U.S. patent application number 11/250899 was filed with the patent office on 2006-03-09 for procedure to block the replication of reverse transcriptase dependent viruses by the use of inhibitors of deoxynucleotides synthesis.
Invention is credited to Andrea Cara, Robert C. Gallo, Wen-Yi Gao, Franco Lori.
Application Number | 20060052317 11/250899 |
Document ID | / |
Family ID | 22065285 |
Filed Date | 2006-03-09 |
United States Patent
Application |
20060052317 |
Kind Code |
A1 |
Lori; Franco ; et
al. |
March 9, 2006 |
Procedure to block the replication of reverse transcriptase
dependent viruses by the use of inhibitors of deoxynucleotides
synthesis
Abstract
A method for inhibiting replication of reverse transcriptase
dependent virus in plant or animal cells, comprising the step of
administering to said cells a compound that depletes the
intracellular pool of deoxyribonucleoside phosphate in an amount
effective to inhibit replication of said virus. Hydroxyurea is one
such suitable compound. Also disclosed is a method for producing
incomplete reverse-transcriptase dependent viral DNA, by
administering a deoxyribonucleoside phosphate-depleting drug to
cells infected with such a virus.
Inventors: |
Lori; Franco; (Bethesda,
MD) ; Cara; Andrea; (Rockville, MD) ; Gao;
Wen-Yi; (Rockville, MD) ; Gallo; Robert C.;
(Bethesda, MD) |
Correspondence
Address: |
KNOBBE, MARTENS, OLSON & BEAR, LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
22065285 |
Appl. No.: |
11/250899 |
Filed: |
October 14, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09756411 |
Jan 8, 2001 |
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11250899 |
Oct 14, 2005 |
|
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|
09497700 |
Feb 3, 2000 |
6194390 |
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09756411 |
Jan 8, 2001 |
|
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08245259 |
May 17, 1994 |
6046175 |
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09497700 |
Feb 3, 2000 |
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08065814 |
May 21, 1993 |
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08245259 |
May 17, 1994 |
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Current U.S.
Class: |
514/23 ; 514/45;
514/49; 514/575 |
Current CPC
Class: |
A61K 31/7072 20130101;
A61K 31/17 20130101; A61P 31/18 20180101; A61K 31/70 20130101; A61K
31/70 20130101; A61P 31/12 20180101; A61P 31/14 20180101; A61K
31/7076 20130101; A61K 31/7076 20130101; A61K 31/19 20130101; A61K
31/17 20130101; A61K 31/19 20130101; A61K 31/708 20130101; A61K
31/17 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 31/17 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 31/7088 20130101;
A61K 31/70 20130101; A61K 31/7072 20130101; A61K 45/06 20130101;
A61K 31/708 20130101; A61K 31/7088 20130101; A61P 43/00
20180101 |
Class at
Publication: |
514/023 ;
514/045; 514/049; 514/575 |
International
Class: |
A61K 31/7072 20060101
A61K031/7072; A61K 31/7076 20060101 A61K031/7076; A61K 31/19
20060101 A61K031/19 |
Claims
1. A method for inhibiting replication of reverse transcriptase
dependent virus in animal cells, comprising the step of
administering to said cells a compound that depletes the
intracellular pool of deoxyribonucleoside phosphate in an amount
effective to inhibit replication of said virus.
2. The method of claim 1, wherein said virus is a retrovirus.
3. The method of claim 1, wherein said deoxynucleoside phosphate
depleting compound is a deoxynucleotide synthesis inhibitor.
4. The method of claim 1, wherein said deoxynucleoside phosphate
depleting compound is an inhibitor of ribonucleotide reductase.
5. The method of claim 4, wherein said compound is hydroxyurea.
6. The method of claim 1, wherein said cells are in vitro.
7. The method of claim 1, wherein said animal cells are mammalian
cells.
8. The method of claim 1 wherein said virus is the human
immunodeficiency virus (HIV) and said cells are human cells.
9. A method for inhibiting replication of reverse transcriptase
dependent virus in animal cells, comprising the steps of
administering to said cells a compound that depletes the
intracellular pool of deoxyribonucleoside phosphate, in conjunction
with administering to said cells an antiviral nucleoside phosphate
analog.
10. The method of claim 9, wherein said deoxynucleotide phosphate
depleting compound is an inhibitor of ribonucleotide reductase.
11. The method of claim 10, wherein said compound is
hydroxyurea.
12. A method for inhibiting replication of reverse transcriptase
dependent viruses in animal cells, comprising the steps of
administering to said cells a first compound that depletes the
intracellular pool of deoxyribonucleoside phosphate, in conjunction
with a second compound that serves to inhibit replication of said
virus by terminating DNA chain elongation.
13. The method of claim 12, wherein said second compound inhibits
replication by premature termination of viral DNA synthesis to
produce incomplete viral DNA.
14. The method of claim 12, wherein said first compound is an
inhibitor of ribonucleotide reductase.
15. The method of claim 14, wherein said first compound is
hydroxyurea.
16. The method of claim 15, wherein said second compound is
selected from the group consisting of ddI, ddC, 2'-F-dd-ara-A,
2'-F-dd-ara-I and 2'-F-dd-ara-G.
17. The method of claim 12, wherein said second compound is
selected from the group consisting of a dideoxynucleoside and
AZT.
18. The method of claim 16, wherein said dideoxy nucleoside is a
2'-fluoro purine dideoxynucleoside.
19. The method of claim 16, wherein said dideoxynucleoside is
selected from the group consisting of ddI, ddC, 2'-F-dd-ara-A,
2'-F-dd-ara-I and 2'-F-dd-ara-G.
20. A method of producing incomplete viral DNA from a reverse
transcriptase dependent virus in animal cells, comprising the step
of administering to said cells a compound that depletes the
intracellular pool of deoxyribonucleoside phosphate in an amount
effective to inhibit replication of said virus.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/756,411, filed Jan. 8, 2001, which is a
continuation of U.S. patent application Ser. No. 09/497,700, filed
Feb. 3, 2000, now U.S. Pat. No. 6,194,390, which is a continuation
of U.S. patent application Ser. No. 08/245,259, filed May 17, 1994,
now U.S. patent No.: U.S. Pat. No. 6,046,175, which is a
continuation-in-part of U.S. patent application Ser. No.
08/065,814, filed May 21, 1993 now abandoned.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
reverse transcriptase dependent viruses. More specifically, the
invention relates to the use of agents which reduce intracellular
concentrations of deoxyribonucleosides as a means to inhibit the
replication of reverse transcriptase dependent viruses.
BACKGROUND OF THE INVENTION
[0003] Viruses are microorganisms that depend, to some degree, on
host cell components for their growth and replication. Viral
infection and replication in host cells generally results in
disease, whether the host is an animal or plant. Human diseases
caused by viral infections include the acquired immunodeficiency
syndrome (AIDS) and hepatitis. A general discussion of this field
is presented in Fundamental Virology, Second Edition, (ed. B. N.
Fields, D. M. Knipe, R. M. Chanock, M. S. Hirsh, J. L. Melnick, T.
P. Monath, and B. Roizman, Raven Press, Ltd., New York, N.Y.
1991).
Retrovirus Replication
[0004] Retroviruses comprise a large family of viruses that
primarily infect vertebrates. Many diseases, including the
induction of some tumors, are associated with retroviral infection
(see Fundamental Virology, supra, pp. 645-708). All retroviruses,
regardless of their clinical manifestations, have related
structures and modes of replication.
[0005] Retroviruses contain an RNA genome that is replicated
through a DNA intermediate. Inside the cell, the viral genome
serves as a template for the synthesis of a double-stranded
deoxyribonucleic acid (DNA) molecule that subsequently integrates
into the genome of the host cell. This integration occasionally
results in the induction of a tumor in the infected host organism.
Following integration, a complex sequence of events leads to the
production of progeny virions which are released from the infected
cell.
[0006] Early in the retroviral life cycle, the RNA genome is copied
into DNA by the virally encoded reverse transcriptase (RT). This
enzyme can use both RNA and DNA templates, thereby producing the
first strand of DNA (the negative strand) from the infecting RNA
genome and a complementary second strand (the positive strand) of
DNA using the first DNA strand as a template. To synthesize these
DNA strands, the RT utilizes cellular substrates called
deoxynucleoside triphosphates (dNTP).
[0007] Human retroviruses can be grouped into the leukemia viruses
(HTLV type viruses) and the immunodeficiency viruses (HIV type
viruses). HTLV infection may lead to one form of leukemia. Acquired
immunodeficiency syndrome (AIDS) is caused by a form of HIV, with
HIV-1 being more virulent than HIV-2. Both HTLV and HIV infect
peripheral blood lymphocytes (PBL).
[0008] Other animal retroviruses include feline leukemia virus
(FeLV) and lentiviruses. Virulent FeLV infection generally results
in fatal aplastic anemia in cats. Lentiviruses cause a variety of
neurological and immunological diseases such as visna in sheep and
infectious anemia in horses.
HIV Infection
[0009] HIV-1 was first identified as the causative agent of AIDS in
1983. The AIDS pandemic is now one of the most serious health
problems worldwide. Catastrophic medical and social consequences
are likely to extend into the next century. The World Health
Organization (WHO) has estimated that between eight and ten million
people are currently infected with HIV, and that approximately ten
times as many individuals will be affected in the next decade. The
large pool of HIV carriers makes the development of effective
antiviral treatments a medical priority.
Hepatitis B Infection
[0010] Hepatitis B virus (HBV) is one of at least three (A, B and
C) viruses that selectively infect liver cells (for a general
discussion of HBV see Fundamental Virology, supra, pp. 989-1021).
HBV infections tend to be persistent with minimal liver damage or
with chronic hepatitis that may lead to cirrhosis or liver cancer
(hepatocellular carcinoma or HCC). Worldwide, more than 200 million
people infected with HBV.
Other Viruses
[0011] Several other viruses that infect humans, animals and plants
also depend on reverse transcriptase for replication. These include
retroviruses such as the leukemia viruses known to exist in several
species, including HTLV-1 in humans, as well as reverse
transcriptase dependent DNA viruses, such as the cauliflower mosaic
virus (a plant virus).
Antiviral Therapies
[0012] There is a critical need to develop effective drug
treatments to combat RT dependent viruses such as HIV. Such efforts
were recently urged in the United Kingdom-Irish-French Concorde
Trial conclusions which reported that the nucleoside analog
zidovudine (AZT), a mainstay in the treatment of patients infected
with HIV-1, failed to improve the survival or disease progression
in asymptomatic patients. Other nucleoside analogs like didanosine
(ddI) are currently under evaluation. The effects of ddI on disease
progression and patient survival endpoints have not been adequately
investigated. Non-competitive HIV-1 RT inhibitors and HIV-1
protease inhibitors have also been recently developed. Despite the
high efficacy of these compounds, the initial in vitro/in vivo
testing has been characterized by the rapid onset of variants of
HIV-1 resistant to these drugs (escape mutants). Despite having
different antiviral activities and pharmacokinetics properties, the
drugs mentioned here all directly target HIV-1 proteins.
[0013] Although this latter approach must be continued, we have
developed a different antiviral strategy that targets one or more
cellular components that are required for the replication of
reverse transcriptase dependent viruses.
SUMMARY OF THE INVENTION
[0014] The present invention is based on the discovery that drugs
which reduce the intracellular concentration of deoxynucleoside,
phosphates inhibit the replication of reverse transcriptase
dependent viruses: Such drugs act either by inhibiting the
intracellular synthesis of deoxynucleoside phosphates or by
depleting the intracellular pool of deoxynucleoside phosphates.
Viruses sensitive to growth inhibition by limiting deoxynucleoside
phosphates are retroviruses, including HIV which causes AIDS,
hepatitis B virus, cauliflower mosaic virus, and other reverse
transcriptase dependent viruses. As one example, hydroxyurea limits
synthesis of the intracellular deoxynucleoside phosphates by
inhibiting enzymatic activity of ribonucleoside reductase. Other
compounds are known that similarly inhibit accumulation of
intracellular deoxynucleoside phosphates by this mechanism or by
affecting other biosynthetic steps that lead to production of
intracellular deoxynucleoside phosphates. Compounds that limit
intracellular deoxynucleoside phosphates can be used in conjunction
with antiviral nucleoside phosphate analogs, which are themselves
therapeutic as competitive inhibitors of native nucleosides, to
increase the effectiveness of antiviral treatment. Compounds that
deplete intracellular deoxynucleoside phosphates may be used as an
alternative to treatment with antiviral nucleoside phosphate
analogs, especially when a virus has become refractory to
nucleoside analog treatment.
[0015] One aspect of the present invention is a method for
inhibiting replication of reverse transcriptase dependent virus in
animal cells, comprising the step of administering to the cells a
compound that depletes the intracellular pool of
deoxyribonucleoside phosphate in an amount effective to inhibit
replication of the virus. The virus can, for example, be a
retrovirus, or a reverse transcriptase-dependent DNA virus. The
deoxynucleoside phosphate depleting compound in one embodiment is a
deoxynucleotide synthesis inhibitor. In another embodiment, the
deoxynucleoside phosphate depleting compound is an inhibitor of
ribonucleotide reductase. One preferred compound is
hydroxyurea.
[0016] The invention can be used on cells in vitro or in vivo. In
various preferred embodiments, the animal is a mammal or a bird.
Preferably, the animal is a human.
[0017] In one specific embodiment, the virus is the human
immunodeficiency virus (HIV), such as HIV-1 or HIV-2, and the cells
are human cells. In another specific embodiment, the virus is
hepatitis B and the cells are human cells.
[0018] The method of the present invention may be practiced by
depleting the intracellular pool of deoxynucleoside phosphates to
limit viral replication by limiting the rate of DNA chain
elongation. For example, AZT and dideoxynucleosides, such as ddI,
ddC and 2'-fluoro dideoxynucleosides, so limit viral replication.
This can result in premature termination of viral DNA synthesis to
produce incomplete viral DNA.
[0019] Another aspect of the present invention is a method for
inhibiting replication of reverse transcriptase dependent virus in
animal cells, comprising the steps of administering to the cells a
compound that depletes the intracellular pool of
deoxyribonucleoside phosphate, and coadministering to the cells
antiviral nucleoside phosphate analogs which compete with the pool
of deoxyribonucleoside phosphates. Preferred antiviral nucleoside
phosphate analogs include AZT, ddI, and ddC.
[0020] A different aspect of the invention relates to a method of
producing incomplete viral DNA from reverse transcriptase dependent
virus in animal cells, comprising the step of administering to the
cells a compound that depletes the intracellular pool of
deoxyribonucleoside phosphate in an amount effective to inhibit
replication of the virus.
[0021] Finally, the invention includes a method for inhibiting
replication of reverse transcriptase dependent virus in plant
cells, comprising the step of administering to the cells a compound
that depletes the intracellular pool of deoxyribonucleoside
phosphate in an amount effective to inhibit replication of the
virus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1a graphically depicts p24 expression in HIV-1 infected
PBL as a function of the hydroxyurea and ddI concentrations.
[0023] FIG. 1b graphically depicts the number of viable PBL in an
HIV-1 infected culture as a function of the hydroxyurea and ddI
concentrations.
[0024] FIG. 1c shows HIV-1 p24 expression normalized to the number
of viable cells as a function of the HU and ddI concentrations.
[0025] FIG. 2 graphically depicts p24 expression in HIV-1 infected
human primary macrophages as a function of hydroxyurea and AZT
concentrations.
[0026] FIG. 3a graphically depicts a time course of p24 inhibition
by hydroxyurea and/or by ddI in activated PBL isolated from an
HIV-1 infected patient.
[0027] FIG. 3b graphically depicts the number of viable cells
isolated from an HIV-1 infected patient that survived in culture
with treatment by hydroxyurea and/or ddI.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0028] The present invention is based on the discovery that a
reduction of the intracellular deoxynucleoside triphosphate (dNTP)
concentration selectively inhibits the replication of reverse
transcriptase dependent viruses. An approach to virus inhibition
that is based on this strategy advantageously avoids triggering the
formation of viral escape mutants. Conversely, direct selective
pressure against viral proteins would be expected to promote the
formation of such mutants.
[0029] In the practice of the present invention, hydroxyurea is one
preferred compound that depletes intracellular dNTP levels. This
compound is one of many inhibitors of ribonucleotide reductase, an
enzyme catalyzing the reduction of ribonucleoside diphosphates to
their deoxyribonucleoside counterparts for DNA synthesis. Other
ribonucleotide reductase inhibitors include guanazole,
3,4-dihydroxybenzo-hydroxamic acid,
N,3,4,5-tetrahydroxybenzimidamide HCl, 3,4-dihydroxybenzamidoxime
HCl, 5-hydroxy-2-formylpyridine thiosemicarbazones, and
.alpha.-(N)-heterocyclic carboxaldehyde thiosemicarbazones,
4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone,
N-hydroxy-N'-amino-guanidine (HAG) derivatives,
5-methyl-4-aminoisoquinoline thiosemicarbazone, diaziquone,
doxorubicin, 2,3-dihydroxylbenzoyl-dipeptides and
3,4-dihydroxylbenzoyl-dipeptides, iron-complexed 2-acetylpyridine
5-[(2-chloroanilino)-thiocarbonyl]-thiocarbonohydrazone (348U87),
iron-complexed
2-acetylpyridine-5-[(dimethylamino)thiocarbonyl]-thiocarbonohydrazone
(A1110U), 2'-deoxy-2'-methylenecytidine 5'-diphosphate (MdCDP) and
2'-deoxy-2',2'-difluorocytidine 5'-diphospahte(dFdCDP),
2-chloro-9-(2-deoxy-2-fluoro-.beta.-D-arabinofuranosyl)-adenosine
(Cl--F-ara-A), diethyldithiocarbamate (DDC),
2,2'-bipyridyl-6-carbothioamide, phosphonylmethyl ethers of acyclic
nucleoside analogs, [e.g., diphosphates of
N-(S)-(3-hydroxy-2-phosphonylmethoxypropyl and
N-2-phosphonylmethoxyethyl) derivatives of purine and pyrimidine
bases], nitrosourea compounds, acylclonucleoside hydroxamic acids
(e.g.,
N-hydroxy-.alpha.-(2-hydroxyethoxy)-1(2H)-pyrimidineacetamides 1-3,
and 2-acetylpyridine 4-(2-morpholinoethyl)thio-semicarbazone
(A723U)).
[0030] Compounds that inhibit dNTP synthesis or that otherwise
deplete the intracellular pool of at least one dNTP may be
administered by any conventional route. Where treated cells are in
vitro, the compound may simply be introduced into the medium in
which the cells are growing. On the other hand, where cells to be
treated are part of a larger organism, that is, where treatment is
in vivo, administration to an animal may be via the oral route, or
may be intravenous, intraperitoneal, intramuscular, subcutaneous,
transdermal, transmucosal (e.g., by inhalation or by means of a
suppository), or by any other suitable route. Administration to
plants may be accomplished by spraying, dusting, application in
irrigation water, or by any other conventional means.
[0031] It should be noted that depletion of the intracellular pool
of any one of the four deoxynucleoside phosphates is considered to
be within the scope of the present invention. Furthermore,
depletion of mono-, di-, or triphosphates of nucleosides is also
within the scope of this invention.
[0032] The particular dosage, toxicity, and mechanism for delivery
of the dNTP-depleting drugs of the present invention are either
already known, or can be readily determined by conventional
empirical techniques. Although some of the dNTP-depleting compounds
may exhibit limiting toxicity or difficulties in intracellular
delivery, others (such as hydroxyurea) have been extensively
studied and found to have favorable pharmacological properties.
[0033] Suitable human dosages for these compounds can vary widely.
However, such dosages can readily be determined by those of skill
in the art. For example, dosages to adult humans of from about 0.1
mg to about 1 g or even 10 g are contemplated.
[0034] In one preferred embodiment, the dosage is such that the
intracellular dNTP pool is depleted to a concentration that is
below the K.sub.m of the viral reverse transcriptase, but above the
K.sub.m of endogenous cellular polymerases, such as DNA polymerase
.alpha., .beta., and .gamma.. This permits selective inhibition of
viral replication without significant cellular toxicity.
[0035] Hydroxyurea has been widely used in cancer therapy as a
broad spectrum antineoplastic drug (R. C. Donehower, Seminars in
Oncology 19 (Suppl. 9), 11 (1992)). Hydroxyurea is readily absorbed
after oral ingestion, rapidly distributed in the body fluids,
including the cerebrospinal fluid, and enters cells efficiently by
passive diffusion (Id.). Its toxic effects are less profound and
easier to control than other chemotherapeutic drugs (Id.).
[0036] In human chemotherapy, hydroxyurea is currently administered
using two basic schedules: (a) a continuous daily oral dose of
20-40 mg per kg per day, or (b) an intermittent dose of 80 mg per
kg per every third day. Either schedule could be used in the
treatment of viral infections. However, because response to
treatment is variable, peripheral white blood cell counts must be
monitored so that treatment can be stopped when leukopenia occurs.
Similar dosage ranges may be used in the practice of the present
invention.
[0037] Given that viral reverse transcriptase is generally quite
sensitive to decreased levels of dNTP, lower dosages of hydroxyurea
may also be effective in treating viral infections. Such low
dosages of hydroxyurea would reduce the toxicity to white blood
cells. Any dosage that effectively decreases the replication of
RT-dependent viruses would be useful in chronically treating AIDS
patients.
[0038] In the practice of the present invention, the inhibition of
reverse transcriptase activity and the impairment of HIV-1 DNA
synthesis are accomplished by treating cells with hydroxyurea.
Under the specified conditions, incomplete HIV-1 DNA was formed
without apparent toxic effects to the cells. Incomplete viral DNA
has been shown to be rapidly degraded in PBL (Zack, et al., supra,
1990 and 1992). Therefore, the present invention provides a new
method to inhibit HIV replication by modulating intracellular dNTP
pools. This is accomplished by employing drugs such as hydroxyurea
at pharmacological ranges.
[0039] The present invention also encompasses antiviral therapies
that are based on the use of dNTP-depleting drugs in conjunction
with conventional or novel nucleoside phosphate analogs. By
depleting the intracellular dNTP pool, drugs such as hydroxyurea
are expected to increase the therapeutic effect of treatment of HIV
infection by nucleoside phosphate analogs such as AZT, ddI, ddC,
2'-F-dd-ara-A, 2'-F-dd-ara-I and 2'-F-dd-ara-G. These analogs act
as competitors of cellular dNTP according to an antiviral mechanism
that is distinct from that of hydroxyurea. A description of the
2'-fluoro nucleosides has been presented by Marquez et al. in 1990
J. Med. Chem. 33:978-985. Currently, antiviral therapy requires
doses of AZT or ddI at 500 mg per day or ddC at 2 mg per day for an
adult human. Similar dosages may be used in the present invention.
However, use of dNTP depleting drugs may increase the effectiveness
of these nucleoside phosphate analogs so that they can be used at
lower dosages or less frequently.
[0040] One of the problems in using antiviral nucleoside phosphate
analogs is the appearance of escape mutants. Such variants usually
derive from mutations in the gene that encodes RT. We believe the
appearance of RT mutants that can function using low levels of
nucleotides will be an unlikely event. Hence, we believe that
antiviral drugs, such as hydroxyurea, which deplete intracellular
dNTP pools will be unlikely to favor the evolution of RT escape
mutants. Furthermore, drugs that deplete the intracellular dNTP
pool could be of value in the treatment of viral disease in cases
where RT escape mutants have appeared.
[0041] Because dNTP-depleting drugs and nucleoside phosphate
analogs have different inhibitory mechanisms, we predict that
combinations of these agents will result in synergistic inhibitory
effects. By depleting the intracellular nucleotide pool with
hydroxyurea or a similarly acting drug, the therapeutic effects of
nucleoside phosphate analogs, which act as competitors of dNTP, are
expected to increase. Such a combination drug treatment may also
result in decreased toxicity since lower dosages of nucleoside
phosphate analogs would be rendered more effective.
[0042] Because HIV-1 RT is a distributive enzyme, we expected that
low levels of dNTPs induced by drugs such as hydroxyurea would
affect RT more than the cellular DNA polymerases .alpha., .beta.,
and .gamma., which are known to be processive enzymes (Huber, et
al., supra; Kati, et al., supra; U. Hubscher, supra). This
selective effect on RT may result in lower cellular toxic effects
than occur with other antiviral drugs.
[0043] Unlike retroviruses, HBV is a DNA virus with a partially
double-stranded and partially single-stranded genome. However, like
retroviruses, reverse transcription is required early in the
process of HBV genome replication. The RT is specified by the HBV
genome and synthesized in the infected host liver cell where viral
replication occurs.
[0044] Because replication of the HBV viral genome is dependent on
RT, it is expected that the method of limiting dNTP pools by
treating people with therapeutic drugs that inhibit dNTP synthesis
would also be effective in limiting HBV viral replication. Drugs
such as hydroxyurea that diffuse into nearly all cell types would
be particularly advantageous in controlling hepatic replication of
HBV.
[0045] Limiting HBV replication has two important effects. First,
it limits the spread of infectious virions from carriers to
uninfected individuals. Second, it decreases the symptoms such as
chronic hepatitis in infected individuals. Generally, liver
function improves after HBV replication ceases. Also, because the
incidence of HCC is much higher in HBV-infected humans, decreased
infection in the population presumably would result in a decreased
incidence of liver cancer.
[0046] As described above, the use of hydroxyurea (or similar
dNTP-limiting drugs) in conjunction with antiviral drugs, such as
adenine arabinoside, ara-monophosphate, acyclovir,
6-deoxyacyclovir, and .alpha., .beta., and .gamma. interferons,
that act via other mechanisms could also increase the effectiveness
of these anti-HBV drugs. This is especially predicted for adenine
arabinoside which acts as a competitive inhibitor in a mechanism
analogous to that of antiviral nucleoside phosphate analogs used to
treat HIV infections. Furthermore, treatment with hydroxyurea (or
similar dNTP-limiting drugs) could make antiviral drugs more
effective at lower doses than required for treatment solely using
antiviral drugs.
[0047] As described above, the method of using hydroxyurea (or
similar dNTP-limiting drugs) on people whose HBV infections have
become refractory to antiviral drugs is also anticipated in the
present invention.
[0048] Other viruses that infect animals or plants are also
dependent on RT activity for their replication. Cauliflower mosaic
virus is one example of a virus that uses a RT in replication of
its DNA genome.
[0049] The botanical use of compounds that limit intracellular dNTP
pools to inhibit viral replication of other reverse transcriptase
dependent viruses is within the scope of the present invention, as
is the use of such compounds on animals, including humans, infected
with a wide variety of RT-dependent viruses.
[0050] The rationale for the present invention and the practice of
the present invention may be better understood by reference to the
following nonlimiting examples.
[0051] A key step of HIV-1 infection of PBL is the conversion of
the viral RNA genome into double-stranded DNA by the action of
HIV-1 RT. Viral DNA synthesis differs in different states of
infected PBL. In quiescent PBL, viral DNA synthesis can be
initiated as efficiently as in mitogen-stimulated PBL. However, in
contrast to the stimulated cells, DNA synthesis in quiescent PBL
may terminate prematurely (J. A. Zack, et al. 1990 Cell 61:213; J.
A. Zack, et al. 1992 Virology 66:1717) producing no HIV-1 progeny
(Zack, et al., supra; M. Stevenson, et al., 1990 EMBO J. 9:1551; M.
I. Bukrinsky, et al. 1991 Science 254:423). This process results in
a pool of unintegrated viral DNA (Stevenson, et al., supra;
Bukrinsky, et al., supra), which can remain latent in both in vitro
infected quiescent PBL and in vivo infected resting PBL (Zack, et
al., supra, 1990 & 1991; Stevenson, et al., supra; Bukrinsky,
et al., supra). Stimulation of these cells can rescue HIV-1 DNA,
leading to integration and production of viral progeny (Id.).
Incomplete viral DNA has also been found associated with HIV-1
mature infectious particles, but the biological role of this DNA is
unclear (F. Lori, et al. 1992 J. Virol. 66:5067; D. Trono 1992
ibid. 66:4893).
[0052] Example 1 illustrates a method that can be used to
quantitate the replication of the HIV-1 genome in infected cells.
In this example, the rates of HIV-1 DNA synthesis in infected
quiescent and stimulated PBL were quantitatively analyzed using a
polymerase chain reaction (PCR) assay.
EXAMPLE 1
HIV Replication
[0053] The PCR assay, previously applied to quantitate HIV-1 DNA in
mature HIV-1 virions (F. Lori et al., supra; D. Trono, supra), was
used to amplify several regions of the HIV-1 genome. The primer
pairs used to amplify the viral DNA were M667/AA55, M667/BB301, and
M667/M661 (M. Stevenson et al., supra; M. I. Bukrinsky, et al.,
supra). M667 is a sense primer in the R region of the long terminal
repeat (LTR). AA55 is an antisense primer immediately 5' to the PB
(tRNA primer binding) region. The M667/AA55 primer pair amplifies
the negative strand region initially synthesized by RT to yield a
product called R-U5. BB301 is complementary to the PB region.
Amplification by M667/BB301 can be achieved in the presence of
positive stand DNA which has been synthesized starting at the
polypurine tract upstream from the right LTR and, after jumping to
the other end of the template, extended up to the PB region to
yield a product called R-PB (H. E. Varmus and R. Swanstrom, in
Replication of Retroviruses, RNA Tumor Viruses. R. Weiss, N. Teich,
H. Varmus, J. Coffin, Eds., Cold Spring Harbor Laboratory, Cold
Spring Harbor, 1984, pp. 369-512). The negative strand, which is
not fully completed, is not expected to be amplified because the
RNA sequences which are complementary to the PB region have been
digested in these experiments. M661 is an antisense primer in the
gag region. Amplification by M667/M661 reflects the presence of
complete negative strand DNA to yield a product called R-gag. These
primers were designed to estimate the extent of reverse
transcription at three different replicative steps: R-U5, initial
negative strand synthesis; R-PB, initial positive strand synthesis
up to the tRNA primer binding region; and R-gag, complete negative
strand synthesis. These steps occur in subsequent order during
reverse transcription (Varmus and Swanstrom, supra). If the DNA
carried by the virus was a full-length negative strand DNA, the
three regions analyzed by quantitative PCR should be amplified to
equivalent levels. .beta. globin sequences were amplified from the
same DNA extracts in order to normalize the amount of DNA used as
described in J. A. Zack et al., supra (1990); and J. A. Zack et
al., supra (1992).
[0054] Viral DNA was detected immediately after infection of
quiescent PBL and the amount of DNA observed at that time was
proportional to the initial multiplicity of infection (MOI) of the
HIV-1 IIIB strain (M. Popovic, et al. 1984 Science 224:497). MOI of
1 and 10 were used and viral DNA was detected comparable to HIV-1
DNA standards corresponding to about 100 and 1000 copies,
respectively, of HXB2(RIP7) plasmid DNA (J. M. McCune, et al. 1988
Cell 53:55) for the R-U5, R-PB and R-gag regions.
[0055] This DNA was incompletely replicated, the typical form
associated with the mature HIV-1 particles (F. Lori et al., supra;
D. Trono supra). These results suggest that a portion of the
incomplete DNA observed in PBL at early phases of infection was
contributed by the DNA carried by the infectious viruses. Viral DNA
synthesis for 72 hours after infection was next analyzed. HIV-1 DNA
synthesis in quiescent PBL was significantly slower and less
efficient than in stimulated PBL. In particular, in quiescent PBL
the initial synthesis of viral DNA at the origin of retroviral DNA
replication (immediately upstream to the tRNA primer binding
region, represented by the R-U5 product of the PCR reaction) was
achieved relatively early after infection (after 10 hours), while
the completion of full-length negative strand DNA synthesis was
significantly delayed (between 48 and 72 hours post-infection,
represented by the R-gag product of the PCR assay). In contrast,
synthesis of full-length negative strand DNA was completed within
10 hours after infection in stimulated PBL.
[0056] Moreover, in stimulated PBL the DNA synthesis progressively
increased during the time course at much higher levels than in
quiescent PBL.
[0057] In summary, we found the total amount of viral DNA produced
in quiescent PBL was significantly less than that produced in
stimulated PBL. Even at 72 hours, the amount of viral DNA in
quiescent PLB was about 10-fold less than the amount produced in
stimulated PBL after 10 hours of growth. After 72 hours of growth,
the total amount of viral DNA produced in stimulated PBL was at
least 100-fold more than the amount produced in quiescent PBL.
[0058] Conflicting observations have been reported previously
regarding the form of HIV-1 DNA in infected quiescent lymphocytes.
An incomplete DNA in infected quiescent cells was reported by Zack
et al. (supra, 1990 and 1992). On the other hand, Stevenson et al.
(supra) showed latent complete DNA was present in quiescent PBL,
but this DNA was unintegrated. These discrepancies could be
explained by our findings that DNA synthesis proceeds in a slow and
inefficient manner in quiescent PBL.
[0059] Previous studies have shown that cellular enzymes which are
responsible for dNTP synthesis, such as thymidine kinase and
deoxycytidine kinase, have extremely low activities in quiescent
PBL, that increase dramatically in activated PBL (L. Pegoraro and
M. G. Bernengo, 1971 Exp. Cell Res. 68:283). Low levels of dNTP
synthesis and the high turnover rate of dNTP during DNA replication
(J. Ji and C. K. Mathews, 1991 Mol. Gen. Genet. 226:257) would
deplete the intracellular dNTP pool. In steady-state kinetics, if
the dNTP pools were significantly lower than the Michaelis
constant, K.sub.m, most of the catalytic potential of HIV-1 RT
would be wasted and the rate of the viral DNA synthesis would be
expected to be very sensitive to changes in dNTP concentrations (I.
H. Segel, in Biochemical Calculations, John Wiley & Sons, New
York, 1975).
[0060] Example 2 illustrates the correlation between the low levels
of dNTP in quiescent PBL and the low rate of viral DNA synthesis
that was described above.
EXAMPLE 2
Correlation Between dNTP Pool and HIV Replication
[0061] PBLs were cultured in the presence or absence of
phytohemagglutinin A (PHA) at 10 .mu.g/ml for 48 hours.
Intracellular dNTP were extracted with 60% methanol and were
examined by an enzyme assay using synthetic oligonucleotides (P. A.
Sherman and A. J. Fyfe, 1989 Anal. Biochem. 180:222). Data
represent the mean value of three experiments. K.sub.m values were
determined using a 600-base globin mRNA as template and were 3.8,
4.0, 3.9, and 2.6 .mu.M for dCTP, dTTP, dGTP, and dATP,
respectively. The cellular volume of PBL was measured using a
Coulter counter analizer and found to be approximately 0.25
.mu.l/10.sup.6 cells for quiescent PBL and 0.38 .mu.l/10.sup.6
cells for stimulated PBL, respectively.
[0062] As shown in Table 1, the levels of dNTP in quiescent PBL
were significantly lower than in the stimulated PBL. The latter
were significantly higher than the K.sub.m of HIV-1 RT. Similar
results were obtained after infection with HIV-1. TABLE-US-00001
TABLE 1 Comparison of deoxyribonucleoside triphosphate pools
(.mu.M) in quiescent and PHA stimulated PBL cells. Treatment dATP
dGTP dCTP dTTP PBL 0.32 .+-. 0.04 0.52 .+-. 0.12 1.48 .+-. 0.40
5.60 .+-. 0.80 PBL + 3.24 .+-. 0.08 8.00 .+-. 2.67 18.13 .+-. 1.86
26.13 .+-. 1.60 PHA
[0063] We also assessed the in vitro activity of recombinant HIV-1
RT at dNTP concentrations that were equivalent to those found in
quiescent and stimulated PBL. DNA was synthesized using a globin
mRNA template and an oligo dT.sub.16 primer (a primer extension
assay). The HIV-1 RT reaction mixture contained 50 mM Tris-HCl (pH
8.0), 6 mM MgCl.sub.2, 76 mM KCl, 0.5 mM DTT, 80 nM globin mRNA
primed with oligo dT.sub.16 in 1:5 ratio, and dNTP at (a) the
concentrations equivalent to quiescent cells and (b) the
concentrations equivalent to stimulated cells as described in Table
1. Recombinant HIV-1 RT (obtained from American Biotechnologies)
was used at 5 U/ml.
[0064] Under the nucleotide concentrations that characterized
quiescent conditions, the rate and yield of total DNA synthesis
were profoundly lower than those corresponding to the stimulated
condition. The rates of dTMP incorporation by HIV-1 RT for
quiescent conditions and stimulated conditions are presented in
Table 2. This could explain why DNA synthesis was slower and less
efficient in quiescent than in stimulated PBL. TABLE-US-00002 TABLE
2 Rates of dTMP incorporation in vitro (.rho.mol per unit of HIV-1
RT) in quiescent (-PHA) and stimulated (+PHA) PBL Incubation Time
(min) PBL 0 30 60 90 120 -PHA 0 1.2 .+-. 0.1 4.2 .+-. 0.8 7.6 .+-.
0.8 12.8 .+-. 1.6 +PHA 0 21.6 .+-. 2.2 49.6 .+-. 5.8 104.4 .+-.
13.6 153.0 .+-. 10.6 ratio of +PHA/-PHA 0 18.0 11.8 13.7 12.0
[0065] The mode of action of HIV-1 RT and the size of the DNA
products were further examined using the primer extension assay
described above except that the template-primer was a 600-base
globin mRNA primed with oligo(dT).sub.16 that was .sup.32[p]
labeled at the 5' end. Aliquots were harvested at 0, 15, 30, 60,
and 120 minutes. Reaction products were separated by (a) 15% and
(b) 6% polyacrylamide gel electrophoresis.
[0066] Two types of HIV RT activities were evident: an initial
distributive activity and a later processive activity. In the
initial distributive phase, the RT often became dissociated after
incorporation of a dNTP into the nascent chain, giving rise to
discrete molecular weight DNA products. This was particularly
evident at dNTP concentrations characteristic of quiescent PBL. In
the gel lanes, this gave rise to the ladder appearance of products
ranging in size from the 1 6-mer primer (at time 0) to
approximately a 70-mer (after 60 minutes under quiescent
conditions). After 120 minutes incubation at quiescent PBL
conditions, the longest DNA products measured approximately 70-100
nt. After approximately 70 new nucleosides (nt) were added, the
processivity of HIV-1 RT increased and higher molecular weight DNA
was synthesized. Processivity was observed primarily at dNTP
concentrations similar to those in stimulated PBL. Processivity was
seen after 15 minutes incubation under stimulated PBL conditions
resulting in DNA products over 70-100 nt; and it continued
throughout the experiment resulting in full-length transcripts
after 120 minutes incubation. In contrast, little or no
processivity was seen under quiescent PBL conditions, even after
120 minutes of incubation. These results suggest that low
concentrations of endogenous dNTP alone are sufficient to explain
the impaired DNA elongation observed in quiescent PBL.
[0067] This biphasic pattern of HIV-1 RT activity is in agreement
with the enzyme kinetics studies from others (H. E. Huber et al.
1989 J. Biol. Chem. 264:4669; W. M. Kati, K. A. Johnson, L. F.
Jirva, K. S. Anderson, 1992 ibid. 267:25988) and differs from the
action of most of the replicative DNA polymerases which are
processive polymerases, such as E. coli pol I and IIII, HSV DNA
polymerase, and mammalian DNA polymerases a and y (Huber, et al.,
supra; Kati, et al., supra; U. Hubscher, 1983 Experientia
39:1).
[0068] Example 3 illustrates both that hydroxyurea can be used to
deplete the intracellular dNTP concentration, and that such
suboptimal concentrations of dNTP cause incomplete HIV-1 DNA
synthesis in PBL. The 1 mM hydroxyurea concentration used in these
procedures approximates the blood concentration of this drug during
standard clinical protocols in humans (R. C. Donehower, 1992
Seminars in Oncology 19 (Suppl. 9):11). Notably, hydroxyurea did
not directly inhibit RT enzymatic activity even at a 200-fold
higher concentration (200 mM).
EXAMPLE 3
Use of Hydroxyurea to Inhibit HIV Replication
[0069] IV-1 DNA synthesis was measured after infection of mitogen
(PHA) stimulated PBL in the presence or absence of hydroxyurea.
After 48 hours of PHA stimulation and 24 hours pretreatment with 1
mM hydroxyurea, cells were infected with HIV-1 IIIB (Popovic et
al., supra) in the presence of hydroxyurea. Control cells were
treated similarly, but hydroxyurea treatment was omitted. Cell
aliquots were harvested 24, 48 and 72 hours after infection and
analyzed for the rate of dNTP synthesis inhibition (Sherman and
Fyfe, supra) measured as the percentage of dNTP levels compared to
the control cells.
[0070] The results of this study, illustrated in Table 3, show that
dNTP pools were substantially depleted in stimulated PBL incubated
in the presence of 1 mM hydroxyurea. TABLE-US-00003 TABLE 3 Effect
of hydroxyurea on dNTP pools in treated PBL relative to untreated
control PBL (% of untreated control amount). Incubation Time
(hours) dNTP 0 24 48 72 dATP 100 .+-. 10 19 .+-. 2 19 .+-. 1 7 .+-.
0.5 dGTP 100 .+-. 10 70 .+-. 5 45 .+-. 3 32 .+-. 3 dCTP 100 .+-. 10
82 .+-. 9 61 .+-. 6 35 .+-. 4 dTTP 100 .+-. 10 115 .+-. 10 34 .+-.
2 23 .+-. 3
[0071] In addition, HIV-1 DNA synthesis was measured after
infection of PHA-stimulated PBL in the presence or absence of
hydroxyurea using the PCR analysis as described above. Standards
used for comparison were serial dilutions of HXB2(RIP7) plasmid DNA
(number of copies; McCune et al., supra) and f, globin DNA
(nanograms).
[0072] Depletion of dNTP significantly affected the HIV-1 DNA
synthesis rate and inhibited the completion of viral DNA synthesis
in stimulated PBL. Furthermore, dNTP depletion delayed production
of full-length negative strand viral DNA which was seen in only
limited amounts (approximately 10-fold to 100-fold less over a 72
hour period) relative to cells that were not treated with
hydroxyurea. The pattern of inhibition was quite similar to that
observed in quiescent PBL. After 72 hours, cell viability was
comparable between hydroxyurea treated and untreated cells.
[0073] Because most circulating lymphocytes in vivo are quiescent,
the relevance of the population of quiescent infected PBL serving
as a reservoir of inducible HIV-1 has been recognized (Zack, et
al., supra, 1990 and 1992; Stevenson, et al., supra; Bukrinsky, et
al., supra). The latent viral DNA pool in these cells clearly plays
a role in viral rescue after mitogenic stimulation (Id.). Our
results suggest a mechanism of inefficient reverse transcription
and subsequent formation of latent HIV-1 DNA in quiescent PBL.
Although other mechanisms may also block HIV-1 replication in
quiescent PBL, naturally occurring low levels of dNTP are
sufficient to inhibit reverse transcription.
[0074] Example 4 illustrates that treatment of target cells with
hydroxyurea, alone or in combination with the nucleoside phosphate
analog ddI, inhibits HIV-1 viral expression. In particular, viral
expression of RT in HIV-1 infected PHA-stimulated PBL was
significantly reduced by pre-infection treatment with 1 mM
hydroxyurea.
EXAMPLE 4
Use of Hydroxyurea and/or ddI to Inhibit HIV Expression
[0075] PBL were stimulated with PHA for 48 hours and treated with 1
mM hydroxyurea for 24 hours prior to infection with HIV-1 IIIB
(Popovic, et al., supra) in the presence of- the drug. Control
cells were treated similarly except that hydroxyurea treatment was
omitted. Cell supernatant aliquots were harvested two, five and
nine days after infection and assayed for RT activity. RT activity
was monitored as in Example 2. The results of this procedure are
presented in Table 4. TABLE-US-00004 TABLE 4 Inhibition of HIV-1 RT
expression in infected PBL treated with 1 mM hydroxyurea (HU) RT
Activity (cpm/ml .times. 1000) at Days Treatment 2 5 9 +HU 0.892
0.589 0.434 -HU control 0.863 3.951 81.263
[0076] When PBL cells were treated with a combination of
hydroxyurea and ddI, HIV protein p24 expression detected in cell
supernatants significantly decreased (Table 5). TABLE-US-00005
TABLE 5 Viral expression of p24 protein after HIV-1 infection of
PHA-stimulated PBL in the presence of .mu.M of hydroxyurea (HU)
and/or ddI (ddI). p24 (ng/ml) after infection (days) Treatment 4 8
12 none 5 142 195 (control) ddI 0.2 4 116 197 1 3 90 196 5 1 58 125
20 1 25 51 HU 10 4 100 204 50 3 113 148 100 2 111 116 HU + ddI 10 +
0.2 3 144 200 10 + 1 2 95 158 10 + 5 2 74 127 10 + 20 0 19 44 50 +
0.2 2 109 146 50 + 1 1 70 128 50 + 5 0 12 24 50 + 20 0 0 0 100 +
0.2 2 105 95 100 + 1 0 55 71 100 + 5 0 2 4 100 + 20 0 0 0
[0077] Furthermore, the hydroxyurea and ddI synergistically
inhibited HIV-1 p24 expression. That is, p24 expression decreased
more when cells were treated with both drugs rather than one or the
other drug alone. For example, Table 5 shows that treatment of
cells with 5 .mu.M ddI yielded 125 ng of p24 per ml of cell
supernatant after 12 days of infection, while treatment with 50
.mu.M hydroxyurea yielded 148 ng/ml after 12 days of infection.
However, when cells were treated with both 5 .mu.M ddI and 50 .mu.M
hydroxyurea, only 24 ng/ml of p24 were detected at day 12 after
infection. Because of this synergistic effect, lower concentrations
of hydroxyurea and ddI in combination could effectively eliminate
p24 expression compared to treatment with only hydroxyurea or ddI
at the same concentrations.
[0078] Whereas the procedure in Example 4 involved the
pre-treatment of target cells with hydroxyurea and/or ddI before
infection, we also investigated the effect of drug treatment in
cells that were already infected with the HIV-1 IIIB virus. In the
latter procedure, we measured p24 production during the course of
an in vitro infection to assess the inhibition of HIV-1 replication
in activated PBL.
[0079] Example 5 illustrates how hydroxyurea, either alone or in
combination with ddI effects the production of p24 in HIV-1 IIIB
infected cells.
EXAMPLE 5
Effect of Hydroxyurea on P24 Production
[0080] PBL from healthy donors were infected for 2 hour at
37.degree. C. with HIV-1 [HTLV-IIIB] (m.o.i.=1) after 2 days
stimulation with PHA and Interleukin-2 (IL-2). After washing out
the residual virus, cells were treated with hydroxyurea and/or ddI
at the concentrations indicated (supernatant with no drug was used
as control). Every 3-4 days, supernatant was harvested for p24
analysis, cells were counted and fresh supernatant and drugs were
added. Samples were analyzed for (a) p24 production in the
supernatant, (b) count of viable cells, and (c) ratios between the
values expressed in (a) and (b). The results from this procedure
are presented in FIGS. 1a-1c.
[0081] As shown in FIG. 1a, when used alone at low concentrations,
or in combination with ddI, HIV-1 replication was inhibited in a
dose-dependent manner.
[0082] Notably, the combination of hydroxyurea and ddI completely
blocked HIV-1 replication (>99.9%), thus illustrating the
powerful synergistic effect of this drug combination. Cell toxicity
analysis reflected the known properties of the two drugs.
[0083] Hydroxyurea is known to act mainly as a cytostatic drug.
However, continuous drug exposure eventually results in some
cytotoxic effects (Yarbro, J. W. 1992 Semin. Oncol. 19:1). This was
also observed in our experiments at 0.1 mM hydroxyurea
concentrations (FIG. 1b). However, both cytostatic and cytotoxic
effects virtually disappeared when the drug concentration was
decreased (FIG. 1b).
[0084] Cytotoxicity of ddI is known to be low at the doses used in
our experiments, which correspond to the plasma concentrations
observed in AIDS treated patients (Faulds, D. and Brogden, R. N.
1992 Drugs 44:94). The combination of the two drugs did not
significantly change the cytotoxicity compared to the use of
hydroxyurea alone.
[0085] This represents a further advantage of the hydroxyurea/ddI
combination since the antiviral effects were synergistically
augmented without a significant increase in cytotoxicity. To
understand whether the different number of viable cells observed at
different drug concentrations or during the course of infection
could have affected our data (less cells alive yielding less virus
production), we normalized the p24 expression to the number of
viable cells (FIG. 1c). Our results showed the antiviral effect of
hydroxyurea at low concentrations is not mediated by
cytotoxicity.
[0086] We also investigated the inhibitory effects of hydroxyurea,
either alone or in combination with AZT, on HIV-1 infection of
primary human macrophages. Although we found greater variability
among different experiments that employed macrophages compared to
our results with primary PBL (note that in each experiment the same
donor was used as a source of both PBL and macrophages), the
dose-dependent inhibition of HIV-1 production by hydroxyurea was
nonetheless consistently more potent than with primary PBL.
[0087] Example 6 illustrates the effectiveness of hydroxyurea as an
inhibitor of HIV infection in macrophages. Moreover, this example
illustrates the powerful synergistic effect of hydroxyurea and AZT
as inhibitors of HIV infection of macrophages.
EXAMPLE 6
Time Course of HIV-1 Inhibition by Hydroxyurea and/or AZT in
Macrophages
[0088] Macrophages were obtained by cell adhesion after
purification of PBL from healthy donors. After 14 days treatment
with granulocyte-macrophage-colony-stimulating factor, cells were
infected overnight with the HIV-1 strain Ba-L (Gartner et al.,
Science 233:215 (1986)). Cells were subsequently washed and treated
with hydroxyurea and/or with AZT at the indicated concentrations.
Supernatants were harvested ever 4-5 days for p24 analysis and
fresh supernatant and drugs were added. We noted that no cytotoxic
effects were observed in this experiment (also see Table 6). The
results of these experiments are presented in FIG. 2.
[0089] Our results show that concentrations of hydroxyurea as low
as 0.05 mM blocked HIV-1 replication (>99.9%). Use of lower
doses of hydroxyurea and AZT, at concentrations at which each of
the two drugs were only partial effective, resulted in complete
inhibition (>99.9%). The synergistic effects of hydroxyurea and
AZT that were observed in macrophages were therefore consistent
with the results obtained using primary PBL treated with
hydroxyurea and ddI.
[0090] Our demonstration that hydroxyurea inhibited two different
HIV-1 strains in primary human cells suggested this drug, either
alone or in combination, could also be effective in vivo. To
further test this possibility we confirmed the previous
observations by employing another in vitro system for drug testing.
This in vitro system made use of primary cells isolated from
HiV-1-infected individuals. We believe this model of HIV-1
inhibition closely approximates in vivo conditions, since it
combines the use of primary cells and viral isolates, in the
setting of an infection that was established in vivo.
[0091] Example 7 illustrates that hydroxyurea, either alone or in
combination with nucleoside analogs, inhibits HIV-1 replication in
cells isolated directly from an HIV-1 infected patient.
EXAMPLE 7
Inhibition of HIV-1 in Activated PBL from an HIV-1 Infected
Patient
[0092] PBL were isolated and stimulated for 2 days with PHA and
IL-2. Subsequently, hydroxyurea and ddI were added at the specified
concentrations. The extent of HIV-1 infection was analyzed as
described in Example 5. Samples were tested for (a) p24 production
in the supernatant, (b) viable cell count.
[0093] Once again, hydroxyurea inhibited HIV-1 replication in a
dose-dependent manner and, in combination with ddI, showed strong
synergistic effects (FIG. 3a). However, in some instances, both the
pharmacologic and the cytotoxic effects of hydroxyurea were more
pronounced (FIGS. 3a, 3b), and lower doses of hydroxyurea (compared
to the experiments on PBL derived from healthy donors and
illustrated in FIG. 1 were used, especially with 6ells from
HIV-1-infected patients in the advanced stages of AIDS. Also note
that at the lowest levels both hydroxyurea and ddI in some cases
(as illustrated in FIG. 3) stimulated HIV-1 replication, but only
when used individually.
[0094] This phenomenon was not confined to the use of cells from
infected patients, since it was also occasionally observed when
cells from a healthy donor were used. Independent of the viral or
cellular source, however, stimulatory effects were not observed
when hydroxyurea and either of the nucleoside analogs were used in
combination (not shown).
[0095] Example 8 illustrates the effect of high doses of
hydroxyurea on HIV-1 infection of activated PBL and macrophages
that were isolated from healthy donors.
EXAMPLE 8
The Effect of High Concentrations of Hydroxyurea on HIV-1
Infection
[0096] Experiments were conducted as described in FIGS. 1 (for PBL)
and 2 (macrophages) with 1 mM hydroxyurea. Percentages of HIV-1
inhibition were calculated based on p24 production compared to the
untreated control. Drug treatment of macrophages was suspended
after 14 days. The results of this experiment are presented in
Table 6. TABLE-US-00006 TABLE 6 1 mM Hydroxyurea drug suspension no
drug Days after infection 4 7 10 14 21 28 35 4 7 10 14 21 28 35
PBMC HIV-1 100 100 100 100 n.d. n.d. n.d. 0 0 0 0 n.d n.d n.d
inhibition, % Viable cells, 500 185 87 36 n.d. n.d. n.d. 500 610
1500 1300 n.d n.d n.d thousands/ml Macrophages HIV-1 100 100 100
100 100 100 100 0 0 0 0 0 0 0 inhibition, % Viable cells, 300 280
270 270 260 240 190 300 310 310 290 270 230 200 thousands/cm.sup.2
PBMC = peripheral blood mononuclear cells n.d. = not done
[0097] Continuous treatment with 1 mM hydroxyurea completely
blocked HIV-1 replication both in activated PBL and macrophages. In
activated PBL, however, toxic effects at these concentrations were
observed early, in contrast with the lack of significant toxicity
in macrophages. Furthermore, in some experiments the absence of
HIV-1 replication in infected macrophages was documented even
several weeks after discontinuing the drug treatment.
[0098] Our finding that hydroxyurea, alone or in combination with
nucleoside analogs, efficiently inhibited HIV-1 replication in
primary human cells in vitro suggests this drug will also be useful
in human therapy.
[0099] Example 9 describes the use of hydroxyurea in a protocol
designed to control in vivo HIV-1 replication, thereby benefiting
the treated individual.
EXAMPLE 9
Administration of Hydroxyurea to HIV Infected Humans
[0100] One or more HIV-1 seropositive volunteers are first
identified. Blood samples drawn from the volunteers are assayed for
CD4.sup.+T-cells using any suitable quantitation means. Such
quantitation means include, but are not limited to, the flow
cytometer. Over a period of from several weeks to months, the
number of CD4.sup.+T-cells is observed to decrease steadily as an
indicator of disease progression.
[0101] The HIV-1 infected volunteers are then put on a regimen of
drug therapy that includes hydroxyurea, either alone or in
combination with nucleoside analogs. The nucleoside analogs can be
any of ddI, ddC or AZT, or combinations thereof.
[0102] Hydroxyurea is combined with a pharmaceutically acceptable
excipient and is administered in dosages of from 20-40 mg per kg
per day. The drug dosage is adjusted to result in a stable
hydroxyurea blood concentration of approximately 1 mM. This
concentration is chosen because it approximates the blood
concentration of hydroxyurea during standard clinical protocols in
humans. When hydroxyurea is used in conjunction with a nucleoside
analog, the dosage of the analog is determined according to
convention in the medical and pharmaceutical arts.
[0103] After one month of drug treatment blood samples are again
drawn and assayed for CD4.sup.+T-cells. The T-cell population has
stabilized or increased as an indication of the therapeutic
effectiveness of the antiviral activity of hydroxyurea.
[0104] The most dramatic improvements are observed in volunteers
who received the combination of hydroxyurea together with a
nucleoside analog.
[0105] The preceding Examples have presented results obtained using
combinations of hydroxyurea and certain chain-terminating
compounds, such as nucleoside analogs, to inhibit reverse
transcriptase dependent viral replication. We also expect the
chain-terminating efficiency of other dideoxynucleoside phosphate
analogs, and derivatives thereof, to be enhanced by combination
drug therapy involving hydroxyurea. Hence, fluorinated derivatives
of purine dideoxynucleosides, such as those described by Marquez et
al. in 1990 J. Med. Chem. 33:978-985), are expected to exhibit
particularly potent antiviral activities when administered in
combination with hydroxyurea. These fluorinated derivatives include
2'-F-dd-ara-A, 2'-F-dd-ara-I and 2'-F-dd-ara-G. Advantageously,
such fluorinated derivatives are expected to be useful as oral
medications because of their chemical stability under acidic
conditions.
[0106] Example 10 describes an experiment that can be used to
assess the in vitro anti-viral effects of hydroxyurea and various
fluorinated derivatives of chain terminating nucleoside
analogs.
EXAMPLE 10
Use of Hydroxyurea and Fluorinated Derivatives of Chain Terminating
Nucleoside Analogs to Inhibit HIV Expression
[0107] PBL isolated from healthy donors are stimulated with PHA and
IL-2 for 48 hours using standard protocols. At the same time, the
cells are pre-treated with hydroxyurea alone or in combination with
either ddI or fluorinated derivatives of chain-terminating
nucleosides. The use of ddI in this procedure serves as a positive
control for hydroxyurea-enhanced inhibition of p24 production. At
the end of the 48 hour period, samples of the treated cells are
infected with HIV-1 IIIB (Popovic et al., supra). Aliquots of the
cell supernatants are then harvested at various time points
post-infection and analyzed for the presence of p24 antigen as an
indicator of HIV-1 infection. Example results expected in this
procedure are qualitatively presented in Table 7. TABLE-US-00007
TABLE 7 Viral expression of p24 protein after HIV-1 infection of
PHA-stimulated PBL in the presence of .mu.M of hydroxyurea (HU)
and/or nucleoside analogs p24 Expression after Infection (Days)
Treatment 4 8 12 Untreated Low High Very High HU, 50 Low Medium
High ddI, 20 Low Medium Medium 2'-F-dd-ara-A, 20 Low Medium Medium
2'-F-dd-ara-I, 20 Low Medium Medium 2'-F-dd-ara-G, 20 Low Medium
Medium HU + ddI, Low Low Low 50 + 20 HU + 2'-Fdd-ara-A, Low Low Low
50 + 20 HU + 2'-F-dd-ara-I, Low Low Low 50 + 20 HU + 2'-F-dd-ara-G,
Low Low Low 50 + 20
[0108] Results such as those presented in Table 7 will confirm that
the antiviral activities of fluorinated chain-terminating
nucleoside analogs are enhanced when used in combination with
hydroxyurea.
[0109] We have demonstrated that hydroxyurea is an effective HIV-1
inhibitor. Significantly, these antiviral properties were not
solely mediated by the cytostatic or cytotoxic effects of the drug
in non-stimulated PBL and macrophages. We believe this was true
because these cells were either quiescent (PBL) or terminally
differentiated (macrophages), and therefore did not require high
levels of dNTP synthesis. Even after PBL activation, when dNTP
synthesis was required for cell cycling, the antiviral and
cytotoxic effects could be distinguished at low drug
concentrations. The selective anti-HIV-1 activity of hydroxyurea in
activated PBL may be partly explained by the distributive
properties of HIV-1 RT. Compared to cellular polymerases, the
distributive property of RT may render it more sensitive to low
intracellular concentrations of dNTP. In activated PBL, the
cytostatic properties of hydroxyurea probably contributed to its
antiviral activity, since viral replication in lymphocytes requires
cell division.
[0110] By decreasing the intracellular concentration of dNTP while
increasing the uptake and metabolism of nucleoside analogs, such as
ddI or AZT, hydroxyurea decreased the ratio between intracellular
dNTP and nucleoside analogs, thus enhancing their antiviral
effects.
[0111] Combinations of hydroxyurea and either ddI or AZT proved to
be extremely effective antiviral treatments. In particular, this
combination decreased the drug concentrations necessary to obtain
>99.9% inhibition of HIV-1 replication, and gave clear
synergistic effects over the use of the individual drugs without
increasing their cytotoxicities. The phenomenon of viral
stimulation that is sometimes observed when low doses of drugs are
used individually was also eliminated. The combined use of these
drugs may therefore be beneficial and safe for asymptomatic,
seropositive individuals.
[0112] The use of hydroxyurea in the treatment of AIDS offers
several advantages. After more than 30 years in human use, the
properties of this drug are well established. As a result of its
extreme diffusiblity, this drug can enter all tissues, including
cells of the central nervous system, with a V.sub.max that appears
infinite (Morgan, J. S., Creasey, D. C. and Wright, J. A. 1986
Biochem. Biophys. Res. Commun. 134:1254). In view of the fact that
hydroxyurea is highly effective at inhibiting HIV-1 replication in
macrophages, we expect this drug to be effective against the
neurological manifestations of AIDS, which are believed due to the
effects of viral replication in macrophages (Koenig, S., et al.
1986 Science 233:1089).
[0113] The activity of hydroxyurea does not depend on the
metabolism of the drug within cells. Thus, in contrast with
nucleoside analogs, hydroxyurea is expected to be effective in all
cells, independent of their activation state. Hydroxyurea is
classified as a mildly toxic drug and does not cause
immunodepression. Myelotoxicity is hydroxyurea's dose-limiting
toxicity. However, such toxicity can be easily monitored and it is
constantly and rapidly reversible after decreasing the dose or
suspending the treatment (Donehower, R. C., 1992 Semin. Oncol.
19:11). By monitoring simple parameters like peripheral cell
counts, hydroxyurea can be administered for years, and sometimes
decades. Furthermore, bone marrow toxicity is severe only when
hydroxyurea is used at very high doses, such as those used in
leukemia treatment (approximately 0.5-2.5 mM) (Belt, R. J. et al.
1980 Cancer 46:455). In most of our experiments, hydroxyurea
concentrations that were 2-3 logs lower than these levels still
were adequate to completely inhibited HIV-1 replication.
Hydroxyurea can be orally administered and is much less expensive
than other drugs that are presently used for AIDS therapy.
Hydroxyurea does not inhibit HIV-1 directly, but via the inhibition
of the cellular enzyme ribonucleotide reductase. Cellular enzymes
do not mutate under physiological conditions and one could expect
that HIV-1 resistance to hydroxyurea would be far less likely to
occur than with conventional drugs. This could circumvent the onset
of HIV-1 escape mutants. To date, none of the anti-HIV-1 drugs that
have been tested have prevented the evolution of escape mutants.
This failure represents a major frustration in the battle against
AIDS. Moreover, the onset of escape mutants that arise during
treatment of AIDS victims with nucleoside analogs, should also be
reduced when these drugs are used in combination with hydroxyurea.
Since the synergistic effect of the combination of a nucleoside
analog and hydroxyurea inhibits virus replication, which may be a
requisite step in the process of virus mutation that leads to the
development of escape mutants.
[0114] In our opinion, two main strategies utilizing hydroxyurea as
AIDS therapies may be followed. The first is the use of low doses
of hydroxyurea. Drug combinations are recommended in this case, for
the reasons above illustrated, and trials could safety include
asymptomatic seropositive individuals. The second strategy would
use high levels of hydroxyurea, with protocols similar to those
used in leukemia. This strategy would be more potent against HIV-1
and would also kill the replicating PBL producing virus. However,
one could design a combination of both strategies by alternating
high doses of hydroxyurea for purging purposes, followed by lower
maintenance doses.
[0115] While particular embodiments of the invention have been
described in detail, it will be apparent to those skilled in the
art that these embodiments are exemplary rather than limiting, and
the true scope of the invention is that defined by the claims that
follow.
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