Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. We have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration (Jefferies, W. A., Brandon, M. R., Hunt, S. V., Williams, A. F., Gatter, K. C. & Mason, D. Y. (1984) Nature (London) 312, 162-163), labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling ofbrain capillaries becomes extremely punctate =4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a timedependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier.Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabelod methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26.The levels of various substances in the blood, such as hormones, amino acids, and ions, undergo frequent small fluctuations that can be brought about by activities such as eating and exercise (1). If the brain were not protected from these variations in serum composition, the result could be uncontrolled neural activity. The blood-brain barrier (BBB) functions to ensure that the homeostasis of the brain is maintained. Specialized characteristics of the endothelial cells that form brain capillaries are responsible for this barrier (1, 2). Brain capillary endothelial cells are joined together by tight intercellular junctions that form a continuous wall against the passive movement of substances from the blood to the brain (3, 4). These cells lack continuous gaps or channels connecting the luminal and abluminal membranes, which, in other endothelial cells, allow relatively unrestricted passage of blood-borne molecules into tissue.The isolation of the brain from the bloodstream is not complete; were this the case, the brain would be unable to function properly due to a lack ofnutrients and because ofthe need to exchange hormones and other compounds with the rest of the body. The presence of specific transport systems within the capillary endothelial cells, such as those for amino acids, transferrin, glucose, and insulin (2,(5)(6)(7)(8) A problem posed by the BBB is that, in the process of protecting the brain, it also excludes many potentially useful therapeutic agents. Currently, only sub...

Through the analysis of a series of 25 peptides composed of various portions of the histatin 5 sequence, we have identified P-113, a 12-amino-acid fragment of histatin 5, as the smallest fragment that retains anticandidal activity comparable to that of the parent compound. Amidation of the P-113 C terminus increased the anticandidal activity of P-113 approximately twofold. The three histidine residues could be exchanged for three hydrophobic residues, with the fragment retaining anticandidal activity. However, the change of two or more of the five basic (lysine and arginine) residues to uncharged residues resulted in a substantial loss of anticandidal activity. A synthetic D-amino-acid analogue, P-113D, was as active against Candida albicans as the L-amino-acid form. In vitro MIC tests in low-ionic-strength medium showed that P-113 has potent activity against Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis. These results identify P-113 as a potential antimicrobial agent in the treatment of oral candidiasis.

Nerve growth factor (NGF) is essential for the survival of both peripheral ganglion cells and central cholinergic neurons of the basal forebrain. The accelerated loss of central cholinergic neurons during Alzheimer's disease may be a determinant of dementia in these patients and may therefore suggest a therapeutic role for NGF. However, NGF does not significantly penetrate the blood-brain barrier, which makes its clinical utility dependent on invasive neurosurgical procedures. When conjugated to an antibody to the transferrin receptor, however, NGF crossed the blood-brain barrier after peripheral injection. This conjugated NGF increased the survival of both cholinergic and noncholinergic neurons of the medial septal nucleus that had been transplanted into the anterior chamber of the rat eye. This approach may prove useful for the treatment of Alzheimer's disease and other neurological disorders that are amenable to treatment by proteins that do not readily cross the blood-brain barrier.

LEU3 of Saccharomyces cereviswae encodes an 886-amino-acid polypeptide that regulates transcription of a group of genes involved in leucine biosynthesis and has been shown to bind specifically to a 114-base-pair DNA fragment of the LEU2 upstream region (P. Friden and P. Schimmel, Mol. Cell. Biol. 7:2707-2717, 1987 (4,17). Because a-IPM synthase is feedback inhibited by leucine, the levels of a-IPM are directly related to the levels of leucine in the cell.Recent work revealed that mutations in LEU3 resulted in decreased levels of LEU1-and LEU2-specific transcripts (5). This provided direct evidence for a role for LEU3 in the pathway-specific control of these genes. Moreover, evidence was also obtained for LEU3-dependent regulation of LEU4 expression. This was the first demonstration that LEU3 exerts control over a gene other than LEUI or LEU2 and raised the possibility that additional genes are also regulated by LEU3.The LEU3 gene product (886 amino acids) has near its amino terminus a sequence that resembles the "zinc finger" motif of the'Xenopus laevis transcription factor IIIA and of other transcriptional regulatory proteins (5,18,22,23,37). The possibility that LEU3 interacts with DNA was supported by the detection of a LEU3-dependent DNA-binding activity whose target site was localized to within a 114-basepair (bp) region of the LEU2 promoter (5). We show here that LEU3 binds to a specific decanucleotide sequence element that is found in the promoter regions of at least five genes and that this element is sufficient to confer LEU3-dependent and leucine-sensitive expression to a downstream promoter. The results suggest an expanded role for LEU3 such that it regulates the synthesis of all three branchedchain amino acids through a sequence-specific interaction with a set of promoters that are dissimilar in sequence except for the LEU3-binding site. MATERIALS AND METHODSStrains and genetic methods. The following yeast strains were used in these studies: PDY102-1A (a

Antimicrobial peptides are a source of novel agents that could be useful for treatment of the chronic lung infections that afflict cystic fibrosis (CF) patients. Efficacy depends on antimicrobial activity against the major pathogens of CF patients, Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in the environment of the CF patient's airway. We describe the in vitro efficacies of derivatives of histatins, which are histidine-rich peptides produced by the salivary glands of humans and higher primates. P-113, a peptide containing 12 of the 24 amino acid residues of the parent molecule, histatin 5, retained full antibacterial activity and had a good spectrum of activity in vitro against the prominent pathogens of CF patients. However, P-113 was not active in the presence of purulent sputum from CF patients. In contrast, P-113D, the mirrorimage peptide with the amino acid residues in the D configuration, was stable in sputum, was as active as P-113 against pathogens of CF patients in the absence of sputum and retained significant activity in the presence of sputum from CF patients. Recombinant human DNase, which effectively liquefies sputum, enhanced the activity of P-113D in undiluted sputum against both exogenous (added) bacteria and endogenous bacteria. Because of its properties, P-113D shows potential as an inhalant in chronic suppressive therapy for CF patients.

Intraventricular administration of nerve growth factor (NGF) in rats has been shown to reduce age-related atrophy of central cholinergic neurons and the accompanying memory impairment. Intraventricular administration of NGF is necessary because NGF will not cross the blood-brain barrier (BBB). Here we have used a novel carrier system, consisting of NGF covalently linked to an anti-transferrin receptor antibody (OX-26), to transport biologically active NGF across the BBB. In our experiment, aged (24 months old) Fischer 344 rats received intravenous injections of the OX-26-NGF conjugate or a control solution (a mixture of unconjugated OX-26 and NGF) twice weekly for 6 weeks. The OX-26-NGF injections resulted in a significant improvement in spatial learning in previously impaired rats but disrupted the learning ability of previously unimpaired rats. Neuroanatomical analyses showed that OX-26-NGF conjugate treatment resulted in a significant increase in cholinergic cell size in the medial septal region of rats initially impaired in spatial learning. These results indicate the potential use of the transferrin receptor antibody delivery system for treatment of CNS disorders with neurotrophic proteins.

Photodynamic therapy (PDT) has historically been used as a means to treat cancerous tumors but has recently been used to kill bacterial cells through the use of targeted photosensitizers. PDT is a potential adjunct to scaling and root planing in the treatment of periodontal disease. However, the effectiveness of porphyrin derivatives against microorganisms has been limited because some gram-negative bacteria are refractory to photodynamic treatment with these agents. We have designed a porphyrin derivative conjugated to a pentalysine moeity that endows the molecule with activity against gram-positive and gram-negative bacteria. Whereas the porphyrin, chlorin e6, showed in vitro activity against a limited spectrum of bacteria, chlorin e6 conjugated to pentalysine showed in vitro activity against all oral microorganisms tested, including Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum subsp. polymorphum, Actinomyces viscosus, and the streptococci. Potent antimicrobial activity (>5-log-unit reduction in the numbers of CFU per milliliter) was retained in the presence of up to 25% whole sheep blood. The use of potent, selective agents such as this chlorin e6-pentalysine conjugate to more effectively reduce the pathogenic bacteria in the

Metal ions were evaluated as potential antimicrobial agents suitable for local delivery in the oral cavity for the treatment of periodontitis. Silver nitrate, copper chloride, and zinc chloride were tested for antimicrobial activity in in vitro killing assays conducted in phosphate buffered saline with a series of oral bacteria including gram-negative periodontal pathogens and gram-positive streptococci. Copper and zinc salts failed to exhibit strong and consistent activity against periodontal pathogens. In contrast, silver at a concentration of 0.5 microg/mL produced a 3 log10 reduction in colony forming units (CFU)/mL or greater against all periodontal pathogens tested including Porphyromonas gingivalis, Prevotella intermedia, Prevotella denticola, Bacteroides forsythus, Fusobacterium nucleatum vincentii, Campylobacter gracilis, Campylobacter rectus, Eikenella corrodens, and Actinobacillus actinomycetemcomitans. In comparison, substantially higher concentrations of silver nitrate failed to kill oral streptococci. A silver nitrate concentration of 25 microg/mL produced log10 reductions in CFU/mL of 3.5-5 in killing assays performed in human serum against P. gingivalis, demonstrating the ability of silver to retain activity in a biological medium similar to that encountered in vivo in the periodontal pocket. These results identify silver nitrate, an antimicrobial that may possess advantages over traditional antibiotics, as a potential agent for controlled release local delivery in the oral cavity for the treatment of periodontitis.