Antiretroviral Drug Transporters and Metabolic Enzymes in Human Testicular TissueBilly Huang1, Md. Tozammel Hoque1, Kishanda Vyboh2, Mohammad-Ali Jenabian3, Nancy Sheehan4, Pierre Brassard5, Maud Bélanger5, Nicolas Chomont6, Jean-Pierre Routy2, and Reina Bendayan1

1Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, Toronto, Canada. 2Chronic Viral Illness Service, McGill University Health Centre, Montréal, Canada. 3Department of Biological Sciences, Université du Québec à Montréal (UQAM) Montréal, Canada. 4Québec Antiretroviral Therapeutic Drug Monitoring Program, McGill University Health Centre, Montréal, Canada. 5Metropolitan Centre of Plastic Surgery, Montréal, Canada. 6Vaccine and Gene Therapy Institute of Florida, Florida, USA.

Recent studies have suggested that low drug permeability in tissue sanctuary sites could contribute to persistent HIV-1 infection (Fletcher et al., PNAS, 2014). Drug distribution into tissues is regulated by a dynamic interplay between ATP-binding Cassette (ABC) drug efflux transporters, solute-carrier (SLC) drug influx transporters and drug metabolic enzymes i.e., cytochrome P450 (CYP450). These proteins interact with many antiretroviral drugs (ARVs) used both in first line and alternative treatment regimens (Kis et al., TIPS, 2010) and their expression at key tissue barrier sites can result in sub-therapeutic ARV tissue concentrations, possibly contributing to the formation of viral reservoirs.

Very few studies have characterized the expression and localization of ABC transporters, SLC transporters and CYP450 metabolic enzymes in human testicular tissue. The presence of these proteins both in the testes and at the blood-testis barrier (BTB) could result in poor ARV penetration and contribute to the formation of a sanctuary site for HIV-1 in this tissue. This study aims to characterize the expression and localization of key drug transporters and metabolic enzymes in the testes and quantify ARV concentrations in testicular tissue relative to blood in order to better understand ARV permeability in this tissue.

The expression of drug transporters and metabolic enzymes in the testes of HIV-1 infected men is associated with a decrease in ARV concentrations in testicular tissue compared to blood plasma.

Figure 1. qPCR analysis of selected ABC transporters, SLC transporters and CYP450 metabolic enzymes known to be involved in the disposition of ARVs, in testicular tissue collected from eight uninfected and five HIV-1 infected, treated men. The results are expressed as mean relative mRNA expression +/- S.E.M. normalized to the housekeeping gene GAPDH.

Figure 2: Western blots of selected ABC transporters, SLC transporters and CYP450 metabolic enzymes known to be involved in the disposition of ARVs, in testicular tissue collected from five uninfected and five HIV-1 infected, treated men.

Figure 3: Immunofluorescence localization of selected ABC transporters, SLC transporters and CYP450 metabolic enzymes known to be involved in the disposition of ARVs.

Table 2. Drug concentrations in plasma and testes of HIV-infected, treated individuals. TFV: tenofovir, FTC: emtricitabine, 3TC: lamivudine, EFV: efavirenz, ATV: atazanavir, DRV: darunavir, RTV: ritonavir. Testicular tissue concentrations are expressed as ng/mL of testicular tissue, with the assumption that 1g of tissue is equivalent to 0.96mL.

Table 1. Patient Characteristics

We observed high gene expression of MRP1 and OATP2B1, moderate expression of BCRP, and low expression of P-gp, MRP2, OATP1A2, OATP1B1, OAT1, OCT1, CNT1, ENT2, CYP2D6 and CYP3A4. However, we detected protein expression for all transporters and metabolic enzymes analysed with the exception of OATP1A2 and OCT1. Overall, gene and protein expression levels did not differ significantly between the uninfected and HIV-infected, treated patient groups, and showed significant inter-individual differences. Fluorescence microscopy results also demonstrated that these proteins are not only localized at the BTB but can be found throughout the testicular tissue.

We also quantified ARV concentrations in both the plasma and testicular tissues of HIV-infected, treated patients. Overall, we obtained similar [plasma] and [testes] concentrations for tenofovir, lamivudine and emtricitabine, higher [testes] for ritonavir, and lower [testes] for efavirenz, atazanavir and darunavir. The differences we observe between [plasma] and [testes] appear to reflect predicted drug-drug interactions in the context of our drug transporter and metabolic enzyme data.

We would like to thank Dr. Courtney Fletcher and his laboratory members, especially Lee C. Winchester for helping us generate the ARV quantification data in plasma and testicular tissues.

This study is supported by a CIHR Catalyst operating grant. Dr. Reina Bendayan is a Career Scientist of the OHTN. Mr. Billy Huang is a recipient of an Ontario Graduate Scholarship (OGS) award.

Study PopulationIn collaboration with plastic surgeons Dr. P Brassard and Dr. M Belanger from the Metropolitan Centre for Plastic Surgery in Montréal, we have obtained testicular tissues from eight uninfected men and five HIV-1 infected, HAART-treated men electing to undergo orchiectomy for gender modification.

Tissue ProcessingDr. Routy’s research group at McGill University in Montréal, Quebec, collected and processed blood and testicular samples from enrolled patients. Samples were cut into small pieces, snap frozen in liquid nitrogen and stored at -80°C until used.

ABCB1

(P-g

p)ABCC1

(MRP1)

ABCC2

(MRP2)

ABCG2

(BCRP)

0.00

0.01

0.02

0.03

0.04

0.050.1

0.2

0.3

0.4

0.5

Expr

essi

onR

elat

ive

to G

APD

H

ABC Transporters

UninfectedInfected

SLCO1A2

(OATP1A

2)

SLCO1B1

(OATP1B

1)

SLCO2B1

(OATP2B

1)

SLC22A6

(OAT1)

SLC22A1

(OCT1)

SLC28A1

(CNT1)

SLC29A2

(ENT2)

0.000

0.001

0.002

0.003

0.004

0.005

0.1

0.2

0.3

0.4

Expr

essi

on

Rel

ativ

e to

GA

PDH

SLC Transporters

UninfectedInfected

CYP3A4

CYP2D6

0.0000

0.0005

0.0010

0.0015

0.0020

CYP450 Metabolic Enzymes

Expr

essi

on

Rel

ativ

e to

GA

PDH

UninfectedInfected

Cell CultureAll cell lines used as positive controls for immunoblots (MDA-MDR1, MX100, HeLa-MRP1, MDCKII-OATP2B1, HEK-WT, HepG2) were cultured according to previously published protocols in our laboratory (Robillard et al., JPET, 2012; Banerjee et al., JPET, 2012) and maintained at 37°C humidified 5% CO2-95% air with fresh media replaced every 2-3 days. Cells were subcultured with 0.25% trypsin-EDTA upon reaching 80 – 90% confluency.

Quantification of Selected Drug Transporters and Metabolic EnzymesDrug transporters and metabolic enzymes known to be involved in ARV disposition were examined in this study. Gene expression was quantified using TaqMan qPCR. Total RNA was prepared from frozen testicular tissue using Trizol as reported previously (Banerjee et al., JPET, 2012). Validated TaqMan primers were used to probe for the genes of interest and expression

levels were normalized to the GAPDH housekeeping gene.

Protein quantification was assessed applying immunoblotting. Whole tissue lysates were extracted from frozen testicular samples using a modified RIPA lysis buffer as reported previously (Robillard et al., JPET, 2012). Protein expression levels of drug transporters and drug metabolic enzymes were determined using commercially available primary antibodies and their corresponding HRP-labelled secondary antibodies. Cell-lines previously shown to overexpress the proteins of interest were used as positive controls and ß-actin was used as a loading control.

Localization of Selected Drug Transporters and Drug Metabolic EnzymesLocalization of proteins was assessed using immunofluorescence confocal microscopy. A

sample of testicular tissue was fixed in 10% neutral buffered formalin for 48 hours and then fixed in paraffin wax before it was sectioned and mounted on glass slides. Primary antibodies for the proteins of interest were used to probe the mounted sections along with the appropriate corresponding fluorescently labelled secondary antibody. Confocal microscopy work was performed at the Advanced Optical Microscopy Facility at the Toronto MARS Discovery Tower using a LSM700 confocal microscope (Zeiss).

ARV Quantification using LCMS/MSTesticular tissue lysates and plasma samples from HIV-infected, treated patients were analysed in Dr. C. Fletcher’s laboratory according to previously validated methods (Fletcher et al., PNAS, 2014). Briefly, tissue lysates and plasma were extracted using 70:30 methanol/water, mixed with 13C internal standards (IS), and proteins were precipitated out using an

acetonitrile preparation step. Final extracts were quantified using a Shimadzu Nexera ultra high-performance liquid chromatograph attached to an AB Sciex 5500 qTrap mass spectrometer. Ion pairs (TFV/FTC/ATV/DRV/RTV, positive; EFV, negative) were monitored in multiple reaction-monitoring mode. Analyte peaks were normalized to the corresponding IS peak, with the exception of EFV, for which no IS was available and was instead normalized to the FTC-IS. The linear range of quantification for each analyte was 2 – 400 fmol total mass on column, with the exception of ATV, which was 0.2 – 40 fmol total mass on column. To compare between [plasma] and [testes] in ng/mL, we assumed that 1g of tissue was equivalent to 0.96mL (Mardirossian et al., J. Nucl. Med, 1999).

METHODS

HYPOTHESIS

RATIONALE &GOALS

SUMMARY &CONCLUSIONS

BACKGROUND

RESULTS

qPCR Results Immunoblotting Results Immunofluorescence Results - Uninfected Immunofluorescence Results - HIV-1 Infected ARV Quantification Results

534

Patient ID

6

7

8

9

10

Drug

DRVRTV

TFVFTC

3TCEFV

3TC

DRVRTV

TFV3TCATVRTV

2043.748.9

61.7214.4

207.87448.9

125.8

2375.8236

51.01136.71300.2271.8

396.594389.294

44.71251.786

147.0951856.445

140.362

523.109683.824

43.51221.3391029.762523.81

[Plasma](ng/mL)

[Testes](ng/mL)

ACKNOWLEDGEMENTS

Patient ID

AgeViral Load

(copies/mL)ARV Regimen Other Drugs

6 46

<40

ritonavir, darunavirmaraviroc

dolutegravircitalopram

7 27

<40

emtricitabine, tenofovirrilpivirine

cyproterone acetatefolic acidestradiol

8 N/A <40 abacavir, lamivudineefavirenz

levothyroxineestradiol

9 24

<40

ritonavir, darunavirlamivudine, zidovudine

raltegravirN/A

10 47

<40

ritonavir, atazanavirtenofovir, abacavir, lamivudine

N/A