h, corresponding to a total dose rate of 0.72 MR. After irradiation, the solvent is stripped off, the polymer is boiled several times with small amounts of acetone and then dried in the drying gun

 Yield = 0.26g

Melting point greater than 25O ⁰ C (decomposition) Monomer ^ratio in the polymer: vC ^dhp : vPyrro = 1: 0.9

3. terpolymerization of vC ^{dd with} vPyrround 1-vinylcytosine: _f

0.10g (0.45mmol) vC * ¹ , 0.35g (3.15mmol) vPyrro and 0.35g (2.55mmol) 1-vinylcytosine (vC) are dissolved in 5ml DMSO and 1ml 1 η HCl, with argon rinsed and melted in an ampoule. The reaction takes place at 15 ° C for 8 hours \ with a radiation dose of 90KR / H, this corresponds to a total dose rate of 0.72 MR. It is then filled with acetone, boiled several times with small quantities and dried in the drying gun.

Yield = 0.10g

Melting point greater than 25O ⁰ C (decomposition)

Monomer ratio in the polymer: vCivC ^ ivPyrro = 1: 0.5: 1.9 Hybridization of the interferon inducers

From the uni-, bi- and terpolymers stock solutions are prepared with sterile water. These stock solutions are subjected to a gravity field in an ultracentrifuge at 100,000G to remove interfering bacteria and fungi. For the hybridization of the dsRNA analogues, use is made of the sterile supernatants of the precipitates. The (I) _n stock solution is incubated for 15 minutes at 5O ⁰ C. During this time, the secondary structure of the homopolyrl nucleotide breaks down. It is then cooled rapidly to 4 ⁰ C and mixed with the equivalent of the same pretreated Uni, Bi- or Terpolymerlsat stock solution. The storage of these entirely salt-free solutions is carried out at 4 ⁰ C. After two weeks, the sample solution by means of a 10-fold PBS buffer to the physiological salt concentration of 0.01 M phosphate and 0.15 M NaCl (pH 7.15) is placed

 Induction of interferon

Humsne mononuclear leukocytes are prepared from the venous blood of healthy donors according to the principle of B0YUM (A. B0yum, Scand J. Immunol.5 [1976] 9-15) by density gradient centrifugation, washed twice with PBS, and finally in RPMI 1640 supplemented medium of 10% fetal calf serum. This cell suspension contains an average of 90% lymphocytes and 10% monocytes.

For induction of interferon, 1.5 × 10 ⁷ cells per ml are incubated for 2 hours at 20 ° C. in the presence of the inducers, then centrifuged at 800 × g for 10 minutes, the supernatant is removed and the cells are resuspended in fresh medium. This is followed by a further incubation of 20 hours at 37 ^° C. in glass bottles made of glass. Thereafter, the lymphocytes not adhering to the glass surface are removed and centrifuged at 800 χ g for 10 minutes. In the resulting cell-free supernatants, the interferon concentration is determined. The lymphocytes are resuspended in fresh medium and used in the lymphocyte transformation assay (LTT), as well as to determine the activity of natural killer cells. The gold-adherent monocytes in the culture flasks are washed twice with PBS and used to determine phagocytosis activity.

Non-inducer treated cells are incubated and processed in parallel. They serve for the control regulations. Determination of interferon

Microtiter plates (flat bottom) are used to grow monolayers of human, dumb-on fibroblasts. These cells are incubated with serial dilutions of the Interferonhaitigen material (volume 200 μΙ) at 37 ⁰ C for 24 hours, then with 100 pfu (plaque forming units) of vesicular stomatitis virus. After further incubation at 37 ⁰ C of 24 hours duration, the microscopic assessment of CPE (Cytopathogenic effect) takes place in the individual approaches. The interferon value is the reciprocal of the highest dilution of interferon-containing material at which the CPE is at least 50% prevented. In parallel with each determination approach, a standardized interferon preparation (calibrated in International Units = IE) is determined. The specific endpoint of the titration of this internal standard is used to calculate the titres of unknown samples in IE

 Lymphocyte Transform Ratio Test (LTT)

Pretreated with the interferon inducers lymphocytes are incubated C for 3 days in microtiter plates (2 x 10 ⁶ cells per 200μΙ approach) at 37 ^0, parallel, aliquots of this pre-treated cells in the presence of 20 mg / ml concanavalin A (Con A). 16 hours before the end of the incubation period, an addition of 37 kBq 'H-thymidine to mark the cells. The incorporation of tritium into the precipitate, insoluble in trichloroacetic acid, which represents the DNA of the cells, is determined by scintillation spectrometry in Ultrabeta (LKB-Wallac, Sweden).

As a measure of lymphocyte transformation, the stimulation rate of DNA synthesis in the cells. It is the mean ratio of pulses (cpm = counts per minute) of stimulated cells to those of unstimulated controls in triplicate determinations.

Determination of the activity of natural killer cells (NK cells)

(K.Winkke, R. v.Baehr, G. Wolf, Allerg Immunol 33 (1987) 27-34)

As "targets" of the cytotoxic activity of NK cells serve cells of the line K562 (C. B. Lozzio, B. B. Lozzio, Blood 45 (1975) 321-327). They are incubated with 5 MBq of Na ₂ ⁵¹ CrO ₄ at 37 ^° C. for one hour, washed three times with PBS, then

suspended in RPMI 1640 medium and labeled in this way.

The test kits contain 4 x 10 ⁴ labeled K562 cells mixed with 2 x 10 * lymphocytes {corresponding to a ratio

of 1:50 targets to effector cells) suspended in 1 ml of RPMI 1640 medium supplemented with 10% fetal calf serum. After prior centrifugation at 200 χ g of 6 minutes duration, incubation is carried out for 4 hours at 37 ⁰ C. Thereafter

after centrifugation for 5 minutes at 800 χ g of 500 μΙ aliquot taken from the cell-free supernatants to determine the activity of the Cr released from the targets.

The expression of cytotoxicity is the proportion of released ⁶¹ Cr from the specifically lysed targets:

./ ·, ., > T! .A. cpm test batch - cpm spontaneous

% Cytotoxicity = - _: ---

cpm total - cpm spontaneous

(cpm = pulses per minute)

The determination of the spontaneous release of ³¹ Cr is carried out in batches of target cells without Lyrnphozyten, parallel to the Test approaches are treated. The total radioactivity is expressed in corresponding allotments of suspended targets (4 χ 1O ⁴ AnI)

certainly.

The different individual NK activity of individual donors for the LympnozytonprSparationen makes the application of the Terminus of "relative cytotoxicity" to compare the Induktorwirkung between the individual test approaches necessary. It corresponds to the ratio of the percentage cytotoxicity in interferon-inducible treated samples to that in

untreated control approaches of an individual assay.

Determination of phagocytosis

(K.Winkke, J. Bellach, R. v.Baehr, Z. Klin.Med. 41, No. 21 (1986) 1741-1744)

The bacteria E. coli 075: K95: H 5 serving as phagocytosis objects are incubated in a nutrient broth (pancreatic peptone 20 g / l, NaCl 5 g / l, glucose 1 g / l) with fresh air at 37 ⁰ C freshly grown overnight, centrifuged (2CH) xg, 5 min.),

washed twice with PBS and finally in RPMI 1640 medium supplemented with 10% native human AB serum (for

Opsonlerung mittete complement) suspended. The glass-adherent monocytes in the culture bottles are mixed with 4 ml of this bacterial suspension, 10 ⁷ bacteria per ml

containing, overlaid. The ratio of monocytes to bacteria is 1:50. During incubation at 37 ⁰ C the removal of aliquots (100μΙ) is carried out at the start and continue at intervals of 20 minutes for the determination of the extracellular (not phagocytosed) bacteria in a standard colony assay.

The number of bacteria is expressed in the form of colony-forming units: CFU (colony forming units). The bacterial growth occurring during the incubation period is determined in parallel runs without monocytes.

Claims (2)

1. A process for the preparation of high molecular weight Cytokindnterferonjinduktoren and activators of natural killer cells, characterized in that semiplastics of polyinosinic acid [PoIy (I) or (I) _n ] and a potentially bioactive, partially sequence and stereoregulär uni-, bi- or terpolymer with partially or completely linker-modified nucleobases of the general formula 1: I ¹ ι ² ι I ¹ ι ² PoIy (I) · Poly [> CH; f VT 2 'X * ^CH i in which: B ₁ = left-modified nucleobase B ₂ = left-unmodified nucleobase, Nucleobases = adenine, 2-aminoadenine, cytosine, guanine, iso-guanine, thymine, uracil R = pyrrolidin-2-one-i-yl, -COOH, -CONH ₂ , -COO (alkyl), -NH ₂ , -NH (Alkyl), -N (alky!) ₂ , -OH, -O (alkyl) with alkyl: methyl, ethyl, propyl, isopropyl x, y, ζ = monomer content sm polymer with