U.S. patent application number 10/150690 was filed with the patent office on 2003-05-29 for drug combination for the treatment of viral diseases.
This patent application is currently assigned to UNIVERSITY OF MEDICINE & DENTISTRY OF NEW JERSEY. Invention is credited to Medina, Daniel, Strair, Roger, Tung, Peter.
Application Number | 20030100533 10/150690 |
Document ID | / |
Family ID | 27410539 |
Filed Date | 2003-05-29 |
United States Patent
Application |
20030100533 |
Kind Code |
A1 |
Strair, Roger ; et
al. |
May 29, 2003 |
Drug combination for the treatment of viral diseases
Abstract
This invention pertains to a method for treating a human with
human immunodeficiency virus infection which comprises
administering to the human a therapeutically effective amount of a
thymidine analog, which analog acts as an inhibitor of viral
reverse transcriptase necessary for viral replication of human
immunodeficiency virus, and a thymidylate synthase inhibitor, or
pharmaceutically acceptable salts thereof.
Inventors: |
Strair, Roger; (Skillman,
NJ) ; Medina, Daniel; (Monmouth Junction, NJ)
; Tung, Peter; (New Haven, CT) |
Correspondence
Address: |
PERKINS COIE LLP
POST OFFICE BOX 1208
SEATTLE
WA
98111-1208
US
|
Assignee: |
UNIVERSITY OF MEDICINE &
DENTISTRY OF NEW JERSEY
New Brunswick
NJ
|
Family ID: |
27410539 |
Appl. No.: |
10/150690 |
Filed: |
May 17, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10150690 |
May 17, 2002 |
|
|
|
08929249 |
Sep 10, 1997 |
|
|
|
08929249 |
Sep 10, 1997 |
|
|
|
08585287 |
Jan 11, 1996 |
|
|
|
08585287 |
Jan 11, 1996 |
|
|
|
08403320 |
Mar 14, 1995 |
|
|
|
Current U.S.
Class: |
514/49 ; 514/151;
514/251; 514/50; 514/575 |
Current CPC
Class: |
A61K 31/70 20130101;
A61K 31/70 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/49 ; 514/50;
514/251; 514/151; 514/575 |
International
Class: |
A61K 031/7072; A61K
031/655; A61K 031/525; A61K 031/19 |
Claims
We claim:
1. A method for treating a human with human immunodeficiency virus
infection which comprises administering to the human a
therapeutically effective amount of a thymidine analog, which
analog acts as an inhibitor of viral reverse transcriptase
necessary for viral replication of human immunodeficiency virus,
and a thymidylate synthase inhibitor, or pharmaceutically
acceptable salts thereof.
2. The method according to claim 1, wherein the thymidine analog is
selected from the group consisting of 3'-azido-3'-deoxythymidine,
and D4T.
3. The method according to claim 2, wherein the thymidine analog is
3'-azido-3'-deoxythymidine.
4. The method according to claim 1, wherein the thymidylate
synthase inhibitor is selected from the group consisting of
5-fluorouracil, 5-fluoro-2-pyrimidone, and floxuridine.
5. The method according to claim 4, wherein the thymidylate
synthase inhibitor is floxuridine.
6. The method according to claim 1, further comprising a
therapeutically effective amount of a folate antagonist, or a
pharmaceutically acceptable salt thereof.
7. The method according to claim 1, wherein the folate antagonist
is selected from the group consisting of methotrexate and
trimetraexate.
8. The method according to claim 1, wherein the folate antagonist
is methotrexate.
9. The method according to claim 1, further comprising a
therapeutically effective amount of hydroxyurea, or a
pharmaceutically acceptable salt thereof.
10. The method according to claim 1, wherein the thymidine analog
is administered in an amount from about 5 mg to 250 mg per kilogram
body weight per day.
11. The method according to claim 10, wherein the thymidine analog
is administered in an amount from about 7.5 mg to 100 mg per
kilogram body weight per day.
12. The method according to claim 1, wherein the thymidylate
synthase inhibitor is administered in an amount from about 0.01 mg
to 25 mg per kilogram body weight per day.
13. The method according to claim 12, wherein the thymidylate
synthase inhibitor is administered in an amount from about 0.01 mg
to 10 mg per kilogram body weight per day.
14. The method according to claim 1, wherein the folate antagonist
is administered in an amount from about 0.05 mg to 25 mg per
kilogram body weight per day.
15. The method according to claim 14, wherein the folate antagonist
is administered in an amount from about 0.05 mg to 10 mg per
kilogram body weight per day.
16. The method according to claim 1, wherein hydroxyurea is
administered in an amount from about 5 mg to 250 mg per kilogram
body weight per day.
17. The method according to claim 16, wherein hydroxyurea is
administered in an amount from about 7.5 mg to 100 mgper kilogram
body weight per day.
Description
BACKGROUND OF THE INVENTION
[0001] This is a continuation application of U.S. patent
application Ser. No. 08/929,249 filed Sep. 10, 1997 (Lyon &
Lyon Docket No. 266/298), which is a continuation-in-part
application of U.S. patent application Ser. No. 08/585,287 (Lyon
& Lyon Docket No. 266/297), filed on Jan. 11, 1996, which is a
continuation-in-part application of U.S. patent application Ser.
No. 08/403,320 filed on Mar. 14, 1995 (Lyon & Lyon Docket No.
266/303), all entitled "New Drug Combination for the Treatment of
Viral Diseases" by Strair et al, and all hereby incorporated by
reference herein in its entirety, including the drawings.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for treating a
human with human immunodeficiency virus infection. The method
comprises administering to the human a therapeutically effective
amount of a thymidine analog, which analog acts as an inhibitor of
viral reverse transcriptase necessary for viral replication of
human immunodeficiency virus, and a thymidylate synthase inhibitor.
In other embodiments, the method further comprises administering to
the human a therapeutically effective amount of a folate antagonist
or hydroxyurea, or both.
DESCRIPTION OF THE BACKGROUND
[0003] The disclosures referred to herein to illustrate the
background of the invention and to provide additional detail with
respect to its practice are incorporated herein by reference. For
convenience, the disclosures are referenced in the following text
and respectively grouped in the appended bibliography.
Sanctuary Growth of HIV in the Presence of AZT
[0004] Acquired immunodeficiency syndrome (AIDS) is believed to be
caused by the human immunodeficiency virus (HIV). Human
immunodeficiency virus is a retrovirus which replicates in a human
host cell. The human immunodeficiency virus appears to
preferentially attack helper T-cells (T-lymphocytes or OKT4-bearing
T-cells). When the helper T-cells are invaded by the virus, the
T-cells become a human immunodeficiency virus producer. The helper
T-ells are quickly destroyed causing the B-cells and other T-cells,
normally stimulated by helper T-cells, to no longer function
normally or produce sufficient lymphokines and antibodies to
destroy the invading virus or other invading microbes.
[0005] Although the human immunodeficiency virus does not
necessarily cause death, the virus generally causes the immune
system to be so depressed that the human develops secondary
infections such as herpes, cytomegalovirus, pneumocystis carinni,
toxoplasmosis, tuberculosis, other mycobacteria, and other
opportunistic infections. Kaposi's sarcoma, lymphomas, and cervical
cancer may also occur. Some humans infected with the human
immunodeficiency virus appear to live with little or no symptoms,
but appear to have persistent infections, while others suffer mild
immune system depression with symptoms such as weight loss,
malaise, fever, and swollen lymph nodes. These syndromes have been
called persistent generalized lymphadenopathy syndrome (PGL) and
AIDS related complex (ARC) and generally develop into AIDS. Humans
infected with the AIDS virus are believed to be persistently
infective to others.
[0006] Human immunodeficiency virus is an extremely heterogeneous
virus. The clinical significance of this heterogeneity is evidenced
by the ability of the virus to evade immunological pressure,
survive drug selective pressure, and adapt to a variety of cell
types and growth conditions. A comparison of isolates among
infected patients has revealed significant diversity, and within a
given patient, changes in the predominant isolate over time have
been noted and characterized. In fact, each patient infected with
human immunodeficiency virus harbors a "quasispecies" of virus with
a multitude of undetected viral variants present and capable of
responding to a broad range of selective pressures, such as those
imposed by the immune system or antiviral drug therapy. Therefore,
diversity is a major obstacle to pharmacologic or immunologic
centrol of human immunodeficiency virus infection. Human
immunodeficiency virus infection has multiple mechanisms to
maximize its potential for genetic heterogeneity. These mechanisms
result in an extremely diverse population of virus capable of
responding to a broad range of selective pressures, including the
immune system and antiretroviral therapy, with the outgrowth of
genetically altered virus.
[0007] When a patient with human immunodeficiency virus infection
is initiated on antiretroviral therapy, there is generally a
virologic response characterized by declining virernia and
antigenemia (5,7,19,20,25). Unfortunately, the currently available
antiretroviral agents which have undergone clinical evaluation have
only limited benefit because most patients will ultimately have
evidence of worsening disease and increasing viral burden.
[0008] This progression often occurs in association with the
emergence of drug-resistant human immunodeficiency virus. For
example, most patients who are treated with
3'-azido-3'-deoxythymidine (AZI) will have initial evidence of
improvement of clinical and laboratory parameters of human
immunodeficiency virus infection (7,20). The duration of this
benefit varies from patient to patient and is likely to be disease
stage related (21).
[0009] Ultimately, however, most patients will have progressive
disease and genotypic or phenotypic evidence of the appearance of
AZT-resistant human immunodeficiency virus (9,12). Since clinical
failure and the appearance of virus with high level resistance to
AZT both occur with evidence of increasing levels of viremia and
changes in viral tropism, it has been difficult to ascribe the
clinical failure solely to the development of AZT resistance
(2,11). Nevertheless, it seems likely that AZT resistance
ultimately contributes to the clinical failure seen in most
patients receiving prolonged AZT therapy.
[0010] While the development of viral-encoded drug resistance may
contribute to the limited efficacy of currently used antiretroviral
agents, it cannot explain all of the in vitro and in vivo phenomena
associated with viral replication in the presence of an
antiretroviral agent. For example, many patients will have
continued evidence of viral replication after initiation of AZT
therapy, but the isolated virus will remain sensitive to AZT when
analyzed in tissue culture (7,20). In contrast, high level human
immunodeficiency virus resistance to many of the non-nucleoside
reverse transcriptase inhibitors develops very rapidly in culture
and in patients (13,16,22,23). Some of these differences may relate
to the complexity and prevalence of viral variants harboring
pre-existing drug resistance mutations prior to the application of
the selective pressure. However, some of the differences may be due
to cellular heterogeneity in the uptake or metabolism of the
antiretroviral agents, that is, each cell population may have some
cells that are refractory to the antiviral effects of the drug.
This would allow a subset of the cellular population to be
successfully infected by genetically drug-sensitive human
immunodeficiency virus in the presence of the antiviral drug.
Depending upon the prevalence of drug-resistant human
immunodeficiency virus in the initial population, the relative
rates of replication of drug-resistant and drug-sensitive virus,
and the percentage of cells refractory to the antiviral effects of
the drug, different patterns of viral breakthrough would emerge.
Notably, the non-nucleoside reverse transcriptase inhibitors do not
undergo cellular metabolism and cellular effects of uptake or
metabolism may be less likely in this setting. This is consistent
with the observation that viral-encoded drug resistance to the
non-nucleoside reverse transcriptase inhibitors develops very
rapidly under selection in tissue culture and in patients. In fact,
the rapid development of resistance in patients suggests that the
blood and plasma compartment of virus is subjected to drug
selective pressure. The presence of human immunodeficiency virus,
but lack of AZT-resistant human immunodeficiency virus, early after
the initiation of AZT suggests that a component of this viral pool
may be capable of averting selective drug pressure in vivo.
Continued viral replication in cells in which AZT is an ineffective
antiretroviral agent could conceivably result in the continued
growth of virus that is sensitive to AZT. An increase in the number
of these cells over time could also alter viral growth kinetics in
the presence of AZT without the emergence of virus with high level
AZT resistance. Therefore, many mechanisms may contribute to the
inability of an antiviral agent to completely suppress human
immunodeficiency virus infection. Viral growth in the presence of
the non-nucleoside reverse transcriptase inhibitors appears due to
the rapid selection of genetically resistant virus. In contrast,
genetic viral drug resistance does not appear to be the major
mechanism contributing to early viral growth in the presence of
AZT.
[0011] The use of recombinant human immunodeficiency virus encoding
reporter genes has been reported to analyze viral breakthrough
infection in the presence of antiretroviral agents (26). In that
study, to determine the prevalence of viral variants spontaneously
resistant to the non-nucleoside reverse transcriptase inhibitor
TIBO R82150, HeLa-T4 cells were infected in the presence of drug
with replication defective HIV-gpt (18,26) or HIV-LacZ (26). The
recombinant virus used for these infections was produced by
infection of cell lines containing an integrated copy of the
defective recombinant virus with replication-competent human
immunodeficiency virus. The replication-competent human
immunodeficiency virus provided the necessary gene products to
rescue the defective virus. The prevalence of viral variants
containing mutations encoding resistance to TIBO R82150 was
reflected by the prevalence of recombinant viruses capable of
infecting HeLa-T4 cells in the presence of TIBO R82150. The
presence of reporter genes in the recombinant viruses allowed for a
quantitative analysis of a single cycle of infection on a single
cell basis.
[0012] U.S. Pat. No. 4,724,232 (Rideout et al.) discloses a method
for treating a human having acquired immunodeficiency syndrome
which comprises administering to the human
3'-azido-3'-deoxythymidine.
[0013] Cancer, Dec. 15, 1992, vol. 70, no. 12, pp. 2929-2934
(Posner et al.) discloses the use of 3'-azido-3'-deoxythymidine and
5-fluorouracil in the treatment of cancer.
Early HIV Breakthrough Infection in the Presence of Stavudine
[0014] The measurement of plasma HIV RNA copy number after the
initiation of antiviral therapy has provided several insights into
the kinetics and dynamics of HIV infection. Initial studies
quantitating HIV RNA after the initiation of a non-nucleoside
reverse transciptase inhibitor. (NNRTI), nevirapine, indicated a
very rapid turnover of plasma HIV (28). In those studies there was
an initial decline in HIV RNA followed by a rapid increase in
plasma viral RNA. The studies with nevirapine demonstrated that the
rapid rebound in HIV RNA levels was a consequence of the outgrowth
of HIV with phenotypic and genotypic resistance to nevirapine (28).
An in vitro model of HIV infection after the initiation of a
different NNRTI (TIBO) has also indicated a similarly high
prevalence of variants capable of infection in the presence of the
drug (26).
[0015] Similar clinical and laboratory studies analyzing early HIV
infection in the presence of AZT have also been undertaken (29). In
contrast to the clinical studies with nevirapine, early HIV
infection in the presence of AZT does not appear to be predominated
by the early outgrowth of drug-resistant HIV. While the amount of
virus circulating in plasma shortly after the initiation of AZT
rapidly declines, the remaining circulating virus after this
decline does not contain mutations known to encode resistance to
AZT (29). Laboratory infections using an in vitro model of
infection in the presence of AZT have demonstrated a similar
pattern: early breakthrough infection independent of the presence
of genetic resistance (31). These more complex dynamics may be a
consequence of a variety of pharmacologic, cellular and viral
features. The mutations associated with AZT-resistance may be
present in the initial (unselected) viral population but mutant HIV
with high level AZT-resistance generally contain multiple mutations
associated with AZT-resistance and the emergence of these variants
often occurs over months-years. While the slow emergence of these
high level resistant variants can be explained by a low prevalence
of AZT-resistant variants, the need for superimposed mutations, or
selection against the emergence of these variants, the early
outgrowth of AZT-sensitive virus in the presence of AZT must be
explained by virologic, cellular or pharmacologic features that
result in the ability of HIV-1 that is genotypically and
phenotypically sensitive to AZT to replicate in the presence of
AZT.
[0016] A quantitative in vitro model of HIV infection which
utilizes recombinant HIV has been used to characterize some of the
mechanisms responsible for HIV kinetics after the initiation of
antiviral drugs (26,31). In that model a replication-defective HIV
encoding a selectable marker is used to assess a single cycle of
infection in the absence of either repeated cycles of infection or
selection of virus in the presence of antiviral drugs. The use of a
replication-defective virus allows an assessment of mechanisms of
early HIV breakthrough infection in the presence of antiviral drugs
and has been used to quantitate HIV breakthrough infection.
Similarly, this system has been used to determine that such
infection in the presence of a NNRTI is likely due to infection by
genetically resistant virus while early infection in the presence
of AZT is due to the infection by virus without genetic drug
resistance (26,31). These in vitro results mimic those described in
clinical studies of HIV dynamics after the initiation of a NNRTI
(28) or AZT (29).
[0017] Another feature of the replication-defective recombinant HIV
system is that cells infected with HIV in the presence of the
antiviral drug can be readily isolated and characterized. Using
this approach it has been possible to determine that some of the
cells infected in the presence of AZT had metabolic features that
rendered AZT an ineffective antiviral drug. Attempts to reverse
these metabolic features has resulted in the development of new
drug combinations designed to modulate the antiviral efficacy of
AZT. One such combination has improved antiviral efficacy in both
cells demonstrated to be refractory to the antiviral effects of AZT
and primary blood mononuclear cells (35).
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 is a schematic representation of the production of
recombinant HIV-gpt by COS cell transfection or rescue from the
H9/HIV-gpt cell line.
[0019] FIG. 2 is a schematic representation of the analysis of
colonies arising after COS cell derived HIV-gpt infection of
HeLa-T4 cells in the presence of 10 .mu.M AZT.
[0020] FIG. 3 is a graph showing the infection of a clone of
HeLa-T4 cells "print resistant" to the antiviral effects of AZT
(clone R116) and a control clone (S1) with replication-competent
HIV-1IIIB in the presence of 0.1 .mu.M AZT.
[0021] FIGS. 4A and 4B are graphs illustrating thymidine
metabolism-HPLC analysis of clones obtained after infection of
HeLa-T4 cells with HIV-gpt in the presence and absence of AZT.
[0022] FIG. 5 is a graph showing a comparison of thymidine kinase
MRNA levels (A) and enzyme activity (B) in cell lines sensitive and
persistently resistant to the antiretroviral effects of AZT.
[0023] FIG. 6 is a graph showing cellular toxicity of AZT.
[0024] FIG. 7A and FIG. 7B are graphs showing the suppression of
viral breakthrough in cells sensitive and refractory to the
antiviral effects of AZT.
[0025] FIG. 8 is a graph illustrating FUdR cytotoxicity in cells
sensitive and refractory to the antiretroviral activity of AZT.
[0026] FIG. 9 is a graph showing AZT-FUdR cytotoxicity in JE6.1
cells sensitive and resistant to the antiviral effects of AZT.
[0027] FIG. 10 is a graph showing that the AZT-FUdR combination
inhibits HIV-1 infection of PBMC.
[0028] FIG. 11 is a graph showing the infection of JE6.1 cell
clones persistently resistant to the antiviral effects of d4T (D4T
bulk, D4TR1, D4TR3) and a control clone of JE6.1 cells with
HIV-IIIB in the presence of various concentrations of d4T.
[0029] FIG. 12 is a graph showing that the D4T-FUdr combination
inhibits HIV-1 infection of PBMCs.
SUMMARY OF THE INVENTION
[0030] This invention pertains to a method for treating a human
with human immunodeficiency virus infection (acquired
immunodeficiency syndrome) which comprises administering to the
human a therapeutically effective amount of a thymidine analog,
which analog acts as an inhibitor of viral reverse transcriptase
necessary for viral replication of human immunodeficiency virus,
and a thymidylate synthase inhibitor, or pharmaceutically
acceptable salts thereof. In other embodiments, the method further
comprises administering to the human a therapeutically effective
amount of a folate antagonist or hydroxyurea, or both.
DETAILED DESCRIPTION OF THE INVENTION
Sanctuary Growth of HIV in the Presence of AZT
[0031] Human immunodeficiency virus resistance to the
non-nucleoside reverse transcriptase inhibitors emerges very
rapidly under selection in culture and in patients. In contrast,
AZT-resistant HIV generally emerges in patients only after more
prolonged therapy. Although HIV can be cultured from many patients
shortly after the initiation of AZT treatment, characterization of
the virus that is cultured generally indicates that it is sensitive
to AZT. To initiate an evaluation of the mechanisms contributing to
early HIV breakthrough in the presence of AZT and other nucleoside
analogs, replication-defective HIV encoding reporter genes were
utilized. These recombinant HIV allow a quantitative analysis of a
single cycle of infection. Results with these defective HIV
indicate that early infection in the presence of AZT often results
from the infection of a cell which is refractory to the
antiretroviral effects of AZT.
[0032] Characterization of cell lines derived from such infected
cells has demonstrated decreased accumulation of AZT, increased
phosphorylation of thymidine to TTP, and increased levels of
thymidine kinase activity. In addition, AZT inhibition of
replication-competent HIV infection is also significantly impaired
in this cell line.
Early HIV Breakthrough Infection in the Presence of Stavudine
[0033] By utilizing replication-defective HIV encoding reporter
genes, applicants have demonstrated that early HIV breakthrough
infection in the presence of Stavudine results from infection of
cells which are refractory to the antiviral effects of the drug. In
addition, applicants demonstrate that the combination of Stavudine
and Floxuridine has potent antiviral activity in cells refractory
to the antiviral activity of Stavudine alone. Data is presented
indicating that the predominant mechanism of HIV breakthrough early
after the initiation of stavudine (d4T) is related to the
inefficacy of d4T as an antiviral agent in a subset of the host
population. This inefficacy is demonstrated to be independent of
the presence of HIV with genetic drug resistance and has also been
demonstrated in several cell types and with retroviruses other than
HIV. These results may help explain several features of the in vivo
and in vitro selection of d4T resistant virus and contribute to an
understanding of the mechanisms responsible for HIV kinetics after
the initiation of antiviral drugs.
[0034] The present invention relates to a method for treating a
human with human immunodeficiency virus infection. The method
comprises administering to the human a therapeutically effective
amount of a thymidine analog, which analog acts as an inhibitor of
viral reverse transcriptase necessary for viral replication of
human immunodeficiency virus, and a thymidylate synthase inhibitor.
Thymidine analogs, such as 3'-azido-3'-deoxythymidine (AZT), are
prodrugs in the treatment of acquired immunodeficiency syndrome.
3'-Azido-3'-deoxythymidine is converted by cellular enzymes to
3'-azido-3'-deoxythymidine monophosphate (AZTMP).
[0035] The monophosphate is then converted by cellular enzymes to
3'-azido-3'-deoxythymidine diphosphate (AZTDP) and
3'-azido-3'-deoxythymidine triphosphate (AZTTP). In human cells
infected with HIV, 3'-azido-3'-deoxythymidine triphosphate is an
inhibitor of the viral reverse transcriptase necessary for viral
replication. Some cells, however do not efficiently metabolize AZT
to the triphosphate and may overproduce the natural thymidine
triphosphate, which competes with the antiviral activity of AZTIP.
Studies have demonstrated that these cells contribute to the early
failure of the antiviral activity of AZT. By coadministering a
thymidylate synthase inhibitor with the thymidine analog,
applicants have found that that the thymidine analog is a more
effective inhibitor of HIV replication. The thymidylate synthase
inhibitor may function by resulting in lower levels of thymidine
triphosphate to compete with the phosphorylated thymidine analog
reverse transcriptase inhibition.
[0036] In another embodiment, the method further comprises
administering to the human a therapeutically effective amount of a
folate antagonist together with the thymidine analog and the
thymidylate synthase inhibitor to modulate the effects of the
thymidine analog. In yet another embodiment, the method further
comprises administering to the human a therapeutically effective
amount of hydroxyurea together with the thymidine analog and the
thymidylate synthase inhibitor to modulate the effects of the
thymidylate synthase inhibitor. In still yet another embodiment,
both the folate antagonist and hydroxyurea may be administered with
the thymidine analog and the thymidylate synthase inhibitor.
Use of Floxuridine to Modulate the Antiviral Activity of AZT
[0037] Recent clinical studies have demonstrated that early HIV
replication after initiation of AZT is generally a consequence of
the replication of AZT-sensitive virus (29). A prior in vitro
analysis of this early breakthrough replication in the presence of
AZT has demonstrated the infection of cells in which AZT was an
ineffective antiviral agent (31). A metabolic characterization of
these cells has led to the development of a novel combination
therapy designed to potentiate the antiviral efficacy of AZT. The
present invention describes the antiviral effects of the
combination of floxuridine and AZT. This combination suppresses
early viral breakthrough, lowers the IC.sub.50 of AZT, and has
particular antiviral efficacy in the subset of cells that are
infected with AZT-sensitive virus in the presence of AZT. The
antiviral efficacy of this combination in peripheral blood
mononuclear cells suggests potential clinical utility.
[0038] In an attempt to explain the ability of
genetically-sensitive HIV to replicate in the presence of AZT,
applicants have initially utilized recombinant
replication-defective HIV to quantitate infection in the presence
of AZT (31). In those studies, replication-defective HIV encoding a
selectable marker was used to infect target cells in the presence
of 10 .mu.M AZT. The cells infected with the defective HIV were
isolated by expression of the selectable marker. A subset of these
infected cells was demonstrated to be readily infected with another
HIV in the presence of 10 .mu.M AZT. These cells were persistently
refractory to the antiviral effects of AZT and were demonstrated to
have excessive phosphorylation of thymidine to TTP, increased
thymidine kinase activity and decreased accumulation of AZTTP.
[0039] These data suggested that a component of early infection
with AZT-sensitive HIV in the presence of AZT was a consequence of
the infection of cells which were refractory to the antiviral
effects of AZT. Some of these cells had metabolic factors resulting
in reduced AZTTP/TTP ratios in the cells. These data also suggest
that it may be possible to overcome this reduced antiviral efficacy
of AZT by biochemical modulation of TTP pool sizes. One way to
potentially modulate these cells is with fluoropyrimidines such as
5-fluorodeoxyuridine (FUdR). These compounds are known to reduce
cellular thymidine pools by the inhibition of thymidylate
synthase.
[0040] In the present invention, applicants demonstrate the
suppression of early HIV infection in the presence of AZT with
FUdR. FUdR will be shown to potentiate the antiviral effects of AZT
in whole cell populations (including peripheral blood mononuclear
cells [PBMC]) as well as in subsets of cells isolated by infection
with recombinant HIV in the presence of AZT. Infection of these
latter cells will be shown to be extremely sensitive to combined
AZT-FUdR therapy.
[0041] The term "prodrug", as used herein refers to compounds which
undergo biotransformation prior to exhibiting their pharmacological
effects. The chemical modification of drugs to overcome
pharmaceutical problems has also been termed "drug latentiation."
Drug latentiation is the chemical modification of a biologically
active compound to form a new compound which upon in vivo enzymatic
attack will liberate the parent compound. The chemical alterations
of the parent compound are such that the change in physicochemical
properties will affect the absorption, distribution and enzymatic
metabolism. The definition of drug latentiation has also been
extended to include nonenzymatic regeneration of the parent
compound. Regeneration takes place as a consequence of hydrolytic,
dissociative, and other reactions not necessarily enzyme mediated.
The terms prodrugs, latentiated drugs, and bioreversible
derivatives are used interchangeably. By inference, latentiation
implies a time lag element or time component involved in
regenerating the bioactive parent molecule in vivo. The term
prodrug is general in that it includes latentiated drug derivatives
as well as those substances which are converted after
administration to the actual substance which combines with
receptors. The term prodrug is a generic term for agents which
undergo biotransformation prior to exhibiting their pharmacological
actions.
[0042] As set out above, the present invention relates to a method
for treating a human with human immunodeficiency virus infection
which comprises administering to the human a therapeutically
effective amount of a thymidine analog, which analog acts as an
inhibitor of viral reverse transcriptase necessary for viral
replication of human immunodeficiency virus, and a thymidylate
synthase inhibitor.
[0043] The thymidine analogs, and prodrugs thereof, which may be
employed in the present invention are compounds which act as
inhibitors of viral reverse transcriptase necessary for viral
replication of human immunodeficiency virus. In general, the
thymidine analogs are prodrugs which are converted by cellular
enzymes to their respective active monophosphates, diphosphates,
and/or triphosphates which are inhibitors of viral reverse
transcriptase. Nonlimiting examples of thymidine analogs may be
selected from the group consisting of 3'-azido-3'-deoxythymidine,
and D4T. In a preferred embodiment, the thymidine analog is
3'-azido-3'-deoxythymidine.
[0044] 3'-Azido-3'-deoxythymidine (AZT, azidothymidine, zidovudine,
Retrovir.TM.), is an antiretroviral drug active against human
immunodeficiency virus. 3'-Azido-3'-deoxythymidine is an inhibitor
of the replication of retroviruses including HIV also known as HTLV
111, LAV, or ARV. 3'-Azido-3'-deoxythymidine is a thymidine analog
in which the 3'-hydroxy (--OH) group of thymidine is replaced by an
azido (--N.sub.3) group. Cellular thymidine kinase converts
3'-azido-3'-deoxythymidine into AZT monophosphate. The
monophosphate is further converted into AZT diphosphate by cellular
thymidylate kinase and to the AZT triphosphate derivative by other
cellular enzymes. 3'-Azido-3'-deoxythymidine triphosphate
interferes with the HIV viral RNA dependent DNA polymerase (reverse
transcriptase) and thus, inhibits viral replication.
3'-Azido-3'-deoxythymidine is useful in treating humans Identified
as having HIV infection. 3'-Azido-3'-deoxythymidine is disclosed in
J. R. Horwitz et al., J. Org. Chem. 29, July 1964, pp. 2076-2078;
M. Imazawa et al., J. Org. Chem., 43(15) 1978, pp. 3044-3048; also
see Biochemical Pharmacology, Vol. 29, pp. 1849-1851; C. J. Kreig
et al., Experimental Cell Research 116 (1978) pp. 21-29; W.
Ostertag et al, Proc. Nat. Acad. Sci. U.S.A. 71 (1974).
[0045] The thymidine analogs which act as an inhibitor of viral
reverse transcriptase necessary for viral replication of human
immunodeficiency virus, may be administered as the free base or in
the form of a pharmaceutically acceptable salt, e.g., an alkali
metal salt such as sodium or potassium, an alkaline earth salt or
an ammonium salt (all of which are hereinafter referred to as a
pharmaceutically acceptable base salt). The salts of the thymidine
analog are converted to the free base after being administered to
the human and are thus prodrugs.
[0046] The amount of thymidine analog which acts as an inhibitor of
viral reverse transcriptase present in the therapeutic compositions
of the present invention is a therapeutically effective amount. A
therapeutically effective amount of thymidine analog is that amount
necessary to inhibit viral reverse transcriptase. All prodrugs or
precursors are administered to a human in a therapeutically
effective amount sufficient to generate an effective amount of the
compound which inhibits viral reverse transcriptase necessary for
viral replication of human immunodeficiency virus. In general, a
suitable effective dose of the thymidine analog or its
pharmaceutically acceptable basic salts will be in the range of
about 5 mg to 250 mg per kilogram body weight of recipient per day,
preferably in the range of 7.5 mg to 100 mg per kilogram body
weight per day, and most preferably in the range 10 mg to 40 mg per
kilogram body weight per day.
[0047] The thymidylate synthase inhibitors, and prodrugs thereof,
which may be employed in the present invention are compounds which
are antimetabolites which interfere with the synthesis of
deoxyribonucleic acid (DNA) and to a lesser extent inhibit the
formation of ribonucleic acid (RNA). In general, the thymidylate
synthase inhibitors inhibit the synthesis of thymidine triphosphate
so that the phosphorylated thymidine analog which acts as an
inhibitor of the viral reverse transcriptase can compete more
effectively with thymidine triphosphate and will more effectively
inhibit viral reverse transcriptase necessary for viral replication
of human immunodeficiency virus. Nonlimiting examples of
thymidylate synthase inhibitors may be selected from the group
consisting of 5-fluorouracil, 5-fluoro-2-pyrimidone (a prodrug of
5-fluorouracil), and floxuridine. Preferably, the thymidylate
synthase inhibitor is floxuridine. These drugs may inhibit HIV
infection by other mechanisms as well.
[0048] 5-Fluorouracil (5-FU) is a fluorinated pyrimidine
antineoplastic antinetabolite. The metabolism of 5-fluorouracil in
the anabolic pathway blocks the methylation reaction of
deoxyuridylic acid to thymidylic acid and interferes with the
synthesis of deoxyribonucleic acid (DNA) and to a lesser extent
inhibits the formation of ribonucleic. acid (RNA). Since DNA and
RNA are essential for cell division and growth, the effect of
fluorouracil may be to create a thymine deficiency which provokes
unbalanced growth and death of the cell. The effects of DNA and RNA
deprivation are most marked on those cells which grow more rapidly
and which take up fluorouracil at a more rapid pace.
[0049] Floxuridine (FUdr) is a fluorinated pyrimidine
antineoplastic antimetabolite. Chemically, floxuridine is
2'-deoxy-5-fluorouridine. FUdr produces the same toxic and
antimetabolic effects as does 5-fluorouracil. The primary effect is
to interfere with the synthesis of deoxyribonucleic acid (DNA) and
to a lesser extent inhibit the formation of ribonucleic acid
(RNA).
[0050] Derivatives of 5-fluorouracil and floxuridine may also be
incorporated into DNA or RNA.
[0051] The amount of thymidylate synthase inhibitor present in the
therapeutic compositions of the present invention is a
therapeutically effective amount. A therapeutically effective
amount of thymidylate synthase inhibitor is that amount necessary
to improve the antiviral efficacy of the thymidine analog so that
the phosphorylated thymidine analog which acts as an inhibitor of
the viral reverse transcriptase can compete more effectively in the
inhibition of viral reverse transcriptase necessary for the
replication of HIV. In general, a suitable effective dose of the
thymidylate synthase inhibitor or its pharmaceutically acceptable
salts will be in the range of about 0.01 mg to 25 mg per kilogram
body weight of recipient per day, preferably in the range of 0.01
mg to 10 mg per kilogram body weight per day, and most preferably
in the range 0.01 mg to 5 mg per kilogram body weight per day.
[0052] As set out above, the method of the present invention may
further comprise administering to a human a therapeutically
effective amount of a folate antagonist together with the thymidine
analog which acts as an inhibitor of viral reverse transcriptase
and the thymidylate synthase inhibitor to modulate the effects of
the thymidine analog. The folate antagonists, and prodrugs thereof,
which may be employed in the present invention are compounds which
are antimetabolites which interfere with the synthesis of
deoxyribonucleic acid (DNA) and to a lesser extent inhibit the
formation of ribonucleic acid (RNA). Nonlimiting examples of folate
antagonists may be selected from the group consisting of
methotrexate and trimetraexate. Preferably, the folate antagonist
is methotrexate.
[0053] Methotrexate (Amethopterin) is an antimetabolite used in the
treatment of certain neoplastic diseases, severe psoriasis, and
adult rheumatoid arthritis. Chemically methotrexate is
N-[4-[[(2,4-diamino-6-pt-
eridinyl)-methyl]methylamino]benzoyl]-L-glutamic acid. Methotrexate
inhibits dihydrofolic acid reductase. Dihydrofolates must be
reduced to tetrahydrofolates by this enzyme before they can be
utilized as carriers of one carbon groups in the synthesis of
purine nucleotides and thymidylate. Therefore, methotrexate
interferes with DNA synthesis, repair, and cellular
replication.
[0054] The amount of folate antagonist present in the therapeutic
compositions of the present invention is a therapeutically
effective amount. A therapeutically effective amount of folate
antagonist is that amount necessary to modulate the effects of the
thymidine analog. In general, a suitable effective dose of folate
antagonist or its pharmaceutically acceptable salts will be in the
range of about 0.05 mg to 25 mg per kilogram body weight of
recipient per day, preferably in the range of 0.05 mg to 10 mg per
kilogram body weight per day, and most preferably in the range 0.05
mg to 4 mg per kilogram body weight per day.
[0055] As set out above, the method of the present invention may
further comprise administering to a human a therapeutically
effective amount of hydroxyurea, and prodrugs thereof, together
with the thymidine analog and the thymidylate synthase inhibitor to
modulate the effects of the thymidylate synthase inhibitor.
Hydroxyurea has the structural formula H.sub.2N--CO--NHOH. The
precise mechanism by which hydroxyurea produces cytotoxic effects
is not known but it is believed that hydroxyurea causes an
immediate inhibition of DNA synthesis without interfering with the
synthesis of ribonucleic acid or of protein.
[0056] The amount of hydroxyurea present in the therapeutic
compositions of the present invention is a therapeutically
effective amount. A therapeutically effective amount of hydroxyurea
is that amount necessary to modulate the effects of the thymidylate
synthase inhibitor. In general, a suitable effective dose of
hydroxyurea or its pharmaceutically acceptable salts will be in the
range of about 5 mg to 250 mg per kilogram body weight of recipient
per day, preferably in the range of 7.5 mg to 100 mg per kilogram
body weight per day, and most preferably in the range 10 mg to 40
mg per kilogram body weight per day.
[0057] Administration may be by any suitable route including oral,
rectal, nasal, topical (including buccal and sublingual), vaginal,
and parenteral (including subcutaneous, intramuscular, intravenous
and intradermal), with oral or parenteral being preferred. The
preferred route may vary with the condition and age of the
recipient.
[0058] While it is possible for the administered ingredients to be
administered alone, it is preferable to present them as part of a
pharmaceutical formulation. The formulations of the present
invention comprise the administered ingredients, as above defined,
together with one or more acceptable carriers thereof and
optionally other therapeutic ingredients. The carrier(s) must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the
recipient.
[0059] The formulations include those suitable for oral, rectal,
nasal, topical (including buccal and sublingual), vaginal or
parenteral (including subcutaneous, intramuscular, intravenous and
intradermal) administration. The formulations may conveniently be
presented in unit dosage form, e.g., tablets and sustained release
capsules, and may be prepared by any methods well known in the art
of pharmacy.
[0060] Such methods include the step of mixing the ingredients to
be administered with the carrier which constitutes one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association the active
ingredient with liquid carriers or finely divided solid carriers or
both, and then if necessary shaping the product.
[0061] Formulations of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets, or tablets each containing a predetermined amount of the
active ingredient; as a powder or granules; as a solution or a
suspension.
[0062] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active ingredient
in a free-flowing form such as a powder or granules, optionally
mixed with a binder, lubricant, inert diluent, preservative,
surfactant or dispersing agent. Molded tablets may be made by
molding in a suitable machine a mixture of the powdered compound
moistened with an inert liquid diluent. The tablets may optionally
be coated or scored and may be formulated so as to provide slow or
controlled release of the active ingredient therein.
[0063] Formulations suitable for topical administration include
lozenges comprising the ingredients in a flavored base, usually
sucrose and acacia or tragacanth; pastilles comprising the active
ingredient in an inert basis such as gelatin and glycerin, or
sucrose and acacia; and mouthwashes comprising the ingredient to be
administered in a suitable liquid carrier.
[0064] Formulations suitable for topical administration to the skin
may be presented as ointments, creams, gels and pastes comprising
the ingredient to be administered and a pharmaceutically acceptable
carrier. A preferred topical delivery system is a transdermal patch
containing the ingredient to be administered.
[0065] Formulations for rectal administration may be presented as a
suppository with a suitable base comprising, for example, cocoa
butter or a salicylate.
[0066] Formulations 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 close
up to the nose. Suitable formulations wherein the carrier is a
liquid, for administration, as for example, a nasal spray or as
nasal drops, include aqueous or oily solutions of the active
ingredient.
[0067] Formulations suitable for vaginal administration may be
presented as pessaries, tampons, creams, gels, pastes, foams or
spray formulations containing in addition to the active ingredient
such carriers as are known in the art to be appropriate.
[0068] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain antioxidants, buffers, bacteriostats and solutes which
render the formulation isotonic 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 presented in unit dose or multidose containers,
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 to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the kind previously described.
[0069] Preferred unit dosage formulations are those containing a
daily dose or unit, daily subdose, or an appropriate fraction
thereof, of the administered ingredient.
[0070] The present invention is further illustrated by the
following examples which are presented for purposes of
demonstrating, but not limiting, the preparation of the compounds
and compositions of this invention.
EXAMPLES
Sanctuary Growth of HIV in the Presence of AZT
Methods
Construction of Recombinant Proviral DNA
[0071] The HIV construct encoding LacZ has been described (26). It
contains the LacZ gene driven by an SV40 promoter inserted into a
large deletion in the HIV genome extending from the 5' end of the
pol gene to the 3' end of the env gene. The HIV-gpt and HXB2env
plasmids were kindly provided by Kathleen Page (University of
California, San Francisco, Calif.) (18).
[0072] The HIV-gpt plasmid contains an HXB2 provirus into which an
SV40 promoter gpt (E. coli guanine phosphoribosyl transferase) gene
was inserted into the env region. The HXB2 env plasmid contains the
HXB2 gp160 gene driven by an SV40 promoter.
Production of "Plasmid Derived" Recombinant Retroviruses
[0073] All transfections and cell culture were performed in an
approved facility using BSL3 techniques. Plasmid DNA
co-transfections into COS cells were performed as described by Page
et al. (18). Supernatants from COS cells were collected 40 hours
after transfection and assayed for infectious recombinant HIV-LacZ
virus by inoculating 2.times.10.sup.5 HeLa-T4 cells with 0.1 ml of
filtered (0.45 .mu.m) supernatant. Cells were stained for
beta-galactosidase activity with X-gal 48 hours after infection as
described (26,27). To assay for infectious recombinant HIV-gpt
virus, the infected cells were split 1:10 into gpt selective media
as described (26). Medium changes were performed every 3 days and
colonies were counted 10-14 days post-infection after staining with
1% crystal violet in 10% formalin.
Cell Lines Containing Defective HIV-gpt and HIV-LacZ
[0074] The H9/HIV-gpt cell line and HeLa T4/HIV-LacZ cell line were
prepared and used as previously described (26). Rescue of defective
retroviruses from the H9/HIV-gpt cell line and the HeLa T4/HIV-LacZ
cell line were performed as previously described (26). Following
each rescue infection, the resultant titer of HIV-LacZ or IUV-gpt
was determined and the inoculum used to infect HeLa-T4 cells was
adjusted depending upon the number of infectious events to be
analyzed.
HPLC Analysis of Clones
[0075] Cell lines were incubated with .sup.3H-thymidine or
.sup.3H-AZT for 4 hours. Dried methanol extracts of the clones were
redissolved in 60 .mu.l of distilled water and centrifuged to
remove undissolved material. Twenty microliters of the sample was
injected and separated on a 10.times.100 mm Rainin Hydropore anion
exchange column. The nucleosides were eluted from the column with a
linear gradient of potassium phosphate (5 mM to 1 M, pH 4.0) at a
flow rate of 1 ml/min. The samples were collected (0.5 ml), mixed
with 5 ml Packard scintillation fluid, and quantitated using a
liquid scintillation counter. Phosphorylated derivatives of
thymidine and AZT were identified with authentic standards.
Cytotoxicity Assay
[0076] AZT-mediated cytotoxicity was assayed in cells persistently
refractory to the antiviral effects of AZT (R116) and in cells
sensitive to the antiviral effects of AZT (HT4, S pool and S1)
using a standard assay (14). Triplicate wells of 24--well plates
containing 3.times.10.sup.4 cells were cultured in the absence or
presence of various concentrations of AZT. Three days later, drug
cytotoxicity was quantitated with a standard MTT assay in which the
uptake and metabolism of 3-[4,5-dimethylthiazol-2-yl]
2,5-dephenyltetrazolium bromide (MTT) by cells was measured (14).
The amount of formazan produced in 2 hours was determined by
dissolving the product in 100% DMSO and then measuring the
absorbance at 570 nm.
Northern Blot Analysis
[0077] Total RNA from S1 and R116 cells were extracted as described
previously (4). Equal amounts of total RNAs were electrophoresed on
an agarose gel containing 1% formaldehyde and blotted onto a nylon
membrane. The RNAs were hybridized with a .sup.32P-labled human
thymidine kinase probe (3). The labeled bands were visualized using
autoradiography and quantitated using a Molecular Dynamics Personal
Densitometer.
Thylnidine Kinase Assay
[0078] Thymidine kinase activity was determined in cell lines
sensitive and resistant to AZT. Cellular extracts of S1 and R116
cells were prepared according to Sherley and Kelly (24) and assayed
for thymidine kinase activity as described by Lee and Cheng (10).
Protein concentration of each extract was determined using Biorad
Protein Reagent.
Use of Floxuridine to Modulate the Antiviral Activity of AZT
Materials and Methods
[0079] Cells. Cell line R116 is a derivative of HeLa-T4 cells that
was isolated after infection of HeLa-T4 cells with HIV-gpt in the
presence of 10 .mu.M AZT (31). This cell line was demonstrated to
be refractory to the antiviral effects of AZT by virtue of
reinfection with either recombinant or replication-competent HIV
infection in the presence of AZT. Cell line S1 is a derivative of
HeLa-T4 cells that was isolated after infection of HeLa-T4 cells
with HIV-gpt in the absence of AZT (31). Cells were cultured in
Dulbecco's modified Eagle's medium supplemented with antibiotics, 2
mM L-glutamine, and 10% fetal bovine serum (FBS). H9 cells, JE6.1
cells and MT-2 cells were cultured in RPMI 1640 medium supplemented
with antibiotics, 2 mM L-glutamine, and 10% FBS. Peripheral blood
mononuclear cells (PBMC) isolated from healthy HIV-1 seronegative
donors were activated with PHA (10 ug/ml) for 72 hours prior to
HIV-1 infection. PBMC were maintained in RPMI 1640 supplemented
with 10% interleukin-2 (Advanced Biotechnologies, Columbia, Md.),
20% FBS, 2 mM L-glutamine and antibiotics.
Virus
[0080] Stock preparations of HIV-1 IIIB were harvested from H9
cells by the "shake off method" (13). An AZT sensitive clinical
isolate (HIV-1.sub.preAO8) (9) was prepared in MT-2 cells. Stock
virus infectivity was determined by end-point dilution in MT-2
cells (32). Virus-induced cytopathic effect (syncytium formation)
was scored 7 days post-infection and the TCID.sub.50 was calculated
with the Reed and Muench equation (33).
Compounds
[0081] Azidothymidine (AZT) and Floxuridine (FUDR) were purchased
from Sigma Chemical Co. (St. Louis, Mo.) and were dissolved in
phosphate buffered saline, sterile filtered (0.22 um) and stored at
-20.degree. C.
HIV RT Assay
[0082] HIV-1 production in infected cultures was determined by a
.sup.32P-based assay as described (34). RT activity was determined
by qualification of .sup.32P bound to the DE81 paper by using a
Molecular Dynamics phosphorimager. The results are reported as
pixel units per microliter of the reaction mixture.
Cytotoxicity Assay
[0083] A checkerboard analysis of the cytotoxicity of AZT and FUDR
alone and in combination was assayed. Triplicate wells of of
24-well plates containing 1.times.10.sup.5 cells were cultured in
the absence or presence of various concentrations of each drug
alone and in combination. Samples were taken every two days for
8-10 days. Drug cytotoxicity was quantitated by the MTT reduction
assay (14). The amount of formazan produced in 4 hours was
determined by dissolving the product in 0.1N HCl made with
2-propanol and then measuring the A.sub.570.
Early HIV Breakthrough Infection in the Presence of Stavudine
Materials and Methods
[0084] Cells: The lymphoid cell lines H9 and JE6.1 were cultured in
RPMI 1640 medium supplemented with antibiotics, 2 mM L-glutamine
and 10% FBS. Peripheral blood mononuclear cells (PBMC) isolated
from healthy HIV-1 seronegative donors were activated with PHA (10
ug/ml) for 72 hours prior to infection. After PHA stimulation,
PBMCs were maintained in RPMI 1640 supplemented with 10%
interleukin-2 (Advanced Biotechnologies, Columbus, Md.), 20% FBS, 2
mM L-glutamine and antibiotics.
[0085] Virus: Production of recombinant HIV-gpt has been described
elsewhere (26). The amphotropic cell line PA317 was transfected
with the recombinant murine retrovirus pLXSN (36) and was used as
the source of the recombinant MLV-neo virus. Stock preparations of
HIV-1IIIB were harvested from H9 cells by the "shake off method"
(13). Stock virus infectivity was determined by end-point dilution
in MT-2 cells (32). Virus induced cytopathic effect was scored 7
days post-infection and the TCID50 was calculated with the Reed and
Muench equation (33).
[0086] Compounds: Stavudine (D4T) and Floxuridine (FUdr) were
purchased from Sigma Chemical Co. (St. Louis, Mo.) and were
dissolved in phosphate buffered saline, sterile filtered and stored
at -20.degree. C.
[0087] HIV-1 RT assay: HIV-1 production in infected cells was
determined by a .sup.32P-based assay as described (37). RT activity
was determined by quantification of .sup.32P-bound to DE81 paper by
using a Molecular Dynamics phosphorimager. The results are reported
as pixel units per microliter of the reaction mixture.
[0088] Cytotoxicity assay: A checkerboard analysis of the
cytotoxicity of D4T and FUdr alone and in combination was assayed.
Triplicate wells of 24 well plates containing 1.times.105 cells
were cultured in the absence or presence of various concentrations
of each drug alone or in combination. Samples were taken every two
days for 8-10 days. Drug cytotoxicity was quantitated by the MTT
reduction assay (14). The amount of formazan produced in 4 hours
was determined by dissolving the product in 0.1N HCL made with
2-propanol and the measuring the A570.
Results
Sanctuary Growth of HIV in the Presence of AZT
HIV-gpt Infection of Cells in the Absence and Presence of AZT
[0089] HeLa-T4 cells were infected with a recombinant HIV, HIV-gpt,
in the presence or absence of 10 .mu.M AZT (FIG. 1). Two separate
populations of HIV-gpt were utilized for these infections. One
population of HIV-gpt was produced in COS cells by co-transfection
of the HIV-gpt plasmid with a plasmid encoding the HXB2 env gene.
The infectious virions produced by this co-transfection have little
genetic diversity in that they are produced from products encoded
by plasmids in COS cells. The second population of HIV-gpt was
genetically more diverse, being produced by rescue from the
H9/HIV-gpt cell line with replication competent HIV-1IIIB that had
been propagated in culture (26). After infection, the HeLa-T4 cells
were placed in gpt selective media and the number of colonies
developing by day 10 was used as an indicator of the number of
cells initially infected in the absence or presence of 10 .mu.M
AZT. As can be seen in Table 1, the prevalence of colony formation
after infection in the presence of AZT was similar
(-5.times.10.sup.4) with the two preparations of HIV-gpt. This
similarity is very distinct from the results of infections
performed in the presence of a normucleoside reverse transcriptase
inhibitor, TIBO R82150. In those studies, the prevalence of
infection with the COS-cell derived virus was twenty fold lower
than infection with HIV-gpt rescued by replication-competent virus
(26). Since the HIV-gpt produced in COS cells would not be expected
to be genetically diverse, this relatively high rate of infection
in the presence of AZT was not likely due to the detection of viral
encoded-AZT resistance. Similarly, the absence of more prevalent
infection in the presence of AZT when HIV-gpt was produced by
rescue with a propagated stock of replication-competent HIV,
implies that true genetic resistance was not being detected in
these experiments. These data suggest that other mechanisms may
contribute to this early viral breakthrough in the presence of
AZT.
Identification of Cells Refractory to the Antiviral Effects of
Nucleoside Analogs
[0090] To characterize further the mechanism(s) of viral infection
accounting for the high frequency of colony formation after
infection in the presence of 10 .mu.M AZT, the experiment depicted
in FIG. 2 was performed. HeLa-T4 cells were infected with HIV-gpt
(prepared in COS cells) in the absence or presence of AZT. Infected
cells were selected in gpt selective media and colonies were
isolated and expanded into cell lines. Twelve cell lines developing
after infection in the presence of AZT were further characterized.
To determine if these cell lines were refractory to the
antiretroviral effects of AZT they were infected with HIV-LacZ in
the presence of 10 .mu.M AZT. Three days after infection, the cells
were stained with X-gal to detect 13-galactosidase activity. Nine
of these twelve cell lines behaved like wild type HeLa-T4 cells
with complete inhibition of infection in the presence of AZT.
However, three of these cell lines demonstrated greater than 50% of
control infection (-AZT) despite the presence of 10 .mu.M AZT.
These cell lines were labeled as "persistently resistant" to the
antiretroviral effects of AZT.
[0091] Infection of these "persistently resistant" cell lines with
replication-competent HIV confirmed the relative inefficacy of AZT
in these cells. For example, a clinically relevant concentration of
0.1 .mu.M AZT was much less effective in inhibiting HIV-1IIIB in
the "persistently resistant" cell line than in the control cells
(FIG. 3). No such cells resistant to the antiviral effects of AZT
were obtained when colonies derived from HIV-gpt infections in the
absence of AZT were studied (see Table 2).
[0092] None of the "persistently resistant" cell lines were cross
"resistant" to the antiretroviral effects of ddl or ddC.
Interestingly, cells with persistent resistance to AZT showed
partial cross resistance to the antiretroviral effects of 50 .mu.M
d4T. In addition to this evaluation for cellular cross-resistance,
it was possible to use a similar experimental protocol to
demonstrate the independent selection of cells refractory to the
antiretroviral effects of a variety of other nucleoside analogs
(Table 2). In contrast, no cells "persistently resistant" to the
antiretroviral effects of the non-nucleoside reverse transcriptase
inhibitor TIBO R82150 could be selected using identical techniques.
These results indicate that HeLa-T4 cells have subpopulations of
cells that are independently refractory to the antiretroviral
effects of a variety of nucleoside analogs.
Comparison of Thymidine and AZT Phosphorylation in Isolated
Clones
[0093] To initiate an analysis of the mechanisms responsible for
this cellular resistance, a persistently resistant cell line was
compared to a control cell line obtained by HIV-gpt infection in
the absence of AZT. Each of these cell lines was incubated with
.sup.3H-thymidine and thymidine metabolites were assayed by HPLC.
As shown in FIGS. 4A and 4B, the persistently resistant cell line
(R116) had a greater phosphorylation of thymidine into TTP compared
to the non-resistant cell line (SI). An identical experiment with
.sup.3H-AZT indicated a nearly 2 fold reduction in AZTTP in R116
cells compared to Si cells (Table 3). Therefore, a component of the
resistance may be related to a diminished AZTTP/TTP ratio. These
results suggest that alterations in nucleotide metabolism may
underlie some of the differences between these cell lines. To
further characterize the basis for these differences, thymidine
kinase mRNA levels and thymidine kinase activity were compared in
the two cell lines. Although there were no differences in the
thymidine kinase mRNA levels on a Northern blot analysis, the R116
cell line had 3 times greater thymidine kinase activity than the S1
cell line (FIG. 5).
Tolerance of the Clones to Very High Concentrations of AZT
[0094] In additional studies of these cell lines, tolerance of high
concentrations of AZT was tested. As shown in FIG. 6, the
persistently resistant cell line (R116) was much more tolerant of
high concentrations of AZT. The cytotoxic concentration of AZT that
killed 50% of a variety of control cell lines was approximately 100
.mu.M. In contrast, the cytotoxic concentration of AZT that killed
50% of the persistently resistant clone R116 was greater than 1 mM.
This implies that the mechanisms that protect HIV from AZT in the
resistant cell lines also protect these cell lines from the
cytotoxic effects of even higher concentrations of AZT. This
demonstrates another AZT-related difference amongst these clones
derived from the same parental cell line.
Use of Floxuridine to Modulate the Antiviral Activity of AZT
Inhibition of Early Viral Breakthrough in Cells Sensitive and
Refractory to the Antiretroviral Effects of AZT
[0095] A previous study has utilized replication-defective HIV to
quantitate early infection in the presence of AZT (31). In that
study, HIV-gpt (a recombinant HIV encoding a selectable marker) was
used to infect HeLa-T4 cells in the presence of AZT. Infected cells
were isolated in gpt selective media and expanded into cell lines.
Several such cell lines were refractory to the antiviral effects of
AZT as evidenced by the ability of replication-defective or
replication-competent HIV to infect these cells in the presence of
AZT. Several control cell lines were obtained by infection of
HeLa-T4 cells with HIV-gpt in the absence of AZT. Cell line R116 is
a cell line that was determined to be refractory to the antiviral
effects of AZT. Cell line SI is a control cell line. A prior
metabolic analysis of these cell lines indicated that cell line
R116 had a reduced accumulation of AZTTP and an increased
phosphorylation of thymidine to TTP in comparison to the SI control
cell line (31). To determine if the addition of a fluoropyrimidine
to AZT increased the antiviral efficacy of AZT in the R116 cell
line, cells were cultured in the absence or presence of 0.1 .mu.M
AZT or 0.01 .mu.M FUdR alone or in combination prior to infection
with HIV-1 IIIB at an input multiplicity of infection of 1. As
demonstrated in FIG. 7A, 0.1 .mu.M AZT had potent antiviral
efficacy in the control S1 cell line. In contrast, in the cell line
refractory to the antiviral effects of AZT (R116) there was
significant HIV replication in the presence of 0.1 .mu.M AZT (FIG.
7B). However, the addition of 0.01M FUdR to 0.1 .mu.M AZT
suppressed this viral breakthrough. At these concentrations, no
cytotoxicity was observed. To further characterize these different
cell lines, cytotoxicity to various concentrations of FUdR were
determined. As shown in FIG. 8, the R116 cell line had an ED.sub.50
of 0.7 .mu.M FUdR whereas the S1 cell line, parental HeLa-T4 cells
and a pool of control cell lines all had an ED50 of 7 .mu.M. These
results further substantiate the presence of metabolic differences
in cells refractory to the antiviral effects of AZT as opposed to
cells sensitive to the antiviral effects of AZT.
Efficacy of AZT in Combination with FUdR in Inhibiting HIV-1
Infection of Lymphoid cells Sensitive and Refractory to the
Antiviral Activity of AZT
[0096] To extend the analysis of the antiviral efficacy of AZT in
combination with FUdR to lymphoid cells, similar experiments were
performed in Jurkat JE6.1 cells. In these experiments, three
populations of cells were studied. The parental JE6.1 cells were
compared to populations of JE6.1 cells that were isolated after
infection with a replication-defective recombinant Moloney Leukemia
virus containing the Tn5 neo gene (MLV-neo) in the absence or
presence of AZT. JE6.1 cells were infected in the absence or
presence of 10 .mu.M AZT and two days later the cells were placed
in media containing 1 mg/ml G418 to allow the growth of infected
cells. JE6.1AZTR is the cell population that was infected with
MLV-neo in the presence of AZT. JE6.1con is the control population
of cells infected with MLV-neo in the absence of AZT.
[0097] The efficacy of AZT in combination with FUdR in inhibiting
HIV-1 infection of these cell populations was determined by
infection in the absence of AZT or in the presence of 0.001 .mu.M,
0.01 .mu.M, 0.1 .mu.M, 1 .mu.M or 10 .mu.M AZT in combination with
no FUdR, 0.005 .mu.M FUdR, 0.01 .mu.M FUDR or 0.025 .mu.M FUdR.
This experiment allowed a detailed analysis of the IC.sub.50 of AZT
in each population with different concentrations of FUdR. These
results are shown in Table 4. Based upon prior results in HeLa-T4
cells (31), it is likely that the JE6.1AZTR cells represent a
mixture of cells, some of which require an increased concentration
of AZT to inhibit HIV infection. This is reflected by a 2 fold
increase AZT IC.sub.50 when analyzing the entire population.
[0098] Strikingly, the combination of FUdR with AZT dramatically
suppresses HIV infection of this population. A greater than 600
fold reduction of AZT IC.sub.50 is seen during infection of these
cells in the presence of AZT and FUdR. In fact, these cells, which
were initially isolated as cells infected in the presence of AZT,
were more sensitive to the antiviral effects of the AZT-FUdR
combination than were control or parental cells. These results
suggest that this population of cells has metabolic features that
renders them highly susceptable to the antiviral effects of the
AZT-FUdR combination. Of note, there is a 10-fold reduction of AZT
IC.sub.50 when the parental and control cell populations were
infected with HIV-1 in the presence of AZT and FUdR. No
cytotoxicity was observed in the AZT-FUdR combination except at the
highest drug concentrations used (10 .mu.M AZT plus 0.025 .mu.M
FUdR, FIG. 9).
Efficacy of AZT in Combination with FUdR in Inhibiting HIV-1
Infection of Primary Blood Mononuclear Cells
[0099] The antiviral efficacy of the combination AZT and FUdR was
also assessed in PBMC. These studies are shown in FIG. 10 and
demonstrate that the combination of AZT and FUdR has potent
antiviral activity in PBMC. Similar results were obtained with a
primary HIV isolate known to be genetically sensitive to AZT.
Therefore, FUdR potentiates the antiviral efficacy of AZT in PBMC
infected with either HIVIIIB or a clinical isolate. Of note,
cytotoxicity in the AZT-FUdR combination was similar to that seen
for the JE6.1 cells in that cytotoxicity was only observed when 1 0
.mu.M AZT was combined with 0.025 .mu.M FUDR.
Early HIV Breakthrough Infection in the Presence of Stavudine
Preliminary Characterization of Mechanisms Allowing HIV Infection
in the Presence of d4T
[0100] To undertake a preliminary characterization of the
predominant mechanisms responsible for early HIV infection after
the initiation of d4T, we utilized several populations of
recombinant viruses. Recombinant replication-defective HIV was
prepared by transfection of COS cells with complementing plasmids
encoding the RNA and proteins necessary for the production of a
recombinant HIV encoding gpt (26). Such viruses are produced
without major genetic heterogeneity as the predominant mechanisms
responsible for the generation of heterogeneity (e.g., cycles of
reverse transcription) are not involved in the production of these
viruses. Even if there was heterogeneity as a consequence of errors
during plasmid transcription, the resulting mutated proteins would
be greatly diluted in a population of proteins. In contrast,
replication defective HIV-gpt made by rescue with
replication-competent HIV will contain proteins encoded by the
replication-competent virus used for rescue. Prior experiments have
demonstrated the close relationship between the drug sensitivity
phenotype of the recombinant virus and the drug sensitivity
phenotype of the virus used to rescue the recombinant virus (26).
Therefore, recombinant virus produced by rescue with
replication-competent virus will be heterogeneous and may reflect
the drug sensitivity profile of the virus used for rescue. In a
prior experiment, the heterogeneity introduced by the
replication-competent virus used for rescue resulted in a
calculation of the prevalence of HIV resistant to a NNRTI in an
unselected population (26). This calculated value was very similar
to the prevalence subsequently calculated from in vivo studies of
HIV dynamics after the initiation of a NNRTI (38).
[0101] A comparison of the two virus populations described above
demonstrated very similar rates of infection in the presence of
high concentrations of d4T (Table 5). This high level of infection
with a virus produced from plasmid transcripts (HIV-gpt) suggested
a high rate of infection with a homogenous population of a
recombinant virus whose proteins were generated by translation of
plasmid transcripts and thus not anticipated to have a high level
of genetic heterogeneity. To confirm this high rate of infection in
the absence of genetic drug resistance, we used several other
recombinant viruses and host cells. MLV based recombinant viruses
showed a similar high rate of infection in the presence of d4T
(Table 6). Similar infections in the presence of high
concentrations of other nucleoside reverse transcriptase inhibitors
have consistently demonstrated a rate of infection in the presence
of high concentrations of d4T greater than that seen in the
presence of high concentrations of the other nucleoside analogs
(Table 6). A high rate of infection of Jurkat cells in the presence
of d4T has also been seen.
[0102] The similar rates of infection with virus prepared by
plasmid transfection and virus prepared by rescue with
replication-competent HIV suggested that genetic resistance was not
the major mechanism of early HIV breakthrough being detected. This
interpretation was supported by evidence of high rates of infection
with MLV based recombinant virions (also anticipated to have a low
level of genetic heterogeneity). Infections of Jurkat cells
indicated that the high rate of infection in the presence of high
concentrations of d4T was not cell line specific. These data
suggested that early HIV breakthrough infection in the presence of
d4T was not due to infection by d4T-resistant virus.
Isolation of the Cells Infected with HIV in the Presence of d4T
[0103] The presence of a selectable marker gene in the recombinant
HIV allowed the isolation of cells infected by the recombinant
viruses in the presence of d4T. These cells were characterized by
infection with both additional recombinant viruses and by
replication-competent viruses. As demonstrated in Table 6,
approximately 37% of the isolated cells were repeatedly refractory
to the antiviral effects of d4T (i.e., they could be readily
re-infected with recombinant HIV, recombinant MLV, or
replication-competent HIV in the presence of high concentrations of
d4T). An even higher percentage of the Jurkat cells infected in the
presence of d4T were persistently refractory to the antiviral
effects of d4T (Table 7). Infections with replication-competent HIV
demonstrated that the refractoriness to infection detected in these
clones was not a phenomena solely associated with recombinant
viruses (FIG. 11). A subset of these persistently refractory cells
(approximately 20%) were also refractory to the antiviral effects
of AZT.
Combined d4T-FUdR Antiviral Activity
[0104] As has been demonstrated previously for AZT, a component of
the refractoriness to the antiviral effects of d4T can be reversed
by the addition of FUdR (Table 8). The antiviral efficacy of the
combination therapy, as measured by the d4T IC50, is markedly
improved with combination therapy. Prior studies of the antiviral
efficacy of FUdR have demonstrated limited antiviral efficacy of
FUDR alone, but marked antiviral efficacy of combined AZT and FUdR.
Table 8 shows the capacity of the FUdR-d4T combination to reverse
some of the cellular refractoriness to d4T described above. The
FUdR-d4T combination has marked antiviral activity in cells
demonstrated to be refractory to the antiviral effects of d4T. The
antiviral efficacy of the combination has also been studied in
unselected PBMC (FIG. 12).
Discussion
Sanctuary Growth of HIV in the Presence of AZT
[0105] The studies described above indicate that sanctuary growth
of HIV may occur in the presence of AZT and that early in treatment
cellular resistance may make a large contribution to viral
breakthrough. In fact, there was no quantitative difference in HIV
breakthrough when HIV-gpt prepared by transfection in COS cells was
compared to HIV-gpt produced by rescue with replication-competent
HIV. This suggests that a large part of early infection in the
presence of AZT may be a consequence of cellular effects. At least
two types of such sanctuary growth were detected. Nine of the
twelve cell lines analyzed did not have persistent resistance to
the antiviral effects of AZT and may have had epigenetic
alterations such as those that might occur at specific points in
the cell cycle. In contrast, three of the twelve cell lines had
persistent resistance to the antiviral effects of AZT, with both
recombinant and replication-competent HIV. In studies with
replication-competent HIV, virtually complete inhibition of the
infection of control cells was obtained with a concentration of AZT
that only reduced viral production in a persistently resistant
clone by 50%.
[0106] These cell lines refractory to the antiviral effects of AZT
are likely to have specific alterations that render AZT less
effective. Metabolic studies suggest that some of this resistance
may be due to differences in nucleotide metabolism resulting in a
reduction of AZTTP in the resistant cells. It will be important to
further characterize and define the mechanisms responsible for
cellular resistance because reversal of this resistance may greatly
reduce viral burden and delay the outgrowth of virus with genetic
resistance. It is important to emphasize that the cells that were
detected as refractory to the antiviral effects of AZT were only
exposed to AZT for a short period of time. There was no
preselection of cells prior to infection with the recombinant
viruses.
[0107] Recent reports on nucleotide pool sizes in resting as
opposed to stimulated blood mononuclear cells and different cell
lines derived from different blood cell lineages have demonstrated
marked differences that might translate into variable efficacies of
nucleoside analogs within populations of blood cells (6,15).
Furthermore, other investigators have grown cells in high
concentrations of AZT for prolonged periods of time and
demonstrated the selection of cells with reduced levels of
thymidine kinase activity (17). Additional data about metabolic
differences occuring in the lymphocytes of patients treated with
prolonged courses of AZT also suggests that cellular resistance may
contribute to HIV breakthrough (1). Thus, cellular resistance is
likely to contribute to viral breakthrough during an in vivo
infection and multiple mechanisms may contribute to cellular
resistance. The prevalence of resistant cells detected in single
cell lines derived during infection in these studies raises
interesting speculation concerning the prevalence of similar
resistant cells during an in vivo infection involving multiple cell
types.
[0108] Earlier studies with recombinant viruses indicated that
there is a high prevalence of genetically TIBO resistant HIV in an
unselected HIV population. As a consequence of this high prevalence
and the lack of cellular metabolism for TIBO, genetically resistant
virus is rapidly selected in vivo and in vitro. In contrast, AZT is
metabolized in cells, a subpopulation of which is refractory to the
antiretroviral effects of AZT. Early growth of "non-genetically
resistant" virus can occur in these sanctuary cells ("cellular
resistance"). With continued growth there is amplification of
pre-existing (or emerging) viral variants with genetic resistance
because the truly resistant virus can infect any suitable target
cell, not just those cells in which AZT is ineffective. This gives
a relative growth advantage to the genetically resistant virus.
Subsequent additional mutations or recombination events may result
in viruses with multiple mutations. The initial "cellular
resistance" may allow a population of non-resistant or partially
resistant virus to replicate, providing a pool of virus in which
additional mutations and recombination events can occur. Reversal
of cellular resistance could conceivably delay, or even prevent,
the outgrowth of highly resistant virus with multiple mutations by
not allowing non-resistant or partially resistant virus (with
single mutations) to replicate.
[0109] FIG. 1 is a schematic representation of the production of
recombinant HIV-gpt by COS cell transfection or rescue from the
H9/HIV-gpt cell line.
[0110] FIG. 2 is a schematic representation of the analysis of
colonies arising after COS cell derived HIV-gpt infection of
HeLa-T4 cells in the presence of 10 .mu.M AZT. Twelve such colonies
were expanded and infected with HIV-LacZ in the presence and
absence of 10M AZT. Ten control colonies derived from HIVgpt
infection of HeLa-T4 cells in the absence of AZT were studied in
parallel. "Persistent" cellular resistance was defined by a high
level infection with HIVLacZ in the presence of AZT, as shown for
colony number 2. HIV-LacZ contains the LacZ gene driven by an SV40
promoter inserted into a large deletion in the HIV genome extending
from the pol gene to the 3' end of the env gene. HIVLacZ virus
production has been previously described (16).
[0111] FIG. 3 is a graph showing the infection of a clone of
HeLa-T4 cells "persistently resistant" to the antiviral effects of
AZT (clone R116) and a control clone (S1) with replicationcompetent
HIV-1IIIB in the presence of 0.1 .mu.M AZT. P24 was assayed,
compared to a control infection in the absence of AZT and plotted
as a function of time. P24 values in the absence of AZT were
1857+104 ng/ml for S1 and 1717+113 ng/ml for R116.
[0112] FIGS. 4A and 4B are graphs illustrating thymidine
metabolism-HPLC analysis of clones obtained after infection of
HeLa-T4 cells with HIV-gpt in the presence and absence of AZT. FIG.
4A illustrates the SI cell line derived from HeLa-T4 cells after
infection with HIV-gpt in the absence of AZT. FIG. 4B illustrates
the R116 cell line, which was persistently resistant to the
antiviral effects of AZT. The earliest peak represents thymidine
and the subsequent peaks represent TMP, TDP, and TTP.
[0113] FIG. 5 is a graph showing a comparison of thymidine kinase
mRNA levels (A) and enzyme activity (B) in cell lines sensitive and
persistently resistant to the antiretroviral effects of AZT. The
mRNA levels of S1 and R116 were 8390 and 8500 densitometry units,
respectively. Thymidine kinase activity was based upon three
independant experiments performed in triplicate.
[0114] FIG. 6 is a graph showing cellular toxicity of AZT. The cell
lines were grown in the presence of the indicated concentrations of
AZT. Cellular toxicity was then determined in cells persistently
refractory to the antiviral effects of AZT (R116) and in cells
sensitive to the antiviral effects of AZT (HT4, S pool and S1)
using a standard MTT assay. S pool was a pool of colonies derived
from HIV-gpt infection of HeLa-T4 cells in the absence of AZT.
HeLa-T4 is the parental cell line.
[0115] Table 1 shows the frequency of HIV-gpt colony formation in
the presence and absence of AZT. Table 1 also shows a comparison of
HIV-gpt produced in COS cells by transfection with plasmids and
HIV-gpt produced by rescue from the H9/HIV-gpt-cell line after
infection with HIV-1lilB (see FIG. 1).
1TABLE 1 Number of colonies Source of HIV-gpt -AZT +AZT Frequency
Plasmid-derived (COS cells) 3.1 .times. 10.sup.4 16 5.2 .times.
10.sup.-4 Rescue with HIV-1 1.8 .times. 10.sup.4 9 5.0 .times.
10.sup.-4
[0116] Table 2 shows colony formation and "persistent resistance"
after HeLa-T4 infection with plasmid derived HIV-gpt (produced in
COS cells) in the presence of high doses of the indicated
antiretroviral agents.
[0117] Concentrations of the antiretroviral agents were: AZT-10
.mu.M, DDI-50 .mu.M, D4T-50 .mu.M and DDC-10 .mu.M.
2TABLE 2 Number of Number of "persistently resistance" Drug
colonies colonies Frequency No Drug (Control) 8800 0/10 0 AZT 12
3/12 3.4 .times. 10.sup.-4 D4T 50 not done -- DDI 16 2/16 2.3
.times. 10.sup.-4 TIBO 3 0/3 0
[0118] Table 3 shows the concentration of phosphorylated AZT
metabolites in the "persistently resistant" (R116) and sensitive
(SI) cell lines. Pool sizes were determined by incubation of cells
with .sup.3H-AZT for 4 hours followed by cellular extraction and
HPLC. The numbers are expressed as pmoles/10.sup.6 cells. The
numbers in parentheses represent the percentage of total
radioactive species in that pool.
3TABLE 3 Clone AZT AZTMP AZTDP AZTTP S1 0.0206(12.2) 0.1212(71.6)
0.0116(6.9) 0.0158(9.3) R116 0.0155(7.7) 0.1575(78.6) 0.0193(9.6)
0.0083(4.2)
Use of Floxuridine to Modulate the Antiviral Activity of AZT
[0119] Preliminary studies from our laboratory have demonstrated
that early HIV infection of various cell lines in the presence of
AZT is not the consequence of infection with AZT-resistant virus.
In both HeLa-T4 cells and a lymphoid cell line (Jurkat JE6.1), the
predominant component of early HIV infection in the presence of AZT
is a consequence of infection with AZT-sensitive virus (31).
Clinical studies also demonstrate that early HIV infection in the
presence of AZT occurs with AZT-sensitive virus (29). To
characterize the mechanisms allowing the replication of
AZT-sensitive HIV in the presence of AZT, a metabolic analysis of
some of the cells infected with HUV in the presence of AZT in vitro
was previously undertaken (31). Those studies demonstrated that a
component of early infection with drug-sensitive virus was occuring
in a subpopulation of cells with features that would be anticipated
to decrease the antiviral efficacy of AZT. These studies were
important because they indicated that the reversal of early HIV
infection in the presence of AZT required interventions directed at
features other than viral drug-resistance. Based upon a prior study
demonstrating increased phosphorylation of thymidine to TTP and
decreased AZTTP in a subset of cells infected with drug-sensitive
HIV in the presence of AZT, applicants have attempted to modulate
the antiviral efficacy of AZT by combining AZT therapy with
floxuridine. These initial studies have demonstrated the
suppression of early viral breakthrough infection in the presence
of AZT with drug combinations that are readily achievable in vivo
and are non-cytotoxic. In addition, there is a clear
concentration-response relationship when FUdR is added to AZT.
[0120] In addition to the determination that the AZT-FUdR
combination suppressed HIV infection of cells that were infected
with HIV in the presence of AZT, the combination was much more
effective than AZT alone at inhibiting HIV infection of an
unfractionated lymphoid cell line and PBMC. This increased efficacy
was also demonstrated with a clinical isolate. Therefore, the
enhanced antiviral activity of the combination therapy is not
restricted to cell lines, recombinant viruses, or laboratory
strains of virus and may therefore have clinical utility.
[0121] The increased efficacy of AZT-FUdR in suppressing HIV
infection of cells readily infected with HIV in the presence of AZT
is particularly striking. Since this population of cells is a
mixture of cells with and without persistent refractoriness to the
antiviral effects of AZT (i.e., infection of a subset of this
population is repeatedly refractory to the antiviral effects of
AZT), the AZT IC.sub.50 for this population is only minimally
elevated. Nevertheless, infection of this entire population is
extremely sensitive to inhibition by the AZT-FUDR combination. The
supersensitivity of infection of this population of cells to
combination therapy was unanticipated and is likely to be explained
by metabolic features that are responsible for the efficacy of the
combination. Determination of the mechanisms responsible for this
supersensitivity to combined AZT-FUdR therapy must await metabolic
analysis of thymidine, AZT and FUdR phosphorylated intermediates in
populations of cells and individual clones. It is important to note
that in all of these studies FUdR has moderate antiviral activity
when used by itself. The mechanisms by which this inhibition occurs
are also currently unknown and may also be related to perturbations
of normal thymidine metabolite pools, direct inhibition of viral or
cellular processes or by incorporation into the viral DNA during
reverse transcription.
[0122] It is very likely that the long term ability of HIV to
replicate in the presence of AZT is a consequence of the emergence
of AZT-resistant virus. Multiple mutations in RT are necessary for
the development of this genetic AZT-resistance and these mutations
emerge over several months-years. Suppression of early HIV
replication with AZTsensitive virus in the presence of AZT could
delay, or even prevent the emergence of AZT resistant virus by
diminishing the substrate for subsequent genetic changes.
Therefore, studies that define the mechanisms of early viral
breakthrough infection have potential long term therapeutic
implications.
[0123] The clinical feasibility of combined fluoropyrimidine-AZT
therapy needs to be evaluated. At low concentrations the
fluoropyrimidines are often well tolerated by oncology patients
with few significant neurologic, gastrointestinal or hematologic
toxicities. The in vivo dose necessary to improve the antiviral
efficacy of AZT will need to be determined, however extrapolation
from in vitro studies indicates that cytotoxic concentrations of
fluoropyrimidines will not be needed. Phase I clinical studies of
FUdR combined with AZT in patients with HIV-1 infection will
provide information about the feasability of combination therapy.
In addition, other drugs with the ability to decrease Tm levels
will also be evaluated in pre-clinical studies.
[0124] FIG. 7 is a graph showing the suppression of viral
breakthrough in cells sensitive and refractory to the antiviral
effects of AZT. Cells sensitive (S1) and refractory (R116) to the
antiretroviral effects of AZT were infected with HIV-1 IIIB in the
absence of drug (open squares), 0.1 .mu.M AZT (solid squares), 0.01
.mu.M FUdR (solid triangle) or a combination of 0.1 .mu.M AZT plus
0.01 .mu.M FudR (open triangle). Cell free supernatants were
assayed for RT activity every two days. Results are the mean of
triplicate cultures. Standard deviations were <15%.
[0125] FIG. 8 is a graph illustrating FUDR cytotoxicity in cells
sensitive and refractory to the antiretroviral activity of AZT.
Cells sensitive, parental HT4 (open circle), S1 (solid square),
Spool (solid circle) and refractory, R116 (open square) were grown
in the presence of various concentrations of FUdR. Three days
latter, cell viability was determined by the MTT reduction method.
Spool cells are a population of control cells obtained by infection
with HIV-gpt in the absence of AZT (7).
[0126] FIG. 9 is a graph showing AZT-FUdR cytotoxicity in JE6.1
cells sensitive and resistant to the antiviral effects of AZT.
Cytotoxicity of 10 .mu.M AZT in combination with 0.025 .mu.M FUdR
was determined in JE6.1 cells sensitive (solid circle), JE6.1con
(open circle) and resistant, JE6.1AZTR (open triangle) to the
antiviral effects of AZT as described in Materials and Methods.
[0127] FIG. 10 is a graph showing that the AZT-FUdR combination
inhibits HIV-1 infection of PBMC. PBMC were infected with HIV-1 in
the absence of drug (cross) with AZT alone (x), with various
concentrations of FUdR alone, [0.005 .mu.M FUdR (open circle), 0.01
.mu.M FUdR (open square), 0.025 .mu.M FUdR (open triangle)], or
with combinations of FUdR and AZT [AZT+0.005 .mu.M FUdR (closed
circle), AZT+0.01 .mu.M FUdR (solid square) AZT+0.025.mu.M FUdR
(solid triangle). Panel A, [AZT]=0.001 .mu.M; Panel B, [AZT]=0.01
.mu.M.
[0128] Table 4. Jurkat JE6. 1 cells, Jurkat JE6. 1 cells refractory
to the antiviral effects of AZT (JE6. AZTR) and control JE6.1 cells
(JE6.1con) obtained by infection with MLV-neo in the absence of AZT
were infected with HIV-1 IIIB in the presence of 0.001 .mu.M AZT,
0.01 .mu.M AZT, 0.1 .mu.M AZT, 1 .mu.M AZT or 10 .mu.M AZT in the
presence of 0.05 .mu.M FUdR, 0.01 .mu.M FUdR or 0.025 .mu.M FUdR.
IC.sub.50 represents the concentration of AZT required for 50%
inhibition of reverse transcriptase activity at day 6 of
infection.
4TABLE 4 AZT/FUdR Susceptibility In Cells Sensitive And Refractory
To The Antiretroviral Activity Of AZT JE6.1 JE6.1.sub.AZTR
JE6.1.sub.Con IC.sub.50 Sensitivity IC.sub.50 Sensitivity IC.sub.50
Sensitivity Treatment (.mu.M) (fold) (.mu.M) (fold) (.mu.M) (fold)
AZT 0.3 0.6 0.2 AZT + .005F 0.3 0 0.003 20 0.2 0 AZT + .01F 0.1 3
0.001 600 0.03 7 AZT + .025F 0.03 10 <.001 >600 0.02 10
Early HIV Breakthrough Infection in the Presence of Stavudine
[0129] Recent clinical analyses have emphasized the potential of
prolonged suppression of HIV viremia when antiviral drug
combinations are used (39,42). However, eradication of virus has
not yet been demonstrated and virus regrowth with cessation of
antiviral drugs is likely. In addition, the propensity of HIV with
resistance to triple drug combinations (e.g., AZT, 3TC and a
protease inhibitor) to emerge is still unclear. In the face of this
uncertainty more detailed information concerning the mechanisms
contributing to HIV breakthrough in the presence of antiviral drugs
is needed.
[0130] In this report we utilize an in vitro model of HIV infection
to provide two lines of evidence that early HIV breakthrough
infection in the presence of d4T is not a consequence of infection
by HIV with genetic drug resistance. Initial studies demonstrated
that the frequency of HIV infection in the presence of d4T was very
similar with several stocks of virus predicted to have significant
differences in genetic heterogeneity. These studies suggested that
any pre-existing unselected d4T resistant HIV in the population of
HIV used to rescue the replication-defective HIV was not detected
above the very high level of infection occurring with the other
virus populations. The fact that nearly 50% of the cells infected
with HIV in the presence of d4T are readily re-infected in the
presence of high concentrations of d4T provides additional evidence
supporting a high level of infection in the absence of genetic d4T
resistance. These results are not limited to recombinant HIV and
were also demonstrated with MLV based viruses as well as
replication-competent HIV.
[0131] A subset of the cells infected with HIV in the presence of
d4T do not have a persistent phenotype of being refractory to d4T.
They may have been infected as a consequence of cell cycle related
phenomena, intravirion reverse transcription (40) or other features
that are not characterized by persistent cellular phenotypic
change. The mechanisms underlying the refractoriness of both of the
populations of cells (those with and those without persistent
refractoriness to the antiviral effects of d4T) are currently being
assessed with metabolic analyses.
[0132] Previous studies have demonstrated that the combination of
FUdR with AZT or d4T has significant antiretroviral activity (41).
Furthermore, the combination of FUDR and AZT has potent
antiretroviral activity in cells refractory to the antiretroviral
activity of AZT alone (31). These studies demonstrate the capacity
to improve the antiviral efficacy of d4T by the addition of drugs
such as FUdR with the capacity of interacting with the biochemical
mechanisms responsible for AZT and/or d4T metabolic activation.
[0133] Several investigators have noted clinical features which are
consistent with the results presented above. For example, the
definition of genetic changes associated with clinical d4T
resistance has been more difficult than the definition of genetic
changes associated with AZT resistance. In addition, the selection
of d4T resistant HIV in tissue culture has also more difficult than
the selection of AZT resistant virus. The high level of early
infection with drug sensitive virus might contribute to these
features.
[0134] In summary, we have demonstrated that early HIV breakthrough
infection in the presence of d4T is not a consequence of infection
with virus that is resistant to d4T. As with AZT, infection with
drug-sensitive virus predominates early after the initiation of the
drug. Further analyses of the mechanisms responsible for HIV
breakthrough in the presence of antiviral drugs are essential to
efforts to define drug combinations that provide durable
suppression of HIV infection and viremia (42). Clinical studies
with FUdr may be warranted for selected patients intolerant of or
not optimally responsive to current combination antiretroviral
regimens containing either d4T or AZT.
[0135] FIG. 11 is a graph illustrating the infection of JE6.1 cell
clones persistently resistant to the antiviral effects of d4T (D4T
bulk, D4TR1, D4TR3) and a control clone of JE6.1 cells with
HIV-IIIB in the presence of various concentrations of d4T. RT
activity was assayed and compared with those for a control
infection in the absence of d4T.
[0136] FIG. 12 is a graph showing that D4T-FUdr combination
inhibits HIV-1 infection of PBMCs. PBMCs were infected with HIV-1
in the absence of drug [cross], with d4T alone (0.01 uM) [X], with
various concentrations of FUdr alone (0.005 uM FUdr [open circle],
0.01 uM FUdr [open square], 0.025 uM FUdr [open triangle]) or with
combinations of FUdr and d4T (d4T+0.005 uM FUdr [solid circle],
d4T+0.01 uM FUdr [solid square], d4T+0.025 uM FUdr [solid
triangle]).
[0137] Table 5. HIV-gpt produced in COS cells by transfection with
plasmids (plasmid derived) is compared with HIV-gpt produced by
rescue from H9/HIV-gpt cell line after infection with HIV-IIIB and
cells infected with MLV-neo.
5TABLE 5 Frequency of HIV-gpt and MLV-neo Colony Formation in the
Presence and Absence of D4T No. of colonies without with Sources of
Virus D4T D4T Frequency plasmid derived 4.7 .times. 10.sup.4 80 1.7
.times. 10.sup.3 Rescue with HIV 3.4 .times. 10.sup.4 53 1.6
.times. 10.sup.3 MLV-neo 9.2 .times. 10.sup.4 101 1.1 .times.
10.sup.3
[0138] Table 6. Colony formation and persistent resistance after
Hela-T4 cell infection with MLV-neo virus in the presence and
absence of various antiviral agents.
6TABLE 6 Colony Formation and Persistent Resistance After Hela-T4
Infection With MEV-neo Virus in the Presence and Absence of Various
Antiretroviral Agents* No. of No. of persistently Drug colonies
resistant colonies Frequency AZT 12 2/12 2.2 .times. 10.sup.-4 DDI
19 4/19 4.4 .times. 10.sup.-4 DDC 17 2/17 2.2 .times. 10.sup.-4 D4T
63 23/63 2.5 .times. 10.sup.-3 Control 9050 0/15 0 *Concentrations
of the antiviral agents were as follows: AZT, 10 uM; DDI, 50 uM;
DDC, 10 uM; and D4T, 50 uM.
[0139] Table 7. Infection of Jurkat JE6.1 cell clones sensitive and
resistant to D4T.
7TABLE 7 Infection of Jurkat cell clones Sensitive and Resistant to
D4T* Clone** No Drug 50 uM D4T 100 uM D4T Bulk ++++ ++ + R1 ++++
++++ ++++ R3 ++++ +++ ++ R4 ++++ - - R7 ++++ - - R12 ++++ ++++ ++++
R14 ++++ + - R15 ++++ ++++ ++++ R16 ++++ + - R19 ++++ - - R20 ++++
++++ +++ R21 ++++ ++++ ++++ R22 ++++ ++ + R26 ++++ + - R29 ++++ - -
R33 ++++ +++ + R36 ++++ ++++ ++ C2 ++++ - - C23 ++++ - - C26 ++++ -
- C27 ++++ - - JE6.1 ++++ - - These data represent the percentage
of cells infected with MLV-LacZ in the presence of D4T in
comparison with cells infected in the absence of D4T ([% cell
infected in the presence of D4T/% cell infected in the absence of
D4T] .times. 100). *(-), 20%; (+) 21-40%; (++) 41-60%; (+++),
61-80%; (++++), 81-100%. **Bulk = bulk culture; R = resistant
clone; C = control clone; JE6.1 = parental cell line.
[0140] Table 8. Jurkat JE6.1 cells refractory to the antiviral
activity of d4T (D4T bulk, D4TR1, D4TR3) and control JE6.1 cells
were infected with HIV-IIIB in the presence of 0.001 uM d4T, 0.01
uM d4T, 0.1 uM d4T, 1 uM d4T, 10 uM d4T, 100 uM d4T and 1000 uM d4T
in the presence of 0.005 uM FUdr, 0.01 uM FUdr or 0.25 uM FUdr. The
IC50 represents the concentration of D4T required for 50%
inhibition of RT activity on day 6 of infection.
8TABLE 8 AZT/FUdr Susceptibility in Cells Sensitive and Refractory
to the Antiretroviral Activity of D4T JE6.1 D4TBuIk D4TR1 D4TR3
IC.sub.50 Sensitivity IC.sub.50 Sensitivity IC.sub.50 Sensitivity
IC.sub.50 Sensitivity Treatment (uM) (fold) (uM) (fold) (uM) (fold)
(uM) (fold) D4T 0.02 -- 3 -- 74 -- 2 -- D4T + 0.005F 0.003 3 0.8 4
0.9 82 0.1 20 D4T + 0.01F 0.001 20 0.04 75 0.2 370 0.03 67 D4T +
0.025F 0.0008 25 0.009 333 0.01 7400 0.007 286
REFERENCES
[0141] 1. Avramis, V. I; Kwock, R.; Solorzano, M. M.; Gomperts, E.
Evidence of in vitro development of drug resistance to
azidothymidine in T-lymphocytic leukemia cell lines (Jurkat
E6-1/AZT-100) and in pediatric patients with HIV-1 infection.
Acquir Immune Defic Syndr 1993 6:1287-96.
[0142] 2. Boucher, C. A., Lange, J. M., Miedema, F. F., et al.
HIV-1 biological phenotype and the development of zidovudine
resistance in relation to disease progression in asymptomatic
individuals during treatment. AIDS 1992;6:12591264.
[0143] 3. Bradshaw, H. D. and P. L. Deininger. 1984. Human
thymidine kinase gene: molecular cloning and nucleotide sequence of
a cDNA expressible in mammalian cells. Mol. Cell. Biol
4:2316-2320.
[0144] 4. Chomozynski, P. and Sacchi, N. 1987. Single-step method
of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction. Anal. Biochem. 162:156-159.
[0145] 5. Fischl, M. A., Richman, D. D., Grieco, M. H., et al. The
efficacy of azidothymidine AZT in the treatment of patients with
AIDS and the AIDS related complex. A double blind placebo
controlled trial. N. Engl. J. Med. 1987;317:185-191.
[0146] 6. Gao W-Y, Shirasaka T., Johns, D. G., Broder, S., Mitsuya,
H. Differential phosphorylation of azidothymidine, dideoxycytidine
and dideoxyinosine in resting and activated peripheral blood
mononuclear cells. J. Clin. Invest. 1993;91 :2326-2333.
[0147] 7. Johnston, M. I., McGowan, J. J. Strategies and Progress
in the Development of Antiretroviral Agents. In: DeVita V T,
Hellman S, Rosenberg S A, ed. AIDS: Etiology, Diagnosis, Treatment
and Prevention. Philadelphia: J. B. Lipincott Company, 1992:
357-372.
[0148] 8. Larder, B. A., Coates, K. E., Kemp, S. D.
Zidovudine-resistant human immunodeficiency virus selected by
passage in cell culture. J. Virol. 1991: 65:5232-5236.
[0149] 9. Larder, B. A., Darby, G., Richman, D. D. HIV with reduced
sensitivity to zidovudine (AZT) isolated during prolonged therapy.
Science 1989;243:17311734.
[0150] 10. Lee, L-S. and Y-C. Cheng. 1976. Human deoxythymidine
kinase. J. Biol. Chem. 251: 2600-2604.
[0151] 11. Mayers, D. L. Clinical significance of in vitro
zidovudine resistance. Third Workshop on Viral Resistance.
Gaithersburg, Md.: 1993.
[0152] 12. Mayers, D. L., McCutchan, F. E., Sanders, B. E., et al.
Characterization of HIV isolates arising after prolonged zidovudine
therapy. J Acquir Immune Defic Syndr 1992;5(8):749-59.
[0153] 13. Mellors, J., Dutschman, G., Im, G., Tramontano, E.,
Winkler, S. R., Cheng, Y. C. In vitro selection and molecular
characterization of HIV-1 resistant to normucleoside inhibitors of
reverse transcriptase. Mol. Pharmacol. 1992;41:446451.
[0154] 14. Mosmarn, T. Rapid colorimetric assay for cellular growth
and survival:application to proliferation and cytotoxicity assays.
J Immunol Methods 1983 65: 55-63.
[0155] 15. Mukherji, E., Au, J. L. S., Mathes, L. E. Differential
antiviral activities and intracellular metabolism of
3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine in human cells.
Antimicrob. Agents Chemother. 1994,38:1573-1579.
[0156] 16. Nunberg, J. H., Schleif, W. A., Boots, E. J., et al.
Viral resistance to to HIV-1 specific pyridinone reverse
transcriptase inhibitors. J. Virol. 1991;65:4887-92.
[0157] 17. Nyce, J., Leonard, S., Canupp, D., Schulz, S., Wong, S.
Epigenetic mechanisms of drug resistance: Drug induced DNA
hypermethylation and drug resistance. Proc. Natl. Acad. Sci. 1993
;90:2960-2964.
[0158] 18. Page, K. A., Landau, N., Littman, D. R. Construction and
use of a human immunodeficiency virus vector for analysis of of
viral infectivity. J. Virol. 1990,34:5270-5276.
[0159] 19. Richman, D. D., Fischl, M. A., Grieco. M. H., et al. The
toxicity of azidothymidine in the treatment of patients with AIDS
and A/DS-related complex: A double blind placebo controlled study.
N. Engl. J. Med. 1987;322:941.
[0160] 20. Richman, D. D. Antiretroviral Therapy: Azidothymidine
and Other Deoxynucleoside Analogues. In: DeVita V T, Hellman S,
Rosenberg S A, ed. AIDS: Etiology, Diagnosis, Treatment and
Prevention. Philadelphia: J. B. Lippincott, 1992: 373-387.
[0161] 21. Richman, D. D., Grimes, J. M., Lagakos, S. W. Effect of
stage of disease and drug dose on zidovudine susceptibilities of
isolates of human immunodeficiency virus. J. Acquired Immune Defic.
Syndr. 1990;3:743-746.
[0162] 22. Richman, D., Shih, C. K., Lowy, I., et al. Human
immunodeficiency virus type 1 mutants resistant to normucleoside
inhibitors of reverse transcriptase arise in tissue culture. Proc
Natl Acad Sci USA 1991; 88 (24):11241-5.
[0163] 23. Saag, M. S., Emini, E. A., Laskin, O. L. A short terrn
clinical evaluation of L697,661, a non-nucleoside inhibitor of
HIV-1 reverse transcriptase. N. Engl. J. Med. 1993;329:
1065-1072.
[0164] 24. Sherley, J. L. and T. J. Kelly. 1988. Human cytosolic
thymidine kinase. J. Biol. Chem. 263:375-382.
[0165] 25. Spector, S. A., Kennedy, C., McCutchan, J. A. The
antiviral effect of zidovudine and and ribavirin in clinical trials
and the use of p24 antigen as a virologic marker. J. Infect. Dis.
1989;159:822.
[0166] 26. Strair, R. K., Medina, D. J., Nelson, C. J., Graubert,
T., Mellors, J. W. Recombinant retroviral systems for the analysis
of drug-resistant HIV. Nucl. Acids Res. 1993; 21 :4836-42.
[0167] 27. Strair, R. K., Nelson, C. J., Mellors, J. W. Use of
recombinant retroviruses to characterize the activity of
antiretroviral compounds. J. Virol. 1991;65:63396342.
[0168] 28. Wei, X., Ghosh, S. K., Taylor, M. V., Johnson, V. A.,
Emini, E. A., Deutsh, P., Lifson, J. D., Bonhoeffer, S. Nowak, M.
A., Hahn, B. H., Saag, M. S., Shaw, G. M. (1995) Nature
373:117.
[0169] 29. Loveday, C., Kaye, S., Tenant-Flowers, M., Semple, M.,
Ayliffe, U., Weller, I. D., Tedder, R. S. (1995) Lancet
345:820-825.
[0170] 30. Ho, D. D., Neumann, A. U., Chen, W.-, Leonard, J. M.,
Markowitz, M. (1995) Nature 373:123-126.
[0171] 31. Medina, D. J., Tung, P. P., Lerner-Tung, M. B., Nelson,
C. J., Mellors, J. W., Strair, R. K. (1995) J. Virol.
69:1606-1611.
[0172] 32. Haertle, T., Carrera, C. J., Wasson, D. B., Sowers, L.
C., Richman, D. D., Carson, D. A. (1988) J. Biol. Chem.
262:5870-5875.
[0173] 33. Reed, L., and Muench, H. (1938) Am. J. Hyg.
27:493496.
[0174] 34. Antoni, B. A., Sabbatini, P., Rabson, A. B., While, E.
(1995) J. Virol. 69:2384-2392.
[0175] 35. Medina, D. J., P. P. Tung, B. Sathya, and R. K. Strair.
1996. Use of floxuridine to modulate the antiviral activity of
zidovudine. AIDS Res. Hum. Retroviruses. 12:965-968.
[0176] 36. Miller, A. D., and G. T. Rosman. 1989. Improved
retroviral vectors for gene transfer and expression. Biotechniques.
7:980-990.
[0177] 37. Willey, R. L., D. H. Smith, L. A. Lasky, T. S. Theodore,
P. L. Earl, P. Moss, D. J. Caponi, and M. A. Martin. 1988. In vitro
mutagenesis identifies a region within the envelope gene of the
human immunodeficiency virus that is critical for infectivity. J.
Virol. 62:139-147.
[0178] 38. Havlir, D., S. Eastman, and D. D. Richman. 1995. HIV-1
kinetics: Rates of production and clearance of viral populations in
asymptomatic patients treated with nevirapine. 2nd Natl. Conf. on
human Retroviruses and Related Infections. Washington D.C. January
1995. Abstract 229.
[0179] 39. Gulick, R., J. Mellors, D. Havlir, J. Eron, et al. 1996.
Safety and activity of indinavir in combination with zidovudine and
lamivudine. 3rd Natl. Conf. on Human retroviruses and related
infections. Washington D.C. January 1996.
[0180] 40. Zhang, H., O. Bagasra, M. Nikura, B. J. Poiesz, and R.
J. Pomerantz. 1994. Intravirion reverse transcripts in the
peripheral blood plasma of human immunodeficiency virus type-1
infected individuals. J. Virol. 68:7591-7597.
[0181] 41. Ahluwalia, G. A., W. Gao, H. Mitsuya, and D. G. Johns.
1996. 2',3'-didehydro-3'-deoxythymidine: Regulation of its
metabolic activation by modulators of thyrnidine-5'-triphosphate
biosynthesis. Mol. Pharm. 50:160-165.
[0182] 42. Havlir D., and D. D. Richman. 1994. Viral dynamics of
HIV: Implications for drug develpoment and therapeutic strategies.
Ann. Int. Med. 124:984-989.
[0183] It will be understood that the embodiments described herein
are merely exemplary and that a person skilled in the art may make
many variations and modifications without departing from the spirit
and scope of the invention. All such modifications and variations
are intended to be included within the scope of the invention as
defined in the appended claims.
* * * * *