EP1135157A1 - Procede de regulation de la permeabilite de la barriere hemato-encephalique - Google Patents

Procede de regulation de la permeabilite de la barriere hemato-encephalique

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Publication number
EP1135157A1
EP1135157A1 EP99953234A EP99953234A EP1135157A1 EP 1135157 A1 EP1135157 A1 EP 1135157A1 EP 99953234 A EP99953234 A EP 99953234A EP 99953234 A EP99953234 A EP 99953234A EP 1135157 A1 EP1135157 A1 EP 1135157A1
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EP
European Patent Office
Prior art keywords
nos
permeability
nitric oxide
brain barrier
blood brain
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EP99953234A
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German (de)
English (en)
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EP1135157A4 (fr
Inventor
Carol Shoshkes Reiss
Takashi Komatsu
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New York University NYU
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New York University NYU
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Publication of EP1135157A1 publication Critical patent/EP1135157A1/fr
Publication of EP1135157A4 publication Critical patent/EP1135157A4/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method for regulating the permeability or integrity of the blood brain barrier and a method for delivering a compound into the central nervous system by increasing the local permeability of brain microcapillary endothelial cells constituting the blood brain barrier.
  • the central nervous system has been traditionally considered an "immunologically privileged site" because of the inadequacy of immune response under normal conditions.
  • the CNS is protected by the bones of the skull, meninges, the cerebrospinal fluid (CSF) , and the blood brain barrier (BBB) , a highly-selective vascular compartment which limits the flow of many biologically active molecules into the CNS.
  • the CNS has no well defined lymphatic system or mechanism for antibody production and is isolated from the immune system in the absence of disease (Leibowitz et al, 1983) . This "immunological privilege" may prevent the CNS from being damaged by excessive immune responses and may deter entry of pathogens in circulating cells.
  • the CNS has been shown to be constantly under immune surveillance and is capable of terminating neurotropic infections by initiating effective antigen specific and non-specific response (Cserr et al, 1992; Fabry et al 1994; Lotan et al, 1994) .
  • the BBB functions to regulate the constitution of the brain microenvironment essential for normal cerebral functions.
  • the permeability of the BBB is determined by complex tight intercellular junctions between a highly- specialized group of microvascular endothelial cells located within the brain which restrict passage of macromolecules between the blood and the brain (Brightman et al, 1969) .
  • This highly-selective group of microvascular endothelial cells are characterized not only by extremely tight junctions between cells, but are also surrounded by the end-feet of astrocytes, and, more rarely, by perivascular pericytes.
  • This capillary endothelial bed is distinct from capillaries in the periphery which are not fenestrated and have underlying smooth muscle cells.
  • nitric oxide has been implicated in this process (Boje, 1996; Buster et al, 1995; Chi et al , 1994; Johnson et al, 1995;
  • VSV vesicular stomatitus virus
  • BBB Perturbations of the BBB have been reported in a wide variety of CNS disorders and diseases, and the disruption of the integrity of the BBB selectivity can lead to drastic consequences to the individual. Brain vessels are normally impermeable to serum proteins due to the presence of tight junctions. Infection of brain endothelial cells may cause perturbations in BBB function, allowing toxic substances to cross into the normally inaccessible CNS. Modern understanding of brain pathophysiology has led to the provocative thought that many diseases of the CNS are associated with a failure of BBB integrity (Pardridge, 1986) .
  • Altered BBB permeability is commonly observed during ischemia, inflammation, trauma, neoplasia, hypertension, dementia and epilepsy (Buster et al, 1995; Chi et al, 1994; Mayhan, 1995; Prado et al, 1992; Shukla et al, 1995; Zhang et al, 1995) .
  • the extravasation of plasma proteins with BBB dysfunction may occur through a number of different transcellular or paracellular routes. This includes altered tight junctions, induction of fluid-phase or non-specific pinocytosis and transcytosis, formation of transendothelial channels or by disruption of the endothelial cell membrane (Durieu-Trautmann et al , 1993; Gross et al, 1991).
  • the selectivity of the BBB serves to prevent the entry into the CNS of therapeutic drugs.
  • AZT and protease inhibitors are excluded by the BBB.
  • Chemotherapeutic drugs are also excluded by the BBB and conventionally require administration intraventricularly, i.e., by catheter.
  • Viral infections of the CNS which disrupt the integrity of the BBB include viral encephalitis, such as from polio, measles, herpes, VSV, rabies, etc.
  • viral encephalitis such as from polio, measles, herpes, VSV, rabies, etc.
  • the earliest host responses to viral infections are non-specific and involve the induction of cytokines, among them interferons (IFNs) and tumor necrosis factor alpha (TNF- ⁇ ) .
  • IFNs interferons
  • TNF- ⁇ tumor necrosis factor alpha
  • NOS-2, iNOS NO synthase type 2
  • NOS-1 the isoform expressed in neurons, NOS-1, and the isoform expressed in astrocytes and endothelial cells, NOS-3, are IFN- ⁇ , TNF-c and interleukin-12 (IL-12) inducible.
  • IL-12 interleukin-12
  • NO which is the smallest, lightest molecule known to act as a biological messenger in mammals, was first identified as an endothelial cell relaxing factor (Furchgott et al, 1980; Palmer et al, 1987).
  • NOS nitric oxide synthases
  • All three enzymes have binding domains for calmodulin, flavin monocludeotide, flavin adenine dinucleotide, NADPH and a heme-binding site near the N-terminus (Table 1)
  • NO has an unpaired electron; thus, its effects are mediated through other molecules that accept or share this odd electron (Butler et al, 1995; Gaston et al, 1994) .
  • Target molecules include oxygen, other free radicals, thiol groups and metals.
  • NO is relatively less reactive than other oxygen radicals, such as superoxide anion (0 2 ⁇ ) and hydroxyl radical (OH " ) , making it a more stable carrier of unpaired electrons.
  • NO has a short half-life, in the range of a few seconds or less, and reacts readily with reduced cysteine moieties, yielding S-nitrosothiols that are somewhat stable with a half-life of minutes to hours.
  • the amino acid L- arginine, a substrate for NO synthesis contains two guanidine nitrogens that accept five electrons in an oxidation-reduction pathway, which results in the formation of L-citrulline and NO (Yun et al, 1996) ( Figure 1) .
  • NO is produced by the enzymatic modification of L- arginine to L-citrulline and requires many cofactors, including tetrahydrobiopterine, calmodulin, NADPH and 0 2 . NO rapidly reacts with proteins or with H 2 0 2 to form ONOO " , peroxynitrite, which is highly toxic ( Figure 1) . NO also readily binds heme proteins, including Hb and its own enzyme.
  • NO peroxynitrite
  • the neuronal NOS isoform (ncNOS, bNOS, NOS-1) is constitutively expressed and postranscriptionally regulated. Activity is dependent on calcium and calmodulin. It exists as a cytosolic homodimer under native conditions (Marietta, 1994) . Enzyme levels are cytokine inducible (Barna et al, 1996; Komatsu et al, 1996).
  • the acrophage form (NOS-2, iNOS) is rapidly induced by lipopolysaccharide (LPS) , TNF-c., IL-12 and IFN- ⁇ treatment, and is independent of calcium.
  • NOS-2 is a cytosolic dimer under native conditions (Marietta, 1994) . In the CNS, it is expressed in some astrocytes, microglia and inflammatory monocytes (Amin et al, 1995a; Galea et al, 1994; Merill et al , 1993; Zielasek et al, 1992).
  • the endothelial form (NOS-3 ecNOS) is constitutively expressed by posttranslationally regulated and PI linked membrane associated. Like NOS-1, it is dependent on calcium and calmodulin.
  • NOS activity has been found in autoimmune diseases, such as multiple sclerosis, associated with demyelinating lesions (DeGroot et al, 1997) and arthritic joints (Shiraishi et al, 1997) and are thought to contribute to disease pathogenesis .
  • NOS is frequently observed to be induced during the immune response (Barna et al, 1996) .
  • NOS activity has been observed to be essential in eliminating pathogens, such as Plasmodium falciparum (Anstey et al, 1996) .
  • NO has been demonstrated to be a key component in host defense against a variety of pathogens, including protozoan parasites, fungi, bacteria and viruses (Harris et al, 1995; Karupiah et al , 1993; Lee et al, 1994; Seguin et al, 1994; Stenger et al, 1994; and reviewed by Reiss et al, 1998) . It has inhibitory effects on ectromelia, vaccinia and herpes simplex type-1 viruses in macrophages (Karupiah et al, 1993) and the murine Friend leukemia virus (Akarid et al, 1995) . It also has an inhibitory effect on HIV replication (Mannick et al, 1996) .
  • Boje (1995) disclosed that LPS injected into ventricles induced meningeal NO production and BBB permeability.
  • Nakano et al indicate that the selective permeability increase in brain tumor microvessels after bradykinin infusion is mediated by NO and speculate that the absence of high levels of NOS in normal brain may account for the attenuated permeability response to bradykinin in normal brain microvessels.
  • the results reported in these publications on altered BBB permeability were all obtained in disease models in which the effector molecules for BBB permeability were present systemically in the animal model. There is, furthermore, no disclosure or suggestion of delivering a therapeutic compound into the CNS through increased BBB permeability by activating NOS-3.
  • the present invention is based on the discovery that the constitutive endothelial isoform of nitric oxide synthase (NOS-3) is central to the integrity of the blood brain barrier and provides a method for regulating the permeability of this barrier.
  • One aspect of the method according to the present invention reduces the increased permeability of the blood brain barrier as a result of a pathological condition by locally administering a NOS-3 inhibitor, and another aspect increases the permeability of the blood brain barrier by locally administering a NOS-3 activator or nitric oxide donor, thereby avoiding the problems associated with the systemic administration of NOS-3 inhibitors or activators.
  • a further aspect of the method according to the present invention is to provide for systemic administration of a NOS-3 inhibitor which is associated with a targeting molecule specific for cells forming the blood brain barrier.
  • the association of the NOS-3 inhibitor with the targeting molecule delivers the NOS-3 inhibitor directly to the blood brain barrier and moreover avoids the problems associated with systemic administration of NOS-3 inhibitors and their prolonged presence in the circulation.
  • a method for delivering a neurologically active therapeutic compound or diagnostic compound into the central nervous system by the contemporaneous local administration of a NOS-3 activator or a nitric oxide donor or by the systemic administration where the therapeutic or diagnostic compound is in association with both a targeting molecule and a NOS-3 activator or nitric oxide donor.
  • Figure 1 shows the enzymatic formation of NO and its reaction with proteins and other compounds.
  • Figures 2A-2C show the kinetics of the IFN- ⁇ augmented NOS activity in NB41A3 neuroblastoma, C6 rat glioma and RAW cells. Aliquots of supernatant were removed from triplicate wells of 2 x 10 5 NB41A3 (Fig. 2A) , RAW (Fig. 2B) or C6 (Fig. 2C) cells, cultured with medium or with 5 ng IFN- ⁇ for up to 72 hours. The medium was assayed for the presence of N0 2 using the Griess reagent, and expressed as nM N0 2 ⁇ S.D. present .
  • Figure 3 shows the role of type 1 NOS in IL-12 inhibition of VSV in vivo.
  • the amount of virus in individual samples was determined by plaque assay on CHO monolayers . Geometic mean titers ⁇ SEM are shown.
  • Figure 4 demonstrates how IL-12 treatment increased survival from VSV infection.
  • Groups of ten mice were injected i.p. with either the control medium or 200 ng IL-12 on days 0-7 post infection. All mice were infected intranasally with VSV (2 x 10 5 PFU/10 ⁇ L) . The number of survivors was greater in mice treated with IL-12 in WT mice but not in NOS-1 KO mice.
  • Figure 5 shows that IL-12 significantly increased weight loss recovery from VSV infection. The average weight ⁇ SEM of the surviving mice from Fig. 4 was recorded. Mice receiving IL-12 treatment were found to rapidly recover from the weight loss from the viral infection, as determined by the Student's t test in WT mice but not in NOS-1 KO mice.
  • FIG. 6 shows that IL-12 treatment significantly inhibited VSV infection in the CNS.
  • Groups of six mice were injected i.p. with either the control medium or 200 ng IL-12 on days 0-4 post infection. All mice were infected intranasally with VSV (2 x 10 5 PFU/10 ⁇ L) . On day four post infection, six mice of each group were sacrificed, and the mouse brains were homogenized for determination of viral titers on CHO cells. Viral titers of mice receiving IL-12 treatment were found to be significantly lower than those of the control mice, as determined by the Student's t test (P ⁇ 0.01) .
  • Figures 7A-7D show the IL-12 treatment-enhanced expression of NOS-1.
  • Figure 8 shows that the levels of VSV protein production is inhibited in cells treated with IL-12.
  • NB41A3 cells were stimulated with media or 5 ng of IL-12 for 72 hours prior to 2.5 or 5-hour infection with VSV at 1 moi .
  • Cells were lysed and the proteins were run on 7.5% SDS-acrylamide gel and a Western Blot was performed.
  • Figure 9 shows the relative density levels of VSV protein production. The relative density of the bands from Fig. 8 was measured. The data reveals there is an approximately 80% difference in the relative amounts of viral protein between the treated and untreated samples.
  • Figure 10 shows that the VSV proteins are nitrosylated. Cultures of NB41A3 cells were stimulated with media or 5 ng of IL-12 for 72 hours prior to 2.5 to 5-hour infection with VSV at 1 moi .
  • Figures 11A and 11B show the dual staining of the gels.
  • the gels from Figs. 8 and 9 were simultaneously stained for VSV (Fig. 11A) and nitrosine (Fig. 11B) .
  • Figure 12 shows that the levels of VSV mRNA production is inhibited in cells treated with IL-12.
  • NB41A3 cells were stimulated with media or 5 ng of IL-12 for 72 hours prior to one-hour infection of VSV at 1 moi. Cells were lysed and the mRNA were run on 2% agarose/formaldehyde gel and a Northern Blot was performed for the mRNA encoding the N gene.
  • Figure 13 shows the relative density levels of VSV mRNA production.
  • the relative density of the bands from Fig. 12 was measured. The data reveals that there is an approximately 20% difference in the relative amounts of viral mRNA between the treated and untreated samples.
  • Figure 14 shows that IL-12 treatment increased survival from VSV infection.
  • Groups of ten mice were injected i.p. with either the control medium or 200 ng IL-12 on days 0-7 post infection. All mice were infected intranasally with VSV (2 x 10 5 PFU/10 ⁇ L) . The number of survivors was greater in mice treated with IL-12, even in the NOS-3 KO mice.
  • FIG 15 shows that IL-12 treatment significantly increased weight loss recovery from VSV infection.
  • the average weight, ⁇ SEM, of the surviving mice from Fig. 14 was recorded. Mice receiving IL-12 treatment were found to rapidly recover from the weight loss from the viral infection, as determined by the Student's t test, even in NOS-3 KO mice.
  • Figure 16 shows that IL-12 treatment significantly inhibited VSV infection in the CNS. Groups of six mice were injected i.p. with either the control medium or 200 ng IL-12 on days 0-4 post infection. All mice were infected intranasally with VSV (2 x 10 5 PFU/10 ⁇ L) .
  • mice from each group were sacrificed, and the mouse brains were homogenized for determination of viral titers on CHO cells.
  • Viral titers of mice receiving IL-12 treatment were found to be significantly lower than those of the control mice, even in the NOS-3 -KO mice, as determined by the Student's t test (P ⁇ 0.01).
  • FIG 17 shows the breakdown of the BBB following VSV infection.
  • Three mice from each group were injected intravenously with 200 mL of 2% Evans blue at various time points. One hour later, the mice were sacrificed and perfused with normal saline. Brains were removed, and photos taken. One representative brain from each group is sown.
  • VSV-infected WT mice showed breakage of the BBB by day eight post infection, but not the infected NOS-3 KO mice.
  • Figure 18 is a diagrammatical depiction of areas from which data points were collected.
  • Figures 19A-19D show micrographs of the blood vessels of WT mice: WT + Med (Fig. 19A) ; WT + IL-12 (Fig. 19B) ; WT + VSV + Med (Fig. 19C) ; and WT + VSV + IL-12 (Fig. 19D) .
  • the mice were sacrificed at various time points, and the gap junctions were measured. Measurement areas are noted by an arrow.
  • Figures 20A-20D show micrographs of the blood vessels of NOS-3 KO (N3-K0) mice: N3-K0 + Med (Fig. 20A) ; N3-K0 + IL-12 (Fig. 20B) ; N3-K0 + VSV + Med (Fig. 20C) ; and N-3K0 + VSV + IL-12 (Fig. 20D) .
  • the mice were sacrificed at various time points, and the gap junctions were measured. Measurement areas are noted by an arrow.
  • Figures 21A and 21B are graphical depictions of the average distance of the intercellular junction's gap between the endothelial cell lining of the blood vessel.
  • Fig. 21A shows WT mice and Fig. 21B shows N3-KO mice. All of the groups, except WT + VSV, showed no statistical difference in comparison to each other. All of these groups show statistical difference from the WT + VSV group.
  • Figures 22A-22D are micrographs of the ependymal cells lining the fourth ventricle in WT mice: WT + Med (Fig. 22A) ; WT + IL-12 (Fig. 22B) ; WT + VSV + Med (Fig. 22C) ; and WT + VSV + IL-12 (Fig. 22D) .
  • the mice were sacrificed at various time points, and the gap junctions were measured.
  • Figures 23A-23D are micrographs of the ependymal cells lining the fourth ventricle in NOS-3 KO mice: N3-KO + Med (Fig. 23A) ; N3-KO + IL-12 (Fig. 23B) ; N3-KO + VSV + Med (Fig. 23C) ; and N3-KO + VSV + IL-12 (Fig. 23D) .
  • the mice were sacrificed at various time points, and the gap junctions were measured.
  • Figures 24A and 24B are graphical depictions of the average distance of the intercellular junction's gap between the ependymal cells which line the fourth ventricle of the CNS. All of the groups except WT + VSV showed no statistical difference in comparison to each other. All of these groups showed statistical difference from the WT + VSV group.
  • the present inventors have conducted experiments that provided a novel insight into some of the important features of the BBB which have otherwise been overlooked by investigators in the field.
  • the present invention is based on the discovery of the present inventors, using the viral encephalitis model, that a specific enzyme system, the constitutive endothelial cell isoform NOS-3, is central to the integrity of the BBB.
  • the laboratory of the present inventors has found ultrastructural changes in the BBB associated with infection. For example, capillary diameter increases substantially and the tight junctions of brain microvascular capillary endothelial cells increase in distance. There were also changes in the ependymal cells lining the fourth ventricle, which showed increased distances in the tight junctions.
  • the present inventors then conducted survival, CNS viral titers, Evans blue dye exclusion and immunohistochemical studies in three knock-out mouse strains (IFN- ⁇ deficient, NOS-1 deficient and NOS-3 deficient) and their appropriate control strains of mice. Based on the substantially increased viral titers and the immunohistochemical analyses, NOS-3 deficient mice were expected to succumb to acute infection.
  • the BBB involves the administration of a NOS-3 regulating agent.
  • the NOS-3 regulating agent is a NOS- 3 activator or NO donor.
  • the NOS-3 regulating agent is a NOS-3 inhibitor/antagonist.
  • Such a pathological condition is any abnormal condition which causes BBB permeability to increase as a result of said condition, examples of which include, but are not limited to, stroke (ischemia) , peripheral gram negative bacterial infections (via cytokine storm), bacterial toxins (e.g., LPS, pertussis toxin) and CNS infections (i.e., viral infections, which include lymphocytic choriomeningitis, VSV, bacterial infections; fungal infections; parasitic infections, such as malaria) .
  • stroke ischemia
  • peripheral gram negative bacterial infections via cytokine storm
  • bacterial toxins e.g., LPS, pertussis toxin
  • CNS infections i.e., viral infections, which include lymphocytic choriomeningitis, VSV, bacterial infections; fungal infections; parasitic infections, such as malaria
  • Non- limiting examples of NOS-3 inhibitors/ antagonists include analogs of L-arginine, such as N 3 - Monomethyl-L-Arginine (L-NMMA) , L-N-Methyl Arginine (L-NMA) ,
  • the NOS-3 inhibitor/antagonist be selective for the NOS-3 isoform.
  • the NOS-3 inhibitor/antagonist preferably has a negligible or low K ⁇ with the NOS-1 and NOS-2 isoforms relative to its K with the NOS-3 isoform. Using such a selective inhibitor of NOS-3 would avoid or limit any unintended inhibition of NOS-1 and NOS-2 activities.
  • the NOS-3 regulating agent is administered either locally, such as injection into the cervical artery, close to the BBB so that there is little or no exposure outside of the local area of administration to the NOS-3 regulating agent, or systemically in a manner which targets the NOS-3 regulating agent specifically to cells forming the BBB, such as the microvascular endothelial cells of the brain.
  • a further embodiment of the method of the present invention is to contemporaneously deliver a neurologically active therapeutic compound or a diagnostic compound into the CNS through the permeable BBB.
  • the increased permeability of the BBB is temporary, and more preferably, the increased permeability of the BBB is of a short duration, just sufficient for delivering the therapeutic or diagnostic compound across the BBB.
  • the NOS-3 activator or NO donor is preferably co-administered together with the therapeutic or diagnostic agent sought to be delivered to the CNS.
  • pathological conditions in which it would be desirable to deliver therapeutic or diagnostic compounds to the CNS include infections (i.e., highly-active anti-retroviral therapy for HIV or antibiotics for bacterial infection) , primary CNS tumors or secondary metastases (i.e., chemotherapeutic drugs to treat primary gliomas, astrocytomas and meningiomas, and secondary lymphomas, and breast, liver, pancreatic and colon metastatic cells), Alzheimer's Disease, etc.
  • infections i.e., highly-active anti-retroviral therapy for HIV or antibiotics for bacterial infection
  • primary CNS tumors or secondary metastases i.e., chemotherapeutic drugs to treat primary gliomas, astrocytomas and meningiomas, and secondary lymphomas, and breast, liver, pancreatic and colon metastatic cells
  • Alzheimer's Disease etc.
  • the method of the present invention provides a means of delivering therapeutic as well as diagnostic compounds, which can be imaged, across the BBB.
  • Non-limiting examples of NOS-3 activators and NO donors include nitroglycerin, histamine, L-glutamic acid, calcimycin, sodium nitroprusside (SNP) , S-nitroso-L- acetylpenicillamine (SNAP), 3-morpholino-sydononimine (SIN- 1) , cytokines which trigger Ca ++ flux and also induce neosynthesis of NOS-3 (i.e., IFN- ⁇ , TNF- ⁇ , IL-12), etc.
  • NOS inhibitors/antagonists, activators and NO donors there is a wealth of NOS inhibitors/antagonists, activators and NO donors known to those of skill in the art, many of which are available commercially from suppliers, such as Calbiochem/Oncogene Research Products (San Diego, CA and Cambridge, MA), Sigma-Aldrich Co. (St. Louis, MO), Cayman Chemical (Ann Arbor, MI), Oxford Biomedical Research, Inc. (Oxford, MI), Alexis Corp. (San Diego, CA) , etc.
  • the NOS-3 regulating agent may be administered systemically to regulate the permeability of the BBB
  • the NOS-3 regulating agent is associated with a targeting molecule which is specific for the cells forming the BBB.
  • a targeting molecule which is specific for the cells forming the BBB.
  • Such an association may be by conjugation or by the formation of a complex, etc., where the association is stable to transport from the site of administration to the targeted cells of the BBB.
  • targeting molecule which is specific for the cells forming the BBB
  • the “targeting molecule” be any molecule which specifically recognizes or is recognized by a cell surface marker on cells forming the BBB.
  • this recognition involves binding.
  • the “targeting molecule” can be, for example, a ligand for a cell surface receptor or a molecule having the antigen-binding portion of an antibody which recognizes and binds to an epitope of a cell surface marker.
  • antibody or “antibodies”
  • this is intended to include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs) , as well as proteolytic fragments thereof such as the Fab or F(ab') 2 fragments.
  • mAbs monoclonal antibodies
  • proteolytic fragments thereof such as the Fab or F(ab') 2 fragments.
  • the DNA encoding the variable region of the antibody can be inserted into other antibodies to produce chimeric antibodies (see, for example, U.S. Patent
  • Single-chain antibodies can also be produced and used.
  • Single-chain antibodies can be single-chain composite polypeptides having antigen-binding capabilities and comprising a pair of amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain (linked V H -V L or single- chain F v ) .
  • Both V H and V L may copy natural monoclonal antibody sequences or one or both of the chains may comprise a CDR-FR construct of the type described in U.S. Patent 5,091,513.
  • the separate polypeptides analogous to the variable regions of the light and heavy chains are held together by a polypeptide linker.
  • a "molecule which includes the antigen-binding portion of an antibody” is intended to include not only intact immunoglobulin molecules of any isotype and generated by any animal cell line or microorganism, but also the antigen-binding reactive fraction thereof, including, but not limited to, the Fab fragment, the Fab' fragment, the F(ab') 2 fragment, the variable portion of the heavy and/or light chains thereof, and chimeric humanized or single-chain antibodies incorporating such reactive fraction, as well as any other type of molecule in which such antibody reactive fraction has been physically inserted or molecules developed to deliver therapeutic moieties by means of a portion of the molecule containing such a reactive fraction.
  • Such molecules may be provided by any known technique, including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques, such as phage display libraries.
  • an antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitope is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody.
  • Epitopes or "antigenic determinants” usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains and have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
  • Monoclonal antibodies are a substantially homogeneous population of antibodies to specific antigens.
  • MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler et al (1975); U.S. Patent No. 4,376,110; Ausubel et al (1987-94); Harlow et al (1988); and Coligan et al (1993) .
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAbs may be cultivated in vi tro or in vivo .
  • High titers of mAbs can be obtained in in vivo production where cells from the individual hybridomas are injected intraperitoneally into pristane-primed BALB/c mice to produce ascites fluid containing high concentrations of the desired mAbs.
  • MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art .
  • Chimeric antibodies are molecules, the different portions of which are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric (humanized) mAbs are used.
  • An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared.
  • the immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, for example, U.S. Patent No. 4,699,880.
  • the anti-Id antibody may also be used as an " immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the anti-anti-Id may bear structural similarity to the original mAb which induced the anti-Id.
  • antibody is also meant to include both intact molecules, as well as fragments thereof, such as, for example, Fab and F(ab') 2 , which are capable of binding antigen.
  • Fab and F(ab') 2 fragments are preferably used as targeting molecules because they lack the Fc fragment of intact antibody, clear more rapidly from the circulation and may have less non-specific tissue binding than an intact antibody (Wahl et al, 1983) .
  • an in vi tro technique uses the V gene repertoires harvested from populations of lymphocytes or assembled in vi tro for cloning and display of associated heavy and light chain variable domains on the surface of a filamentous bacteriophage (Winter et al, 1994) . From a phage library containing the V gene repertoire, phage which bind to an antigen from the surface of cells forming the BBB are selected.
  • Nucleic acid isolated from the selected phage which bind specifically to the surface of the cells forming the BBB are then introduced into host cells to express soluble antibody fragments.
  • the affinity of the soluble antibody fragments for the cell surface antigen can be improved by mutagenesis of the DNA coding for the soluble antibody fragments in the host cells.
  • a specific example of a method for panning antibodies against cell surface antigens using in vi tro antibody phage display is a method derived from Palmer et al (1997) , where a single pot of human Fv semi-synthetic filamentous phage display library is to be constructed in the pHENl vector according to the procedure of Nissim et al (1994) .
  • the library will be rescued with VC3M13 helper phage (Stratagene, La Jolla, CA) , and the phage will be purified using polyethylene glycol.
  • BMEC brain microcapillary endothelial cells
  • approximately 10 13 transducing units of phage in PBS with 5% milk powder (for non-specific blocking) will be added to target BMEC and incubated overnight at 4°C. Cells will then be washed with PBS, 1% albumin, to remove unbound phage. Bound phage will be eluted from BMEC by adding 300 ⁇ l of 76 mM citric acid in PGS (pH 2.5), and the fluid neutralized with 400 ⁇ l 1M Tris-HCl, pH 7.4. The phage will be subsequently expanded overnight in E. coli TGI cells.
  • Phage particles will be enriched for specific high- affinity antigen binding phage through a further five rounds of binding to BMEC, and screening for binding to a panel of cell types, such as dermal microcapillaries, foreskin microcapillaries, umbilical vein endothelial cells, aorta and standard human cell lines derived from cornea, keratinocytes, kidney, etc., to determine cell and tissue specificity. Only those phage which exclusively bind BMEC will be used for further experiments.
  • a panel of cell types such as dermal microcapillaries, foreskin microcapillaries, umbilical vein endothelial cells, aorta and standard human cell lines derived from cornea, keratinocytes, kidney, etc.
  • the plasmid carried by the selected phage which encode the Fv segment (s) will be isolated, characterized and cloned for expression in bacterial host cells to produce a soluble Fv segment (s) that can be purified and used for derivatization with cross-linkers for drug delivery.
  • the soluble antibody fragments produced according to the above procedure can be used as targeting molecules for delivering a NOS-3 regulating agent to the BBB upon systemic administration to a subject.
  • the association of a NOS-3 regulating agent with an antibody as a targeting molecule is preferably by conjugation.
  • physiological ligands of cell surface receptors specific for brain microvascular endothelial cells constituting the BBB can be identified without undue experiment according to well-known screening techniques, etc., once a cell surface receptor specific to brain microvascular endothelial cells has been characterized, i.e., using antibodies from a phage display library that binds specifically to cell surface antigens as discussed above.
  • the conjugation or cross-linking of a therapeutic agent to one of the above-mentioned protein or peptide molecules can be accomplished in a manner so that the ability of the protein or peptide to bind to its cell surface marker is not significantly altered, nor is the bioactivity of the therapeutic agent or NOS-3 regulating agent significantly affected by the cross-linking procedure.
  • Numerous considerations, such as reactivity, specificity, spacer arm length, membrane permeability, cleavability and solubility characteristics need to be evaluated when choosing an appropriate cross-linker.
  • a recent review of the "Chemistry of Protein Conjugation and Cross-Linking" can be found by Shan S. Wong, CRC Press, Ann Arbor (1991) .
  • a homobifunctional sulfhydryl reactive cross- linker would be appropriate. If carboxyls and amines are available, carbodiimide works well. Furthermore, if there are no readily reactive groups, a photoactivatible cross- linker can be used. If lysines are important for the functionality of the molecule, then a cross-linker that will couple through sulfhydryls, carboxyls or non-specifically can be used.
  • Conjugation or coupling reagents have at least two reactive groups and can be either homobifunctional with two identical reactive groups or heterobifunctional with two more different reactive groups. Trifunctional groups also exist and can contain three functional groups. Most homobifunctional cross-linkers react with primary amines commonly found on proteins. Other homobifunctional cross- linkers couple through primary sulfhydryls. Homobifunctional cross-linkers can be used in a one-step reaction procedure in which the compounds to be coupled are mixed and the cross- linker is added to the solution. The resulting cross-linking method may result in self-conjugation, intermolecular cross- linking, and/or polymerization. The following are examples of suggested cross- linking approaches and are not meant to be inclusive .
  • Imido esters are the most specific acylating reagents for reaction with the amine groups whereby in mild alkaline pH, imido esters react only with primary amines to form imidoamides .
  • the product carries a positive charge at physiological pH, as does the primary amine it replaces and, therefore, does not affect the overall charge of the protein.
  • Homobifunctional N-hydroxysuccinimidyl ester conjugation is also a useful cross-link approach to crosslink amine-containing proteins.
  • Homobifunctional sulfhydryl reactive cross-linkers include bismaleimidhexane (BMH) , 1,5- difluoro-2,4-dinitrobenzene (DFDNB) and 1, 4-di- (3 ' , 2 ' - pyridyldithio) propionamido butane (DPDPB) .
  • heterobifunctional cross-linkers are commercially available with the majority containing an amine- reactive functional group on one end and a sulfhydryl- reactive group on the other end.
  • Multiple heterobifunctional haloacetyl cross-linkers are available, as are pyridyl disulfide cross-liners.
  • heterobifunctional cross-linking reagents which react with carboxylic groups involve the carbodiimides as a classic example for coupling carboxyls to amines resulting in an amide bond.
  • Another embodiment according to the present invention is to further associate a neurologically active therapeutic compound or diagnostic compound with a targeting molecule and a NOS-3 activator or NO donor for targeted delivery into the CNS.
  • the association is preferably by conjugation or by formation of a complex.
  • a pharmaceutical composition containing the active ingredients can be advantageously administered systemically, as well as locally.
  • the presence of the targeting molecule in association with the NOS-3 activator or NO donor and the therapeutic or diagnostic compound targets its delivery to the BBB and thereby beneficially limits the systemic exposure of the subject to the NOS-3 regulating agent, as well as to the therapeutic or diagnostic compound.
  • this pharmaceutical composition can be administered by any means that achieves its intended purpose and is not limited to local administration in the vicinity of the BBB.
  • administration may be by various parenteral routes, such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal or buccal routes.
  • parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal or buccal routes.
  • administration may be by the oral route.
  • Parenteral administration can be by bolus injection or by gradual perfusion over time.
  • Preparations for parenteral, as well as local administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions, which may contain auxiliary agents or excipients which are known in the art and which may facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • Pharmaceutical compositions, such as tablets and capsules can also be prepared according to routine methods.
  • Suitable formulations for administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspension of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions that may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • VSV Vesicular stomatitis virus
  • Wagner 1987
  • VSV encodes a single variable glycoprotein, a conserved matrix protein, a nucleoprotein, a large protein and phosphoproteins in overlapping reading frame (Wagner, 1987) .
  • the natural host of VSV is the cow, and it is transmitted by an arthropod vector, commonly the sandfly. In cows, the infection is mild and causes the characteristic vesicle lesions in the oral cavity.
  • VSV intranasal instillation could lead to lethal infection of the CNS (Sabin et al, 1937) , which has led to the use of VSV as a model for studies of neurotropic viral infections (Andersson et al,
  • VSV When administered intranasally to mice, VSV infects olfactory receptor neurons (Plakhov et al, 1995), is transmitted to neurons within the olfactory bulb, and then to more caudal regions of the CNS (Forger et al, 1991; Huneycutt et al, 1994; Lundh et al, 1987) . Surviving mice completely clear the virus from the CNS by day 12 post infection (Forger et al, 1991) . The laboratory of the present inventors has previously shown that VSV infection activates both innate immunity and acquired immunity (Bi et al , 1995a) and that recovery from infection requires T cell immunocompetence (Huneycutt et al, 1993) . However, the mechanism (s) of host defense and recovery during VSV infection of the CNS remain unclear, and the experiments in this example were conducted to elucidate the role of NO and NOS isoforms in the CNS.
  • VSV Indian serotype San Juan strain
  • Chinese Hamster Ovary (CHO) cells twice purified using a sucrose gradient.
  • Viral titers were determined on monolayers of CHO cells as previously described by Huneycutt et al (1993) .
  • Herpes Simplex virus-1 was kindly provided by Dr. Priscilla A. Schaffer (Dana-Farber Cancer Institute) .
  • Influenza virus A/WSN/33 was provided by Dr. Peter Palese (Mount Sinai School of Medicine) .
  • Sindbis AR339 virus was the gift of Dr. Beth Levine (Columbia University) .
  • mice were lightly anesthetized for one minute in a closed container containing HalothaneTM (Halocarbon Lab, North Augusta, SC) , followed by intranasal infection with 2 x 10 5 plaque-forming unit of VSV in a total volume of 0.01 mL administered equally through each nostril, as previously described by Bi et al (1995a) .
  • HalothaneTM Halocarbon Lab, North Augusta, SC
  • 2 x 10 5 plaque-forming unit of VSV in a total volume of 0.01 mL administered equally through each nostril, as previously described by Bi et al (1995a) .
  • mice in each treatment group were reserved for evaluation of morbidity (weight changes) and mortality (Plakhov et al, 1995) .
  • At each time point at least 8 mice per treatment group were given a lethal dose of ketamine-xylazine .
  • Five brains from each group were homogenized and frozen for later determination of viral titers, as previously described (Bi et al, 1995a; Forger et al , 1991) .
  • the lower limit of detection of the assay is 100 PFU/mL homogenate .
  • mice in each group were perfused transcardially with 20 to 30 mL of normal saline, and the whole brains were removed and quick-frozen in isopentene kept on dry ice before being stored at -70°C until sectioning, as previously described (Bi et al, 1995a) . Protocol of In Vivo Treatment of IL-12
  • Murine rIL-12 was generously provided by Genetics Institute (Cambridge, MA) . On the day of infection, groups of mice were injected i.p. daily with medium alone or 200 ng of IL-12/mouse; this was continued daily from 0 to 7 days post infection. Immunohistochemical Reagents
  • TBS Tris buffered saline
  • Sections were then washed twice in 0.1M TBS, and background staining was blocked by preincubation in 1% bovine serum albumin (BSA) (Fisher Scientific, Pittsburgh, PA) in 0.1M TBS for 45 minutes. Sections were then incubated with primary Abs for specific Ags for one hour at room temperature, except for VSV staining which was conducted overnight. Sections were washed again in 0.1M TBS twice and then incubated in biotinylated secondary Ab (ABC Kit, Vector Laboratory) , followed by avidin- biotinylated peroxidase for another 30 minutes. Sections were then incubated with DAB in 0.01% H 2 0 2 for five minutes. Dried sections were coverslipped with Permount (Fisher Scientific) .
  • BSA bovine serum albumin
  • the Mouse IFN- ⁇ ELISA kit was purchased from BioSource International (Camarillo, CA) .
  • the Mouse TNF- ⁇ ELISA kit was purchased from Genzyme (Cambridge, MA) . Cells and Viruses
  • NB41A3 neuroblastoma cells and C6 astrocytoma cells were obtained from ATCC.
  • RAW murine macrophage cells were generously provided by Dr. Ashok Amin, Hospital for Joint Diseases, NYU.
  • CHO cells obtained from Dr. Alice S. Huang, were maintained as previously described (Huneycutt et al, 1994) .
  • Infectious virus was quantified on CHO cell monolayers.
  • Monolayers were prepared by inoculating 20 x 10 4 cells in 1 ml per well (234-2311 plate, Nunc) and incubated for two days at 37°C. The medium was removed, 0.1 ml of each dilution of samples (ten-fold serial dilutions) was added to each well, in duplicate/triplicate, and the wells were then incubated for 30 minutes at 37°C. The medium was removed, 1 ml of the mixture of equal volumes of 1.8% agar (kept at 45°C) and 2 x culture medium (kept at 37°C) were added to each well, and the wells were then incubated at 37°C for 24 hours. Plaques were fixed with 10% formaldehyde for 30 minutes and stained with 0.5% methylene blue. Chemicals and Cytokines
  • L-N-Methyl Arginine (Sigma) and 7- nitroindazole (7-NI) (Calbiochem) were used at 400 ⁇ M as was the control compound, indazole (Aldrich) .
  • L- and D-arginine were purchased from Sigma and were used at 5 ⁇ M.
  • Indomethacin was purchased from Sigma and was used at 10 ⁇ g/kg.
  • Bayer Aspirin (ASA) was used at 100 mg/kg.
  • Recombinant mouse IFN- ⁇ was purchased from Genzyme. Recombinant mouse IL-12 was provided by Genetics Institute. In some preliminary experiments, rat conA supernatant
  • the concentration of NO in culture supernatants was determined by assaying its stable end-product, N0 2 (Bredt et al, 1989) . Briefly, equal volumes of experimental sample and Griess reagent (1% sulfanilamidide, 0.1% N-l- naphthylethylene-diamine, 5% H 3 P0 4 ) (Sigma) were incubated at room temperature for ten minutes. The reaction produces a pink color, which was quantitated at 540 nm against standards in the same buffer, using an automated plate reader (Bio-Tek, Inc., model EL309) . The data is expressed as mM. Immunoprecipitation
  • NB41A3 cells (5 x 10 5 ) were cultured in culture medium with our without IFN- ⁇ for 72 hours. Cells were mock infected with media or infected with VSV (1 MOI) for 2.5 hours or 5 hours. Cells were then chilled on ice for ten minutes and lysed with 0.5 mL of lysis buffer (0.5% NP-40, 300 mM NaCl, 50 mM Tris, 100 ⁇ g/ml PMSF, 1 ⁇ g/ml leupeptin, pH 7.4) for 20 minutes. Cell lysate was centrifuged at
  • the cells were then either mock infected with medium or infected with VSV at 1 moi for 2.5 hours or 5 hours, upon which they were lysed with lysis buffer (0.5% NP-40, 300 mM NaCl, 50 mM Tris, 100 ⁇ g/ml PMSF, 1 ⁇ /ml Leupeptin, pH 7.4.
  • lysis buffer (0.5% NP-40, 300 mM NaCl, 50 mM Tris, 100 ⁇ g/ml PMSF, 1 ⁇ /ml Leupeptin, pH 7.4.
  • the proteins were run on a 7.5% SDS-PAGE gel and electrophoretically transferred onto a nitrocellulose membrane. After transfer, the blot was washed in PBS-0.05% Tween-20 for ten minutes. The blot was blocked using PBS containing 3% nonfat dry milk for 20 minutes.
  • Anti-VSV Ab (1:5000) or anti-nitrotyrosine Ab (Upstate Biotechnology, NY) (1:10000) was added, and the incubation was carried out at room temperature for two hours .
  • secondary antibody anti-sheep for VSV, anti-rabbit for nitrotyrosine
  • the blot was incubated with Enhanced Chemiluminescence substrate (ECL) (Amersham) based on the manufacturer's directions. Film exposure was on Kodak Bio-Max MR film for two minutes. Phosphorimaging analysis of the gel was applied with Bio-Rad Model GS-250 Molecular ImagerTM .
  • PCR-Dig Labelling of the Probes The plasmids encoding the five VSV proteins were generously provided by Dr. John Rose (Yale University) (Lawson et al, 1995) . These clones were used to generate the probes for the Northern Blots.
  • the PCR Dig Probe Synthesis Reaction (Boehringer Mannheim) was used to label the fragments. Briefly, this required two sets of PCR reactions. The first reaction generated a concentrated batch of double- stranded DNA encoding the region of interest . The second reaction generated single-stranded (either 5' or 3') Dig- labelled DNA fragments, which were used as probes.
  • the reactions conditions were:
  • VSV-N gene (Base pairs 77-1136) :
  • NB41A3 cells (5 x 10 5 ) were cultured in culture medium with or without IL-12 for 72 hours. The cells were then infected with VSV or mock infected with medium for one hour, upon which they were lysed using the Poly (A) pure mRNA purification kit (Ambion) . The mRNA were run on 2% formaldehyde/agarose gel and transferred onto a nylon membrane. After transfer, the membrane was cross-linked using a UV Crosslinker (Stratagene) . The membrane was then incubated in pre-hybridization buffer for two hours at 40°C and hybridized overnight with the probes at the same temperature .
  • the membrane was incubated in blocking solution for 30 minutes. The membrane was then incubated with anti- dig/alkaline phosphatase conjugated antibody solution for 30 minutes. After the washes, the membrane was incubated with CSPD, which reacts with the alkaline phosphatase.
  • mice Normal male BALB/c mice and NOS-3 KO mice, 5-7 weeks of age were used for this experiment. Some mice were intranasally infected with 2 x 10 5 PFU of VSV and injected with either 200 ng of IL-12 or medium alone daily until the time of sacrifice. Some uninfected mice were treated with IL-12 or medium. After a lethal dose of ketamine-xylazine, the mice were perfused with 5 mL of 0.9% saline/1% heparin solution, then with 200 mL of 2% paraformaldehyde/2% gluteraldehyde solution. After the brains were extracted, they were post-fixed over night in 2% paraformaldehyde/2% gluteraldehyde solution. The brains were sectioned coronally (50 ⁇ M) using a vibrotome, and the sections were placed in 0.1M PBS. The sections were then fixed in 1% Os0 4 for one hour. The tissues were dehydrated sequentially with:
  • epon acetone mixture
  • the epon concentration was as follows: EM-Bed 812 (24 mL) , DDSA (15 ml), NMA (13.5 mL) , and DMP (0.525 mL) .
  • the tissues were placed in full epon for two more hours with gentle agitations.
  • the tissue was flat embedded onto clean aclar sheets and baked overnight in an oven at 65 °C until the epon was fully polymerized.
  • an area of interest was cut out and placed onto an epon block for sectioning on the ultramicrotome to a thickness of approximately 900 angstroms. These sections were then transferred to copper grids and stained with lead citrate for one minute before transferring them to the TEM.
  • mice were infected with VSV and were injected with either indazole or with 7-NI.
  • half of the mice were injected with IL-12, which was shown by the laboratory of the present inventors to have profound recovery-promoting effect (s) in this experimental system.
  • the mice were sacrificed, and brain homogenates were tested for the presence of virus.
  • Figure 3 shows the results of the plaque assay on homogenates .
  • the geometric mean titer (GMT) of virus in individuals within each group was compared. 7-NI treatment of mice resulted in a ten-fold greater GMT compared to indazole-treated mice.
  • IFN- ⁇ Induced Upregulation of Type I NOS Activity Inhibits VSV Replication Whether viral replication in NB41A3 cells could be inhibited by IFN- ⁇ -induced type I NOS was investigated. Treatment of NB41A3 cells for 72 hours prior to infection significantly inhibited VSV and HSV-1 replication (Table 3) . Replication of influenza virus A/WSN/33 and Sindbis virus in NB41A3 cells was also significantly inhibited. In other experiments, the IFN- ⁇ -mediated reduction in viral propagation was prevented by addition of anti-IFN- ⁇ R mAb GR- 20 (results not shown) .
  • VSV and HSV-1 are sensitive to nitric oxide-mediated inactivation, but influenza and Sindbis viruses are resistant to NOS-inhibition
  • 7-Nitroindazole (7-NI) is a selective inhibitor of type I, but not types II or III NOS (Moore et al, 1993) . Therefore, cells expressing the three isoforms of NOS were incubated with IFN- ⁇ , NMDA or medium and two inhibitors, L-NMA and 7-NI, and the cells were infected with VSV, and the progeny virus was determined eight hours later by plaque assay. L-NMA antagonized NOS-associated inhibition of viral replication in all three cell lines, whether NOS activated by triggering of the cells through their glutamate receptors, or by IFN- ⁇ treatment (Table 4) . 7-NI treatment was controlled with indazole incubation. Only neuronal NOS was antagonized with 7-NI, the resultant virus produced in RAW and C6 cells was indistinguishable from medium- or indazole-treated activated cells.
  • Underlined data points are significantly different from uninhibited viral replication, P ⁇ . 05 or better .
  • IL- 12 treatment resulted in twice the survival rate from VSV infection in WT mice than observed in control mice (Fig . 4 ) .
  • both groups of VSV- infected mice lost weight .
  • IL- 12 -treated mice rapidly gained weight and exceeded their initial measures by 12 days after infection, while the control infected mice showed more signs of morbidity (decreased appetite and activity) and remained below the initial level throughout the two-week observation period (Fig . 5) .
  • IL-12 treatment was not able to rescue the NOS- 1 KO mice , suggesting that NOS- 1 is important for host defense (Figs . 4 and 5 ) .
  • mice treated with IL- 12 were lower in WT, but not in NOS- 1 KO mice , than the control groups (Fig . 6 ) .
  • Inj ection of 200 ng of IL- 12 /mouse per day decreased the VSV titer about 100 - fold for the WT mice .
  • Immunohistochemical staining of VSV Ags on frozen sections from brains of other mice confirmed this observation .
  • MHC class I Ags Neither uninfected B6 WT nor NOS- 1 KO brain sections expressed MHC Ags above the background level of immunohistochemical staining .
  • OB olfactory bulb
  • MHC class II was barely detected in the OB in the control groups, consistent with our earlier observations (Christian et al, 1996) .
  • IL-12 treatment expression of MHC Ags was significantly increased in both the wild-type and the knockout groups.
  • MHC class I Ags was found in the OB, the hippocampal formation, and along the fourth ventricle.
  • MHC class II Ags were increased in many areas, particularly in the OB and the hippocampal formation (Tables 5A and 5B) . In the absence of NOS-1, MHC was induced well above baseline levels, though it was lower than that of IL-12 treated WT mice. IL-12 Treatment Induces Activation of Astrocytes and Microglia
  • GFAP glial fibrillary acidic protein
  • Mac-1 Ag expressed by microglial cells.
  • IL-12 Treatment resulted in more numerous and heavier-staining cells, suggesting astrocytosis and microgliosis (Tables 5A and 5B) .
  • the most pronounced astrocytosis and microgliosis was observed to coincide with VSV Ag + areas.
  • NOS-1 expression was poorly detected in neurons in the uninfected and control groups, as previously observed by Komatsu et al (1996) . Following IL-12 treatment, the expression was substantially increased in WT, but was undetectable in NOS-1 KO mice (Tables 5A and 5B, Figs. 7A- 7D) .
  • NOS-2 expression by microglia and macrophates was found at low levels in the uninfected and control groups, consistent with previous data (Bi et al, 1994; Bi et al,
  • NOS-3 was previously observed in astrocytes and endothelial cells (Barna et al, 1996) .
  • IL-12 treatment induced the expression of NOS-3 in both WT and NOS- 1 KO mice, although it was lower in NOS-1 KO mice (Tables 5A and 5B) .
  • Infiltration of VSV Infected Brains by T Cells and NK Cells T cells were detected very infrequently in media- treated infected B6 and uninfected B6 mouse brains.
  • IL-12 treatment resulted in the accumulations of CD4 and CD8 expressing T cells in the VSV- infected areas, such as the OB and HC (Tables 5A and 5B) .
  • NKl.l expressing cells were detected at relatively higher frequency in the infected media-treated than in uninfected brains (Tables 5A and 5B) .
  • a profound increase in the number of NK cells was found in the OB and other areas following L-12 treatment in both WT and NOS-1 KO sections.
  • the laboratory of the present inventors investigated whether viral replication in NB41A3 cells for 72 hours prior to infection significantly inhibited VSV protein replication (Figs. 8 and 9) .
  • Analysis of the data revealed an approximately 80% difference in the relative amounts of viral protein between the treated and the untreated samples . These results were consistent at both 2.5 hours and 5 hours post infection and were uniform for each of the five viral proteins.
  • the VSV control showed protein levels similar to those of the untreated samples .
  • Levels of Nitrosylation To determine whether the viral proteins from the above samples are nitrosylated, the viral proteins were immunoprecipitated, and a Western Blot was run for a- Nitrotyrosine residues.
  • IL-12 treatment resulted in twice the survival rate from VSV infection than observed in control mice (Fig. 14) .
  • both groups of VSV-infected mice lost weight.
  • IL- 12-treated mice rapidly gained weight and exceeded their initial measurements by 12 days after infection, while the control infected mice showed more signs of morbidity (decreased appetite and activity) and remained below the initial level throughout the two-week observation period (Fig. 15) .
  • IL-12 treatment was associated with an acute and transient cytokine-induced physiological response, possibly due to increased cytokine levels, such as TNF-o. (see below), which resulted in weight loss.
  • mice treated with IL-12 were lower for both WT and NOS-3 KO mice than the control groups (Fig. 16) .
  • Injection of 200 ng of IL-12/mouse per day decreased the VSV titer about 100 -fold for the WT mice and
  • MHC class I coincided with VSV Ag + areas . Induced expression of MHC class II was barely detected in the OB in the control groups, consistent with the earlier observations of the laboratory of the present inventors (Christian et al, 1996) . Following IL-12 treatment, expression of MHC Ags was significantly increased in both the wild-type and the knockout groups. MHC class I Ags was found in the OB, the hippocampal formation and along the fourth ventricle. Expression of MHC class II Ags was increased in many areas, particularly in the OB and the hippocampal formation (Tables 6A and 6B) . In the absence of NOS-3, MHC was induced well above baseline levels, although it was lower than that of IL-
  • Microglia The brain sections were stained for either glial fibrillary acidic protein (GFAP) , a marker of astrocytes, or Mac-1 Ag expressed by microglial cells. IL-12 treatment resulted in more numerous and heavier-staining cells, suggesting astrocytosis and microgliosis (Tables 6A and 6B) . The most pronounced astrocytosis and microgliosis was observed to coincide with VSV Ag + areas.
  • GFAP glial fibrillary acidic protein
  • nitric oxide synthase (NOS) isoforms are induced and increased in immunohistochemical staining (Barna et al, 1996; Bi et al, 1995a; Bi et al 1995b; Christian et al, 1996; Komatsu et al, 1996) .
  • IFN- ⁇ can activate NOS gene expression for all three isoforms (Barna et al , 1996; Kamijo et al, 1994; Komatsu et al, 1996) . Therefore, the effects of IL-12 on NOS isoform expression during VSV infection were examined.
  • NOS-1 expression was poorly detected in neurons in the uninfected and control groups, as previously observed ( Komatsu et al , 1996) . Following IL-12 treatment, the expression was substantially increased in both the WT and NOS-3 KO mice (Tables 6A and 6B) , although it was lower in NOS-3 KO mice.
  • NOS-2 expression by microglia and macrophages was found at low levels in the uninfected and control groups, consistent with the previous data from the laboratory of the present inventors (Bi et al, 1995a; Bi et al, 1995b) .
  • IL-12 treatment resulted in the higher NOS-2 expression in both WT and NOS-3 KO mice (Tables 6A and 6B) ; albeit lower in NOS-3 KO mice.
  • NOS-3 was previously observed in astrocytes and endothelial cells (Barna et al, 1996) .
  • IL-12 treatment induced the expression of NOS-3 in WT mice but was undetectable in NOS-3 KO mice (Tables 6A and 6B) .
  • Infiltration of VSV-Infected Brains by T Cells and NK Cells T cells were detected very infrequently in media- treated infected B6 and uninfected B6 mouse brains.
  • IL-12 treatment resulted in the accumulations of CD4 and CD8 expressing T cells in the VSV-infected areas, such as the OB and HC (Tables 6A and 6B) .
  • NKl.l expressing cells were detected at relatively higher frequency in the infected media-treated than in uninfected brains (Tables 6A and 6B) .
  • a profound increase in the number of NK cells was found in the OB and other areas following IL-12 treatment in both WT and NOS-3 KO sections. Breakdown of the BBB During VSV Infection Some dyes, such as Evans blue, are normally excluded from the brain by intact BBB but can enter the brain when the integrity of the BBB is broken. This method is often used to assess simple alteration of the BBB (Bi et al, 1995; Doherty et al , 1974; Kandel et al , 1991) .
  • mice Eight groups of mice were tested:
  • mice were sacrificed at various time points (days 6 and 8 post infection for WT; days 8 and 10 post infection for NOS-3 KO) .
  • IFN- ⁇ was demonstrated in these studies to inhibit VSV replication through induction of the synthesis and activity of type I NOS in neurons in vi tro (Tables 3 and 4) and in vivo (Fig. 3) .
  • This antiviral effect in culture was shown to be limited to VSV, but can be extended to HSV-1 (Table 3) .
  • IFN regulatory factor (IRF) -1 is required in iNOS induction in mouse macrophage (Kamijo et al, 1994) . IFN- ⁇ signal transduction in neurons, however, may or may not be similar to that in other types of cells.
  • IRF IFN regulatory factor
  • Two closely linked, but separable, promoters of human type I NOS have been identified (Xie et al, 1995) . The analysis of the sequence of the promoter region of human type I NOS suggested a STAT core element and possible sites for PIE and GAS. IRS, IRF-1, IFN- ⁇ responsive sequence and interferon stimulation responsive elements were not found.
  • the human type II NOS gene behaves differently than the mouse gene and is not readily inducible by IFN- ⁇ , TNF-c, or LPS (Reiling et al , 1994) . There may be other cytokine response elements in the 5 ' region of the gene .
  • IFN- ⁇ can inhibit several viral infections in macrophages through iNOS induction (Karupiah et al, 1993) .
  • the results presented here are the first to report that IFN- ⁇ can inhibit VSV in neurons through inducing type I NOS (Table 4) .
  • NO inhibits replication of HSV-1 in neurons (Table 3) .
  • NO-generating neurons are selectively resistant to neurotoxicity of NO (Dawson et al, 1994)
  • one more advantage can be attributed to IFN- ⁇ -mediated activation of OS in neurons in inhibiting viral infections in the CNS, rather than simply just induction of iNOS in neighboring neuroglial cells .
  • IL-12 treatment was associated consistently and significantly decreased VSV titers in CNS (Figs. 6 and 16), and VSV protein is detected in brain tissues (Tables 5A, 5B, 6A and 6B) of NOS-3 KO mice but not in NOS-1 KO mice. This was also observed in the survival and morbidity experiments as well (Figs. 4, 5, 14 and 15).
  • IL-12 had a positive effect on the immune response in both types of KO mice. This intervention was associated with induced expression of MHC class I and class II Ags, as well as the
  • NOS isoforms not knocked out, albeit not to the levels in WT mice (Tables 5A, 5B, 6A and 6B) .
  • Astrocytosis and microgliosis was detected in the VSV Ag + areas (Tables 5A, 5B, 6A and 6B) .
  • IL-12 treatment resulted in the rapid infiltration of T cells and NK cells into the VSV- infected brains, although the levels in KO mice was not at the level of WT mice.
  • the replication of VSV in NB41A3 cells was inhibited by the NO production of the cells (Figs. 8 and 9) . This anti-VSV effect may partially be due to the nitrosylation of the viral proteins (Fig. 10) .
  • VSV is a negative sense RNA virus which first transcribes its genome into mRNAs after uncoating and has to bear a complete set of viral enzymes in the virion to initiate a new round of the life cycle in infected cells.
  • NO may achieve its biological functions inside the cell by covalent and/or oxidative modifications of target molecules (Stamler et al, 1992; Stamler, 1994) .
  • NO has an inhibitory effect on a variety of virus infections (Reiss et al, 1998) . It is frequently difficult to distinguish whether the inhibitory effect of NO is the consequence of the inhibition of cellular metabolism or of virus replication or both.
  • NO may be influencing several steps in the VSV life cycle to inhibit viral replication. It may be blocking viral RNA synthesis and decreasing viral protein accumulation. It may be nitrosylating the viral proteins, making them inactive. This anti-VSV effect of NO is unlikely due to the direct cytotoxic effect of NO on infected cells (Lin et al, 1997) . NO has been demonstrated to directly (Lancaster et al, 1990; Nathan, 1992; Pellat et al, 1990; Stamler et al, 1992) or indirectly (Drapier et al , 1986; Granger et al, 1980; Granger et al, 1982; Hibbs et al, 1990; Johnson et al, 1985) inhibit numerous cellular enzymes.
  • NO may inactivate the viral enzymes required for viral RNA synthesis and may be blocking viral protein synthesis because the virus cannot sufficiently amplify viral mRNA. NO had a single unpaired electron, making it a free radical. Most eukaryotic cells respond to stress, such as free radicals, by increasing the rate of intracellular proteolysis (Ciechanover et al , 1994) . Thus, the IL-12- treated cells may be undergoing proteolysis, which increases the degradation of viral proteins accumulated in the cells . This may inhibit viral RNA synthesis by decreasing the amount of RNA-dependent RNA polymerase.
  • the neurons normally do not express MHC class I and II antigens.
  • NO an antiviral component
  • the host may rely on NO to clear virus from the CNS during the early stages of infection without the cytolytic effects of NK and T cells (Bi et al , 1995a) .
  • NK cells can indirectly restrict viral replication without lysis of the virus-infected cells by stimulating NO production in macrophages (Karupiah et al, 1995) .
  • this type of inhibitory mechanism may furnish what is lacking in acquired immunity for virus clearance from the CNS (Lin et al, 1997) .
  • NO has been implicated in the impairment of the integrity of the BBB in many types of clinical conditions (Boje, 1996; Buster et al, 1995; Chi et al, 1994; Hurst et al, 1996; Johnson et al, 1995; Thompson et al, 1992) .
  • MS a disease where one of the early crucial events is the perturbation of the BBB
  • elevated mRNA for NOS has been detected in postmortem brain sections (Bo et al, 1994) .
  • NADPH diaphorase activity has been observed in astrocytes from demyelinating lesions and the levels of nitrate and nitrite (stable end products of NO) are raised in the CSF.
  • Cytokines such as TNF-o; and various interleukins, have also been implicated in the BBB breakdown during bacterial sepsis (Goldblum et al, 1990; Tracey et al, 1990) . Cytokines induce a disruption of the BBB at the level of the cerebral endothelial cells, in vi tro (DeVries et al, 1995) . These effects can be abolished in the presence of Indomethacin, a cyclooxygenase inhibitor (DeVries et al, 1996) . In the present study, these inhibitors are shown to abolish the effects of the breakdown of the BBB in vivo, as well.
  • cytokines are activating the cerebral endothelial cells to produce eicosanoids, which subsequently induce the breakdown of the BBB.
  • IL-1 and IL-6 have been shown to induce rat cerebral endothelial cells to produce large quantities of eicosanoids, mainly prostaglandin E 2 and thromboxane A 2 (Clark et al, 1988; DeVries et al, 1995) , which may give rise to vasodilatory substances.
  • TxA 2 receptor on endothelial cells has been associated with vasodilatory effects (Amin et al, 1997; Kent et al, 1993) .
  • Sodium salicylate can inhibit TNF- induced p42/p44 mitogen-activated protein kinase (Schewenger et al, 1996) . Furthermore, the TNF-induced injury to aortic endothelial cells could be reduced in the presence of eicosanoid synthesis inhibitor BW 755c (Clark et al, 1988) . Thus, cytokines released during inflammatory diseases of the CNS can exert a direct effect on the integrity of the BBB. The formation of eicosanoids by the cerebral endothelial cells are likely to play a key role in this process, which suggests a potential therapeutic effect of cyclooxygenase inhibitors on the BBB integrity during CNS inflammatory diseases.
  • cytokines have been shown to enhance NOS activity (Durieu-Trautmann et al, 1993; Gross et al ; 1991; Komatsu et al, 1996). Thus it is possible that cytokines may mediate BBB breakage through the generation of NO in the cells that constitute the BBB.

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Abstract

La présente invention se rapporte à un procédé de régulation de la perméabilité de la barrière hémato-encéphalique, qui consiste à administrer un inhibiteur NOS-3 pour réduire la perméabilité de la barrière hémato-encéphalique accrue en raison d'un trouble pathologique ou à administrer un activateur de NOS-3 ou un donneur de monoxyde d'azote de manière à accroître cette perméabilité de la barrière hémato-encéphalique. Cet accroissement de la perméabilité de la barrière hémato-encéphalique permet d'administrer un composé thérapeutique ou diagnostique destiné au système nerveux central.
EP99953234A 1998-10-19 1999-10-19 Procede de regulation de la permeabilite de la barriere hemato-encephalique Withdrawn EP1135157A4 (fr)

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US7211561B2 (en) 2001-10-12 2007-05-01 Cedars-Sinai Medical Center Method for inducing selective cell death of malignant cells by activation of calcium-activated potassium channels (KCa)
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