CA2142831A1 - Treatment of human viral infections - Google Patents

Abstract

Treatment of cells or humans carrying or infected with a virus capable of causing an immunodeficiency disease with compounds of formula (I), wherein R, R1 and R2 are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unsubstituted alkenyl, provided at least two of the substituents of R, R1 and R2 are other than hydrogen.

Description

~2831 ~V0 94/(~161~ l'C~ S93/(~793~ ~

=.

TREATMENT OF HUM/~ VIRAL INFECTIONS

BACKGROUND OF THE INVENTION

The present application is a continua~ion-in-part o U.S.
Serial No. 07/933,460, filed on August 21, 1992.
s The human immunodeficiency virus type 1 (HIV-l, also referred to as HTLV-III, LAV or HTLV-III/LAV) ~nd, to a lesser extent, h~an immunodeficiency virus type 2 (HIV-2) is the etiological agent o the acquired immune deficiency syndrome tAIDS) and related disorders. Barre-Sinoussi, et al., Science, 220:868-871 (1983); Gallo, et al., Science, 224:500-503 (1984);
Levy, ec al., Science, 2~5:840-842 (1984); Popovic, et al., Science, 224:4~7-500 (1984); Sarngadharan, et al., sclence, 224:506-508 (1984); Siegal, et al., N._En~L. J Med., 305:1439-1444 (1981); Clavel, F., AIDS, 1:135-140. This disease is characterlzed by a long asy~ptomatic period followed by the progressive degeneration of the immune system and the central nervous system. Studies of the virus indicate that replication ` I . i5 Ihighly regulated, and both latent and lytic infection of the ;
CD4 positive helper subset of T-lymphocytes occur in ~issue culture. Zagury, et al., Scienc~, 231:850-853 ~lY86). The expression of the virus in infected patients also appears to ~e regulated as the titer of infectious virus remains low , throughout ehe course o~ the disease. Both HIV-l and 2 share a slmilar structural and function genomlc organ.i~ation, having ~ W O 94l0~160 ~ PCT/~S93/07934 regulatory genes such as tat, rev, nef, in addition to structural genes such as env, ~a~ and ~l.
'.' While AIDS, itself, does not necessarily cause death, in many individuals the immune system is so severely depressed that various other diseases (secondary infections or unusual tumors) ~ such as herpes, cy~omegalovirus, Kaposi's sarcoma and ¦ Epstein-Barr virus related lymphomas among others occur, which ultimately results in death. These secondary infec~ions may be treated USillg other medications. However, such treatment can be adversely affected by the weakened immune system. Some humans in~ected with the AIDS virus seem to live many years with lit~le or no s`ymptoms, b~lt appear to have persistent infections.
Another group of humans suffers mild immune system depression with various symptoms such as we`ight loss, malaise, fever and swollen lymph nodes. These syndro~es have been called persistent generaliæed lymphadenopathy syndrome (PCL) and AIDS
r~lated complex (ARC) and may or may not develop into AIDS. In all cases, chose infected with the HIV are beli~ved to be persistently infeccive to others.

~ The activation of che latent HIV provirus from the ; .
;~ asympto~atic period has been reported to be governed by long : .
ter~inal repeats (LTR) in the viral DNA. See Ranki, A., et al., Lancet ii: 589-S93 (1987); Fauci, A.S., et al., Science, 239:617-622 (1988); Zagury, D., et al., Science, 231:850-~53 (1985); Mosca, J.D., ~5~ (London), 325:67-70 (1987). The activity of HIV-l is determined by the complex interac~ion of positive and negative transcriptional regula~ors that bind eO
speci~ic sequences within the LTR. ~ Cullen, B.R., et al., Cell, -:
58:423-426 ~1989). Changes in the quantity or quality of ehese ~ --factors may underlie the activation of transcription of HIV 1 .-and HIV-2 laten~ provirus by a myriad of stimuli. See Fauci, A.S., Science~ 239:617-622 (1988); Griffin, G.E., et al., Na~ure (London), 339:70-73 (1989); Nabel, G., et al., Science, :

2`8 3 1 ~0 94/WI~,Q l~cr/~iss3/07s . i, 239:1299-1302 (1988). Specifically, p~orbol 12-myristate-13-acetate (PMA) and T~mor Necrosis Factor-,~
(TNF~) are believed to be poten,~ activators. In particular, TNF~ is pre~sent in ~arkedly enhanced levels in HIV infected individuals, suggesting that the cytokine plays an icportant role in the pathogenesis of AIDS~ Lahdevir,a, J., A~. J. .~ed., 85:2~,9-291 (1988).

Most known methods for treatin,g individuals infected by HIV
have focused on preventing integration of the virus into the host cell's chromosome or on stages other than provirus. Thus, one area of interest has been drugs that affect reverse transcriptase. Many of the proposed therapeutic methods, however, have not proven clinically effective. Indeed, even ~rea~,~ments thac have resulted in clinical utility such as ~ZT
(zidovudine) have not been reported to prevent the breakdo~wn of the immune system in many patients after a number of years of treatment~ Few m,ethods have been reported to inhibit both expression of integrated provirus and chronic infection of HIV-l. Reverse transcriptase inhibitors e.g. AZT, ddC, ddI have not been reported to have inhibitory effect on chronic infeceions. Ro3-3335 was reported to be effective on chronic infection. See Hsu, M-C, Science, 254:1799-1800 (1992).

It thus would be desirable to have a new compound that can inhibie expression of provirus of HIV in HIV infected cells and inhibit chronic infections. It would be particularly desirable to have a new therapy that can be used to treae already infected ' I cèlls by means of inhibiting expression of pr,-,virus, or a means to keep the provirus dormant within infected cells.
,, ~ . .

SUMMARY OF THE INVENTION
We have now discovered that compounds of the following formula I are inhibitory on expression of an immunodeficiency provirus, such as HIV, prererably HIV-l and thus are useful for \~'0 94~16() PCT/~'S93/0793 treating ce.lls that have been acutely or chronically infected ~y immunodeiciency viruses such as HIV:
i : a~ 3 0 wherein R, Rl and R2 are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unqubstituted alkenyl, provided at least two of the substituents of R, Rl ;
and R2 are other than hydrogen. These compounds ha~ at least one chiral center. The present invention includes use of , ~
racemic mixtures of stereoisomers of the above compounds.
Preferably the compounds are optically enriched, i.e., substaneially more of one enantiomer (or diastereomer) than the other stereoisomer(s). The biological activity of the optically active stereoisomer can be determined empirically as disclosed herein. Typically the compounds where the carbon at the 4 position has the (R) configuration are more preferred.
Preferred compaunds of formula I include those where R is alkyl, Rl i5 hydroxyl and/or R2 is heteroalkyl. Especially ;~ 25 preferred compounds include those where R is hydrogen, Rl is hydroxyl and R2 is heteroalkyl, particularly alkyleneamino . ;~ including monoalkyl- or dialkyl(alkylene)amino. Specifically preferred compounds include Topotecan, i.e., the compound of ' formula I where R i5 hydrogen j Rl iS 9-hydroxyl, and R2 i5 ~; 30 lO-N-N-dimethyl(methylene)amino (i.e., (CH3)2NCH2-), and ~;~
the compound of formula I where;R is ethyl, Rl is hydrogen and R2 ls 9-hydroxyl, wherein said 9- and lO- prefixes refer ~o positions of ring members as indicated in formula I abo~e.

~ ~0 94/Q~l6() PCI/~S93/()793~ ~ ~
~j !

-The co~pounds of formula I can inhibi. activity of an LTR of an immunodeficiency virus such as the HIV LTR and reduce or ~.
inhibit expression of genes operably linked to the HIV LTR.

In one embodiment, the compounds of formula I can treat cells infected by i~unodeficiency viruses, for example, HIV, preferably HIV-l, and thus can be used to trea~ humans infected by HIV. For example, tr~atment of those diagnosed as having ~ AIDS as well as those having ARC, PGL and those not yet ¦ 10 e~hibiting such conditions.

These compounds can be used against a different target than the conventional drugs being used to treat humans infected by --HIV, e.g., re.verse transcriptase inhibitors such as zidovudine (AZT~, 2',3'-dideoxyinosine (ddI) and 2',3'-dideoxycytidine (ddC). Using such drugs in combination with the present compounds is anticipated to result in a synergistic resul,.
Similarly, the pressent compounds should be effective in cells ~ that are resistant to such compounds. For example, compounds of 1 20 the present invention can be used to block HIV-l LTR direc~ed expression in AZT resistant cell lines.

The invention also provldes pharmaceutical compositions comprising a co~pound of formula I and a suitable carrier therefor for use in the conditions referred to above.

BRIEF DESCRIPTION OF THE DRAWINGS
. . _ ,. .
Figure 1 shows inhibition of cytokine stimulated HIV LTR j ` -directed gene expression by Topotecan (varying concentrations).
. :
Figure 2 shows the effect of Topotecan on cell survival. ~.

Figure 3 shows inhibitory effects of Topotecan on activ~ted HIV LT~.

~O 94/04l60 2 1 4 2 8 ~ 1 PCT/~S93/()7934 Figure 4 shows inhibition of HIV-l replication by Topotecan ~ (varying concentrations) in acutely infected human peripheral j blood mononuclear cells.

Figure 5 shows lnhibition of HIV-l replication by Topotecan, as determined by levels of mRNA of reverse transciptase in acutely infected human peripheral blood mononuclear cells.

Figure 6 shows inhibition of HIV-l expression by Topotecan (varyLng concentrations) in chronically infected human T cells.

Figure 7 shows selective effects of Topotecan on the accumulation of HIV LTR directed mRNA and cellular ~ actin mRNA.
~`~ 15 Figure 8 shows dose dependent Inhibition by Topotecan (varying concentrations) an the accumulation of HIV-l LTR
directed mRNA.

Figure 9 shows the effect of Topotecan (varying ~` concentrations) on total RNA synthesis of cell.
:
DETAILED DESCRIPTION OF THE INVENTION
~ We have discovered that compounds of the following formula I
: 25 can be used to inhibit expression of an immunodeficiency provirus, for example, to treat cells infected with an immunodeficiency virus such as HIV and ehus can be used for treatment in HIV infected individuals:

; 2~ ~ o ,~ :

\VO 9~/Wl6() 2142~3~ PC~/~S93/~?793~ ~
$
,.

wherein R, R1 and R2 are each independently seleGted from the group consisting of hydrogen, substitu~ed and unsubstituced alkyl, noncycli.c he~eroalkyl, and substituted and unsubsLituted alkenyl, provided at least two of the substituents of R, Rl and R2 are other than hydrogen. The alkyl groups preferably have from l to about 12 carbon atoms, more preferably from about l to 6 carbon atoms. As used herein, the term alkyl unless otherwise modified refers to both cyclic and noncyclic groups, although of course cyclic groups will comprise at least three carbon ring members. Straigh~ or branched cnain noncvclic groups are generally more preferred than cyclic groups.
Straight chain groups are still more preferred. The alkenyl substituents preferably have from 2 to about 12 carbon a~oms, more preferably from 2 to about 6 carbon atoms. Heteroalkyl groups include those noncyclic groups that comprise one or more hetero atoms and each hetero atom has one or more alkyl linkages such as alkyl linkages having .rom 1 to 8 car~on atoms or l ~o 4 carbon atoms. Thus suitable heceroalkyl groups include those ~; where a hetero atom is directly bonded to ehe general ring system of formuLa I as well as those groups where a hetero atoms is spaced from the ring system by an alkylene linkage of, e.g., one to four carbon atoms. Particularly preferred heteroalkyl groups include aminoalkyl groups including primary, secondary and tertiary alkylamines, and especially preferred are N-N^dialkyl(alkylene~amino groups, e.g., groups of the formula (alkyl)2N(CH2)n- where n is an integer of l to 4. A
particularly perferred gruop is N-N-dimethylmethyleneamino. The term "noncylic" heteroalkyl, is intended to include straight a7d branched chain moieties, but not groups that compxise a closed '' ring structure including those groups that form a ring ~r comprising two or more aromatic carbons at positions 7 to 10 as s.
depicted above in formula I.
. .
Said substituted R, R1 and R2 groups may be substituted at one or more available posi;ion by one or more suitable groups .

~5~ r~

\VO ~4/0416~1 Z ~ 42 ~ ~ ~ PCr/l S93/~1793 -8- i such as, for example, ~alogen such as fluoro, chloro or bromo,~
alkyl such as alkyl having from l to 12 carbon atoms or from l to 6 carbon ato~s, alkenyl such as alkenyl having from 2 ~o 10 carbon atoms or 2 ~o 6 carbon atoms, aryl having from 6 tO IO
carbon atoms, and N, O, S, including heceroalkyl, e.g., heteroalkyl alkyl having one or more of said hetero a~o~s and from 1 to 10 carbon atoms or from 1 to 6 carbon atoms.

The present invention includes use of both racemic mixtures and optically active s~ereoiso~ers of compounds of for~ula I.
The optically active stereoisomers of formula I are preferred.
Typically still more preferred are the optically active compounds where the chiral carbon of the lac~one ~,oiety (i.e., the ring carbon of position 4 as depicted above) is of the (R) lS configuration according to the Cahn-Ingold-Prelog nomenclature system. See Carey, F.A., Advanced Organic Chemistrv, Part A, p.
65-66 (2d ed., Plenum Press 1984).

It is believed chac the co~pounds of formula I provide eective therapy of chronically infected celIs (i.e. cells infected by a virus ~hich is an immunodeficiency virus such as FIV, SIV, HIV, etc.) as evidenced by a ~eduction in, preîerably a co~,plete repre~sion of, e.g. HIV ~TR directed gene expression. Thus, in an HIV infected cell addi~ion of an , ~
effcc~ive amount of a compound of formula I will reduce the expression of a gene operably linked eo the HIV LTR. Preferably the gene is operably linked to an HIV-l LTR.` As used~herein, the ter~ operably linked means that the gene is under the control of the HIV LT~ and positioned in a nucleotide sequence to acco=plish this. Typically, the gene is downstream of the $
LTR, which acts as a promoter. Preferably, t~e gene corresponds , ~
to a viral gene such as the HIV env gene, HIV tat genç, HIV rev j .-~ gene, etc. ~.

;`~`." ~ 3 5 : ~ ~
: : :
~:`: :
:

"``~ , ,~i~2~31 ~i O 9 ~ 1 1 fi O P ~ / 1~ ~i 9 3 / 0 7 9 3 ~i ? ~
.

i Hence, in one preferred embodiment the present invention can be used in treating a serum positive but asy~ptomatic patient.
For e~ample, as a preven~ative, it can also be used prophylactically as a preventative for high risk individuals.
This, method of protection comprised administering an effective amount of the compound of formula I is by means as set forth below.

Compounds of formula I can be used to .reat cells, especially mammalian .ells and in particular human cells, infected by an immunodeficiency virus such as HIV infected cells. As a result of .reatment with compounds of formula I
viral e~pression is significantly reduced.

lS For ex mple, viral expression of HIV can be studied by a number of methods such as looking at the expression of a marker gene, e.g. CAT, Lac Z, e~c., operably linked to the HIV LTR, which acts as the promoter. Use o the present compounds such as Topotecan can significantly reduce expression of such a marker. HIV-l viral expression is turned on and enhanced by HIV
LTR stimulators such as tumor necrosis factor~ (TNFct) or phorbol-12-myristate-13-acetate (PMA). One product of this expression, i.e. tat can further augment HIV-l gene expression.
Using a marker gene such as Lac Z operably-linked ta the HIV LTR
in HIV infected cells, the addition of an effective amount of compounds of formula I significantly inhibits expression of lac Z gene, thereby indicating that HIV expression under the control of the HIV LTR such as HIV en~elope glycoprotein expression has been inhibited if not completely stopped.
p2 4, a major structural protein (product of gag), has been widely used for monitoring HIV-l replication in cells and ~, vireamia in indi~iduals. Use of present compounds such as ; Topocecan, at concentrations that do not significantly adversely affec~ cells, can dramatically reduce HIV-l replication, as : ~

3 ~

0 94/0~16(~ PCT/~'S93/0793~ :

., -10-determined by P~ 4 levels, i.e., preferably a reduction of more than 25~ as determined by p2 4 levels, more preferably a reduction of more than 50%, and still more preferably a reduction of HIV-l replication of more than 80~ as de~ermined by p2 4 levels.

The effective amount used to obtain such a result is at micromolar and even nanomolar concentrations. Furthermore, ~he administration of the compounds of ~he present invention at effective concentrations, which inhibit for example, HIV
expression, do noc adversely affect the cell.

The compounds of the present invention can be administered to HIV infected individuals or to individuals at high risk for HIV infection. For example, those having sexual relations with an HIV infected partner, intravenous drug users, etc. Because of its inhibieory effect, the virus of the present invention can ,~ be used prophylactically for such individuals to minimize their ~ risk.
^~ 20 Compounds of formula~I can readily be mads or obtained.
See, generally, U.S. Patent No. 5 ! 004,758. Other compounds of formula I can be prepared by procedures well known ~o those ;~ skilled in the synthesis art.
! `~` 25 ~ ~
i ~ As demonstrated in the examples which folIow, compounds offormula I block activation or suppress activity of HIV-l LTR and thus expression of genes under its control in both chronically ~' ` and acutely infected cells. In particular, it has been found ;~ 30 that co~p~unds of formula (I) in a dose~dependen~ fashion inhibit HIV LTR directed TNF~ and PMA stimulated gene expression. Moreover, such inhihition is provided with ,.
~`` essentially no adverse effects on cell survival or cellular mRNA .
or total cellular RNA synthesis. Thus, it is believed compounds 3S of formula I~uill have utilicy in inhibiting the prcgression of : .
' ~, ':; :

~:0 ~4/1);~1~.0 i~ 31 Pcr/uss3/o7s34 l~
~ 7 - 1 1 - ', an HIV infection and o~her retroviral infections in cells and in a human, includin~ utility in extending the latency of an HIV
infection in a human. `-While not wishing to be bound by theory, the absence of cytotoxicity of the compounds of formula I indicates that these compounds affect positive or negative regulators of HIV LTR, preferably HIV-l LTR, that are nore critical to the re~rovirus than the host cell.
1~
In general for the immunodeficiency infections, for example an HIV infectic,n, a suitable effective dose of one or mGre compounds of formula I will be in the range 0.4 to 10,000 ~g per kilogram body weight of recipient per day, preferably in the range of 1 to 1,000 ~g per kilogram, still more preferably ; in the range of S ~g to 500 ~g per kilogram body weight per day. The desired dose is suitably administered once or several nore sub-doses adminis~ered at appropriate intervals ; throughout the day, or other appropriate schedule These sub-doses may be administered as unit dosage forms, for example, containing 0.1 to 250 ~g, preferably 5 to 250 ~g.

Administration of the compounds of the invention may be by any suitable rouce including oral, rectal, nasal, topical (including buccal and sublingual), ~aginal and parenteral (including subcutaneous, intramuscular, intra~enous and lntradermal) with oral or parenteral being preferred. It will i be appreciated that the preferred route may vary with, for j ,~
example, the condition and age of the recipient.
~`~ The administered in~redients nay be used Ln therapy in '.
conjunction with other medicaments such as reverse transcriptase ., inhibitors such as dideoxynucleosides, e.g. zidovudine (AZT), 2',3'-dideoxyinosine (ddI) and 2',3'-dideoxycytidine (ddC), TAT
3S antagonises such a: Ro 3-3335, and Ro 24-7429, protease :

~ W 0 9 1/0~16(~ 83 1 PCT/~'593/0793~

1.
inhibitors and other agents such as 9-(2-hydroxyethoxymethyl)guanine ~acyclovir), interferon, e.g., alpha interferon, interleukin II, and phosphonofor~ate (Foscarnet) or in conjunction with other immune modulation agents includin~, bone marrow or lymphocyte transplants or other medications such as levamisol or thymosin which would increase lymphocyte num~ers and/or function as is appropriate. Because many of these drugs are directed to dif~erent targets, e.g., reverse transcription, it is anticipated that a synergistic result will be obtained by this combination.
;;' ~
Similarly, the present compounds may be effective when the above-described drugs are not or are no longer effective. For example, compounds of the present in~ention can be used in cells ;~ 15 that are resistant to reverse transcriptase inhibitors such as AZT, ddI, and ddC. For instance, the compounds of for~ula (I), can be used to block HIV-l LTR directed LTR expression in such resistant cell lines and for trea~ment of such resistant strains. For example, the present compounds can block HIV-l LTR
;, ~; 2Q directed expression in an AZT resistant strain of HIV-l.
~ ~ , Accordingly, the present invention can be used therapeutically in an individual as that individual develops resistance to drugs ; that act on different targets such as AZT, ddI, ddC, R03-3335, etc. It is expected that the present invention can be used for z5 treatment of HI~-l infected individuals who develop resistance ~ to any drug tha~ targets a different state~in ~he v~ral life i~ cycle than the present compounds.

While one or more compounds of formula~I may be administered~ ' 30 ~alonc, they also may be present as part of a pharmaceutical ii .
;~ compositi~on. The compositions of the inveneion comprise at least one compound of formula I together with one or more .
acceptable carriers, e.g. liposomes, and optionally other ` '~
therapeutic ingredients, including~hose therapeutic agents discussed su~ra. The carrier(s) must be "accepeable" in the ~o 94/oJI6n 21 i 2 8 3 1 Pcr/~sg3/0l7~3~ .~

~ 13- ll t sense of being compatible with the other ingredients of the ..
for~ulation and no~ deleterious to the recipient thereof.
i,"
The co~positions include those sui~able for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, in~ravenous and intradermalj adminis~ration. The formulations may conveniently be presented in unit dosa~e form, e.g., tablets and sustained reiease eapsules, and in liposomes and may be prepared by any methods well known in the art of pharmacy.
:
Such methods include the step of bringing into associa~ion ` the to be ad~inistered ingredients with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, }iposomes or finely divided solid carriers or both, and then if necessary shaping the product.
, Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or table~s each containing a predetermined ~ amount of the active ingredient; as a powder or granules; as a I solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion or packed in liposomes and a~ a bolus, etc.

A tablet may be made by compression or molding, op~tionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredïent in a free-flowing form such as a powder or granules, ; optionally mixed with a binder, lubricant, inert diluen~, preservatl~e, surface-active or dispersing agent. Molded ;~ tablets may be made by molding in a suitable machine a mixture of the powdered compound mois~ened with an inert liquid s~
~0 9~ 160 : PCT/~S93/079~
~ 1 4 2 8 3 1 diluent. The tablets may optionally be coated or scored and may be for~ulated so as to provide slow or controlled release of the active ingredient therein.

Composieions suitable for topical administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an iner~ basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be adminis~ered in a suitable liquid carrier.

Compositions suitable for topical administration to the skin may be presen~ed as ointments, creams, gels and pastes comprising one or more compounds of formula I and a pharmaceutically acceptable carrier. A suitable topical delivery system is a transdermal patch containing the ingredient eo be administered.

Compositions suitable for rectal administration may be preseneed as a suppository with a suitable base comprising, for example, cocoa but~er or a salicylate.
.
Compositions suitable. for nasal administration wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range 20 to 500 microns which is : administered in the manner in which snuff is taken, i~e., by rapid inhalation through the nasal passage from a container of the powder held clos;e up to the nose. Suitable formulatiops wherein the carrier is a liquidl for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.
j~
Compositlons suitable for vaginal administration ~ay be presented as pessaries, tampons, creams, gels, pastes, foams or 3 5 spray formulations con~aLning in addition to the acti~e :

~ \~;O 9~/0~160 ~ 2 ~ 3 1 ~CT/~S93/()793~ ~'''`' !

ingredient such carriers as are known in the art to be appropriate.
j, .
¦ Composi~ions suitable for paren~eral adminis~ration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation iso~onic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presen~ad in uni~-dose or multi-dose ~ concainers, 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, for example water for injections, immediately prior ~o use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and table~s of the kind previously described.

It should be underscood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventîonal in the art having regard to the type of formulation in question, for exa~ple, those `, suitable for oral administ~-ation may include flavoring agents.

All documents mentione.d herein are incorporated herein by reference.
`.
The present invention is further illustrated by the ~.`
, I , following examples. These examples are provided to aid in the }
understanding of the invention and are not to be construed as limitations thereof. ~ -:

GENERAL COMMENTS r~
The following reagents and procedure-c were employed as , .
specified in the exa~ples, .

~ .

'O ~/0416() ~ ~ 4 2 8 3 1 PCl/l~`S93/07~3 ~ Tirus. HIV-l was obtained from the culture superna~ant of HTLV-IIIB-producing H9 (H9/HTLV-IIIB~ cells. During the exponential phase of grow~h, cell free supernatant was harvested, standardized for reverse transcriptase (RT) activity, and frozen in aliquots at -70C. Clinical isolates of HIV-l were prep~red from patients testing positive for the human immunodeficiency virus, and standardized for RT activity.

Calls. Cell clone 293.27.2, obtained from L.A. Herzenberg 3 10 (Stanford University), was derived from human embronic kidney ~ epithelial cells, which were cultured in Dulbecco's modified ¦ Eagle's medium (GI~C0) supplemented wi~h 10% Fetal Calf Serum (FCS, Sigma) plus L-glutamine. See Roederer, M., et al., Proc.
Natl, Acad. Sci. USA, 87:4884-4888 (1990). This cell clone had been stably transfected with PNA~, which is an expression construcc of lacZ gene driven by HIV-~l LTR. Expression of ~-galactosidase can be greatly induced by P~ or TNF~.
Human peripheral blood mononuclear cells (PBMC) were prepared by - Ficoll-Hypaque gradient centrifugation of blood from HIV-seronegative individuals, and cultured in RPMI 1640 supplemented with 20% FCS (Sigma), penicillin, streptomycin, and L-glutamine in the presence of PHA (3 ~g/ml). RPMI 8402 cell line, recei~ed from Toshiwo Ando (Aichi Cancer Research Institute, Nagoya, Japan), is a ~uman T cell line. It was grown in RPMI 1640 medium supplemented with 15% FCS and L-glutamine.
' i Seock Solution. A stock solution was prepared in Topotecan in water at concentration of 20 mM. Aliquots of the stock solution were stored frozen at -20C.
~ .
Qu~antitation of ~IV^l LTR directed gene ex~ssion. .
Exponentially growing 293.27.2 (L.A. Herzenberg of Stanford Universi~y) cells were plated in ~ well plates at 2 x 105 cells per well in 2 ml of growth medium. After 48 hours, cells were stimulated with 40 u/ml of TNF~ (Genzyme, Cambridge, ~1~2~
,~, .`. ~,~.
`~ W O 94/~16() ` ~ P~T/~:S~3/0793~ ~`

MA) or 2 ng~ml of PMA (Sigma). Various concentra~ions of the~ ' specified compound of formula I were added to the medium at ~.
designated times after stimulation. Controls were run with final concentrations of ethanol, less than 0.1% (vol/vol).
After 6-8 hours incubation at 37C, medium was aspirated, cells were harvested, washed 4 times with PBS, and lysed in lacZ
buffer (60 mM Na2HP04, 40 mM NaH2P04, l0 mM KCl, 1 mM
MgSO~ -Galactosidase activities o~ cell lysates were quantieated by using ONPG as substrate. See Herbomel, et al., Cell, 39:653-662 (1984). Protein concentration was measured.
Cell visibility was determined by the colony formation assay af~er cells were erea~ed as above.

Total cellular RNA svnthesis. 293.27.2 cells were plated at 1 X 106 cells per Pl50 plate in 20 ml of growth medium.
After 48 hours incubation, cells were treated with different concentrations of Topotecan for 2 hours. Cells were then incubated for another l hour in the presence of 3H-uridine ~; at 1 uCi/ml. Total cellular RNA was prepared by the guanidium-CsCl step gradient method as described in Ausubel, F., et al., Current Pro~ocols in Molecular Biology, 1 (Wlley Interscienc0 l990~. Newly synthesized RNA was quanticated by scintillation counting, which was adjusted against RNA
concentrations measured at 260 nm.

Northern blot analysis. 293.27.2 cells in log phase growth were stimulated with TNF~ at 40 u/ml in the presence or absence of~drugs. ~fter 2 to 6 hours incuba~ion, cells were harvested. Tocal cellular RNA was prepared by the guanidium-CsCl s~ep gradient method by procedures described supra. RNA was equally loaded, fractionated and Northern transferred, and then hybridized with a lacZ gene fragmen~
tHindIII/EcoR I fro~ PHSV lac l9) or a ~-actin probe. j~

.;

:

!..~ .

\V0 94/()~l60 ~ l'Cr/~S93/()7934 ,, Tre~tment in acute HIV-1 inrectio . Peripheral blood ;~i mononuclear cells (PBMC), after 72 hours sti~ulation with 3 ~g/ml PHA, were infected with either HTLV-IIIB or a clinical isolate of HIV-l at 1 reverse transcriptase unit (K~U) per lO cells. Infection was carried out at 37C for 2 hours. Then PBMC were washed with PBS to remove free virus and replated at 4.5 X Io6 cells per well in 2 ml medium in the absence or presence of differenc concentra~ions of drugs. The ) cells were then continuously exposed to drugs for 6 days. Qn i 10 day 3, 1 ml of medium was removed from each well and replaced with 1 ml of fresh media containing drug at the previous concentration. On day 6, cells and media were harvested. Cell ! viability was decermined by the trypan blue exclusion method and I MTT metabolic assays. See Mosman, T., J. I~munolo~ical Methods, 1 15 65:55-63 (1983). P~4 levels in the culture supernatant were quan~itated by ELISA assay with HIV-l p24 Antigen Kinetics Assay Kit (Coulter, Hialeah, FL) according to the manufacturer's protocol. RNA or HIV-l reverse transcriptase was assayed with HIV-l RNA Detection Kit (GeneTrak, Framingham, MA) according to manufacturer's instructions. Bri.efly, to~al cellular ~NA was prepared, dot blotted onto a nitrocellulose membrane, and hybridized with 3 2p labeled probe for RT RNA.

Treatment in chronic HIV-l infection. RPMI 8402, cells have been chronically infected with HTLV-IIIB, was cultured at 4 X
105 cells per well in 2 ml of medium in the presence or absence of Topotecan. On day 3 and 6, p2 4 levels in the culture supernatant were measured with the procedure described above. Cell viability was deter~ined by trypan blue exclusion and MTT metabolic assay as described by Mosman, T., J ¦:
Immunoloeical Methods, 65:55-63 (1983). . I

-The HIV LTR activicy of 293.27.2 cells were simulated with TNF~ and PMA to greater than a 20 fold increase in a period ~1~2931 ' ~VO 9~/~1160 PC~/~;S93/t)793~
, .

!
of 6 hours, The cells were treated with varying concentrations of Topotecan as indictaed in Figure l. The Topote~an effectively inhibited the LTR stimulations at a Topotecan dose ~-of 0.5 ~M. In Figure l, enzymatic activi.y is expressed as percentage of ma~imum e~pression in drug untreated samples (taken as lO0). Basal ~-galactosidase activi~y is defined as in samples wi.thout cytokine sti~ulation. Each data point is the average of triplicate culture wells, Experiments were repeated on at least three independent occasions. The IC50 was approxi~ately 50 nM. A si~ilar inhibitory effect was observed upon similar treatment of RPMI 8402 ~a CD4+ T-cell line transiently transfected with PNAZ) wi~h Topotecan.
Specifically, an inhibi~ion of gene expression 62~ relative ~o the untreated con~rol was observed at a Topotecan concentration o~ 125 nM (data not depicted in Figures).
:
,~YAMPLE 2 293.27.2 cells were treated with Topotecan as described in . . Exa~ple l above... After treatment with varying concen~rations of Topotecan for 6 hours, cells were trypsinized and replaced in triplicate at lO0, 1,000, lO,000 cells per 60 ~m plate. After 7 day incubation, cell colonies (more than 50 cells) were counted. Plating efficiency was around 20%. The results are depicted in Figure 2 where each data point is exprassed as percen~ of control survival, i.e. from drug-untreated plates (taken as lO0).

,, ' ~ 293.27.2 cells were plated as described in Example l above.
TNF~ (40 ~/ml) was added to culture ~edia after 48 '~`, hours . Topotecan (0.5 ~M) was added to the cells 0.5, 2, -and 5,5 hours after addition of TNF~, i.e., Topoteca~ was , , added to the cells after HIV LTR activation. The cells were harvested 6 hours after the addition of TNF~.
~ 35 ~-Galactosidase was assayed as depicted in Figure 3 where .

`;:: ~ :

~ 9~ 16() . ~'CT/US93/0793~ :`

~ -20-¦ each data poi~t represents an average of triplicate culture wells~ Experi~ents were repeated twice independently. As shown I in Figure 3, ~-galactosidase accumulated time-dependently j after stimulation with TNF~ in the avse11ce oE Topotecan.
I 5 Treatment of the TNF~ stimulated cells with Topotecan at 2.0 I and 5.5 hours as described rapidly and ~horoughly stopped accumulation. When cells were treated with Topotecan at a concentration of 0.5 uM for 2.0 hours before TNF~
stimulation and washing (3X) with fresh medium showed 60~
inhibition of ~-galactosidase accumulation. Substantially no ~-galactosidase accumulation was observed upon simultaneous trea~men~ of the cells with Topotecan and TNF~
(See Figure 3).

Ex(~l?LE 4 The inhibition of HIV-l repLication in acutely infected human PBMC by compounds of formula I was investigated. The effect of Topotecan on viral replication was measured by p2 4 release onto the culture supernatant. Human PBMC were infected with HIV-l (HTLV-IIIB or clinical isolate) at 37C for 2 hours. Free virus was removed by washing with PBS. Infected PBMC were then aliquoted at 4.5 X lOs cells/well. Various concentrations of Topotecan were added to culture media. After 6 days, cell viability was determined by trypan blue exclusion.
p2 4 levels were determined and RT ~RNa analyzed as described in the General Comments abo~e. p2 4 levels were expressed as percent of p24 concentration of drug treated samples versus untreated samples (taken as lO0) and are depicted in ~igure 4 where results are representative of two independent studies where each data point was obtained from duplicate samples. As L~
shown in Figure 4, Topotecan dramatically decreased HIV-l replication as indicated by p2 4 production for both the lab strain (HTLV-IIIB) and clinical isolate of the virus. The median HIV replication inhibition concentration was approximately 20 nM1 similar to the effective HIV LTR activity ;~

28~1 ~
~VO 94/()~16n P ~ /~!S93/()793~ ~
.~ ~

inhibition concentration of Toptecan thus suggestin~ that HIV
LTR is a target of the co~pounds of formula I. This inhibition '~
was achieved in the absence of significant adverse effec~ on 3 cell survival. Cell viability o~ stimulated PBMC was about 90 S as determined by trypan blue exclusion. Cell viability was 100 in unstimulated PBMC treated with sao ~M Topotecan. At a concentration of 31 nMi, Topotecan reduced RT MRNA to an essentially undetectable level (See results depicted in Figure 5) E~9~fiPI.E 5 RPMI 8402 cells were infected with HTLV-IIIB. Topotecan was added to the culture media of the chronically infeeted cells (?
X 105 cell/ml). Three and si~ days after addition of Topotecan, p24 in the culture supernatant was quantitated as described in the General Comments. The thus determined p2 ~
levels are depicted in Figure 6. In particular, it was found that Topotecan at concentrations of 31 nMi reduced HIV-l replication by more than 80~. Cell viability as shown in Figure 20 ~ 5 was determined by MTT assay as described in the General Comments.

E~AMPLE 6 293.27.2 cells were plated at I X 106 cells~plate in 175 mm plates. After 48 hours, cells were treated with TNF~ (40 u/ml) in the presence of different concentrations oE Topotecan Total cellular RNA was prepared at 0 hours, 2 hours, or 6 hours ¦ after addition of TNF~. Northern blot analysis was carried out as described in the General Comments and the results depicted in Figure 7.
~ e ~ EXAMPLE 7 -i 293.27.2 cells were plated and treated with differen~
~ concentrations of Topotecan, as described in Example 6 above.
';~ 35 Total cellular RNA was prepared at 6 hours after addition of :

: , .
~ ~, z 8 3 ~
~ ~0 ~/0~160 PCT/US93/()793 ! - 22-TNF~. Northern blot analysis was done as described in the General Comments. The autoradiograms were scanned. HIV LTR
directed ~-galactosidase mRNA was determined by densitometry. Results are depicted in Figure 8.

293.27.2 cells were plated as described in Example 6 above.
After 48 hours, cells were treated with varying concentrations of Topotecan as shown in Figure 9 for 3 hours. 3H-uridine was added to the culture media (1 ~Ci/~l) at the beginning of the third hour. Total cellular RNA was prepared by the end of the third hour. Newly synthesized total cell RNA was quantitated by scintillation counting, and RNA ooncentrations were ~easured spectrophotometricalIy and resuLts depicted in Figure 9 where each data point is an average of triplicate dishes of a representative study of two independent experiments.

E~MPLE 9 The inhibition of HIV-l replication in ~IV-l infected cells ehat are resistant ~o a reverse transcriptase inhibitor was investigated. Topotecan (TPT) at a dose of about 0.006-0.008 ~M (IC50) was measured in PBMCs acutely infected with an AZT resistant HIV-l strain. The AZT resistant strain was inhibited by TPT at such doses as measured by the production of - 25 HIV p24 antigen and HIV-l LTR directed ~-galactosidase gene RNA expression.

A chronically infected T-lymphocyte line and the promyelocytic cell line OM10.1 also showed marked inhibition at IC50 ~ 0.008 ~M following treatment with TPT whereas AZT
and ddI had little effect. The promyelocytic cell lina OM10.1 shows activation of HIV-l p24 antigen following treatments with 20 units of TNF ~ and this is efectively blocked by treatment with TPT. Thus, TPT inhibits HIV-l LTR directed expresslon and viral Pi4 antigen and ~IV RNA production in :

:
.~ :

~ VO 94/04160 Z l `~2~31 PCT/~iS93/0793~ ~
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i such acutely infected and chronically in~ec~ed cell lines with minimal cell toxici.ty. TPT also prevents TNF ~ ~ediated activation of HIV p2 4 and OM10.1 cells.

; Topotecan (TPT) was added to cultures of OM10.1 cells (105 cells/ml) at the sa~e time as TNF~ (20 units/ml). OM10.1 cells in log growth phase were resuspended in RPMI 1640 ~edium IO and aliquoted into a 24-well ~icro-culture plate at 3 x 105 cells/well. A range of 0.002-0.031 ~M TPT was applied to the cell culture in duplicate. OM10.1 cells were stimulated with TNF-o (Endogen Inc., Boston, ~A) for 36 hours and p2 4 antigen was determined by Coulter p2 4 kinetics assay with 1:250 dilution o~ ~he specimen. The assay was run in : duplicates with results shown in the below Table, where values ` are the mean of two reprcsentative assays. The pro~yelocytic cell line OM10.1 contains one copy of HIV-l per cell integrated into its genome and continually produces a low level of HIV-l ~' 20 proteins. This cell line was kindly supplied by NIH-AIDS
Research and Reference Reagent Program.

TABLE
.
TOPOTECAN PREVENTS ACTIVATION OF HIV p24 ~ EXPRESSION BY TNF-~ IN OM10.1 CELLS

Trea~ment p24 (pg/~l) % Inhibition % Cell #

i~ 28 I00~ 1 30 TN~-~ 624 0 100 ' TNF-~ + TPT .004 ~M 386 ~ ; 40 89 TNF-~ + TPT .008~M 326 50 98 TNF-~ + TPT .016 ~M243 ; 64 154 35TNF-~ + TPT .031 ~M 70 93 131 W O 94/0~l6U ~ 3~ PCT/~-93~07-3~

¦ This invention has been described in detail with reference ¦ to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of l~.the disclosure, may make modification and improvements with the 5 sp~rit and scope of the invention.

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Claims (16)

What is claimed is:

1. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective antiviral treatment amount of a compound of formula I

I

wherein R, R1 and R2 are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unsubstituted alkenyl, provided at least two of the substituents of R, R1 and R2 are other than hydrogen, adapted for treating a mammal having an immunodeficiency virus.

2. A method of inhibiting or reducing the expression of genes operably linked to a LTR of an immunodeficiency virus which comprises administering an effective gene expression reduction amount of a compound of the formula I:

I

wherein R, R1 and R2 are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unsubstituted alkenyl, provided at least two of the substituents of R, R1 and R2 are other than hydrogen.

3. A method for treating cells infected with a virus capable of causing an immunodeficiency disease in a human, comprising administering to the cells an effective antiviral treatment amount of a compound of the following formula I:

I

wherein R, R1 and R2 are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unsubstituted alkenyl, provided at least two of the substituents of R, R1 and R2 are other than hydrogen.

4. A method of treating humans having an immunodeficiency disease, wherein the immunodeficiency disease is resistant to a reverse transcriptase inhibitor, comprising administering to said human an effective immunodeficiency disease treatment amount of a compound of the following formula I:

I

wherein R, R1 and R2 are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unsubstituted alkenyl, provided at least two of the substituents of R, R1 and R2 are other than hydrogen.

5. The method of claim 1 wherein the cell line is a reverse transcriptase inhibitor resistant cell line.

6. The method of claims 1, 2, 4, and 5 wherein the reverse transcriptase inhibitor is selected from the group consisting of zidovudine (AZT), 2',3'-dideoxyinosine (ddI), and 2',3'-dideoxycytidine (ddC).

7. The method of claim 6, wherein the reverse transcriptase inhibitor is AZT.

8. The method of claim 1 or 2 wherein the LTR is an HIV
LTR.

9. The method of claim 8 wherein the LTR is an HIV-1 LTR.

10. The compound described in claims 1, 2, 3, or 4 where R
is ethyl, R1 is hydrogen, and R2 is 9-hydroxyl.

11. The compound described in claims 1, 2, 3 or 4, wherein the compound of formula I is Topotecan.

12. The method of claim 2 wherein the virus infecting the cells is resistant to a reverse transcriptase inhibitor.

13. The method of claim 2 or 12 where the cells are human cells.

14. The method of claims 1, 2, 4, 5, 12 where the virus is capable of causing in a human, acquired immune deficiency syndrome or an acquired immune deficiency syndrome related complex.

15. The method of claim 14 where the virus is HIV.

16. The method of claim 15 where the virus is HIV-1.