Drug delivery to the brain using an anti-transferrin receptor antibody.
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Animals↗
Biomedical subjects
B Malfroy
Publications and source records attributed to B Malfroy.
Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier.
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 of brain capillaries becomes extremely punctate approximately 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 time-dependent 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 radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26.
Animals↗
Characterization of endopeptidase 3.4.24.11 ("enkephalinase") activity in human plasma and cerebrospinal fluid.
The presence of the neutral metallo-endopeptidase 3.4.24.11 ("enkephalinase") activity was investigated by fluorimetric assay in human body fluids. Although the enzyme was previously known to occur exclusively in membrane bound form in the human or animal central nervous system, its activity was detected in human cerebrospinal fluid (CSF), plasma and amniotic fluid. Although the endopeptidase 3.4.24.11 activity found in human body fluids has properties closely related to the membrane bound enzyme such as affinity constant for the inhibitors, optimal pH and Km for the substrate, the Vmax values were in CSF, plasma and amniotic fluid, respectively, 2 x 10(3), 10(2), 10 lower than mouse brain homogenate.
Adult↗
Substance P contracts the human iris sphincter: possible modulation by endogenous enkephalinase.
Substance P-immunoreactive neurons have been found in the irides of many species including humans. In several species, substance P has been shown to induce contraction of the sphincter muscle but this action of substance P has not been previously demonstrated in the human eye. Using an eye cup model in which the sensitivity of the iris muscle to substance P is increased compared to the isolated sphincter muscle, we have observed that nanomolar amounts of substance P induced contraction of the sphincter in the human iris. This contractile response was enhanced in eyes pretreated with thiorphan, an enkephalinase inhibitor, suggesting that endogenous enkephalinase (E.C. 3.4.24.11) may modulate the substance P contraction in the human iris. Further support for this hypothesis was the finding of enkephalinase-like immunoreactivity and enzyme activity in the human iris sphincter muscle.
Aged↗
Recombinant enkephalinase effectively inhibits substance P-induced miosis in the rabbit eye cup model.
Enkephalinase (EC 3.4.24.11) is a naturally occurring, membrane-bound peptidase that degrades substance P in vivo and in vitro. Addition of this neutral endopeptidase to a rabbit eye cup model partially inhibits substance P-induced contraction of the iris sphincter muscle. Inactivation of substance P is reversed by thiorphan, a specific inhibitor of enkephalinase. These results show that enkephalinase degradation of substance P produces metabolites that are physiologically inactive in iris contraction. We also observed that atropine acts synergistically with enkephalinase to completely abolish substance P-induced iris contraction suggesting that the action of substance P on the iris contains an acetylcholine-stimulatory effect which is not lost by enkephalinase treatment.
Animals↗
Administration of recombinant enkephalinase (neutral endopeptidase) prevents capsaicin-induced miosis in the rabbit eye in vivo.
Substance P induced a dose-dependent contraction of iris sphincter muscles when applied in the presence of atropine to the isolated rabbit iris in vitro as evidenced by a decreased pupil diameter. Pretreatment of the iris with 20 micrograms of recombinant enkephalinase (neutral endopeptidase; EC 3.4.24.11) totally abolished the contractile response to substance P. Injection of 10 micrograms of capsaicin into the anterior chamber of atropine-treated rabbit eyes in vivo induced an immediate and intense miosis. Injection of 100 micrograms of recombinant enkephalinase, 1 or 5 min before capsaicin injection, significantly inhibited this miosis. This effect of enkephalinase was totally abolished by preincubating the enzyme with thiorphan, a high-affinity enkephalinase inhibitor. These results show that enkephalinase, which is known to hydrolyze substance P in vitro with high efficiency, also hydrolyzes endogenously released substance P in vivo. Furthermore, our results suggest that enkephalinase application may represent a novel therapeutic approach to treat substance P-mediated pathologies.
Animals↗
Elevated synovial tissue concentration of the common acute lymphoblastic leukaemia antigen (CALLA)-associated neutral endopeptidase (3.4.24.11) in human chronic arthritis.
The cell-surface neutral endopeptidase 3.4.24.11 (NEP) activity of the common acute lymphoblastic leukaemia antigen (CALLA) cleaves diverse peptide mediators at specific sites and it has been postulated that it regulates immune responses. The concentration of NEP was quantified in detergent extracts of synovial tissues by the percentage hydrolysis of [3H-D-Ala]-Leu enkephalin/hr/100 mg of tissue. The synovial tissue concentration of NEP was higher in all patients with rheumatoid arthritis (n = 7; group mean +/- SD = 29.4 +/- 20.2%), and was higher with degenerative joint disease (n = 6 of 8; group mean +/- SD = 11.9 +/- 10.4%) than with traumatic arthropathy (n = 3; 1.1 +/- 0.7%). The lack of direct relationship between synovial tissue NEP concentration and leukocytic infiltration suggests that the cellular source of NEP may be synoviocytes or fibroblasts, and that NEP may have distinctive pathogenetic roles in human arthritis.
Adult↗
Substance P primes human neutrophil activation: a mechanism for neurological regulation of inflammation.
The neuropeptide substance P (SP), a member of the tachykinin family, has stimulatory effects on various cell types at nanomolar concentrations. SP has also direct effects on polymorphonuclear leukocytes (PMNs). However, unlike other cells, stimulation of PMNs requires extremely high concentrations of the peptide (greater than 10 microM), suggesting that direct PMN activation by SP is not physiologically relevant. By measuring primed stimulation of PMNs, we now demonstrate potent synergistic effects of nanomolar doses of SP on the migratory and cytotoxic functions of human PMNs stimulated by fMLP and C5a. This synergism between SP and chemotactic peptides reveals a new regulatory activity of SP and suggests that neurogenic stimuli may prepare neutrophils for an exaggerated inflammatory response to other phlogistic mediators.
Cell Movement↗
Molecular cloning and amino acid sequence of rat kidney aminopeptidase M: a member of a super family of zinc-metallohydrolases.
Using a polyclonal antibody, a partial cDNA clone for rat aminopeptidase M was identified in a lambda gt11 library from rat kidney. A synthetic oligonucleotide probe derived from the sequence of the insert was used to screen a randomly primed lambda gt10 library. This allowed the identification of several overlapping clones encoding the full sequence of the enzyme. The reading frame, 2898 base pairs in length, encodes a 966 amino acid polypeptide. A highly hydrophobic segment, 24 amino acids in length, located close to the aminoterminus, is proposed to serve as the membrane-spanning domain for this membrane-bound enzyme. The sequence includes nine potential N-linked glycosylation sites and one potential sulfation site. In addition, the rat aminopeptidase M sequence contains an eight amino acid consensus sequence believed to serve as the zinc binding domain in a family of zinc-metallohydrolases. Rat aminopeptidase M shows 77% similarity with the recently cloned human enzyme, as well as weaker but significant similarity with aminopeptidase N from E. coli (18%) and with human leukotriene A4 hydrolase (21%).
Amino Acid Sequence↗
Preferential cleavage of amino- and carboxyl-terminal oligopeptides from vasoactive intestinal polypeptide by human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11).
Human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) cleaved synthetic vasoactive intestinal peptide (VIP1-28) with time-and peptidase concentration-dependence, which left less than 30% intact after 30 micrograms was incubated at 37 degrees C with 0.1 micrograms and 10 micrograms of peptidase for 120 min and 15 min, respectively. The rank order of relative rates of peptidolysis amino-terminal to hydrophobic amino acids was Ala4 and Val5 greater than Tyr22 and Ile26 much greater than Leu13 and Met17. The many effects of VIP1-28 on epithelial cell and leukocyte functions thus may be influenced by degradation of the mediator by enkephalinase at the surface of target cells.
Glycopeptides↗
Expression of enzymatically active enkephalinase (neutral endopeptidase) in mammalian cells.
A cDNA encoding the rat enkephalinase protein (neutral endopeptidase; EC 3.4.24.11) has been constructed from overlapping lambda gt10 cDNA clones. This cDNA was inserted into an expression plasmid containing the cytomegalovirus enhancer and promoter. When transfected with this plasmid, Cos 7 cells transiently expressed the enkephalinase protein in a membrane-bound state. Recombinant enkephalinase recovered in solubilized extracts from transfected Cos 7 cells was enzymatically active and displayed properties similar to those of the native enzyme with respect to sensitivity to classical enkephalinase inhibitors.
Animals↗
Effects of bombesin on human small cell lung cancer cells: evidence for a subset of bombesin non-responsive cell lines.
The effects of bombesin on three human small cell lung carcinoma cell (SCLC) lines (NCI-H69, NCI-H128, and NCI-H345) have been examined and compared to the effects of the peptide on the mouse fibroblast cell line Swiss 3T3, and the rat pituitary tumor cell line GH3W5. While all three SCLC lines expressed messenger RNA encoding pro-gastrin releasing peptide (GRP), only the NCI-H345 cells expressed detectable membrane receptors for GRP and responded to nanomolar concentrations of bombesin as shown by 125I-GRP binding, total inositol phosphate accumulation, and increased clonal growth in soft agarose. These data show that some SCLC lines are insensitive to bombesin and do not express detectable membrane receptors for GRP.
Animals↗
Recombinant human enkephalinase (neutral endopeptidase) prevents cough induced by tachykinins in awake guinea pigs.
To determine whether recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) prevents cough induced by exogenously applied and endogenously released neuropeptides, we measured cough responses to aerosolized solutions of substance P or of capsaicin for 2 min in random-source guinea pigs before or after exposing them to aerosolized recombinant human enkephalinase. Substance P (10(-16) M) increased coughing compared with its vehicle. Enkephalinase (120 micrograms) inhibited cough induced by subsequent exposure to substance P compared with the response to substance P alone, but after further exposure to the enkephalinase inhibitor leucine-thiorphan (10(-5) M), substance P increased cough significantly. Similar results were obtained for capsaicin-induced cough. In pathogen-free guinea pigs, after they inhaled inactive recombinant enkephalinase (33 micrograms), capsaicin (10(-13) M) increased cough significantly. In contrast, after they inhaled active recombinant enkephalinase (33 micrograms), capsaicin increased cough only slightly. These results suggest that aerosolized enkephalinase reaches the sites of release or actions of endogenous neuropeptides and, by degrading them, prevents cough induced by their release. Furthermore, these studies suggest that recombinant enkephalinase might be useful in the treatment of cough and other symptoms of diseases involving peptides cleaved by this enzyme.
Aerosols↗
Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase).
We have isolated a cDNA clone encoding human enkephalinase (neutral endopeptidase, EC 3.4.24.11) in a lambda gt10 library from human placenta, and present the complete 742 amino acid sequence of human enkephalinase. The human enzyme displays a high homology with rat and rabbit enkephalinase. Like the rat and rabbit enzyme, human enkephalinase contains a single N-terminal transmembrane region and is likely to be inserted through cell membranes with the majority of protein, including its carboxy-terminus, located extracellularly.
Amino Acid Sequence↗
Cellular catabolism of recombinant tissue-type plasminogen activator. Identification and characterization of a novel high affinity uptake system on rat hepatocytes.
The uptake, internalization, and intracellular degradation of 125I-labeled recombinant tissue-type plasminogen activator (rt-PA) by isolated rat hepatocytes was investigated. Incubation at 37 degrees C resulted in internalization of 125I-rt-PA, followed by the appearance of labeled trichloroacetic acid-soluble material in the incubation media due to degradation of rt-PA. Degradation of rt-PA was inhibited by the presence of NH4Cl (10 mM) or chloroquine (1 mM) (lysosomal tropic agents) in the incubation media. This suggests that rt-PA degradation occurs intracellularly, perhaps within the lysosomes. 125I-rt-PA was taken up by rat hepatocytes through a specific, high affinity mechanism with half-maximal uptake at 10 nM. Uptake of 125I-rt-PA was not inhibited by glycopeptides isolated from rt-PA nor by several other glycoproteins known to be cleared by identified hepatic receptors. These results suggest that the uptake of rt-PA by rat hepatocytes involves a receptor specific for t-PA and is not mediated by a carbohydrate-specific receptor.
Animals↗
Molecular cloning and amino acid sequence of rat enkephalinase.
cDNA clones encoding rat enkephalinase (neutral endopeptidase, EC 3.4.24.11) have been isolated in lambda gt10 libraries from both brain and kidney mRNAs and the complete 742 amino acid sequence of rat enkephalinase is presented. The enzyme possesses a single transmembrane spanning domain near the N-terminal of the molecule but lacks a signal sequence. Because enkephalinase has it active site located extracellularly and is thus an ectopeptidase, we suggest that the N-terminal transmembrane region of the enzyme anchors the protein in membranes and that the majority of the protein, including the carboxy terminus, is extracellular. Enkephalinase, a zinc-containing metallo enzyme, displays homology with other zinc metallo enzymes such as carboxypeptidase A, B and E, suggesting enzymatic similarities in these enzymes.
Amino Acid Sequence↗
New substrates for enkephalinase (neutral endopeptidase) based on fluorescence energy transfer.
Novel fluorescent substrates for enkephalinase (neutral endopeptidase; EC 3.4.24.11) have been developed. These new assays are based on the disappearance of energy transfer between a tryptophan or a tyrosine residue and the 5-dimethylaminonaphthalene-1-sulfonyl group (dansyl) in the substrates dansyl-Gly-Trp-Gly or dansyl-Gly-Tyr-Gly upon hydrolysis of their Gly-Trp or Gly-Tyr amide bond by enkephalinase. No significant difference in Km or kcat values were found for dansyl-Gly-Trp-Gly and dansyl-Gly-Tyr-Gly, indicating that, in contrast to thermolysin, the active site of enkephalinase easily accommodates tryptophan residues. Both tryptophan and tyrosine-containing substrates can be used for continuous recording of enkephalinase activity and should prove useful for detailed study of the substrate specificity of this enzyme.
Animals↗
Brain-specific gene expression.
The brain of an adult rat expresses approximately 30,000 different brain-specific mRNAs. To investigate their encoded proteins, we have selected cDNA clones corresponding to mRNAs expressed exclusively in rat brain, determined their nucleotide sequences and generated antisera against synthetic peptides mimicking short regions of the deduced protein sequences. The clone plB236 encodes a protein that defines a widely distributed neuronal system and may be the precursor for a family of novel neuropeptides. A second clone, plB208, encodes rat brain proteolipid protein, the major protein component of central nervous system myelin. These studies have also identified an 82 nucleotide genetic element called an ID (identifier) sequence that may be involved in the regulation of transcription of brain-specific genes.
Amino Acid Sequence↗