Characterization of Genotypic and Phenotypic Assays
Well-characterized genotypic and phenotypic assays can provide the basis for the analysis of the emergence of resistant virus during the development of investigational drugs. Phenotypic and genotypic assays used in clinical practice need more extensive validation than exploratory assays used for the characterization of antiviral activity and/or the resistance profile of the investigational drug. Commercially available assays that are routinely used should be identified, but it may not be necessary to provide the performance characteristics. The amount and nature of validation necessary for an assay and mechanisms of submitting assay performance characteristics should be discussed with the division --Antiretroviral Drugs Using Plasma HIV RNA Measurements
1. Genotypic Assays
The performance characteristics of genotypic assays should be described, including elaboration of the following characteristics:
• Minimum plasma viral RNA level with a standard isolate to assess PCR sensitivity
• Purification methodology for viral nucleic acids
• Amplification methodology and primers
• PCR controls
• Clade differences
• Nucleic acid sequencing methodology
• Description of sequencing primers
• Range of mutant and WT ratios detectable
• Interpretation criteria for mutant scoring
The entire coding sequence of the gene for the target protein should be determined in the early stages of resistant variants analysis. Once the mutations leading to resistance are identified, only the relevant portions of the genome need to be sequenced. The pattern of mutations leading to resistance of an investigational drug should be documented and compared with the pattern of mutations of other drugs in the same class.
Reporting the details of methodologies is important. Sponsors should identify sequencing primers and state how many bases from them can be read accurately. Sponsors also should define the sensitivity of the genotypic assay used for detecting minority viral subpopulations.
2. Phenotypic Assays
The performance characteristics (accuracy, precision, limits of detection and quantification, specificity, linearity, range, robustness, stability) of an investigational phenotypic assay should be well documented. The sources of viruses (e.g., blood, plasma), their storage and stability, and cell culture procedures should be described. For definitions on assay validation, refer to Bioanalytical Method Validation. Sponsors are encouraged to use a previously characterized and validated assay.
The utility of a phenotypic assay will depend upon its sensitivity (i.e., its ability to measure shifts in susceptibility (fold-changes) in comparison to baseline clinical isolates) and on the drug concentrations achieved. Shifts in susceptibility for a clinical isolate are measured by determining the EC50 values for the isolate and a WT standard virus done under the same conditions and at the same time. Simultaneous testing provides for absolute comparisons between assays. Readout of phenotypic assays can be detected with any standard virus assay, such as p24, viral RNA, RT assay, MTT cytotoxic assay, and reporter gene expression.
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.
Source:
Drug Resistance Assay and Antiretroviral drug development guidance--->
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm071173.pdf
------------------------------------------------------------------------------------------------------------
This website provideds complete information about hiv virus , hiv infection , early hiv symptoms , hiv treatment,hiv test and hiv testing centers, aids symptoms, symptoms of hiv immideately 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
Last update : 28-Oct-2010
Well-characterized genotypic and phenotypic assays can provide the basis for the analysis of the emergence of resistant virus during the development of investigational drugs. Phenotypic and genotypic assays used in clinical practice need more extensive validation than exploratory assays used for the characterization of antiviral activity and/or the resistance profile of the investigational drug. Commercially available assays that are routinely used should be identified, but it may not be necessary to provide the performance characteristics. The amount and nature of validation necessary for an assay and mechanisms of submitting assay performance characteristics should be discussed with the division --Antiretroviral Drugs Using Plasma HIV RNA Measurements
1. Genotypic Assays
The performance characteristics of genotypic assays should be described, including elaboration of the following characteristics:
• Minimum plasma viral RNA level with a standard isolate to assess PCR sensitivity
• Purification methodology for viral nucleic acids
• Amplification methodology and primers
• PCR controls
• Clade differences
• Nucleic acid sequencing methodology
• Description of sequencing primers
• Range of mutant and WT ratios detectable
• Interpretation criteria for mutant scoring
The entire coding sequence of the gene for the target protein should be determined in the early stages of resistant variants analysis. Once the mutations leading to resistance are identified, only the relevant portions of the genome need to be sequenced. The pattern of mutations leading to resistance of an investigational drug should be documented and compared with the pattern of mutations of other drugs in the same class.
Reporting the details of methodologies is important. Sponsors should identify sequencing primers and state how many bases from them can be read accurately. Sponsors also should define the sensitivity of the genotypic assay used for detecting minority viral subpopulations.
2. Phenotypic Assays
The performance characteristics (accuracy, precision, limits of detection and quantification, specificity, linearity, range, robustness, stability) of an investigational phenotypic assay should be well documented. The sources of viruses (e.g., blood, plasma), their storage and stability, and cell culture procedures should be described. For definitions on assay validation, refer to Bioanalytical Method Validation. Sponsors are encouraged to use a previously characterized and validated assay.
The utility of a phenotypic assay will depend upon its sensitivity (i.e., its ability to measure shifts in susceptibility (fold-changes) in comparison to baseline clinical isolates) and on the drug concentrations achieved. Shifts in susceptibility for a clinical isolate are measured by determining the EC50 values for the isolate and a WT standard virus done under the same conditions and at the same time. Simultaneous testing provides for absolute comparisons between assays. Readout of phenotypic assays can be detected with any standard virus assay, such as p24, viral RNA, RT assay, MTT cytotoxic assay, and reporter gene expression.
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.
Source:
Drug Resistance Assay and Antiretroviral drug development guidance--->
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm071173.pdf
------------------------------------------------------------------------------------------------------------
This website provideds complete information about hiv virus , hiv infection , early hiv symptoms , hiv treatment,hiv test and hiv testing centers, aids symptoms, symptoms of hiv immideately 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
Last update : 28-Oct-2010
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