HU221497B1 - Antisens hiv splice donor oligodeoxynucleotides, containing 5-(n-alkin-1-yl)-pyrimidine bases, inhibiting hiv-proliferation, process for producing medicaments containing the same and the medicaments - Google Patents

Abstract

SUMMARY OF THE INVENTION The present invention relates to oligodezoxynucleotides having a nucleotide sequence of 17 to 26 nucleotides, the nucleotide sequence of which is complementary (antisense) to the nucleotide sequence of the 275-305 nucleotide portion of the HIV viral RNA splice donor portion, and in which oligodeoxynucleotides are 4- (5 n-n-chain) instead of thymine bases. -alkin-1-yl) -uracil, preferably 5- (n-hexin-1-yl) -uracil, or, instead of cytosine bases, (C5-C10) 5- (n-alkin-1-yl) cytosine, preferably 5 - (n-hexin-1-yl) -cytosine, instead of any pyrimidine base, 5- (n-alkin-1-yl) -pyrimidine (4-10 carbon atoms), preferably 5- (n-hexin-1-yl) -pyrimidine and in the azoligodoxynucleotides the nucleotide linking group is a phosphorus diester or thiophosphoric diester. These antisense oligodeoxynucleotides have a significant anti-HIV activity in vitro in MT-4 cell culture and can therefore be used as a drug active agent for the treatment of HIV infections. ŕ

Description

The present invention relates to oligodeoxynucleotides having 17-26 nucleotide units whose nucleotide sequence is complementary (antisense) to the human immunodeficiency virus (HIV) RNA splice donor portion 275-305. which in the oligodeoxynucleotides is 5- (nalkin-1-yl) -uracil, preferably 5- (n-hexin-1-yl) -uracil, instead of the thymine bases, or 4-10 instead of the cytosine bases. C 5 -alkyl 5- (n-alkynyl-1-yl) -cytosine, preferably 5- (n-hexin-1-yl) -cytosine, or instead of both pyrimidine bases

The C4-C10 alkyl chain is 5- (n-alkin-1-yl) -pyrimidine, preferably 5- (n-hexin-1-yl) -pyrimidine and the nucleotide linker in the oligodeoxynucleotides is a phosphorous diester or thiophosphoric diester. These antisense oligodeoxynucleotides have significant anti-HIV activity in vitro in MT-4 cell culture. The invention further relates to pharmaceutical compositions containing such compounds and to processes for the preparation of such compounds and pharmaceutical compositions. The present invention also provides the use of the above compounds for the preparation of a medicament for the treatment of HIV infections.

It is known that technology based on the design and application of so-called antisense oligonucleotides is the latest and most selective way of inhibiting gene expression. It also represents a new level of drug design. An antisense oligonucleotide is an oligonucleotide complementary to a particular region of the gene to be inhibited.

The gene to be inhibited may be, for example, a gene of a virus that is pathogenic to humans. Obviously, a particular region of a gene is a critical region of the gene, usually viral DNA or RNA or mRNA, which is sterically accessible to the antisense oligonucleotide and by binding to it will inhibit gene expression. Complementarity refers to the principle of Watson-Crick base pairing (pairs of adenine-thymine and guanine-cytosine). Antisense oligonucleotides are usually 15 to 30 nucleotides in length (see, for example, J. L. Tonkinson & C. A. Stein Antiviral Chem. &Amp; Chemother. 4: 193-200 (1993); Μ. K. Ghosh & J. S. Cohen Progress in Nucleic Acid Rés. Mole. Biol. 42: 79-126 (1992); or C. Héléne & J.-J. Toulmé Biochim. Biophys. Acta 1049, 99-125 (1992).]

Antisense oligonucleotides designed for therapeutic purposes must have the following characteristics:

1. High biological stability, that is, they must be resistant to nucleic acid-degrading enzymes, both in blood and in cells.

2. They must enter the cells at the site of action at a sufficiently high concentration, and

3. create a strong and long-lasting complex, called a duplex, with the intended nucleic acid sequence of the selected gene to achieve the effect.

If the antisense oligonucleotide enters the cells with the appropriate biological stability, at the right concentration, and produces a strong and durable duplex with the appropriate region of the selected gene, it can produce sequence-specific antisense effects through two so-called termination events:

1. The resulting duplex is stable enough to physically inhibit gene expression (e.g., protein synthesis on the mRNA is permanently blocked), and

2. If the resulting duplex is an RNA DNA called a hybrid duplex, i.e. the target gene region is an RNA (virus RNA or mRNA) and the antisense is a deoxy oligonucleotide, the RNA portion of this hybrid duplex is the endogenous RNase-H enzyme. hybrid duplex decomposition is the specificity, decomposes.

With the antisense oligonucleotides constructed from the natural nucleotides, both ribo- and 2'-deoxyribonucleotides, the above requirement is not fulfilled as their biological stability is very low. They have a serum half-life of less than one hour. Therefore, at present, only chemically modified, amplified oligonucleotides are used for biological purposes.

Since the spread of antisense technology, in the last 6 to 7 years, numerous chemical modifications have been described and applied (see, for example, P. Danish Cook Anti-Cancer Drug Design 6: 585-607 (1991) and E. Uhlmann & A. Peyman Chem. Port. 90, 543-584 (1990)]. Chemical modification of the phosphate and / or sugar moiety of the nucleotide units (Formula 1) naturally increases the stability to the nucleases, since these parts of the molecules are the attack sites of the enzymes and their immediate environment. Some of these modifications, such as nonionic oligonucleotides, which abolish the polyanion nature of the oligonucleotides, also increase cellular uptake. However, the vast majority of sugar-phosphate modifications reduce the tendency for duplex formation and the strength of duplex formation. A further disadvantage is that the above second terminating event does not occur with the nonionic and also with many sugar-modified oligomers which are RNA analogs.

In order to increase the antisense effect, chemical modification of the third component of the nucleotides, the heterocyclic bases, was limited until the early 1990s. It has been speculated that modifying these may be counter-productive and may reduce the antisense effect (see P. Dan Cook and Uhlmann and Peymann, supra). However, subsequent research to prevent or cure AIDS disease has shown that when Rev is able to bind to the Rev Response Element (RRE) of the HIV virus in some oligodeoxynucleotides, the phosphate moiety is converted to the thiophosphate moiety and the thymidine base (i.e., 5-methyl 5-propynyluracil base is incorporated in place of uracil base), the antiviral activity of the compounds observed in vitro is significantly increased (Biochemistry 33: 8391-8398 (1994); Antiviral Research 28, 28-91 (1995)]. The effect was attributed primarily to the incorporation of the propynyl group; conversion of the phosphate moiety to the thiophosphate moiety alone resulted in only a small increase in activity. However, in our experience, these compounds are nucleic

They are not sufficiently stable against algae and are therefore not sufficiently potent or long-lasting to be degraded by nucleases in the living organism.

SUMMARY OF THE INVENTION It is an object of the present invention to provide antisense oligodeoxynucleotides having anti-HIV activity which effectively inhibit the growth of HIV in human T cells without toxic side effects.

The present invention is based on the discovery that HIV RNA (for the complete nucleotide sequence of HIV RNA see, for example, Y. Li et al., J. Virol. 66, 6587-6600 (1992)], starting from nucleotide 290 of the splice donor region in the 5 'direction 7-15. and nucleotide 291 complementary to the 5 'to 14', i.e. 17 to 26 nucleotide (more particularly 275-299, 280-299, 283-299, and 280-296 and 280-305) sequences in the 3 'direction, antisense oligodeoxynucleotides constructed from natural nucleotides showed weak but measurable anti-HIV activity in MT-4 cell cultures that were infected with HIV-1 virus. It has now been found that the incorporation of C4-C10 n-alkin-1-yl groups at the 5-position of the pyrimidine bases in these oligodeoxynucleotides and / or the exchange of the phosphorus diester linking nucleotides to thiophosphoro diester the anti-HIV activity of the compounds increases. It has further been found that compounds containing C4-C10 n-alkynyl-1-yl groups at the 5-position of the pyrimidine bases are substantially more stable to nucleases than their corresponding propin-1-yl analogs, and thus have a substantially longer effect.

By way of example, splice donor RNA 280-299 is disclosed. sequence complementary antisense oligodeoxynucleotide IC ₅₀ -value of 63 pg / ml. When this oligodeoxynucleotide (a) is replaced with 5- (n-hexin-1-yl) ylcyl base instead of the three thymine bases, the IC ₅₀ is reduced to 38 pg / ml;

(b) replacing the nine cytosine bases with 5- (n-hexin-1-yl) -cytosine base, the IC _{50 being} reduced to 9 pg / ml;

(c) replacing all thymine and cytosine bases with 5- (nhexin-1-yl) uracil and 5- (n-hexin-1-yl) cytosine base with an IC _{50 of} 3 pg / ml; decreases;

(d) replacing the phosphorus diester linkages linking the nucleotides with thiophosphorus diester linkages, decreasing the IC _{50 to} 2 pg / ml, and at a concentration of 20 pg / ml in an MT-4 cell culture acutely infected with HIV-1 18. no HIV multiplication was observed for days;

(e) replacing the phosphorus diester linkages linking the nucleotides with thiophosphoro diester linkages, while introducing 5- (n-hexin-1-yl) uracil bases in place of the thymine bases with an IC _{50 of} 1.2 pg / ml and no growth of HIV was detected at 5 pg / ml in MT-4 cells cultured in acute infection with HIV-1 virus for 30 days from infection [by comparison, 5- (prop-1-inyl) uracil analogue inhibited HIV proliferation for only 15 days];

(f) replacing the phosphorus diester linkages linking the nucleotides with thiophosphoro diester linkages, and adding 5- (n-hexin-1-yl) cytosine bases in place of the cytosine bases with an IC _{50 of} 0.6 pg / ml and no growth of HIV was observed in MT-4 cell cultures of acute infection with HIV-1 virus at 5 pg / ml for 18 days after infection;

(g) replacing the phosphorus diester linkages linking the nucleotides with thiophosphoro diester linkages, while replacing the thymine bases with 5- (n-hexin-1-yl) uracil bases and the cytosine bases with 5- (n-hexinyl-yl) cytosine bases were added, the IC ₅₀ was reduced to 2 pg / ml and no HIV proliferation was observed in MT-4 cell culture acutely infected with HIV1 at 5 pg / ml for 18 days after infection;

(h) exchanging the phosphorus diester linkages linking the nucleotides with thiophosphoro diester linkages, and adding 5- (n-octin-1-yl) uracil bases in place of the thymine bases with an IC _{50 of} 2.5 pg / ml and at 5 pg / ml, no HIV multiplication was observed in MT-4 cell culture acutely infected with HIV-1 virus by day 22 of infection.

In contrast to the cytotoxicity observed with AZT as a control, the above modified antisense oligodeoxynucleotides up to 100 pg / ml did not show cytotoxic activity.

Thus, the present invention relates to antisense oligodeoxynucleotides comprising 17-26 nucleotide units having a nucleotide sequence of 7-15 nucleotides 5 'to 5' and 5 'to 14' nucleotides 291 to 291 of the splice donor region of HIV RNA. and wherein at least a portion of the pyrimidine bases in the oligodeoxynucleotides have 5 (C 4 -C 10) n-alkynyl-1-yl pyrimidine bases and / or the phosphorus diester groups linking the nucleotides have thiophosphoro diester groups. . These antisense oligodeoxynucleotides have significant and long-lasting anti-HIV activity in MT-4 cell cultures in vitro, without cytotoxic side effects.

In a preferred group of antisense oligodeoxynucleotides of the invention, thymine bases are substituted with 5- (C 4 -C 10) n-alkynyl-1-yl uracil bases.

In another preferred group of antisense oligodeoxynucleotides of the invention, the thymine bases are substituted with 5- (n-hexin-1-yl) uracil bases.

In another preferred group of antisense oligodeoxynucleotides of the invention, the cytosine bases are substituted with 5- (C 4 -C 10) n-alkin-1-yl cytosine bases, preferably 5- (n-hexin-1-yl) cytosine bases.

In another preferred group of antisense oligodeoxynucleotides of the invention, both the cytosine bases and the thymine bases have the appropriate 5- (C 4 -C 10) n-alkin-1-yl [preferably 5- (nhexin-1-yl)] bases. .

A further preferred group of antisense oligodeoxynucleotides of the present invention has the sequence complementary to the HIV viral RNA 275-299, 280-299, 283-299, and 280-296 and 280-305.

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The invention also relates to the preparation of the above antisense oligodeoxynucleotides. These compounds are prepared in a manner known per se by coupling the appropriate nucleotides in the appropriate order.

The present invention also provides pharmaceutical compositions comprising the above antisense oligodeoxynucleotides. These pharmaceutical compositions comprise the antisense oligodeoxynucleotides of the invention in combination with one or more pharmaceutically acceptable carriers and / or other excipients.

The present invention also provides the use of the above antisense oligodeoxynucleotides in the manufacture of a medicament for the treatment of HIV infections.

The invention further relates to a process for the preparation of pharmaceutical compositions comprising admixing the antisense oligodeoxynucleotides of the invention with non-toxic, inert, solid or liquid carriers, diluents and / or other excipients commonly used in the preparation of pharmaceutical compositions and conventional dosage forms for enteral or parenteral administration. Suitable carriers, diluents, or binders for these purposes include water, gelatin, lactose, sucrose, starch, pectin, stearic acid, magnesium stearate, talc, various vegetable oils, and glycols ( such as propylene glycol or polyethylene glycol).

Pharmaceutical excipients include preservatives, such as methyl 4-hydroxybenzoate, various natural or synthetic emulHIV RNA: 3'-r (CGCA1 299 ........

deoxy-antisense: 5'-d (GCG TAC TCA CCA GTC GCC GC) -3 ', with thiophosphoro diester linkages (s) and 5- (n-hexin-1-yl) uracil (instead of thymine) ( hU) modified 20-membered antisense deoxyoligonucleotide of formula (2):

5'-d (GsCsGs hUsAsCs hUsCsAs CsCsAs GshUsCs GsCsCs GsC) -3 '

A Biosearch 8700 DNA synthesizer was used to couple the 2'-deoxynucleotide units.

5'-O- (p, p'-Dimethoxy) trityl-5- (h-hexin-1-yl) -2'-deoxyoxyuridin-3'-O- (2-cyanoethyl) ) A glass container containing 0.03 M acetonitrile (water content <0.002%) in N, N-diisopropylphosphoramidite (DMT-2'-deoxy-n-hexin-1-yl-CED-phosphoramidite, Formula 3) was synthesized in the synthesizer. Attached to position 13 (U) In position 9-12, solutions of the 3'-phosphorus-N, N- (diisopropyl) amide of nucleosides with protecting groups containing natural bases were placed (9 = A, 10 = C, 11 = G, 12 = T) in the same concentration as above The 1-H-tetrazole used as the activating agent in the condensation reaction in the tank at position 8 is in 0.47 M acetonitrile.

The first member of the sequence is covalently linked via a spacer-controlled pore glass surface (CPG) via its 3'-hydroxyl group. Carrier-bound 5'-dimethoxy-60 gelling, dispersing and wetting agents, coloring agents, flavoring agents, buffering agents, and disintegrating and dissolving agents.

Conventional dosage forms include those prepared by the aforementioned pharmaceutical excipients which may be solid dosage forms such as tablets, capsules, powders, dragees, pills or granules, or liquid dosage forms such as syrups, solutions, emulsions or suspensions, and parenteral preparations such as injectables. and infusion solutions, and rectal preparations such as suppositories. The daily dose of the compositions of the invention will depend on many factors, such as the condition of the patient and the mode of treatment. Suitably, 0.5 to 1200 mg of active ingredient per kg of body weight per day is administered to adults. Accordingly, it is preferable to administer a tablet, capsule or dragee containing from 0.01 to 0.2 g of active ingredient 1 to 3 times daily.

The antisense oligodeoxynucleotides of the present invention are illustrated by the following examples.

Example 1

A H1V-RNS 280-299. Synthesis of 25 antisense oligodeoxynucleotides (KKKI538) complementary to the nucleotide sequence of SEQ ID NO: 280-299 containing thiophosphoro diester linkage and 5- (n-hexin-1-yl) uracil instead of thymine. nucleotide sequence and the complementary antisense oligodeoxynucleotide have the following nucleotide sequence (triplicate for a better overview):

G AGU GGU CAG CGG CG) -5 '

.................................. 1 micromolar (20-25 mg of the bound nucleoside) of 280 trityl protecting nucleosides (38-40 microns nucleoside / g carrier) into a 1 mm x 30 mm reactor, sealed at each end with a filter cap. Program the target sequence into the device by replacing the T symbols with U (Sequence menu) and then starting the synthesis (Run menu).

The first step is to remove the dimethoxytrityl protecting group from the nucleoside bound to the support with 90% dichloroacetic acid in dichloromethane for 90 s, followed by condensation with the following member of the target sequence after acetonitrile washing: approx. 15 micromolar, from position 8 approx. 75 microliters of reactant was added to the reactor and allowed to stand for 3 minutes. After washing with acetonitrile, the phosphite bond was oxidized to a thiophosphate solution with a 0.5 M solution of tetraethyl thiuram disulphide in thiophosphate (20 min, then unreacted 5'-hydroxyl groups were acetonitrile / collidine / dimethylaminopyridine / acetic anhydride (73:15). : 2: 10) After washing, the cycle starting with detritilization is repeated until all members of the sequence are sequenced in the condensation reaction, removing the dimethoxytrityl group from the last member as above.

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The oligonucleotide containing the protected heterobases bound to the support is cleaved from the support with 30% aqueous ammonia solution at 20 ° C for 1 hour. The resulting solution was heated to 60 ° C and held at this temperature for 8 hours to remove the other protecting groups from the free 3'-hydroxyl containing oligomer. The solution was evaporated, extracted with ether and the aqueous solution was purified by HPLC. The crude oligonucleotide mixture contains, in addition to the main component, oligomers of shorter chain lengths as impurities.

Purification of the principal component was performed by reverse phase chromatography on HPLC using two Jasco PU980 HPLC pumps and a Jasco UV-975 UV / VIS detector. The pumps and detector can be individually programmed. UV absorption data is transferred from the detector via a BDS card to an ARL AT386 computer using the BDS Barspec Data System software. The chromatographic separation can be followed on the screen. Separation was performed on a Shandon Hypersil ODS (Cl8), 5 micrometer, 120,, 250 χ 8 mm (BST, Budapest) with a linear gradient of acetonitrile (eluent: 0.1 M potassium phosphate buffer, pH 7.5% acetonitrile). Eluent B: 0.1 M potassium phosphate buffer, pH 7.50% acetonitrile, 100% A to 100% B over 45 minutes). Upon preliminary analysis of the image 100-1000 ₂₆ p-units, optimally loaded unit 400 (this is 16 mg and 20 nucleotides in length tiooligonukleotid potassium salt) of the crude oligomer is up to 250x8 mm semipreparative column. The amount of components was determined by UV spectroscopy on a Hewlett-Packard HP8452A photometer.

The main peak fraction was then removed by vacuum rotary evaporation at room temperature and further concentrated to a volume of 10-100 mL. It is then desalted by dialysis (Spectrapor dialysis membrane, molecular weight transfer limit: below 3500, Serva). Dialysis was performed against high purity deionized water (Milli-Q), and the salt flow was monitored by conductivity measurement. The desalted oligomer solution was lyophilized and stored at -25 ° C.

The yield per step of condensation is approx. 97-98%, based on which a 20-membered oligomer is ca. It is obtained in 60% yield. This is approx. 100 _{Corresponds to 260} raw units. Of this, on average, ca. ₂₆₀ units (about 2 mg of potassium salt) of pure oligonucleotide were obtained.

The nucleotide sequence (sequence) of the synthesized oligonucleotides is guaranteed by the DNA synthesizer program. The structure of the synthesized and purified oligonucleotides was confirmed by the following analyzes.

1. Composition Analysis: The oligonucleotide was cleaved to nucleosides with phosphoesteresterase and alkaline phosphatase and the hydrolysis mixture was analyzed by HPLC. Analysis yielded the expected nucleoside composition: 45% C, 25% G, 15% A and 15% hU, ± 2.5% error, corresponding to 9 C, 5 G, 3 A in the above structure and 3 pieces of hU.

2. Sequence Analysis: For this purpose, the oligonucleotide was labeled at the 5 'end with gamma [ ³² P] ATP and analyzed by the known Maxam-Gilbert technique.

The sequence read from the sequencing gel was the same as that described in the above formula.

3. The purity of the oligonucleotide was assayed by analytical reverse phase HPLC. According to the chromatogram, the oligonucleotide was 95% pure.

4. Thiophosphoro diester oligonucleotides were analyzed by ³¹ P-NMR. Based on the spectrum, 98% of the intemucleotide bonds in the oligonucleotide were thiophosphoric diester.

5. The tendency to duplex formation was measured by determining the strength of the complex with the synthesized sense deoxyoligonucleotide. The stability of the light absorption temperature curve at 260 nm for each oligonucleotide was significantly higher than 37 ° C.

Example 2

HIV-RNA 280-299. synthesis of antisense oligodeoxynucleotide (KKKI 515, 516) complementary to its nucleotide sequence

Nucleotide sequence of the 20-membered oligomer:

5'-d (GCG TAC TCA CCA GTC GCC GC> 3 '

The oligomer was prepared, purified and analyzed by the procedure described in Example 1 with the following modifications:

- the original T symbol was not changed when programming the sequence,

- the program was changed by acetylation of unreacted 5'-hydroxy groups after the condensation step and oxidizing the phosphite bond to phosphate with 0.01 M iodine (dissolved in acetonitrile collidin-water = 64: 6: 30). This compound is outside the scope of the compounds of the invention; the example is provided as a reference.

Example 3

HIV-RNA 280-299. synthesis of antisense oligodeoxynucleotide (KKKI 513) complementary to its nucleotide sequence and containing 5- (n-hexin-1-yl) uracil instead of thymine

Nucleotide sequence of the 20-membered oligomer:

5'-d (GCG hUAC hUCA CCA GhUC GCC GC) -3 '

The preparation, purification and analysis of the oligomer was carried out as in Example 2, except that the synthesizer 13 (U) was replaced by 5'-O- (p, p'-dimethoxy) trityl-5- ( Glass container containing a solution of 0.03 M acetonitrile (water content <0.002%) in N-N-N-diisopropylphosphoramidite (-hexm-1-yl) -2'-deoxyuridine-3'-O- (2-cyanoethyl) and the target sequence was programmed into the device by replacing the T symbols with U (Sequence menu).

Example 4

HIV-RNA 280-299. synthesis of antisense oligodeoxynucleotide (KKKI 520, 552) complementary to its nucleotide sequence and containing 5- (n-hexin-1-yl) cytosine instead of cytosine

Nucleotide sequence of the 20 membered oligomer (abbreviated as hC)

Means 5- (n-hexin-1-yl) -cytosine):

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5'-d (GhCG TAhC ThCA hChCA GThC GhChC GhC) -3 '

The preparation, purification and analysis of the oligomer was carried out as in Example 2, except that the arrangement was changed by replacing the synthesizer with 10 '(C) at 5'-O- (p, p'dimethoxy). -trityl-5- (n-hexin-1-yl) -2'-deoxycytidin-3'O- (2-cyanoethyl) -N, N-diisopropylphosphoramide (Formula 4), A glass container containing 03 M acetonitrile (water content <0.002%) was placed.

Example 5

HIV-RNA 280-299. Synthesis of an antisense oligodeoxynucleotide (KKKI554) complementary to its nucleotide sequence and containing 5- (n-hexin-1-yl) uracil instead of thymine and 5- (n-hexin-1-yl) cytosine instead of cytosine The nucleotide sequence of the 20-membered oligomer is:

5'-d (GhCG hUAhC hühCA hChCA GhUhC GhChC GhC) -3 '.

The preparation, purification and analysis of the oligomer was carried out as described in Example 4, except that the program and the arrangement were changed by replacing the synthesizer 13 (U) with 5'-O- (p, p). '-dimethoxy) -trityl-5 - (' - hexm-1-yl) -2'-deoxyuridine-3 '- (2-cyanoethyl) -N, N-diisopropylphosphoramide (0.03 M in acetonitrile). water content <0.002%) and the target sequence was programmed into the apparatus by replacing the T symbols with U (Sequence menu).

Example 6

HIV-RNA 280-299. synthesis of antisense oligodeoxynucleotide (KKKI 517, 545) complementary to the nucleotide sequence of

Nucleotide sequence of the 20-membered oligomer:

5'-d (GsCsGs TsAsCs TsCsAs CsCsAs GsTsCs GsCsCs GsC) -3 '

The oligonucleotide was prepared, purified and analyzed by the method of Example 1 with the exception that the original T symbol was not altered in sequence programming.

Example 7

HIV-RNA 280-299. Synthesis of an antisense oligodeoxynucleotide (KKKI 583) complementary to its nucleotide sequence and containing 5- (n-octin-1-yl) uracil and thiophosphoro diester linkage instead of cytosine The nucleotide sequence of the 20-membered oligomer (the abbreviation oU is 5- (n-octin-1). -il) means uracil:

5'-d (GsCsGs oUsAsCs oUsCsAs CsCsAs GsoUsCs GsCsCs GsC) -3 '

The preparation, purification and analysis of the oligomer was carried out as in Example 1, except that the program and the arrangement were changed by replacing the synthesizer 13 (U) with 5'0- (p, p '). -dimethoxy) trityl-5 - ((- octin-1-yl) -2'-deoxyuridine-3 '- (2-cyanoethyl) -N, N-diisopropylphosphoramidite 0.03 M acetonitrile (water content) A <RTIgt; glass </RTI> container containing <0.002%) solution was placed and the target sequence programmed into the apparatus by replacing the T symbols with U (Sequence menu).

Example 8

HIV-RNA 280-299. Synthesis of an antisense oligodeoxynucleotide (KKKI 549) complementary to its nucleotide sequence and containing 5- (n-hexin-1-yl) cytosine and thiophosphoro diester linkage instead of cytosine The nucleotide sequence of the 20-membered oligomer:

5'-d (GshCsGs TsAshCsTshCsAs hCshCsAs GsTshCs GshCshCs GshC)

The oligonucleotide was prepared, purified and analyzed by the method of Example 6 with the modification that the synthesizer at position 10 (C) was 5'-O- (p, p'-dimethoxy) trityl-5- (M-). Hexin-1-yl) -2'-deoxycytidine-3 '- (2-cyanoethyl) -N, N-diisopropylphosphoramidite was placed in a glass container containing 0.03 M acetonitrile (water content <0.002%).

Example 9

HIV-RNA 280-299. synthesis of 5- (n-hexin-1-yl) cytosine, 5- (n-hexin-1-yl) uracil and thiophosphoro diester linkage (KKKI 542) complementary to its nucleotide sequence, containing cytosine instead of cytosine

Nucleotide sequence of the 20-membered oligomer:

5'-d (GshCsGs hUsAshCs hUshCsAs hCshCsAs GshUshCs GshCshCs GshC> 3 '

The oligonucleotide was prepared, purified and analyzed by the method of Example 8 with the modification that the synthesizer 13 (U) was replaced by 5'-O- (p, p'-dimethoxy) trityl-5 - (- Hexin-1-yl) -2'-deoxyuridine-3 '- (2-cyanoethyl) -N, N-diisopropylphosphoramidite was placed in a glass container containing 0.03 M acetonitrile (water content <0.002%) and the The target sequence was programmed into the device by replacing the T symbols with U (Sequence menu).

Example 10

HIV-RNA 280-305. synthesis of antisense oligodeoxynucleotide (5 '+ 6 base, KKKI 577) complementary to its nucleotide sequence and containing thiophosphoro diester linkage and 5- (n-hexin-1-yl) uracil instead of thymine

Nucleotide sequence of the 26-membered oligomer:

5'-d (hUshUshUs hUshüsGs_GsCsGs hüsAsCs hUsCsAs CsCsAs GshUsCs GsCsCs GsC) -3 '(The beginning of the sequence given in Example 1)

The oligonucleotide was prepared, purified and analyzed by the method of Example 1 with the exception that the oligonucleotide 5 'up to 5' in length based on the HIV-RNA sequence was used in the sequence programming.

Example 11

HIV-RNA 275-299. Synthesis of an antisense oligodeoxynucleotide complementing its nucleotide sequence with a thiophosphoro diester linkage and 5- (n-hexin-1-yl) uracil instead of thymine (+ 3 bases + 5 base, KKKI 593)

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Nucleotide sequence of the 25-membered oligomer:

5'-d (GsCsGs hUsAsCs hUsCsAs CsCsAs GshUsGs

GsCsCs GsC_CsCsCs hUsC) -3 '(The beginning of the sequence given in Example 1)

The oligonucleotide was prepared, purified, and analyzed by the method of Example 1 with the exception that the oligonucleotide 3 'up to 5' was used in the sequence programming based on the HIV RNA sequence.

Example 12

HIV-RNA 280-296. antisense oligodeoxynucleotide complementary to its nucleotide sequence, containing a thiophosphoro diester linkage and 5- (n-hexin-1-yl) uracil instead of thymine (base 5 'in the 5' direction, KKK1

584)

Nucleotide sequence of the 17-membered oligomer:

5'-d (hUsAsCs hUsCsAs CsCsAs GshUsCs GsCsCs

GSC> 3 '

The oligonucleotide was prepared, purified, and analyzed by the method of Example 1 with the modification that the oligonucleotide 5'-down by 5 'based on the HIV-RNA sequence was used in programming the sequence.

Example 13

HIV-RNA 283-299. antisense oligodeoxynucleotide complementary to its nucleotide sequence, containing a thiophosphoro diester linkage and 5- (n-hexin-1-yl) uracil instead of thymine (base 3 ', 3', KKK1

585)

Nucleotide sequence of A17 oligomer:

5'-d (GsCsG hUsAsCs hUsCsAs CsCsAs GshUsCs

GsCs) -3 '

The oligonucleotide was prepared, purified and analyzed by the method of Example 1 with the exception that the 3 'shorter oligonucleotide based on the HIV RNA sequence was used in programming the sequence.

The present invention relates to the anti-HIV activity of antisense oligodeoxynucleotides

The substances were tested at concentrations of 1, 5.20 and 100 micrograms / ml on the MT-4 cell line. MT4 cells were 20 TCID ₅₀ Ηΐν-1 _Ν | _Β (NIH, USA). Cells were treated with antisense oligonucleotides 4 hours before HIV infection, and 24 and 48 hours after HIV infection (acute infection). The effect on virus production was assessed by reverse transcriptase enzyme activity assay from culture supernatants. The degree of reverse transcriptase activity was determined by [ ³ H] dTTP incorporation and the activity of the samples was expressed in cpm / ml.

At concentrations of 5.20 and 100 micrograms / ml, treatment was also performed on uninfected MT-4 cells to test the cytotoxic activity of the test compounds. Cell viability was assessed by light microscopy and trypan blue supavavital staining. The compounds were also tested for their effect on cell proliferation.

Figure 1 shows the anti-HIV activity of the antisense oligonucleotides of Examples 1, 6, 8 and 9 in MT-4 cells as a function of the time of infection based on reverse transcriptase activity. As a control, virus growth (untreated HIV control) was measured in infected but untreated cells. For reference, the effect of AZT measured under the same conditions is also indicated.

The peak of viral replication in our system generally occurred on the seventh day. The antisense concentration (IC ₅₀ ) required for 50% inhibition was determined by measuring the activity at the peak of viral growth.

The IC ₅₀ values are determined as shown in Figures 1-13. All antisense oligonucleotides given in Example 1A were performed as described above. The results are summarized in Table 1, supplemented by the duration of the effect.

Table 1

Anti-HIV activity of antisense oligonucleotides of the invention in MT-4 cells

Antisense oligomer (CCI number) HIV RNA sequence Chemical amendment ic ₅₀ (micrograms / ml) Duration of action (day / antisense concentration pg / ml) Example 1 (538) 280-299 thiophosph., hU 1.2 30/5 Example 1 2 (515,516) 280-299 - 63 6/100 Example 3 (513) 280-299 hU 38 10/100 Example 4 (520,552) 280-299 hC 2 18/20 Example 5 (554) 280-299 Wow, hC 2.8 18/20 Example 6 (517,545) 280-299 thiophosphate. 2 18/20 Example 7 (583) 280-299 thiophosph., oU 2.5 22/5 Example 8 (549) 280-299 thiophosph., hC 0.6 18/5 Example 9 (542) 280-299 thiophosph., hU, hC 2 18/5 Example 10 (577) 280-305 thiophosph., hU 6.5 30/10

HU 221 497 Al

Table 1 (continued)

Antisense oligomer (CCI number) HIV RNA sequence Chemical amendment IC ₅₀ (micrograms / ml) Duration of action (day / antisense concentration pg / ml) Example 11 (593) 275-299 thiophosph., hU 2.5 22/20 Example 12 (584) 280-296 thiophosph., hU 2.4 22/5 Example 13 (585) 283-299 thiophosph., hU 2.6 22/5

Abbreviations: hU = 5- (n-hexin-1-yl) uracil hC = 5- (n-hexin-1-yl) cytosine oU = 5- (n-octin-1-yl) uracil thiophosph. = thiophosphoro diester intemucleotide bond

The antisense oligonucleotide constructed from natural nucleotides of Example 2 showed a weak anti-HIV activity. The effect was enhanced with the antisense oligomer of Example 3 containing 5- (n-hexin-1-yl) uracil instead of thymine base. The anti-HIV activity was further enhanced with the antisense of Example 4, which contained 5- (n-hexin-1-yl) cytosine as a modified pyrimidine base. The antisense of Example 5, modified in all pyrimidine bases, and the antisense of Example 6, containing unmodified base but having thiophosphoric diester linkages instead of phosphorus diester intemucleotide bonds, had the same effect. With the latter two, the effect lasted for 18 days at a concentration of 20 micrograms / ml. The antisense oil gonucleotide of Example 1 which contained both a 5- (n-hexin-1-yl) uracil and a thiophosphoro di modification was surprisingly large and sustained.

This is shown by its low IC ₅₀ value and persistence of 5 micrograms / ml for 30 days for the duration of the study (Figure 3). 35

Similarly, the antisense of Example 7 mixed with 5 (n-octin-1-yl) uracil and thiophosphoro diester linkages showed similar antiviral activity, as well as the antisense of Examples 8-9. Examples are the modified antisense oligonucleotides also exemplified by example. The same effects were observed in Figures 12-13. Examples include the shorter chain antisense, also mixed.

No cytotoxic effect was observed with any of the antisense oligonucleotides of the invention at the above concentrations. AZT above 5 micrograms / ml was toxic.

1-13. The retention times (tpm) of the compounds prepared in Example 1c are shown in Table 2. The ς values were determined by HPLC under the conditions described in Example 1 for purification of the major component, except that the column used for separation was 4.6 mm instead of 8 mm.

Table 2

Compound (example number) tjperc) 1 ^l 20.5 1 ² - 16.0

Compound (example number) tr (min) Third 16.8 4th 17.9 5th 18.5 6th 19.4 7th 21.0 8th 23.2 9th 24.1 10th 24.7 11th 25.2 12th 18.0 13th 18.3

PATENT CLAIMS

Claims (11)

Claims 1. An antisense oligodeoxynucleotides comprising 17-26 nucleotide units, the nucleotide sequence of which is 7 'to 15' from nucleotide 290 of the splice donor region of the HIV RNA. and nucleotides 291 and 5 'to 14' in the 3 'direction, wherein at least a portion of the pyrimidine bases are 5- (C4-C10) n-alkin-1-yl) pyrimidine bases, and / or the phosphorus diester groups linking the nucleotides are substituted with thiophosphorus diester groups. The antisense oligodeoxynucleotides of claim 1, wherein the thymine bases are substituted with 5- (C 4 -C 10) n-alkynyl-1-yl uracil bases. The antisense oligodeoxynucleotides according to claim 1 or 2, wherein the thymine bases are 5- (n-hexin-1-yl) uracil bases. The antisense oligodeoxynucleotides of claim 1, wherein the cytosine bases are 55 are 5- (C 4 -C 10) n-alkynyl-1-yl cytosine bases, preferably 5 (n-hexin-1-yl) cytosine bases. The antisense oligodeoxynucleotides of claim 1, wherein each pyrimidine base is replaced by a 5- (C 4 -C 10) n-alkin-1-yl pyrimidine base, Preferably, the base is 5- (n-hexin-1-yl) pyrimidine base. HU 221 497 Al 6. The antisense oligodeoxynucleotides of any one of claims 1 to 5, wherein the nucleotide linker is a phosphorus diester group. 7. The antisense oligodeoxynucleotides of any one of claims 1 to 5, wherein the antisense oligodeoxynucleotides are HIV RNA 275-299, 280-299, 283-299, and 280-296. 280-305. complementary to its sequence. Pharmaceutical compositions comprising, in addition to a pharmaceutically acceptable carrier and / or other excipient, at least one of claims 1-7. An antisense oligodeoxynucleotide according to any one of claims 1 to 3. 9. Use of antisense oligodeoxynucleotides according to any one of claims 1 to 6 for the preparation of a medicament for the treatment of HIV infections. 10. Procedure 1-7. Antisense oligodeoxynucleotides 5 according to any one of claims 1 to 5, characterized in that the corresponding nucleotides are linked in a known manner in the appropriate order. 11. A process for the preparation of a pharmaceutical composition according to claim 8, characterized in that the method according to claims 1-7. The antisense oligodeoxynucleotide of any one of claims 10 to 10 is mixed with a carrier and / or other excipient commonly used in the preparation of a medicament and formulated into a pharmaceutical composition.