Selection of Drug-Resistant HIV-1 Variants in Cell Culture
Selection of resistant viruses in cell culture may indicate whether development of resistance to a drug is likely to involve a few (1 to 2) or several (more than 2) mutations. The ability of an investigational drug to select HIV-1 variants with reduced drug susceptibility (phenotypic resistance) should be determined in cell culture systems. Selection of variants resistant to the investigational drug should be repeated multiple times (e.g., with different strains of WT, with resistant strains, under high and low selective pressure) to determine if the same or different patterns of resistance mutations develop.
Two basic methods have been developed to identify mutations conferring a reduction in susceptibility to a drug.
1. In the first method, a high initial virus inoculum is propagated for several passages at a fixed drug concentration, using multiple cultures to test different concentrations.
2. In the second method, a low initial inoculum of virus is passaged in the presence of increasing drug concentrations starting near the EC50 value for the parental virus.
Virus production is monitored to detect the outgrowth of resistant virus by characterizing intermediate and ending isolates with respect to genotype and phenotype.
1. Genotype
Mutations responsible for reductions in susceptibility to a drug can be identified by DNA sequence analysis of the relevant portions of the virus genome. The complete coding sequence of the gene for the target protein should be determined in the early stages of characterization of mutations associated with reduced drug susceptibility. Once mutations are identified, their ability to confer phenotypic resistance should be evaluated in a recombinant virus system (e.g., by using site-directed mutagenesis or polymerase chain reaction (PCR) amplification of relevant portions of the virus genome to introduce these mutations into a standard laboratory HIV genetic background) or other suitable system, such that the mutations necessary to reproduce the resistant phenotype are identified. If site-directed mutagenesis experiments within the target gene fail to recapitulate the resistance phenotype, then the potential effects of mutations elsewhere in the viral genome should be examined. In the case of studying mutations in the envelope gene, which is highly variable, a possible option is to introduce mutations into the parental envelope to assess their contribution to the resistance phenotype. Recombinant virus should then be tested for drug susceptibility in cell culture. Shifts in drug susceptibility (fold-increases in EC50 value) for recombinant virus relative to WT should be determined (see section IV.G., Characterization of Genotypic and Phenotypic Assays).
2. Phenotype
Drug susceptibility (EC50 values) for resistant variants and the fold change in EC50 values relative to the parent virus should be determined (see section IV.G., Characterization of Phenotypic and Genotypic Assays).
A number of drugs targeting gp120, gp41, and the CCR5 and CXCR4 chemokine co-receptors are in development. A potential concern with CCR5 inhibitors is that loss of effectiveness may develop by the virus switching to the CXCR4 co-receptor. The evolution of HIV to a CXCR4-utilizing virus has been proposed to result in a more virulent virus. Therefore, sponsors should monitor co-receptor usage in cell culture drug selection experiments and in clinical trials evaluating drugs targeting the co-receptors gp120 and gp41.----Ad-
Source:
Drug Resistance Assay and Antiretroviral drug development guidance--->
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm071173.pdf
IV. NONCLINICAL STUDIES
A. Mechanism of Action
B. Antiviral Activity in Cell Culture
C. Cytotoxicity and Therapeutic Indexes
D. Protein Binding
E. Selection of Drug-Resistant HIV-1 Variants in Cell Culture
1. Genotype
2. Phenotype
F. Cross-Resistance
G. Characterization of Genotypic and Phenotypic Assays
1. Genotypic Assays
2. Phenotypic Assays
V. CLINICAL STUDIES: USE OF RESISTANCE TESTING IN CLINICAL PHASES OF DRUG DEVELOPMENT
A. General Considerations
B. Data Collection
C. Methods and Types of Analyses
1. Baseline Genotype and Virologic Response
2. Development of HIV Mutations
3. Baseline Phenotype and Virologic Response
4. Genotypic and Phenotypic Correlations: Changes in Susceptibility from Baseline
5. Cross-Resistance
6. Additional Analyses
D. Other Considerations
1. Role for Supporting Initial Activity and Dose-Finding Studies
2. Data Collection from Dose-Finding Trials
3. Use of Resistance Data to Establish an Indication
4. Use of Resistance Data for Study Enrollment Criteria, Background Regimen Selection, and Stratification Factors
5. Nonclade B Subtypes
E. Monitoring during Phase 4
REGULATORY REQUIREMENTS FOR SUBMITTING HIV RESISTANCE DATA
SUMMARY
There is much more information on HIV virus new findings available on this blog.
Here are given details about symptoms of AIDS , all new symptoms of AIDS and HIV virus infection are updated.
------------------------------------------------------------------------------------------------------------
This website provides complete information about hiv virus , hiv infection , early hiv symptoms , hiv treatment,hiv test and hiv testing centers, aids symptoms, symptoms of hiv immediately after infection.
------------------------------------------------------------------------------------------------------------
Following are the most read topic on this website you should find them on blog archive of this website
Stem cell treatment for AIDS
AIDS symptoms
SYMPTOMS OF HIV
HIV infection symptoms
Latest treatment for AIDS
How anti retroviral medicines are developed
How Chemotherapeutic drugs are developed to treat HIV virus infection
New drug developments against HIV Virus AIDS
Vaccines research for HIV AIDS Why it is difficult to develop a vaccine against HIV
Mother to child prevention of HIV virus infection.
HIV virus infection prevention and education bloggers
Information about HIV Virus AIDS symptoms
Last updated : 28-Oct-2010
Selection of resistant viruses in cell culture may indicate whether development of resistance to a drug is likely to involve a few (1 to 2) or several (more than 2) mutations. The ability of an investigational drug to select HIV-1 variants with reduced drug susceptibility (phenotypic resistance) should be determined in cell culture systems. Selection of variants resistant to the investigational drug should be repeated multiple times (e.g., with different strains of WT, with resistant strains, under high and low selective pressure) to determine if the same or different patterns of resistance mutations develop.
Two basic methods have been developed to identify mutations conferring a reduction in susceptibility to a drug.
1. In the first method, a high initial virus inoculum is propagated for several passages at a fixed drug concentration, using multiple cultures to test different concentrations.
2. In the second method, a low initial inoculum of virus is passaged in the presence of increasing drug concentrations starting near the EC50 value for the parental virus.
Virus production is monitored to detect the outgrowth of resistant virus by characterizing intermediate and ending isolates with respect to genotype and phenotype.
1. Genotype
Mutations responsible for reductions in susceptibility to a drug can be identified by DNA sequence analysis of the relevant portions of the virus genome. The complete coding sequence of the gene for the target protein should be determined in the early stages of characterization of mutations associated with reduced drug susceptibility. Once mutations are identified, their ability to confer phenotypic resistance should be evaluated in a recombinant virus system (e.g., by using site-directed mutagenesis or polymerase chain reaction (PCR) amplification of relevant portions of the virus genome to introduce these mutations into a standard laboratory HIV genetic background) or other suitable system, such that the mutations necessary to reproduce the resistant phenotype are identified. If site-directed mutagenesis experiments within the target gene fail to recapitulate the resistance phenotype, then the potential effects of mutations elsewhere in the viral genome should be examined. In the case of studying mutations in the envelope gene, which is highly variable, a possible option is to introduce mutations into the parental envelope to assess their contribution to the resistance phenotype. Recombinant virus should then be tested for drug susceptibility in cell culture. Shifts in drug susceptibility (fold-increases in EC50 value) for recombinant virus relative to WT should be determined (see section IV.G., Characterization of Genotypic and Phenotypic Assays).
2. Phenotype
Drug susceptibility (EC50 values) for resistant variants and the fold change in EC50 values relative to the parent virus should be determined (see section IV.G., Characterization of Phenotypic and Genotypic Assays).
A number of drugs targeting gp120, gp41, and the CCR5 and CXCR4 chemokine co-receptors are in development. A potential concern with CCR5 inhibitors is that loss of effectiveness may develop by the virus switching to the CXCR4 co-receptor. The evolution of HIV to a CXCR4-utilizing virus has been proposed to result in a more virulent virus. Therefore, sponsors should monitor co-receptor usage in cell culture drug selection experiments and in clinical trials evaluating drugs targeting the co-receptors gp120 and gp41.----Ad-
Source:
Drug Resistance Assay and Antiretroviral drug development guidance--->
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm071173.pdf
IV. NONCLINICAL STUDIES
A. Mechanism of Action
B. Antiviral Activity in Cell Culture
C. Cytotoxicity and Therapeutic Indexes
D. Protein Binding
E. Selection of Drug-Resistant HIV-1 Variants in Cell Culture
1. Genotype
2. Phenotype
F. Cross-Resistance
G. Characterization of Genotypic and Phenotypic Assays
1. Genotypic Assays
2. Phenotypic Assays
V. CLINICAL STUDIES: USE OF RESISTANCE TESTING IN CLINICAL PHASES OF DRUG DEVELOPMENT
A. General Considerations
B. Data Collection
C. Methods and Types of Analyses
1. Baseline Genotype and Virologic Response
2. Development of HIV Mutations
3. Baseline Phenotype and Virologic Response
4. Genotypic and Phenotypic Correlations: Changes in Susceptibility from Baseline
5. Cross-Resistance
6. Additional Analyses
D. Other Considerations
1. Role for Supporting Initial Activity and Dose-Finding Studies
2. Data Collection from Dose-Finding Trials
3. Use of Resistance Data to Establish an Indication
4. Use of Resistance Data for Study Enrollment Criteria, Background Regimen Selection, and Stratification Factors
5. Nonclade B Subtypes
E. Monitoring during Phase 4
REGULATORY REQUIREMENTS FOR SUBMITTING HIV RESISTANCE DATA
SUMMARY
There is much more information on HIV virus new findings available on this blog.
Here are given details about symptoms of AIDS , all new symptoms of AIDS and HIV virus infection are updated.
------------------------------------------------------------------------------------------------------------
This website provides complete information about hiv virus , hiv infection , early hiv symptoms , hiv treatment,hiv test and hiv testing centers, aids symptoms, symptoms of hiv immediately after infection.
------------------------------------------------------------------------------------------------------------
Following are the most read topic on this website you should find them on blog archive of this website
Stem cell treatment for AIDS
AIDS symptoms
SYMPTOMS OF HIV
HIV infection symptoms
Latest treatment for AIDS
How anti retroviral medicines are developed
How Chemotherapeutic drugs are developed to treat HIV virus infection
New drug developments against HIV Virus AIDS
Vaccines research for HIV AIDS Why it is difficult to develop a vaccine against HIV
Mother to child prevention of HIV virus infection.
HIV virus infection prevention and education bloggers
Information about HIV Virus AIDS symptoms
Last updated : 28-Oct-2010
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