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\begin{document}

\title{Inflammation and Intracellular Exposure of Dolutegravir, Darunavir,
Tenofovir and Emtricitabine in People living with HIV}



\author[1]{Micol Ferrara}%
\author[1]{Elena Salvador }%
\author[2]{Alice Trentalange}%
\author[3]{Chiara Alcantarini}%
\author[4]{Mattia Trunfio}%
\author[5]{Elvira Stefania Cannizzo}%
\author[6]{Valeria Bono}%
\author[7]{Silvia Nozza}%
\author[1]{Amedeo De Nicolò}%
\author[2]{Alice Ianniello}%
\author[2]{Elisa De Vivo }%
\author[1]{Antonio D'Avolio}%
\author[2]{Giovanni Di Perri}%
\author[8]{Stefano Bonora}%
\author[5]{Giulia Marchetti }%
\author[4]{Andrea Calcagno}%
\affil[1]{University of Turin}%
\affil[2]{Department of Infectious Diseases, University of Torino}%
\affil[3]{Università degli Studi di Torino}%
\affil[4]{University of Torino}%
\affil[5]{University of Milan}%
\affil[6]{Affiliation not available}%
\affil[7]{San Raffaele Hospital}%
\affil[8]{Department of Infectious Disases, University of Torino}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: Antiretroviral therapy reduces systemic inflammation and
immune activation, but not to levels like HIV-negative. Limited drug
penetration within tissues has been argued as potential mechanism of
persistent inflammation. Data on the role of inflammation on
plasma/intracellular (IC) pharmacokinetics (PK) of ARV drugs through to
downregulation/expression of cytochrome P450 3A/membrane transport
proteins are limited. Aim of this study was to investigate the
correlation between inflammation markers and plasma/IC PK of different
ARVs regimen in HIV-positive patients. Methods: We included in the study
ART-treated HIV+ pts switching to 3 different ARV regimens: 1) DTG-based
dual-therapy plus boosted-PIs, 2) DTG-based triple-therapy without PIs,
3) DRV/c-based triple-therapy. Plasma and IC ARV drugs concentration
means at the end of dosing interval (T0), IM on samples concomitantly
with ARV PK determination: sCD14, CRP, IL-6 and LPS were analysed.
Results: 60 samples from pts included in the switching study were used
for measuring plasma and IC concentrations of HIV drugs. No
significative differences between CRP, sCD14, IL-6 and LPS values in 3
arms of therapy were observed. Significant correlation was observed
between tenofovir plasma concentrations and sCD14 (p\textless{}0.001),
DRV plasma concentration and sCD14 (p=0,07) and DRV IC/plasma ratio and
Log10 IL-6 concentrations (p=0.04). Furthermore, in 24 pts on DTG-TT, we
observed a negative trend between DTG IC concentrations and sCD14
(p=0.09). Conclusions: Our preliminary data support the hypothesis of
lower IC concentrations of DRV and DTG in pts with higher plasma IM,
suggesting an interplay between HIV drug penetration and persistent
inflammation in cART-treated HIV-positive patients.%
\end{abstract}%



\sloppy


Inflammation and Intracellular Exposure of Dolutegravir, Darunavir,
Tenofovir and Emtricitabine in People living with HIV

\selectlanguage{ngerman}\textbf{M. Ferrara\textsuperscript{1}*, E. Salvador\textsuperscript{1},
A. Trentalange\textsuperscript{1}, C. Alcantarini\textsuperscript{1}, M.
Trunfio\textsuperscript{1},E. S. Cannizzo\textsuperscript{2}, V.
Bono\textsuperscript{2}, S. Nozza\textsuperscript{3}, A. De
Nicolò\textsuperscript{4}, A. Ianniello\textsuperscript{4}, E. De
Vivo\textsuperscript{4},} \textbf{A. D'Avolio\textsuperscript{4}, G. Di
Perri\textsuperscript{1}, S. Bonora\textsuperscript{1}, G.
Marchetti\textsuperscript{2}, A. Calcagno\textsuperscript{1}}1.Unit of
Infectious Diseases, Department of Medical Sciences, University of
Turin, 10149 Turin, Italy; 2. Department of Health Sciences, Clinic of
Infectious Diseases, ASST, Santi Paolo e Carlo, University of Milan,
20142 Milan, Italy ; 3. Department of Infectious Diseases, San Raffaele
Scientific Institute, 20127 Milan, Italy; 4. Laboratory of Clinical
Pharmacology and Pharmacogenetics, Department of Medical Sciences,
University of Turin, 10149 Turin, Italy. *Corresponding
author:\emph{micol.ferrara29@gmail.com}. Department of Medical Sciences,
University of Turin, Corso Svizzera 164, 10149 Turin, Italy WHAT IS
ALREADY KNOWN ABOUT THIS SUBJECT A valid intracellular penetration of
antiretroviral drugs is required to reach drugs target for virological
control. Penetration within reservoirs and in the intracellular
compartment is mediated by membrane protein drug transporters and during
chronic and systemic inflammation defence mechanisms are enhanced such
as higher expression of drug transport membrane protein responsible of
the uptake/excretion of antiretroviral drug with a potential reduction
of antiretroviral concentration within cells. In this study we wanted to
better evaluate the role of markers of inflammation on intracellular
antiretroviral concentration. WHAT THIS STUDY ADDS We wanted to explore
the potential interplay between inflammation and antiretroviral
pharmacokinetics. Our preliminary results show for the first time how
the penetration of some of the antiretrovirals in study is significantly
lower in presence of higher plasma inflammation
biomarkers.\textbf{Background}: Antiretroviral therapy reduces systemic
inflammation and immune activation, but not to levels like HIV-negative.
Limited drug penetration within tissues has been argued as potential
mechanism of persistent inflammation. Data on the role of inflammation
on plasma/intracellular (IC) pharmacokinetics (PK) of ARV drugs through
to downregulation/expression of cytochrome P450 3A/membrane transport
proteins are limited. Aim of this study was to investigate the
correlation between inflammation markers and plasma/IC PK of different
ARVs regimen in HIV-positive patients.\textbf{Methods}: We included in
the study ART-treated HIV+ pts switching to 3 different ARV regimens: 1)
DTG-based dual-therapy plus boosted-PIs, 2) DTG-based triple-therapy
without PIs, 3) DRV/c-based triple-therapy. Plasma and IC ARV drugs
concentration means at the end of dosing interval (T0), IM on samples
concomitantly with ARV PK determination: sCD14, CRP, IL-6 and LPS were
analysed.\textbf{Results}: 60 samples from pts included in the switching
study were used for measuring plasma and IC concentrations of HIV drugs.
No significative differences between CRP, sCD14, IL-6 and LPS values in
3 arms of therapy were observed. Significant correlation was observed
between tenofovir plasma concentrations and sCD14 (p\textless{}0.001),
DRV plasma concentration and sCD14 (p=0,07) and DRV IC/plasma ratio and
Log\textsubscript{10} IL-6 concentrations (p=0.04). Furthermore, in 24
pts on DTG-TT, we observed a negative trend between DTG IC
concentrations and sCD14 (p=0.09).\textbf{Conclusions}: Our preliminary
data support the hypothesis of lower IC concentrations of DRV and DTG in
pts with higher plasma IM, suggesting an interplay between HIV drug
penetration and persistent inflammation in cART-treated HIV-positive
patients.\textbf{Background}Despite the antiretroviral therapy
(ART)-associated increase in life expectancy, people living with HIV
(PLWH) continue to have increased morbidity and mortality compared to
the general population, often due to non-AIDS-related events (1-3).
There is evidence that heightened systemic inflammation and persistent
immune activation, including monocyte activation, play an important role
in the pathogenesis of various diseases (4-5). Reducing viremia below
the limits of detection, regardless of the timing of ART initiation, has
been associated with decline in immune activation (6), even though to an
extent still higher than what is observed in accurately matched
controls. Aim of ART is to maintain a persistent plasma undetectability
and a valid penetration within intracellular (IC) compartment.
Antiretroviral (ARV) drug targets are mostly intracellular, and
different IC concentrations have been associated with distinct antiviral
effects. The uptake and excretion of endogenous and exogenous compounds
is expressed in the intestine, liver and kidney, and contribute to the
intestinal absorption, liver metabolism and renal secretion of ionic
drugs mediated respectively by drug transporters, belonging to solute
carriers (SLC) or ATP-binding cassette (ABC) transporters. (7) The
former is mediated in part by SLC transporters, such as organic cation
transporter (OCT) (8,9) organic anion transporter (OAT) (10,11) and
organic anion transporting polypeptides (OATP1B1, OATP1B3, and OATP2B1)
(12,13) located at the basolateral membrane of cells, while the latter
is mediated by ABC transporters, such as P-glycoprotein (P-gp) encoded
by multidrug resistance (MDR) 1 gene, multidrug resistance associated
protein (MRP) 2, and breast cancer resistance protein (BCRP), located at
the apical membrane of the cells (14). These results indicate that the
expression levels as well as single nucleotide polymorphisms (SNPs) of
drug transporters are responsible for individual variation in
pharmacokinetics (15). Efflux transporters, such as P-gp, often protect
from treatment cellular and anatomical reservoirs causing a decrease in
IC drug concentrations. During chronic inflammation, cellular protective
mechanisms are usually enhanced, such as with a higher expression of
P-gp or organic anion transporters, and they have been associated with
lower ARVs concentrations within cells, despite virological suppression
(16). Even expression changes of many hepatic drug transporter genes
have been observed in some pathophysiological conditions such as
inflammation-induced primary biliary cirrhosis (PBC), and obstructive
cholestasis. \textbf{(}16). Limited drug penetration within tissues and
presence of immune sanctuaries has been argued as potential mechanisms
of persistent inflammation (17). Few data are available on the role of
inflammation on plasma and intracellular (IC) pharmacokinetics (PK) of
ARV drugs through to downregulation of cytochrome P450 3A (Cyp 3A4),
protein expression\textsuperscript{1} and suppression of expression of
P-gp in several tissues. (18) Therefore, aim of this study was to
investigate the correlation between inflammation markers (IM), microbial
translocation markers and plasma and IC PK of different ARVs regimen in
stable HIV-positive patients (pts).\textbf{Material and Methods} We
included in the study PLWH on stable ARV therapy with plasma HIV RNA
\textless{}50 copies/mL since at least six months enrolled in different
switch studies; all protocols received Ethics Approval and participants
signed a written informed consent before being enrolled. Three different
ARV regimens were assessed: i) Dolutegravir based dual therapy (DTG-DT)
plus boosted Protease Inhibitors {[}PI, Atazanavir (ATV) or Darunavir
(DRV){]}, ii) DTG based triple therapy (DTG-TT; without PI), and iii)
DRV/c based triple therapy (DRV/c-TT). Pharmacokinetic analyses on
plasma and intra-PBMCs ARV drugs concentration at the end of dosing
interval (C\textsubscript{trough}) were performed at the Laboratory of
Clinical Pharmacology and Pharmacogenetics, University of Turin at the
``Amedeo di Savoia'' Hospital using UHPLC-MS/MS validated methods. We
measured C-reactive protein (CRP), Inteleukin-6 (IL-6), soluble CD14
(sCD14) and Lipopolysaccharide (LPS) on frozen serum samples collected
on the same day of PK measurement. For inflammation biomarkers analysis,
samples were acquired on a Labscan 200 analyzer (Luminex, Austin, Texas)
using Bio-Plex manager software (Bio-Rad, Hercules, California).
Baseline demographics, clinical and immunological characteristics were
described using median values (interquartile ranges) for continuous
variables and number (percentage) for categorical variables.
Non-compartmental plasma and IC PK parameters were calculated and
reported as ng/ml and expressed as geometric means (with 95\% confidence
intervals, 95-CI). Non parametric analysis were performed for bivariate
correlations (Spearman's rank correlation); all statistical analyses
were performed using SPSS Statistics version 23 (IBM). Tests were all
two-sided, and a~\emph{p}-value \textless{}.05 was considered
statistically significant.\textbf{Results} Sixty pts were enrolled,
respectively divided in three groups: 9 on DTG-DT, 25 on DTG-TT and 26
on DRV/c-TT. Participants were mostly male (80\%), with median age and
BMI of 53 years (48-59) and 23 Kg/m\textsuperscript{2} (20-24). Median
time on ART exposure and time from HIV diagnosis were respectively 22,5
(16-29) and 17,5 (12-23) years. At the time of the analysis 78,3\% of
pts had undetectable viremia (HIV-RNA\textless{} 20 cp/mL) and
lymphocytes CD4+ 598 (417-754) cells/\selectlanguage{greek}µ\selectlanguage{english}L, 29 (20-38)\% and, CD4+/CD8+
ratio 0,8 (0,4-1,1). 83\% of pts presented at least one comorbidity, in
particular 33\% hypertension, 46\% dyslipidemia, 26\% hipovitaminosis D,
6\% diabetes mellitus type 2, 10\% chronic kidney impairment. Median DTG
and DRV plasma, IC and ratio IC/plasma PK were respectively 1308,4
(808,2-1808,6) ng/ml, 230.0 (123.0-336,8) ng/mL, ratio 0,174
(0,127-0,221) and 1885,2 (1316,9-2453,4) ng/ml, 829,0 (489,5-1168,4)
ng/mL and ratio 0,436 (0,283-0,588) (Table 1 with baseline and PK
characteristics according to study arm). Median IM markers as sCD14,
IL-6, CRP and LPS in DTG-TT, DTG-DT and DRV/c-TT arms were respectively
1.33 (1.08-1.58), 1.49 (1.29-1.69) and 1.39 (1.25-1.52); 2.33
(0.82-3.85), 2.67 (2.15-3.20) and 2.70 (0.52-4.88); 3.08 (0.97-5.2),
3.12 (1.03-5.20) and 2.78 (0.33-5.22); 180.6 (115.3-245.9), 163.0
(113.3-212.7) and 171,6 (127.0-216.3) (Supplementary Table 1 --
stratified by study arm) No significative difference in biomarkers among
study arms was observed. We observed a significant inverse correlation
between tenofovir and sCD14 concentrations (rho=-0.79,
p\textless{}0.001), between DRV and sCD14 concentrations (rho=0,31,
p=0,07) and between DRV IC/plasma ratio and Log\textsubscript{10}
transformed IL-6 concentrations (rho= -0.36, p=0.04). In the 34
participants receiving DTG the drug IC concentrations were associated
with age (rho=0.4, p=0.01). Furthermore, in 24 pts on DTG-TT, we
observed a trend towards lower IC DTG concentrations with higher sCD14
levels (rho=-0.34, p=0.09). Table 2 summarizes bivariate correlations
(with rho and p values) while Figure 1 includes scatter-dot plots of
these 4 described correlations.\textbf{Discussion}In our study we
observed a correlation between some inflammation biomarkers and plasma
and IC concentrations of the ARVs we assessed. In particular, sCD14
resulted to have different correlation with different ARVs: inverse and
strongly significant with TDF plasma concentration, an inverse trend
with DTG IC concentration when dosed with backbone in DTG-TT and a
linear trend with DRV plasma concentration. Also IL-6 resulted to have
an inverse and significant correlation with DRV IC/plasma ratio. By
showing a negative correlation between plasmatic inflammatory biomarkers
and ARV concentration, our study would altogether suggest a possible
negative effect of a pro-inflammatory milieu on intracellular
penetration of ARVs. Few manuscripts has so far analyzed the
interactions between inflammation biomarkers and ARVs PKs, mostly
focusing on ARVs penetration within tissue and its impact on viral
parameters such as residual HIV viremia and reservoir (19) Moreover,
data on the influence of different ART regimens on inflammation and
immune activation have been controversial so far (20. Various authors
reported different and mixed results on changes in systemic and vascular
inflammation, monocyte activation, T-cell senescence and inflammation
biomarkers based on the exposure to different ART regimens, as the
comparison between a INSTI- vs NNRTI or PI-based ART (21-24). One of the
potential mechanisms of HIV persistence is ongoing viral replication
despite ART which can result from insufficient penetration of a number
of antiretroviral drugs into tissues and anatomic sanctuaries (25).
Importantly, heightened immune activation and inflammation has been
previously suggested as both a cause and a consequence of HIV reservoir
persistence and residual viral replication, mainly in tissues (26, 27).
In chronic inflammatory conditions cellular protective mechanism are
usually raised, such as a higher expression P-gP or organic anion
transporters, and they have been associated with lower ARVs
concentrations within cells. Moreover na\selectlanguage{ngerman}ïve and memory T cells have been
shown to differently express P-gP: those with higher expression can
eliminate intracellular Raltegravir faster than those with low
expression (16). In microglial cells, a dose dependent decrease in
Saquinavir accumulation following treatment with LPS-induced
inflammation was observed and on the other hand an accumulation of the
same drug was increased significantly by a potent P-glycoprotein
inhibitor (PSC833), confirming that the presence of neuroinflammation
within the brain parenchymal compartment can exacerbate the ability to
extrude antiretroviral agents from glial cells (28). Another instance
has been shown in the context of liver cirrhosis, where a significantly
decreased expression of mRNA levels of some drug transporters including
OCT1 and OATP1B1 has been observed in cirrhotic (F4) versus
non-cirrhotic patients (F0-F3).The reason could be explained with the
presence of elevated levels of reactive oxygen species (ROS) and
decreased antioxidant levels during HCV infection. In particular, ROS
production within hepatocytes leads to the release of proinflammatory
cytokines, such as tumor necrosis factor (TNF)-alpha, in turn decreasing
mRNA levels of some SLC transporters including OCT1, OATP1B1, and
OATP1B3, suggesting that TNF-a is in part responsible for the
downregulation of SLC transporters in HCV-related cirrhosis (16). Our
preliminary data support the hypothesis of lower IC concentrations of
the ARVs analysed in patients with higher plasma IM, suggesting an
interplay between HIV drug penetration and persistent inflammation in
cART- treated HIV positive pts. We want to acknowledge the limitations
of this work: the small sample size of the included participants was
limited to those who underwent a TDM and inflammatory biomarkers
analysis at the time of regular follow-up visits, retrospectively
collected. Neither data on duration of virological suppression nor
burden of comorbidities were available for the analysis. In the context
of HIV curative interventions it will be important to include
inflammatory biomarkers concomitantly to ARVs penetration in order to
assess the role of inflammation/microbial translocations in HIV
persistence and include appropriate interventions in such
studies.\textbf{Data availability statement}Research data are not
shared\textbf{Funding} This work was supported by Italian Ministry of
Health -- ``Ricerca Finalizzata 2013'' Grant. The funding sponsors had
no role in: the design of the study; the collection, analyses or
interpretation of data; the writing of the manuscript; or the decision
to publish the results.\textbf{Transparency Declaration} None to declare
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