N-, alpha-, and beta-Substituted 3-Aminopropionic acids: design,syntheses and antiseizure activities

A treatment for epilepsy is proposed based on analogues of 3-aminopropionic acid (beta-alanine), a putative neurotransmitter in the central nervous system (CNS). A model three point pharmacophore was proposed based on modelling data obtained from the study of antagonists for both the glial gamma-aminobutyric acid (GABA)-uptake site and the glycine co-agonist site of N-methyl-D-aspartate (NMDA) receptor. Three series of 3-aminopropionic acids containing, N-, alpha-, and beta-substituents, were designed and synthesized to probe the position and the size of a lipophilic binding pocket within the proposed pharmacophore. These analogues were tested in vivo for both their antiseizure activities and their neurologic toxicities. Among the fourteen novel 3-aminopropionic acids synthesized, eight were found to have promising antiseizure activity. This study shows that substitution on the N-terminus confers the greatest antiseizure activity, particularly against pilocarpine-induced seizures.     

Influence of T-2 and HT-2 Toxin on the Blood-Brain Barrier In Vitro: New Experimental Hints for Neurotoxic Effects

The trichothecene mycotoxin T-2 toxin is a common contaminant of food and feed and is also present in processed cereal derived products. Cytotoxic effects of T-2 toxin and its main metabolite HT-2 toxin are already well described with apoptosis being a major mechanism of action. However, effects on the central nervous system were until now only reported rarely. In this study we investigated the effects of T-2 and HT-2 toxin on the blood-brain barrier (BBB) in vitro. Besides strong cytotoxic effects on the BBB as determined by the CCK-8 assay, impairment of the barrier function starting at low nanomolar concentrations were observed for T-2 toxin. HT-2 toxin, however, caused barrier disruption at higher concentrations compared to T-2 toxin. Further, the influence on the tight junction protein occludin was studied and permeability of both toxins across the BBB was detected when applied from the apical (blood) or the basolateral (brain) side respectively. These results clearly indicate the ability of both toxins to enter the brain via the BBB.     

4-Aminobutyraldehyde as a precursor convertible to gamma-aminobutyric acid in vivo

It was found that 4-aminobutyraldehyde (ABAL) is a precursor convertible to gamma-aminobutyric acid (GABA) in vivo. [2,3-3H]ABAL was synthesized from [2,3-3H]putrescine. After the subcutaneous administration of [3H]ABAL at the dose of 1 mumol/g body weight, [3H]GABA was produced in the mouse brain in an amount of about 350 nmol/g brain in 10 min. After oral administration of [3H]ABAL at the dose of 2 mumol/g body weight, [3H]GABA was also produced in the brain in an amount of about 530 nmol/g brain in 30 min. It seems that peripherally administered ABAL penetrates the blood-brain barrier into the central nervous system and is rapidly metabolized to GABA in the brain.     

Perineurium in the Drosophila (Diptera : Drosophilidae) embryo and its role in the blood-brain/nerve barrier

A monolayer of perineurial cells overlies glia and neurons, and this stratum of the central nervous system is the principal site of the Drosophila (Diptera : Drosophilidae) blood-brain barrier. Perineurial cells are bonded together by pleated-sheet septate junctions that are the anatomical correlate of the vertebrate tight junction. The blood-brain barrier maintains the ionic homeostasis necessary for proper nerve function. It was known that a functioning blood-brain barrier is present in mature (Stage 17) Drosophila embryos, but the genesis of this barrier was not known. We surveyed the central nervous system of late stage embryos (15 through 17) to determine when perineurial cells could first be detected. These cells take their place in (on) the central nervous system and are joined together by pleated-sheet septate junctions, during Stage 17. Those septate junctions are quickly occlusive to lanthanum tracer. This development step occurs during the same time as when chemical synapses first become functional. Such concurrent maturation is far from coincidental, because partitioning nerves and their synapses from hemolymph (with its variable ionic constitution) are essential for normal electrophysiology. We discuss details of the germ line derivation of perineurial cells, their first detection in the embryonic central nervous system, their functional properties, and the polygonal cell-packing pattern seen in the larval central nervous system.     

中枢神经系统疾病治疗性抗体药物应用进展

单克隆抗体药物是一种新兴的治疗药物,具有高选择性,被用于多种疾病的治疗,如肿瘤、免疫疾病等,也可以用于中枢神经系统疾病,如阿尔茨海默病、帕金森病、中风和脑肿瘤等。然而,因为血脑屏障低通透性,限制了抗体药物在中枢神经系统疾病治疗中的应用,在很多神经系统疾病临床试验中,抗体药物并没有取得预期效果。如今,人们利用血脑屏障上内源性转运蛋白介导,设计了可以通过血脑屏障的抗体药物。对通过血脑屏障治疗性抗体药物研发进展及其应用前景进行了综述。     

Minireview. Peptides and the blood-brain barrier

Most neuropeptides are known to occur both in the central nervous system and in blood. This, as well as the occurrence of central nervous peptide effects after peripheral administration, show the importance of studying the relationships between the peptides in the two compartments. For many peptides, such as the enkephalins, TRH, somatostatin and MIF-1, poor penetration of the blood-brain barrier was shown. In other cases, including beta-endorphin and angiotensin, peptides are rapidly degraded during or just after their entry into brain or cerebrospinal fluid. Some peptides, such as insulin, delta-sleep-inducing peptide, and the lipotropin-derived peptides, enter the cerebrospinal fluid to a slight or moderate extent in the intact form. Many peptide hormones, such as insulin, calcitonin and angiotensin, act directly on receptors in the circumventricular organs, where the blood-brain barrier is absent. Oxytocin, vasopressin, MSH, and an MSH-analog alter the properties of the blood-brain barrier, which may result in altered nutritient supply to the brain. In conclusion, the diffusion of most peptides across the brain vascular endothelium seems to be severely restricted. There are, however, several alternative routes for peripheral peptides to act on the central nervous system. The blood-brain barrier is a major obstacle for the development of pharmaceutically useful peptides, as in the case of synthetic enkephalin-analogs.     

Entry of EGF into brain is rapid and saturable.

Epidermal growth factor (EGF) is a neurotrophic peptide produced both in the central nervous system and the periphery. Peripheral administration of EGF causes central nervous system-mediated changes. The central nervous system effects could be explained by the permeation of EGF across the blood-brain barrier (BBB). In this report, we show that 125I-EGF crosses the BBB rapidly, with an influx rate of about 2 microl/g x min, much faster than that for neurotrophins, cytokines, and most other bioactive peptides tested. The 125I-EGF was recovered intact in the brain 10 min after i.v. injection, and the majority of the peptide reaching the brain was present in the parenchyma. The fast rate of influx was significantly decreased by co-administration of nonradiolabeled EGF and transforming growth factor alpha, peptides that share the EGF receptor. By contrast, a monoclonal antibody against the EGF receptor failed to inhibit the entry of EGF. Furthermore, mice with a mutation in the EGF receptor had no significant decrease in the rapid rate of entry of 125I-EGF. By contrast to the fast rate of entry, 125I-EGF injected intracerebroventricularly (i.c.v.) only exited the brain with the bulk flow of cerebrospinal fluid. Thus, EGF has a saturable transport system at the BBB for rapid, unidirectional influx. The transport system does not require the entire EGF receptor and is susceptible to possible therapeutic manipulation.     

Facilitated Transport of the Neurotoxin, β-N-Methylamino-l-Alanine, Across the Blood-Brain Barrier

beta-N-Methylamino-L-alanine (BMAA) is a neurotoxic plant amino acid that has been implicated in the pathogenesis of the high incidence amyotrophic lateral sclerosis and related parkinsonism dementia of the western Pacific. Previous studies have demonstrated that BMAA is taken up into brain following intravenous or oral administration. To examine the kinetics and mechanism of brain transfer, BMAA influx across the blood-brain barrier was measured in rats using an in situ brain perfusion technique. BMAA influx was found to be saturable with a maximal transfer rate (Vmax) of 1.6 +/- 0.3 x 10(-3) mumol/s/g and a half-saturation constant (Km) of 2.9 +/- 0.7 mM based on total perfusate BMAA concentration. Uptake was sodium independent and inhibitable by excess L-leucine, but not by L-lysine, L-glutamate, or methylaminoisobutyric acid, indicative of transfer by the cerebrovascular large neutral amino acid carrier. L-BMAA competitively reduced brain influx of L-[14C]leucine, as expected for cross-inhibition. The results demonstrate that BMAA is taken up into brain by the large neutral amino acid carrier of the blood-brain barrier and suggest that uptake may be sensitive to the same factors that affect neutral amino acid transport, such as diet, metabolism, disease, and age.     

Atrial natriuretic peptide in the central nervous system of the rat

1. Studies of the presence of atrial natriuretic peptide immunoreactivity and receptor binding sites in the central nervous system have revealed unusual sites of interest. 2. As a result, numerous studies have appeared that indicate that brain atrial natriuretic peptide is implicated in the regulation of blood pressure, fluid and sodium balance, cerebral blood flow, brain microcirculation, blood-brain barrier function, and cerebrospinal fluid production. 3. Alteration of the atrial natriuretic peptide system in the brain could have important implications in hypertensive disease and disorders of water balance in the central nervous system.     

Retrograde axonal transport of an exogenous enzyme covalently linked to B-IIb fragment of tetanus toxin.

Attempt to replace enzymes in a number of fatal lysosomal storage disease involving the central nervous system have as yet been unsuccessful owing to the impermeability of the blood/brain barrier to macromolecules. In order to treat storage disease due to enzyme deficiencies, we investigated the feasibility of transporting an enzyme into the central nervous system without crossing the blood/brain barrier. Using the B-IIb fragment of tetanus toxin (because it is involved in recognition by the nerve-cell endings), retrograde axonal transport toward the spinal cord and trans-synaptic movement, and glucose oxidase as a marker, we demonstrated that a non-toxic enzyme-vector conjugate was taken up by axon terminals. After injection into the gastrocnemius muscle, the B-IIb-glucose oxidase conjugate was detected, both histologically and electrochemically, distally to a ligature on the sciatic nerve. Thus the B-IIb fragment could serve as a vector for glucose oxidase transport into the central nervous system. It was also verified that the transported enzyme retained its activity. Transport of this 150 kDa molecule by fragment B-IIb of tetanus toxin suggests that other enzymes of a lesser molecular mass may also be transported.     

A cell culture model of the blood-brain barrier

Endothelial cells that make up brain capillaries and constitute the blood-brain barrier become different from peripheral endothelial cells in response to inductive factors found in the nervous system. We have established a cell culture model of the blood-brain barrier by treating brain endothelial cells with a combination of astrocyte-conditioned medium and agents that elevate intracellular cAMP. These cells form high resistance tight junctions and exhibit low rates of paracellular leakage and fluid-phase endocytosis. They also undergo a dramatic structural reorganization as they form tight junctions. Results from these studies suggest modes of manipulating the permeability of the blood-brain barrier, potentially providing the basis for increasing the penetration of drugs into the central nervous system.     

Blood–Brain Barrier Transport of Kynurenines: Implications for Brain Synthesis and Metabolism

To evaluate the potential contribution of circulating kynurenines to brain kynurenine pools, the rates of cerebral uptake and mechanisms of blood-brain barrier transport were determined for several kynurenine metabolites of tryptophan, including L-kynurenine (L-KYN), 3-hydroxykynurenine (3-HKYN), 3-hydroxyanthranilic acid (3-HANA), anthranilic acid (ANA), kynurenic acid (KYNA), and quinolinic acid (QUIN), in pentobarbital-anesthetized rats using an in situ brain perfusion technique. L-KYN was found to be taken up into brain at a significant rate [permeability-surface area product (PA) = 2-3 x 10(-3) ml/s/g] by the large neutral amino acid carrier (L-system) of the blood-brain barrier. Best-fit estimates of the Vmax and Km of saturable L-KYN transfer equalled 4.5 x 10(-4) mumol/s/g and 0.16 mumol/ml, respectively. The same carrier may also mediate the brain uptake of 3-HKYN as D,L-3-HKYN competitively inhibited the brain transfer of the large neutral amino acid L-leucine. For the other metabolites, uptake appeared mediated by passive diffusion. This occurred at a significant rate for ANA (PA, 0.7-1.6 x 10(-3) ml/s/g), and at far lower rates (PA, 2-7 x 10(-5) ml/s/g) for 3-HANA, KYNA, and QUIN. Transfer for KYNA, 3-HANA, and ANA also appeared to be limited by plasma protein binding. The results demonstrate the saturable transfer of L-KYN across the blood-brain barrier and suggest that circulating L-KYN, 3-HKYN, and ANA may each contribute significantly to respective cerebral pools. In contrast, QUIN, KYNA, and 3-HANA cross the blood-brain barrier poorly, and therefore are not expected to contribute significantly to brain pools under normal conditions.     

Effect of palmitate on carbohydrate utilization and Na/K-ATPase activity in aortic vascular smooth muscle from diabetic rats

Immediately after bacterial endotoxin (LPS) enters the circulatory system there is increased production of free oxygen radicals by cells of the reticulo-endothelial system, followed by the release of cytokines considered as putative endogenous pyrogens. Fever originates by central nervous system activities, but neither exogenous nor endogenous pyrogens are able to cross the blood-brain barrier and the true signal which is transmitted to structures inside the blood-brain barrier is still unknown. To study the role of oxygen radicals in fever, we pretreated rats with methylene blue, an inhibitor of superoxide and hydroxyl radical production and investigated the febrile response to LPS in conscious rats by measuring malondialdehyde formation as an index of lipid peroxidation by oxygen radicals. Methylene blue lowered resting malondialdehyde levels to near detection level and significantly suppressed its rise which was regularly found following LPS in the untreated state. Pretreatment with methylene blue completely blocked the febrile response. Since fever is a central nervous system-mediated response these results indicate that the brain is able to sense oxidative stress and vicinal thiol groups of the redox-modulatory site of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor-channel complex could function as a possible receptive structure. To test this hypothesis we injected rabbits with the disulfide reducing agent dithiothreitol (DTT), known to penetrate the blood-brain barrier, and monitored its effect on normal and febrile body temperatures. DTT induced, independently of ambient temperature, within minutes and dose-dependently the full pattern of heat loss responses causing a fall of core temperature, indicative of a lowered thermoregulatory setpoint. Pretreatment with a bolus dose of 5 mg/kg DTT, followed by a continuous infusion of 5 mg/kg/h for 3 h completely prevented LPS-induced fever. A bolus dose of 20 mg/kg DTT, starting 30 min after LPS, immediately reversed the febrile cold defense pattern and lowered body temperature. We conclude that DTT reduces in the central nervous system oxidized vicinal thiol groups of NMDA receptors, thereby augmenting glutamate-induced nitric oxide synthase activation, and, thus, enhanced formation of NO, which, in turn, lowers the thermoregulatory setpoint. Reduction of other disulfide-containing molecules, especially oxidized glutathione and thiol-containing enzymes, by DTT by might additionally contribute to preventing fever.     

Cationization, a process for the delivery of antibodies to the central nervous system. Problems encountered in its application for immunotherapy strategies such as those for clostridial poisoning

The central nervous system is separated from the rest of the body by the blood-brain barrier. This barrier prevents many substances, such as the antibodies, to penetrate into the brain making it difficult to use them for the treatment of brain diseases, such as tetanus and botulism. These two diseases are caused by the development of bacilli of the genus Clostridium which release neurotropic toxins. Specific antibodies can neutralize toxin activity when the toxin is in the blood but are ineffective when it is transported into nerve cells. Various invasive strategies have been used to deliver antibodies to the brain. However, they can induce seizures and transient neurologic deficits and may be applicable only for diseases restricted to the brain surface. Physiologically based strategies utilizing transport systems naturally present at the blood-brain barrier appear to be a more promising approach to brain delivery of antibodies. Cationization is a chemical treatment that causes the conversion of superficial carboxyl groups on a protein into extended primary amino groups. This is used to increase interactions of this protein with the negative charges at the luminal plasma membrane of the brain endothelial cells. The cationized protein can then undergo adsorptive mediated transcytosis through the blood-brain barrier. There are many problems yet to be solved in successfully carrying out in vivo applications of cationized antibodies. One of these problems is that cationization can cause damage to an antibody molecule and, thus, can compromise its binding affinity. Depending on the radiolabelling of the cationized antibodies, a serum inhibition phenomenon can possibly alter the pharmacokinetics and the organ distribution of these molecules. The antibodies can be cationized using various, synthetic (hexamethylenediamine) or naturally occuring (e.g., putrescine) polyamines. Hexamethylenediamine-induced and putrescine-induced brain uptakes of various antibodies and proteins have been shown, but the results obtained suggest that cationization with putrescine may be a more efficient approach to blood-brain barrier delivery. The development of animal or cellular models to check for therapeutic efficacy of cationized antibodies is necessary. In spite of the difficulties, the studies described in this paper indicate that cationization can be a realistic delivery strategy for carrying antibodies across the blood-brain barrier. The advances made in antibody technologies help generate more appropriate immunological structures for brain transfer with better effector functions and decreased immunogenicity or toxicity. Taken together, these two aspects can lead to further developments in treatment of intoxications caused by the clostridial neurotoxins.     

LYSINE METABOLISM IN THE RAT BRAIN: BLOOD—BRAIN BARRIER TRANSPORT, FORMATION OF PIPECOLIC ACID AND HUMAN HYPERPIPECOLATEMIA

Through the use of intravenous pulse injection of L-[U-¹⁴C] lysine, the blood-brain barrier transport of L-lysine was studied. The uptake of L-lysine plus metabolites in the brain remained essentially unchanged at approx 0.002–0.005 nmol/g in the low dose (3μg per kg body weight) injection, and 20–40 nmol/g in the high dose (30 mg/kg) injection throughout the time intervals of up to 60 min. The uptake of L-lysine plus metabolites in the heart, however, decreased substantially from 0.03 to 0.003 nmol/g in the low dose injection and from 320 to 62 nmol/g in the high dose injection. The plasma to heart uptake ratio only decreased slightly through the 60 min period: from 6 to 2 in either the low or high dose L-lysine injection. The plasma to brain uptake ratio, however, decreased rapidly from a high of 62 to a low of about 4 in either the low or high dose injection throughout the 60-min time course. Study of labeled L-pipecolate formation in the plasma and individual organs indicates that this compound was formed only in the brain to a significant level within 0.5 min of ¹⁴C-L-lysine intravenous pulse injection. Labeled pipecolate was recovered from heart, liver, kidney and plasma in significant quantities only at 2 min or later after pulse-injection. It is concluded that the blood-brain barrier of L-lysine in the rat is not particularly strong and that the rat brain may be primarily responsible for L-pipecolate synthesis from L-lysine. The possible etiology of human hyperpipecolatemia is also discussed in light of the current findings.     

A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration

A new system for lineage ablation is based on transgenic expression of a diphtheria toxin receptor (DTR) in mouse cells and application of diphtheria toxin (DT). To streamline this approach, we generated Cre-inducible DTR transgenic mice (iDTR) in which Cre-mediated excision of a STOP cassette renders cells sensitive to DT. We tested the iDTR strain by crossing to the T cell- and B cell-specific CD4-Cre and CD19-Cre strains, respectively, and observed efficient ablation of T and B cells after exposure to DT. In MOGi-Cre/iDTR double transgenic mice expressing Cre recombinase in oligodendrocytes, we observed myelin loss after intraperitoneal DT injections. Thus, DT crosses the blood-brain barrier and promotes cell ablation in the central nervous system. Notably, we show that the developing DT-specific antibody response is weak and not neutralizing, and thus does not impede the efficacy of DT. Our results validate the use of iDTR mice as a tool for cell ablation in vivo.     

4-Aminobutyraldehyde as a Substance Convertible In Vivo to GABA

Abstract: [2,3-³H]4-Aminobutyraldehyde ([³H]ABAL) was injected subcutaneously into mice, which were sacrificed at various intervals following injection. [³H]γ-Aminobutyric acid ([³H]GABA) synthesized in vivo from [³H]ABAL was extracted from the brains, separated, and quantitated. The results showed that in the brain, injected [³H]ABAL was rapidly transformed into [³H]GABA. [³H]ABAL may penetrate the blood-brain barrier into the central nervous system and then be oxidized to [³H]GABA.     

AN ANTIGENIC POLYPEPTIDE FRAGMENT ISOLATED FROM TETANUS TOXIN: CHEMICAL CHARACTERIZATION, BINDING TO GANGLIOSIDES AND RETROGRADE AXONAL TRANSPORT IN VARIOUS NEURON SYSTEMS

Abstract— The isolation and purification of an antigenic polypeptide fragment from tetanus toxin is described. The main physico-chemical, chemical, immunological, and pharmacological characteristics of this fragment, designated as B-II_b fraction, are reported. It is a polypeptide with a molecular weight close to 46,000. Its amino acid composition is on the whole comparable with that of the toxin. It contains one disulphide link and two free sulphhydryl groups which are not directly available for reaction. Tyrosine and lysine were found to be the two N -terminal groups. However, that B-II_b fraction has a subchain structure is still to be demonstrated. There is some evidence that B-II_b fraction may consist of 'isofragments'. This toxin fragment shows a cross-reaction with intact toxin and a specific flocculating activity of about three times that of the latter. In contrast, however, B-II_b fraction exhibits a specific toxicity approximately one hundred thousand times lower than the intact toxin. Although practically atoxic, this toxin fragment is still able to bind to gangliosides with an affinity which is even greater than that of the toxin. It is also capable of migrating towards the central nervous system by a mechanism of retrograde axonal transport as shown in peripheral adrenergic, sensory and motoneurons.
These unique features of B-II_b fraction are discussed in relation to the use of such fragments both for competing in vivo with the attachment of the toxin in the central nervous system and for specifically carrying therapeutic and pharmacological drugs into the central nervous system by neural route.     

Analog of vertebrate anionic sites in blood-brain interface of larval Drosophila

The blood-brain barrier ensures brain function in vertebrates and in some invertebrates by maintaining ionic integrity of the extraneuronal bathing fluid. Recent studies have demonstrated that anionic sites on the luminal surface of vascular endothelial cells collaborate with tight junctions to effect this barrier in vertebrates. We characterize these two analogous barrier factors for the first time on Drosophila larva by an electron-dense tracer and cationic gold labeling. Ionic lanthanum entered into but not through the extracellular channels between perineurial cells. Tracer is ultimately excluded from neurons in the ventral ganglion mainly by an extensive series of (pleated sheet) septate junctions between perineurial cells. Continuous junctions, a variant of the septate junction, were not as efficient as the pleated sheet variety in blocking tracer. An anionic domain now is demonstrated in Drosophila central nervous system through the use of cationic colloidal gold in LR White embedment. Anionic domains are specifically stationed in the neural lamella and not noted in the other cell levels of the blood-brain interface. It is proposed that in the central nervous system of the Drosophila larva the array of septate junctions between perineurial cells is the physical barrier, while the anionic domains in neural lamella are a charge-selective barrier for cations. All of these results are discussed relative to analogous characteristics of the vertebrate blood-brain barrier.     

Mechanisms of microbial traversal of the blood-brain barrier

Central nervous system (CNS) infections continue to be an important cause of morbidity and mortality. Microbial invasion and traversal of the blood-brain barrier is a prerequisite for CNS infections. Pathogens can cross the blood-brain barrier transcellularly, paracellularly and/or in infected phagocytes (the so-called Trojan-horse mechanism). Consequently, pathogens can cause blood-brain barrier dysfunction, including increased permeability, pleocytosis and encephalopathy. A more complete understanding of the microbial-host interactions that are involved in microbial traversal of the blood-brain barrier and the associated barrier dysfunction should help to develop new strategies to prevent CNS infections.