IE19940570A1 - Use of 2',3'-dideoxyguanosine for the treatment of AIDS - Google Patents

Abstract

ABSTRACT The present invention relates to a method of alleviating the cytopathic effects of HLTV— HI virus on cells which comprises contacting the virus with an effective amount of a purine base, which is 2’,3’ —dideoxyguanosine, or a phosphate thereof.

Description

The present invention relates to the inhibition of in vitro infectivity and cytopathic effect of HTLV— lll/LAV by 2’,3'—dideoxyguammine Since 1983 it has developed that the HTLV—IlI is the etiologic agent of acquired immunodefi- virus ciency syndrome (AIDS) and related diseases. Medicine is currently endeavoring to find agents which will check the ravages of the disease. In this connection, a number of filed one patent has patent applications have been naming as a co- issued, U.S.

Patent No. 4,520,113, which mainly relates to the detec- sera of AIDS and PRE-AIDS inventor Robert C. Gallo; tion of antibodies in the patients.

Further in Science (1983) Vol 220 P.506-508 Montagnier similarly discloses the investigation and isolation of an agent subsequently defined as HTLV-III.

The present invention, however, lies in a method of alleviating the effects of HTLV-III by the in vitro contacting of the virus with a particular puri e base, namely, 2' ,3‘ -dideoxyguanosine, and related mono- and tri-phosphates of this base.

The following references are relevant to determining the state of the art prior to the present invention.

Furmanski, et al, "Inhibition by 2',3'-Dideoxy— of Mouse 1980.

"Effects of 2',3‘-Dideoxynucleo- Infection 8:307-315, thymidine of Retroviral and Human Cells," Cancer Letters, Wagar, et al, sides on Nmnmalian Cells and Viruses," Journal of Cellu- lar Physiology, l21:402—408 (1984).

Rosen, et al, "The Location of Regulatory Sequence in the Human T Cell Type III (HTLV—III/LAV) ggll, 4l:8l3-823, July 1985. , p. 42 (column 3). 4,434,788 to Nakataugawa the utilization of 3'—deoxyguanosine and 3'—deoxyuridine Cis-Acting Lymphotropic Virus Long Terminal Repeat," Time, August 12, US Patent No. shows as anti—tumor agents.

US Patent No. 4,081,534 to Elion et al various purine derivatives utilized against HSV. of the HTLV~IIl by known antiviral shows The uniqueness virus and its resistance to treatment agents has been stated in Time magazine as follows: "Research con- ducted by Harvard's Haseltine and published in the July issue of the [Cell, 4l:8l3-823, 1985} veals that the virus has a unique genetic component that allows it to reproduce itself a thousand times as fast as any other kind of virus. journal Cell re- The mechanimn for this repro- duction is ‘one of biggest effects I‘ve seen in biology,‘ says Haseltine. ‘It helps explain why AIDS is such and why it fast.‘ a devastating disease can spread so the AIDS Thus it is a In the process of rampant replication, the T cell. peculiar feature of this disease that as virus destroys its home, it progresses, the helper T cells disappear and so does the virus. By then, however, the patient is invariably beyond recov— ery." EP—A—0206497 is an intervening publication which reveals in the relevant priority document the utilization of a 2' ,3'~dideoxynucleoside exemplified solely by dideoxyribofuranoside. use of the compound of our invention is not supported in this disclosure.

The Journal of Cellular Physiology 1984, Vol 121. NO- 2. NOVEWDBT 1984 reveals that 2', 3‘-dideoxyguanosine inhibits the growth of MuLV virus at about 50 um. The disclosure is silent on the application of dideoxyguanosine to alleviate the symptoms of HTLV—lll.

It has been noted that the chronicity of infec- tion and the propensity of the virus to infect the brain make it necessary to develop new classes of remedies and drugs administration In this the capacity of purine and pyrimidine nucleoside inhibit the infectivity and effect of HTLV—III/LAV in vitro were explored. ribose moiety of the molecule the nucleosides tested suppressed the virus. which have the potential for oral and penetration across the blood—brain barrier. study, derivatives to cytopathic With the in a 2',3'—dideoxy—config- uration, guanosine In general, it was observed that complete suppresion of the virus occurs at doses that were 10- to 20-fold lower than those needed to inhibit the proliferation of the target T-cells and the imnune reactivity of normal T-cells in vitro. An analysis of five adenosine congeners, which differed only in the sugar moiety, revealed that reduction (an absence of hydroxyl determinants) at both the 2' and 3' of the ribose was necessary for an anti-viral effect, and carbons an additional reduction at the 5‘ carbon nullified the anti—viral activity.

Brief Description of the Drawings Figure l illustrates the potent protec- tive effect of 2',3‘—dideoxynucleosides on the survival and growth of ATH8 cells when exposed to HTLV—III/LAV.

HTLV—III/LAV p24 gag protein in H9 cells.

Figure 2 illustrates expression of the Detailed Description of the Drawings In Figure l, which shows the inhibition of cytopathic effect of HTLV—III/LAV by 2',3‘—dideoxynucleo— ATH8 2x105 ATH8 exposed to polybrene, exposed to HTLV-IIIB (2,000 virus in the sides against cells, Cells Were Dre‘ particles/cell) in culture tubes (solid columns) presence or absence of various concentrations of 2‘,3‘~ rA0579 .4. Ext dideoxyadenosine, 2',3'—dideoxyinosine, (P.L. ‘,3’-dideoxythymidine- ',3‘-dideoxy— guanosine Biochemicals Inc.), 2‘,3‘-dideoxycyti— dine, or Control cells (open columns) were similarly treated but were not exposed to On day 5, the virus. total viable cells were counted as described in Example 1.

In Figure 2,‘ which shows the inhibition of the infectivity and replfcation of HTLV—III/LAV in H9 cells by 2',3‘—dideoxynucleosides, l05 H9 cells were exposed to various concentrations of 2‘,3‘-dideoxynucleosides for 4 then to 2 Hg/ml f0P 30 pelleted, and exposed to HTLV—IlIB (3,000 virus parti- Cells hours, polybrene minutes, cles/cell) for 1.5 hours. were resuspended in fresh complete mediwn and cultured in tubes at 37°C in 5% The cells were contin- On days 8 the per- CO2—containing humidified air. uously exposed to 2‘,3'—dideoxynucleosides. (left), 9 (middle), and 10 (right) centage of the target H9 cells expressing p24 gag protein of HTLV-III/LAV was indirect innmnofluor— escence microscopy by using anti—HTLV—III/LAV p24 murine monoclonal antibody (M26 provided by Drs. and R.C. Gallo). in culture, determined by F.D. Veronese Specific Description In the HTLV—III/LAV (as cell-free virus) exerts a profound cytopathic effect on the target T-cells by day 4 and by day 10, 98% of the cells are killed by the virus, The killing of cells can be monitored quantitatively as a function of the starting dose of virus (Table 1). When ATH8 cells virus particles/cell repre- the nfinimwn cytopathic the ,000 or 3,000 virus parti- in order system, in culture, are used in a 7-day assay, sented dose of virus. In experiments reported below, cles/cell were used to test various compounds under conditions of substantial virus excess.

Figure 1 illustrates the potent protective effect of 2‘,3'—dideoxynucleosides on the survival and growth of ATH8 cells when exposed to HTLV-III/LAV. It has been found that concentrations greater than 0.5 UM of 2',3'-dideoxycytidine completely protected ATH8 This compound exhibited a strong anti—viral effect to 20-fold lower than those that inhibited growth of the target cells when no virus was 2‘,3'— dideoxythwnidine required relatively high concentrations cells and enabled then) to survive and grow. at doses that were 10- present. In the present experimental conditions, to exert a protective effect, and unlike the other com- parable dideoxynucleosides tested, its capacity to nulli- fy the cytopathic effect of the virus was lost on day 10 of the culture (Figure l,bottmm.

These anti—viral effects were confirnwd in a different system, using the expression of the HTLV~ III/LAV p24 gag protein in H9 cells (Figure 2). The H9 cells are relatively resistant to the cytopathic effect of HTLV~III/LAV, and p24 gag protein expression following exposure to HTLV-III/LAV virions may be used as an index of The 2',3'—dideoxy-derivatives of cytidine were potent inhibi- while 2',3'- relatively higher viral infectivity and replication ip_ vitro. tors of viral replication in the systenn dideoxythymidine required concentra- tions to mediate an anti—viral effect, and this compound allowed a resumption of viral bottom). tested were replication by day 10 of culture (Figure 2.

Also 2',3'~dideoxynuc1eosides the the lectin-induced reactivity of normal (Table 2). cific antigen-specific effects the various on and A normal clone (TM3) of tetanus-toxoid spe- T-cells the the compounds on antigen—driven activation, helper/inducer of and normal was used to monitor effects circulating lymphocytes were used to monitor effects and on pokeweed mitogen phytohaemagglutinin— Concentrations of these compounds, - to 20~fold higher than block the _in_ vitro infectivity and of HTLV-III/LAV left the innmne reactivity of normal lymphocytes basically intact. driven activation. including those that were those necessary to cytopathic effect in_ vitro lwnphocytes in vitro :5 g%05?0 The mechanisnw by which 2',3'-dideoxynucleo- sides suppress the replication of HTLV—IlI/LAV are not known. It is known that the 5'-triphosphate product of ',3‘—dideoxy 2‘,3'-dideoxyadenosine can adenosine, 2‘,3'-dideoxyguanosine, and inhibit cellular DNA polymer- ases beta and gmnna, as well as viral reverse transcrip- DNA poly- synthetic tase, but not mwnnalian DNA polymerase alpha. to be the key DNA enzmne for DNA replication during cell division, and Of interest, Herpes sim- plex type I DNA polymerase is reported to be as resistant merase alpha is assumed also has a role in DNA repair. to 2‘,3'-dideoxythymidine as cellular DNA polymerase alpha. Unphosphorylated dideoxynucleosides have rather negligible effects on the growth of cultured inmnnalian cells (a phenomenon which is confirmed here in human T- This intra—cel1ular is believed to be so because of inefficient the 5'- resistance of DNA poly- cells). conversion to corresponding triphosphates coupled with the merase alpha to low levels of the 5'-triphosphates.

Example l With specific T—cell reference to Table 1, a tetanus toxoid— line, established by repeated cycles of stimulation with antigen as described in Mitsuya, et al, Science, 225:l484—l486 (1984), was cloned in the presence of lethally irradiated (12,000 rad) human T-lmnphotropic virus type I (HTLV—I)-producing MJ—tumor cells in 96-well microtiter culture plates (Costar, Cambridge, MA). Clone ATH8 well) was selected for drug screening on the basis of its (obtained frmn a culture plated at 0.5 cells per and ex- of HTLV- III/LAV. of HTLV—I blot hybridization using a radiolabelled HTLV—I cDNA probe but of HTLV—l p24 gag to 2 ug/ml interleukin-2) effect distinct rapid growth (in the presence of quisite sensitivity to the ATH8 cytopathic clone bears several copies in genome when assessed by Southern does not produce detectable mnounts 105 ATH8 ininutes, protein. cells were pre-exposed polybrene for pelleted, exposed to various E 949570 -7 _ amounts of HTLV-IIIB, resuspended in 2 ml complete medium (RPMI supplemented with 15% undialysed, heat inactivated fetal calf serwn, 4 mM L-glutamine, 5xl0"5 2-mercapto- ethanol, 50 U/ml penicillin, % Products and 50 pg/ml streptomycin) (vol/vo1)—inter1eukin2 Buffalo, NY), Oxnard, containing (lectin-depleted; Cellular Inc., culture tubes (3033, CO2-containing hmnidified air. incubated in at 37°C in 5% 7, the total viable cells were counted by the trypan blue dye exclu- sion Inethod. Table 1 shows of HTLV-III/LAV on the ATH8 cells; data are expressed as the and CA) On day Falcon, the cytopathic effect arithmetic means standard deviation for duplicate determinations.

Table l Cytopathic Effect of HTLV—III/LAV on ATH8 Cells Number of HTLV-III? Number of Viablg 1 ) Virus Particles/Ce ATH8 Cells (x10

Claims (3)

CLAIMS:

1. The use of an effective amount of a 2’,3’-dideoxyguanosine or a mono— or tri- phosphate thereof in the preparation of‘ a medicament ‘for the treatment of AIDS and AIDS related diseases.

2. The use according to claim 1 wherein the‘ medicament is in dosage form adapted to give an in vivo concentration of 10 to 100 pm.

3. The use according to claim 1 and substantia11y as described herein by way of example. TOMKINS & C0.