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Dendritic spine injury induced by the 8-hydroxy metabolite of Efavirenz
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JPET Fast Forward. Published on September 19, 2012
Jnl of Pharmacology & Experimental Therapeutics
Luis B. Tovar-y-Romo, Namandje N. Bumpus, Daniel Pomerantz, Lindsay B.
Avery, Ned Sacktor, Justin McArthur and Norman J. Haughey
Departments of Neurology, Richard T. Johnson Division of Neuroimmunology
and Neurological Infections (L.B.T-y-R., D.P., N.S., J.M., N.J.H.), Pharmacology
and Molecular Sciences (N.N.B., L.B.A.) and Psychiatry (N.J.H), The Johns
Hopkins University School of Medicine, Baltimore, MD
"EFV is metabolized primarily by CYP2B6 to produce a series of metabolites of which 8-hydroxyefavirenz (8-OH-EFV) is the most abundant (Ward et al., 2003; Bumpus et al., 2006; Ogburn et al., 2010). In this study we provide evidence that 8-OH-EFV is a potent neurotoxin that dysregulates neuronal calcium homeostasis, and damages dendritic spines in the low nM range. The effects of this metabolite are distinct from the parent drug, and highlight the importance of screening not only antiretroviral medications for neurotoxicity, but also drug metabolites......These data suggest that the metabolism of EFV produces a highly neurotoxic metabolite (8-OH-EFV) that is capable of damaging neurites at very low concentration. Thus, cognitive impairments associated with EFV may involve synaptic damage mediated by its major metabolite 8-OH-EFV......
These findings contribute to our understanding of the mechanism for neurotoxicity associated with EFV therapy. Since the anticipated length of therapy for cART is the lifetime of the patient, these data highlight the importance of screening antiretroviral drugs and drug metabolites for neurotoxic potential. This principle may be especially important if brain penetration is desired to reduce CNS viral replication.......there is also evidence that some antiretroviral drugs (ARVs) are toxic to neurons (Liner et al., 2010), and that ARVs with increased brain penetration are associated with poor cognitive performance (Tozzi et al., 2007; Marra et al., 2009). Therefore, the effectiveness of brain penetrating cART regimens is currently in question (Koopmans et al., 2009)."
"The widespread use of combination antiretroviral therapy (cART) dramatically decreased the mortality rate of HIV-infected individuals, and decreased the incidence of HIV-associated dementia (HAD (Heaton et al., 2011). Although cART decreased the incidence HAD, it appears to have had little impact on the prevalence of milder forms of cognitive impairments that are collectively known as HIV-Associated Neurocognitive Disorders (HAND) (Heaton et al., 2010; Letendre et al., 2010; McArthur et al., 2010; Heaton et al., 2011; Valcour et al., 2011b). Currently available data suggests that 50% of HIV-infected subjects will develop a neurologic disorder (Chang et al., 2004; Ernst and Chang, 2004; Valcour et al., 2004; Chang et al., 2008; Valcour et al., 2011a). Moreover, the occurrence of HAND is associated with an increased risk of death (Vivithanaporn et al., 2010). Although the mechanism(s) for this residual cognitive impairment and association with increased mortality are not completely understood, continued viral replication in the brain due to insufficient central nervous system (CNS) penetration of cART is though to be an underlying mechanism (Robertson et al., 2007). Therefore, cART regimens with increased brain penetration have been proposed to combat HAND (Letendre et al., 2008). While there is evidence that this approach reduces CSF viral load (Marra et al., 2009) and may improve cognitive function (Letendre et al., 2004; Smurzynski et al., 2011), there is also evidence that some antiretroviral drugs (ARVs) are toxic to neurons (Liner et al., 2010), and that ARVs with increased brain penetration are associated with poor cognitive performance (Tozzi et al., 2007; Marra et al., 2009). Therefore, the effectiveness of brain penetrating cART regimens is currently in question (Koopmans et al., 2009)."
Popular HIV drug may cause memory declines
http://www.eurekalert.org
Johns Hopkins study suggests the commonly prescribed anti-retroviral drug efavirenz attacks brain cells
The way the body metabolizes a commonly prescribed anti-retroviral drug that is used long term by patients infected with HIV may contribute to cognitive impairment by damaging nerve cells, a new Johns Hopkins research suggests.
Nearly 50 percent of people infected with HIV will eventually develop some form of brain damage that, while mild, can affect the ability to drive, work or participate in many daily activities. It has long been assumed that the disease was causing the damage, but Hopkins researchers say the drug efavirenz may play a key role.
People infected with HIV typically take a cocktail of medications to suppress the virus, and many will take the drugs for decades. Efavirenz is known to be very good at controlling the virus and is one of the few that crosses the blood-brain barrier and can target potential reservoirs of virus in the brain. Doctors have long believed that it might be possible to alleviate cognitive impairment associated with HIV by getting more drugs into the brain, but researchers say more caution is needed because there may be long-term effects of these drugs on the brain.
"People with HIV infections can't stop taking anti-retroviral drugs. We know what happens then and it's not good," says Norman J. Haughey, Ph.D., an associate professor of neurology at the Johns Hopkins University School of Medicine. "But we need to be very careful about the types of anti-retrovirals we prescribe, and take a closer look at their long-term effects. Drug toxicities could be a major contributing factor to cognitive impairment in patients with HIV."
For the study led by Haughey and described online in the Journal of Pharmacology and Experimental Therapeutics, researchers obtained samples of blood and cerebrospinal fluid from HIV-infected subjects enrolled in the NorthEastern AIDS Dementia study who were taking efavirenz. Researchers looked for levels of the drug and its various metabolites, which are substances created when efavirenz is broken down by the liver. Performing experiments on neurons cultured in the lab, the investigators examined the effects of 8-hydroxyefavirenz and other metabolites and found major structural changes when using low levels of 8-hydroxyefavirenz, including the loss of the important spines of the cells.
Haughey and his colleagues found that 8-hydroxyefavirenz is 10 times more toxic to brain cells than the drug itself and, even in low concentrations, causes damage to the dendritic spines of neurons. The dendritic spine is the information processing point of a neuron, where synapses - the structures that allow communication among brain cells - are located.
In the case of efavirenz, a minor modification in the drug's structure may be able block its toxic effects but not alter its ability to suppress the virus. Namandje N. Bumpus, Ph.D., one of the study's other authors, has found a way to modify the drug to prevent it from metabolizing into 8-hydroxyefavirenz while maintaining its effectiveness as a tool to suppress the HIV virus.
"Finding and stating a problem is one thing, but it's another to be able to say we have found this problem and here is an easy fix," Haughey says.
Haughey says studies like his serve as a reminder that while people infected with HIV are living longer than they were 20 years ago, there are significant problems associated with the drugs used to treat the infection.
"Some people do seem to have this attitude that HIV is no longer a death sentence," he says. "But even with anti-retroviral treatments, people infected with HIV have shortened lifespans and the chance of cognitive decline is high. It's nothing you should treat lightly."
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Investigator: Dr. Norman Haughey
http://www.hopkinsmedicine.org
Director, JHU NIMH Surrogate Markers Core
Associate Professor of Neurology, Johns Hopkins University
Dr Norman Haughey received his PhD in Pharmacology from the University of Manitoba in 1998. He then completed one year of post-doctoral training at the University of Kentucky's Center on Aging before moving to the National Institute on Aging, Gerontology Research Center for an additional two years of fellowship. Dr Haughey joined the faculty of the Department of Neurology at Johns Hopkins in 2002.
Now an Associate Professor at Hopkins, Dr Haughey is a member of the JHU NIMH Center with specialization in Neurovirology and Neuroregeneration. His research focuses on signaling pathways that promote the dysfunction and death of neuronal cells. In this context, he is interested in the mechanisms that promote neurotoxic processes in HIV and Alzheimer's related dementias.
Dr Haughey and his colleagues use a multidisciplinary approach including tissue culture, molecular reconfiguration, transgenic or knock-out mice and biochemical approaches in order to identify and modify signal transduction pathways that are associated with neurodegenerative processes. Using these model systems, they have described pathogenic modifications of calcium permeable receptors and transporters that promote excessive calcium accumulation.
Current research is focused on mapping perturbations in lipid raft based signal transduction pathways that lead to the oxidative modification of subcellular targets involved in apoptotic and anti-apoptotic signaling.
A second research interest is in the biology of neural stem and neural progenitor cells. In particular, he is interested in the mechanisms that govern the function and survival of neural stem and neural progenitor cells and how these processes are disrupted in neurodegenerative conditions. These interests relate directly to diagnostics, therapeutics, cellular and molecular dynamics.
The study was supported by grants from the National Institute on Alcohol Abuse and Alcoholism (AA0017408), the National Institute of Mental Health (MH077543, MH075673 and MH71150), the National Institute on Aging (AG034849) and the National Institute of Neurological Disorders and Stroke (NS049465).
Other Hopkins researchers involved in the study include Luis B. Tovar y Romo, Ph.D.; Lindsay B. Avery, Ph.D.; Ned Sacktor, M.D.; and Justin McArthur, M.B.B.S., M.P.H.
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Dendritic spine injury induced by the 8-hydroxy metabolite of Efavirenz
Luis B. Tovar-y-Romo, Namandje N. Bumpus, Daniel Pomerantz, Lindsay B. Avery, Ned Sacktor, Justin McArthur and Norman J. Haughey
Departments of Neurology, Richard T. Johnson Division of Neuroimmunology and Neurological Infections (L.B.T-y-R., D.P., N.S., J.M., N.J.H.), Pharmacology
and Molecular Sciences (N.N.B., L.B.A.) and Psychiatry (N.J.H), The Johns Hopkins University School of Medicine, Baltimore, MD
ABSTRACT
Despite combination antiretroviral therapies (cART), a significant proportion of HIV-infected patients develop HIV-associated neurocognitive disorders (HAND). Ongoing viral replication in the CNS due to poor brain penetration of cART may contribute to HAND. However, it has also been proposed that toxic effects of long-term cART may contribute to HAND. A better understanding of the neurotoxic potential of cART is critically needed in light of the use of CNS penetrating cART to contend with virus reservoir in brain. The Efavirenz (EFV) metabolites 7-hydroxyefavirenz (7-OH-EFV) and 8-hydroxyefavirenz (8-OH-EFV) were synthesized, purified, and chemical structures confirmed by mass
spectrometry and NMR. The effects of EFV, 7-OH-EFV and 8-OH-EFV on calcium, dendritic spine morphology, and survival were determined in primary neurons. EFV, 7-OH-EFV and 8-OH-EFV each induced neuronal damage in a dose dependent manner. However, 8-OH-EFV was at-least an order of magnitude more toxic than EFV or 7-OH-EFV, inducing considerable damage to dendritic spines at a 10 nM concentration. The 8-OH-EFV metabolite evoked calcium flux in neurons, which was primarily mediated by L-type voltage operated calcium channels. Blockade of L-type VOCCs protected dendritic spines from 8-OH-EFV-induced damage. Concentrations of EFV and 8-OH-EFV in the cerebral
spinal fluid of HIV-infected subjects taking EFV were within the range that damaged neurons in culture. These findings demonstrate that the 8-OH metabolite of EFV is a potent neurotoxin and highlight the importance of directly
Introduction
The widespread use of combination antiretroviral therapy (cART) dramatically decreased the mortality rate of HIV-infected individuals, and decreased the incidence of HIV-associated dementia (HAD (Heaton et al., 2011). Although cART decreased the incidence HAD, it appears to have had little impact on the prevalence of milder forms of cognitive impairments that are collectively known as HIV-Associated Neurocognitive Disorders (HAND) (Heaton et al., 2010; Letendre et al., 2010; McArthur et al., 2010; Heaton et al., 2011; Valcour et al., 2011b). Currently available data suggests that 50% of HIV-infected subjects will develop a neurologic disorder (Chang et al., 2004; Ernst and Chang, 2004; Valcour et al., 2004; Chang et al., 2008; Valcour et al., 2011a). Moreover, the occurrence of HAND is associated with an increased risk of death (Vivithanaporn et al., 2010). Although the mechanism(s) for this residual cognitive impairment and association with increased mortality are not completely understood, continued viral replication in the brain due to insufficient central nervous system (CNS) penetration of cART is though to be an underlying mechanism (Robertson et al., 2007). Therefore, cART regimens with increased brain penetration have been proposed to combat HAND (Letendre et al., 2008). While there is evidence that this approach reduces CSF viral load (Marra et al., 2009) and may improve cognitive function (Letendre et al., 2004; Smurzynski et al., 2011), there is also evidence that some antiretroviral drugs (ARVs) are toxic to neurons (Liner et al., 2010), and that ARVs with increased brain penetration are associated with poor cognitive performance (Tozzi et al., 2007; Marra et al., 2009). Therefore, the effectiveness of brain penetrating cART regimens is currently in question (Koopmans et al., 2009).
Few studies have directly determined the effects of antiretroviral drugs on neuronal function (Schweinsburg et al., 2005; Cardenas et al., 2009), and no studies have determined potential neurotoxic effects of antiretroviral drug metabolites. Most xenobiotics are metabolized by the cytochrome P450 (CYP) superfamily of enzymes that catalyze Phase 1 reactions (oxidation, reduction, hydrolysis). CYPs are concentrated in liver, but are also expressed in brain (Gervot et al., 1999; Bhagwat et al., 2000; Miksys et al., 2003). CNS effects of the non-nucleoside reverse transcriptase inhibitor Efavirenz ((S)-(-)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-2,4-dihydro-1H-3,1-benzoxazin-2-one;
EFV), have been reported that include sleep disturbances, cognitive and mood disorders(Marzolini et al., 2001; Perez-Molina, 2002; Lochet et al., 2003; Rihs et
al., 2006), but to date there are no studies that have directly determined the effects of EFV or its metabolites on neuronal function.
EFV is metabolized primarily by CYP2B6 to produce a series of metabolites of which 8-hydroxyefavirenz (8-OH-EFV) is the most abundant (Ward et al., 2003; Bumpus et al., 2006; Ogburn et al., 2010). In this study we provide evidence that 8-OH-EFV is a potent neurotoxin that dysregulates neuronal calcium homeostasis, and damages dendritic spines in the low nM range. The effects of this metabolite are distinct from the parent drug, and highlight the importance of screening not only antiretroviral medications for neurotoxicity, but also drug metabolites.
RESULTS
The 8-hydroxyefavirenz metabolite evokes calcium influx in neurons.
EFV and the 7-OH-EFV and 8-OH-EFV metabolites used for these studies were isolated by HPLC-fractionation and the structures confirmed using mass spectrometry and NMR according to published spectral information (Mutlib et al., 1999) (Fig 1). We first investigated whether EFV or its hydroxylated metabolites could alter intracellular Ca2+ homeostasis in primary rat hippocampal neurons. Two-minute applications of EFV (10 μM), or 7-OH-EFV (10 μM) had no effect on intracellular calcium (Fig 2A-C). However, applications of 8-OH-EFV (10 μM) induced immediate increases of intraneuronal Ca2+ (Fig 2D). With a 10 μM dose of 8-OH-EFV, approximately half of the neurons appeared undergo a catastrophic loss of membrane integrity and release of the calcium probe. The remaining neurons exhibited clear signs of damage including beading of neurites and vacuolization of the soma (data not shown). We then conducted a dose response for 8-OH-EFV (0.1-10 μM) and found that 1 μM was the minimum effective dose to consistently increase intraneuronal Ca2+ (Fig 2E) within 1-5 sec of application (Fig 2E inset). Applications of 8-OH-EFV induced the entry of calcium from an extracellular source, since the removal calcium from the buffer blocked this effect (Fig 2F, G).
To determine the mechanism of Ca2+ influx we used pharmacological inhibitors to block the major calcium permeable ion channels in neurons including NMDA (MK-801; 20 μM), AMPA (NBQX; 20 μM), purinergic (PPADS; 10 μM and Suramin; 100 μM), or voltage-gated calcium channels (VOCC; nifedipine; 10 μM) and exposed neurons to 8-OH-EFV (1 μM; Fig 3A). Blockade of NMDA or AMPA receptors had no effects on 8-OH-EFV-induced calcium influx (Fig 3B-C). Blockade of purinergic receptors (Fig 3D, E), or VOCC (Fig 3F) each partially reduced 8-OH-EFV-evoked calcium influx. Blocking both purinergic receptors and VOCC slowed the rise of intraneuronal Ca2+ that followed 8-OH-EFV exposures, but resulted in a similar peak level within 100 sec. compared with either drug alone (Figure 3G).
8-hydroxyefavirenz is potent neurotoxin.
We exposed primary neuronal cultures to EFV, 7-OH-EFV, or 8-OH-EFV (0.01-10 μM) for 24 h and determined apoptosis by nuclear morphology using the fluorescent DNA binding dye Hoescht 33342. The dose-response relationships for EFV and 7-OH-EFV were similar, with each compound exhibiting a minimum toxic dose of 0.1 μM that produced 41.3 ± 14.0% (EFV), and 41.2 ± 15.0 % (7-OH EFV) increases in apoptotic nuclei (Fig 4). At the highest dose tested (10 μM) EFV produced a 66.6 ± 4.2% and 7-OH-EFV produced a 63.5 ± 12.2 % increase in apoptotic nuclei (Fig 4A) In contrast, 8-OH-EFV was at-least an order of magnitude more toxic, and increased the percent of apoptotic nuclei to 36.4% ± 7.1% at a 0.01 μM dose (Fig 4A). Neurotoxicity induced by 8-OH-EFV was not prevented by inhibition of purinergic receptors with PPADS (37.9 ± 11.8 % death), but was partially prevented by inhibition of L-type VOCCs (24.5 ± 8.0 % death). Inhibition of both purinergic receptors and L-type VOCCs did not further improve neuronal survival (27.6 ± 8.0; Fig. 4B).
We next determined if low concentrations of EFV, or its hydroxylated metabolites (0.01 and 0.1 μM) damaged dendritic spines. EFV and the metabolites 7-OH-EFV and 8-OH-EFV each produced considerable loss of dendritic spines at a 0.1 μM dose. In control conditions there were 9.5 ± 0.9 secondary dendrites per 10 μm. Spine density was reduced to 4.0 ± 1.4 for EFV, 4.9 ± 1.5 for 7-OH-EFV, and to 4.7 ± 1.6 for 8-OH-EFV with a 0.1 μM dose. In contrast, the 8-OH metabolite of EFV was considerably more toxic, and reduced the number of dendritc spines to 5.2 ± 1.7 per 10 μm at a dose of 0.01 μM (Fig 5B). EFV and 7-OH-EFV at the dose of 0.001 μM did not appreciably alter the number of dendritic spines (Fig 5A). Blockade of purinergic receptors with PPADS did not rescue dendritic spine damage induced by 8-OH-EFV (5.2 ± 2.0 per 10 μm), while inhibition of L-type VOCCs with nifedipine partially protected from 8-OH-EFV induced spine loss (8.9 ± 2.4 per 10 μm)(Fig 5B). Inhibition of both purinergic receptors and VOCCs did not offer additional protection (7.5 ± 2.2 per 10 μm) compared with inhibition of VOCCs alone (Fig 5B). These data demonstrate that the EFV metabolite 8-OH-EFV in the low nM range induces damage to dendritic spines.
Plasma and CSF concentrations of Efavirenz and 8-hydroxyefavirenz
We determined plasma and CSF concentrations of EFV and 8-OH-EFV in human subjects on stable cART regimens that included EFV. In these subjects the median plasma concentration of EFV was 2170 ng/ml (range 1010-7510), and 166 ng/ml (range 69.3-621) for 8-OH-EFV (Fig 6A). The median CSF concentration of EFV was 18.8 ng/ml (range 6.28-52.9) and 3.37 (range 0.35-32.7) for 8-OH-EFV (Fig 6B). These median values correspond to CSF concentrations of 59.6 nM for EFV and 10.2 nM for 8-OH-EFV.
DISCUSSION
Long-term cART has dramatically decreased the mortality rate of HIV-infected individuals, owing to the ability of these drugs to suppress viral replication. Most ARVs do not enter into the CNS in appreciable amounts, and thus brain has remained a reservoir for HIV (Langford et al., 2006). Ongoing (although likely low-level) viral replication in brain is thought to contribute to the pathogenesis of HAND (Masliah et al., 2000; Neuenburg et al., 2002; Langford et al., 2003) and targeting ARVs to inhibit viral replication in brain to treat or prevent HAND has been suggested (May et al., 2007). However, this approach has raised concerns that some ARVs may damage neurons. Unfortunately, very little experimental data is available on the potential of ARVs or drug metabolites to damage neurons.
The CNS side effects of EFV that have been described include dizziness, vivid dreams, headaches, disturbances in attention and sleep, psychotic events and hallucinations, with the majority of these events regarded as mild. The most evident CNS side effects of EFV generally occur and resolve within the first month of therapy and rarely lead to discontinuation of the therapy (Moyle, 1999). However, recent data have associated EFV with a higher risk of neurocognitive impairment, particularly on tasks requiring a high degree of attention and executive function (Ciccarelli et al., 2011). It has also been reported that cART regimens containing EFV may lead to a worsening of cognitive performance after several weeks of treatment (Winston et al., 2010), and that these impairments in cognitive performance are significantly improved when patients discontinued treatment (Robertson et al., 2010). As would be expected, the severity of these CNS symptoms correlates to EFV plasma concentrations (Marzolini et al., 2001). However not all studies have confirmed this relationship (Clifford et al., 2005).
In this study we sought to directly determine the effects of EFV and its hydroxylated metabolites on neurons. Our findings suggest that 8-OH-EFV is at least an order of magnitude more toxic to neurons compared to the parent compound EFV or 7-OH-EFV. Damage to dendritic spines was produced with 100 nM EFV or 7-OH-EFV. The 8-OH-EFV metabolite was approximately 10-fold more toxic compared to EFV and caused considerable dendritic damage10 nM with frank cell death at a 100 nM dose. We found that concentrations of EFV and 8-OH-EFV in CSF from human subjects taking cART appear to be within this neurotoxic dose range. The median concentration of EFV in the CSF of HIV-infected subjects taking cART-containing EFV was 59.6 nM, similar to previously reported CSF concentrations of 35 nM (0.5% of that in plasma) (Moyle, 1999; Tashima et al., 1999; Best et al., 2011). The median concentration of 8-OH-EFV in CSF was 10.2 nM. Together these data suggest that concentrations of the parent drug EFV in brain may be within the range that can damage neurons, and that concentrations of 8-OH-EFV could be three times the minimal dose that produced dendritic damage to cultured neurons. Moreover, there may be a genetic susceptibility to the neurotoxic effects of EFV that is related to its rate of metabolism. Extensive EFV metabolizers were recently identified who express the *1/1 haplotype of CYP2B6 (Ngaimisi et al., 2010). Thus, a genetic susceptibility in some individuals may exaggerate the neurotoxic effects of EFV due to a rapid metabolism of EFV with accumulations of the 8-OH-EFV metabolite.
This enhanced toxicity of 8-OH-EFV appears to be due to the ability of this metabolite to perturb calcium homeostasis. The 8-OH-EFV metabolite, but not 7-OH-EFV or the parent compound EFV induced rapid calcium influx in neurons that was partially inhibited by a general antagonist of purinergic receptors, and was almost completely eliminated by blockade of L-type VOCC. Calcium permeable NMDA and AMPA-type glutamate receptors were not activated by 8-OH-EFV. Consistent with a prominent role for VOCCs, inhibition of L-type calcium channels partially protected densritic spines from 8-OH-EFV-induced damage. These data suggest that the metabolism of EFV produces a highly neurotoxic metabolite (8-OH-EFV) that is capable of damaging neurites at very low concentration. Thus, cognitive impairments associated with EFV may involve synaptic damage mediated by its major metabolite 8-OH-EFV. It is interesting to note that the parent compound EFV, and the 7-OH-EFV metabolite also displayed a neurotoxic potential that appeared to be calcium-independent. The mechanisms of these separate toxic effects are currently being studied and may interact with the toxicity produced by 8-OH-EFV.
It is interesting that position of the OH group on the 7 vs 8 carbon of the benzoxazine ring produces such a dramatic difference in evoked calcium flux and neurotoxicity. These data suggest that the proximity of the OH group at position 8 to the nitrogen group in the benzoxazine structure is the critical determinant for forming a highly neurotoxic metabolite of EFV. Therefore, substituting the carbon at position 8 so that EFV cannot be hydroxylated at this position should produce a compound with decreased neurotoxicity. Such a drug with a fluorine substituent at carbon 8 which retains inhibitory activity at reverse transcriptase (with an IC90= 7.19 nM that is close to the IC90 = 2.03 nM of EFV) has been synthesized (Patel et al., 1999). This drug may offer an alternative to EFV with reduced neurotoxicity resulting from secondary metabolite production.
These findings contribute to our understanding of the mechanism for neurotoxicity associated with EFV therapy. Since the anticipated length of therapy for cART is the lifetime of the patient, these data highlight the importance of screening antiretroviral drugs and drug metabolites for neurotoxic potential. This principle may be especially important if brain penetration is desired to reduce CNS viral replication.
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