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WO1992003052A1 - Procede pour supprimer par l'ascorbate la replication du vih en cas d'infection chronique et aigüe par le vih - Google Patents

Procede pour supprimer par l'ascorbate la replication du vih en cas d'infection chronique et aigüe par le vih Download PDF

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Publication number
WO1992003052A1
WO1992003052A1 PCT/US1991/005895 US9105895W WO9203052A1 WO 1992003052 A1 WO1992003052 A1 WO 1992003052A1 US 9105895 W US9105895 W US 9105895W WO 9203052 A1 WO9203052 A1 WO 9203052A1
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WIPO (PCT)
Prior art keywords
ascorbate
hiv
infection
cells
ascorbic acid
Prior art date
Application number
PCT/US1991/005895
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English (en)
Inventor
Raxit J. Jariwalla
Steve M. Harakeh
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Jariwalla Raxit J
Harakeh Steve M
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jariwalla Raxit J, Harakeh Steve M filed Critical Jariwalla Raxit J
Publication of WO1992003052A1 publication Critical patent/WO1992003052A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof

Definitions

  • 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.
  • 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)).
  • HTLV-1 latent human retrovirus
  • 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) .
  • 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.
  • 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.
  • interferon- ⁇ 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.
  • 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.
  • 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.
  • RT values on day 2 and day 4 were, respectively, 55 x 10 4 and 267 x 10 A cpm/10 6 cells; average background value in blanks (i.e., reactions without enzyme) was 1530 cpm per ml culture supernatant.
  • the mean of three samples was 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/10 6 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 OD 570 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 35 S-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 CD 4 + 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.
  • 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.
  • the above-described compounds will be administered in a therapeutically-effective amount to the HIV-infected subject.
  • 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 ⁇ g/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.
  • 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.
  • 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 ⁇ g 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.
  • L-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.
  • Fresh working solutions (lOx strength) of ascorbate were prepared daily by diluting the stock in complete growth medium.
  • 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 ⁇ g/ l.
  • the controls received 0.1ml of growth medium. Plates were incubated at 37°C in 5% C0 2 /95% air humidified atmosphere for various time intervals. At periodic intervals, 0.5 ml aliguots of cell suspension were collected, mixed with 50 ⁇ l trypan blue, and tested for viability.
  • 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.
  • 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 ⁇ l ) were assayed for RT activity as described by Hoffman, et jal.. , using fresh batches of [methyl- 3 H]-dTTP (5A ⁇ 80 Ci/ mol, NEN/Du Pont research products) .
  • RT activity was expressed as the amount of [ ⁇ Hj-dTMP incorporated (cpm/10 6 cells) .
  • H9 cells (3 x 10 5 cells per well in microtiter plates) were grown in the presence of 0, 75, 100 and 150 ⁇ g/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 ⁇ Ci of 35 S-Translabel (5A 1013 Ci/Mm, ICNRadiochemicals) .
  • Metabolic Activity Assayed by MTT Determination 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 ⁇ g/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).
  • Infectious HIV stock was 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 10 5 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-a ⁇ corbate solution was added daily and the cells were incubated.
  • 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.
  • H9/HTLV-III B cells 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 ⁇ g/ml) for a period of four days. Viability of control and ascorbate-treated cultures was determined using the trypan blue exclusion test.
  • RT activity was assayed in cell-free supernatant (Hoffman, et al., Virolo ⁇ v. 147, 326 (1985)) harvested from cultures grown in non-toxic ascorbate concentrations (0 to 150 ⁇ g/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.
  • RT titer The first noticeable drop (64% inhibition) in RT titer occurred on day 2 at 50 ⁇ g/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 ⁇ g/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 ⁇ g/ml) on day 2. However, increase in virus production was transient, as these effects did not persist on day 4 of incubation.
  • 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 ⁇ g/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 ⁇ g/ml ascorbate were reduced to 13% of the control.
  • H9 cells grown in the presence of different concentrations of ascorbate (0 to 150 ⁇ g/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 ⁇ g/ml.
  • 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 ⁇ g/ml ascorbate, RT activity was reduced respectively to 31.5% and 7.0% of control (Table 1).
  • chronically infected cells were exposed to 100 and 150 ⁇ g/ml ascorbate for 4 days. The RT levels in supernatant were reduced to 4.0 and 0.6% of control (Table 1).
  • 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 ⁇ g/ml ascorbate and incubated at 37 * C with daily addition of fresh compound.
  • 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.

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Abstract

Procédé pour traiter des états symptomatiques ou non symptomatiques associés à l'infection par le VIH par inhibition de la réplication de ce dernier, en administrant à un sujet infecté une dose thérapeutiquement efficace d'ascorbate, acide ascorbique, leurs produits métaboliques, des dérivés ou des mélanges de ceux-ci.
PCT/US1991/005895 1990-08-23 1991-08-23 Procede pour supprimer par l'ascorbate la replication du vih en cas d'infection chronique et aigüe par le vih WO1992003052A1 (fr)

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US57208690A 1990-08-23 1990-08-23
US572,086 1990-08-23

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2715301A1 (fr) * 1994-01-24 1995-07-28 Aromafarm Ltd Application d'au moins un sel métallique de l'acide ascorbique comme antiviral administrable par voie générale et composition pharmaceutique.
EP0643775A4 (fr) * 1992-05-28 1995-12-27 Univ Monash Compositions therapeutiques.
EP1013272A1 (fr) * 1998-12-23 2000-06-28 Biomedical Primate Research Centre (BPRC) Manipulation de l'activité d'une voie de production du radical oxyde d'azote pour le traitement de maladies liées à la présence de radicaux libres oxygénés
US7354906B2 (en) 1999-01-21 2008-04-08 Samaritan Pharmaceuticals, Inc. Composition of anti-HIV drugs and anti-cortisol compounds and method for decreasing the side effects of anti-HIV drugs in a human
WO2021209800A1 (fr) * 2020-06-25 2021-10-21 Elhayesh Mohamed Khairy Abd Ellatif Traitement d'une infection humaine par le coronavirus et le virus de la grippe (h1n1) (grippe porcine.)

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CA: 110(6): 44968 r, "Vaccines containing mycobacterium phlei FU and diisopropyl-ammonium-dichlorate and ascorbic acid for the treatment of HIV infections". See entire document. *
CA: 111 (19): 167396 f, "Treatment acquired immunodeficiency especially AIDS, with 17-(cyclopropylmethyl) -4,5-epoxy-3,14-dihydroxymorphenon -6-one and pharmaceutical compositions containing it". See entire document. *
CATHCART, BIOLOGISK MEDICINE, 3:6 (1983), See entire statement. *
CATHCART, R.F. MEDICAL HYPOTHESIS, 14:423-433 (1984). See entire article. *
GARLAND, D. et al., ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 251:7710776 (1986). See entire article. *
NAKANISHI, Y. et al., EUROPEAN JOURNAL OF BIOCHEMISTRY 152:337-342 (1985), see entire document. *
ORTWERTH, B.J. et al., EXP. EYE RES., 47: 155-168 (1988). See entire article. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0643775A4 (fr) * 1992-05-28 1995-12-27 Univ Monash Compositions therapeutiques.
US5981601A (en) * 1992-05-28 1999-11-09 Centre For Molecular Biology And Medicine Method for enhancing cellular bioenergy
FR2715301A1 (fr) * 1994-01-24 1995-07-28 Aromafarm Ltd Application d'au moins un sel métallique de l'acide ascorbique comme antiviral administrable par voie générale et composition pharmaceutique.
EP1013272A1 (fr) * 1998-12-23 2000-06-28 Biomedical Primate Research Centre (BPRC) Manipulation de l'activité d'une voie de production du radical oxyde d'azote pour le traitement de maladies liées à la présence de radicaux libres oxygénés
WO2000038662A3 (fr) * 1998-12-23 2001-08-23 Stichting Biomedical Primate R Manipulation de l'activite d'un trajet de production d'un radical de monoxyde d'azote pour le traitement de maladies associees a la presence de radicaux libres d'oxygene
US7354906B2 (en) 1999-01-21 2008-04-08 Samaritan Pharmaceuticals, Inc. Composition of anti-HIV drugs and anti-cortisol compounds and method for decreasing the side effects of anti-HIV drugs in a human
WO2021209800A1 (fr) * 2020-06-25 2021-10-21 Elhayesh Mohamed Khairy Abd Ellatif Traitement d'une infection humaine par le coronavirus et le virus de la grippe (h1n1) (grippe porcine.)

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