Evaluation of the RBE4 Cell Line to Explore Carrier-mediated Drug Delivery to the CNS Via the L-system Amino Acid Transporter At the Blood-Brain Barrier

The L-system amino acid transporter on the RBE4 cell line, a well established in vitro model of the blood-brain barrier (BBB), was characterised with the aim to evaluate this in vitro BBB model as tool for the systematic exploration of this endogenous carrier system for drug delivery to the CNS. Transport of l - [3 H]-leucine in RBE4 cells was rapid, Na + -independent, bidirectional and followed the principles of trans -stimulation. The inhibition profile of l -leucine uptake was consistent with transport mediated by the L-system amino acid carrier with strong inhibition by large neutral amino acids (LNAA) such as l -phenylalanine and 2-aminobicyclo-heptanecarboxylic acid (BCH), whereas small neutral, basic and acidic amino acids had no significant effect. The transport of l -leucine into the RBE4 cells was saturable and followed single carrier Michaelis-Menten kinetics with K m 107 ± 10 μ M, V max 9.13 ± 0.45 nmol/min/mg protein and K D 1.36 ± 0.13 μ l/min/mg protein. The kinetic constants of l -leucine transport, as well as the ranking of the kinetic constants of the transport of other LNAA investigated, correspond to those of the BBB in vivo. The characteristics of the LNAA transport in RBE4 cells suggest that transport is mediated by a system with characteristics similar to the L 1 subtype of amino acid transporter, with carrier specificity equivalent to the L 1 carrier system at the BBB in vivo. The study shows that the RBE4 cell line is a very suitable tool for the detailed examination of structure-transport relationships with respect to carrier-mediated drug delivery to the CNS via the L-system amino acid carrier at the BBB. The strength of this in vitro BBB model lies in the combination of the advantages of a cell line, being inexpensive, reproducible and easy to maintain, with the brain endothelium-specific expression of transport systems, to produce an efficient assay for the screening of potential neuropharmaceuticals targeted to specific transport routes to enhance CNS drug delivery.     

Apolipoprotein-mediated Transport of Nanoparticle-bound Drugs Across the Blood-Brain Barrier

Recent studies have shown that drugs that are normally unable to cross the blood-brain barrier (BBB) following intravenous injection can be transported across this barrier by binding to poly(butyl cyanoacrylate) nanoparticles and coating with polysorbate 80. However, the mechanism of this transport so far was not known. In the present paper, the possible involvement of apolipoproteins in the transport of nanoparticle-bound drugs into the brain is investigated. Poly(butyl cyanoacrylate) nanoparticles loaded with the hexapeptide dalargin were coated with the apolipoproteins AII, B, CII, E, or J without or after precoating with polysorbate 80. In addition, loperamide-loaded nanoparticles were coated with apolipoprotein E alone or again after precoating with polysorbate 80. After intravenous injection to ICR mice the antinociceptive threshold was measured by the tail flick test. Furthermore, the antinociceptive threshold of polysorbate 80-coated dalargin-loaded nanoparticles was determined in ApoEtm1Unc and C57BL/6J mice. The results show that only dalargin or loperamide-loaded nanoparticles coated with polysorbate 80 and/or with apolipoprotein B or E were able to achieve an antinociceptive effect. This effect was significantly higher after polysorbate-precoating and apolipoprotein B or E-overcoating. With the apolipoprotein E-deficient ApoEtm1Unc mice the antinociceptive effect was considerably reduced in comparison to the C57BL/6J mice. These results suggest that apolipoproteins B and E are involved in the mediation of the transport of drugs bound to poly(butyl cyanoacrylate) nanoparticles across the BBB. Polysorbate 80-coated nanoparticles adsorb these apolipoproteins from the blood after injection and thus seem to mimic lipoprotein particles that could be taken up by the brain capillary endothelial cells via receptor-mediated endocytosis. Bound drugs then may be further transported into the brain by diffusion following release within the endothelial cells or, alternatively, by transcytosis.     

Predicting Blood-Brain Barrier Permeability of Drugs: Evaluation of Different In Vitro Assays

The ability of a drug to penetrate the blood-brain barrier (BBB) is essential for its use in the pharmaceutical treatment of CNS disorders. Five different in vitro methods to predict BBB permeability of drugs were compared and evaluated in the present study. All assays were performed with a consistent set of seven compounds and in the same physiological buffer to provide a basis for direct comparison of the results. Octanol-buffer and liposome-buffer partition coefficients were most conveniently obtained but failed to predict BBB permeability for certain drugs. The incorporation of drugs into lipid monolayers at the air-buffer interface was found to be a poor predictor of BBB permeability and was furthermore not considered suitable for screening due to the demanding experimental requirements. Permeability studies using Caco-2 cell monolayers provided a good correlation to an in vitro model of the BBB, which was based on primary cultured porcine brain capillary endothelial cells (PBCEC). However, differences in drug permeability between the intestine and brain derived cells were detected, limiting the advantages of the easy handling of the Caco-2 cell line compared to the more time-consuming primary culture of the BCEC.     

Pharmacokinetics and Brain Uptake of Biotinylated Basic Fibroblast Growth Factor Conjugated to a Blood-Brain Barrier Drug Delivery System

Human basic fibroblast growth factor (bFGF) is a potent neuroprotective agent. The clinical efficacy of this neurotrophin, however, is restricted by poor permeability across the blood-brain barrier (BBB). This study was designed to test the hypotheses that bFGF will retain its biological activity and have an enhanced BBB transport after re-formulation and conjugation to a BBB peptide drug delivery vector. The BBB delivery vector is comprised of a conjugate of streptavidin (SA) and the murine OX26 monoclonal antibody against the rat transferrin receptor, and the conjugate of biotinylated bFGF (bio-bFGF) bound to a vector is designated bio-bFGF/OX26-SA. A radioreceptor binding assay shows that the native bFGF, bio-bFGF, and bio-bFGF/OX26-SA conjugate have IC 50 values of 0.12, 0.40, and 0.56 nM, respectively. After an IV bolus injection to the rat, [125 I]-bio-bFGF is avidly taken up by peripheral organs, with low brain uptake at 60 min, 0.010 ± 0.004% of injected dose (ID)/g brain. By contrast, the brain uptake of the [125 I]-bio-bFGF/OX26-SA is increased 5-fold to 0.050 ± 0.011%ID/g, although the uptake of the conjugate by peripheral tissues was decreased relative to the unconjugated bio-bFGF. In conclusion, conjugation of bio-bFGF to a BBB drug delivery vector (a) causes only a minor decrease in affinity for the bFGF receptor, (b) decreases the peripheral organ uptake of the bFGF, and (c) increases the brain uptake of the neurotrophin. The re-formulation of bFGF to enable receptor-mediated transcytosis across the BBB may improve the therapeutic index of this neurotrophin as a neuroprotective agent.     

Blood-Brain Barrier Transport of L-iyrosine Conjugates: a Model Study for the Brain Targeting using Large Neutral Amino Acid Transport System

Abstract

We examined the relation ship between the charge of the amino or carboxylic function of a substrate and the substrate recognition by the large neutral amino acid (LNAA) transport carrier, using the in situ brain perfusion technique. Glucose-coupled L-tyrosine (GcpY), which has a free carboxylic function, and 2-(L-tyrosylamide)-2-deoxy-D-glucose (Y-2DG), which has a free amino function were synthesized. The inhibitory effect of GcpY on [³H]L-tyrosine uptake was larger than that of N-methly-L-phenylalanine or N-acetyl-L-phenylalanine, whereas Y-2DG did not affect it. These results indicate that a free amino group is not required for recognition, provided that the modified amino group is able to take a positive charge. Steric factors appeared to be relatively unimportant.     

药物经鼻向中枢神经系统转运的试验方法及研究进展

综述了药物经鼻向中枢神经系统转运的机理、试验方法及研究进展。     

The Effect of RMP-7 and its Derivative on Transporting Evens Blue Liposomes into the Brain

To investigate the effect of RMP-7 and its derivative on drug transport across blood brain barrier (BBB), RMP-7 and DSPE-PEG-NHS [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)]-hydroxy succinamide, PEG M 3400] were conjugated under mild conditions and the reaction ratio was determined using MALDI–TOF-MS (matrix-assisted laser desorption-ionization time-of-flight mass spectrometry). An endothelial cell monolayer in vitro BBB model was established and used to determine the bioactivity of RMP-7 and its derivative “opening BBB.” Horse radish peroxide (HRP), liposome (HRP-L-PEG), and Evens blue (EB) liposome (EB-L-PEG) were prepared using the reverse-phase evaporation method. HRP-L-PEG-RMP-7 and EB-L-PEG-RMP-7 were obtained by inserting DSPE-PEG-RMP-7 into the surface of liposome. The bioactivity of RMP-7 and DSPE-PEG-RMP-7 opening BBB were evaluated to determine their effect on the permeation ratio of HRP and HRP liposome across the in vitro BBB model. To evaluate the in vivo bioactivity of RMP-7 and DSPE-PEG-RMP-7 on EB transport across BBB into the brain, the indicated compounds were administered to rats. Then, brain slices were analyzed using confocal laser scanning microcopy and the EB concentration in the brain, liver, spleen, lung, and kidney was determined using the formamide–extraction–ultraviolet-spectrophotometric method. The results demonstrated that RMP-7 was conjugated with DSPE-PEG-NHS at the molecular ratio of 1:1 and the product is DSPE-PEG-RMP-7. Compared with adding HRP alone, RMP-7 and DSPE-PEG-RMP-7 improved 2- to 3-fold the transport of HRP in the in vitro BBB model. The in vivo experiments showed that DSPE-PEG-RMP-7 was better at facilitating EB transport into brain than RMP-7. The reason may be that DSPE-PEG-RMP-7 can “open BBB” as soon as the EB-L-PEG-RMP-7 reaches BBB.     

Inhibition of P-Glycoprotein: Rapid Assessment of Its Implication in Blood-Brain Barrier Integrity and Drug Transport to the Brain by an In Vitro Model of the Blood-Brain Barrier

Purpose. The objective of this work was to assess, in vitro, the passage of P-glycoprotein dependent drugs across brain capillary endothelial cells, when these drugs are associated with a reversing agent. Methods. An in vitro model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes was used. Results. We demonstrate that P-glycoprotein expression is upregulated by the presence of astrocytes. Uptake in the cells and transport across endothelial cell monolayers of vincristine, cyclosporin A and doxorubicin were studied. Using S9788 or verapamil as reversing agents, we found an increase in vincristine transport across the endothelial cell monolayers. On the other hand, the association of S9788 or verapamil with cyclosporin A failed to increase the transport of this drug. An increase in the transport of doxorubicin from luminal to abluminal compartment was also observed, due to endothelial cell monolayer breakdown. Conclusions. Using this model, it is possible to predict the passage of a P-glycoprotein dependent drug to the brain or its sequestration in brain capillary endothelial cells when this drug is associated with a reversing agent, or its toxicity on the blood-brain barrier integrity.     

中职生在学习药事法规中的学习障碍及其教学对策

通过对中职生在学习药事法规过程中的障碍分析，实施相应的教学对策，以提高药事法规的教学质量。     

Carrier-Mediated Transport of H1-Antagonist at the Blood–Brain Barrier: Mepyramine Uptake into Bovine Brain Capillary Endothelial Cells in Primary Monolayer Cultures

The transport mechanism of the H₁antagonist mepyramine at the blood-brain barrier (BBB) was studied by using primary cultured monolayers of bovine brain capillary endothelial cells (BCEC). The initial uptake of [³H]mepyramine into the BCEC showed strong temperature and concentration dependency, indicating that it involves both saturable and nonsaturable processes. Transport at the luminal membrane may be the rate-limiting process in the transcellular transport, since the values of the uptake coefficient of [³H]mepyramine at the luminal membrane (609 µl/mg protein/min) and the transcellular permeability coefficient (488 µl/mg protein/min) are very similar. The initial uptake of [³H]mepyramine was not affected by metabolic inhibitors, but was stimulated by preloading with the drug. Mepyramine appears to be transported into the BCEC by a carrier-mediated transport system which does not require metabolic energy, probably via a facilitated diffusion mechanism.     

Drug transport to the brain: comparison between in vitro and in vivo models of the blood-brain barrier

The aim of the present studies was to compare the transport of drugs using two in vitro models routinely used in our laboratories: a primary culture of brain microvessel endothelial cells and a coculture of brain capillary endothelial cells and astrocytes. For this purpose we selected a set of compounds corresponding to a wide range of lipid solubility. Additionally the in vitro results were compared to in vivo results obtained with the single carotid injection, and a good correlation between in vivo extraction ratios (E_t) and in vitro permeability coefficients (P_e) was shown as indicated by the Spearman's correlation coefficient (r = 0.90 andr = 0.96 for primary culture and coculture, respectively). The studies show that the use of brain capillary endothelial cells together with astrocytes is slightly more predictive and significantly easier than the use of primary cultured brain microvessels.     

The effect of RMP-7 and its derivative on transporting Evans blue liposomes into the brain

To investigate the effect of RMP-7 and its derivative on drug transport across blood brain barrier (BBB), RMP-7 and DSPE-PEG-NHS [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)]-hydroxy succinamide, PEG M 3400] were conjugated under mild conditions and the reaction ratio was determined using MALDI-TOF-MS (matrix-assisted laser desorption-ionization time-of-flight mass spectrometry). An endothelial cell monolayer in vitro BBB model was established and used to determine the bioactivity of RMP-7 and its derivative “opening BBB.” Horse radish peroxide (HRP), liposome (HRP-L-PEG), and Evens blue (EB) liposome (EB-L-PEG) were prepared using the reverse-phase evaporation method. HRP-L-PEG-RMP-7 and EB-L-PEG-RMP-7 were obtained by inserting DSPE-PEG-RMP-7 into the surface of liposome. The bioactivity of RMP-7 and DSPE-PEG-RMP-7 opening BBB were evaluated to determine their effect on the permeation ratio of HRP and HRP liposome across the in vitro BBB model. To evaluate the in vivo bioactivity of RMP-7 and DSPE-PEG-RMP-7 on EB transport across BBB into the brain, the indicated compounds were administered to rats. Then, brain slices were analyzed using confocal laser scanning microcopy and the EB concentration in the brain, liver, spleen, lung, and kidney was determined using the formamide-extraction-ultraviolet-spectrophotometric method. The results demonstrated that RMP-7 was conjugated with DSPE-PEG-NHS at the molecular ratio of 1:1 and the product is DSPE-PEG-RMP-7. Compared with adding HRP alone, RMP-7 and DSPE-PEG-RMP-7 improved 2- to 3-fold the transport of HRP in the in vitro BBB model. The in vivo experiments showed that DSPE-PEG-RMP-7 was better at facilitating EB transport into brain than RMP-7. The reason may be that DSPE-PEG-RMP-7 can “open BBB” as soon as the EB-L-PEG-RMP-7 reaches BBB.     

The solute carrier transporters and the brain: Physiological and pharmacological implications

Solute carriers(SLCs)are the largest family of transmembrane transporters that determine the exchange of various substances,including nutrients,ions,metabolites,and drugs across biological membranes.To date,the presence of about 287 SLC genes have been identified in the brain,among which mutations or the resultant dysfunctions of 71 SLC genes have been reported to be correlated with human brain disorders.Although increasing interest in SLCs have focused on drug development,SLCs are currently still under-explored as drug targets,especially in the brain.We summarize the main substrates and functions of SLCs that are expressed in the brain,with an emphasis on selected SLCs that are important physiologically,pathologically,and pharmacologically in the blood-brain barrier,astrocytes,and neurons.Evidence suggests that a fraction of SLCs are regulated along with the occurrences of brain disorders,among which epilepsy,neurodegenerative diseases,and autism are representative.Given the review of SLCs involved in the onset and procession of brain disorders,we hope these SLCs will be screened as promising drug targets to improve drug delivery to the brain.     

The Transport Barrier of Epithelia: A Comparative Study on Membrane Permeability and Charge Selectivity in the Rabbit

The transport barrier of the epithelia presents one of the major problems limiting the effective use of these tissues as alternate delivery routes for macromolecules such as peptides and proteins. In the present study, two membrane transport properties, namely, the permeability and permselectivity of the shunt pathway, were investigated and compared in various tissues including the nasal, tracheal, bronchial, buccal, rectal, vaginal, corneal, epidermal, duodenal, jejunal, ileal, and colonic epithelia. Membrane permeability was evaluated using a combined method based on electrical conductance and flux measurements of a hydrophilic fluorescent probe, 6-carboxy fluorescein (CF). Membrane permselectivity or the charge discriminating ability of the membrane was evaluated by KCl diffusion potential measurements. The results indicate that all epithelia under investigation possess a relatively high degree of permeation barrier and are highly selective for the absorption of positively charged solutes. Shunt path permeability was found to vary greatly among tissues from different epithelia, whereas membrane charge selectivity was relatively constant in these tissues. A good correlation was observed between membrane electrical conductance and steady-state flux of CF, indicating a paracellular transport of the compound. The rank order of the intrinsic membrane permeability was as follows: intestinal nasal bronchial tracheal > vaginal rectal > corneal > buccal > skin. Membrane permselectivity, expressed as the ratio of transport number (positive over negative), ranges from 1.78 for the buccal to 1.33 for the rectal epithelium. These results suggest that, for effective delivery purposes, permeation enhancing methods, by either increasing tissue permeability or modifying drug-membrane charge selectivity, are generally required. The permeation data also suggest that the respiratory epithelia represent good alternate routes for drug delivery, particularly for those that are orally ineffective, i.e., due to extensive gastrointestinal tract degradation or first-pass metabolism.     

Brain uptake and distribution of the potential memory enhancer CL 275,838 and its main metabolites in rats: relationship between brain concentrations and in vitro potencies on neurotransmitter mechanisms

The kinetics, brain uptake and distribution of CL 275,838, a potential memory enhancer, and its main metabolites (II and IV) were evaluated in rats after intraperitoneal doses of 5, 10 and 20 mg/kg. Brain maximum concentrations (C_max) of the three compounds after pharmacologically active doses were then related to the in vitro concentrations affecting some monoaminergic and amino acid receptor sites to examine the relative importance of these neurotransmitter systems in the pharmacological actions of CL 275,838. After 10 mg/kg CL 275,838, the unchanged compound rapidly entered the brain and distributed almost uniformly in various regions inside the blood-brain barrier. Its disappearance from brain and plasma was almost parallel with a comparable elimination half-life (t_1/2) of about 2 h. Metabolite II entered the brain and equilibrated with plasma more slowly than the parent compound, achieving mean C_max (0.2 µM) within 3 h of dosing. Metabolite IV was rapidly detected in rat brain but hardly amounted to 10% (0.1 µM) of the parent compound C_max (1 µM). There was a linear relationship between dose and plasma and brain concentrations of the three compounds up to 20 mg/kg CL 275,838. At micromolar concentrations the parent compound had affinity for serotonin (5-HT) uptake sites, 5-HT₂ and dopamine (DA₂) receptors. Only at much higher concentrations than those achieved in vivo after pharmacologically active doses did it increase the binding of³H-glutamate to NMDA (N-methyl-d-aspartate) receptors. Metabolite II had a similar neurochemical profile. Metabolite IV had no affinity for these neurotransmitter systems, except for benzodiazepine (BDZ) receptor sites where it interacts with micromolar affinity behaving, however, as an agonist as determined by the GABA ratio. Although the effect on NMDA is compatible with favourable effects on memory, it is unlikely that it is involved in the ability of CL 275,838 to attenuate the impairment of a passive avoidance task caused by drugs or aging in rats.     

Neuroimmunity and the Blood–Brain Barrier: Molecular Regulation of Leukocyte Transmigration and Viral Entry into the Nervous System with a Focus on NeuroAIDS

HIV infection of the central nervous system (CNS) can result in neurologic dysfunction with devastating consequences in a significant number of individuals with AIDS. Two main CNS complications in individuals with HIV are encephalitis and dementia, which are characterized by leukocyte infiltration into the CNS, microglia activation, aberrant chemokine expression, blood–brain barrier (BBB) disruption, and eventual damage and/or loss of neurons. One of the major mediators of NeuroAIDS is the transmigration of HIV-infected leukocytes across the BBB into the CNS. This review summarizes new key findings that support a critical role of the BBB in regulating leukocyte transmigration. In addition, we discuss studies on communication among cells of the immune system, BBB, and the CNS parenchyma, and suggest how these interactions contribute to the pathogenesis of NeuroAIDS. We also describe some of the animal models that have been used to study and characterize important mechanisms that have been proposed to be involved in HIV-induced CNS dysfunction. Finally, we review the pharmacologic interventions that address neuroinflammation, and the effect of substance abuse on HIV-1 related neuroimmunity.     

Drug use and drug markets in the context of political conflict: The case of Northern Ireland

The focus of this article addresses drug use and drug markets in Northern Ireland against the backdrop of the most recent Irish political conflict, e.g., 1969 to the present. Between 1969 and 1999, a total of 3289 individuals had died and more than 40?000 were injured as a result of the Northern Ireland political conflict. Extrapolating the data to Britain, comparable figures would reflect 111?000 fatalities and over one million injured (Hayes and McAllister, 2000). This paper describes how the nature of the Northern Ireland political conflict contributed to low levels of drug use in the 1970s and 1980s. In 1994, the cessation of military operations by the Irish Republican Army (IRA) and subsequently by mainstream Loyalist organizations led to the possibility of widespread political and social change. Use of certain drugs, namely heroin, appeared to increase from the mid-1990s, although the effects of political conflict on drug use are less clear during the post-ceasefire era.     

Legitimacy and modernity via policy transfer: The utility of the 2003 Afghan National Drug Control Strategy

Very much an exercise in historical reconstruction, this article is concerned with the development of the first version of the Afghan NDCS. It is hoped that this domain of enquiry will contribute to discussions around the ‘governance of drug policy’ in this special issue of the International Journal of Drug Policy by focusing on how different policy actors operate in influencing the policy process; or parts thereof. More specifically, exploration of the formulation of the Strategy does much to help us understand not only the origins and shifting nature of ownership of drug policy within Afghanistan but also the relationship between the NDCS and the broader normative expectations of what has been referred to as the global drug prohibition regime (Andreas & Nadelmann, 2006, p. 38). As will be discussed, while indisputably the product of a process of policy transfer involving a number of non-Afghan actors – and as such arguably not always appropriate to the peculiarities of the drug market within the country – it can be argued that the 2003 National Drug Control Strategy fulfilled a useful functional role that in many ways exceeded its utility as a guiding document beyond the confines of Kabul.     

The Relationship Between the Quality of Drug User Treatment and Program Completion: Understanding the Perceptions of Women in a Prison-Based Program

To determine why some women offenders complete prison-based drug user treatment and others leave early, clients' (N = 101) perceptions of various aspects of the quality of the treatment experience were compared. Analyses of both quantitative and qualitative data indicate that clients who completed the program had a more favorable perception of staff and felt empowered by the experience in treatment. Most of the clients who left early did so because of conflicts or disagreements with the program's rules. We discuss how a supportive approach to personal development may enhance client perceptions of program quality and increase retention rates.     

Proteolysis of Human Calcitonin in Excised Bovine Nasal Mucosa: Elucidation of the Metabolic Pathway by Liquid Secondary lonization Mass Spectrometry (LSIMS) and Matrix Assisted Laser Desorption lonization Mass Spectrometry (MALDI)

Purpose. Two calcitonins, i.e. human calcitonin (hCT) and, for comparison, salmon calcitonin (sCT), were chosen as peptide models to investigate nasal mucosal metabolism. Methods. The susceptibility of hCT and sCT to nasal mucosal enzymes was assessed by in-and-out reflection kinetics experiments in an in vitro model based on the use of freshly excised bovine nasal mucosa, with the mucosal surface of the mucosa facing the peptide solution. The kinetics of CT degradation in the bulk solution was monitored by HPLC. Peptide sequences of the main nasal metabolites of hCT were analyzed by using both liquid secondary ionization mass spectrometry (LSIMS), following HPLC fractionation of the metabolites, and matrix-assisted laser desorption ionization mass (MALDI) spectrometry. For sCT, the molecular weights of two major metabolites were determined by LC-MS with electrospray ionization. Results. Both CTs were readily metabolized by nasal mucosal enzymes. In the concentration range studied metabolic rates were higher with hCT than with sCT. Presence of endopeptidase activities in the nasal mucosa was crucial, cleaving both calcitonins in the central domain of the molecules. Conclusions. Typically, initial metabolic cleavage of hCT in nasal mucosa is due to both chymotryptic- and tryptic-like endopeptidases. The subsequent metabolic break-down follows the sequential pattern of aminopeptidase activity. Tryptic endopeptidase activity is characteristic of nasal sCT cleavage.