NZ242264A - Polyadenylic acid/polyuridylic acid complexes in pharmaceutical compositions - Google Patents

Description

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H'F'E&'WS' Publication Dale: ! : .0. Journal, No: N.Z. patent OPF!i"g j Patents Form No. 5 I 7 APR 183^' ! Patents Act 1953 ' COMPLETE SPECIFICATION COMPLEXES OF POLYADENYLIC ACID WITH POLYURIDYLIC ACID We, SOCIETE DE CONSEILS DE RECHERCHES ET D1 APPLICATIONS SCIENTIFIQUES (S.C.R.A.S.) of 51/53 rue du Docteur Blanche, 75016 Paris, France, a French Societe Anonyme, hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: 1 (to be followed by la) / k / / The invention relates to pharmaceutical compositions containing complexes of polyadenylic acid with polyuridylic acid, optionally associated with an anti-Aids drug, and also to the use of such complexes for the treatment of Acquired ImmunoDeficiency Syndrome (Aids).

British Patent No. 2 211 847 describes a process for the preparation of homopolymers and copolymers of polynucleotides and complexes thereof. Although these products were not new, previous processes were not able to produce them at an economically acceptable cost without toxic impurities which made them unsuitable for pharmaceutical use. The process described in aforesaid British Patent 10 leads to products of sufficient purity for pharmaceutical use which were described namely for the treatment of tumours.

The complex of Poly(A).Poly(U) has been described as a poorly anti-viral agent (cf "Effects of polynucleotides on monkeys and man", CLINICAL ASPECTS OF INTERFERONS, 1988, pages 319-331) ; consequently, it was not obvious that 15 Poly(A).Poly(U) might be an efficient anti-retroviral agent, as virus and retro-virus are considered as entirely different species.

It has been found that a complex of Poly(A).Poly(U) is also a potent inhibitory agent of various HIV in cell cultures, by blocking the entry of the virus. The invention provides a therapeutic agent for the treatment of Aids, wherein the major 2 0 active ingredient is Poly(A).Poly(U). Preferably, the Poly(A).Poly(U) is prepared by the process described in said British Patent No. 2 211 847.

It has also been found that Poly(A).Poly(U) is effective when administered with other anti-Aids drugs, such as 3'-azido-3'-deoxythymidine (AZT), Dideoxyinosine (DDI) or Dideoxycytidine (DDC), and enhances the effect of the latter. 25 Accordingly, the invention further provides a pharmaceutical composition comprising Poly(A).Poly(U), associated with a sufficient amount of another anti-Aids drug and pharmaceutically acceptable diluents or carriers. 2^22 64 The invention provides, finally, a method for the treatment of Aids, the method comprising administering an effective amount of Poly(A).Poly(U), alone or in association with any anti-Aids drug, to a patient suffering from Aids.

Accordingly, this invention provides a therapeutical composition for the treatment 5 of Acquired Immuno Deficiency Syndrome (Aids) and related infections, said composition comprising of from O.lto 100 % of complex of Poly(A).Poly(U), and an other anti-Aids agent acting on the HIV virus according to a different mechanism of the one of the complex of Poly(A).Poly(U), and pharmaceutically acceptable diluents or carriers.

Preferably, said composition comprises of from 0.1 to 99 I of complex of Poly(A).Poly(U), and an other anti-Aids agent acting on the HIV virus according to a different mechanism of the one of the complex of Poly(A).Poly(U), and pharmaceutically acceptable diluents or carriers.

According to an embodiment of the invention, the anti-Aids agent acting on the 15 HIV virus is selected from within AZT, DDIandDDC.

The interest of Poly(A).Poly(U) in the treatment of Aids will appear clearly from the various experiments hereafter described.

CYTOPATHOLOOY Two batches of CEM cells (T cells rich in CD4 receptors) infected with Human 20 Immunodeficiency Virus HIV-1 Bru (LAI) were treated with Poly(A).Poly(U) 24 hours after infection. Two similar batches of HIV infected CEM cells were used as controls, and not treated. The cell cultures were regularly inspected under the microscope to observe the onset of cytopathic effects (fusion of cells and formation of syncitia) resulting from the HIV. 2 5 The experimental protocols were as follows : 5 x 10^ CEM cells were incubated with 1 ml of supernatent containing HIV with a reverse transcriptase activity of 1.0 x 10^ cpm. One hour afterwards, the cells were centrifuged and the cellular residue (0.5 x 10^ cells) was suspended in RMP1 medium containing 10 % fetal serum and 2ng/ml polybrene. Twenty four hours later, Poly(A).Poly(U) was added at a concentration 3 0 of 200 ng/ml. The cell cultures were maintained without further addition of Poly(A).Poly(U). ^ The results were as follows : Day 6 Day 7 Day 8 Day 11 Controls ++ ++++ ++++ cells dead Treated Cells - + + ++ The symbol "+" indicates the appearance of cytopathic effects ; the number of such symbols used is an approximate quantification. These results show that the cytopathic effects are significantly reduced by the treatment.

Cultures were prepared as above described, and during the night of days 7 and 8 were incubated with ^-methionine for electrophoretic analysis on SDS of the viral 15 proteins synthesised, both in the cellular mass and in the supernatent. The results are shown in the accompanying drawing. In the drawing, indicates a sample from an untreated control and "+" a sample from a culture treated with Poly(A).Poly(U). The identifiers on the right of spots shown are as follows : gp 120 the envelope protein 2 0 p68 reverse transcriptase p55 precursor of the core protein p40 precursor of the core protein, partially cleaved p32 endonuclease p25 the major core protein The results show an almost total reduction of the presence of viral proteins in the cells and supernatents of the cultures treated with Poly(A).Poly(U).

The quantity of p24/p25 antigen in the supernatent was measured 8 days after infection, by the ELISAtest. 4 Controls Treated Cells 1.01 (ig/ml 1.27 ng/ml 0.03 (ig/ml 0.07 (ig/ml This shows suppression of 96.5 % of the HIV production in the cultures treated by Poly(A).Poly(U).

The action of Poly(A).Poly(U) on the inhibition of the production of HIV in cultured cells has been confirmed by the following experiments. 1. Effect of variation of the dose of PoIv(A).Polv(U): CEM cells were infected with HIV. 24 hours later, different concentrations of Poly(A).Poly(U) were added. The production of the virus in the supernatent of the 15 culture was determined on day 5 after infection, by quantifying the amount of p24/p25 antigen present. Control cells, which had been infected but to which Poly(A).Poly(U) had not been added, had by then ruptured.

The obtained results were as follows : Poly(A).Poly(U) (|ig/ml) p24/p25 (|ig/ml) 0 1.10 0.53 50 0.14 100 0.10 200 0.07 2. Effect of Repeated Treatment: The operating procedure was similar to that described in experiment 1, but addition of PoIy(A).Poly(U) was at a concentration of 200|ig/ml at 24 and 96 hours after 242264 infection. An untreated control was also run ; as before, the cells in the control had ruptured by day 5.

The concentration (|ig/ml) of p24/p25, at the days 4, 5, 6 and 7 after infection has been measured in the untreated control and in the cells treated at days 1 and 4.

The obtained results were as follows : p24/p25 (|ig/ml) Day 4 Day 5 Day 6 Day 7 Control (untreated) 0.72 1.25 Treated cells 0.05 0.10 0.20 0.26 3. The effect of Polv(A).Polv(U) is at an early step in virus infection : HIV infected cells were treated with 200 (ig/ml of Poly(A).Poly(U) at different times before, during and after the infection with HIV. On day 6 after infection, the 15 number of syncitia were counted. On day 7, the level of p25 in the culture medium was assayed.

P- L2£ & A.

Poly(A).Poly(U) Syncitia p25 Control (untreated) 90% 1320 ng/ml Cells treated at: . 3 days before 85% 1215 ng/ml . 2 days before 72% 965 ng/ml . 1 day before 45% 280 ng/ml . 1 hour before 6 ng/ml . Togetherwith HIV <5 ng/ml . 1 hour after % 110 ng/ml . 4 hours after % 120 ng/ml 242264 6 STUDY OF USE OF POLY(A).POLY(U) WITH AZT : Cells were infected with HIV 24 hours before a single treatment with : (a) Poly(A).Poly(U) alone, at the concentration of 100 jig/ml; (b) AZT alone, at the various concentration of 100, 250, 1 000, 5 000 and 5 10 000 ng/ml, (5 samples); (c) AZT at the various concentration of 100, 250, 1 000, 5 000 and 10 000 ng/ml, each with Poly(A).Poly(U) at a concentration of 100 |ig/ml (5 samples).

A control (non-treated) was also kept. The levels of antigen p25 in the supernatents were measured at days 7, 12 and 14 after infection. The results, expressed in ng/ml, 10 were as follows : P. L. B. & A. per t° I Day 7 p25 Day 12 Day 14 Control 1250 Poly(A).Poly(U) 135 980 1200 AZT 100 38 380 1200 AZT 100 Poly(A).Poly(U) 16 75 900 AZT 250 18 170 1200 AZT 250 Poly(A).Poly(U) 100 40 840 AZT 1000 <5 52 590 AZT 1000 Poly(A).Poly(U) 100 <5 17 200 AZT 5000 27 275 AZT 5000 Poly(A).Poly(U) 100 <5 48 AZT 10000 220 AZT 10000 Poly(A).Poly(U) 100 <5 8 39 These results show that the association AZT/Poly(A).Poly(U) is more effective than either alone.

DISCUSSION : The results show that Poly(A).Poly(U) has an inhibitory effect on HIV. This has been observed in different tests : A : metabolic synthesis of viral proteins in infected cells B : activity of reverse transcriptase in the culture medium of infected cells C : amounts of the major core protein p25 of HIV in the culture medium of infected cells D : cell fusion The inhibitory action of Polv(A).Polv(U) on different types and isolates of HIV : In all of the experiments presented above, the HIV-1 Bru isolate (now referred to as HIV-1 LAI) was employed. In order to demonstrate that the inhibitory action of Poly(A).Poly(U) is not restricted to the HIV-1 species used, another species of HIV-1 referred to as ELI (31) and two different HIV-2 species ROD and EHO were tested. Poly(A).Poly(U) added 6 hours before infestation of CEM cells with these viruses resulted in more than 90 % inhibition of virus production.

In all these tests, Poly(A).Poly(U) at a dosage of 200 ng/ml exercises an inhibitory action which can be estimated as between 85 and 90 %. An amount per single injection of from 200 to 4 000 mg may be suitable in man ; doses of from 300 to 1 000 mg may be efficiently injected in man, preferably of from 400 to 600 mg, the preferred dose being 500 mg. The maximal effect is obtained by treatment as close as possible in time to the infection, but treatment before of after infection is also effective. The treatment may be repeated at intervals of 3 to 5 days.

Moreover, now it has been demonstrated a potent anti-retroviral action. It appears that Poly(A).Poly(U) seems to work at the level of penetration of the virus into the cell, whereas AZT works at the level of intra-cellular transcription. Using Poly(A).PoIy(U) combined treatment with another anti-retroviral agent gives far better results than use of either alone. 8 CLAIMS

Claims (3)

WHAT WE CLAIM IS;-

1. A therapeutical composition for the treatment of Acquired Immuno Deficiency Syndrome (Aids) and related infections, said composition comprising of from 0.1 to 99% by weight of a complex of Poly (A).Poly(U), (this percentage being of the total composition) and another anti-Aids agent acting on the HIV virus according to a different mechanism from the one of the complex of Poly (A). Poly (U) , and one or more pharmaceutically acceptable diluents or carriers wherein said Poly(A).Poly(U) complex and said other anti-Aids agent react f synergistically.

2. A therapeutical composition according to claim 1 wherein the anti-Aids agent acting on the HIV virus according to a mechanism different from the one of the Poly(A).Poly(U) complex, is selected from AZT, DDI and DDC.

3. A therapeutical composition as claimed in either claim 1 or claim 2 and substantially as hereinbefore described with reference to and as shown in any one of the foregoing Examples. SOCIETE DE CONSEILS DE RECHERCHES ET D7APPLICATIONS SCIENTIFIOUES (S.R.A.S^V by their authorised agents P.L. BERRY & ASSOCIATES per* ;