Brain tumours—Problems and perspectives

The interpretation of carcinogenesis bioassay data is a complex process that requires a ‘weight of evidence’ approach rather than a rigid decision based solely on statistics. This is particularly true for rat brain tumours, since recent bioassays have demonstrated rather marked variations in the incidence of these neoplasms. The objective of this paper is to point out some of the considerations that should be included in such a ‘weight of evidence’ approach and to provide a few examples relevant to brain tumours. Areas for future research on mechanisms of neuro-oncology should provide additional information relevant to determining the chemical causes of brain tumours.     

Brain metallothionein and neuroprotection against metal toxicity

Metuls axe important etiology factors in neurodegenerative diseases. Metallothionein （MT） , a cystein - rich, metal - binding protein, has been proposed to play important roles in neurobiology. MT - 1 and MT -2 are dramatieally increased by neuroinflammation and oxidative stress, and are an important adaptive meehanism against toxic insults. MT -3 has been implicated in Alzheimer disease. Metallothioneins can be induced by metals, herbal medicines, and various stimuli, and induetion of brain metallothionein ean be envisioned as an important meehanism against oxidative stress and neurodegeneratire diseases. Cadmium is neurotoxie and has been implicated in Parkinson＇s diseases in humans. Tile role of oxidative stress and metallothionein induetion in eadmium neurotoxicity was studied in rat primary mesencencephalic neuron - glia euhures. Cadmium aceumulated in neuron - glia cultures and produced cytotoxicity in a dose - dependent manner. Cadmium damage to dopaminergic neurons was evidenced by decreased ^3 H - dopamine uptake. Cadmium induced oxidative stress were further demonstrated by increases in 2＇7＇- dechlorofluorescein signals and electron spin resonance signals with 5, 5 - dimethyl -1 -pyrroline N -oxide （DMPO） as a spin trap agent. Cadmium activation of redox sensitive transeription factor NF - kB and AP - 1 was demonstrated by the gel - shift assays. The expression of brain MT - 1 mRNA was dose - dependently increased up to 60 - fold, along with other genes against oxidative stress including heine oxygenase - 1 and glutathione S - transferases. The dramatic induction of metallothionein could well play a protective role against eadmium toxicity to dopaminergie neurons and against cadmium - induced oxidative stress.     

Lomustine Nanoparticles Enable Both Bone Marrow Sparing and High Brain Drug Levels – A Strategy for Brain Cancer Treatments

Purpose

The blood brain barrier compromises glioblastoma chemotherapy. However high blood concentrations of lipophilic, alkylating drugs result in brain uptake, but cause myelosuppression. We hypothesised that nanoparticles could achieve therapeutic brain concentrations without dose-limiting myelosuppression.

Methods

Mice were dosed with either intravenous lomustine Molecular Envelope Technology (MET) nanoparticles (13 mg kg^?1) or ethanolic lomustine (6.5 mg kg^?1) and tissues analysed. Efficacy was assessed in an orthotopic U-87 MG glioblastoma model, following intravenous MET lomustine (daily 13 mg kg^?1) or ethanolic lomustine (daily 1.2 mg kg^?1 - the highest repeated dose possible). Myelosuppression and MET particle macrophage uptake were also investigated.

Results

The MET formulation resulted in modest brain targeting (brain/ bone AUC_0-4h ratios for MET and ethanolic lomustine?=?0.90 and 0.53 respectively and brain/ liver AUC_0-4h ratios for MET and ethanolic lomustine?=?0.24 and 0.15 respectively). The MET formulation significantly increased mice (U-87 MG tumours) survival times; with MET lomustine, ethanolic lomustine and untreated mean survival times of 33.2, 22.5 and 21.3 days respectively and there were no material treatment-related differences in blood and femoral cell counts. Macrophage uptake is slower for MET nanoparticles than for liposomes.

Conclusions

Particulate drug formulations improved brain tumour therapy without major bone marrow toxicity.

     

Brain and immune system - a complex relation

The interactions between central nervous system (CNS) and immune system are manifold and diverse. First, neuronal function is known to actively modulate immune reactivity, either directly by acting on T- or B- lymphocytes, or indirectly by affecting the cellular microenvironment. Second,and     

Does Bosentan Protect Diabetic Brain Alterations in Rats? The Role of Endothelin‐1 in the Diabetic Brain

Diabetes mellitus (DM) is a major problem all over the world, affecting more people in recent years. Individuals with diabetes are more prone to disease than non‐diabetics, especially vascular complications. The aim of this study was to examine the roles of the endothelin (ET)‐1 in brain damage formed in a streptozocin (STZ)‐induced diabetes model, and the effect of bosentan, which is the non‐specific ET1 receptor blocker in the prevention of the diabetes‐induced brain damage. To examine the effects of bosentan (50 mg/kg and 100 mg/kg) in this study, the rats were given the drug for 3 months. The rats were divided into four groups: the sham group (n = 10), the diabetic control group (n = 10), the group of diabetic rats given bosentan 50 mg/kg (n = 10) and the group of diabetic rats given bosentan 100 mg/kg (n = 10). Diabetes was induced in the rats by STZ (60 mg/kg i.p.). On day 91, all rats were killed. Brain tissues of the rats were measured by molecular, biochemical and histopathological methods. Antioxidant levels in the therapy groups were observed as quite near to the values in the healthy group. In this study, while the brain eNOS levels in the diabetic groups decreased, the ET1 and iNOS levels were found to be increased. However, in the diabetes group, hippocampus and cerebellum, pericellular oedema and a number of neuronal cytoretraction were increased in neuropiles, whereas these results were decreased in the therapy group. Based on all of these results, ET1 will not be ignored in diabetes‐induced cerebral complications.     

Effect of Insulin Receptor-Knockdown on the Expression Levels of Blood–Brain Barrier Functional Proteins in Human Brain Microvascular Endothelial Cells

Pharmaceutical Research - The insulin receptor (INSR) mediates insulin signaling to modulate cellular functions. Although INSR is expressed at the blood–brain barrier (BBB), its role in the...     

Blood–Brain Barrier Transport of Therapeutics via Receptor-Mediation

Drug delivery to the brain is hindered by the presence of the blood-brain barrier (BBB). Although the BBB restricts the passage of many substances, it is actually selectively permeable to nutrients necessary for healthy brain function. To accomplish the task of nutrient transport, the brain endothelium is endowed with a diverse collection of molecular transport systems. One such class of transport system, known as a receptor-mediated transcytosis (RMT), employs the vesicular trafficking machinery of the endothelium to transport substrates between blood and brain. If appropriately targeted, RMT systems can also be used to shuttle a wide range of therapeutics into the brain in a noninvasive manner. Over the last decade, there have been significant developments in the arena of RMT-based brain drug transport, and this review will focus on those approaches that have been validated in an in vivo setting.     

In Situ Blood–Brain Barrier Transport of Nanoparticles

PURPOSE: Two novel types of nanoparticles were evaluated as poten tial carriers for drugs across the blood-brain barrier (BBB). METHODS: Nanoparticles were composed of biocompatible materials including emulsifying wax (E. Wax) or Brij 72. Brij 78 and Tween 80 were used as surfactants for E. Wax nanoparticles (E78 NPs) and Brij 72 nanoparticles (E72 NPs), respectively. Both nanoparticle formulations were prepared from warm microemulsion precursors usin melted E. Wax or Brij 72 as the oil phase. Nanoparticles were radio-labeled by entrapment of [3H]cetyl alcohol, and entrapment efficiency and release of radiolabel were evaluated. The transport of E78 and E72 NPs across the BBB was measured by an in situ rat brai perfusion method. RESULTS: Both formulations were successfully radiolabeled by entrapment of [3H]cetyl alcohol; -98% of radiolabel remained associated with nanoparticles at experimental conditions. The transfer rate (Kin) of E78 NPs from perfusion fluid into the brain was 4.1 +/- 0.5 x 10(-3) ml/s/g, and the permeability-surface area product (PA) was 4.3 +/- 0.7 x 10(-3) ml/s/g. The values for Kin and PA for E72 NPs were 5.7 +/- 1.1 x 10(-3) ml/s/g and 6.1 +/- 1.4 x 10(-3) ml/s/g, respectively. CONCLUSIONS: For both nanoparticle types, statistically significant uptake was observed compared to [14C]sucrose, suggesting central nervous system uptake of nanoparticles. The mechanism underlying th nanoparticle brain uptake has yet to be fully understood.     

Brain stimulation reward „Thresholds” self-determined in rat

Summary The self-stimulation technique of Olds is modified to permit a continuous determination of the smallest current levels that will produce a rewarding effect by stimulating positive brain sites. The animal receives brief brain shocks that decrease in intensity in small steps by operating one lever, and indicates which current step stimulated insufficiently by resetting the current at a second lever. Preliminary data suggest that the technique will provide a valuable tool for the investigation of the central nervous system actions of drugs.Public Health research fellow of the National Institute of Mental Health.     

Construction and Functional Evaluation of a Three-Dimensional Blood–Brain Barrier Model Equipped With Human Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells

Purpose

The purpose of this study was to construct and validate an in vitro three-dimensional blood–brain barrier (3DBBB) model system equipped with brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs).

Methods

The 3D-BBB system was constructed by seeding hiPS-BMECs onto the capillary lane of a MIMETAS OrganoPlate^® 3-lane coated with fibronectin/collagen IV. hiPS-BMECs were incubated under continuous switchback flow with an OrganoFlow^® for 2 days. The 3D capillary structure and expression of tight-junction proteins and transporters were confirmed by immunocytochemistry. The mRNA expression of transporters in the 3D environment was determined using qRT-PCR, and the permeability of endogenous substances and drugs was evaluated under various conditions.

Results and Discussion

The expression of tight-junction proteins, including claudin-5 and ZO-1, was confirmed by immunohistochemistry. The permeability rate constant of lucifer yellow through hiPS-BMECs was undetectably low, indicating that paracellular transport is highly restricted by tight junctions in the 3D-BBB system. The mRNA expression levels of transporters and receptors in the 3D-BBB system differed from those in the 2D-culture system by 0.2- to 5.8-fold. The 3D-cultured hiPS-BMECs showed asymmetric transport of substrates of BCRP, CAT1 and LAT1 between the luminal (blood) and abluminal (brain) sides. Proton-coupled symport function of MCT1 was also confirmed.

Conclusion

The 3D-BBB system constructed in this study mimics several important characteristics of the human BBB, and is expected to be a useful high-throughput evaluation tool in the development of CNS drugs.

     

Transport Characteristics of Tramadol in the Blood–Brain Barrier

Tramadol is a centrally acting analgesic whose action is mediated by both agonistic activity at opioid receptors and inhibitory activity on neuronal reuptake of monoamines. The purpose of this study was to characterize the blood–brain barrier (BBB) transport of tramadol by means of microdialysis studies in rat brain and in vitro studies with human immortalized brain capillary endothelial cells (hCMEC/D3). The K_p,uu,brain value of tramadol determined by rat brain microdialysis was greater than unity, indicating that tramadol is actively taken up into the brain across the BBB. Tramadol was transported into hCMEC/D3 cells in a concentration‐dependent manner. The uptake was inhibited by type II cations (pyrilamine, verapamil, etc.), but not by substrates of organic cation transporter OCTs or OCTN2. It was also inhibited by a metabolic inhibitor but was independent of extracellular sodium or membrane potential. The uptake was altered by changes of extracellular pH, and by ammonium chloride‐induced intracellular acidification, suggesting that transport of tramadol is driven by an oppositely directed proton gradient. Thus, our in vitro and in vivo results suggest that tramadol is actively transported, at least in part, from blood to the brain across the BBB by proton‐coupled organic cation antiporter.     

Characteristics of Substance P Transport Across the Blood–Brain Barrier

Purpose Substance P (SP; NH₃⁺-Arg⁺-Pro-Lys⁺-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂) belongs to a group of neurokinins that are widely distributed in the central nervous system and peripheral nervous system. The biological effects mediated by SP in the central nervous system include regulation of affective behavior, emesis, and nociception. Many of these actions are believed to be the result of the binding of SP to the neurokinin-1 (NK-1) receptor and subsequent transport across the blood–brain barrier (BBB). The objective of the study was to investigate the involvement of the NK-1 receptor in the permeation of SP across the BBB. Methods Transport of ³H SP (1–13 nM) was investigated using BBMEC monolayers grown on polycarbonate membranes mounted on a Side-bi-Side™ diffusion apparatus. ³H SP samples were analyzed by scintillation spectrometry. Liquid chromatography-tandem mass spectrometry was used to monitor the transport at higher concentrations (micromolar). Results SP transport across BBMEC monolayers was found to be saturable (K_m = 8.57 ± 1.59 nM, V_max = 0.017 ± 0.005 pmol min⁻¹ mg⁻¹ protein) in the concentration range of 0–13 nM. Significant (p < 0.05) decline in ³H SP permeation was observed in the presence of unlabeled SP and at 4°C, indicating that the transport process is carrier-mediated. High-performance liquid chromatography analysis showed no significant metabolism of ³H SP in either the donor or receiver chambers. ³H SP transport was inhibited by 2–11 SP (p < 0.05) but not by any other fragments, indicating that both the C- and N-terminal regions are essential for molecular recognition by the receptor. Endocytic inhibitors (chloroquine, phenylarsine oxide, monensin, and brefeldin) did not inhibit SP transport, suggesting the involvement of a nonendocytic mechanism in SP permeation. Pro⁹ SP, a high-affinity substrate for the NK-1 major subtype receptor, significantly (p < 0.05) inhibited the transport of SP. However, Sar⁹Met(O₂)¹¹ SP, a high-affinity substrate for the NK-1 minor subtype receptor, septide, and neurokinin A, inhibitors of NK-1 and neurokinin-2 (NK-2) receptors, respectively, did not produce any inhibition of SP transport. Western blot analysis confirmed the presence of the NK-1 receptor in BBMEC monolayers. Conclusions The above results provide functional and molecular evidence for the existence of a carrier-mediated mechanism in the transport of SP across the BBB. The effects of specific inhibitors and the results of Western blot analyses demonstrate the involvement of the NK-1 receptor in the transport of SP across the BBB.     

Influence of P-Glycoprotein Inhibition or Deficiency at the Blood–Brain Barrier on 18F-2-Fluoro-2-Deoxy-d-glucose (18F-FDG) Brain Kinetics

The fluorinated d-glucose analog ¹⁸F-2-fluoro-2-deoxy-d-glucose (¹⁸F-FDG) is the most prevalent radiopharmaceutical for positron emission tomography (PET) imaging. P-Glycoprotein’s (P-gp, MDR1, and ABCB1) function in various cancer cell lines and tumors was shown to impact ¹⁸F-FDG incorporation, suggesting that P-gp function at the blood–brain barrier may also modulate ¹⁸F-FDG brain kinetics. We tested the influence of P-gp inhibition using the cyclosporine analog valspodar (PSC833; 5 μM) on the uptake of ¹⁸F-FDG in standardized human P-gp-overexpressing cells (MDCKII-MDR1). Consequences for ¹⁸F-FDG brain kinetics were then assessed using (i) ¹⁸F-FDG PET imaging and suitable kinetic modelling in baboons without or with P-gp inhibition by intravenous cyclosporine infusion (15 mg kg⁻¹ h⁻¹) and (ii) in situ brain perfusion in wild-type and P-gp/Bcrp (breast cancer resistance protein) knockout mice and controlled d-glucose exposure to the brain. In vitro, the time course of ¹⁸F-FDG uptake in MDR1 cells was influenced by the presence of valspodar in the absence of d-glucose but not in the presence of high d-glucose concentration. PET analysis revealed that P-gp inhibition had no significant impact on estimated brain kinetics parameters K₁, k₂, k₃, V_T, and CMR_Glc. The lack of P-gp effect on in vivo¹⁸F-FDG brain distribution was confirmed in P-gp/Bcrp-deficient mice. P-gp inhibition indirectly modulates ¹⁸F-FDG uptake into P-gp-overexpressing cells, possibly through differences in the energetic cell level state. ¹⁸F-FDG is not a P-gp substrate at the BBB and ¹⁸F-FDG brain kinetics as well as estimated brain glucose metabolism are influenced by neither P-gp inhibition nor P-gp/Bcrp deficiencies in baboon and mice, respectively.KEY WORDS: ABC transporters, blood–brain barrier, cyclosporine, glucose, multidrug resistance, nonhuman primate, positron emission tomography     

Small-Volume Analysis of Cell–Cell Signaling Molecules in the Brain

Modern science is characterized by integration and synergy between research fields. Accordingly, as technological advances allow new and more ambitious quests in scientific inquiry, numerous analytical and engineering techniques have become useful tools in biological research. The focus of this review is on cutting edge technologies that aid direct measurement of bioactive compounds in the nervous system to facilitate fundamental research, diagnostics, and drug discovery. We discuss challenges associated with measurement of cell-to-cell signaling molecules in the nervous system, and advocate for a decrease of sample volumes to the nanoliter volume regimen for improved analysis outcomes. We highlight effective approaches for the collection, separation, and detection of such small-volume samples, present strategies for targeted and discovery-oriented research, and describe the required technology advances that will empower future translational science.     

Transferrin Receptor Binding BBB-Shuttle Facilitates Brain Delivery of Anti-Aβ-Affibodies

Pharmaceutical Research - Affibodies targeting amyloid-beta (Aβ) could potentially be used as therapeutic and diagnostic agents in Alzheimer’s disease (AD). Affibodies display suitable...     

Nanoparticle Surface Charges Alter Blood–Brain Barrier Integrity and Permeability

Purpose: The blood–brain barrier (BBB) presents both a physical and electrostatic barrier to limit brain permeation of therapeutics. Previous work has demonstrated that nanoparticles (NPs) overcome the physical barrier, but there is little known regarding the effect of NP surface charge on BBB function. Therefore, this work evaluated: (1) effect of neutral, anionic and cationic charged NPs on BBB integrity and (2) NP brain permeability.

Methods: Emulsifying wax NPs were prepared from warm oil-in-water microemulsion precursors using neutral, anionic or cationic surfactants to provide the corresponding NP surface charge. NPs were characterized by particle size and zeta potential. BBB integrity and NP brain permeability were evaluated by in situ rat brain perfusion.

Results: Neutral NPs and low concentrations of anionic NPs were found to have no effect on BBB integrity, whereas, high concentrations of anionic NPs and cationic NPs disrupted the BBB. The brain uptake rates of anionic NPs at lower concentrations were superior to neutral or cationic formulations at the same concentrations.

Conclusions: (1) Neutral NPs and low concentration anionic NPs can be utilized as colloidal drug carriers to brain, (2) cationic NPs have an immediate toxic effect at the BBB and (3) NP surface charges must be considered for toxicity and brain distribution profiles.     

Correction to: Construction and Functional Evaluation of a Three-Dimensional Blood–Brain Barrier Model Equipped With Human Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells

Pharmaceutical Research -     

Brain IL-1β was involved in reserpine-induced behavioral depression in rats

AIM: To investigate the mechanism of brain interleukin-1β(IL-1β) in reserpine-induced behavioral depression in rats. METHODS: Porsult swim test was used in the measurement of depressive behavior and ELISA was used in measurement of brain IL-1β. RESULTS: Intraperitoneal injection of reserpine (0, 4, 6, and 8 mg/kg, ip) increased floating time in the Porsult swim test in a dose-and time-dependent manner in rats. Intracerebroventricular injection(icv) of IL-1β receptor antagonist (IL-1ra, 6 mg/kg) blocked the increment of floating time in Porsult swim test at 48 and 72 h after reserpine injection, but not at 1 and 24 h after injection. Brain IL-Iβ increased after reserpine treatment in posterior cortex, hippocampus, and hypothalamus. The increase of IL-1β concentration starts at 24 hours after injection of reserpine and reached the peak at 48 h. CONCLUSION: Reserpine induced behavioral depression partially via brain interleukin-1β generation.     

Brain natriuretic peptide: Disease marker or more in cardiovascular medicine?

Brain natriuretic peptide (BNP) is predominantly a cardiac ventricular hormone that promotes natriuresis and diuresis, inhibits the renin-angiotensin-aldosterone axis, and is a vasodilator. Plasma BNP levels are raised in essential hypertension, and more so in left ventricular (LV) hypertrophy and heart failure. Plasma BNP levels are also elevated in ischemic heart disease. Attempts have been made to use plasma BNP levels as a marker of LV dysfunction, but these have shown that plasma BNP levels are probably not sensitive enough to replace echocardiography in the diagnosis of LV dysfunction. Pericardial BNP or N-BNP may be more suitable markers of LV dysfunction. Plasma BNP levels are also elevated in right ventricular dysfunction, pregnancy-induced hypertension, aortic stenosis, age, subarachnoid hemorrhage, cardiac allograft rejection and cavopulmonary connection, and BNP may have an important pathophysiological role in some or all of these conditions. Clinical trials have demonstrated the natriuretic, diuretic and vasodilator effects, as well as inhibitory effects on renin and aldosterone of infused synthetic human BNP (nesiritide) in healthy humans. BNP infusion improves LV function in patients with congestive heart failure via a vasodilating and a prominent natriuretic effect. BNP infusion is useful for the treatment of decompensated congestive heart failure requiring hospitalization. The clinical potential of BNP is limited as it is a peptide and requires infusion. Drugs that modify the effects of BNP are furthering our understanding of the pathophysiological role and clinical potential of BNP. Increasing the effects of BNP may be a useful therapeutic approach in heart failure involving LV dysfunction. The levels of plasma BNP are increased by beta-blockers, cardiac glycosides and vasopeptidase inhibitors, and this may contribute to the usefulness of these agents in heart failure. (c) 2001 Prous Science. All rights reserved.     

Pharmacokinetic Consequences of Active Drug Efflux at the Blood–Brain Barrier

Purpose The objective of this simulation study was to investigate how the nature, location, and capacity of the efflux processes in relation to the permeability properties influence brain concentrations. Methods Reduced brain concentrations can be due to either influx hindrance, a gatekeeper function in the luminal membrane, which has been suggested for ABCB1 (P-glycoprotein), or efflux enhancement by transporters that pick up molecules on one side of the luminal or abluminal membrane and release them on the other side. Pharmacokinetic models including passive transport, influx hindrance, and efflux enhancement were built using the computer program MATLAB. The simulations were based on experimentally obtained parameters for morphine, morphine-3-glucuronide, morphine-6-glucuronide, and gabapentin. Results The influx hindrance process is the more effective for keeping brain concentrations low. Efflux enhancement decreases the half-life of the drug in the brain, whereas with influx hindrance the half-life is similar to that seen with passive transport. The relationship between the influx and efflux of the drug across the blood–brain barrier determines the steady-state ratio of brain to plasma concentrations of unbound drug, K_p,uu. Conclusions Both poorly and highly permeable drugs can reach the same steady-state ratio, although the time to reach steady state will differ. The volume of distribution of unbound drug in the brain does not influence K_p,uu, but does influence the total brain-to-blood ratio K_p and the time to reach steady state in the brain.