Age, Inflammation, Blood Brain Barrier Permeability and Single Nucleotide
Polymorphisms in Transporters May Influence Cerebrospinal Fluid Antiretrovirals’
Concentrations
Calcagno A, Cusato J, Scarvaglieri E, Audagnotto S, De Nicolò A, Imperiale D, Mighetto L, D’Avolio A, Di Perri G, Bonora S.
19th International Workshop on Clinical Pharmacology of Antiviral Therapy, 22 - 24 May 2018, Baltimore, Maryland, USA
Potential relevance of CNS exposure of ARVs
Nightingale S, et al. Lancet Neurol 2014; Estes J, et al. Nature Med 2017
What did we learn in the past two years?
What did we learn in the past two years?
Curley P et al, AAC 2017; Thompson CG, et al. AAC 2015; Srinivas et al, IAS 2017, Abstract WEAB0105; Fletcher
CV, et al. PNAS 2014
1. Cerebrospinal fluid is a poor predictor of brain concentrations of different drugs• EFV Brain PK 3.7-12.7 times higher than plasma in a
macaque• EFV Brain PK to plasma ratios 9.5 (rodents) - 15.8 (PBPK
modelling) 2. CSF concentrations correlate with brain concentrations
• Individual PK in macaques3. Inflammation is associated with the induction of several
transporters involved in drug distribution• SIV infected animals have higher expression of P-gp and
BCRP4. Methods matter
• Tissue homogenate vs. mononuclear cells
What did we learn in the past two years?
Curley P et al, AAC 2017; Thompson CG, et al. AAC 2015; Srinivas et al, IAS 2017, Abstract WEAB0105; Fletcher
CV, et al. PNAS 2014
1. Cerebrospinal fluid is a poor predictor of brain concentrations of different drugs• EFV Brain PK 3.7-12.7 times higher than plasma in a
macaque• EFV Brain PK to plasma ratios 9.5 (rodents) - 15.8 (PBPK
modelling) 2. CSF concentrations correlate with brain concentrations
• Individual PK in macaques3. Inflammation is associated with the induction of several
transporters involved in drug distribution• SIV infected animals have higher expression of P-gp and
BCRP4. Methods matter
• Tissue homogenate vs. mononuclear cells
AcknowledgementsWe acknowledge the following funding sources: RO1 GM66940, RO1GM09697, AFPE pre-doctoral fellowship and the Royster Society ofFellows
SHIV Infection and Drug Transporters Influences Brain Tissue Concentrations of EfavirenzN. Srinivas1, J. Fallon1, C. Sykes1, N. White1, A. Schauer1, Michelle Matthews1, L. Adamson2, P. Luciw2, P. Smith1, A.D.M. Kashuba1
1University of North Carolina, Chapel Hill, United States, 2University of California at Davis, Davis, United States,
• Despite antiretroviral (ARV) therapy, there is a high prevalenceof HIV-associated neurocognitive disorder (HAND) in HIV-infected individuals1.
• It has been theorized that inadequate ARV concentrations maycontribute to the persistence of HAND2. However, there is noconclusive relationship between ARV concentration in the CSFand HAND3,4. Concentration of ARVs in the brain may be amore accurate pharmacokinetic measure but the data on braintissue concentrations is sparse.
• This study compared the concentration of ARVs in four regionsof brain tissue with CSF in uninfected and SHIV-infected rhesusmacaques and investigated the effect of drug transporters oninfluencing ARV concentrations in brain tissue
Background
MethodsConcentration of ARVs in brain tissue and CSF• In 12 male macaques (6 uninfected and 6 SHIV-infected) dosed
to steady-state, concentrations of 6 ARVs –tenofovir (TFV),emtricitabine (FTC), efavirenz (EFV), raltegravir (RAL), maraviroc(MVC) and atazanavir (ATZ) were measured by LC-MS/MS in theCSF, where LLOQ was 0.5 ng/mL and in the frontal cortex,cerebellum, basal ganglia and parietal cortex regions of the brainwhere LLOQ of homogenate ranged from 0.002-0.01 ng/mL.
• Tissue concentrations were converted to ng/g using density of1.06.
Concentration of BCRP and P-gp in brain tissue• To assess the influence of drug transporters on ARV
concentration, protein was extracted from the brain tissue andanalyzed for Pgp and BCRP efflux transporter concentration byLCMS/MS proteomics method called Quantitative TargetedAbsolute Proteomics (QTAP)5. The LLOQ was 0.1 pMol/mgprotein).
Data Analysis• Statistical analysis was performed by Kruskal-Wallis test using
Sigmaplot®
Results
Conclusions and future directions
• Antiretroviral concentrations in brain tissue were significantly greater than CSF for TFV, FTC and EFV (Figure 1): ranging from 5-times (FTC)to 769-times (EFV) higher. Brain tissue concentration of EFV was 4.1 times higher in uninfected animals. Brain tissue concentrations ofefavirenz correlated with CSF over a range of concentrations (Figure 2).
• BCRP concentration (Figure 3) was 1.7 times higher in infected animals (p=0.02) while Pgp concentration did not differ with infection status(p=0.06). There was a trend noted for correlation between concentration of BCRP transporter and efavirenz in brain tissue (Figure 4).
• CSF concentrations need to be interpreted cautiously as surrogate for brain tissue concentration for several ARVs. Further, increase in BCRPconcentration may lead to lower EFV concentration in infected rhesus macaques.
• In future analyses, additional animals will be evaluated, and unbound concentration of EFV in brain tissue will be quantified since EFVexhibits a high degree of non-specific binding (>99.5%) in the brain tissue. Further investigations are needed to determine the influence ofbrain tissue concentrations on HAND
References1. Marra CM. HIV-associated neurocognitive disorders and central nervous system drug penetration: what next? AntivirTher. 2015. doi:10.3851/IMP2951.
2. Decloedt EH, Rosenkranz B, Maartens G, Joska J. Central Nervous System Penetration of Antiretroviral Drugs:Pharmacokinetic, Phharmacodynamic and Pharmacogenomic Considerations. Clin Pharmacokinet. 2015 Jun;54(6):581-98
3. Caniglia EC, Cain LE, Justice A, et al. Antiretroviral penetration into the CNS and incidence of AIDS-defining neurologicconditions. Neurology. 2014;83(2):134-141.
4. Scott Lentendre. Randomized Clinical Trial of Antiretroviral Therapy for Prevention of Hand (Oral Presentation). In: CROI2015..
5. Uchida Y et al. A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application forinter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at theblood–brain barrier in ddY, FVB, and C57BL/6J mice. Fluids Barriers CNS. 2013; 10: 21
Figure 1. Concentrations of Tenofovir, Emtricitabine, Efavirenz, Raltegravir,
Maraviroc and Atazanavir in brain tissue and CSF of rhesus macaquesFigure 3. Concentration of BCRP and P-gp transporter
proteins in brain tissue of rhesus macaques by QTAP
Figure 4.
Correlation
analysis between
brain tissue
concentration of
antiretroviral and
concentration of
BCRP transporter
protein in rhesus
macaques
Figure 2. Correlation between brain tissue and CSF
concentrations of Antiretrovirals
What did we learn in the past two years?
Curley P et al, AAC 2017; Thompson CG, et al. AAC 2015; Srinivas et al, IAS 2017, Abstract WEAB0105; Fletcher
CV, et al. PNAS 2014
1. Cerebrospinal fluid is a poor predictor of brain concentrations of different drugs• EFV Brain PK 3.7-12.7 times higher than plasma in a
macaque• EFV Brain PK to plasma ratios 9.5 (rodents) - 15.8 (PBPK
modelling) 2. CSF concentrations correlate with brain concentrations
• Individual PK in macaques3. Inflammation is associated with the induction of several
transporters involved in drug distribution• SIV infected animals have higher expression of P-gp and
BCRP4. Methods matter
• Tissue homogenate vs. mononuclear cells
AcknowledgementsWe acknowledge the following funding sources: RO1 GM66940, RO1GM09697, AFPE pre-doctoral fellowship and the Royster Society ofFellows
SHIV Infection and Drug Transporters Influences Brain Tissue Concentrations of EfavirenzN. Srinivas1, J. Fallon1, C. Sykes1, N. White1, A. Schauer1, Michelle Matthews1, L. Adamson2, P. Luciw2, P. Smith1, A.D.M. Kashuba1
1University of North Carolina, Chapel Hill, United States, 2University of California at Davis, Davis, United States,
• Despite antiretroviral (ARV) therapy, there is a high prevalenceof HIV-associated neurocognitive disorder (HAND) in HIV-infected individuals1.
• It has been theorized that inadequate ARV concentrations maycontribute to the persistence of HAND2. However, there is noconclusive relationship between ARV concentration in the CSFand HAND3,4. Concentration of ARVs in the brain may be amore accurate pharmacokinetic measure but the data on braintissue concentrations is sparse.
• This study compared the concentration of ARVs in four regionsof brain tissue with CSF in uninfected and SHIV-infected rhesusmacaques and investigated the effect of drug transporters oninfluencing ARV concentrations in brain tissue
Background
MethodsConcentration of ARVs in brain tissue and CSF• In 12 male macaques (6 uninfected and 6 SHIV-infected) dosed
to steady-state, concentrations of 6 ARVs –tenofovir (TFV),emtricitabine (FTC), efavirenz (EFV), raltegravir (RAL), maraviroc(MVC) and atazanavir (ATZ) were measured by LC-MS/MS in theCSF, where LLOQ was 0.5 ng/mL and in the frontal cortex,cerebellum, basal ganglia and parietal cortex regions of the brainwhere LLOQ of homogenate ranged from 0.002-0.01 ng/mL.
• Tissue concentrations were converted to ng/g using density of1.06.
Concentration of BCRP and P-gp in brain tissue• To assess the influence of drug transporters on ARV
concentration, protein was extracted from the brain tissue andanalyzed for Pgp and BCRP efflux transporter concentration byLCMS/MS proteomics method called Quantitative TargetedAbsolute Proteomics (QTAP)5. The LLOQ was 0.1 pMol/mgprotein).
Data Analysis• Statistical analysis was performed by Kruskal-Wallis test using
Sigmaplot®
Results
Conclusions and future directions
• Antiretroviral concentrations in brain tissue were significantly greater than CSF for TFV, FTC and EFV (Figure 1): ranging from 5-times (FTC)to 769-times (EFV) higher. Brain tissue concentration of EFV was 4.1 times higher in uninfected animals. Brain tissue concentrations ofefavirenz correlated with CSF over a range of concentrations (Figure 2).
• BCRP concentration (Figure 3) was 1.7 times higher in infected animals (p=0.02) while Pgp concentration did not differ with infection status(p=0.06). There was a trend noted for correlation between concentration of BCRP transporter and efavirenz in brain tissue (Figure 4).
• CSF concentrations need to be interpreted cautiously as surrogate for brain tissue concentration for several ARVs. Further, increase in BCRPconcentration may lead to lower EFV concentration in infected rhesus macaques.
• In future analyses, additional animals will be evaluated, and unbound concentration of EFV in brain tissue will be quantified since EFVexhibits a high degree of non-specific binding (>99.5%) in the brain tissue. Further investigations are needed to determine the influence ofbrain tissue concentrations on HAND
References1. Marra CM. HIV-associated neurocognitive disorders and central nervous system drug penetration: what next? AntivirTher. 2015. doi:10.3851/IMP2951.
2. Decloedt EH, Rosenkranz B, Maartens G, Joska J. Central Nervous System Penetration of Antiretroviral Drugs:Pharmacokinetic, Phharmacodynamic and Pharmacogenomic Considerations. Clin Pharmacokinet. 2015 Jun;54(6):581-98
3. Caniglia EC, Cain LE, Justice A, et al. Antiretroviral penetration into the CNS and incidence of AIDS-defining neurologicconditions. Neurology. 2014;83(2):134-141.
4. Scott Lentendre. Randomized Clinical Trial of Antiretroviral Therapy for Prevention of Hand (Oral Presentation). In: CROI2015..
5. Uchida Y et al. A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application forinter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at theblood–brain barrier in ddY, FVB, and C57BL/6J mice. Fluids Barriers CNS. 2013; 10: 21
Figure 1. Concentrations of Tenofovir, Emtricitabine, Efavirenz, Raltegravir,
Maraviroc and Atazanavir in brain tissue and CSF of rhesus macaquesFigure 3. Concentration of BCRP and P-gp transporter
proteins in brain tissue of rhesus macaques by QTAP
Figure 4.
Correlation
analysis between
brain tissue
concentration of
antiretroviral and
concentration of
BCRP transporter
protein in rhesus
macaques
Figure 2. Correlation between brain tissue and CSF
concentrations of Antiretrovirals
AcknowledgementsWe acknowledge the following funding sources: RO1 GM66940, RO1GM09697, AFPE pre-doctoral fellowship and the Royster Society ofFellows
SHIV Infection and Drug Transporters Influences Brain Tissue Concentrations of EfavirenzN. Srinivas1, J. Fallon1, C. Sykes1, N. White1, A. Schauer1, Michelle Matthews1, L. Adamson2, P. Luciw2, P. Smith1, A.D.M. Kashuba1
1University of North Carolina, Chapel Hill, United States, 2University of California at Davis, Davis, United States,
• Despite antiretroviral (ARV) therapy, there is a high prevalenceof HIV-associated neurocognitive disorder (HAND) in HIV-infected individuals1.
• It has been theorized that inadequate ARV concentrations maycontribute to the persistence of HAND2. However, there is noconclusive relationship between ARV concentration in the CSFand HAND3,4. Concentration of ARVs in the brain may be amore accurate pharmacokinetic measure but the data on braintissue concentrations is sparse.
• This study compared the concentration of ARVs in four regionsof brain tissue with CSF in uninfected and SHIV-infected rhesusmacaques and investigated the effect of drug transporters oninfluencing ARV concentrations in brain tissue
Background
MethodsConcentration of ARVs in brain tissue and CSF• In 12 male macaques (6 uninfected and 6 SHIV-infected) dosed
to steady-state, concentrations of 6 ARVs –tenofovir (TFV),emtricitabine (FTC), efavirenz (EFV), raltegravir (RAL), maraviroc(MVC) and atazanavir (ATZ) were measured by LC-MS/MS in theCSF, where LLOQ was 0.5 ng/mL and in the frontal cortex,cerebellum, basal ganglia and parietal cortex regions of the brainwhere LLOQ of homogenate ranged from 0.002-0.01 ng/mL.
• Tissue concentrations were converted to ng/g using density of1.06.
Concentration of BCRP and P-gp in brain tissue• To assess the influence of drug transporters on ARV
concentration, protein was extracted from the brain tissue andanalyzed for Pgp and BCRP efflux transporter concentration byLCMS/MS proteomics method called Quantitative TargetedAbsolute Proteomics (QTAP)5. The LLOQ was 0.1 pMol/mgprotein).
Data Analysis• Statistical analysis was performed by Kruskal-Wallis test using
Sigmaplot®
Results
Conclusions and future directions
• Antiretroviral concentrations in brain tissue were significantly greater than CSF for TFV, FTC and EFV (Figure 1): ranging from 5-times (FTC)to 769-times (EFV) higher. Brain tissue concentration of EFV was 4.1 times higher in uninfected animals. Brain tissue concentrations ofefavirenz correlated with CSF over a range of concentrations (Figure 2).
• BCRP concentration (Figure 3) was 1.7 times higher in infected animals (p=0.02) while Pgp concentration did not differ with infection status(p=0.06). There was a trend noted for correlation between concentration of BCRP transporter and efavirenz in brain tissue (Figure 4).
• CSF concentrations need to be interpreted cautiously as surrogate for brain tissue concentration for several ARVs. Further, increase in BCRPconcentration may lead to lower EFV concentration in infected rhesus macaques.
• In future analyses, additional animals will be evaluated, and unbound concentration of EFV in brain tissue will be quantified since EFVexhibits a high degree of non-specific binding (>99.5%) in the brain tissue. Further investigations are needed to determine the influence ofbrain tissue concentrations on HAND
References1. Marra CM. HIV-associated neurocognitive disorders and central nervous system drug penetration: what next? AntivirTher. 2015. doi:10.3851/IMP2951.
2. Decloedt EH, Rosenkranz B, Maartens G, Joska J. Central Nervous System Penetration of Antiretroviral Drugs:Pharmacokinetic, Phharmacodynamic and Pharmacogenomic Considerations. Clin Pharmacokinet. 2015 Jun;54(6):581-98
3. Caniglia EC, Cain LE, Justice A, et al. Antiretroviral penetration into the CNS and incidence of AIDS-defining neurologicconditions. Neurology. 2014;83(2):134-141.
4. Scott Lentendre. Randomized Clinical Trial of Antiretroviral Therapy for Prevention of Hand (Oral Presentation). In: CROI2015..
5. Uchida Y et al. A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application forinter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at theblood–brain barrier in ddY, FVB, and C57BL/6J mice. Fluids Barriers CNS. 2013; 10: 21
Figure 1. Concentrations of Tenofovir, Emtricitabine, Efavirenz, Raltegravir,
Maraviroc and Atazanavir in brain tissue and CSF of rhesus macaquesFigure 3. Concentration of BCRP and P-gp transporter
proteins in brain tissue of rhesus macaques by QTAP
Figure 4.
Correlation
analysis between
brain tissue
concentration of
antiretroviral and
concentration of
BCRP transporter
protein in rhesus
macaques
Figure 2. Correlation between brain tissue and CSF
concentrations of Antiretrovirals
AcknowledgementsWe acknowledge the following funding sources: RO1 GM66940, RO1GM09697, AFPE pre-doctoral fellowship and the Royster Society ofFellows
SHIV Infection and Drug Transporters Influences Brain Tissue Concentrations of EfavirenzN. Srinivas1, J. Fallon1, C. Sykes1, N. White1, A. Schauer1, Michelle Matthews1, L. Adamson2, P. Luciw2, P. Smith1, A.D.M. Kashuba1
1University of North Carolina, Chapel Hill, United States, 2University of California at Davis, Davis, United States,
• Despite antiretroviral (ARV) therapy, there is a high prevalenceof HIV-associated neurocognitive disorder (HAND) in HIV-infected individuals1.
• It has been theorized that inadequate ARV concentrations maycontribute to the persistence of HAND2. However, there is noconclusive relationship between ARV concentration in the CSFand HAND3,4. Concentration of ARVs in the brain may be amore accurate pharmacokinetic measure but the data on braintissue concentrations is sparse.
• This study compared the concentration of ARVs in four regionsof brain tissue with CSF in uninfected and SHIV-infected rhesusmacaques and investigated the effect of drug transporters oninfluencing ARV concentrations in brain tissue
Background
MethodsConcentration of ARVs in brain tissue and CSF• In 12 male macaques (6 uninfected and 6 SHIV-infected) dosed
to steady-state, concentrations of 6 ARVs –tenofovir (TFV),emtricitabine (FTC), efavirenz (EFV), raltegravir (RAL), maraviroc(MVC) and atazanavir (ATZ) were measured by LC-MS/MS in theCSF, where LLOQ was 0.5 ng/mL and in the frontal cortex,cerebellum, basal ganglia and parietal cortex regions of the brainwhere LLOQ of homogenate ranged from 0.002-0.01 ng/mL.
• Tissue concentrations were converted to ng/g using density of1.06.
Concentration of BCRP and P-gp in brain tissue• To assess the influence of drug transporters on ARV
concentration, protein was extracted from the brain tissue andanalyzed for Pgp and BCRP efflux transporter concentration byLCMS/MS proteomics method called Quantitative TargetedAbsolute Proteomics (QTAP)5. The LLOQ was 0.1 pMol/mgprotein).
Data Analysis• Statistical analysis was performed by Kruskal-Wallis test using
Sigmaplot®
Results
Conclusions and future directions
• Antiretroviral concentrations in brain tissue were significantly greater than CSF for TFV, FTC and EFV (Figure 1): ranging from 5-times (FTC)to 769-times (EFV) higher. Brain tissue concentration of EFV was 4.1 times higher in uninfected animals. Brain tissue concentrations ofefavirenz correlated with CSF over a range of concentrations (Figure 2).
• BCRP concentration (Figure 3) was 1.7 times higher in infected animals (p=0.02) while Pgp concentration did not differ with infection status(p=0.06). There was a trend noted for correlation between concentration of BCRP transporter and efavirenz in brain tissue (Figure 4).
• CSF concentrations need to be interpreted cautiously as surrogate for brain tissue concentration for several ARVs. Further, increase in BCRPconcentration may lead to lower EFV concentration in infected rhesus macaques.
• In future analyses, additional animals will be evaluated, and unbound concentration of EFV in brain tissue will be quantified since EFVexhibits a high degree of non-specific binding (>99.5%) in the brain tissue. Further investigations are needed to determine the influence ofbrain tissue concentrations on HAND
References1. Marra CM. HIV-associated neurocognitive disorders and central nervous system drug penetration: what next? AntivirTher. 2015. doi:10.3851/IMP2951.
2. Decloedt EH, Rosenkranz B, Maartens G, Joska J. Central Nervous System Penetration of Antiretroviral Drugs:Pharmacokinetic, Phharmacodynamic and Pharmacogenomic Considerations. Clin Pharmacokinet. 2015 Jun;54(6):581-98
3. Caniglia EC, Cain LE, Justice A, et al. Antiretroviral penetration into the CNS and incidence of AIDS-defining neurologicconditions. Neurology. 2014;83(2):134-141.
4. Scott Lentendre. Randomized Clinical Trial of Antiretroviral Therapy for Prevention of Hand (Oral Presentation). In: CROI2015..
5. Uchida Y et al. A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application forinter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at theblood–brain barrier in ddY, FVB, and C57BL/6J mice. Fluids Barriers CNS. 2013; 10: 21
Figure 1. Concentrations of Tenofovir, Emtricitabine, Efavirenz, Raltegravir,
Maraviroc and Atazanavir in brain tissue and CSF of rhesus macaquesFigure 3. Concentration of BCRP and P-gp transporter
proteins in brain tissue of rhesus macaques by QTAP
Figure 4.
Correlation
analysis between
brain tissue
concentration of
antiretroviral and
concentration of
BCRP transporter
protein in rhesus
macaques
Figure 2. Correlation between brain tissue and CSF
concentrations of Antiretrovirals
What did we learn in the past two years?
Curley P et al, AAC 2017; Thompson CG, et al. AAC 2015; Srinivas et al, IAS 2017, Abstract WEAB0105; Fletcher
CV, et al. PNAS 2014
1. Cerebrospinal fluid is a poor predictor of brain concentrations of different drugs• EFV Brain PK 3.7-12.7 times higher than plasma in a
macaque• EFV Brain PK to plasma ratios 9.5 (rodents) - 15.8 (PBPK
modelling) 2. CSF concentrations correlate with brain concentrations
• Individual PK in macaques3. Inflammation is associated with the induction of several
transporters involved in drug distribution• SIV infected animals have higher expression of P-gp and
BCRP4. Methods matter
• Tissue homogenate vs. mononuclear cells
Brain tissue homogenate Mononuclear cells extracted from tissues
Brain distribution is not homogeneous
Bumpus N, et al. CROI 2015 #436; Srinivas et al, CROI 2018 #472
nOverall
Mean
WM
(mean)
GP
(mean)
CGM
(mean)
Concentrations Similar to Historical CSF Concentrations
Atazanavir
(ATV)2 < 25 < 25 < 25 < 25
Efavirenz
(EFV)2 38.6 45.2 34.8 35.9
Emtricitabine
(FTC)4 181.3 230.4 173.2 140.3
Lamivudine
(3TC)3 196.9 205.5 209.8 175.4
Concentrations in White Matter Higher than Historical CSF
Concentrations
Lopinavir
(LPV)4 153.3 410.6 < 25 < 25
Concentrations Higher than Historical CSF Concentrations
Tenofovir
(TDF)6 206.0 220.0 212.1 185.8
WM = White Matter; GP = Globus Pallidus (Deep Gray Matter);
CGM = Cortical Gray Matter
Background
Despite ongoing antiretroviral (ARV) therapy, HIV inflammation continues to cause
issues in the central nervous system (CNS), as demonstrated by HIV-associated
neurocognitive disorder.1
HIV persistence in the brain may be due to inadequate drug exposure in HIV-target cells;
however, there is little information on brain distribution of ARVs.
In this study, we have quantified the concentration of 4 ARVs in brain tissue by liquid
chromatography mass spectroscopy (LC-MS/MS) and infrared matrix-assisted laser
desorption electrospray ionization (IR-MALDESI) while mapping their distribution
relative to expression of CD4+ T-cells and CD11b+ microglia.
Methods
Concentration of ARVsin cerebellum by LC-MS/MS
• Four male rhesus macaques were dosed to steady-state with a combination of 4
ARVs –tenofovir (TFV), emtricitabine (FTC), efavirenz (EFV) and raltegravir
(RAL). Two animals were left uninfected and two were infected with SHIV for 4
weeks prior to ARV dosing. Concentrations of the ARVs were measured by LC-
MS/MS2 in a 10 μm thick section of the cerebellum region of the brain tissue where
LLOQ of homogenate ranged from 0.002-0.01 ng/mL.
• Tissue concentrations were converted to ng/g using density of 1.06.
Massspectrometry imaging (MSI)
• 10 μm thick frozen slices of discrete cerebellum regions were thaw-mounted onto a
glass slide, covered with an ice layer and ablated with 2 mid-IR laser pulses with a
100 μm spot-to-spot distance.3,4 Ablated molecules were ionized by electrospray
and sampled into a Thermo Q-Exactive Plus mass spectrometer.
• Raw data from each volumetric pixel were converted to the imzMl format to
evaluate using MSiReader.5 Quantification of ARV concentration was achieved by
spotting calibration standards of known concentration onto a non-dosed tissue slice.
Immunohistochemistry (IHC) staining
• IHC staining of CD11b+ microglia and CD4+ T-cells was performed on contiguous
slices.
Imageanalysis
• Analysis of co-registered MSI and down-sampled IHC images was performed in
MATLAB®.
Conclusion and Future Directions
Figure 1. Cholesterol and efavirenz MSI in cerebellar
brain tissue in a) uninfected and b) RT-SHIV-infected
animals
Figure 2. Overlay between efavirenz MSI and CD11b+ cell distribution in the
cerebellum tissue in a) uninfected and b) RT-SHIV-infected animals
Distribution of EFV and CD11b+ cells in the cerebellum and the overlay of
distribution are shown in a) uninfected and b) RT-SHIV-infected animals.
Fractional coverage of the CD11b+ cells with EFV (FrC) was higher in the
uninfected animals compared to the RT-SHIV infected animals.
• EFV accumulation was 12- to 60-fold greater in brain tissue compared to other ARVs in RT-SHIV+ animals but only 14% to 59% of CD11b+ and CD4+ brain cells in these animals were colocalized with
detectable EFV.
• This low fractional coverage suggests that ARV exposure may be incomplete for cell populations that harbor, or can become infected, with HIV.
• This approach has the potential to provide ARV concentration-effect relationships in the brain at the cellular level with improvements in the spatial resolution of the MSI.
• TFV, FTC, and RAL were not detected by
MALDESI and were <100 ng/g by LC-MS/MS
(range of concentrations were 9.4-61.2 ng/g).
• EFV concentrations by IR-MALDESI had a
standard deviation of 663 ng/g for all samples and
was 86% lower in RT-SHIV-infected than
uninfected brain tissue (median = 1596 and 723
ng/g, respectively).
• EFV concentrations were up to 3-fold higher in the
white matter versus gray matter (Figure 1).
• The fractional coverage of CD11b+ cells co-
localized with EFV (FrC) differed based on
infection status: for CD11b+ cells FrC was 22-59%
(RT-SHIV+) and 76-81% (RT-SHIV-) and for
CD4+ T-cells FrC was 14-59% (RT-SHIV+) and
73-77% (SHIV-). However, FrC of total CD11b+
cells exposed to EFV concentrations above the
upper limit of the in-vitro IC50 reported from the
literature (0.5 ng/g) was considerably smaller: 0-
3.3%, regardless of infection status (Figure 2).
• Overlay results between the EFV concentration
above the IC50 and CD4+ T-cell distribution
showed a similarly low FrC.
1.Marra CM. Antivir. Ther., 2015; 20(4): 365-367.
2.Srinivas N, Fallon JK, Sykes C, White N, et al. 9th International AIDS Society Conference on HIV Sciences 23-26 July 2017: Abstract WEAB0105.
3.Barry JA, Robichaud G, Bokhart MT, Thompson C, Sykes C, Kashuba AD et al. J. Am. Soc. Mass Spectrom., 2014; 25(12): 2038–2047.
4.Thompson CG, Bokhart MT, Sykes C, Adamson L, Fedoriw Y, Luciw PA, Muddiman DC, et al. Antimicrob. Agents Chemother., 2015; 59(5): 2944–2948.
5.Robichaud G, Garrard KP, Barry JA, and Muddiman DC, J. Am. Soc. Mass Spectrom., 2013; 24(5):718–721.
Acknowledgements
We acknowledge the following funding sources: the National Institutes of Health
under grants: RO1 AI111891, S10 RR024595, RO1 GM66940, RO1 GM09697,
the UNC center for AIDS research under grant: P30 AI50410, AFPE pre-doctoral
fellowship and the Royster Society of Fellows, UNC Chapel Hill.
Mapping the Distribution of Efavirenz Relative to Brain CellsN. Srinivas1, E. P. Rosen1, G. De La Cruz1, C. Sykes1, A. Schauer1, L. Adamson2, P. Luciw2, A.D.M. Kashuba1
1University of North Carolina, Chapel Hill, United States. 2University of California at Davis, Davis, United States.
Results
Ion maps of cholesterol (left) indicate distinction between
white matter (WM) and gray matter (GM) in the brain tissue.
The EFV heat maps (right) show the amount of efavirenz per
voxel based on accompanying scale bar. (a) In the uninfected
animals, clear preferential distribution of efavirenz was noted
in the WM compared to the GM (b) In RT-SHIV-infected
animals, efavirenz concentrations were 86% lower.
References
Poster Number 472
Background
Despite ongoing antiretroviral (ARV) therapy, HIV inflammation continues to cause
issues in the central nervous system (CNS), as demonstrated by HIV-associated
neurocognitive disorder.1
HIV persistence in the brain may be due to inadequate drug exposure in HIV-target cells;
however, there is little information on brain distribution of ARVs.
In this study, we have quantified the concentration of 4 ARVs in brain tissue by liquid
chromatography mass spectroscopy (LC-MS/MS) and infrared matrix-assisted laser
desorption electrospray ionization (IR-MALDESI) while mapping their distribution
relative to expression of CD4+ T-cells and CD11b+ microglia.
Methods
Concentration of ARVsin cerebellum by LC-MS/MS
• Four male rhesus macaques were dosed to steady-state with a combination of 4
ARVs –tenofovir (TFV), emtricitabine (FTC), efavirenz (EFV) and raltegravir
(RAL). Two animals were left uninfected and two were infected with SHIV for 4
weeks prior to ARV dosing. Concentrations of the ARVs were measured by LC-
MS/MS2 in a 10 μm thick section of the cerebellum region of the brain tissue where
LLOQ of homogenate ranged from 0.002-0.01 ng/mL.
• Tissue concentrations were converted to ng/g using density of 1.06.
Massspectrometry imaging (MSI)
• 10 μm thick frozen slices of discrete cerebellum regions were thaw-mounted onto a
glass slide, covered with an ice layer and ablated with 2 mid-IR laser pulses with a
100 μm spot-to-spot distance.3,4 Ablated molecules were ionized by electrospray
and sampled into a Thermo Q-Exactive Plus mass spectrometer.
• Raw data from each volumetric pixel were converted to the imzMl format to
evaluate using MSiReader.5 Quantification of ARV concentration was achieved by
spotting calibration standards of known concentration onto a non-dosed tissue slice.
Immunohistochemistry (IHC) staining
• IHC staining of CD11b+ microglia and CD4+ T-cells was performed on contiguous
slices.
Image analysis
• Analysis of co-registered MSI and down-sampled IHC images was performed in
MATLAB®.
Conclusion and Future Directions
Figure 1. Cholesterol and efavirenz MSI in cerebellar
brain tissue in a) uninfected and b) RT-SHIV-infected
animals
Figure 2. Overlay between efavirenz MSI and CD11b+ cell distribution in the
cerebellum tissue in a) uninfected and b) RT-SHIV-infected animals
Distribution of EFV and CD11b+ cells in the cerebellum and the overlay of
distribution are shown in a) uninfected and b) RT-SHIV-infected animals.
Fractional coverage of the CD11b+ cells with EFV (FrC) was higher in the
uninfected animals compared to the RT-SHIV infected animals.
• EFV accumulation was 12- to 60-fold greater in brain tissue compared to other ARVs in RT-SHIV+ animals but only 14% to 59% of CD11b+ and CD4+ brain cells in these animals were colocalized with
detectable EFV.
• This low fractional coverage suggests that ARV exposure may be incomplete for cell populations that harbor, or can become infected, with HIV.
• This approach has the potential to provide ARV concentration-effect relationships in the brain at the cellular level with improvements in the spatial resolution of the MSI.
• TFV, FTC, and RAL were not detected by
MALDESI and were <100 ng/g by LC-MS/MS
(range of concentrations were 9.4-61.2 ng/g).
• EFV concentrations by IR-MALDESI had a
standard deviation of 663 ng/g for all samples and
was 86% lower in RT-SHIV-infected than
uninfected brain tissue (median = 1596 and 723
ng/g, respectively).
• EFV concentrations were up to 3-fold higher in the
white matter versus gray matter (Figure 1).
• The fractional coverage of CD11b+ cells co-
localized with EFV (FrC) differed based on
infection status: for CD11b+ cells FrC was 22-59%
(RT-SHIV+) and 76-81% (RT-SHIV-) and for
CD4+ T-cells FrC was 14-59% (RT-SHIV+) and
73-77% (SHIV-). However, FrC of total CD11b+
cells exposed to EFV concentrations above the
upper limit of the in-vitro IC50 reported from the
literature (0.5 ng/g) was considerably smaller: 0-
3.3%, regardless of infection status (Figure 2).
• Overlay results between the EFV concentration
above the IC50 and CD4+ T-cell distribution
showed a similarly low FrC.
1.Marra CM. Antivir. Ther., 2015; 20(4): 365-367.
2.Srinivas N, Fallon JK, Sykes C, White N, et al. 9th International AIDS Society Conference on HIV Sciences 23-26 July 2017: Abstract WEAB0105.
3.Barry JA, Robichaud G, Bokhart MT, Thompson C, Sykes C, Kashuba AD et al. J. Am. Soc. Mass Spectrom., 2014; 25(12): 2038–2047.
4.Thompson CG, Bokhart MT, Sykes C, Adamson L, Fedoriw Y, Luciw PA, Muddiman DC, et al. Antimicrob. Agents Chemother., 2015; 59(5): 2944–2948.
5.Robichaud G, Garrard KP, Barry JA, and Muddiman DC, J. Am. Soc. Mass Spectrom., 2013; 24(5):718–721.
Acknowledgements
We acknowledge the following funding sources: the National Institutes of Health
under grants: RO1 AI111891, S10 RR024595, RO1 GM66940, RO1 GM09697,
the UNC center for AIDS research under grant: P30 AI50410, AFPE pre-doctoral
fellowship and the Royster Society of Fellows, UNC Chapel Hill.
Mapping the Distribution of Efavirenz Relative to Brain CellsN. Srinivas1, E. P. Rosen1, G. De La Cruz1, C. Sykes1, A. Schauer1, L. Adamson2, P. Luciw2, A.D.M. Kashuba1
1University of North Carolina, Chapel Hill, United States. 2University of California at Davis, Davis, United States.
Results
Ion maps of cholesterol (left) indicate distinction between
white matter (WM) and gray matter (GM) in the brain tissue.
The EFV heat maps (right) show the amount of efavirenz per
voxel based on accompanying scale bar. (a) In the uninfected
animals, clear preferential distribution of efavirenz was noted
in the WM compared to the GM (b) In RT-SHIV-infected
animals, efavirenz concentrations were 86% lower.
References
Poster Number 472
EFV up to 3-fold higher in the WM vs. GM
22-59 of CD11b+
microglialcells with EFV and only 3.3
with EFV>0.5
ng/g
Background
Despite ongoing antiretroviral (ARV) therapy, HIV inflammation continues to cause
issues in the central nervous system (CNS), as demonstrated by HIV-associated
neurocognitive disorder.1
HIV persistence in the brain may be due to inadequate drug exposure in HIV-target cells;
however, there is little information on brain distribution of ARVs.
In this study, we have quantified the concentration of 4 ARVs in brain tissue by liquid
chromatography mass spectroscopy (LC-MS/MS) and infrared matrix-assisted laser
desorption electrospray ionization (IR-MALDESI) while mapping their distribution
relative to expression of CD4+ T-cells and CD11b+ microglia.
Methods
Concentration of ARVsin cerebellum by LC-MS/MS
• Four male rhesus macaques were dosed to steady-state with a combination of 4
ARVs –tenofovir (TFV), emtricitabine (FTC), efavirenz (EFV) and raltegravir
(RAL). Two animals were left uninfected and two were infected with SHIV for 4
weeks prior to ARV dosing. Concentrations of the ARVs were measured by LC-
MS/MS2 in a 10 μm thick section of the cerebellum region of the brain tissue where
LLOQ of homogenate ranged from 0.002-0.01 ng/mL.
• Tissue concentrations were converted to ng/g using density of 1.06.
Massspectrometry imaging (MSI)
• 10 μm thick frozen slices of discrete cerebellum regions were thaw-mounted onto a
glass slide, covered with an ice layer and ablated with 2 mid-IR laser pulses with a
100 μm spot-to-spot distance.3,4 Ablated molecules were ionized by electrospray
and sampled into a Thermo Q-Exactive Plus mass spectrometer.
• Raw data from each volumetric pixel were converted to the imzMl format to
evaluate using MSiReader.5 Quantification of ARV concentration was achieved by
spotting calibration standards of known concentration onto a non-dosed tissue slice.
Immunohistochemistry (IHC) staining
• IHC staining of CD11b+ microglia and CD4+ T-cells was performed on contiguous
slices.
Image analysis
• Analysis of co-registered MSI and down-sampled IHC images was performed in
MATLAB®.
Conclusion and Future Directions
Figure 1. Cholesterol and efavirenz MSI in cerebellar
brain tissue in a) uninfected and b) RT-SHIV-infected
animals
Figure 2. Overlay between efavirenz MSI and CD11b+ cell distribution in the
cerebellum tissue in a) uninfected and b) RT-SHIV-infected animals
Distribution of EFV and CD11b+ cells in the cerebellum and the overlay of
distribution are shown in a) uninfected and b) RT-SHIV-infected animals.
Fractional coverage of the CD11b+ cells with EFV (FrC) was higher in the
uninfected animals compared to the RT-SHIV infected animals.
• EFV accumulation was 12- to 60-fold greater in brain tissue compared to other ARVs in RT-SHIV+ animals but only 14% to 59% of CD11b+ and CD4+ brain cells in these animals were colocalized with
detectable EFV.
• This low fractional coverage suggests that ARV exposure may be incomplete for cell populations that harbor, or can become infected, with HIV.
• This approach has the potential to provide ARV concentration-effect relationships in the brain at the cellular level with improvements in the spatial resolution of the MSI.
• TFV, FTC, and RAL were not detected by
MALDESI and were <100 ng/g by LC-MS/MS
(range of concentrations were 9.4-61.2 ng/g).
• EFV concentrations by IR-MALDESI had a
standard deviation of 663 ng/g for all samples and
was 86% lower in RT-SHIV-infected than
uninfected brain tissue (median = 1596 and 723
ng/g, respectively).
• EFV concentrations were up to 3-fold higher in the
white matter versus gray matter (Figure 1).
• The fractional coverage of CD11b+ cells co-
localized with EFV (FrC) differed based on
infection status: for CD11b+ cells FrC was 22-59%
(RT-SHIV+) and 76-81% (RT-SHIV-) and for
CD4+ T-cells FrC was 14-59% (RT-SHIV+) and
73-77% (SHIV-). However, FrC of total CD11b+
cells exposed to EFV concentrations above the
upper limit of the in-vitro IC50 reported from the
literature (0.5 ng/g) was considerably smaller: 0-
3.3%, regardless of infection status (Figure 2).
• Overlay results between the EFV concentration
above the IC50 and CD4+ T-cell distribution
showed a similarly low FrC.
1.Marra CM. Antivir. Ther., 2015; 20(4): 365-367.
2.Srinivas N, Fallon JK, Sykes C, White N, et al. 9th International AIDS Society Conference on HIV Sciences 23-26 July 2017: Abstract WEAB0105.
3.Barry JA, Robichaud G, Bokhart MT, Thompson C, Sykes C, Kashuba AD et al. J. Am. Soc. Mass Spectrom., 2014; 25(12): 2038–2047.
4.Thompson CG, Bokhart MT, Sykes C, Adamson L, Fedoriw Y, Luciw PA, Muddiman DC, et al. Antimicrob. Agents Chemother., 2015; 59(5): 2944–2948.
5.Robichaud G, Garrard KP, Barry JA, and Muddiman DC, J. Am. Soc. Mass Spectrom., 2013; 24(5):718–721.
Acknowledgements
We acknowledge the following funding sources: the National Institutes of Health
under grants: RO1 AI111891, S10 RR024595, RO1 GM66940, RO1 GM09697,
the UNC center for AIDS research under grant: P30 AI50410, AFPE pre-doctoral
fellowship and the Royster Society of Fellows, UNC Chapel Hill.
Mapping the Distribution of Efavirenz Relative to Brain CellsN. Srinivas1, E. P. Rosen1, G. De La Cruz1, C. Sykes1, A. Schauer1, L. Adamson2, P. Luciw2, A.D.M. Kashuba1
1University of North Carolina, Chapel Hill, United States. 2University of California at Davis, Davis, United States.
Results
Ion maps of cholesterol (left) indicate distinction between
white matter (WM) and gray matter (GM) in the brain tissue.
The EFV heat maps (right) show the amount of efavirenz per
voxel based on accompanying scale bar. (a) In the uninfected
animals, clear preferential distribution of efavirenz was noted
in the WM compared to the GM (b) In RT-SHIV-infected
animals, efavirenz concentrations were 86% lower.
References
Poster Number 472
Background
Despite ongoing antiretroviral (ARV) therapy, HIV inflammation continues to cause
issues in the central nervous system (CNS), as demonstrated by HIV-associated
neurocognitive disorder.1
HIV persistence in the brain may be due to inadequate drug exposure in HIV-target cells;
however, there is little information on brain distribution of ARVs.
In this study, we have quantified the concentration of 4 ARVs in brain tissue by liquid
chromatography mass spectroscopy (LC-MS/MS) and infrared matrix-assisted laser
desorption electrospray ionization (IR-MALDESI) while mapping their distribution
relative to expression of CD4+ T-cells and CD11b+ microglia.
Methods
Concentration of ARVsin cerebellum by LC-MS/MS
• Four male rhesus macaques were dosed to steady-state with a combination of 4
ARVs –tenofovir (TFV), emtricitabine (FTC), efavirenz (EFV) and raltegravir
(RAL). Two animals were left uninfected and two were infected with SHIV for 4
weeks prior to ARV dosing. Concentrations of the ARVs were measured by LC-
MS/MS2 in a 10 μm thick section of the cerebellum region of the brain tissue where
LLOQ of homogenate ranged from 0.002-0.01 ng/mL.
• Tissue concentrations were converted to ng/g using density of 1.06.
Massspectrometry imaging (MSI)
• 10 μm thick frozen slices of discrete cerebellum regions were thaw-mounted onto a
glass slide, covered with an ice layer and ablated with 2 mid-IR laser pulses with a
100 μm spot-to-spot distance.3,4 Ablated molecules were ionized by electrospray
and sampled into a Thermo Q-Exactive Plus mass spectrometer.
• Raw data from each volumetric pixel were converted to the imzMl format to
evaluate using MSiReader.5 Quantification of ARV concentration was achieved by
spotting calibration standards of known concentration onto a non-dosed tissue slice.
Immunohistochemistry (IHC) staining
• IHC staining of CD11b+ microglia and CD4+ T-cells was performed on contiguous
slices.
Image analysis
• Analysis of co-registered MSI and down-sampled IHC images was performed in
MATLAB®.
Conclusion and Future Directions
Figure 1. Cholesterol and efavirenz MSI in cerebellar
brain tissue in a) uninfected and b) RT-SHIV-infected
animals
Figure 2. Overlay between efavirenz MSI and CD11b+ cell distribution in the
cerebellum tissue in a) uninfected and b) RT-SHIV-infected animals
Distribution of EFV and CD11b+ cells in the cerebellum and the overlay of
distribution are shown in a) uninfected and b) RT-SHIV-infected animals.
Fractional coverage of the CD11b+ cells with EFV (FrC) was higher in the
uninfected animals compared to the RT-SHIV infected animals.
• EFV accumulation was 12- to 60-fold greater in brain tissue compared to other ARVs in RT-SHIV+ animals but only 14% to 59% of CD11b+ and CD4+ brain cells in these animals were colocalized with
detectable EFV.
• This low fractional coverage suggests that ARV exposure may be incomplete for cell populations that harbor, or can become infected, with HIV.
• This approach has the potential to provide ARV concentration-effect relationships in the brain at the cellular level with improvements in the spatial resolution of the MSI.
• TFV, FTC, and RAL were not detected by
MALDESI and were <100 ng/g by LC-MS/MS
(range of concentrations were 9.4-61.2 ng/g).
• EFV concentrations by IR-MALDESI had a
standard deviation of 663 ng/g for all samples and
was 86% lower in RT-SHIV-infected than
uninfected brain tissue (median = 1596 and 723
ng/g, respectively).
• EFV concentrations were up to 3-fold higher in the
white matter versus gray matter (Figure 1).
• The fractional coverage of CD11b+ cells co-
localized with EFV (FrC) differed based on
infection status: for CD11b+ cells FrC was 22-59%
(RT-SHIV+) and 76-81% (RT-SHIV-) and for
CD4+ T-cells FrC was 14-59% (RT-SHIV+) and
73-77% (SHIV-). However, FrC of total CD11b+
cells exposed to EFV concentrations above the
upper limit of the in-vitro IC50 reported from the
literature (0.5 ng/g) was considerably smaller: 0-
3.3%, regardless of infection status (Figure 2).
• Overlay results between the EFV concentration
above the IC50 and CD4+ T-cell distribution
showed a similarly low FrC.
1.Marra CM. Antivir. Ther., 2015; 20(4): 365-367.
2.Srinivas N, Fallon JK, Sykes C, White N, et al. 9th International AIDS Society Conference on HIV Sciences 23-26 July 2017: Abstract WEAB0105.
3.Barry JA, Robichaud G, Bokhart MT, Thompson C, Sykes C, Kashuba AD et al. J. Am. Soc. Mass Spectrom., 2014; 25(12): 2038–2047.
4.Thompson CG, Bokhart MT, Sykes C, Adamson L, Fedoriw Y, Luciw PA, Muddiman DC, et al. Antimicrob. Agents Chemother., 2015; 59(5): 2944–2948.
5.Robichaud G, Garrard KP, Barry JA, and Muddiman DC, J. Am. Soc. Mass Spectrom., 2013; 24(5):718–721.
Acknowledgements
We acknowledge the following funding sources: the National Institutes of Health
under grants: RO1 AI111891, S10 RR024595, RO1 GM66940, RO1 GM09697,
the UNC center for AIDS research under grant: P30 AI50410, AFPE pre-doctoral
fellowship and the Royster Society of Fellows, UNC Chapel Hill.
Mapping the Distribution of Efavirenz Relative to Brain CellsN. Srinivas1, E. P. Rosen1, G. De La Cruz1, C. Sykes1, A. Schauer1, L. Adamson2, P. Luciw2, A.D.M. Kashuba1
1University of North Carolina, Chapel Hill, United States. 2University of California at Davis, Davis, United States.
Results
Ion maps of cholesterol (left) indicate distinction between
white matter (WM) and gray matter (GM) in the brain tissue.
The EFV heat maps (right) show the amount of efavirenz per
voxel based on accompanying scale bar. (a) In the uninfected
animals, clear preferential distribution of efavirenz was noted
in the WM compared to the GM (b) In RT-SHIV-infected
animals, efavirenz concentrations were 86% lower.
References
Poster Number 472
Aim of the study
• To characterize ARVs’ cerebrospinal fluid concentrations according to patients’ demographic, treatment and genetic features
• HIV-positive patients receiving lumbar punctures for clinical reasons and participating to specific study protocols were included after signing a written informed consent.
• CSF and plasma were withdrawn less than 15 minutes apart and analyzed using validated HPLC/MS-MS methods.
• BBB permeability was estimated using Reibergrams(CSAR) and CSF neopterin trough ELISA methods.
• Multivariate linear regression analysis were performed including age, CSAR, CSF neopterin and plasma concentrations besides SNPs with univariate p values <0.20.
Material and Methods
BBB impairment
Intrathecalproduction of IgGs
• Genomic DNA was extracted using QIAamp whole blood mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Genotyping was conducted by real time-based allelic discrimination
• including the following SNPs:– ABCB1 (rs1045642, rs1128503,
rs2032582),– ABCC2 (rs717620), – SLC22A6 (rs4149170), – SLCO1A2 (rs10841795, rs11568563), – ABCG2 (rs2231142, rs13120400), – HNF4α (rs1884613).
Material and Methods (PG)
abundantly expressed in the kidney, with only modest or low expression in the liver. In
contrast, OCTN2 (SLC22A5) and OAT2 (SLC22A7) were highly expressed in both kidney
and liver. OCT1 (SLC22A1) was most strongly expressed in the liver and OCT3
(SLC22A3) in the prostate, salivary gland and skeletal muscle. Expression of the
concentrative and equilibrative nucleoside transporters (CNTs, ENTs) was ubiquitous,
with the exception of CNT3 (SLC28A3), which was restricted to pancreas and skin, with
lower levels detected in mammary gland, jejunum and foetal liver.
ABCB1 (MDR1)ABCB4 (MDR3)
ABCB11 (BSEP)ABCC1 (MRP1)ABCC2 (MRP2)ABCC3 (MRP3)ABCC4 (MRP4)ABCC5 (MRP5)ABCC6 (MRP6)
ABCC10 (MRP7)ABCC11 (MRP8)ABCC12 (MRP9)ABCG2 (BCRP)
OSTalphaSLC10A1 (NTCP)SLC10A2 (ASBT)
SLC15A1 (PEPT1)SLC15A2 (PEPT2)SLC16A1 (MCT1)SLC16A7 (MCT2)SLC17A1 (NaPi1)SLC22A1 (OCT1)SLC22A2 (OCT2)SLC22A3 (OCT3)
SLC22A4 (OCTN1)SLC22A5 (OCTN2)
SLC22A6 (OAT1)SLC22A7 (OAT2)SLC22A8 (OAT3)
SLC22A11 (OAT4)SLC22A12 (URAT1)
SLC28A1 (CNT1)SLC28A2 (CNT2)SLC28A3 (CNT3)SLC29A1 (ENT1)SLC29A2 (ENT2)SLC29A3 (ENT3)SLC29A4 (ENT4)
SLC6A14 (ATB(0+))SLCO1A2 (OATP-A)SLCO1B1 (OATP-C)SLCO1B3 (OATP-8)SLCO1C1 (OATP-F)
SLCO2A1 (PGT)SLCO2B1 (OATP-B)SLCO3A1 (OATP-D)SLCO4A1 (OATP-E)SLCO4C1 (OATP-H)SLCO5A1 (OATP-J)SLCO6A1 (OATP-I)
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Absolute Intensity -- Percentile
0 50 100
Figure 1. Average absolute gene-expression intensity of multiple probes for human xenobiotictransporter genes. White represents missing data due to erroneous measurements. Percentiles arebased on a reference set of intensities of 19 000 genes in over 100 tissues.
Tissue distribution of transporter genes 971
Xen
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For
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ly.
abundantly expressed in the kidney, with only modest or low expression in the liver. In
contrast, OCTN2 (SLC22A5) and OAT2 (SLC22A7) were highly expressed in both kidney
and liver. OCT1 (SLC22A1) was most strongly expressed in the liver and OCT3
(SLC22A3) in the prostate, salivary gland and skeletal muscle. Expression of the
concentrative and equilibrative nucleoside transporters (CNTs, ENTs) was ubiquitous,
with the exception of CNT3 (SLC28A3), which was restricted to pancreas and skin, with
lower levels detected in mammary gland, jejunum and foetal liver.
ABCB1 (MDR1)ABCB4 (MDR3)
ABCB11 (BSEP)ABCC1 (MRP1)ABCC2 (MRP2)ABCC3 (MRP3)ABCC4 (MRP4)ABCC5 (MRP5)ABCC6 (MRP6)
ABCC10 (MRP7)ABCC11 (MRP8)ABCC12 (MRP9)ABCG2 (BCRP)
OSTalphaSLC10A1 (NTCP)SLC10A2 (ASBT)
SLC15A1 (PEPT1)SLC15A2 (PEPT2)SLC16A1 (MCT1)SLC16A7 (MCT2)SLC17A1 (NaPi1)SLC22A1 (OCT1)SLC22A2 (OCT2)SLC22A3 (OCT3)
SLC22A4 (OCTN1)SLC22A5 (OCTN2)
SLC22A6 (OAT1)SLC22A7 (OAT2)SLC22A8 (OAT3)
SLC22A11 (OAT4)SLC22A12 (URAT1)
SLC28A1 (CNT1)SLC28A2 (CNT2)SLC28A3 (CNT3)SLC29A1 (ENT1)SLC29A2 (ENT2)SLC29A3 (ENT3)SLC29A4 (ENT4)
SLC6A14 (ATB(0+))SLCO1A2 (OATP-A)SLCO1B1 (OATP-C)SLCO1B3 (OATP-8)SLCO1C1 (OATP-F)
SLCO2A1 (PGT)SLCO2B1 (OATP-B)SLCO3A1 (OATP-D)SLCO4A1 (OATP-E)SLCO4C1 (OATP-H)SLCO5A1 (OATP-J)SLCO6A1 (OATP-I)
Retin
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eta
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Testis
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land
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Skin
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l leukocyte
s
Tum
or: b
reast
Tum
or: co
lon
Tum
or: lu
ng
Absolute Intensity -- Percentile
0 50 100
Figure 1. Average absolute gene-expression intensity of multiple probes for human xenobiotictransporter genes. White represents missing data due to erroneous measurements. Percentiles arebased on a reference set of intensities of 19 000 genes in over 100 tissues.
Tissue distribution of transporter genes 971
Xen
ob
ioti
ca D
ow
nlo
aded
fro
m i
nfo
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m b
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For
per
sonal
use
on
ly.
Results
• 259 patients providing 405 paired CSF/plasma samples
• Asymptomatic (29.6%), HAND (21.5%), CNS Opportunistic infections or neoplasms (17.8%) or White matter hyperintensities (4.2%)
• Altered BBB (<6.5 if age <=40, <8 if >40) in 21.5%
• High CSF neopterin (>1.5 ng/mL) in 24.4%
N or median
or IQR
Male 185 71.4
European Ancestry
194 74.9
Age 48 41-55
BMI 22.4 20.1-24.6
CD4 321 145-549
Nadir CD4 97 24-208
Plasma VL<50 267 68.8
CSF VL<50 228 60.5
Results (2)
n 57 34 12 107 104 9 14 14 6 28 78 31 116 4 21 80 4 12
Q1 75 18 6,8 0 57 269 25 5,4 1 13 14 1,9 0 2,3 0,41 19 2,6 3,3
Med 122 88 15 5 103 1062 41 17 2 25 29 7,2 0,36 6,5 2,2 30 3,9 6,5
Q3 162 234 35 10 155 3051 82 23 3 49 47 22 0,90 7,8 5,5 57 5,7 11
3TC
ABC
AZT
TFVFTC
NVP
EFVETV
RPV
LPVDRV
ATV
RTV
CO
BM
VCRAL
EVG
DTG
1
10
100
1000
10000
CS
F c
on
ce
ntr
atio
ns
(L
og
10 n
g/m
L)
CSF concentrations below the LOD were observed
in patients receiving
TDF31.8%
AZT16.8%
ATV12.9%
RTV28.4%
n 54 33 12 99 102 9 13 14 6 28 77 31 112 4 19 64 4 11
Q1 (%) 18 38 4,6 0 16 4 0,8 0,8 1 0,1 0,4 0,5 0 0,6 0,4 2,2 0,4 0,3
Med (%) 37 92 97 7,7 39 45 1,5 2,7 1,3 0,3 0,7 1,3 0,1 8,9 2,4 12 0,4 0,6
Q3 (%) 58 200 150 20 54 51 2,8 3,6 1,7 0,8 1,1 1,4 0,3 28 5,8 27 0,4 1
Results (3)
3TC
ABC
AZT
TFVFTC
NVP
EFVETV
RPV
LPVDRV
ATV
RTV
CO
BM
VCRAL
EVGDTG
0.001
0.010
0.100
1
10
CS
F to
pla
sm
a r
atio
s
CSF to plasma correlations
0 500 1000 15000
1000
2000
3000
4000
5000
NRTIs
CSF concentrations
Pla
sm
a c
on
ce
ntra
tio
ns
0 50 100 150 2000
5000
10000
15000
20000
25000
PIs
CSF concentrations
0 1000 2000 3000 4000 50000
2000
4000
6000
8000
10000
NNRTIs
CSF concentrations
Pla
sm
a c
on
ce
ntra
tion
s
0 100 200 3000
5000
10000
15000
20000
25000
CSF concentrations
INSTIs
Rho=0.78P<0.001
Rho=0.72P<0.001
Rho=0.18P=0.115
Rho=0.52P<0.001
A
g
e
NNRTIsNRTIs
INSTIsPIs
BBB and inflammationrho= 0.178, p=0.002
NRTIs
p=0.003
INSTIs
p=0.012
PIs
p=0.088
rho= 0.235, p=0.034
Single nucleotide polymorphisms
NRTIs PIs INSTIs
CSF CPR CSF CPR CSF CPR
ABCB1
rs1045642
rs1128503
rs2032582
ABCC2 rs717620 0.043
SLC22A6 rs4149170 0.065
SLCO1A2rs10841795
rs11568563
ABCG2rs2231142
rs13120400
HNF4α rs1884613 0.020
p value set at 0.005
CSF NRTIs CSF PIs CSF INSTIs
Model1
R2=0.297
p<0.001
Model2
R2=0.353
p<0.001
Model1
R2=0.333
p<0.001
Model2
R2=0.341
p<0.001
Model1
R2=0.450
p<0.001
Model2
R2=0.173
P=0.029
Age
1 year increase0.018 0.003 0.108 0.099 0.913 0.968
CSAR
1 unit increase0.068 0.046 0.998 - 0.350 0.437
Neopterin
1 ng/mL increase0.661 0.771 <0.001 <0.001 0.757 -
Plasma conc.
1 ng/mL increase<0.001 <0.001 0.001 0.001 <0.001 0.020
PI coadministration
yes vs. no0.778 0.397 - - - -
SLCO1A2 516
AC vs. AA- 0.052 - - - -
ABCG2 421
CA/AA vs. CC- 0.014 - - - -
ABCC2 -24
AA/AC vs. CC- - - - - 0.056
Multivariate Linear Regression Analysis
Conclusions
• Several limitations including heterogeneous time after dosing (however mostly “flat” CSF PK), a limited number of samples (NNRTIs and EVG) and different drugs within the same class
• A significant variability in CSF concentrations was explained by plasma concentrations, age/BBB permeability (NRTIs) and CSF immune activation (PIs)
Conclusions and Discussion
• The effect of SNPs in transporters was small but including ABCG2 (BCRP) improved the model performance for NRTIs
• The effect of immune activation, BBB permeability and SNPs needs to be considered when modelling antiretrovirals’ exposure in the CNS
Acknowledgements
Prof. G Di PerriProf. S BonoraLaura TrentiniCristina TettoniRoberto BertucciSabrina AudagnottoLetizia MarinaroIlaria MottaAlice TrentalangeElisabetta ScarvaglieriElisa ScabiniChiara Cardellino
Antonio D’AvolioJessica CusatoMarco SimieleAmedeo de Nicolò
Daniele ImperialeCristiana AtzoriDaniela VaiAlessandra Romito
Prof. P CassoniLuca Bertero Consuelo Valentini
Prof. R SwanstromSarah B JosephLaura P Kincer
Enrica AmasioSebastiano Catera
Valeria GhisettiTiziano Allice