WO1993007881A1

WO1993007881A1 - Inhibition of viruses by the administration of homopolyribonucleotides and/or duplex polymers

Info

Publication number: WO1993007881A1
Application number: PCT/US1992/008863
Authority: WO
Inventors: Arthur D. Broom; Robert W. Sidwell; John H. Huffman
Original assignee: University Of Utah
Priority date: 1991-10-16
Filing date: 1992-10-16
Publication date: 1993-04-29
Also published as: AU2798992A

Abstract

A series of polyribonucleotides containing methyl and/or sulphur substitutions such as poly(1-methyl-6-thioinosinic acid) also [poly(m1s6I)], poly(1-methyl-6-thioguanylic) acid [poly(m1s6G)], and poly(6-thioguanylic acid) complexed with polycytidylic acid [poly(s6G)-poly(C)] are useful in treating human cells infected with AIDS viruses or viral opportunistic pathogens by inhibiting the virus from developing or spreading to unaffected cells. Poly(m1s6I), poly(m1s6G), and poly(s6G)-poly(C) may be administered orally, parenterally or transdermally per se or in the form of a pharmaceutically acceptable salt. As regarding the AIDS virus, these agents function in one or more ways as inhibitors of the viral reverse transcriptase necessary for viral replication, and/or also block the formation of syncytia (giant cells) which are characteristic of HIV infection and facilitate the transmission of the virus to uninfected cells.

Description

INHIBITION OF VIRUSES BY THE ADMINISTRATION OF

HOMOPOLYRIBONUCLEOTIDES AND/OR DUPLEX POLYMERS Background of the Invention.

This invention relates to the inhibition of the AIDS virus by the administration of homopolyribonucleotides and/or duplex polymers. More particularly, this invention relates to the inhibition of the AIDS virus by the administration of

polyribonucleotideβ containing methyl and/or sulphur

substitutions represented by the group consisting of poly(l- methyl-6-thioinosinic acid) or poly(m¹s⁶I), poly(1-methyl-6-thioguanylic) acid, or poly(m¹s⁶G) and poly(6-thioguanylie acid) complexed with polycytidylic acid or poly(s⁶G)-poly(C).

The acquired immunodeficiency syndrome (AIDS) is considered to be caused by the HIV-1 virus (also known as HTLV-III/LAV) or by HIV-2. HIV-1 and HIV-2 are RNA genetically unique retroviruses which may rapidly replicate in a human host cell, collectively HIV.

The HIVs preferentially attack helper T-cells (T-lymphocytes or CD4-bearing T-cells as they are sometimes known) and possibly other human cells, e.g. certain cells within the brain. The helper T-cells are destroyed and their number in the human is depleted to such an extent the body's B-cells, as well as other T-cells, normally stimulated by helper T-cells no longer function normally or produce sufficient lymphokines and antibodies to destroy the invading virus or other invading microbes, etc.

While the HIV virus does not necessarily cause death, per se, it does in many cases cause the human's immune system to be so severely depressed the human falls prey to various other diseases (secondary infections or unusual tumors such as Pneumocytis carinii, herpes, cytomegalovirus, Kaposi's sarcoma, Epstein-Barr virus, and related lymphomas among others). These secondary infections are separately treated using other conventional medication. In some humans infected with HIV-1 or HIV-2 viruses (hereinafter referred to as AIDS viruses and which are meant to include mutants thereof), humans seem to live on with little or no symptoms but appear to have persistent infections. Another group of humans suffer mild immune system depression with various symptoms such as weight loss, malaise, fever, and swollen lymph nodes. These syndromes have been called persistent generalized lymphadenopathy syndrome (PGL) and AIDS-related complex (ARC), and they will probably develop into AIDS. Another complication for some sufferers of AIDS is a progressive dementia

characterized by severe cognitive, behavioral, and motor impairment (Price et al. Science 1988, 239, 586-592). The primary neuronal targets appear to be monocytes, macrophages and glial cells, although other resident cells in CNS, such as endothelial cells, may also be targets (Ho. Ann. Intern . Med. 1989, 111 , 400-410; Koenig et al. Science 1986, 233, 1089-1092; Wigdahl and Kunsch. AIDS Res. Hum. Retro . 1989, 5, 369-374).

In all cases the AIDS virus is persistently infectious through contact with body fluids such as blood, urine, saliva, semen and tears. Contact with such body fluids infected with the AIDS virus causes an infection if the victim has open cuts or the infected fluid comes in contact with tissues that have direct communication with the bloodstream of the victim such as mucous membranes and vaginal tissues.

The life cycle of HIV and the components of that life cycle that might be utilized as targets for AIDS therapy have been analyzed by Mitsuya et al. Science 1990, 233, 1533. Aspects of the viral life cycle which present plausible targets for the active polymers utilized in the present invention include blockade of binding of HIV virions to CD4⁺ target cells and inhibition of virally-induced syncytium formation, inhibition of reverse transcriptase, inhibition of the RNase H activity associated with reverse transcriptase, interferon induction, stimulation of 2',5'-oligoadenylate synthesis, and possible interference with later steps of the processing of viral mRNA. It is probable that more than one of these mechanisms may pertain to the polymers utilized in this invention. Mitsuya et al., supra, point out that the very complexity of HIV would make the virus more vulnerable to coordinated attacks by antiretroviral drugs at multiple stages of its life cycle. Therefore, the discovery of compounds which exhibit multiple sites of action should present positive features in their ability to function as anti-AIDS agents.

Objects and Summary of the Invention

It is an object of this invention to provide a series of polyribonucleotide polymers which are useful in treating human cells infected with AIDS viruses by inhibiting AIDS from developing or spreading to other unaffected cells.

It is also an object of this invention to provide a series of polyribonucleotide polymers which act as inhibitors of the viral reverse transcriptase necessary for replication of the AIDS viruses.

A still further object of this invention is to provide a series of polyribonucleotide polymers which function to block the formation of syncytia which are characteristic of HIV infection and facilitate the transmission of the virus to uninfected cells.

These and other objects may be accomplished by a series of polyribonucleotides containing methyl and/or sulphur

substitutions including poly(1-methyl-6-thioinosinic acid) also referred to as "poly(m¹s⁶I)", poly(1-methyl-6-thioguanylic) acid also referred to as "poly(m¹s⁶G)", and poly(6-thioguanylic acid) complexed with polycytidylic acid also known as "poly(s⁶G)-poly(C)" which have been found to be useful in treating human cells infected with AIDS viruses by inhibiting AIDS from

developing or spreading to other unaffected cells.

Detailed Description of the Invention

Poly(m¹s⁶I), poly(m¹s⁶G), and poly(s⁶G)-poly(C) maybe

administered per se or in the form of a pharmaceutically

acceptable salt, e.g. an alkali metal salt such as sodium or potassium, or an ammonium salt (all of which are hereinafter referred to as a pharmaceutically acceptable base salt).

Poly(m¹s⁶I), poly(m¹s⁶G), and poly(s⁶G)-poly(C) act as inhibitors of the viral reverse transcriptase necessary for viral replication, and/or also block the formation of syncytia (giant cells) which are characteristic of HIV infection and facilitate the transmission of the virus to uninfected cells. The

poly(m¹s⁶I, poly(m¹s⁶G), and poly(s⁶C)-poly(C) or the

pharmaceutically acceptable base salts are administered to a human in an amount sufficient to generate an effective amount of the compound which contacts the AIDS virus, its component enzymes and/or its cell surface receptor, and interact with them to prevent replication of the virus. The above amount may vary from case to case and may need to be empirically determined by those skilled in the treatment of AIDS patients. Hence, the term

"effective amount" as used herein is that amount in a particular patient which is sufficient to contact the AIDS virus, its component enzymes and/or its cell surface receptor, and interact with them to prevent replication of the virus and can be determined by those skilled in the art without undue

experimentation.

In general for the above AIDS virus infections, a suitable effective dose of the poly(m¹s⁶I), poly(m¹s⁶G), and poly(s⁶G)-poly(C), their pharmaceutically acceptable base salts (which areherein referred to as the administered ingredient) will be in the range 1 to 50 mg/Kg body weight of recipient/day, preferably in the range of 2 to 10 mg/K body weight/day, or in other amounts and schedules as determined by clinical trials.

Administration may be by an suitable route including oral, nasal, topical, parenteral (including subcutaneous,

intramuscular, intravenous and intradermal) with parenteral being preferred. It will be appreciated that the preferred route may vary with, for example, the condition and age of the recipient.

The compound poly(m¹s⁶I) is disclosed in E.W. Chan et al., J. Gen. Virol . 1981, 52 , 291-299 as a potent inhibitor of reverse transcriptase. A disclosure of poly(m¹s⁶G) was made in V.

Amarnath and A.D. Broom, Biochim. Biophys . Acta 1977, 479, 16-23. Poly(s⁶G)-poly(C) is disclosed in V. Amarnath et al. (Biochemistry 1976, 15, 4386-4389) as being an antitumor agent.

While it is possible for the ingredients to be administered alone, it is preferable to present them as part of a

pharmaceutical formulation. The formulations of the present invention comprise at least one administered ingredient, as defined above, together with one or more acceptable carriers thereof and optionally, other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

Formulations include those suitable for oral, nasal, topical, subcutaneous, intramuscular, intravenous and intradermal (or transdermal) administration. The formulations may conveniently be presented in unit dosage form, i.e., sterile single-dose lyophilized vials ready for reconstitution with a suitable vehicle (e.g. isotonic saline), and may be prepared by any methods well known in the art of pharmacy.

Such methods include the step of bringing into association the ingredients to be administered with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into

association the active ingredient with liquid carriers and then bringing the ingredients into solution.

Formulations suitable for topical skin administration may be ointments, creams, gels and/or pastes comprising the ingredient to be administered and a pharmaceutically acceptable carrier. A preferred topical delivery system is a transdermal patch containing the ingredient to be administered.

Formulations suitable for nasal administration wherein the carrier is a liquid for administration, as for example, a nasal spray or as nasal drops, including aqueous or oily solutions of the active ingredient.

Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions which may including suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers; for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, such as water, for injections immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, as recited above, or an appropriate fraction thereof of the administered ingredient.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question; for example, those suitable for oral administration.

The following examples and tables illustrate the invention.

Example 1

Preparation of poly(m¹s⁶I).

1-Methyl-6-thioinosine, prepared according, to Broom and. Milne (J". Heterocyclic Chem. 1975, 12, 171-174), is reacted with two equivalents of phosphoryl chloride in trimethyl phosphate as described by Yoshikawa et al. (Ball. Chem. Soc. Japan 1969, 42, 3505-3508). The resulting 5'-monophosphate was converted to the diphosphate in. about 50% yield by the procedure of Michelβon (Bioch±m. Biophys. Acta 1964, 91 , 1-13). The diphosphate was purified by ion exchange methods and catalytically polymerized with polynucleotide phosphorylase (E.C.2.7.7.8, Micrococcus luteus, P-L Biochemicals) to form polytm's⁶!) in 35 to 40% yield. The molecular weight range of this poly(m^Is^eI) polymer was estimated to be between about 100,000 and 500,000.

Example 2

Preparation of poly(m¹s⁶G).

Synthesis of 1-methyl-6-thioguanosine-5'-diphosphate. To a cold, stirred suspension of l-methyl-6-thioguanosine (1.6 g, 5 mmol in 25 mL trimethyl phosphate) was added 1 mL phosphoryl chloride. The solution was stirred at -3 to 0ºC for 2 h. The reaction mixture was poured slowly into excess ice and

neutralized with 1 M LiOH. The solution (~50 mL) was

concentrated in vacuo to -20 mL, then adjusted to pH 12 with cold 1 M LiOH. Lithium phosphate precipitated and was removed by filtration. The filtrate was adjusted to pH 8 with Dowex 50-X8 (H⁺) resin, then concentrated to -5 mL. The barium salt of the monophosphate was precipitated by the addition of 1.2 g barium acetate, 50 mL ethanol, and 50 mL acetone. The barium salt of 1-methyl-6-thioguanosine 5'-monophosphate was collected by centrifugation, washed with 1:1 (v/v) ethanol/acetone (2 × 4 mL) and dried to give 1.7 g (75%).

The 5'-monophosphate was converted to the 5 '-diphosphate according to the procedure of Michelson. The product was purified on a Dowex AG 1-X4 (Cl) column by elution with a linear gradient of 0.003 M HCl to 0.003 M HCl/0.25 M LiCl. Recovery from the resin was very incomplete. The pure product (thin-layer chromatography, SilicAR 7GF, 2-propanol-/NH₄OH/H₂O (7:1:2)) was isolated in 10% yield.

Synthesis of poly(1-methyl-6-thioguanylic acid). The polymerization of m¹s⁶GDP (40 mg) was carried out at 37ºC in a solution containing the diphosphate (0.02 M), 0.2 M TrisΗCl (pH 9), 5 mM magnesium chloride, 0.5 mM EDTA, and polynucleotide phosphorylase (10 units/mL). The phosphate analysis and the isolation of the polymer (20 mg) were done according to Broom, Uchic and Uchic (Biochim. Biophys. Acta 1976, 428, 278-286). A solution of the polymer (12 mg/6 mL) was applied to a column (1.9 × 50 cm) of Sephadex G-75 and eluted with 0.01 M ammonium carbonate buffer (pH 8.6). The high and low molecular weight fractions were individually collected, dialyzed successively against 0.1 M sodium chloride 5 mM EDTA (pH 7.5, 10 L) and distilled deionized water (15 L) and lyophilized. The molecular weight range is estimated to be between about 100,000 to 500,000.

To a mixture of 15 μL venom phosphodiesterase, 15 μL 0.1 M magnesium chloride, 15 μL 2 M TrisHCl buffer (pH 9) and 5 μL alkaline phosphatase, 50 μL of a solution of poly (m¹s⁶G) (2 mg/mL) was added, and the resulting mixture was incubated at 37ºC. The degradation of the polymer was complete within 5 h and the sole nucleoside product was determined to be l-methyl-6- thioguanosine. The ultraviolet spectra of the polymer before and after degradation were compared to calculate its extinction coefficient which was 15.810³.

Example 3

Preparation of poly (s⁶G)-poly(C).

2-Amino-6-chloro-9-(β-D-ribofuranosyl)-9H-purine production described in Gerster et al. (J. Org. Chem. 1963, 28, 945), and Gerster et al. (Synth. Proced. Nucleic Acid Chem. 1968, 1 , 242) was phosphorylated in trimethyl phosphate (2-3 mL for each mmol of the nucleoside) with 2 equivalent of phosphorus oxychloride at 0-2ºC over a period of 3 h (Yoshikawa et al. Bull . Chem. Soc. Japan 1969, 42 , 3505). The yield of the pure 5'-monophosphate was 65%. The conversion of 5'-phosphate to 5'-diphosphate was carried out in a 45% yield according to the method of Michelson (Biochim. Biophys. Acta 1964, 91, 1).

The polynucleotide phosphorylase catalyzed polymerization of the diphosphate to poly(2-amino-6-chloropurinylic acid) was carried out in a reaction mixture (35 mL) containing 0.2 M

TrisΗCl (pH 9), 5 mM magnesium chloride, 0.5 mM EDTA,

polynucleotide phosphorylase (70 mg, 0.94 unit/mg protein) and 20 mM 2-amino-6-chloro-9-(/3-D-ribofuranosyl)purine 5 '-diphosphate at 37ºC for 7 h. After the removal of protein by extraction with 5:2 chloroform-isopentyl alcohol (8 × 40 mL), the aqueous solution was successively dialyzed against 0.3 M NaCl + 5 mM EDTA (5 L, pH 7.5), 0.2 M NaCl + 5 mM EDTA (10 L) and lyophilized to give 120 mg of the polymer having a molecular weight of about 100,000.

To prepare poly(s⁶G), a solution of poly(2-amino-6-chloropurinylic acid) (15 mg) in 0.4 M TrisΗCl (15 mL, pH 8) in a glass tube was frozen by immersion in a dry ice-acetone bath. Hydrogen sul- fide gas was condensed into the tube until 10-15 mL of liquid was collected. The tube was then transferred to a steel bomb which was also kept in a dry ice-acetone bath. The bomb was sealed and transferred to an oil bath; the temperature was maintained at 60- 62ºC for 3 days. Ethanol (150 mL) was added and the reaction mixture was cooled in ice. The Trie salt of poly(s⁶G) precipitated as a white solid which was collected by centrifugation and washed with ethanol. It was then dissolved in water, dialyzed against 0.1 M NaCl + 1 mM EDTA (8 L, pH 7.5) and distilled in deionized water (10 L) and freeze-dried to yield 10 mg of poly(s⁶G) having a molecular weight of about 100,000.

Enzymatic degradation of poly(2-amino-6-chloropurinylic acid) was carried out in accordance with a published procedure by Broom et al. (Biochim. Biophys. Acta 1976, 374 , 407). Poly(s⁶G) was degraded by incubating a mixture of 50 μL of polymer solution (1 mg/mL), 20 μL of 0.1 M MOPS buffer (pH 7.2), 42 mg of urea, 10 μL of venom phosphodiesterase and 5 μL of alkaline phosphatase at 37ºC for 16-20 h.

A complex between poly(s⁶G) and poly (C) was prepared by heating a solution of the two polynucleotides in a suitable buffer at 70-75ºC for 1 h with occasional shaking and then allowing the solution to stand at room temperature for a few hours. The resulting duplex polymer had a molecular weight in the range of about 150,000 to 250,000.

Any desired salt (sodium, potassium, ammonium, etc., is readily formed simply by dialyzing the polynucleotide dissolved in water against 20 L of an aqueous solution of a salt of the desired cation (e.g. NaCl, KCl, NH₄Cl, etc.). The dialyzed solution is then lyophilized to produce the desired salt form of the polynucleotide.

The following summaries of experiments indicate activity of poly(m¹s⁶I) and poly(s⁶G)-poly(C) in assay tests against the AIDS virus. These tests are accepted by virologists as indicating the subject compounds would have utility against the AIDS virus in humans. The following results also show that the poly(m¹s⁶I), poly(m¹s⁶G) and poly(s⁶G)-poly(C) are effective in the control of the AIDS virus in comparison with AZT, one of the drugs that is commonly used in treatment of AIDS.

Example 4

Confirmatory antiviral assays were run using HIV CEM cells testing the activity of polyfm's'l), poly(m¹s⁶G), and AZT, respectively. These studies were carried out at Southern Research Institute using the following standard protocols.

Human Immunodeficiency Virus (HIV). A CPE-inhibition assay is employed to screen compounds for activity against HIV. Briefly, the CPE inhibition assay employs CEM or MT₂ cells, a human T-cell line. The cells are propagated in RPMI 1640 with heat- inactivated fetal calf serum, glutamine and antibiotics at 37ºC in an atmosphere of 5% CO₂ in air. The clone H9 cell line is another CD₄ ⁺ human T-cell line which is permissive for HIV replication but largely resistant to virus-induced CPE. H9 cells productively infected with HTLV-III B Strain of HIV are the source of infectious virus. These cells are propagated in RPMI 1640 medium supplemented with glutamine and antibiotics and with 20% heat-inactivated fetal bovine serum.

CEM or MT₂ cells are resuspended in supernatant virus and incubated for 1 hour at 37ºC. After adsorption, 1 × 10⁴ cells in 0.1 mL of medium are plated per well. An equal volume (0.1 mL) of supplemented RPMI 1640 medium containing test compound is then added. Test compounds are evaluated at 7 half log₁₀ dilutions ranging from 100 μg/mL to 0.32 μg/mL. Triplicate virus-infected cultures and one uninfected compound cytotoxicity control culture are evaluated at each concentration. Cultures are incubated at 37ºC in a humidified atmosphere of 5% CO₂ in air.

On Day 7 post-infection the viable cells are measured by the use of a tetrazolium salt, XTT (final concentration 200 μg/mL) in conjunction with PMS (final concentration 0.02 mM) which is added to the test plates. The optical density (O.D.) value is a function of the amount of formazan produced which is proportional to the number of viable cells. Plates are read at a wavelength of 450-650 nm on a Vmax plate reader.

The following Table 1 shows the inhibitory concentrations (e.g. IC25, IC50, and IC95), cell cytoxicity, (e.g. TC25, TC50 and TC95) and antiviral index Al (TC50/IC50 which is the tissue culture equivalent of therapeutic index) for each of the compounds tested. Table 1

Drug 25% 50% 95% poly(m¹s⁶I)

TC(μG/mL 231.00 >320.00 >320.00

IC(μG/mL) 7.81 14.40 38.70 AI(TC/IC) 29.59 >22.25 >8.27 poly(m¹S⁶G)

TC(μG/mL) 171.00 241.00 >320.00

IC(μG/mL) 5.51 13.20 38.70 AI (TC/IC) 30.97 18.20 >8.27

AZT

TC(μG/mL) 0, .000008 > 0.002670 > 0.002670

IC(μG/mL) 0. .000447 0.000902 - - - - - - - - AI(TC/IC) 0..018663 > 2.091238 - - - - - - - -

It should be noted the poly(m¹s⁶I), poly(m¹s⁶G) polymers have very high molecular weights (-10⁵-10⁶) ; whereas the molecular weight for AZT is 267. On a molar basis, therefore, all three drugs are seen to be active and nontoxic at submicromolar levels.

The antiviral index (Al) of the poly(m¹s⁶G) and poly(m¹s⁶G) polymers is comparable to that of AZT; however, the

polynucleotides act by different mechanisms and provide unique, multiple effects which make these drug potentially much more valuable and less likely to incur resistance than AZT.

Table 2 shows the parallelism between antiviral activity

(reduction in viral cytopathic effect "CPE") and inhibition of reverse transcriptase "RT" in intact cells using poly(m^ls⁶I) and poly(m¹s⁶G).

Table 2

Poly(m^ls⁶Il Poly( m^ls⁶G)

DRUG % REDUCTION * REDUCTION * REDUCTION % REDUCTION CQNC Vbal CPE RT ACΠVΠΎ VITAL CPE RT ACTIVITY

320μg/mL 90 99 46 98

100μglmL 100 99 100 93

32μglmL 94 76 94 68 10μglmL 30 10 36 0

3.2μg/mL 7 0 15 0

1.0μg/mL 4 0 7 0

% reduction in viral CPE was obtained from the XTT assay data sheets

% reduction in RT activity was obtained from the raw RT assay data (cpm per 15 uL of cell free supernatant) virus control was 82,465 cpm (100%)

cell control was 462 cpm (0%)

This establishes that inhibition of this key viral enzyme

(reverse transcriptase), not found in uninfected human cells, is one mechanism by which poly(m¹s⁶I), poly(m¹s⁶G) and related polynucleotides operate.

Table 3 confirms the in vitro potent inhibition of HIV-1 reverse transcriptase by subnanomolar concentrations of

poly(m¹s⁶I) and poly(m¹s⁶G). It may be seen that, even at 0.1 μg/mL, enzyme activity is less than 50% of control values.

Table 3

Compound Concentration % of

(μg/mL) Control (μg/mL) Polyfm's⁶!) 100 3.64 <0.1

10 0

1 7.05

0.1 41.60

Poly(m¹s⁶G) 100 0 <0.1

10 0

1 9.01

0.1 37.48

Poly(s⁶G):poly(C) 100 0 ~0.3

10 0

1 7.05

0.1 41.60

Table 4 illustrates the results of the syncytia reduction assay when treated at 100 μg/mL.

Table 4

Drug # Syncytia Percentage Reduction

Control 87 0

Poly(s⁶G):Poly)C)

Poly(m¹s⁶I) 0 100

Poly(m¹s⁶G) 14 84 92 0 The above data show that polyps*!), in addition to its potent inhibition of reverse transcriptase, is also effective in blocking syncytium formation, presumably by binding to fusogenic sites on infected and or uninfected T-cells. Polytm's^) has no such effect, and poly(s⁶G)-poly(C) potently inhibits syncytium formation, even though it does not inhibit the viral cytopathic effect. This reveals that these polynucleotides may exhibit at least two mechanisms of action, and these may be separated by appropriate alteration of chemical structure.

In Table 5 are presented comparisons of activity of

poly(m¹s⁶I), poly(m¹s⁶G), and AZT (positive control) against several strains of HIV-1 and HIV-2 as well as against various multiplicities of infection of

HIV-1. Table 5

INHIBITION OF HIV-1 AND HIV-2 (IC₅₀μg/mL)

HIV Strain Poly(m¹s⁶I) Poly(m¹s⁶G) AZT

HIV-1

IIIB 8.5 5.2 0.0006

LAV 20.4 13.0 0.0015

RF 17.2 24.9 0.0069

HIV-2

ROD 14.4 4.9 0.0082 MS 14.0 18.9 0.0028

MOI (HIV-1)

1 μL 13.9 5.1 0.0015 10 μL 241.0 44.1 0.0137 20 μL 320.0 (IC₂₅) 22.2 ( IC₂₅) 0.013 ( IC₂₅) 50 μL Inactive Inactive Inactive

It may be seen that the polynucleotides poly(m^ls⁶I), poly(m¹s⁶G) are active against all strains tested above, that their activity parallels that of AZT, and that, given the difference in

molecular weight, they are similar in potency to AZT. For

example, poly(m¹s⁶G) gives 50% inhibition of HIV-1 III B at about 10^-8 M versus about 10^-9 M for AZT. For HIV-2 ROD the molar

concentrations correspond to about 10^-8 M in both cases.

Example 5

Experimentation was conducted to evaluate the effectiveness of the presently described polyribonucleotides in inhibiting or blocking the activity of human cytomegalovirus (HCMV) as compared to one of the current drugs showing activity against HCMV

infections. HCMV is a double stranded DNA virus of the herpes family, as opposed to HIV which is a RNA retrovirus. The HCMV virus has worldwide distribution; in the United States, it is identifiable in an estimated one percent (1%) of newborns. HCMV is sexually transmitted and infections are often benign in

humans, but in the immunosuppressed patient, the clinical course of an HCMV infection can be devastating and life threatening. In patients with compromised immune functions, whether resulting from immunosuppressive agents, such as used to prevent organ transplant rejection, or in AIDS patients, the infection can be manifested as pneumonitis, hepatitis, colitis, retinitis and related conditions.

The drug chosen to conduct this experiment comparison is ganciclovir, specifically 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine or DHPG. DHPG inhibits HCMV DNA

polymerase and has been shown to have benefit in treating HCMV disease in humans, however, it has had significant toxicity problems in treated patients. Foscarnet (phosphonoformate) and HPMPC (hydroxyphosphonylmethoxypropylcytosine) are also drugs used to inhibit HCMV DNA polymerase, but with equally negative toxicity problems. Another drug, acyclovir (ACV) has in vitro activity against HCMV, its in vivo activity against this virus has been disappointing. It is thus desirable to develop drugs with low toxicity to the host, good bioavailability, and possibly, modes of action different from that of the existing drugs. Such drugs could then be used in place of, or in conjunction with, the existing anti-HCMV drugs. There is also a need for new drugs that will be effective against DHPG-resistant and foscarnet-resistant isolates of HCMV as both kinds of resistance have been seen in patients treated with these drugs.

The following experimentation will identify the usefulness of the defined polyribonucleotides in treating cells infected with HCMV.

MRC-5, a continuous line of diploid, human (male) embryonic lung cells obtained from the American Type Culture Collection (ATCC) , Rockville, MD, were the cells used in the experiment. HCMV strain AD-169, was purchased from ATCC. HCMV, strain C8704 and strain C8708 were both provided by Dr. Karen Biron of

Burroughs Wellcome Co., Research Triangle Park, NC. The growth medium for the cells consisted of Basal Medium Eagle (GIBCO BRL, Research Products Division, Life Technologies, Inc., Grand

Island, NY) with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) and 0.035% NaHCO₃, without antibiotics. The viral strain AD-169 has been passaged in the laboratory over many years, the strain C8708 is sensitive to DHPG, having been isolated, in early DHPG therapy, from a patient with symptoms of HCMV disease, and the strain C8704 is resistant to DHPG having been isolated from the same patient but after prolonged DHPG therapy.

The polyribonucleotide, poly(m¹s⁶I) and 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG) (collectively "compounds") were prepared and diluted with and at the concentration indicated in Dulbecco's Modified Eagle Media, containing 2% fetal bovine serum, 0.1% NaHCO₃, and 50 μg gentamicin/mL ("Test Medium"). The growth medium was decanted from established monolayers of the MRC-5 cells in 24-well tissue culture plates (Corning Glass Works, Corning, NY). One mL of virus was diluted in the test medium and was placed in each well except those to be used for cell control (2 wells/plate). The cell control contained 1.0 mL of sterile test medium. The plates were centrifuged at 2200 rpm for 30 minutes at room temperature to allow the virus to adsorb to the cells. The medium was aspirated from each well and 0.8 mL of either poly(m¹s⁶I) or DHPG in test medium was placed in test wells (2 wells/dilution). The test medium without either compound was added (0.8 mL/well) to cell control and virus control wells. The plates were incubated at 37°C in a moist atmosphere of 5% CO₂, 95% air until virus plaques could be distinguished in the virus control wells. The medium was then separately aspirated from all wells and the cells were stained by adding 0.3 mL of 0.2% crystal violet in 10% buffered formalin to each well. After 15 minutes, the stain was aspirated, the plates were rinsed in running tap water until the water was clear, and the plates were inverted and dried at room temperature. Plaques were counted by use of a dissecting microscope.

The following Tables 6 through 9 compare the antirival activity (or plaque reduction) of poly(m¹s⁶I) and DHPG for the various above noted HCMV Strains AD-169, C8708, and C8704.

Comparison of antiviral activity is accomplished through evaluation of respective: 1) ED50's, or effective dose with a 50% endpoint, which is the concentration at which the average number of plagues is reduced to 50% of that seen in the virus controls. 2) CD50's or the concentration of substance calculated to be halfway between the concentration which produces no visible effect on the cells and the concentration which produces complete cytotoxicity; and 3) TI's or therapeutic indexes, for each substance tested, calculated by the formula, TI equals CD50 divided by ED50.

Table 6

Antiviral Activity (Plaques Reduction) of Poly(m¹s⁶l), DHPG vs. Human Cytomegalovirus, Strain AD-169, in MCR1-5 Cells

Poly(m¹s⁶l) DHPG

Compound Number Number DHPG

Concentration of % of % Concentration

(μg/ml) Plaques Reduction Plaques Reduction (μg/ml)

1000 0 100 5.0 91 3.2

316 0 100 50.5 0 1.0

100 4.0 92

32 16.5 69

10 20.0 63

3.2 28.5 47

1.0 38.5 28

ED50^a(μg/ml): 4.1 1.9

CD50^b(μg/ml): 154 >1000 (previous data)

TF; 38 >5

a The concentration at which the average number of plaques is reduced to 50% of that seen in the virus controls (Effective Dose, 50% endpoint).

b The concentration halfway between those at which 100% and 0% cytotoxicity are seen.

c Therapeutic Index (CD_50, ⁺ED₅₀).

Table 7

Antiviral Activity (Plaque Reduction) of Poly(m¹s⁶l), or DHPG vs. Human Cytomegalovirus, Strain C8708, In MCR-5 Cells

Poly(m¹s⁶I) DHPG

Compound Number Number DHPG

Concentration of % of % Concentration

(μg/ml) Plaques Reduction Plaques Reduction (μg/ml)

1000 toxic toxic 0 100 32

316 0 100 4.5 91 10

100 7.5 85

32 17.5 65

10 19.0 62

3.2 37.0 26

1.0 37.5 25

ΕD50^a(μg/ml): 6.8 > 10

CD50^b(μg/ml): 178 > 1000

TT: 26 > 100

a The concentration at which the average number of

plaques is reduced to 50% of that seen in the virus

controls (Effective Dose, 50% endpoint).

b The concentration halfway between those at which

100% and 0% cytotoxicity are seen,

c Therapeutic Index (CD_50, ⁺ED₅₀).

Table 8

Antiviral Activity (Plaque Reduction) of Poly (m^ls⁶l), or DHPG vs. Human Cytomegalovirus, Strain AD-169, in MRC-5 Cells

poly(m¹s⁶I) DHPG

Compound Number Number

Concentration of % of %

(μg/ml) Plaques Reduction Plaques Reduction

316 0 100 0 100

100 10.0 80 0 100

32 14.5 72 0 100

10 26.0 50 0 100

3.2 28.0 46 4.5 91

1.0 29.0 44 26.0 50

0.32 44.5 0 25.5 50

0.1 41.0 0 51.5 0

0.032 51.5 0

ED50^a(μg/ml): -10.0 1.0

CD50^b(μg/ml): -316 >1000

TT: 32 > 100

a The concentration at which the average number of

plaques is reduced to 50% of that seen in the virus

controls (Effective Dose, 50% endpoint).

b The concentration halfway between those at which

100% and 0% cytotoxicity are seen.

c Therapeutic Index (CD_50, ⁺ED₅₀). Table 9

Antiviral Activity (Plaque Reduction) of Poly(m¹s⁶l), or

DHPG vs Human Cytomegalovirus, Strain C8704, in MRC-5 Cells

Poly(m¹s⁶l) DHPG

Compound Number Number

Concentration of % of %

(μg/ml) Plaques Reduction Plaques Reduction

316 0 100 0 100

100 3.0 83 1.0 94

32 8.0 54 4.5 74

10 5.5 68 18.5 0

3.2 16.0 0 20.5 0

1.0 21.0 0 27.0 0

032 21.5 0 26.0 0

0.1 21.0 0 19.5 0

0.032 19.5 0

ED50^a(μg/mI): ~17.4 22

CD50^b(μg/mI): ~178 >1000

TT^c: 24 >45

a The concentration at which the average number of plaques is reduced to 50% of that seen in the

virus controls (Effective Dose, 50% endpoint).

b The concentration halfway between those at which 100% and 0% cytotoxicity are seen,

c Therapeutic Index (CD₅₀, ⁺ED₅₀).

The data described in Tables 6 through 9 represent unique and novel results compared to DHPG. The data describes the effectiveness of the defined polyribonucleotides in inhibiting or blocking the activity of HCMV, over the current standard drug DHPG. Compared to DHPG as the standard positive control, poly(m¹s⁵l) is approximately two orders of magnitude more potent on a molar basis (molecular weight ca. 100,000 compared to ca. 300 for DHPG). More importantly, poly(m¹s⁶I) is fully active against a DHPG resistant strain. The lack of resistance of the DHPG-resistant HCMV strain C8704 to poly(m¹s⁶I) shown in Table 9 is especially interesting since such activity would allow effective poly(m^ls⁶I) treatment of patients who are resistant to therapy with DHPG. This difference in activity also indicates a different mechanism of antiviral action by poly(m¹s⁶I) from that of DHPG. The mechanism by which this class of compounds is active against HCMV is not known, but it seems not to be due to inhibition of virus adsorption to the cells since poly(m¹s⁶I) was added to the cells after the virus had adsorbed to the cells. Equally impressive, and unpredicted based on current knowledge of drug activity and mechanism action, is that the defined

polyribonucleotides are effective against, DNA herpes viruses such as HCMV, as well as RNA retroviruses such as HIV viruses. DNA herpes viruses and retroviruses have different replication mechanisms (in contrast to DNA herpes viruses, the retroviruses utilize reverse transcriptase) . Since the defined

polyribonucleotides have a unique mechanism of action, the possibility exists of utilizing the compounds in a synergistic manner with known agents, or alone, for multiple disease

conditions. Contemporary thought has been that one drug was not effective against DNA herpes viruses and retroviruses, this experimentation runs counter to this till now established consensus.

While certain representative embodiments of the invention have been described herein for purposes of illustration, the invention may be embodied in other specific forms without departing from its spirit or essential characteristics, and the described examples should not be considered restrictive but only

illustrative. The scope of the invention is therefore defined by the appended claims rather than by the foregoing examples.

Claims

1. A method of treating a human being infected with AIDS or AIDS-related viruses by inhibiting HIV from developing or spreading to unaffected cells which comprises administering to said human being an effective amount of an active ingredient which is a homopolyribonucleotide containing polymer or duplex polymer selected from the group consisting of poly(m¹s⁶I), poly(m¹s⁶G) and poly(s⁶G)-poly(C) and pharmaceutically acceptable salts thereof and mixtures of such a polymer or duplex polymer with a pharmaceutically acceptable salt thereof contained in a pharmaceutically acceptable carrier.

2. The method of Claim 1 wherein said administration is by a route selected from the group consisting of oral, nasal, topical and parenteral.

3. The method of Claim 2 wherein said administration is by a parenteral route selected from the group consisting of

subcutaneous, intramuscular, intravenous and intradermal.

4. The method of Claim 2 wherein said administration is oral.

5. The method of Claim 2 wherein said active ingredient is administered in an amount in the range of between about 1 to 50 mg/Kg body weight of human being/day.

6. The method of Claim 3 wherein said active ingredient is poly(m¹s⁶I), a pharmaceutically acceptable salt thereof or a mixture of said poly(m¹s⁶I) and a pharmaceutically acceptable salt thereof.

7. The method of Claim 3 wherein said active ingredient is poly(m¹s⁶G), a pharmaceutically acceptable salt thereof or a mixture of said poly(m¹s⁶G) and a pharmaceutically acceptable salt thereof.

8. The method of Claim 3 wherein said active ingredient is poly(s⁶G)-poly(C), a pharmaceutically acceptable salt thereof or a mixture of said poly(s⁶G)-poly(C) and a pharmaceutically

acceptable salt thereof.

9. The method of Claim 4 wherein said active ingredient is poly(m¹s⁶I), a pharmaceutically acceptable salt thereof or a mixture of said poly(m¹s⁶I) and a pharmaceutically acceptable salt thereof.

10. The method of Claim 4 wherein said active ingredient is poly(m¹s⁶G) , a pharmaceutically acceptable salt thereof or a mixture of said poly(m¹s⁶G) and a pharmaceutically acceptable salt thereof.

11. The method of Claim 4 wherein said active ingredient is poly(s⁶G)-poly(C), a pharmaceutically acceptable salt thereof or a mixture of said poly(s⁶G)-poly(C) and a pharmaceuticily acceptable salt thereof.

12. A method of treating a human being infected with a viral opportunistic pathogen by inhibiting the pathogen from developing or spreading to unaffected cells which comprises administering to said human being an effective amount of an active ingredient which is a homopolyribonucleotide containing polymer or duplex polymer selected from the group consisting of poly(m¹s⁶I), poly(m¹s⁶G) and poly(s⁶G)-poly(C) and pharmaceutically acceptable salts thereof and mixtures of such a polymer or duplex polymer with a pharmaceutically acceptable salt thereof contained in a pharmaceutically acceptable carrier.

13. The method of claim 12 wherein said viral opportunistic pathogen is a DNA virus, of the herpes family.

14. The method of claim 13 wherein said herpes DNA virus is human cytomegalovirus.

15. The method of Claim 12 wherein said administration is by a route selected from the group consisting of oral, nasal, topical and parenteral.

16. The method of Claim 15 wherein said administration is by a parenteral route selected from the group consisting of subcutaneous, intramuscular, intravenous and intradermal.

17. The method of Claim 15 wherein said administration is oral.

18. The method of Claim 16 wherein said active ingredient is poly(m¹s⁶I), a pharmaceutically acceptable salt thereof or a mixture of said poly(m¹s⁶I) and a pharmaceutically acceptable salt thereof.

19. The method of Claim 16 wherein said active ingredient is poly(m¹s⁶G), a pharmaceutically acceptable salt thereof or a mixture of said poly(m¹s⁶G) and a pharmaceutically acceptable salt thereof.

20. The method of Claim 16 wherein said active ingredient is poly(s⁶G)-poly(C), a pharmaceutically acceptable salt thereof or a mixture of said poly(s⁶G)-poly(C) and a pharmaceutically

acceptable salt thereof.

21. The method of Claim 17 wherein said active ingredient is poly(m¹s⁶I), a pharmaceutically acceptable salt thereof or a mixture of said poly(m¹s⁶I) and a pharmaceutically acceptable salt thereof.

22. The method of Claim 17 wherein said active ingredient is polyfm's^G), a pharmaceutically acceptable salt thereof or a mixture of said poly(m's⁶G) and a pharmaceutically acceptable salt thereof.

23. The method of Claim 17 wherein said active ingredient is poly(s⁶G)-poly(C), a pharmaceutically acceptable salt thereof or a mixture of said poly(s⁶G)-poly(C) and a pharmaceuticlly acceptable salt thereof.

24. A method of treating a human being infected with AIDS or AIDS-related viruses and viral opportunistic pathogens by inhibiting the viruses from developing or spreading to

unaffected cells which comprises administering to said human being an effective amount of an active ingredient which is a homopolyribonucleotide containing polymer or duplex polymer selected from the group consisting of polytm's'l), polyfm's^) and poly(s¹G)-poly(C) and pharmaceutically acceptable salts thereof and mixtures of such a polymer or duplex polymer with a

pharmaceutically acceptable salt thereof contained in a

pharmaceutically acceptable carrier.

25. The method of Claim 24 wherein said administration is by a route selected from the group consisting of oral, nasal, topical and parenteral.

26. The method of Claim 25 wherein said administration is by a parenteral route selected from the group consisting of subcutaneous, intramuscular, intravenous and intradermal.

27. The method of Claim 25 wherein said administration is oral.

28. The method of Claim 26 wherein said active ingredient is poly(m¹s⁶I), a pharmaceutically acceptable salt thereof or a mixture of said poly(m¹s⁶I) and a pharmaceutically acceptable salt thereof.

29. The method of Claim 26 wherein said active ingredient is poly(m¹s⁶G), a pharmaceutically acceptable salt thereof or a mixture of said poly(m¹s⁶G) and a pharmaceutically acceptable salt thereof.

30. The method of Claim 26 wherein said active ingredient is poly(s⁶G)-poly(C), a pharmaceutically acceptable salt thereof or a mixture of said poly(s⁶G)-poly(C) and a pharmaceutically

acceptable salt thereof.

31. The method of Claim 27 wherein said active ingredient is poly(m¹s⁶I) , a pharmaceutically acceptable salt thereof or a mixture of said poly(m¹s⁶I) and a pharmaceutically acceptable salt thereof.

32. The method of Claim 27 wherein said active ingredient is poly(m¹s⁶G), a pharmaceutically acceptable salt thereof or a mixture of said poly(m¹s⁶G) and a pharmaceutically acceptable salt thereof.

33. The method of Claim 27 wherein said active ingredient is poly(s⁶G)-poly(C), a pharmaceutically acceptable salt thereof or a mixture of said poly(s⁶G)-poly(C) and a pharmaceuticlly acceptable salt thereof.