WO1992003052A1

WO1992003052A1 - Method for suppression of hiv replication by ascorbate for chronic and acute hiv infection

Info

Publication number: WO1992003052A1
Application number: PCT/US1991/005895
Authority: WO
Inventors: Raxit J. Jariwalla; Steve M. Harakeh
Original assignee: Jariwalla Raxit J; Harakeh Steve M
Priority date: 1990-08-23
Filing date: 1991-08-23
Publication date: 1992-03-05

Abstract

A method is provided for treating symptomatic or nonsymptomatic conditions associated with HIV infection by inhibition of HIV replication, by administering to an infected subject a therapeutically-effective amount of ascorbate, ascorbic acid, their metabolic products, derivatives or mixtures thereof.

Description

METHOD FOR SUPPRESSION OF HIV REPLICATION BY ASCORBATE FOR CHRONIC AND ACUTE HIV INFECTION

The present invention is directed to a method for treating conditions associated with HIV infection by administering to an infected subject a therapeutic amount of ascorbate, or ascorbate in combination with other drugs efficacious for treatment of HIV-infection.

BACKGROUND

Previous studies demonstrated the antiviral activity of ascorbate against a broad spectrum of RNA and DNAviruses in vitro (Murata, et al., A r. Biol. Chem. , 36, 1065; 2597 (1972); Schwerdt, et al.. , Proc. Soc. Exp. Biol. Med.. 148, 1237 (1975) ; Bissell, et al. , P.N.A.S. USA. 77, 2711 (1980)) and in vivo (Klenner, J. Appl. Nutr. , 23, 61 (1971); Cathcart, Bioloαisk Medicin. 3., 6 (1983)). Ascorbate was claimed to have inhibited the activation of a latent human retrovirus (HTLV-1) induced by 5-iodo- 2•-deoxyuridineandN-methyl-N'-nitro-N-nitrosoguanidine (Bla eslee, et al. , Cancer Res., 45, 3471 (1985)). However, it was not established whether ascorbate exerted a virus-specific effect or interacted directly with the activating substances. In addition, the effects of ascorbate on acute infection by human retroviruses or constitutive virus production associated with chronic infection have not heretofore been determined. Oral and intravenous administration of ascorbate is said to have produced clinical improvement inpatients afflictedwith influenza, hepatitis, and herpes virus infections, including infectious mononucleosis (Klenner, supra, and Cathcart, supra) . In one study, some AIDS patients who voluntarily ingested high doses of ascorbic acid manifested clinical improvement (Cathcart, Medical Hypotheses. 14, 423 (1984)). The author attributed the effect to scavenging by ascorbate of free radicals produced by the disease and its associated infections.

Inhibitors of reverse transcriptase (RT) activity have been the focus of intensive investigation for the development of antiretroviral agents. Among these, 3^»-azido-3'-deoxythymidine (AZT) , the first drug approved for AIDS treatment, blocks de novo HIV infection but has recently been shown not to inhibit virus production in cells containing integrated HIV genomes. See Poli, et al. , Science 244. 575 (1989) . In the same study, interferon-α inhibited the budding and release of HIV from chronically infected cells stimulated with cytokines (TNF and PMA) , but did not suppress constitutive virus production in unstimulated cells.

It is therefore an object of the present invention to provide a method for treatment of conditions associated with chronic and acute HIV infection by inhibition of HIV-replication, comprising the step of administering to a subject noncytotoxic ascorbate concentrations which are sufficient to inhibit virus replication.

It is yet a further object of the present invention to provide a method for treating both symptomatic conditions of HIV infection, such as AIDS, and nonsymptomatic conditions, such as ARC. These and other objects will be apparent from the following description and appended claims and from the practice of the invention.

SUMMARY OF THE INVENTION

The present invention provides a method for combatting HIV infection by inhibition of HIV replication in a subject comprising the step of administering to the subject a therapeutically-effective amount of a compound selected from the group consisting of pharmaceutically- acceptable ascorbate salts; ascorbic acid; metabolic products of ascorbic acid or ascorbate salts; derivatives of ascorbic acid, ascorbate salts or the metabolic products thereof; and mixtures of two or more of any of the foregoing compounds.

DESCRIPTION OF THE FIGURES

Fig. 1 is a graph analysis of cytotoxicity of ascorbate for HTLV-III_B-infected H9 T-lymphocytic cells, as determined by trypan blue dye exclusion. Each point is the mean of four cell counts.

Fig. 2 shows the effect of ascorbate on reverse transcriptase (RT) activity in supernatant harvested from H9/HTLV-III_B cultures. In control samples, the RT values on day 2 and day 4 were, respectively, 55 x 10⁴ and 267 x 10^A cpm/10⁶ cells; average background value in blanks (i.e., reactions without enzyme) was 1530 cpm per ml culture supernatant. In each experiment, the mean of three sampleswas determinedand comparedas a percentage of control (taken as 100%) .

Fig. 3 shows the effect of ascorbate on HIV p24 antigen levels in supernatant harvested from H9/HTLV-III_B cultures. Extracellular p24 was assayed by Abbott HIV antigen enzyme immunoassay. In control samples, the p24 levels on days 2 and 4 were, respectively, 244 and 45 nanograms/10⁶ cells. The p24 values of ascorbate-treated cultures are compared as a percentage of control.

Fig. 4 is a graph of metabolic activity in H9 cells, as determined by MTT assay, in the presence and absence of ascorbate. Each point is the mean of four OD₅₇₀ readings. Data are plotted as percentage of control.

Fig. 5 shows the protein synthesis rates in H9 cells in the presence and absence of ascorbate. Each point is the mean of ³⁵S-labeled amino acid incorporation per 10° cells.

Fig. 6 shows the dose-dependent decrease in HIV-induced syncytium formation with ascorbate. Syncytia were counted in CD₄ ⁺ VB cells using a light microscope. Each point represents the mean of at least four samples and is compared as a percentage of the control infected cultures from the same experiment.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The active ingredient according to the present invention may be any pharmaceutically-acceptable ascorbate salt including, but not limited to, calcium, magnesium, potassium, or sodium salt. An active ingredient may also be ascorbic acid. Pharmaceutically-acceptable derivatives of ascorbic acid or ascorbate are also contemplated such as benzoylated ascorbate and other acylated ascorbates, palmitates or stearates. Metabolic products of ascorbic acid or ascorbates are also within the scope of the present invention, which metabolic products include dehydroascorbate, dehydroascorbic acid, gulonolactone or gulonic acid and furan-type compounds that form adducts with amino and hydroxyl groups of p^'roteins. See Nakanishi, et al. , Eur. J. Biochem. , 152, 337 (1985); Garland, et a_l. , Arch. Biochem. Biophvs.. 251, 771 (1986); and Ortwerth, et al., EXP. Eve Res. , 42, 155 (1988).

Mixtures of ascorbate salts, ascorbic acid, metabolic products of ascorbic acid or ascorbate salts, derivatives of ascorbic acid or ascorbate salts or of the metabolic products are also contemplated to be within the scope of the present invention.

The above-described compounds will be administered in a therapeutically-effective amount to the HIV-infected subject. As described below, the effective amount of ascorbate inhibiting replication of HIV in vitro is greater than about 50 micrograms ascorbate/ml of cell growth medium and the cytotoxic amount is greater than about 400 μg/ml. The preferred methods for in vivo use of ascorbate in accordance with the present invention includes oral administration of preferably about 20 to 60 grams/day of ascorbate or other active compoundwithin the scope of the present invention. It will be realized that this dosage level is approximate andmay be exceeded since there is a high bowel tolerance for ascorbate.

Another preferred method of administration is by intravenous administrationbydrips ordirect infusions. The useful dosage for intravenous injection is about 20 to 180 grams/day.

Alternatively, one or a mixture of compounds according to the present invention may be utilized, particularly in less than a therapeutically-effective amount, when used in combination with other drugs used for treatment of HIV infection, such as AZT. The method of the present invention is intended to be used for treatment of any condition associated with HIV infection whether that condition be symptomatic or nonsymptomatic of the infection. The primary symptomatic condition of HIV infection is AIDS whereas the primary nonsymptomatic condition of HIV infection is ARC.

It is surprising that ascorbate, ascorbic acid, their derivatives or metabolic products are useful for treatment of conditions associated with HIV infection. To validate the activity and mode of action of the compounds utilizing the method according to the present invention, the following tests were conducted on acutely and chronically HIV-infected T-lymphocytic cell lines grown continuously in the presence of nontoxic concentrations of ascorbate. Tests were conducted on the action of ascorbate on cell-free virus particles in vitro. The following methodology and experiments are presented for purpose of validation and illustration of the invention, but are not intended to limit the invention in any way.

MATERIALS AMD METHODS

Cells and Cell Viability. H9 and H9/HTLV-III_B cells (Popovic, et al. , Science. 224. 497 (1984)) were originally obtained from Dr. Howard Streicher (National Cancer Institute, National Institute of Health) . In some experiments, batches of the same cell lines provided by Dr. Michael McGrath, University of California at San Francisco, were also utilized, with identical results. Cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum, 2mM L-glutamine, ImM pyruvate and 50 μg of gentamycin/ml. The CD4-positive VB cell line (Lifson, et ai. , Science, 232. 1123 (1986)) was propagated in RPMI-1640 complete growth medium. Cell -7- viability was determined by using the trypan blue exclusion method.

Ascorbate. Stock solution of L-ascorbate was made by dissolving L-ascorbic acid (tissue culture grade from Sigma Chemicals) in RPMI-1640 medium, and was stored at -20'C.

Experimental Protocol. Fresh working solutions (lOx strength) of ascorbate were prepared daily by diluting the stock in complete growth medium. For cytotoxicity assay, 3x10^s cells were suspended in 0.9 ml of growth medium and seeded in 24-well microtiter plates. Fresh solutions of ascorbate (0.1 ml of lOx strength) were added daily to obtain final concentrations of 10, 25, 50, 75, 100, 150, 200, 300, and 400 μg/ l. The controls received 0.1ml of growth medium. Plates were incubated at 37°C in 5% C0₂/95% air humidified atmosphere for various time intervals. At periodic intervals, 0.5 ml aliguots of cell suspension were collected, mixed with 50 μl trypan blue, and tested for viability.

For guantitation of viral and cellular parameters, cell suspensions (in triplicate) were collected, pooled, and centrifuged at 2000 rpm for 10 min. at 4 C. Supernatant was used for assays of extracellular RT activity and p24 antigen. Cell pellets were used for the determination of cellular metabolic activity and protein synthesis rates.

Assay of RT. Virus particles in supernatant were pelleted by centrifugation in a refrigerated microfuge (13,500 rpm, 2 hrs) , then resuspended in l/50th of original volume of TNE buffer. Aliguots (10 μl ) were assayed for RT activity as described by Hoffman, et jal.. , using fresh batches of [methyl-³H]-dTTP (5A ~80 Ci/ mol, NEN/Du Pont research products) . RT activity was expressed as the amount of [^Hj-dTMP incorporated (cpm/10⁶ cells) .

Assay of p2 . Levels of p24 antigen in supernatant were assayed using the Abbott HIV antigen enzyme immunoassay (Goudsmit, Lancet ii. 177 (1986) ; Abbott Laboratories, North Chicago, IL) . The p24 value was expressed as nanograms/10⁶ cells for antigen released from infected cells or nanogram/ml for antigen contained in cell-free virus preparation.

Assay of Protein Synthesis. For radiolabeling, H9 cells (3 x 10⁵ cells per well in microtiter plates) were grown in the presence of 0, 75, 100 and 150 μg/ml ascorbate as described earlier. On days 1, 2 and 4, cells were harvested, washed and resuspended in methionine- and cysteine-free medium and then incubated at 37"C for 30 min in 0.5 ml of the same medium supplemented with 50 μCi of ³⁵S-Translabel (5A 1013 Ci/Mm, ICNRadiochemicals) . Labeled cells were pelleted, washed in phosphate-buffered saline, resuspended in lysis buffer containing 1% NP40, and stored at -70"C. Lysate was thawed and incubated at 100*C for 3-5 min to uncharge transfer RNA. Proteins were precipitated with trichloroacetic acid (TCA) in the presence of bovine serum albumin (0.2 mg per ml) , transferred to nitrocellulose filters (0.45 μm) , dried, suspended in Seta-blend (ICN Radiochemicals) , and counted in a scintillation counter. Protein synthesis was determined on duplicate samples of cells independently grown in the presence of ³⁵S-labeled amino acids.

Metabolic Activity Assayed by MTT Determination. For metabolic activity assay, 3 x 10^s cells were seeded in each well of 24-well microtiter plates and grown in the presence of 0, 75, 100, and 150 μg/ml ascorbate. On days 1, 2 and 4, cells were pelleted, resuspended in 1.0 ml growth medium supplemented with 10% (v/v) MTT (3-(4,5- -9- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, from Sigma Chemicals), incubated for 4 hrs, and treated with acidified isopropanol, and the absorbance at 570 n was measured as described by Mossman, J. Immunol. Methods. .65_. 55 (1983).

Inhibition Assay for the Cytopathic Effect of HTLV-III_B.

Infectious HIV stockwas obtained from supernatant fluid of H9/HTLV-III_B cells cocultivated with VB cells at a 1:7.5 ratio for 3^ days. To guantitate syncytium formation, 2.5 x 10⁵ VB cells in 0.4ml growth medium were mixed with 0.5ml HIV stock and seeded in 24-well microtiter plates. Then 0.1ml of either growth medium or lOx strength fresh L-aεcorbate solution was added daily and the cells were incubated. On specific days after infection, total number of giant cell syncytia in each well were counted under the microscope using xlOO magnification. A giant cell was defined as a cell >4 diameters larger than a single uninfected cell.

Cytotoxicity of Ascorbate Before determining the effect on HIV production, the cytotoxicity of ascorbate on H9/HTLV-III_B cells was evaluated, which are T-lymphocytic H9 cells infected with the AIDS virus (Popovic, supra) . Ascorbate is unstable in solution as in conventional culture conditions, with a short half life, so an experimental protocol was adopted inwhich cell cultures were given daily additions of fresh solutions of ascorbic acid prepared in buffered growth medium (pH 7.3 ± 0.1). Cells were grown in the continuous presence of varying ascorbate concentrations (0 to 400 μg/ml) for a period of four days. Viability of control and ascorbate-treated cultures was determined using the trypan blue exclusion test. No toxicity was observed when cultures were grown in the presence of 5 to 150 μg/ml ascorbate (Fig. 1) . A slight inhibition of cell growth (73-75% survival) was seen on the fourth day of incubation in medium containing ascorbate at 200-300 μg/ml. Cytotoxicity became prominent (>50% cell death) on the fourth day at ascorbate concentration of 400 μg/ml and higher. A slight increase in cell number was noted at concentrations ranging from 10 to 400 μg/ml on the first two days and at 5 to 75 μg/ml ascorbate on day 4. The remaining experiments evaluating ascorbate effects on HIV production were carried out at noncytotoxic concentrations of the compound.

Effects of Ascorbate on HIV Released from

Chronically infected Cells a. Extracellular RT Activity in supernatant. Reverse transcriptase (RT) activity was assayed in cell-free supernatant (Hoffman, et al., Viroloσv. 147, 326 (1985)) harvested from cultures grown in non-toxic ascorbate concentrations (0 to 150 μg/ml) . Fig. 2 shows the average of RT values of ascorbate-treated cultures and controls from 3 independent experiments. In the controls, RT titer manifested a peak of virus production on day 4. In contrast, ascorbate-treated cultures showed a striking inhibition of RT production. The first noticeable drop (64% inhibition) in RT titer occurred on day 2 at 50 μg/ml ascorbate, followed by a progressive decline in a dose-responsive manner. Further decreases in RT level were seen with increase in both ascorbate concentration and time of exposure. On day 4, over 99% inhibition in RT titer was seen at 150 μg/ml ascorbate. A noticeable increase in RT titer consistent with stimulation of cell growth was noted at low concentrations of ascorbate (from 5 to 25 μg/ml) on day 2. However, increase in virus production was transient, as these effects did not persist on day 4 of incubation.

b. p24 Levels in Supernatant. Another parameter of HIV production is the expression of p24 core antigen. Average values computed from three independent experiments are presented in Fig. 3. Control cultures showed a rise in p24 antigen levels at day 2, reaching maximum levels on day 4. In contrast, p24 antigen expression was blocked in ascorbate-treated cultures. Concentrations of ascorbate required to inhibit p24 synthesis were higher than those effective in inhibiting RT production. Thus, the first significant reduction in p24 levels was seen at 150 μg/ml ascorbate on day 2, Higher declines in p24 valueswere observedwith increase in time of exposure to ascorbate. On the fourth day, p24 levels in cultures treated with 150 μg/ml ascorbate were reduced to 13% of the control.

Effect of Ascorbate on Cell Metabolism

The following work addressed the question of whether ascorbate-induced suppression of RT and p24 production in H9/HTLV-III_B cells was a virus-specific effect or an indirect effect due to inhibition of cellular metabolism or protein synthesis. The metabolic activity of uninfected H9 cells in the presence and absence of ascorbate was determined by using a quantitative colorimetric assay that utilizes the tetrazolium salt MTT (Mossman, J. Immunol. Methods. 65, 55 (1983)). This salt measures the activity of various dehydrogenases in viable cells (Montagnier, et al. , AIDS Res. Hum. Retroviruses. 4. (6) , 441 (1988) ) . H9 cells grown in the presence of different concentrations of ascorbate (0 to 150 μg/ml) showed an increase in cellular metabolic activity on day 1 (Fig. 4) . This correlated with stimulation of cell proliferation by ascorbate. On days 2 and 4, no significant change in metabolic activity was noted between control cultures and those exposed to ascorbate at concentrations of 75, 100, and 150 μg/ml.

Effect of Ascorbate on Cellular Protein Synthesis

The effect of ascorbate on cellular protein synthesis was determined by growing uninfected H9 cells for 4 days at concentrations of 0, 75, 100, and 150 μg/ml (So asundaran, et aJL. , Science, 242, 1554 (1988) ) . On day 1, ascorbate was observed to stimulate protein synthesis, consistent with stimulation of metabolic activity and cell growth. On days 2 and 4, there was a less than 2-fold difference in the apparent rates of cellularprotein synthesis inboth ascorbate-treated and control cultures (Fig.5) . Thus the suppressive effects on HIV production could not be ascribed to a general inhibition of cellular metabolism or protein synthesis.

Effect of Ascorbate on Virus Replication in Freshly Infected Cells

To extend these findings to freshly infected cells, we investigated the effects of ascorbate on acute HIV infection of susceptible CD4⁺ T-lymphocytes. Viral infectivity and cytopathic effect in these cells have been correlated with formation of giant-cell syncytia mediated by interaction of HIV envelope glycoprotein with CD4⁺ cell surface receptor. In controls, multinucleated syncytia became visible by day 4, reaching high levels on day 6. The continuous presence of ascorbate in the growth medium of infected cells caused a dose- and time- dependent decrease in syncytium formation. On day 4, approximately 93.3% inhibition in syncytia number was seen at 100 μg/ml ascorbate (Fig. 6) . At this concentration, ascorbate did not inhibit the growth of uninfected VB cells (99% survival by trypan blue dye exclusion) , indicating that the inhibition of virus replication was not due to cytotoxic effect of the compound. Direct Inactivation of Virus Particles in Supernatant

The following work addressed the anti-HIV mechanism to determine whether decrease in RT titer and syncytium formation were due to direct inactivation of virus particles by ascorbate in vitro. Cell-free supernatant containing infectious virus was prepared and incubated in the presence and absence of ascorbate at 37*C for 8 and 18 hrs. Samples were tested for RT activity and syncytium formation was measured in VB cells. After incubation at 37"C for 18 hrs, there was no detectable difference in RT activity between ascorbate-treated virus preparations and controls (Table 1) . Syncytium-forming titer of infectious virus of ascorbate-treated and untreated preparations after incubation at 37 ' C for one day was also approximately equal (2.34-2.70 x 10³

TCID₅₀/ml) . When chronically infected cells were exposed to 150 μg/ml ascorbate for 18 hrs at 37^βC, the RT titer in culture supernatant was reduced to 11.2% of the control (Table 1) . These results indicate that decrease in extracellular RT titer, first seen after overnight treatment of chronically infected cells by ascorbate, was not due to direct inactivation of cell-free virus.

To study RT stability further in the presence of ascorbate upon prolonged incubation (37°C for several days) , the following experiment was carried out. Since thermal inactivation of cell-free virus occurs upon extensive incubation at 37*C, uninfected cells were used to protect virus from heat inactivation. These conditions resemble those present in experiments utilizing HIV-infected cell lines that were grown in the continuous presence of ascorbate for several days.

Accordingly, HIV supernatant was mixed with uninfected

VB cells and incubated with ascorbate for 4 days, with daily addition of fresh compound. Supernatants were harvested and assayed for RT activity. After 4 days in the presence of 100 and 150 μg/ml ascorbate, RT activity was reduced respectively to 31.5% and 7.0% of control (Table 1). In parallel experiments, chronically infected cells were exposed to 100 and 150 μg/ml ascorbate for 4 days. The RT levels in supernatant were reduced to 4.0 and 0.6% of control (Table 1).

In another experiment, the stability of p24 antigen was compared in the presence and absence of ascorbate at 37*C for 4 days. After incubation of cell-free virus with 150 μg/ml ascorbate, the concentration of p24 protein in the ascorbate-treated preparation (283 ng/ml) was not significantly different from that of the control (263 ng/ml) , indicating that the compound does not cause loss of p24 antigenicity in vitro. At the same ascorbate concentration, chronically infected cells exhibited -8- fold reduction in p24 antigen production after 4 days at 37^βC (Fig. 3) .

Table 1. Analysis of RT stability and RT production in the presence of and absence of ascorbate.

.

O

ll HIV virus supernatant was prepared from H9/HTLV-III_B cells. Virus supernatant alone or a suspension of supernatant and uninfected VB cells (3 x 10^s cells per ml) were exposed to 0_f 100 and 150 μg/ml ascorbate and incubated at 37^*C with daily addition of fresh compound. In a parallel experiment, chronically-infected H9/HTLV-III_B were grown under similar conditions. At different time periods, supernatants were collected and assayed for RT activity as described in Materials and Methods. ND — not done.

Claims

-16- WHAT IS CLAIMED IS:

1. A method for combatting HIV-infection in a subject having a condition which is nonsymptomatic of said infection, by inhibition of HIV replication comprising the step of administering to said subject a therapeutically-effective amount of a compound selected from the group consisting of pharmaceutically-acceptable ascorbate salts; ascorbic acid; metabolic products of ascorbic acid or said salts; derivatives of ascorbic acid, said salts or saidmetabolicproducts; andmixtures thereof.

2. A method according to Claim 2 wherein said condition is ARC.

3. Amethod for controlling HIV-infection in a subject having a condition which is symptomatic of said infection, by inhibition of HIV replication, comprising the step of administering to said subject a therapeutically effective amount of a compound selected from the group consisting of metabolic products of ascorbic acid or pharmaceutically acceptable ascorbate salts, derivatives of ascorbic acid or pharmaceutically acceptable ascorbate salts, and mixtures thereof.

4. A method according to Claim 3 wherein said condition is AIDS.

5. A method according to Claim 1 or 3 wherein said administration is oral.

6. A method according to Claim 1 or 3 wherein said administration is intravenous.

7. A method according to Claim 1 or 3 wherein said administration is oral and intravenous.

8. A method according to Claim 5 wherein said therapeutically-effective amount is in the range of about 20 to 180 gm/day.

9. A method according to Claim 6 wherein said therapeutically-effective amount is in the range of about

20 to 180 gm/day of ascorbate.

10. A method for treating a condition associated with HIV infection in a subject by inhibition of HIV replication comprising the step of administering to said subject a therapeutically-effective amount of a combination of:

(a) a compound selected from the group consisting of pharmaceutically-acceptable ascorbate salts; ascorbic acid; metabolic products of ascorbic acid or said salts; derivatives of ascorbic acid, said salts or said metabolic products; and mixtures thereof; and

(b) at least one other drug efficacious for treatment of symptomatic or nonsymptomatic HIV-infection.

11. A method according to Claim 11 wherein said drug comprises AZT.

12. A method according to Claim 10 wherein said condition is nonsymptomatic of HIV-infection.

13. A method according to Claim 12 wherein said condition is ARC.

14. A method according to Claim 10 wherein said condition is symptomatic of HIV infection.

15. A method according to Claim 14 wherein said condition is AIDS.