Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-06-23
2002-12-03
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091330, C514S045000, C514S049000, C514S050000, C536S024300
Reexamination Certificate
active
06489098
ABSTRACT:
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
TECHNICAL FIELD
This invention relates to antiretroviral drug susceptibility and resistance tests to be used in identifying effective drug regimens for the treatment of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). The invention further relates to the means and methods of monitoring the clinical progression of HIV infection and its response to antiretroviral therapy using phenotypic or genotypic susceptibility assays. The Invention also relates to novel vectors, host cells and compositions for carrying out phenotypic susceptibility tests. The invention further relates to the use of various genotypic methodologies to identify patients whose Infection has become resistant to a particular antiretroviral drug regimen. This invention also relates to the screening of candidate antiretroviral drugs for their capacity to inhabit viruses, selected viral sequences and/or viral proteins. More particularly, this invention relates to the determination of nucleoside reverse transcriptase inhibitor resistance using phenotypic susceptibility tests and/or genotypic tests.
BACKGROUND OF THE INVENTION
HIV infection is characterized by high rates of viral turnover throughout the disease process, eventually leading to CD4 depletion and disease progression. Wei X, Ghosh S K, Taylor M E, et al. (1995)
Nature
343, 117-122 and Ho D D, Naumann A U, Perelson A S, et al. (1995)
Nature
373, 123-126. The aim of antiretroviral therapy is to achieve substantial and prolonged suppression of viral replication. Achieving sustained viral control is likely to involve the use of sequential therapies, generally each therapy comprising combinations of three or more antiretroviral drugs. Choice of initial and subsequent therapy should, therefore, be made on a rational basis, with knowledge of resistance and cross-resistance patterns being vital to guiding those decisions. The primary rationale of combination therapy relates to synergistic or additive activity to achieve greater inhibition of viral replication. The tolerability of drug regimens will remain critical, however, as therapy will need to be maintained over many years.
In an untreated patient, some 10
0
new viral particles are produced per day. Coupled with the failure of HIV reverse transcriptase (RT) to correct transcription errors by exonucleolytic proofreading, this high level of viral turnover results in 10
4
to 10
3
mutations per day at each position in the HIV genome. The result is the rapid establishment of extensive genotypic variation. While some template positions or base pair substitutions may be more error prone (Mansky L M, Temin H M (1995)
J Virol
69, 5087-5094) (Schinazi R F, Lloyd R M, Ramanathan C S, et al. (1994)
Antimicrob Agents Chemoter
38, 268-274), mathematical modeling suggests that, at every possible single point, mutation may occur up to 10,000 times per day in infected individuals.
For antiretroviral drug resistance to occur, the target enzyme must be modified while preserving its function in the presence of the inhibitor. Point mutations leading to an amino acid substitution may result in change in shape, size or charge of the active site, substrate binding site or surrounding regions of the enzyme. Mutants resistant to antiretroviral agents have been detected at low levels before the initiation of therapy. (Mohri H, Singh M K, Ching W T W, et al. (1993)
Proc Natl Acad Sci USA
90, 25-29) (Nájera I, Richman D D, Olivares I, et al. (1994)
AIDS Res Hum Retroviruses
10, 479-1488) (Nájera I, Holguin A, Quiñones-Mateu E, et al. (1995)
J Virol
69, 23-31). However, these mutant strains represent only a small proportion of the total viral load and may have a replication or competitive disadvantage compared with wild-type virus. (Coffin J M (1995)
Science
267, 483-489). The selective pressure of antiretroviral therapy provides these drug-resistant mutants with a competitive advantage and thus they come to represent the dominant quasispecies (Frost S D W, McLean A R (1994)
AIDS
8, 323-332) (Kellam P, Boucher C A B, Tijnagal J M G H (1994)
J Gen Virol
75, 341-351) ultimately leading to drug resistance and virologic failure in the patient.
Nucleoside Reverse Transcriptase Inhibitors
Seven nucleoside analogue HIV reverse transcriptase Inhibitors (zidovudine (ZVD: Retrovir, Glaxo Wellcome, Uxbridge, UK), zalcitabine (ddC: HIVID, Hoffman-LaRoche, Basle, Switzerland), didanosine (ddI: Videx, Bristol-Myers Squibb, Syracuse, N.Y., USA), stavudine (d4T: Zerit, Bristol-Myers Squibb, Syracuse, N.Y., USA), and lamivudine (3TC, Epivir), abacavir (ABC, Ziagen, Glaxo Wellcome), and adefovir (ADV, Preveon, Gilead Sciences) are currently licensed in Europe and the USA. Additionally, three NNRTIs, nevirapine (Viramune, Boehringer Ingelheim, Ingelheim am Rhein, Germany) and delavirdine (Rescriptor, Pharmacia & Upjohn, Kalamazoo, Mich., USA) and efavirenz (EPV,) are licensed in the USA. All these agents have demonstrated at least short-term antiviral activity and, therefore, it is not surprising that, as they exert a selective pressure on HIV, drug-resistant mutants arise during therapy. Whilst these drugs are normally used in combination regimens, many of the available resistance data arise from phase I/II monotherapy studies. Mutations observed during monotherapy may not accurately reflect mutations responsible for resistance that develops in the presence of pressure from several agents acting at the same site and, hence, on the same gene.
Novel Mutations
Whilst patterns of genotypic mutations associated with changes in phenotypic resistance to the leading reverse transcriptase inhibitors (RTIs) are established from both in-vitro and in-vivo work, other, rarely reported, resistance mutations may arise occasionally during clinical studies. Isolates with a unique pattern of amino acid substitutions at codons 62, 75, 77, 116, and 115 have been identified in patients receiving prolonged combination therapy with ZDV plus ddI or ddC: these isolates are resistant to both drugs and there is cross-resistance to stavudine and partial cross-resistance to 3TC. No consistent genotypic change has been associated with phenotypic d4T resistance or, indeed, loss of virological effect of this compound.
Mutations to Nucleoside Analogue RT Inhibitors Zidovudine
HIV variants with decreased susceptibility to ZDV were first reported in 1989; in some isolates, greater than 100-fold increases in the concentration of ZDV were required to inhibit viral replication by 50% (Larder B A, Darby G, Richman D D (1989)
Science
243, 1731-1734). The ZDV-resistant phenotype appears to be reasonably stable in vivo, with resistant virus sometimes being detected up to 1 year after cessation of therapy, (Boucher C A, O'Sullivan E, Mulder J W et al. (1992)
J Infect Disease
165, 105-110) and despite treatment with didanosine (Smith M S, Koerber K L, Pagano J S, (1994)
J Infect Disease
169, 184-188).
Nucleotide sequencing of HIV RT has revealed a number of mutations which can influence viral sensitivity to ZDV and which may be used as genotypic markers for the presence of ZDV resistance (Kellam P, Boucher C A B, Tijnagal J M G H et al. (1994)
J Gen Virol
75, 341-351) (Boucher C A B, Tersmette M, Lange J M A, et al. (1990)
Lancet
336, 585-590) (Lopez-Galindez C, Rojas J M, Najera R, et al. (1991)
PNAS
88, 4280-4284). A range of mutants with increasing levels of resistance appear in an ordered manner, with the sequential appearance of these mutations being associated with incremental reductions in viral sensitivity to ZDV (id) (Larder B A, Kellam P, Kemp S D, (1991)
AIDS
5, 137-144). A substitution at codon 70 (Arg70→Lys) may be transiently dominant and appears critical to virological failure during ZDV monotherapy (DeJong M D, Veenstra J, St
Petropoulos Christos J.
Whitcomb Jeannette
Cooper & Dunham LLP
Stucker Jeffrey
ViroLogic, Inc.
White John P.
Winkler Ulrike
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