For the 55 paired CSF and plasma samples, the CSF concentration was below detection in 6 samples (with measurable concentrations in the plasma ranging from 26.1 to 319.8 ng/mL), the plasma concentration was undetectable in 10 samples (with measurable concentrations in the CSF ranging from 1.2 to 32 ng/mL), and 1 subject had undetectable concentrations in both plasma and CSF. The median CSF to plasma tenofovir concentration ratio in the 38 paired specimens with measurable tenofovir in both plasma and CSF was 0.057 or 5.7% (IQR 0.03–0.1) after a median of 6.1 months of therapy. The median (IQR) time between plasma and CSF sample draw times was 0.4 (0.3–0.7) hours. The ratio did not change with time post dose (Fig. 2). One subject had a CSF concentration approximately 84% of the corresponding plasma concentration (CSF = 13.2 ng/mL and plasma = 15.7 ng/mL). The next highest ratios were in the 30%–40% range (n = 2), and all other subjects had ratios of CSF to plasma concentrations of less than 21%. Plasma and CSF concentrations were not significantly correlated (r 2 = 0.04, P = 0.2). Subjects with CSF HIV RNA >50 copies per milliliter (n = 8) had median (IQR) CSF to plasma concentration ratios of 0.045 (0.027–0.123). Subjects with undetectable CSF HIV RNA (n = 30) had CSF to plasma concentration ratios of 0.064 (0.034–0.098, P > 0.1).
From the 77 subjects with measured CSF tenofovir concentrations, CSF HIV RNA concentrations were available for 72. Fifty-six of these 72 subjects (78%) had CSF HIV RNA ≤50 copies per milliliter, whereas the remaining 16 of 72 (22%) had detectable CSF HIV. Rates of detectable CSF HIV were increased in those with tenofovir concentrations in the CSF below 7 ng/mL [14 of 49 (28.6%)] compared to those with higher concentrations [2 of 23 (8.7%), P = 0.05 by a 1-tailed test and P = 0.07 by a 2-tailed test].
In this cohort of subjects, the median plasma concentration of tenofovir in CSF was only 5%, levels of penetration (CSF to plasma concentration ratio) that are much lower than those observed for the nucleoside reverse transcriptase inhibitors. Nucleoside antiretroviral drugs such as abacavir, didanosine, lamivudine, and stavudine have CSF to plasma concentration ratios of approximately 15%–30%.30–34 Similarly, low CNS penetration of tenofovir was found in guinea pigs, which had penetration in the brain tissues of less than 5%, although CSF penetration in guinea pigs was higher than that seen in humans in this study.20 Zidovudine has a median 65% penetration, although variability between patients is high.30,35 Tenofovir does show higher penetration than most protease inhibitors studied, with the exception of indinavir (which has lower protein binding than most protease inhibitors).36 Protease inhibitor CSF to plasma concentration ratios are usually in the range of 1% or less.37,38 Thus, the penetration of this nucleotide agent seems to be intermediate between the penetration of protease inhibitors and nucleoside agents.
More than three quarters of tenofovir concentrations in the CSF were below the lowest reported tenofovir IC50 of 11.5 ng/mL.16 These low concentrations were unable to reliably suppress HIV replication in the CNS. Patients with CSF tenofovir concentrations of 7 ng/mL or less were more likely to have 50 HIV copies or more in CSF compared to those with CSF tenofovir levels higher than 7 ng/mL. Lower plasma tenofovir concentration was associated with significantly increased likelihood of detectable viral replication in the plasma. Although higher plasma and CSF concentrations are associated with suppression of viral replication in the corresponding compartments, plasma and CSF concentrations were not strongly associated with one another. This finding suggests that processes other than simple passive diffusion govern the penetration of tenofovir into the CSF.
Both the blood–CSF barrier and the BBB restrict passage of many small molecules into the CNS. The tight junctions between epithelial cells of the choroid plexus and the brain capillaries largely prevent paracellular diffusion, although the tight junctions of the blood–CSF barrier are quantitatively leakier than those of the BBB. Physicochemical characteristics of the parent compound, tenofovir, may prevent transcellular passage (either passive or facilitated) into the CSF. The molecule has 2 hydroxyl groups, which confer 2 negative charges at the physiologic pH. These negative charges may repel the molecule from the negatively charged surface of the plasma membrane. Furthermore, tenofovir does not have adequate lipophilicity to passively diffuse through the lipophilic components of cell membranes. Because of its high polarity and low lipophilicity, tenofovir likely needs to rely on active transport processes to enter the CSF or CNS.
Tenofovir has been shown to be a substrate for transporter enzymes that contribute to the BBB or blood–CSF barrier, including OATs 1 and 3 and MRPs 2 and 4 but not permeability glycoprotein (P-glycoprotein).21–24,39 These transporters may exclusively efflux tenofovir out of the CSF or brain or may carry tenofovir both into and out of the central compartment, but with efflux processes being the dominant pathways. A limitation of this study was that the penetration of tenofovir across the BBB was not measured. Rather, the CSF penetration was used as an accessible surrogate marker to estimate brain penetration. This may be important due to the differential expression of P-glycoprotein and other transporters at those different interfaces and the fact that tenofovir is not a P-glycoprotein substrate.21,26 This differential expression may lead to limited efflux at the BBB interface and more efflux at the blood–CSF barrier interface, resulting in relatively higher concentrations in brain parenchyma, which unfortunately cannot be measured in vivo in humans.
CSF tenofovir concentrations in this study were measured in subjects taking concomitant protease inhibitors, particularly ritonavir-boosted protease inhibitors. Ritonavir and several other protease drugs are known inhibitors of P-glycoprotein. Protease inhibitor effects on other transport enzymes, such as OATs 1 and 3 and MRP 2 and 4 that are important for tenofovir transport, are less well characterized. Ritonavir may inhibit MRP240 and OAT341 and does not appear to affect OAT1 or MRP4,41 although data are not conclusive and are limited to mainly in vitro studies. If protease inhibitors do affect the transport proteins for which tenofovir is a substrate, then concomitant administration of tenofovir with protease drugs may affect the penetration of tenofovir into the CNS.
Interestingly, one subject with paired plasma and CSF samples had nearly equal tenofovir concentrations in the plasma and in the CSF. One potential explanation could be that the subject has a genetic alteration in the active transport processes governing the transport of tenofovir into or out of the CNS. Also noted among the subjects with paired CSF and plasma samples were undetectable concentrations in either the plasma or the CSF compartments individually, highlighting the high variability of drug exposure between subjects. One subject had undetectable concentrations in both fluids, suggestive of poor adherence. Subjects with undetectable CSF HIV RNA had approximately 40% higher CSF to plasma ratios (6.4%) than subjects who had CSF HIV RNA >50 copies per milliliter (4.5%), although these values were not significantly different.
Tenofovir has been measured in other physiologic compartments. Plasma tenofovir concentration derived from cord blood in women at steady state on standard tenofovir doses was 96% of maternal plasma concentrations.42 Tenofovir freely distributes across the placenta. Tenofovir has also been measured in semen and cervicovaginal fluid. The median semen to plasma concentration ratio in 15 subjects was 3.3 in a study by Lowe et al.43 The median plasma tenofovir concentrations in that study were 112 ng/mL, similar to the value found in this study of 96 ng/mL, whereas the semen concentrations were 250 ng/mL. In cervicovaginal fluid, concentrations range from 75% to over 500% of plasma concentrations.44,45 Several possible explanations could account for the much better penetration of tenofovir into genital tract and fetal compartments. Tenofovir may be able to better diffuse paracellularly in those anatomic sites that do not have the tight junctions between endothelial cells. Also, the other anatomic sites may have different distributions or activity of transporter enzymes that affect tenofovir disposition.46,47
Although the ability of tenofovir to penetrate the CSF is limited, the concentration of the active moiety of tenofovir (intracellular tenofovir diphosphate) was not measured in the cells of the CSF or plasma in this study. Because tenofovir is activated intracellularly, the measurement of the plasma or CSF IC50 of the inactive compound, tenofovir, may be less strongly correlated with viral suppression than are the IC50 of drugs that are directly active such as protease inhibitors. However, the tenofovir IC50 can be assumed to approximate drug potency, recognizing that a certain amount of extracellular inactive drug is in equilibrium with the amount of intracellular drug, which is converted to the active compound. This is supported by our findings in both plasma and CSF of a positive relationship between increasing tenofovir concentrations and a higher likelihood of viral suppression.
Although seemingly ineffective tenofovir CSF concentrations were observed in a majority of subjects, only 20% had detectable CSF viremia. This can be explained by several reasons. First, subjects were taking other antiretrovirals that may have been effective in the CSF. Second, HIV in CSF derives from sources both within and outside the CNS. Tenofovir's effectiveness outside the nervous system could then still be contributing to suppression of that source of HIV. Finally, the IC50 in macrophages, the primary target cell for HIV in the brain, may be lower than the IC50 in lymphocytes.
A limitation of this study is that the CSF to plasma concentration ratio is a dynamic measure, and single ratio measurements may not accurately estimate exposure over time in this sanctuary site. Drugs with very different elimination half-lives in the plasma and CSF will have CSF to plasma ratios that differ markedly depending on time post dose. Therefore, paired samples must be drawn in close time proximity to one another to estimate an accurate ratio at that particular time post dose. In this study, paired plasma and CSF samples were all drawn within 42 minutes or less of one another to obtain an accurate ratio at that particular time post dose. The pairs of samples from this study were drawn at a wide range of postdose sampling times between subjects up to 48 hours after a dose. The ratio did not vary with time post dose between subjects. Only a modest change in plasma concentrations and very little change in CSF concentrations over the dose interval were observed, coinciding with the lack of correlation between the ratio and time post dose. However, paired samples within a subject at different post-dose times may provide a more accurate estimate of tenofovir CSF to plasma ratios.
Despite these limitations, the low tenofovir CSF penetration observed in these subjects is consistent with CNS animal data and with predictions based on physicochemical characteristics of tenofovir. Our finding that higher tenofovir concentrations occurred in samples with suppressed virus indicates that tenofovir CSF penetration (or lack thereof) may be important to antiviral activity and clinical effects. Because plasma and CSF tenofovir concentrations are not related, increasing plasma exposure (eg, by increasing dose) may not reliably increase tenofovir CSF concentrations. Our data suggest that tenofovir taken concomitantly with protease inhibitors may not provide protection against viral replication in the CSF.
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