Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-06-12
2003-11-25
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S005000, C435S008000, C435S091100, C435S091200, C435S069100, C435S235100, C435S238000, C435S441000, C435S442000, C424S188100
Reexamination Certificate
active
06653081
ABSTRACT:
Throughout this application, various publications are referenced by author and date within the text. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims. All patents, patent applications and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
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 less susceptible (“resistant”) to a particular antiretroviral drug regimen. This invention also relates to the screening of candidate antiretroviral drugs for their capacity to inhibit viruses, selected viral sequences and/or viral proteins. More particularly, this invention relates to using phenotypic susceptibility tests and/or genotypic tests to identify patients whose virus/viruses exhibit drug-dependent stimulation of replication in the presence of anti-retroviral agents.
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) (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
10
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
5
mutations per day at each position in the HIV genome. The result is the rapid establishment of extensive genotypic variation. While some template positions 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 Chemother
38, 268-274), mathematical modeling suggests that, at every nucleotide position, mutation may occur 10
4
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 changes 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, 1479-1488) (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 wildtype 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.
A mutation or mutations that results in virus that can not only replicate in the presence of drug (i.e. resistant virus) but could actually replicate more efficiently in the presence of drug than in the absence of drug (i.e. drug-dependent stimulation of virus), would present an especially important phenotype to identify. In this case, a drug could actually accelerate the rate of destruction to the immune system and progression of disease.
Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a chemically diverse group of compounds which are potent inhibitors of HIV-1 Reverse Transcriptase (RT) in vitro. These compounds include pyridinone derivatives, bis(heteroaryl) piperazines (BHAPs) such as delavirdine and atevirdine, the dipyridodiazepinone (nevirapine), the thymine derivative groups (TSAO and HEPT), an a-anilino phenylacetamide (a-APA) compound (loviride), the quinoxaline-class inhibitors (HBY-097), the benzodiazepin-one and -thione (TIBO) compounds, and the pyridinone derivatives (L-697,661). For overviews, see (DeClercq E. (1996)
Rev Med Virol
6, 97-117) (Emini E A (1996)
Antiviral Drug Resistance,
ed. D D Richman, John Wiley & Sons, Ltd). Three NNRTIs: nevirapine (NVP, Viramune, Boehringer Ingelheim, Ingelheim am Rhein, Germany), delavirdine (DLV, Rescriptor, Pharmacia & Upjohn, Kalamazoo, Mich., USA), and efavirenz (EFV, Sustiva, Dupont, Wilmington, Del., USA) are licensed for use in the USA.
High-level resistance to individual compounds appears to develop rapidly, often within a few weeks of initiating monotherapy, frequently involving only single-point mutations, and in many cases leading to considerable cross-resistance to other NNRTIs. Most mutations reported occur in the codon groups 100-108 and 181-190 which encode for the two b-sheets adjacent to the catalytic site of the RT enzyme (Kohlstaedt L A, Wang J, Friedman J M, et al. (1992)
Science
256, 1783-90). The NNRTI binding pocket, as it has been described, is a hydrophobic non-substrate binding region of RT where these agents directly interact with RT. They inhibit activity by interfering with mobility of the ‘thumb’ subdomain, or disrupting the orientation of conserved aspartic acid side chains essential for catalytic activity (D'Aquilla R T. (1994)
Clin Lab Med
14, 393-423) (Arnold E., Ding J., Hughes S H, et al. (1995)
Curr Opin Struct Biol
5, 27-38).
Mutations conferring reduced susceptibility to nevirapine have been described at HIV RT codons 98, 100, 103, 106, 108, 181, 188 and 190 (Richman D D, Havlir D, Corbeil J. (1994)
J Virol
68, 1660-1666). The most frequently selected variant during nevirapine monotherapy is a Tyr
181
àCys (Y181C) mutation, which results in a 100-fold reduction in sensitivity to this agent, and with reduced susceptibility to the pyridinone derivatives (L-696,229 and L-697,661) (Arnold, Ibid). TSAO also has limited activity in the presence of the Y181C mutation, but maintains activity in the presence of mutations HIV RT at codons 100 and 103, and in vitro selects for a unique mutation, GLU
138
àLys (E138K), in the region where it most closely interacts with RT (Richman, D D, Ibid)
Housel James
Li Bao Qun
Pennie & Edmonds LLP
ViroLogic, Inc.
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