L-Arginine and asymmetric dimethylarginine (ADMA) transport across the mouse blood-brain and blood-CSF barriers: Evidence of saturable transport at both interfaces and CNS to blood efflux.

L-Arginine is the physiological substrate for the nitric oxide synthase (NOS) family, which synthesises nitric oxide (NO) in endothelial and neuronal cells. NO synthesis can be inhibited by endogenous asymmetric dimethylarginine (ADMA). NO has explicit roles in cellular signalling and vasodilation. Impaired NO bioavailability represents the central feature of endothelial dysfunction associated with vascular diseases. Interestingly, dietary supplementation with L-arginine has been shown to alleviate endothelial dysfunctions caused by impaired NO synthesis. In this study the transport kinetics of [3H]-arginine and [3H]-ADMA into the central nervous system (CNS) were investigated using physicochemical assessment and the in situ brain/choroid plexus perfusion technique in anesthetized mice. Results indicated that L-arginine and ADMA are tripolar cationic amino acids and have a gross charge at pH 7.4 of 0.981. L-Arginine (0.00149±0.00016) has a lower lipophilicity than ADMA (0.00226±0.00006) as measured using octanol-saline partition coefficients. The in situ perfusion studies revealed that [3H]-arginine and [3H]-ADMA can cross the blood-brain barrier (BBB) and the blood-CSF barrier. [3H]-Arginine (11.6nM) and [3H]-ADMA (62.5nM) having unidirectional transfer constants (Kin) into the frontal cortex of 5.84±0.86 and 2.49±0.35 µl.min-1.g-1, respectively, and into the CSF of 1.08±0.24 and 2.70±0.90 µl.min-1.g-1, respectively. In addition, multiple-time uptake studies revealed the presence of CNS-to-blood efflux of ADMA. Self- and cross-inhibition studies indicated the presence of transporters at the BBB and the blood-CSF barriers for both amino acids, which were shared to some degree. Importantly, these results are the first to demonstrate: (i) saturable transport of [3H]-ADMA at the blood-CSF barrier (choroid plexus) and (ii) a significant CNS to blood efflux of [3H]-ADMA. Our results suggest that the arginine paradox, in other words the clinical observation that NO-deficient patients respond well to oral supplementation with L-arginine even though the plasma concentration is sufficient to saturate endothelial NOS, could be related to altered ADMA transport (efflux).

Drug reformulation for a neglected disease. The NANOHAT project to develop a safer more effective sleeping sickness drug.

BACKGROUND: Human African trypanosomiasis (HAT or sleeping sickness) is caused by the parasite Trypanosoma brucei sspp. The disease has two stages, a haemolymphatic stage after the bite of an infected tsetse fly, followed by a central nervous system stage where the parasite penetrates the brain, causing death if untreated. Treatment is stage-specific, due to the blood-brain barrier, with less toxic drugs such as pentamidine used to treat stage 1. The objective of our research programme was to develop an intravenous formulation of pentamidine which increases CNS exposure by some 10-100 fold, leading to efficacy against a model of stage 2 HAT. This target candidate profile is in line with drugs for neglected diseases inititative recommendations. METHODOLOGY: To do this, we evaluated the physicochemical and structural characteristics of formulations of pentamidine with Pluronic micelles (triblock-copolymers of polyethylene-oxide and polypropylene oxide), selected candidates for efficacy and toxicity evaluation in vitro, quantified pentamidine CNS delivery of a sub-set of formulations in vitro and in vivo, and progressed one pentamidine-Pluronic formulation for further evaluation using an in vivo single dose brain penetration study. PRINCIPAL FINDINGS: Screening pentamidine against 40 CNS targets did not reveal any major neurotoxicity concerns, however, pentamidine had a high affinity for the imidazoline2 receptor. The reduction in insulin secretion in MIN6 ß-cells by pentamidine may be secondary to pentamidine-mediated activation of ß-cell imidazoline receptors and impairment of cell viability. Pluronic F68 (0.01%w/v)-pentamidine formulation had a similar inhibitory effect on insulin secretion as pentamidine alone and an additive trypanocidal effect in vitro. However, all Pluronics tested (P85, P105 and F68) did not significantly enhance brain exposure of pentamidine. SIGNIFICANCE: These results are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine.

The transporter and permeability interactions of asymmetric dimethylarginine (ADMA) and L-arginine with the human blood-brain barrier in vitro.

The blood-brain barrier (BBB) is a biological firewall that carefully regulates the cerebral microenvironment by acting as a physical, metabolic and transport barrier. This selectively permeable interface was modelled using the immortalised human cerebral microvascular endothelial cell line (hCMEC/D3) to investigate interactions with the cationic amino acid (CAA) L-arginine, the precursor for nitric oxide (NO), and with asymmetric dimethylarginine (ADMA), an endogenously derived analogue of L-arginine that potently inhibits NO production. The transport mechanisms utilised by L-arginine are known but they are not fully understood for ADMA, particularly at the BBB. This is of clinical significance giving the emerging role of ADMA in many brain and cerebrovascular diseases and its potential as a therapeutic target. We discovered that high concentrations of ADMA could induce endothelial dysfunction in the hCMEC/D3s BBB permeability model, leading to an increase in paracellular permeability to the paracellular marker FITC-dextran (40kDa). We also investigated interactions of ADMA with a variety of transport mechanisms, comparing the data with L-arginine interactions. Both molecules are able to utilise the CAA transport system y(+). Furthermore, the expression of CAT-1, the best known protein from this group, was confirmed in the hCMEC/D3s. It is likely that influx systems, such as y(+)L and b(0,+), have an important physiological role in ADMA transport at the BBB. These data are not only important with regards to the brain, but apply to other microvascular endothelia where ADMA is a major area of investigation.

Delivery of antihuman African trypanosomiasis drugs across the blood-brain and blood-CSF barriers.

Human African trypanosomiasis (HAT or sleeping sickness) is a potentially fatal disease caused by the parasite, Trypanosoma brucei sp. The parasites are transmitted by the bite of insect vectors belonging to the genus Glossina (tsetse flies) and display a life cycle strategy that is equally spread between human and insect hosts. T.b. gambiense is found in western and central Africa whereas, T.b. rhodesiense is found in eastern and southern Africa. The disease has two clinical stages: a blood stage after the bite of an infected tsetse fly, followed by a central nervous system (CNS) stage where the parasite penetrates the brain; causing death if left untreated. The blood-brain barrier (BBB) makes the CNS stage difficult to treat because it prevents 98% of all known compounds from entering the brain, including some anti-HAT drugs. Those that do enter the brain are toxic compounds in their own right and have serious side effects. There are only a few drugs available to treat HAT and those that do are stage specific. This review summarizes the incidence, diagnosis, and treatment of HAT and provides a close examination of the BBB transport of anti-HAT drugs and an overview of the latest drugs in development.

Global inhibition of reactive oxygen species (ROS) inhibits paclitaxel-induced painful peripheral neuropathy.

Paclitaxel (Taxol®) is a widely used chemotherapeutic agent that has a major dose limiting side-effect of painful peripheral neuropathy. Currently there is no effective therapy for the prevention or treatment of chemotherapy-induced painful peripheral neuropathies. Evidence for mitochondrial dysfunction during paclitaxel-induced pain was previously indicated with the presence of swollen and vacuolated neuronal mitochondria. As mitochondria are a major source of reactive oxygen species (ROS), the aim of this study was to examine whether pharmacological inhibition of ROS could reverse established paclitaxel-induced pain or prevent the development of paclitaxel-induced pain. Using a rat model of paclitaxel-induced pain (intraperitoneal 2 mg/kg paclitaxel on days 0, 2, 4 & 6), the effects of a non-specific ROS scavenger, N-tert-Butyl-α-phenylnitrone (PBN) and a superoxide selective scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were compared. Systemic 100 mg/kg PBN administration markedly inhibited established paclitaxel-induced mechanical hypersensitivity to von Frey 8 g and 15 g stimulation and cold hypersensitivity to plantar acetone application. Daily systemic administration of 50 mg/kg PBN (days -1 to 13) completely prevented mechanical hypersensitivity to von Frey 4 g and 8 g stimulation and significantly attenuated mechanical hypersensitivity to von Frey 15 g. Systemic 100 mg/kg TEMPOL had no effect on established paclitaxel-induced mechanical or cold hypersensitivity. High dose (250 mg/kg) systemic TEMPOL significantly inhibited mechanical hypersensitivity to von Frey 8 g & 15 g, but to a lesser extent than PBN. Daily systemic administration of 100 mg/kg TEMPOL (day -1 to 12) did not affect the development of paclitaxel-induced mechanical hypersensitivity. These data suggest that ROS play a causal role in the development and maintenance of paclitaxel-induced pain, but such effects cannot be attributed to superoxide radicals alone.

Perlecan domain V is neuroprotective and proangiogenic following ischemic stroke in rodents.

Stroke is the leading cause of long-term disability and the third leading cause of death in the United States. While most research thus far has focused on acute stroke treatment and neuroprotection, the exploitation of endogenous brain self-repair mechanisms may also yield therapeutic strategies. Here, we describe a distinct type of stroke treatment, the naturally occurring extracellular matrix fragment of perlecan, domain V, which we found had neuroprotective properties and enhanced post-stroke angiogenesis, a key component of brain repair, in rodent models of stroke. In both rat and mouse models, Western blot analysis revealed elevated levels of perlecan domain V. When systemically administered 24 hours after stroke, domain V was well tolerated, reached infarct and peri-infarct brain vasculature, and restored stroke-affected motor function to baseline pre-stroke levels in these multiple stroke models in both mice and rats. Post-stroke domain V administration increased VEGF levels via a mechanism involving brain endothelial cell α5ß1 integrin, and the subsequent neuroprotective and angiogenic actions of domain V were in turn mediated via VEGFR. These results suggest that perlecan domain V represents a promising approach for stroke treatment.

Intercellular exchange of surface molecules and its physiological relevance.

For many decades, cellular immunologists have relied on the expression of various cell surface molecules to divide cells into different types and subtypes to study their function. However, in recent years, a large and fast-expanding body of work has described the transfer of surface molecules, including MHC class I and II molecules, between cells, both in vitro and in vivo. The function of this process is still largely unknown, but it is likely to have a significant role in the control of the immune system. It is also likely that this process takes place in a regulated rather than stochastic manner, thus providing another way for the immune system to orchestrate its function. In this review we will summarize the key findings so far, examining the mechanisms of transfer, the consequences of this transfer as shown by in vitro experiments, and possible consequences for the wider immune response.