Investigation of distribution, transport and uptake of anti-HIV drugs to the central nervous system

Abstract

The distribution of currently available anti-HIV drugs into the CNS is reviewed with a focus on transport mechanisms. Among these drugs, nucleoside analogs are most well studied for their CNS distribution. The average reported values of the CSF/plasma steady-state concentration or corresponding AUC ratios are 0.23 (AZT), 0.06 (ddI), 0.04 (ddC), 0.49 (d4T), and 0.08 (3TC). Active efflux transport out of the CNS appears to be a predominant mechanism limiting nucleoside access to the CNS, although poor penetration may contribute to some extent for some polar nucleosides. The nature of the efflux pump for these drugs is speculated to be MRP-like transporter(s) in blood–brain and blood–CSF barriers. For non-nucleoside and protease inhibitors, much research remains to be done on the extent, time course, and mechanisms of their CNS distribution. The CNS penetration of some protease inhibitors is restricted by P-glycoprotein. A better understanding of transport mechanisms of anti-HIV drugs in the CNS is essential to develop approaches to enhance CNS delivery of available drugs and to identify new drugs less subject to active efflux transporter(s) in the CNS.

Introduction

Neurologic disorders are common in patients with human immunodeficiency virus (HIV) infection. About 7–20% of patients exhibit neurologic deficits as the first manifestation of symptomatic HIV infection, and the numbers increase to 30–70% in advanced HIV disease [1]. The incidence of pathologic abnormalities in the central nervous system (CNS) is even higher. Autopsy studies have shown that 75–90% of acquired immunodeficiency syndrome (AIDS) patients exhibit CNS pathologic abnormalities [2].

Neurologic dysfunction of AIDS patients involves all levels of the neuroaxis, including the brain, meninges, spinal cord, nerves, and muscles. Among these, AIDS dementia complex has been estimated to affect up to 60% of all individuals in the late stages of HIV disease [3]. AIDS dementia complex is also known as HIV dementia, HIV encephalopathy, or HIV-associated dementia complex. It has been an AIDS-defining condition since 1987 [4]. AIDS dementia complex is uncommon during the early, asymptomatic phases of HIV infection and tends to develop only after severe immunosuppression and AIDS-defining illness [5], [6], [7]. No significant differences in neuropsychiatric testing have been observed between HIV-infected and HIV-free persons [8], [9], [10].

In adults, the clinical manifestation of AIDS dementia complex was reported to be a subcortical dementia [11]. It is characterized by a cognitive impairment, motor dysfunction, and behavioral changes. Without treatment, the average survival time of AIDS dementia patients is about 6 months, less than half the average survival of nondemented AIDS patients [12], [13].

Neuroimaging studies have provided important insight into AIDS dementia complex. Using computed tomography and magnetic resonance imaging, studies have demonstrated that brain atrophy with concomitant enlargement in the cerebral ventricles and subarachnoid space is the most significant anatomical change in the brain of AIDS dementia patients [14]. The extent of the reduction in the brain volume generally increases with severity of the disease. In addition, increased water content and altered metabolic activities have been detected in various brain regions [14].

The cerebrospinal fluid (CSF) in AIDS dementia patients is found to be nonspecifically abnormal with mononuclear pleocytosis and elevated proteins [15], [16]. Despite this non-specificity, CSF examination is useful for excluding other infectious or neoplastic causes of dementia.

While underlying mechanism(s) of AIDS dementia complex are still unclear, most researchers at present favor the general hypothesis that it relates to a fundamental effect of HIV itself rather than to an opportunistic infection by another organism. It has been shown that HIV infection within the CNS involves cells derived from the bone marrow such as macrophages and microglia, not the ‘functional cells’ of the brain, i.e. neurons and oligodendrocytes [17]. In addition, no correlation was found between pathologic and clinical severity. To explain these observations, studies have been focused on the indirect mechanism of brain cell injury instead of the direct cytolytic activity of the virus. It is believed that viral protein gp120, macrophage/astrocyte-derived neurotoxins, and quinolinic acid may play a role in AIDS dementia complex [14], [17]. Cytokines such as tumor necrosis factor alpha (TNF-α) may also contribute to the neurologic injury in AIDS [14], [17].

The development of AIDS dementia complex and other HIV-associated neurologic disorders requires direct penetration of antiviral drugs into the CNS. HIV enters the CNS early, probably concomitant with initial systemic infection [18], [19]; and it has been found in the CNS of persons at all stages of HIV disease [15], [20]. Particularly, as the systemic viral load is greatly reduced by combination therapy, treatment of HIV infection in the CNS becomes even more important to prolong the survival of AIDS patients.

The importance of delivery of anti-HIV drugs into the CNS has recently been addressed by Lipton [21] and by Pialoux et al. [22]. These researchers suggest that the CNS may act as a sanctuary for HIV, rendering the virus less vulnerable to current antiviral treatment. Pialoux et al. [22] treated a patient with the combination therapy of zidovudine, lamivudine, and indinavir. They found that, in spite of plasma viral RNA levels below the detection limit (<200 copies/ml), the patient still developed HIV-related neurologic disorders. These results indicate that successful systemic treatment may not reduce viral load in the CNS, and this probably results from inadequate CNS distribution of these drugs. Accordingly, enhancement of the distribution of available agents into the CNS and development of new drugs with better penetration are keys for successful eradication of virus from this region. Knowledge of the extent and time course, as well as mechanism(s) of distribution of these drugs into the CNS is obviously the first prerequisite in achieving this aim.

The ability of HIV in the CNS to survive antiviral treatment is due to the presence of unique anatomical structures in the CNS which limit the distribution of anti-HIV drugs into this region. These structures are the blood–brain barrier and the blood–CSF barrier.

The blood–brain barrier is located between the blood and brain tissues, and is thought to consist primarily of cerebral capillary endothelial cells with tight junctions [23]. This barrier physically and metabolically protects the brain from many pathogens, immunogens, and toxic substances which may enter the blood, thus providing a stable environment for nerve cell interactions. Because the blood–brain barrier is essentially a lipid bilayer, lipid-soluble substances may enter the brain by passive diffusion. For water-soluble compounds like some nutrients, active transport systems in the blood–brain barrier may be involved in transporting these agents into the brain. For example, glucose, amino acids, and insulin active transport systems have been identified in the blood–brain barrier [24]. L-dopa utilizes an active transport system into the brain [24]. Active efflux transporters also exist in the blood–brain barrier, e.g. P-glycoprotein (P-gp) [25], [26]. These transporters function as an efflux pump to remove or prevent the entry of exogenous substances.

The blood–CSF barrier is primarily formed by the choroid plexus. Transport of a water-soluble drug from the blood into CSF is prevented by tight junctions located at the apical side of epithelium in the choroid plexus [27]. Some active transport systems also exist in the blood–CSF barrier. Physiologically, these carriers are mainly used for the transport of some ‘micronutrients’ into the CSF. Micronutrients are substances which are essential to the brain but only needed in relatively small amounts; these include vitamin C, folate, deoxyribonucleosides, vitamin B₆, and others [27]. These substances can then diffuse into brain tissues from the brain–CSF interface where no barrier exists, except for certain anatomic regions [28]. Some active efflux transporters like the organic anion transporter are located at the blood–CSF barrier [27].

For a drug which passively diffuses into the CNS, a number of factors govern the extent of distribution into the CNS. They are the permeability coefficient of drug through the blood–brain barrier; the luminal surface area; blood flow to the that region of the brain; the free (unbound) fraction of drug in plasma; and the time during which drug circulates to the brain. Some of these factors may not favor the distribution of anti-HIV drugs into the CNS. For example, polar nucleoside analogs may exhibit a low brain permeability coefficient. High plasma protein binding of protease inhibitors may restrict the distribution of these drugs into the CNS.

Methods for studying the transport of drugs into the CNS can be roughly divided into three categories, i.e. in vitro, in situ, and in vivo. Details on these methodologies are beyond the scope of this review. Instead, the general features of each approach and their strengths and limitations will be briefly summarized below.

One of the in vitro approaches is to use primary cultures of brain capillary endothelial cells. This in vitro cell model resembles the basic features of the blood–brain barrier [29] and provides a simple way to study permeability characteristics of compounds across this barrier. Compounds can easily be ranked based on their in vitro blood–brain barrier permeability. The in vitro system also allows one to study transport mechanisms and evaluate the factors which may control such a process. However, system-dependent changes in or a possible lack of expression of active transporters may hinder the use of in vitro models. For example, P-gp expression in cells cultured from human brain microvessels was decreased as compared with isolated microvessels, while multidrug resistance-associated protein exhibited increased expression in cultured brain cells [30]. Downregulation of some nutrient transport systems has been reported by as much as 100-fold [31]. Another disadvantage of the use of cell cultures is that the barrier in vitro is often much more permeable than in vivo. This would result in a much higher in vitro permeability value for a passively diffusing compound than in vivo, making in vitro to in vivo extrapolation difficult since the extent of this difference may depend on lipophilicity, molecular weight, etc. Establishing correlations between in vitro and in vivo permeabilities under carefully established and controlled conditions may allow investigators to overcome this problem [31], [32]. Other in vitro models for the study of CNS transport are isolated brain capillaries [33] and isolated choroid plexus [34].

The in situ methods include brain uptake index [35] and the brain perfusion technique [36]. Compared with in vitro systems, the in situ studies represent a closer approximation of in vivo conditions. Caution, however, should be taken to minimize the backflux from brain to blood in order to estimate permeability accurately, as it is often assumed that there is negligible efflux or diffusion from brain to blood during the study period. Disadvantages of such studies are that normal brain function must be maintained during perfusion, and that the effect of anesthetics on brain uptake is difficult to assess.

Various in situ techniques may provide different advantages. The brain perfusion technique allows absolute control over perfusion composition and flow rate; thus it is useful for studying transport mechanisms of drugs. A longer perfusion time (30 min) has been achieved recently to allow studies of the brain distribution of slowly permeating compounds [37]. Major advantages of the brain uptake index method are simplicity and speed; however, the technique is only suitable for moderately to highly permeable compounds because of the short duration of the experiment (<5 s). In addition, for studying drug transport in the CSF, the ventriculocisternal perfusion technique can be used [38].

The studies for brain distribution in vivo have been traditionally performed using brain tissue homogenization. However, this method measures only “total” drug concentration in the brain, providing no information regarding intra- or extracellular drug concentrations. In addition this technique does not permit an assessment of free drug concentrations, which represent driving force for transport and determine pharmacological activity. Brain tissue homogenization also uses a large numbers of animals. Finally, the interpretation of data from these studies is often complicated by substantial interanimal variability.

Microdialysis represents a relatively new approach to study drug transport in the CNS in vivo [39]. Using this technique, one can follow the extent and time course of distribution of a drug in the CNS in individual animals, thus significantly reducing the number of animals used and allowing assessment of both intra- and interanimal variability. Moreover, microdialysis experiments can be performed in conscious animals, avoiding potential complications of anesthesia. In addition, microdialysis measures unbound drug levels in extracellular fluid (ECF) and does not involve the removal of fluid from sampled tissues.

There are some technical difficulties in using microdialysis. Most notably the efficiency of the probe in sampling the tissue space (recovery) needs to be determined to estimate drug concentration in the probed tissue. Drugs which are highly bound to microdialysis probe membranes or associated tubing are not suitable for microdialysis studies. Finally, the utility of this methodology for high molecular weight compounds may be limited as recovery decreases with molecular size.

Recently, concerns have been raised for using microdialysis in studying drug transport in the CNS [40], [41]. In particular, the integrity of the blood–brain barrier during microdialysis has been questioned. There is no doubt that insertion of microdialysis probes into brain tissue causes disruption of the blood–brain barrier. The key question, however, is how much time is required for functionality of the barrier to return to normal following probe implantation? Evidence indicates that microdialysis experiments conducted within the first 8 h after implantation of a probe may be problematic with respect to local cerebral blood flow, glucose metabolism, and neurotransmitter release [42]. The optimal time for commencing microdialysis studies has been suggested to be at least 16 to 24 h after probe implantation [43]. Fontaine et al. [44] have found that the blood–brain barrier is functional 18 h after probe placement by comparing brain ECF/plasma AUC ratios of ³H₂O and ¹⁴C-mannitol (103% vs. 9%). Likewise, Yang et al. [45] began experiments 18–24 h after probe insertion. AZT steady-state CSF/plasma ratios determined in their study were in excellent agreement with results obtained by direct CSF sampling in rats (0.25±0.04 vs. 0.25±0.14). These studies demonstrate that microdialysis can be a powerful tool for studying drug transport in the CNS. Other techniques such as brain microinjection [46] are also useful in evaluating the CNS distribution of drugs.