Discrepancies between protease inhibitor concentrations and viral load in reservoirs and sanctuary sites in human immunodeficiency virus-infected patients.

Abstract

The variable penetration of antiretroviral drugs into sanctuary sites may contribute to the differential evolution of human immunodeficiency virus (HIV) and the emergence of drug resistance. We evaluated the penetration of indinavir, nelfinavir, and lopinavir-ritonavir (lopinavir/r) in the central nervous system, genital tract, and lymphoid tissue and assessed the correlation with residual viral replication. Plasma, cerebrospinal fluid (CSF), semen, and lymph node biopsy samples were collected from 41 HIV-infected patients on stable highly active antiretroviral therapy regimens to determine drug concentrations and HIV RNA levels. When HIV RNA was detectable, sequencing of the reverse transcriptase and protease genes was performed. Ratios of the concentration in semen/concentration in plasma were 1.9 for indinavir, 0.08 for nelfinavir, and 0.07 for lopinavir. Only indinavir was detectable in CSF, with a concentration in CSF/concentration in plasma ratio of 0.17. Differential penetration into lymphoid tissue was observed, with concentration in lymph node tissue/concentration in plasma ratios of 2.07, 0.58, and 0.21 for indinavir, nelfinavir, and lopinavir, respectively. HIV RNA levels were <50 copies/ml in all CSF samples of patients in whom HIV RNA was not detectable in plasma. HIV RNA was detectable in the semen of three patients (two patients receiving nelfinavir and one patient receiving lopinavir/r), and its detection was associated with multiple resistance mutations, while the viral load in plasma was undetectable. HIV RNA was detectable in all lymph node tissue samples. Differential drug penetration was observed among the three protease inhibitors in the sanctuary sites, but there was no correlation between drug levels and HIV RNA levels, suggesting that multiple factors are involved in the persistence of viral reservoirs. Further studies are required to clarify the role and clinical relevance of drug penetration in sanctuaries in terms of long-term efficacy and drug resistance.

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2003, p. 238–243
0066-4804/03/$08.00?0 DOI: 10.1128/AAC.47.1.238–243.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 1
Discrepancies between Protease Inhibitor Concentrations and Viral
Load in Reservoirs and Sanctuary Sites in Human
Immunodeficiency Virus-Infected Patients
Caroline Solas,1Alain Lafeuillade,2* Philippe Halfon,3Ste ´phane Chadapaud,2
Gilles Hittinger,2and Bruno Lacarelle1
Laboratory of Pharmacokinetics, University Hospital,1and Laboratory of Virology Alphabio,3Marseilles,
and Department of Infectious Diseases, General Hospital, Toulon,2France
Received 12 December 2001/Returned for modification 17 August 2002/Accepted 16 October 2002
The variable penetration of antiretroviral drugs into sanctuary sites may contribute to the differential
evolution of human immunodeficiency virus (HIV) and the emergence of drug resistance. We evaluated the
penetration of indinavir, nelfinavir, and lopinavir-ritonavir (lopinavir/r) in the central nervous system, genital
tract, and lymphoid tissue and assessed the correlation with residual viral replication. Plasma, cerebrospinal
fluid (CSF), semen, and lymph node biopsy samples were collected from 41 HIV-infected patients on stable
highly active antiretroviral therapy regimens to determine drug concentrations and HIV RNA levels. When HIV
RNA was detectable, sequencing of the reverse transcriptase and protease genes was performed. Ratios of the
concentration in semen/concentration in plasma were 1.9 for indinavir, 0.08 for nelfinavir, and 0.07 for
lopinavir. Only indinavir was detectable in CSF, with a concentration in CSF/concentration in plasma ratio of
0.17. Differential penetration into lymphoid tissue was observed, with concentration in lymph node tissue/
concentration in plasma ratios of 2.07, 0.58, and 0.21 for indinavir, nelfinavir, and lopinavir, respectively. HIV
RNA levels were <50 copies/ml in all CSF samples of patients in whom HIV RNA was not detectable in plasma.
HIV RNA was detectable in the semen of three patients (two patients receiving nelfinavir and one patient
receiving lopinavir/r), and its detection was associated with multiple resistance mutations, while the viral load
in plasma was undetectable. HIV RNA was detectable in all lymph node tissue samples. Differential drug
penetration was observed among the three protease inhibitors in the sanctuary sites, but there was no
correlation between drug levels and HIV RNA levels, suggesting that multiple factors are involved in the
persistence of viral reservoirs. Further studies are required to clarify the role and clinical relevance of drug
penetration in sanctuaries in terms of long-term efficacy and drug resistance.
Highly active antiretroviral therapy (HAART) has consid-
erably decreased the rates of morbidity and mortality among
patients infected with human immunodeficiency virus (HIV)
(22). However, therapeutic failure is observed in up to half of
patients after 2 to 3 years of HAART (19). The reasons for
virologic failure are multiple, including adherence problems
and pharmacological factors leading to the presence of sub-
therapeutic concentrations and, consequently, viral resistance
(5, 8). The effects of HAART are usually assessed by use of
blood samples, although several anatomical compartments or
sanctuary sites have been described as viral reservoirs, in which
viral evolution may differ from that in plasma (2, 3, 7, 10, 12,
15, 18, 24, 26). The main sanctuary sites are the central nervous
system, genital tract, and lymphoid tissue. The viral loads and
resistance profiles in these compartments have been described
to be discordant from those in plasma (1, 4, 14, 27, 29).
Therapeutic failure may hence be caused by inefficient drug
penetration in these compartments; variable protease inhibitor
(PI) diffusion in sanctuary sites may contribute to sustained
HIV type 1 (HIV-1) replication, resistance selection, and a
subsequent failure to control the virus in plasma (6, 9, 21, 31).
To date, few studies have analyzed PI concentrations in the
sanctuary sites; no data are available on lopinavir-ritonavir
(lopinavir/r), the most recently licensed PI, or drug concentra-
tions in lymphoid tissue, despite its major role as a viral res-
ervoir. In this study, we evaluated the penetration of indinavir,
nelfinavir, and lopinavir/r in the plasma, cerebrospinal fluid
(CSF), semen, and lymphoid tissue of HIV-infected patients
and analyzed the correlation with residual viral replication in
each compartment.
MATERIALS AND METHODS
Population. Forty-one adult patients with chronic HIV-1 infection were in-
cluded in this cross-sectional study. All patients had been treated for at least 6
months with a combination of two nucleoside reverse transcriptase (RT) inhib-
itors (NRTIs) plus one PI: indinavir (800 mg three times daily) in 16 patients,
nelfinavir (1,250 mg twice daily) in 13 patients, or lopinavir/r (400 and 100 mg,
respectively, twice daily) in 12 patients. All patients provided written informed
consent, and the protocol was approved by the local ethics committee (Centre
Hospitalier Universitaire Timone, Marseilles, France). Adherence to the HAART
regimen was assessed from pill counts, and only patients with adherence rates
?90% were included in the study.
Sampling schedule. Sample collection was performed on the same day for
each compartment. A plasma sample was drawn just before drug intake, about
8 h after the last indinavir dose, and 12 h after the last nelfinavir or lopinavir/r
dose for the determination of trough levels. CSF and semen samples were
collected through lumbar puncture and masturbation, respectively, 8 to 12 h after
drug administration (trough concentration). A lymph node (LN) biopsy was then
performed surgically in superficial areas 3 to 5 h after drug intake. Three addi-
tional plasma samples were drawn concomitantly with CSF, semen, and LN
tissue collection to enable assessment of the ratios of the concentrations in each
compartment. The collection times as they related to the time of prior drug in-
take were documented carefully. All samples were stored at ?80°C until analysis.
* Corresponding author. Mailing address: Unite ´ Infectiologie, Ho ˆ-
pital Chalucet, Rue Chalucet, F-83056 Toulon, France. Phone: 33 4 94
22 77 41. Fax: 33 4 94 92 67 47. E-mail: avps@club-internet.fr.
238

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Drug concentration analysis. Quantification of indinavir, nelfinavir, lopinavir,
and ritonavir concentrations was performed by a sensitive high-performance
liquid chromatography method with UV detection (13, 32). Indinavir, nelfinavir,
ritonavir, and lopinavir were removed from plasma by liquid-liquid extraction.
Chromatographic separation was performed at 210 nm with an Inertsil ODS2,
5-?m column (4.6 by 150 mm; Precision Instrument, Marseilles, France) for
indinavir, ritonavir, and lopinavir and at 220 nm with a Symmetry C185-?m
column (4.6 by 250 mm; Waters, St. Quentin en Yvelines, France) for nelfinavir.
The intra- and interassay variabilities ranged from 0.97 to 3.58% for indinavir,
11.1 to 12.3% for nelfinavir, 3.22 to 9.35% for ritonavir, and 2.9 to 13.8% for
lopinavir for three quality control concentrations. The quantification limits were
20 ng/ml for indinavir and 50 ng/ml for nelfinavir, ritonavir, and lopinavir. The
overall level of recovery of each PI was high, ranging from 80.8 to 93.3%.
Chromatographic data were recorded and analyzed with a Millenium (version
2.0) software system (Waters).
PI concentrations in total LN tissue were measured. Blood contamination was
avoided by at least two washes with cold 0.9% NaCl before homogenization. LN
biopsy specimens were weighed, rinsed again with 500 ?l of 0.9% NaCl, and
homogenized with 1 ml of 0.9% NaCl. Cellular debris was then removed by
centrifugation, and the resulting supernatant was collected and stored at ?80°C
until analysis. Quantification of indinavir, nelfinavir, ritonavir, and lopinavir in
CSF, semen, and LN tissue samples was carried out by the same extraction and
analytic method used for the plasma samples. Due to the lack of availability of
blank CSF, semen, and LN biopsy samples from healthy donors, a reference
curve could not be set up for each of the biologic samples. Therefore, the
amounts of PIs were calculated from the same standard curve used for the
plasma samples. Preliminary assays with 0.9% NaCl as an alternative matrix led
to the same results. The identities of the peaks in the chromatograms for the
various biologic samples were checked on a liquid chromatograph equipped with
a diode array detector.
HIV-1 RNA levels. HIV-1 RNA levels in plasma, CSF, and semen were mea-
sured by using the Amplicor Monitor kit (Roche Diagnostics, Meylan, France).
When HIV-1 RNA levels were undetectable, the ultradirect procedure was used;
that procedure has a lower limit of detection of 50 copies/ml (25).
CSF samples were frozen at ?80°C after centrifugation to remove cells. Semen
samples were processed systematically within 2 h following collection. Samples
were diluted 1:1 in a 5-mg/ml bromeline solution (Sigma-Aldrich, Saint-Quentin,
France) for semen fluidization and dissociation of cellular aggregates. After 10
min at 37°C, diluted specimens were layered over a two-layer Percoll (Sigma-
Aldrich) gradient consisting of 95 and 47% isotonic Percoll solution, centrifuged
at 300 ? g for 20 min, and then collected. For semen samples with volumes
greater than 1.5 ml, one-third of the volume was centrifuged at 3,600 ? g for 10
min to eliminate any cellular component. The internal standard provided with
the Amplicor Monitor kit was added to the lysis buffer to validate the extraction
and amplification steps, and the Amplicor Monitor kit was used to quantify the
HIV-1 RNA as described previously (28).
LN tissue samples were minced with a scalpel, and the cells were teased out in
RPMI 1640 (Eurobio, Les Ulis, France). The LN mononuclear cells (LNMCs)
were then isolated with Lymphocyte Separation Medium (Eurobio, Les Ulis,
France). To quantify the viral load in the LN tissue samples, the LNMCs were
counted and HIV-1 RNA was obtained from a pellet of 106cells after treatment
with RNA-B (Bioprobe Systems, Montreuil sous Bois, France). HIV-1 RNA
levels were measured by using the Amplicor Monitor kit as reported previously
(16); that kit has a limit of detection of 20 copies/106cells.
HIV-1 RNA genotyping. Sequencing of the RT and protease (PR) genes in
samples with detectable HIV-1 RNA levels was done with the TruGene kit
(Visible Genetics, Toronto, Ontario, Canada) according to the instructions of the
manufacturer.
Statistical analysis. HIV-1 RNA levels were log transformed. Comparison of
mean values was done by the Student t test. Nonparametric tests and the Fisher
exact test were used to compare drug levels and HIV RNA levels in semen and
CSF. Correlation between two variables was done by the Pearson test. A P value
?0.05 was considered significant.
RESULTS
Population. Thirty-five men and six women (median age, 41
years) were included in the study. The median duration of the
present HAART regimen was 32 months, and the median
CD4?-cell count was 286 ? 106/liter. Plasma samples were
obtained from all patients. CSF and semen were collected from
40 of 41 and 34 of 41 patients, respectively. The LN biopsy
failed to recover sufficient material for analysis from two pa-
tients (one patient receiving indinavir and one patient receiv-
ing nelfinavir). For other compartments, when sample material
was limited, virologic tests were carried out prior to pharma-
cological evaluation.
PI levels. (i) Plasma. Median trough concentrations in
plasma were 284 ng/ml (interquartile range [IQR], 144 to 504
ng/ml) for indinavir, 1,878 ng/ml (IQR, 1,355 to 3,211 ng/ml)
for nelfinavir, 5,863 ng/ml (IQR, 3,505 to 7,453 ng/ml) for
lopinavir, and 557 ng/ml (IQR, 341 to 741 ng/ml) for ritonavir.
Large interindividual variability was noted (Fig. 1).
(ii) CSF. The indinavir concentration was measured in the
CSF of all 16 patients. The data for two patients were not used
because CSF collection was done 1 h after drug intake, which
corresponded to the time of the peak level in plasma. For the
14 patients whose data were analyzed, the average CSF sam-
pling time was 7 h (range, 7 to 8 h). Indinavir concentrations in
CSF were variable, with a median value of 73 ng/ml (IQR, 52
to 92 ng/ml). The corresponding value in plasma was 357 ng/ml
(IQR, 155 to 914 ng/ml). The median CSF indinavir concen-
tration/plasma indinavir concentration ratio was 0.17 (IQR,
0.10 to 0.49), and a high degree of interindividual variability
was noted (Table 1). Nelfinavir, lopinavir, and ritonavir were
undetectable in CSF samples from 12 patients receiving nelfi-
navir and all patients receiving lopinavir/r.
(iii) Semen. Semen samples were obtained from all men
receiving indinavir (n ? 12), 7 of 11 men receiving nelfinavir,
and 7 of 11 men receiving lopinavir/r. Semen samples were
collected between 8 and 12 h postdosing according to the PI
prescribed (8 h for patients receiving indinavir and 12 h for
patients receiving nelfinavir or lopinavir/r).
The median concentrations of indinavir, nelfinavir, and lopi-
FIG. 1. Distribution of trough concentrations (Ctrough) of indinavir
(IDV), nelfinavir (NFV), lopinavir (LPV), and ritonavir (RTV) in
plasma.
TABLE 1. Ratios (medians) and IQRs of the concentrations of
indinavir, nelfinavir, lopinavir, and ritonavir in CSF, semen,
and LN tissue compared to those in plasma
Sanctuary
site
Concn ratio (IQR)a
IDV NFVLPVRTV
CSF
Semen
LN tissue 2.07 (1.02–3.67) 0.58 (0.27–0.84) 0.21 (0.15–0.26) 0.64 (0–1.15)
0.17 (0.10–0.49)
1.9 (1.07–3.94)
000
00.08 (0.06–0.10) 0.07 (0.01–0.45)
aIDV, indinavir; NFV, nelfinavir; LPV, lopinavir; RTV, ritonavir.
VOL. 47, 2003 DRUG CONCENTRATION AND HIV LOAD IN SANCTUARY SITES239

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navir in semen were 788 ng/ml (IQR, 213 to 1,046 ng/ml), 159
ng/ml (IQR, 94 to 268 ng/ml), and 166 ng/ml (IQR, 84 to 2,353
ng/ml), respectively. We observed a large interindividual vari-
ability in the level of PI penetration into semen (Fig. 2). Nelfi-
navir could be detected in only six of seven semen samples
analyzed. The corresponding concentrations in plasma were
similar to the trough levels in plasma. The median (IQR)
concentration in semen/concentration in plasma ratios are pre-
sented in Table 1. Ritonavir was detectable in only three of
seven samples at levels ranging from 50 to 149 ng/ml.
(iv) Lymphoid tissue. The average sampling time for LN
tissue was 5 to 6 h (range, 4 to 8 h). The median concentration
in LN tissue/concentration in plasma ratios for indinavir, nelfi-
navir, lopinavir, and ritonavir are shown in Table 1. In LN
tissues, the median indinavir concentration was 1,025 ng/g
(IQR, 755 to 1,108 ng/g) in 13 of 16 LN samples, and the
corresponding value in plasma was 321 ng/ml (IQR, 216 to 587
ng/ml). For nelfinavir, the median concentrations were 740
ng/g (IQR, 278 to 2,175 ng/g) in 12 of 13 LN samples and 1,328
ng/ml (IQR, 1,136 to 3,040 ng/ml) in the corresponding plasma
samples. The median lopinavir concentration measured in 11
of 12 LN tissue samples was 1,260 ng/g (IQR, 1,035 to 1,835
ng/g). The corresponding lopinavir level in plasma was 7,333
ng/ml (IQR, 5,400 to 9,458 ng/ml). Ritonavir, which is cofor-
mulated with lopinavir, was also detected in 7 of 11 LN tissue
samples at a median concentration of 410 ng/g. Large interin-
dividual variability was also found for PI penetration into LN
tissues (Fig. 3).
HIV-1 RNA levels and genotyping. (i) Plasma. Plasma
HIV-1 RNA levels remained at ?50 copies/ml in 28 patients
for at least 6 months prior to the trial (on the basis of a
monthly evaluation). HIV-1 RNA was detectable in the plasma
of four patients receiving indinavir (median level, 516 copies/
ml), five patients receiving nelfinavir (median level, 431 copies/
ml), and four patients receiving lopinavir/r (median level,
39,090 copies/ml). The higher HIV-1 RNA levels reported in
patients receiving lopinavir/r reflect the present preferential
use of this drug in France for highly antiretroviral agent-expe-
rienced patients.
(ii) CSF. HIV-1 RNA levels were ?50 copies/ml in 35 of 40
CSF samples obtained, and HIV-1 RNA was detectable in 2
samples from patients receiving nelfinavir (55 and 1,330 copies/
ml, respectively) and 3 samples from patients receiving lopi-
navir/r (median level, 350 copies/ml). For the last five patients,
the detection of HIV-1 RNA in CSF was associated with a
detectable viral load in plasma. In contrast, the CSF of eight
other patients in whom plasma HIV-1 RNA was detectable
(four receiving indinavir, three receiving nelfinavir, and one
receiving lopinavir/r) had HIV-1 RNA levels ?50 copies/ml.
Isolates in plasma from one patient receiving nelfinavir pre-
sented more mutations (RT gene, M41L, D67N, M184V, and
T215Y; PR gene, M36I and V82A) than isolates in CSF (RT
gene, T215Y; PR gene, M36I); isolates from both compart-
ments from another patient presented the same mutation pat-
terns (RT gene, M41L; PR gene, A71V and L90 M). Isolates
from the three patients receiving lopinavir/r presented several
combinations of mutations on the RT and PR genes which
were also detected in isolates from plasma (data not shown).
(iii) Semen. The HIV-1 RNA load in seminal plasma was
?50 copies/ml in 28 of 34 patients whose semen was analyzed,
and HIV-1 RNA was detectable in the semen of 6 patients
(median load, 552 copies/ml): 3 patients receiving nelfinavir
and 3 patients receiving lopinavir/r. Among the six patients in
whom HIV-1 RNA was detectable in seminal plasma, three
had detectable plasma HIV RNA levels (one receiving nelfi-
navir, two receiving lopinavir/r) and three had plasma HIV-1
RNA levels ?50 copies/ml (two receiving nelfinavir, one re-
ceiving lopinavir/r).
Sequencing of the HIV-1 RNA obtained from seminal
plasma of the three patients with undetectable viral loads in
plasma showed resistance mutations in only the PR gene in
one patient treated with nelfinavir, didanosine, and stavudine
(A71V) and in both the RT and the PR genes in two patients:
one patient receiving nelfinavir, lamivudine, and stavudine (an
M184I mutation and M36I and L63P mutations, respectively)
and one patient receiving lopinavir/r, abacavir, didanosine, and
stavudine (M41L and T215Y mutations and L10I, L63P,
A71V, V82A, and L90M mutations, respectively). The last
patient had previously failed three HAART regimens before
the viral load in plasma was controlled with lopinavir/r. The
resistance mutations found in the HIV-1 RNA from the sem-
inal plasma from this patient were already present in the RNA
from plasma analyzed just before the introduction of lopina-
vir/r (data not shown).
(iv) Lymphoid tissue. HIV-1 RNA was detectable in
LNMCs from all patients. The LNMCs of patients with plasma
FIG. 2. Concentrations of indinavir (IDV), nelfinavir (NFV), lopi-
navir (LPV), and ritonavir (RTV) in seminal plasma 8 to 12 h post-
dosing.
FIG. 3. Concentrations of indinavir (IDV), nelfinavir (NFV), lopi-
navir (LPV), and ritonavir (RTV) in lymph node tissue 6 h (range, 4 to
8) after the last drug intake.
240 SOLAS ET AL.ANTIMICROB. AGENTS CHEMOTHER.

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HIV-1 RNA loads ?50 copies/ml had mean HIV-1 RNA levels
of 3.56 ? 0.17 log copies/106cells. No resistance mutations
were found in LNMCs taken from nine patients on first-line
therapy. Conversely, resistance mutations were found in HIV-1
RNA in LNMCs from 15 of 19 patients who had received prior
suboptimal regimens (M41L in the RT gene, 6 patients; D67N
in the RT gene, 1 patient; T69N in the RT gene, 3 patients;
K70R in the RT gene, 11 patients; T215Y in the RT gene, 5
patients; T215F in the RT gene, 5 patients; K219Q in the RT
gene, 5 patients; V82A in the PR gene, 5 patients; L90 M in the
PR gene, 3 patients). Frozen plasma was available from 11
patients before initiation of the present regimen. These muta-
tions were already present in plasma samples taken 24 to 180
weeks beforehand, when the patients had been treated with
suboptimal regimens.
Correlation between PI concentrations and HIV RNA loads
in the different sanctuary sites. In the CSF, there was no
statistical difference between a detectable (patients receiving
indinavir) or a nondetectable (patients receiving nelfinavir and
lopinavir/r) PI concentration and HIV RNA levels (P ? 0.137
by the Fisher exact test).
HIV RNA was not detectable in the semen of 100, 66, and
75% of patients receiving indinavir, nelfinavir, and lopinavir/r,
respectively. Among the three patients who were receiving
nelfinavir and in whom HIV-1 RNA was detectable in semen,
the reported nelfinavir concentrations were very low, ranging
from 0 to 94 ng/ml, but no characteristic nelfinavir resistance
mutations were found in isolates from the semen of these
patients. Furthermore, in four other patients, the HIV-1 RNA
load in seminal plasma was ?50 copies/ml, despite undetect-
able or low nelfinavir concentrations. The nelfinavir concen-
trations were not statistically different between patients in
whom HIV RNA was undetectable and patients in whom
HIV-1 RNA was detectable (P ? 0.20 by the Mann-Whitney
test). Similarly, three patients who were receiving lopinavir/r
and in whom HIV RNA was detectable in seminal plasma had
very low lopinavir concentrations in semen, but three other
patients in whom lopinavir concentrations were undetectable
had seminal plasma HIV RNA loads ?50 copies/ml. As for
nelfinavir, no correlation was found between lopinavir concen-
trations and the detection of HIV-1 RNA in semen.
Among the 28 patients with undetectable viral loads in
plasma, HIV-1 RNA levels in LNMCs were not statistically
different between those treated with indinavir (with a concen-
tration in LN tissue/concentration in plasma ratio ?1) and
those treated with nelfinavir or lopinavir/r (with a concentra-
tion in LN tissue/concentration in plasma ratio ?1): 3.32 ?
0.23 versus 3.80 ? 0.25 log copies/106cells (P ? 0.17 by the
Student t test). Overall, no statistically significant correlation
was found between HIV-1 RNA levels in LNMCs and the
concentration in LN tissue/concentration in plasma ratio for
the effective PI used (r ? ?0.33; P ? 0.1 by the Pearson test).
DISCUSSION
The effectiveness of antiretroviral therapy may depend on
the ability of antiretroviral drugs to reach the sanctuary sites.
Our study analyzed simultaneously viral loads and PI levels in
four different compartments in a population of patients receiv-
ing long-term HAART.
Major differences were demonstrated for PI penetration into
CSF. Nelfinavir and lopinavir were undetectable in the CSF of
all patients, although indinavir was detectable with a concen-
tration in CSF/concentration in plasma ratio of 0.17 and
achieved concentrations in CSF that exceeded the 95% inhib-
itory concentration for the wild-type virus (35 to 70 ng/ml, i.e.,
25 to 100 nM). Our results are concordant with other recently
published data, which showed mean CSF indinavir concentra-
tions ranging from 68 to 137 ng/ml (11, 17, 33). Protein binding
variations appear to be the most likely explanation for these
different levels of penetration into CSF. Indinavir is 61%
bound to plasma protein, whereas nelfinavir and lopinavir/r are
98% bound. Moreover, amprenavir, which is 90% protein
bound, is also present at low levels in CSF (R. Murphy, J.
Currier, J. Gerber, R. D’Aquila, L. Smeaton, J. P. Sommad-
ossi, R. Tung, and R. Gulick, 7th Conf. Retrovir. Opportunist.
Infect., abstr. 314, 2000), further supporting this hypothesis.
Despite these major differences in drug penetration, there was
no correlation between HIV RNA levels and drug levels in
CSF. These observations raise two hypotheses. First, all pa-
tients concomitantly received a combination of two NRTIs that
may suffice to control HIV-1 replication in CSF. Conversely,
the CSF drug concentration may not properly reflect overall
activity in the brain parenchyma.
The control of viral replication in genital fluids during
HAART is of particular importance, as most HIV-1 infections
are transmitted through sexual contact (7). We have shown
that the penetration of PIs into semen was highly variable not
only between the different PIs but also between individuals.
Indinavir had the highest concentration in semen/concentra-
tion in plasma ratio, as reported previously (30), and, as for
CSF, reached concentrations that widely exceed the 95% in-
hibitory concentration for wild-type virus and that are close to
those found in plasma. Indinavir would be expected to be
effective in this compartment since HIV-1 RNA was not de-
tectable in the semen of any of the patients. For the first time,
we have reported on the penetration of nelfinavir into semen,
even though the concentrations were very low (generally below
the 50% effective concentration corrected for in vitro protein
binding [630 ng/ml, i.e., 1.11 ?M]) and highly variable (20).
The same observations were made for lopinavir penetration
into semen, with an important variability detected, despite the
presence of ritonavir as a pharmacokinetic booster. To our
knowledge, no data are available on lopinavir penetration
alone, and we were unable to determine whether ritonavir
enhances the penetration of lopinavir. Genotypic resistance in
an isolate from the semen of one patient treated with lopina-
vir/r was related to the presence of resistant strains previously
selected in plasma by failing regimens. Introduction of lopina-
vir/r in this case was able to control resistant HIV-1 in plasma
but not in semen, where the drug concentrations may have
been too low to be effective against this strain. However, nei-
ther nelfinavir nor lopinavir concentrations were correlated
with HIV RNA levels in semen. Concentrations in semen
should be interpreted with caution, since the percentage of
protein binding of the drugs in this compartment is unknown.
Regarding CSF, the presence of high NRTI concentrations in
semen also makes it difficult to draw conclusions about the
efficacies of PIs for the suppression of viral replication in se-
men (23).
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In lymphoid tissue, residual HIV-1 RNA was detected in all
LN tissue samples of patients who had previously received
suboptimal regimens, even when HIV-1 RNA was undetect-
able in plasma for 2 to 3 years. All three PIs were detectable in
LN tissue samples, but with wide differences in the concentra-
tion in LN tissue/concentration in plasma ratio, suggesting a
better penetration for indinavir. Despite these differences in
drug penetration, similar levels of HIV-1 RNA in LN tissue
samples were found in patients with undetectable viral loads in
plasma, and no correlation between drug penetration and lev-
els of residual viral RNA was found. This suggests that per-
sisting HIV-1 RNA in LN tissue is related to multiple factors
other than PI diffusion, e.g., the existence of a population of T
cells producing viral RNA without new cellular infection cycles
(10).
The present study has some limitations. Indeed, use of a
ratio value to describe PI penetration into CSF, semen, or LN
tissue is not accurate because these ratios tend to change over
time due to different time-concentration profiles in the non-
blood compartments. However, it was not feasible to perform
several lumbar punctures, lymph node biopsies, or semen col-
lections to calculate ratios of areas under the curve. Therefore,
our results did not allow complete evaluation of PI penetration
during a dosing interval and the pharmacokinetic properties of
the respective PIs in the different compartments.
In conclusion, major differences between different PIs in
terms of penetration into nonblood compartments were dem-
onstrated, but we were unable to establish a correlation be-
tween the pharmacological data and the HIV RNA levels.
These discrepancies suggest that PI penetration is not the only
factor which contributes to viral suppression in the different
viral reservoirs. Indeed, protein binding may lead to differ-
ences in drug penetration into the CSF and the genital tract
among the different PIs. Mostly, however, PIs are substrates of
P glycoprotein, which is present in the blood-brain barrier and
the blood-testis barrier and which actively pumps out PIs from
these compartments. Furthermore, other cellular mechanisms
and genetic factors are likely involved in the persistence of
residual viral replication and viral reservoirs. These major dif-
ferences require further study in order to clarify the impor-
tance of the role and the clinical relevance of drug penetration
into these sanctuary sites in terms of long-term efficacy and
drug resistance.
ACKNOWLEDGMENTS
We thank Segolene Durand (INSERM U379 ORS PACA) for con-
tributions to the statistical analysis.
This study was supported in part by grants from Roche, Merck Sharp
& Dohme, and Abbott.
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