Akihiko Urayama

Bioaccumulation and toxicity of gold nanoparticles after repeated administration in mice

Gold nanoparticles (GNPs) offer a great promise in biomedicine. Currently, there is no data available regarding the accumulation of nanoparticles in vivo after repeated administration. The purpose of the present study was to evaluate the bioaccumulation and toxic effects of different doses (40, 200, and 400 microg/kg/day) of 12.5 nm GNPs upon intraperitoneal administration in mice every day for 8 days. The gold levels in blood did not increase with the dose administered, whereas in all the organs examined there was a proportional increase on gold, indicating efficient tissue uptake. Although brain was the organ containing the lowest quantity of injected GNPs, our data suggest that GNPs are able to cross the blood-brain barrier and accumulate in the neural tissue. Importantly, no evidence of toxicity was observed in any of the diverse studies performed, including survival, behavior, animal weight, organ morphology, blood biochemistry and tissue histology. The results indicate that tissue accumulation pattern of GNPs depend on the doses administered and the accumulation of the particles does not produce sub-acute physiological damage.

Mannose 6-Phosphate Receptor–mediated Transport of Sulfamidase Across the Blood–brain Barrier in the Newborn Mouse

Mucopolysaccharidosis type IIIA (MPS IIIA), which is a lysosomal storage disorder (LSD) caused by inherited deficiency of sulfamidase, is characterized by severe, progressive central nervous system (CNS) dysfunction. Enzyme replacement therapy (ERT) to treat CNS storage is challenging, because the access of enzymes to the brain is restricted by the blood-brain barrier (BBB). In a prior study, we found that phosphorylated beta-glucuronidase (P-GUS) could be transcytosed across the BBB in newborn mice by the mannose 6-phosphate (M6P) receptor. In order to determine whether sulfamidase can utilize this pathway, we examined brain influx and the specificity of uptake of sulfamidase after intravenous (i.v.) injection in 2-day-old and 8-week-old mice. [(131)I]Sulfamidase was transported across the BBB in neonates at rates higher than that of simultaneously injected [(125)I]albumin. In contrast, the transport of [(131)I]sulfamidase was negligible in 8-week-old mice, thereby showing that the BBB transport mechanism is developmentally downregulated. Capillary depletion revealed that 83.7% of the [(131)I]sulfamidase taken up by the brain was in the parenchyma, demonstrating transfer across the capillary wall. The uptake of [(131)I]sulfamidase into the brain was significantly reduced by co-injections of M6P and P-GUS. That is, the transport of sulfamidase into the brain parenchyma in early postnatal life is mediated by the M6P receptor, which is shared with P-GUS and is likely accessible to other M6P-containing lysosomal enzymes.

Starvation and Triglycerides Reverse the Obesity-Induced Impairment of Insulin Transport at the Blood-Brain Barrier

Insulin in the brain acts as a satiety factor, reduces appetite, and decreases body mass. Altered sensing by brain of insulin may be a leading cause of weight gain and insulin resistance. A decrease in the transport across the blood-brain barrier (BBB) of insulin may induce brain insulin resistance by inducing obesity. We here report that transport of iv administrated insulin across the BBB of obese mice, as measured by multiple-time regression analysis, was significantly lower than that in thin adult mice. The reduction in obese mice was reversed by starvation for 48 h. There were no differences in insulin transport rates across the BBB of obese, thin, or starved obese mice when studied by the brain perfusion model, demonstrating that BBB transport of insulin is modulated by circulating factors. In the brain perfusion study, the triglyceride triolein significantly increased the brain uptake of insulin, an effect opposite to that on leptin transport, in starved obese mice. Thus, circulating triglycerides are one of the systemic modulators for the transport of insulin across the BBB.

Epinephrine enhances lysosomal enzyme delivery across the blood-brain barrier by up-regulation of the mannose 6-phosphate receptor

Delivering therapeutic levels of lysosomal enzymes across the blood–brain barrier (BBB) has been a pivotal issue in treating CNS storage diseases, including the mucopolysaccharidoses. An inherited deficiency of β-glucuronidase (GUS) causes mucopolysaccharidosis type VII that is characterized by increased systemic and CNS storage of glycosaminoglycans. We previously showed that the neonate uses the mannose 6-phosphate (M6P) receptor to transport phosphorylated GUS (P-GUS) across the BBB and that this transporter is lost with maturation. Induction of expression of this BBB transporter would make enzyme replacement therapy in the adult possible. Here, we tested pharmacological manipulation with epinephrine to restore functional transport of P-GUS across the adult BBB. Epinephrine (40 nmol) coinjected i.v. with 131I-P-GUS induced the transport across the BBB in 8-week-old mice. The brain influx rate of 131I-P-GUS (0.29 μl/g per min) returned to the level seen in neonates. Capillary depletion showed that 49% of the 131I-P-GUS in brain was in brain parenchyma. No increases of influx rate or the vascular space for 125I-albumin, a vascular marker, was observed with epinephrine (40 nmol), showing that enhanced passage was not caused by disruption of the BBB. Brain uptake of 131I-P-GUS was significantly inhibited by M6P in a dose-dependent manner, whereas epinephrine failed to increase brain uptake of nonphosphorylated GUS. Thus, the effect of epinephrine on the transport of 131I-P-GUS was ligand specific. These results indicate that epinephrine restores the M6P receptor-mediated functional transport of 131I-P-GUS across the BBB in adults to levels seen in the neonate.

Transport of prion protein across the blood-brain barrier

The cellular form of the prion protein (PrP(c)) is necessary for the development of prion diseases and is a highly conserved protein that may play a role in neuroprotection. PrP(c) is found in both blood and cerebrospinal fluid and is likely produced by both peripheral tissues and the central nervous system (CNS). Exchange of PrP(c) between the brain and peripheral tissues could have important pathophysiologic and therapeutic implications, but it is unknown whether PrP(c) can cross the blood-brain barrier (BBB). Here, we found that radioactively labeled PrP(c) crossed the BBB in both the brain-to-blood and blood-to-brain directions. PrP(c) was enzymatically stable in blood and in brain, was cleared by liver and kidney, and was sequestered by spleen and the cervical lymph nodes. Circulating PrP(c) entered all regions of the CNS, but uptake by the lumbar and cervical spinal cord, hypothalamus, thalamus, and striatum was particularly high. These results show that PrP(c) has bidirectional, saturable transport across the BBB and selectively targets some CNS regions. Such transport may play a role in PrP(c) function and prion replication.

Neuroprotective effect and brain receptor binding of taltirelin, a novel thyrotropin-releasing hormone (TRH) analogue, in transient forebrain ischemia of C57BL/6J mice

Thyrotropin-releasing hormone (TRH) and some of its stable analogues have been shown to improve neurologic dysfunctions such as brain trauma in both animals and humans. Our previous study revealed that taltirelin, a novel orally active TRH analogue, binds to rat brain TRH receptors in vivo. The present study was undertaken to investigate whether taltirelin has neuroprotective effects in transient brain ischemia of C57BL/6J mice induced by bilateral carotid artery occlusion (2VO). Neuronal cell density in the hippocampal CA1 region of C57BL/6J mice was significantly (39.9%) decreased 1 week after 2VO-reperfusion, compared to the case of the sham group, and this reduction of hippocampal neuronal density was significantly suppressed by an intravenous (i.v.) injection of taltirelin (0.3 mg/kg). The i.v. injection of taltirelin at this dosage produced a significant increase in the dissociation constant (Kd) of specific [3H]MeTRH binding in sham and 2VO-reperfusion groups (33.6 and 51.4%, respectively) compared with the vehicle-treated group. These results indicate that the intravenously injected taltirelin bound to TRH receptors in the ischemic brain. There was little difference in the brain-to-plasma concentration ratio (Kp) of [14C]sucrose between the sham and 2VO groups of C57BL/6J mice, indicating that the tight junction of the blood-brain barrier may be intact in the ischemic brain. In conclusion, the study has shown that taltirelin may have a significant neuroprotective effect on the ischemic brain.

In vitro and ex vivo effects of a selective nociceptin/orphanin FQ (N/OFQ) peptide receptor antagonist, CompB, on specific binding of [3H]N/OFQ and [35S]GTPγS in rat brain and spinal cord

A novel selective nociceptin/orphanin FQ (N/OFQ) peptide receptor antagonist, 1‐[(3R,4R)‐1‐cyclooctylmethyl]‐3‐hydroxymethyl‐4‐piperidyl)‐3‐ethyl‐1,3‐dihydro‐2H‐benzimidazol‐2‐one (CompB), inhibited specific binding of [3H]N/OFQ to crude membranes from the rat brain and spinal cord in a concentration‐dependent manner and their Ki values were 7.11 and 4.02 nM, respectively. Rosenthal analysis indicated that there was a significant increase in the Kd value for [3H]N/OFQ binding in the brain and spinal cord in the presence of CompB (10 nM). There was a dose‐dependent increase in Kd values for [3H]N/OFQ binding in the brain and spinal cord following i.v. injection of CompB at relatively low doses (0.69–6.88 μmol kg−1), compared with the control values. In the spinal cord, enhancement with each dose was constantly greater and the duration of enhancement (6.88 μmol kg−1) was significantly longer. The degree of increase in Kd values for [3H]N/OFQ binding after i.v. injection of CompB (6.88 μmol kg−1) was significantly larger in the lumbar region of the spinal cord compared to other regions. CompB (0.1, 0.3 μM) shifted the concentration–effect curves of N/OFQ‐stimulated [35S]GTPγS binding in the brain and spinal cord to the right. The i.v. injection of CompB (6.88 μmol kg−1) significantly suppressed the N/OFQ‐stimulated [35S]GTPγS binding in the rat spinal cord and shifted the concentration–effect curve to the right, while it produced little inhibitory effect in the brain. The present study has shown that CompB may exhibit pharmacological effects through a predominant blockade of N/OFQ peptide receptors in the spinal cord under in vivo conditions.

Preproenkephalin targeted antisenses cross the blood–brain barrier to reduce brain methionine enkephalin levels and increase voluntary ethanol drinking

Antisense potentially can manipulate target gene expression in the brain if it can cross the blood-brain barrier (BBB). We designed three (10mer, 17mer, and 19mer) phosphorothioated antisenses (PS-ODNs) directed against the precursor molecule of methionine enkephalin (Met-Enk), an opiate peptide which suppresses voluntary ethanol drinking. We measured the ability of the antisenses to cross the BBB, accumulate in the brain and CSF, decrease levels of Met-Enk in brain and blood, and affect voluntary ethanol drinking. Each antisense readily crossed the BBB, with 0.07-0.16% of the i.v. dose accumulating per gram of brain. Capillary depletion and CSF sampling each confirmed that the antisenses entered the CNS. Gel electrophoresis of radioactivity recovered from brain and serum showed intact antisense and a higher molecular weight form likely representing antisense bound to protein, but no degradation products. Each antisense molecule and a cocktail of all three reduced Met-Enk levels in brain and serum. Met-Enk levels in the brain were reduced more rapidly and for a longer duration than Met-Enk levels in the serum, indicating a degree of selective targeting to the CNS. Additionally, administration of the cocktail was more effective in reducing Met-Enk levels than any of the individual antisenses. Each antisense increased voluntary ethanol drinking by about 20% and the cocktail increased it by about 80%. Taken together, these results used pharmacokinetic, immunochemical, and behavioral methods to show that PS-ODN antisenses that readily cross the BBB can decrease brain levels of Met-Enk and increase voluntary ethanol drinking.

Brain receptor binding characteristics and pharmacokinetics of JTP-2942, a novel thyrotropin-releasing hormone (TRH) analogue

JTP-2942 competed with [3H]-Me-TRH for the binding sites in rat brain in vitro, and its inhibitory effect was approximately 17 times less potent than TRH, as shown by Ki values of 673 and 39.7 nM, respectively. Both JTP-2942 and TRH significantly increased apparent dissociation constant (Kd values) for brain [3H]-Me-TRH binding. Intravenous injection of JTP-2942 (0.3-3 mg/kg) and TRH (3 and 10 mg/kg) produced a significant reduction of [3H]-Me-TRH binding sites (Bmax values) in rat brain. Although the decrease by TRH was maximal 10 min after the injection and declined rapidly with time, the decrease by JTP-2942 (1 and 3 mg/kg) tended to be maximal at 30 min later and it lasted until 120 min. The intravenous injection of JTP-2942 was at least 3 times more potent than that of TRH in decreasing Bmax values for brain [3H]-Me-TRH binding. Plasma concentration of JTP-2942 (0.3-3 mg/kg) after intravenous injection in rats rose with the increase of dose, and it peaked immediately after the injection, thereafter decreasing with t1/2 of 19.3-29.9 min. It is concluded that JTP-2942, compared to TRH, may exert fairly potent and sustained occupation of brain TRH receptors under in vivo condition. Thus, JTP-2942 could be clinically useful for the treatment of CNS disorders.

Initial fate of prions upon peripheral infection: half-life, distribution, clearance, and tissue uptake

Prion diseases are infectious neurodegenerative disorders associated with the misfolded prion protein (PrPSc), which appears to be the sole component of the infectious agent (termed prion). To produce disease, prions have to be absorbed into the body and reach sufficient quantities in the brain. Very little is known about the biological mechanisms controlling the initial fate of prions. Here, we studied the systemic pharmacokinetics and biodistribution of PrPSc in vivo. After an intravenous injection of highly purified radiolabeled or native unlabeled PrPSc, the protein was eliminated rapidly from the serum (half‐life of 3.24 h), mostly through tissue uptake. The quantity of intact PrPSc reaching the brain was ~0.2% of the injected dose per gram of brain tissue (ID/g). The highest levels were found in liver (~20% ID/g), spleen (~13% ID/g), and kidney (~7.4% ID/g). Cell surface PrPC does not appear to play a role in PrPSc pharmacokinetics, since the infectious protein distributed similarly in wild‐type and PrP‐null mice. To measure tissue uptake kinetics and biodistribution accurately, vascular space in tissues was measured with radioactively labeled albumin coinjected with radioactively labeled PrPSc. Our results provide a fundamental pharmacokinetic characterization of PrPSc in vivo, which may be relevant to estimate tissue risks and mechanisms of prion neuroinvasion and to identify novel therapeutic strategies.—Urayama, A., Morales, R., Niehoff, M. L., Banks, W. A., Soto, C. Initial fate of prions upon peripheral infection: half‐life, distribution, clearance, and tissue uptake FASEB J. 25, 2792‐2803 (2011). www.fasebj.org