Enhanced survival of glioma bearing rats following boron neutron capture therapy with blood-brain barrier disruption and intracarotid injection of boronophenylalanine

Boronophenylalanine (BPA) has been used for boron neutron capture therapy (BNCT) of brain tumors in both experimental animals and humans. The purpose of the present study was to determine if the efficacy of BNCT could be enhanced by means of intracarotid (i.c.) injection of BPA with or without blood-brain barrier disruption (BBB-D) and neutron irradiation using a rat brain tumor model. For biodistribution studies, F98 glioma cells were implanted stereotactically into the brains of Fischer rats, and12 days later BBB-D was carried out by i.c. infusion of 25% mannitol (1.373 mOsmol/ml), followed immediately by i.c. administration of 300, 500 or 800 mg of BPA/kg body weight (b.w.). At the 500 mg dose a fourfold increase in tumor boron concentration (94.5 g/g) was seen at 2.5 hours after BBB-D, compared to 20.8 g/g in i.v. injected animals. The best composite tumor to normal tissue ratios were observed at 2.5 hours after BBB-D, at which time the tumor: blood (T: Bl) ratio was10.9, and the tumor: brain (T: Br) ratio was 7.5, compared to 3.2 and 5.0 respectively for i.v. injected rats. In contrast, animals that had received i.c. BPA without BBB-D had T: Bl and T: Br ratios of 8.5 and 5.9, respectively, and the tumor boron concentration was 42.7g/g. For therapy experiments, initiated 14 days after intracerebral implantation of F98 glioma cells, 500 mg/kg b.w. of BPA were administered i.v. or i.c. with or without BBB-D, and the animals were irradiated 2.5 hourslater at the Brookhaven Medical Research Reactor with a collimated beam of thermal neutrons delivered to the head. The mean survival time for untreated control rats was 24 ± 3 days, 30 ± 2 days for irradiated controls, 37 ± 3 days for those receiving i.v. BPA, 52 ± 15 days for rats receiving i.c. BPA without BBB-D, and 95 ± 95 days for BBB-D followed by i.c. BPA and BNCT. The latter group had a 246% increase in life span (ILS) compared to untreated controls and a 124% ILS compared to that of i.v. injected animals. These survival data are the best ever obtained with the F98 glioma model and suggest that i.c. administration of BPA with or without BBB-D may be useful as a means to increase the efficacy of BNCT.     

Immunocytochemical studies of protamine-induced blood-brain barrier opening to endogenous albumin

The cellular mechanisms of blood-brain barrier (BBB) opening to endogenous albumin in the mouse brain after intracarotid infusion of solutions of protamine free base (PB) or protamine sulfate (PS) were studied using quantitative immunocytochemistry. Ultrathin sections of brain samples embedded at low temperature in Lowicryl. K4M were exposed to anti-mouse albumin antiserum followed by protein A-gold. Using morphometry, the density of immunosignals (gold particles per m²) was recorded over four compartments: vascular lumen, endothelial profiles, subendothelial space (including the basement membrane), and brain parenchyma (neuropil). In addition, the adsorption of endogenous albumin evidenced by the number of gold particles per m of the endothelial luminal plasmalemma was quantitatively evaluated. In the applied experimental conditions, PB was found to be strongly cytotoxic as indicated by the appearance of rapid degenerative changes and the disruption of the endothelial lining with concomitant clumping of the blood plasma. The action of PS was milder, offering a better opportunity for detailed ultrastructural and morphometric examination of brain samples during consecutive steps of PS action (2, 5, 10 and 30 min). As early as 10 min after infusion of PS solution, the adsorption of blood plasma albumin to the endothelial luminal surface was increased 2.5 times. Simultaneously, the immunolabelling of the endothelial profiles and subendothelial space was significantly increased. These results suggest that BBB disruption occurs through enhanced adsorption of albumin or albumin-protamine complexes to the luminal plasmalemma, followed by transendothelial vesicular transport, rather than through modification of interendothelial junctional complexes. This process appears to be focally disseminated throughout the cerebral vascular network and declines at 30 min following infusion of PS solution.     

The Use of Intracerebral Microdialysis to Determine Changes in Blood-Brain Barrier Transport Characteristics

The aim of this study was to determine whether changes in the transport of drugs into the brain could be determined by in vivo intracerebral microdialysis. Atenolol was used as a model drug to determine blood-brain barrier (BBB) transport characteristics. In rats, unilateral opening of the blood-brain barrier was achieved by infusion of hyperosmolar mannitol (25%, w/v) into the left internal carotid artery. BBB transport, expressed as the ratio of the area under the curve (AUC) of atenolol in brain extracellular fluid over plasma, was three times higher for the mannitol treated hemisphere as compared with the contralateral brain or after infusion of saline, being (mean ± SEM) 0.094 ± 0.024 (n = 16), 0.029 ± 0.007 (n = 12) and 0.030 ± 0.009 (n = 12) respectively. Further evaluation of the data indicated that for experiments performed in the morning the mannitol infusion had little effect on the extent of transport of atenolol into the brain, while in the afternoon BBB transport was about 10-fold higher than in the contralateral and saline group. The mean afternoon ratios ± SEM were 0.155 ± 0.038 (n = 8), 0.012 ± 0.003 (n = 6) and 0.018 ± 0.006 (n = 6) respectively. It is concluded that intracerebral microdialysis is capable of revealing changes in BBB transport and regional and time-dependent differences in drug levels can be demonstrated with the use of this technique.     

Transmission routes of HIV-1 gp120 from brain to lymphoid tissues

The blood-brain barrier (BBB) restricts the entry of antiviral agents into the CNS thereby facilitating the creation of a reservoir of HIV that could potentially reinfect peripheral tissues. We characterized the efflux from brain of radioactively labeled viral coat HIV-1 gp120 (I-gp120) after intracerebroventricular (i.c.v.) injection. The half-time disappearance rate of I-gp120 from brain was 12.6 min, which was faster than could be explained by the reabsorption of cerebrospinal fluid into blood but could not be explained by a saturable transporter. After i.c.v. injection, I-gp120 appeared in the serum and was sequestered by spleen and the cervical nodes, demonstrating a potential for virus within the CNS to reinfect peripheral tissues. However, the amount of I-gp120 appearing in serum was less than that expected based on the efflux rate, whereas uptake by the cervical nodes was much greater after i. c.v. than after i.v. injection of I-gp120. These findings were explained by drainage from the brain directly to the cervical lymph nodes through the brain's primitive lymphatic system. These lymphatics potentially provide a pathway through which CNS reservoirs of HIV-1 could directly reinfect lymphoid tissue without being exposed to circulating antiviral agents.     

P-Glycoprotein on astrocyte foot processes of unfixed isolated human brain capillaries

Sites of immunoreactive P-glycoprotein associated with human brain microvasculature were identified by labeling of unfixed isolated human brain capillaries, allowing visualization of the three-dimensional capillary structure by confocal microscopy. Capillaries isolated from human autopsy brain were dual-labeled with the MRK16 mouse monoclonal antibody (against human P-glycoprotein) and rabbit polyclonal antisera against the human brain microvascular glucose transporter (GLUT1), or glial fibrillary acidic protein (GFAP) on astrocyte foot processes. MRK16 and GLUT1 dual-labeling showed no signal overlap, identical to the staining pattern observed for dual-labeling with anti-GFAP and anti-GLUT1 antibodies: both GFAP and MRK16 labeling were discrete, discontinuous, and not co-localized with continuous GLUT1 labeling of capillary endothelium. In contrast, complete overlap of MRK16 and GFAP labeling demonstrated P-glycoprotein localization on astrocyte foot process remnants at the abluminal face of the brain microvasculature.     

Acute inflammatory responses to mechanical lesions in the CNS: differences between brain and spinal cord

Lesion-induced inflammatory responses in both brain and spinal cord have recently become a topic of active investigation. Using C57BL/6J mice, we compared the tissue reaction in these two central nervous system (CNS) compartments with mechanical lesions of similar size involving both grey and white matter. This evaluation included the quantitative assessment of neutrophils, lymphocytes and activated macrophages/microglia, as well as astrocyte activation, upregulation of vascular cell adhesion molecules (ICAM-1, VCAM-1, PECAM) and the extent of blood-brain barrier (BBB) breakdown. Time points analysed post-lesioning included 1, 2, 4 and 7 days (as well as 10 and 14 days for the BBB). We found clear evidence that the acute inflammatory response to traumatic injury is significantly greater in the spinal cord than in the cerebral cortex. The numbers of both neutrophils and macrophages recruited to the lesion site were significantly higher in the spinal cord than in the brain, and the recruitment of these cells into the surrounding parenchyma was also more widespread in the cord. The area of BBB breakdown was substantially larger in the spinal cord and vascular damage persisted for a longer period. In the brain, as in spinal cord, the area to which neutrophils were recruited correlated well with the area of BBB breakdown. It will be of interest to determine the extent to which the infiltration of inflammatory cells contributes, either directly or indirectly, to the vascular permeability and secondary tissue damage or, conversely, to local tissue repair in the brain and the spinal cord.     

Phenylalanine kinetics are associated with tardive dyskinesia in men but not in women

Rationale: An association between tardive dyskinesia (TD) and severely impaired metabolism of the large neutral amino acid (LNAA), phenylalanine (Phe) was defined in a group of mentally retarded patients. Subsequently, an altered kinetics of Phe was associated with TD in men with schizophrenia based on plasma analyses subsequent to the ingestion of a protein meal. Methods: In the present study, a standardized oral challenge of pure Phe (100 mg/kg in 170 ml orange juice) was administered to psychiatric patients of both sexes (n = 312), with and without TD after an overnight fast. Plasma LNAA levels were assayed both fasting and 2 h subsequent to the ingestion of the challenge. The extent of the increase in plasma Phe levels 2 h following a standardized challenge is determined by the sum of the kinetic processes of plasma absorption, tissue distribution, metabolism and elimination. Results: The study hypothesis, that TD would be associated with significantly higher post-challenge plasma Phe indices of an absolute plasma Phe level and plasma Phe/LNAA ratio (a brain availability measure), was verified for the study men (n = 209), but not for the study women (n = 103). Conclusions: The demonstrated altered kinetics of Phe in men with TD indicates a greater availability of Phe to the brain in these men. We suggest that the disorder may be related to the effects of this greater availability. Such effects could be the direct neurotoxic effects of Phe and its metabolites and/or the modulating effects of these compounds on the synthesis of the monoamine neurotransmitters. The fact that TD (Yes/No) group differences in post-challenge plasma Phe indices were not seen for the study women suggests the possibility of a sex difference in the biology of TD that we propose may be reflective of the young age of the study sample. Received: 28 January 1998/Final version: 14 December 1998     

Current Status and Future Prospects of Transdermal Drug Delivery

Pharmaceutical Research -     

Open-label titration study of the safety of RMP-7 in patients with the acquired immune deficiency syndrome

RMP-7, a nine-amino acid bradykinin analogue, has been shown in animals to temporarily increase the permeability of the blood brain barrier to small molecules including amphotericin B, when administered intravenously. We sought to evaluate the safety of escalating doses of RMP-7 administered to human volunteers with the acquired immune deficiency syndrome (AIDS). Six HIV antibody-positive adults with CD4+ cell counts <50/mm³ received three increasing doses of RMP-7 on successive days: 30 ng/kg, 100 ng/kg and 300 ng/kg infused over 2, 2 and 10 min, respectively. Adverse experiences were dose-related, mild-moderate in intensity, primarily related to vasodilation and resolved rapidly without sequelae. Mean maximum increases in pulse rate at 30 ng/kg, 100 ng/kg and 300 ng/kg were 4.0, 7.8 and 28.2 beats per min, respectively. The maximum changes in average mean arterial pressure were +7.7, +5.6 and −0.2 mmHg from baseline, respectively. Minor increases in liver enzymes were noted in three patients, all with pre-existing enzyme elevations. Despite the high frequency of both occult and overt cardiovascular abnormalities in advanced HIV infection, RMP-7 is shown to be safe in this group of AIDS patients at all dosage levels tested, with adverse effects similar to previous experience in healthy humans.     

Lipopolysaccharide-induced platinum accumulation in the cerebral cortex after cisplatin administration in mice: Involvement of free radicals

The relationship between the accumulation of platinum in the cerebral cortex following cisplatin administration and injury to the blood-brain barrier after lipopolysaccharide (LPS) treatment was investigated. The appearance of intravenously injected fluorescein in the brain was significantly increased 10–24 h after LPS treatment, the effect being dose-dependent. Platinum was detectable in the cerebral cortex of cisplatin-treated mice 24 h after LPS treatment, but not without LPS treatment. In mice pretreated with -tocopherol, LPS administration did not significantly augment fluorescein penetration into the brain, whereas pretreatment with either allopurinol or ascorbic acid did not modify the LPS-induced increase in fluorescein penetration. In contrast, platinum in the cerebral cortex after cisplatin administration was still detectable in the allopurinol-, ascorbic acid-, and -tocopherol-pretreated groups, and the levels of platinum in these groups were not significantly different from those in the group treated with LPS only. Administration of superoxide dismutase (SOD), but not of catalase, tended to inhibit the penetration of fluorescein. Both SOD and catalase significantly lowered platinum content in the cerebral cortex following cisplatin administration in mice treated with LPS. Thus, free radicals may injure the blood-brain barrier in mice challenged with LPS, and allow cisplatin to penetrate into the cerebral cortex, resulting in platinum accumulation.