Most current highly active antiretroviral therapy (HAART) regimens for HIV-positive patients contain two nucleoside reverse transcriptase inhibitors (NRTIs) with either a Protease inhibitor (PIs) or a non-nucleoside reverse transcriptase inhibitors (NNRTI). Notwithstanding the regulatory guidelines recommending therapeutic drug monitoring (TDM) for these drugs, therapeutic failure is a very serious concern implying drug induced toxicity and more importantly viral rebound and viral resistance. Single dose, steady state and dose ranging studies have all more or less demonstrated that there is a positive correlation between plasma concentrations and therapeutic effects of anti-retrovirals (ARVs). However, one of the main challenges still seems to be the target concentrations for these drugs and their relevant inhibitory quotient. In this talk, we are going to examine these issues along with bioanalytical challenges, drug-effect and drug –toxicity relationships and finally drug-drug interactions within different HAART regimes.

1. Therapeutic Drug Monitoring of
Anti-Retroviral Drugs
Track 3-1 Lecture at International Conference and Exhibition on
Analytical and Bioanalytical Techniques, Hyderabad, India
November 1-3, 2010
Dr. Bhaswat S. Chakraborty
Senior VP, Cadila Pharmaceuticals Ltd.
02.11.2010

2. Contents
• Current highly active antiretroviral therapy (HAART)
• Guidelines for administering HAART
• Correlation between plasma concentrations and therapeutic effects
• Drug –toxicity relationships
• Drug-drug interactions within different HAART regimes
• Therapeutic drug monitoring (TDM)
▫ Purpose
▫ Challenges
▫ Approaches
• Bioanalytical challenges
• Conclusions

3. ARV Drugs Darunavir
Enfuvirtide Maraviroc
• NRTI, Nucleoside reverse transcriptase inhibitor; Atazanavir Raltegravir
• NNRTI, Non-nucleoside reverse transcriptase inhibitor; Emtricitabine Tipranavir Etravirine
• PI, protease inhibitor Fosamprenavir
• Integrase Inhibitor
Tenofovir
• CCR5 Antagonist/Entry Inhibitor Nelfinavir
Delavirdine
Lopinavir/r
Ritonavir
Indinavir Amprenavir
Nevirapine Efavirenz
Abacavir
3TC
Saquinavir
ddC d4T
ddl
AZT
Source : Dr. David Back, Univ. of Liverpool

4. Main classes of ARV drugs
• Nucleoside and nucleotide reverse transcriptase inhibitors
(NRTI) inhibit reverse transcription by being incorporated into
the newly synthesized viral DNA and preventing its further
elongation.
• Non-nucleoside reverse transcriptase inhibitors (NNRTI) inhibit
reverse transcriptase directly by binding to the enzyme and
interfering with its function.
• Protease inhibitors (PIs) target viral assembly by inhibiting the
activity of protease, an enzyme used by HIV to cleave
nascent proteins for final assembly of new virons.
• Integrase inhibitors (II) inhibit the enzyme integrase, which is
responsible for integration of viral DNA into the DNA of the
infected cell.

5. Highly active antiretroviral therapy (HAART)
regimens for HIV-positive patients
• Most current HAART regimens consist of three (3) drugs: 2 NRTIs + a PI
or NNRTI or II
▫ Initial regimens use "first-line" drugs with a high efficacy and low
side-effect profile.
• Current preferred initial regimens
• Emtricitabine, tenofovir (both NRTI) and efavirenz (a NNRTI).
• Efavirenz should not be given to pregnant women.
• Emtricitabine, tenofovir and raltegravir (an II)
• Emtricitabine, tenofovir, ritonavir and darunavir (both latter are PI)
• Emtricitabine, tenofovir, ritonavir and atazanavir (both latter are PI)

6. Meaningful inhibitory concentration
• A parameter to estimate in vivo potency of antiretroviral drugs
• Cmin/IC50 is suitable for across-study, across-patient and across-drug
comparison
• ICmin is generated from in vivo pharmacokinetic data
• IC50 or IC95 are generated in vitro, increasing drug concentration
until 50% or 95% of the virus is inhibited
• How close the IC50 and IC95 values are to each other depends on how
steep the curve is (see lower graph)
• How reliable the values are depends on the system used to measure
them
• IQ (inhibitory quotient):
• IQ = trough concentration in plasma/concentration required for
inhibition in vitro
• Gives an index of how far the concentration of a drug in vivo is in
excess of the viral IC50

7. Guideline websites
Country Website
France www.sante.gouv.fr
Germany
and Austria www.rki.de/infekt/aids_std/az_eng/az_e.htm
Italy www.ministerodellasalute/aids/aids.jsp
UK www.bhiva.org
USA www.cdc.org
Netherlands www.NVAB.org

8. Hammer, S. M. et al. JAMA 2008;300:555-570.

9. Therapeutic Drug Monitoring
(DHHS Guidelines 2009)
1. When food-drug and drug-drug interactions lead to decreased efficacy
2. Pathophysiological conditions that impair GI, hepatic function, and
renal function, thereby affecting ADME
3. Treatment-experienced pts with virus with reduced susceptibility to
ARVs (higher concentrations may be required).
4. Treatment-naive pts with suboptimal virologic response
5. In pregnant women due to metabolic and physiological changes that
can affect PK
6. For prevention of ARV-induced concentration-dependent toxicity
7. When using unconventional ARV regimens or dosing not studied in
clinical trials
8. Consider in pediatric pts when there are limited dosing data

10. Recommended trough concentrations
DHHS Guidelines

11. Simulated probabilities of target trough
concentrations of Efavirenz in children
Antivir Ther. 2008;13:77987.

12. Plasma
concentrations and
viral clearance
in 4 drug therapy
Hoetelmans et al. (1998), AIDS. 12:F111-F115

13. Plasma concentrations and viral clearance
in mono & multi therapy
Lotsh et al. (2007), Antimicrob Agents Chemotherap, 51:3264–3272

14. Steady-state saquinavir plasma concentration-
versus-time profiles; n = 56
Very similar
Lotsh et al. (2007), Antimicrob Agents Chemotherap, 51:3264–3272

15. Inter-individual variation in saquinavir
plasma concentrations; n = 56
Lotsh et al. (2007), Antimicrob Agents Chemotherap, 51:3264–3272

16. ADRs vs. Saquinavir plasma concentrations;
n = 56
Log Cmax,saquinavir was predictive (P 0.001 [chi-square after logistic
regression]) of constitutional side effects such as asthenia and
sleepiness (n 7), lymphadenopathy (n 2), orthostatic dizziness (n
2), fever without infection (n 1), weight gain (n 1), peripheral
edema (n 1), and spontaneous pneumothorax (n 1) and GI side
effects.0
Lotsh et al. (2007), Antimicrob Agents Chemotherap, 51:3264–3272

17. Drug-drug interactions
New York State Department of Health AIDS Guidelines

18. Enzyme induction or inhibition
Drug Enzyme Inhibition Enzyme Induction
Atazanavir ++ —
Delavirdine ++ —
Efavirenz + +++
Fosamprenavir + ++
Indinavir ++ —
Lopinavir/ritonavir ++++ ++
Tipranavir/ritonavir ++++ +++
Nelfinavir ++ +
Nevirapine — ++
Ritonavir ++++ ++
Saquinavir — —
Source: Flexner CW. http://clinicaloptions.com/2004PK

19. TDM of antiretroviral drugs – rationale and
purpose
1. Compliance
2. ARV plasma or cell drug concentrations correlate
with antiviral effects
3. Drug concentrations also correlate with excessive
toxicity
4. High variations are present in plasma or cell drug
concentrations
5. Hepatic dysfunction changes clearance of the drug

20. TDM of antiretroviral drugs – approaches
1. Exactly knowing the target concentrations especially multi-
therapy targets
2. Four drug therapy failures do not tell you exactly what
concentrations of which drug should be changed
3. Resistant isolates may require higher drug concentrations
4. Inter- and intra-individual variability
5. Drug-drug and drug food interaction
6. Bioavalilability enhancement
1. e.g., of indinavir by ritonavir
7. Which PK parameter (AUC, Cmin Cmin/IC50)?

21. Bioanalytical Development of antiretroviral
drugs – approaches
1. Many PIs show low nanograms of levels (also do hair and blood
spots)
2. Sensitive and accurate HPLC or LC-MS-MS methods are most
suitable
3. Plasma, dried blood spots, hair, saliva or lysates of peripheral
blood mononeuclear cells (PBMCs) are the drug containing
matrices
4. Simultaneous analysis many drugs and internal standards
1. Selecting MRMs for all is challenging
5. PBMC or dried blood spot concentration for PIs or NNRTIs
would be more relevant as these drugs act intracellularly
6. Sample pre treatments (e.g., PBMC) may be required for CCs and
QCs

22. Acyclovir

23. Case study – LC-MS-MS of multi
ARVs in PBMC samples

24. Selection of IS, RT and MRMs (PBMC
samples)
Heine et al, (2009) J Chromatogr B Analyt Technol Biomed Life Sci. 877: 57580

25. LLOQ
Heine et al, (2009) J Chromatogr B Analyt Technol Biomed Life Sci. 877: 57580

26. Validation of assay
Heine et al, (2009) J Chromatogr B Analyt Technol Biomed Life Sci. 877: 57580

27. Lopinavir
Incurred (clinical)
sample analysis
Ritonavir
Atazanavir
Heine et al, (2009) J Chromatogr B Analyt Technol Biomed Life Sci. 877: 57580

28. PBMC matrix effect
• The number of PBMC cells vary significantly from sample to
sample due to
▫ Natural variation in systemic circulation
▫ Variation in cell recovery
• Therfore, investigation of PBMC ME is required
Heine et al, (2009) J Chromatogr B Analyt Technol Biomed Life Sci. 877: 57580

29. Conclusions

30. Thank You Very Much