Translational research in central nervous system drug discovery

Of all the therapeutic areas, diseases of the CNS provide the biggest challenges to translational research in this era of increased productivity and novel targets. Risk reduction by translational research incorporates the “learn” phase of the “learn and confirm” paradigm proposed over a decade ago. Like traditional drug discovery in vitro and in laboratory animals, it precedes the traditional phase 1–3 studies of drug development. The focus is on ameliorating the current failure rate in phase 2 and the delays resulting from suboptimal choices in four key areas: initial test subjects, dosing, sensitive and early detection of therapeutic effect, and recognition of differences between animal models and human disease. Implementation of new technologies is the key to success in this emerging endeavor.     

The blood-brain barrier and its role in immune privilege in the central nervous system

The blood-brain barrier (BBB) provides both anatomical and physiological protection for the central nervous system (CNS), strictly regulating the entry of many substances and blood borne cells into the nervous tissue. Increased understanding of how the unique microenvironment in the CNS influences the BBB is crucial for developing novel therapeutic approaches to CNS diseases. In this review, we discuss those characteristics of the BBB that play an important role in maintaining immune privilege in the CNS, as well as factors that regulate immune cell invasion through the BBB and thereby modulate immune responses in the nervous tissue. In general, immune cell invasion across the BBB is highly restricted and carefully regulated. A florid invasion of activated white blood cells can create a predominantly proinflammatory local environment in the CNS, leading to immune-mediated diseases of the nervous tissue. Recent developments in cellular and molecular biological methods have allowed closer analysis of BBB function, and led to an improved understanding of the active role of the BBB in immune-mediated diseases of the CNS.     

Positron emission tomography in central nervous system drug discovery and development

Genetics, neuroscience, and imaging science have advanced greatly in the last few years. These advances can be brought together and applied in creative new ways to make available better drugs for treating neuropsychiatric disorders and for getting candidate drugs through the development process faster. One particular approach, built around [18F]fluordeoxyglucose positron emission tomography, is described.     

Investigation of the blood-brain barrier for IgG in inflammatory syndromes of the central nervous system

To investigate whether an intravenously administered compound of the IgG class is able to penetrate the CSF barrier despite its high molecular weight, 12 hepatitis B surface antibody (anti-HBs)-negative patients received 20 ml each of a beta-propiolactone-treated IgG compound with a high anti-HBs titer (1:115,000) as a marker. 8 had an inflammatory CSF syndrome. In cases of inflammatory CSF syndromes, significantly more marked IgG crossed the blood-brain barrier, this could be of considerable clinical importance.     

Patented in vitro blood-brain barrier models in CNS drug discovery

The blood-brain barrier (BBB) is a regulatory interface between the circulation and the central nervous system (CNS). Therapy of neurological diseases is limited due to restricted penetration of pharmacons across the BBB. Models for screening the brain penetration of drug candidates are needed early in drug discovery. Culture-based models are useful tools for both basic research on BBB, and testing the permeability of new therapeutical molecules. This review focuses on patented in vitro BBB models and their potential application in CNS drug discovery. Cell culture models using primary and immortalized brain endothelial cells of non-human and human origin, in co-culture or mono-culture setting, in static or dynamic conditions are discussed, as well as methods to induce BBB properties in such in vitro models. The aim of these models is to reproduce as many aspects as possible of the in vivo BBB. All models should show some elements of general endothelial and specific BBB properties, like physiologically realistic cell architecture, restrictive paracellular pathway, and functional expression of transport mechanisms. Though no "ideal in vitro BBB model" has been constructed yet, the currently available models provide valuable information on BBB permeability and are useful tools in CNS drug discovery.     

Fine structure and blood-brain barrier properties of the central nervous system of a dipteran larva.

Using scanning and transmission electron microscopy, we studied basic ultrastructure, membrane specializations, and blood-brain barrier properties of the ventral ganglion and abdominal nerves of the last (third) instar larva of a dipteran fly, Delia platura. Both ganglion and nerves are covered with a non-cellular neural lamella. A monolayer of flattened perineurial cells lies beneath the neural lamella. Perineurial cells contain stores of metabolites and nutrients and these cells extensively interdigitate with one another. An extensive extracellular series of channels pervades perineurial cells. Glial cells beneath the perineurium envelope but do not entwine axons. In a minority of cases, adjacent axons in nerve and neuropil appear to be contiguous without glial intervention. Extensive (pleated) septate junctions with triangular septa are present between perineurial cells. Hemidesmosomes, half desmosomes (a first report for invertebrates), and desmosomes were also observed. Although no tight junctions were discovered, an effective blood-brain barrier exists, and tracer (ionic lanthanum) in no case reached neuronal surfaces. Extracellular tracer halted within the extensive septate junctions between perineurial cells. We postulate that in the absence of tight junctions the functional blood-brain barrier is effected by the septate junctions in the central nervous system of the Delia larva.     

血脑屏障完整性及鞘内免疫球蛋白合成在中枢神经系统疾病中的意义

目的探讨中枢神经系统疾病血脑屏障(BBB)的完整性和鞘内免疫球蛋白合成的意义。方法将研究对象分为8组(A组为10例正常对照组,B组为10例化脓性脑膜炎患者,C组为10例结核性脑膜炎患者,D组为20例病毒性脑(膜)炎患者,E组为8例脱髓鞘疾病患者,F组为10例格林巴利综合症患者,G组为10例蛛网膜下腔出血和脑出血患者,H组为14例缺血性脑血管病患者),采用速率散射比浊方法测定血清和CSF的白蛋白(Alb)和免疫球蛋白(IgG、IgMI、gA)含量,利用公式计算QAlbI、gG指数、IgG合成率,并用Protis软件进行数据和图形处理分析。结果 (1)各组QAlb异常率均较正常对照组增高,其中B、C、E、G组QAlb明显高于正常对照组(P0.01);(2)B、C、E、G组IgG指数和IgG合成率较正常对照组明显增高(P0.05或0.01),其中IgG指数以E组最高,IgG合成率以B、E组最高;(3)经Protis软件分析82例患者,正常35例,单纯BBB功能障碍20例,仅有鞘内合成8例,BBB功能障碍伴有鞘内合成19例。结论通过对CSF中蛋白质组分定量的分析,并利用Protis软件进行数据和图形处理,可以明确中枢神经系统疾病血脑屏障完整性和鞘内免疫球蛋白的合成,以及对某些中枢神经系统疾病的诊断和鉴别诊断具有重要的临床意义。     

Alterations of the blood-brain barrier after transplantation of autonomic ganglia into the mammalian central nervous system

Autonomic (superior cervical) ganglia, the vessels of which are freely permeable to macromolecules, from mature rat donors (allografts or autografts) were transplanted to different sites in the central nervous system (CNS). Minimal trauma was caused by grafts into the IVth ventricle while grafts to intraparenchymal locations such as cerebral cortex and spinal cord were necessarily traumatic and produced glial scarring. Postoperative periods were between 4 weeks and 30 months. A potentially significant aspect of neural transplantation is the functional vascular connections between host and graft. It is highly likely that grafting procedures alter the blood-brain barrier (BBB) in the recipient brain. In order to determine permanent BBB changes, the glycoprotein horseradish peroxidase (HRP) (M.W. 40,000) was injected intravascularly for circulation periods ranging between 50 seconds and 90 minutes. Protein exudation was monitored by using the chromogens DAB and the highly sensitive TMB. All autonomic ganglia transplants, regardless of postoperative or HRP circulation times, were permeable to the injected protein; no qualitative differences were found between allografts and autografts. The blood-borne protein traversed the autonomic graft and infiltrated into the host brain for distances between 200 micron in intraparenchymal grafts to over 1 mm in intraventricular grafts; a smaller exudate was found in the intraparenchymal model than in the intraventricular site probably due to glial scarring that impeded the protein movement in the interstitial spaces. Significantly, TMB demonstrated that the systemic protein entered the cerebrospinal fluid. HRP was detected on the ventricular floor and in the perivascular spaces of the microvasculature. Transplantation of an autonomic ganglion into the brain provides a biological portal that bypasses normal barriers to macromolecules. The vascular and extracellular confluences between host and graft could provide direct access for systematically administered substances to enter brain regions where they, normally, would be excluded.     

Can SARS-CoV-2 infect the central nervous system via the olfactory bulb or the blood-brain barrier?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China in December 2019. On February 11, the World Health Organization (WHO) announced the name for the new illness caused by SARS-CoV-2: COVID-19. By March 11, the outbreak of COVID-19 was declared a pandemic by the WHO. This virus has extensively altered daily life for many across the globe, while claiming hundreds of thousands of lives. While fundamentally a respiratory illness, many infected individuals experience symptoms that involve the central nervous system (CNS). It is likely that many of these symptoms are the result of the virus residing outside of the CNS. However, the current evidence does indicate that the SARS-CoV-2 virus can use olfactory neurons (or other nerve tracts) to travel from the periphery into the CNS, and that the virus may also enter the brain through the blood–brain barrier (BBB). We discuss how the virus may use established infection mechanisms (ACE2, NRP1, TMPRSS2, furin and Cathepsin L), as well mechanisms still under consideration (BASIGIN) to infect and spread throughout the CNS. Confirming the impact of the virus on the CNS will be crucial in dealing with the long-term consequences of the epidemic.     

Plasma pharmacokinetics, nervous system biodistribution and biostability, and spinal cord permeability at the blood-brain barrier of putrescine-modified catalase in the adult rat.

Free radical-mediated oxidative damage has been proposed to be an underlying mechanism in several neurodegenerative disorders. Previous investigations in our laboratory have shown that putrescine-modified catalase (PUT-CAT) has increased permeability at the blood-brain (BBB) and blood-nerve barriers with retained enzymatic activity after parenteral administration when compared to native catalase (CAT). The goals of the present study were to examine the plasma stability, spinal cord BBB permeability, nervous system biodistribution, and spinal cord enzyme activity of CAT and PUT-CAT after parenteral administration in the adult rat. TCA precipitation and chromatographic analyses revealed that CAT and PUT-CAT were found intact in the plasma and in the central nervous system (CNS) after iv, ip, or sc bolus injections. The highest percentages of intact CAT or PUT-CAT proteins were found in the plasma after iv administration, and similar percentages of intact CAT or PUT-CAT were found in the CNS following all three types of administration. Increases of 2.4- to 4.7-fold in permeability at the BBB and similar increases in the levels of intact PUT-CAT were found in different brain regions compared to the levels of CAT. A 2.4-fold higher level of intact PUT-CAT compared to that of CAT (P < 0.05) was found in the spinal cord 60 min after a sc bolus injection. CAT enzyme activity in the spinal cord was 50% higher (P < 0.05) in rats treated with PUT-CAT continuously for 1 week by subcutaneously implanted, osmotic pumps than the activity found in rats treated with PBS. These results provide evidence that intact, enzymatically active PUT-CAT is efficiently delivered to the nervous system following iv, ip, and sc administration and suggest that sc administration of PUT-CAT may be effective in treating neurodegenerative disorders in which the underlying mechanisms involve the action of free radicals and oxidative damage.     

Horseradish peroxidase as a cytochemical marker of blood-brain barrier integrity in oxygen toxicity in the central nervous system

The ultrastructural integrity of endothelial tight junctions in cerebral microvessels of the rat exposed to hyperbaric oxygen was examined to find whether or not there are alterations of the blood-brain barrier in the initial phase of central nervous system oxygen toxicity. Rats were pre-implanted with cortical electrodes for continuous EEG and provided with two polyethylene cannulae inserted into the common carotid artery. HRP was administered to unanesthetized rats through a cannula directed into the brain, before the onset of O2 pressurization. As soon as the early electrical discharges of oxygen toxicity were recorded (20 to 30 min), the animal was killed by injection with saturated KCl into the heart. Examination of small blood vessels in both LM and EM did not support the possibility that the blood-brain barrier is altered prior to the first electrical discharge appearing in central nervous system oxygen toxicity.     

The blood-brain barrier in the cerebrum is the initial site for the Japanese encephalitis virus entering the central nervous system

Japanese encephalitis (JE) virus is a member of the encephalitic flaviviruses and frequently causes neurological sequelae in a proportion of patients who survive the acute phase of the infection. In the present study, we molecularly identified viral infection in the brain of mice with rigidity of hindlimbs and/or abnormal gait, in which JE virus particles appeared within membrane-bound vacuoles of neurons throughout the central nervous system. Deformation of tight junctions (TJs) shown as dissociation of endothelial cells in capillaries, implying that the integrity of the blood-brain barrier (BBB) has been compromised by JE virus infection. BBB permeability evidently increased in the cerebrum, but not in the cerebellum, of JE virus-infected mice intravenously injected with the tracer of Evans blue dye. This suggests that the permeability of the BBB differentially changed in response to viral infection, leading to the entry of JE virions and/or putatively infected leukocytes from the periphery to the cerebrum as the initial site of infection in the central nervous system (CNS). Theoretically, the virus spread to the cerebellum soon after the cerebrum became infected.     

Drug delivery to tumors of the central nervous system

Contemporary treatment of malignant brain tumors has been hampered by problems with drug delivery to the tumor bed. Inherent boundaries of the central nervous system, such as the blood-brain barrier or the bloodcerebrospinal fluid barrier, and a general lack of response to many chemotherapeutic agents have led to alternative treatment modalities. In general, all these modalities have sought to either disrupt or bypass the physiologic brain barriers and deliver the drug directly to the tumor. This article reviews past, as well as current, methods of drug delivery to tumors of the central nervous system. Special emphasis is placed on biodegradable polymers that can release chemotherapeutic agents against malignant gliomas. A variety of other nonchemotherapeutic drugs, including antiangiogenesis and immunotherapeutic agents, are presented in the context of new polymer technology. Finally, future directions in drug delivery are discussed with an overview on new advances in emerging biotechnology.     

Nanoparticles, a drug carrier system to pass the blood-brain barrier, permit central analgesic effects of i.v. dalargin injections

The Leu-enkephalin dalargin does not normally penetrate the blood brain barrier when given intravenously. Drug targeting to the brain was investigated by using poly(butylcyanoacrylate) nanoparticles which were coated with polysorbate 80. When injected intravenously in mice, dalargin-loaded nanoparticles coated with the polysorbate 80 induced an analgesic effect at doses of 5.0 mg/kg and 7.5 mg/kg dalargin as shown by hindlimb licking on the hot plate. Neither the intravenous injection of dalargin alone at various doses nor the mixture of dalargin-loaded nanoparticles without the polysorbate 80 were able to induce an analgesic activity. This confirms previous observations that nanoparticles provide a convenient method to deliver drugs across the blood-brain barrier.