Current Pharmaceutical Design - Volume 15, Issue 25, 2009

The principal aim of this special issue is to critically and translationally review the pathophysiology of bilirubin-induced neurological disease (BIND). Understanding the mechanisms of BIND can point the way to new diagnostic methods to predict the development of acute BIND and to prevent and treat it more effectively. Kernicterus is a rare disease of the nervous system, seen mostly in severely jaundiced neonates; its incidence is less than 1 in 30,000 jaundiced neonates (<1 per 50,000 neonates) [1]. Kernicterus is characterized by the deposition of the tetrapyrrolic bile pigment, unconjugated bilirubin (UCB) in the basal ganglia, hippocampus, and in several nuclear clusters of the brainstem and cerebellum. This results in a characteristic yellow staining of these brain nuclei, associated with impairment or loss of their function (encephalopathy) that is frequently irreversible. The molecular mechanisms by which UCB affects cellular functions have been elucidated only in the past two decades [2]. Recent major advances have clarified the relationships between the physicochemical structure and biological effects of UCB, which is an antioxidant and cytoprotective at low concentrations and cytotoxic at higher concentrations [3]. Therefore two chapters are devoted to an update of these important dual effects of UCB [4, 5]. Recent discoveries regarding the roles of ATP-binding cassette (ABC) proteins in maintaining low intracellular concentrations of the pigment have been followed by study of the strategic localization of these transporters in different regions of the CNS [2, 6, 7]. The critical role of the ABC proteins and their possible utilization as therapeutic targets are reviewed [8]. In vitro and in vivo studies have revealed the complicated network of sequential reactions that result in UCB cytotoxicity, offering multiple targets for potential therapeutic interventions [9, 10]. This includes also the passage of UCB across the two main barriers separating plasma and cerebrospinal fluid from neurons, astrocytes and glia. The role of these barriers is critically reviewed by Ghersi-Egea [11]. It is becoming increasingly clear that the unbound (free) fraction of plasma and tissue bilirubin (Bf) is the biologically active moiety that determines cytotoxicity [12, 13]. This concept is only just gaining recognition by those caring for jaundiced newborn infants, but additional clinical studies are needed to establish the true prognostic value of measurement of plasma Bf. This important point is reviewed [14,15]. This special issue is designed to rectify the lack of any recent, comprehensive, translational review of the mechanisms of BIND. It was assembled to update the advances of the past two decades and point the way to further investigations. We are grateful to all the authors for their thorough reviews of their individual areas of expertise.

Unconjugated bilirubin (UCB), the principal mammalian bile pigment, is the end product of heme catabolism. Both belong to the superfamily of tetrapyrrolic compounds that serve multiple biological functions in animals and plants. Its six internal hydrogen bonds give UCB a unique structure responsible for its physico-chemical properties and biological effects. Like many weakly-polar, poorly-soluble compounds, UCB is transported in blood tightly bound to albumin, with less than 0.01% of total bilirubin circulating in an unbound form (free bilirubin, Bf). This fraction governs the diffusion of UCB into tissues, and therefore Bf is responsible for both its beneficial and toxic effects on cells. Although, UCB was long thought to be a non-functional waste product, recent studies have shown that the antioxidant effects of mildly elevated serum bilirubin levels, as well as activation of heme oxygenase, may protect against diseases associated with oxidative stress, such as atherosclerosis. By contrast, markedly elevated serum UCB levels may cause severe neurological damage, especially in neonates. The regulation of cellular UCB content, by its conjugation, oxidation, and export, are, therefore of paramount importance to cellular health.

The ATP-Binding Cassette (ABC) superfamily is the largest transporter family known to translocate a wide variety of exogenous and endogenous substrates across cell membranes. In this chapter we review the potential role of three ABC proteins in the transport of unconjugated bilirubin (UCB). These transporters are MRP1, MRP3 and PGP (MDR1). MRP1 is expressed at high levels in most epithelia, usually at the basolateral membrane. Among a multiplicity of substrates, MRP1 mediates the ATP-dependent cellular export of UCB, and its role has been demonstrated in protecting cells from UCB toxicity. MRP3 is an organic anion transporter whose major substrates are GSH conjugates of organic compounds. Among the MRP family members, MRP3 shares the highest degree of amino acid homology with MRP1. Although the hepatic expression of MRP3 has been reported to be up-regulated by bilirubin and bilirubin glucuronides, it is unknown whether MRP3 is also involved in the transport of UCB. PGP is expressed in organs involved in the elimination of endo- and xenobiotics and UCB is one of these substrates. Since the Km of PGP for UCB is well above pathophysiological levels of Bf, it remains uncertain whether it has a role in protecting against UCB cytotoxicity.

The endothelium of the brain microvessels and the choroid plexus epithelium form highly specialized cellular barriers referred to as blood-brain interfaces through which molecular exchanges take place between the blood and the neuropil or the cerebrospinal fluid, respectively. Within the brain, the ependyma and the pia-glia limitans modulate exchanges between the neuropil and the cerebrospinal fluid. All these interfaces are key elements of neuroprotection and fulfill trophic functions; both properties are critical to harmonious brain development and maturation. By analogy to hepatic bilirubin detoxification pathways, we review the transport and metabolic mechanisms which in all these interfaces may participate in the regulation of bilirubin cerebral bioavailability in physiologic conditions, both in adult and in developing brain. We specifically address the role of ABC and OATP transporters, glutathione-S-transferases, and the potential involvement of glucuronoconjugation and oxidative metabolic pathways. Regulatory mechanisms are explored which are involved in the induction of these pathways and represent potential pharmacological targets to prevent bilirubin accumulation into the brain. We then review the possible alteration of the neuroprotective and trophic barrier functions in the course of bilirubin-induced neurological dysfunctions resulting from hyperbilirubinemia. Finally, we highlight the role of the blood-brain and blood-CSF barriers in regulating the brain biodisposition of candidate drugs for the treatment or prevention of bilirubin-induced brain injury.

Unconjugated bilirubin (UCB) is the major degradation product of the heme catabolism. UCB is a potent antioxidant molecule as well as an indirect pro-oxidant generator. Growing evidence suggests that its major cellular effects are mediated by inhibiting proliferation in cancer cell lines and eliciting cytotoxicity, particularly in neurons and glial cells. Here we describe studies showing that alteration of the redox status and generation of oxidative stress are likely early events responsible for UCB-induced cytotoxicity. We then elucidate some of the molecular pathways that govern these effects.

Hyperbilirubinemia is a common condition in neonatal life, where elevated levels of unconjugated bilirubin (UCB) may lead to adverse neurologic outcomes, namely in the presence of inflammatory features. In this review, we summarize recent concepts on UCB damage to brain cells and associated neuroinflammation research. Exposure of astrocytes and microglia to UCB initiates an inflammatory response with the release of proinflammatory cytokines, such as TNF-α, IL-1β and IL-6, accumulation of extracellular glutamate and a time-dependent cell death. Moreover, undifferentiated cells revealed to be particularly susceptible to UCB-induced immunostimulation pointing to a mechanism that may preside to the vulnerability evidenced by premature newborns. Evaluation of intracellular mechanisms of astrocyte and microglia to UCB revealed that TNF-α and IL-1β pathways as well as MAPK and NF-κB signaling cascades are key mediators of both cytokine production and cell toxicity observed upon UCB challenge. Understanding these mechanisms is essential for the development of new strategies targeting UCB-induced neurotoxicity. Thus, a therapeutic approach for the prevention or amelioration of neurological deficits resulting from moderate to severe hyperbilirubinemia, may consist on the use of immunomodulators, such as IL-10 that showed ability to suppress the release of cytokines from astrocytes exposed to UCB, glycoursodeoxycholic acid (GUDCA) that abrogated both UCB-stimulated cytokine secretion and UCBinduced loss of cell survival, and minocycline that evidenced a unique role in preventing neurodegeneration in in vitro and in vivo models. Novel pharmacological strategies may reduce the incidence of UCB encephalopathy and prevent minor cerebral lesions that may result in mental illness.

Severe unconjugated hyperbilirubinemia, seen mainly in neonates, may cause kernicterus and death. Conventional treatment for severe unconjugated hyperbilirubinemia consists of phototherapy and exchange transfusion. Phototherapy, however, has several known disadvantages while exchange transfusion is associated with a significant morbidity, and even mortality. These harmful effects indicate the need to develop alternative pharmacological treatment strategies for unconjugated hyperbilirubinemia. Generally, these strategies aim to decrease the plasma concentration of unconjugated bilirubin (UCB) by inhibiting production, stimulating hepatic clearance, or interrupting the enterohepatic circulation of the pigment. To be considered for routine clinical use, an alternative treatment strategy should be less invasive and at least as effective and safe as phototherapy. Several pharmacological therapies such as metalloporhyrins, clofibrate, bile salts, laxatives and bilirubin oxidase may meet these criteria in the future, but none of them has yet been evaluated sufficiently to allow routine application. This review aims to discuss the state of the art and future perspectives in pharmacological treatment of neonatal jaundice.

The American Academy of Pediatrics has proposed guidelines for treating term/near term infants with hyperbilirubinemia based primarily on maintaining the total serum bilirubin concentration (TSB) below a “critical” level of 25 mg/dL (426 μmol/L). We estimated the sensitivity and specificity of this critical TSB using patient data from published reports. A TSB >25 mg/dL is recorded in about 92% of term/near term infants with kernicterus. When TSB is adjusted lower for risk factors of prematurity, sepsis, and isoimmune hemolytic disease, the guidelines have nearly a 98% sensitivity. The specificity of the critical TSB, however, is very poor; false positive rates exceed 90% if all cases of acute bilirubin toxicity are included, and about 94-96% if the endpoint is mild or severe residual brain injury. Clinically measurable confounders that might explain the large variance in critical TSB include the plasma unbound “free” bilirubin concentration (Bf) and, possibly, the concentration of bilirubin photoisomers. Limited clinical experience supports the proposition that Bf is superior to TSB in identifying patients at risk. Specificity of Bf and TSB was compared in small cohort of babies with acute and permanent bilirubin encephalopathy. The false positive rate for TSB (at 100% sensitivity) was 94%, compared with only 55% for Bf. A more comprehensive comparison of TSB and Bf, controlled for clinical confounders and photoiomers, is needed to formulate intervention guidelines with improved specificity that will reduce hospital admissions for unnecessary treatment.

Virus infection and oncogenesis are two tightly linked processes. Some viruses are endowed with a direct transforming capability and infection activates inflammation that, in turn, favours tumor progression. Also, both inflammation and tumor trigger (and are strongly dependent from) angiogenesis. Finally, some oncogenic viruses release “virokines” that contribute to the development of virus-associated tumors. At a molecular level, viral proteins, cytokines, receptors and adhesion molecules “cross-contribute” to the different processes and, amazingly, many of them bind to heparin and to heparan sulfate proteoglycans to exert their functions. Heparin-like polysulfated (PS) or polysulfonated (PSN) compounds are an heterogeneous group of natural or synthetic molecules whose prototypes are PS heparin and PSN suramin. They vary in their backbone structure, length, number/disposition of sulfated/sulfonated groups. Different combinations of these features confer to PS/PSN the capacity to bind with variable specificity to those heparin-binding proteins that “cross-contribute” to virus infection and tumor progression. Taken together, these considerations suggest that heparin-like PS/PSN antagonists may act as multitarget drugs that may control at once virus infection and tumor progression by targeting different proteins simultaneously. Here we discuss the possibility to exploit PS/PSN compounds for the development of drugs at the cross-road of viral infection and oncogenesis, taking in consideration the past efforts, possible drawbacks and future perspectives.

Dendrimers represent a class of novel polymers having unique molecular architectures characterized by their well-defined structure, with a high degree of molecular uniformity, low polydispersity and properties that make them attractive materials for the development of nanomedicines. The dendrimer drug delivery can be achieved by coupling a drug through one of two approaches. Hydrophobic drugs can be complexed within the hydrophobic dendrimer interior to make them water-soluble or drugs can be covalently coupled onto the surface of the dendrimer. In addition, dendrimers have been shown to be capable of bypassing efflux transporters. A new generation of dendrimer-based delivery systems will enable the efficient transport of drugs across cellular barriers. This review deals principally with the synthesis, characterization and recent applications of dendrimers. In future it will only ever be possible to designate a dendrimer as safe means of drug delivery related to a specific application. However, so far limited clinical experience using dendrimers makes it impossible to designate any particular system which is safe and non toxic. Although there is widespread concern as to the safety of nanosized particles, preclinical and clinical experience gained during the development of polymeric excipients, biomedical polymers and polymer therapeutics showed that judicious development of dendrimer chemistry for each specific application will ensure development of safe and important materials for biomedical and pharmaceutical use.

Malaria continues to devastate much of the tropics and sub-tropics in spite of the availability of a number of antimalarial drugs. Part of this problem is due to the disadvantages of the drugs in use, which include (depending on the drug) side effects, reduced efficacy due to resistance, and high cost. Multiple traditional and novel approaches to the discovery and design of new antimalarial agents are likely to be required to furnish the new drugs necessary for improved malaria control. This review will address one novel and emerging approach, namely the development of hybrid antimalarial agents composed of two distinct antimalarial moieties joined covalently. Particular emphasis will be placed on the properties of the hybrids' design, including biological activity, advantages over other approaches, and the potential to address issues relating to resistance, solubility and formulation/delivery. The review will discuss the synthetic methodology used to form the hybrid and the ease by which it may be cleaved to form the independent components in vivo. The molecules discussed include hybrids of (i) artemisinins or other endoperoxide-based agents and quinolines (e.g. trioxaquines), (ii) chloroquine or other aminoquinolines and resistance-reversing or other agents, and (iii) peptidase inhibitors and other agents. The actual and potential advantages and disadvantages of such hybrids in relation to established single drugs or drug combinations will be discussed critically and promising future directions highlighted.