Patent Application
20110274749
1. Field of the Invention
The invention relates to novel nucleic acids and polypeptides encoded thereby, whose expression is modulated in brain microvascular endothelial cells undergoing early dynamic inflammation-induced changes in blood-brain bather functionality. These polypeptides are designated herein as “lipopolysaccharide-sensitive” polypeptides (LPSS polypeptides). The invention further relates to methods useful for controlling blood-brain barrier properties in mammals in need of such biological effects. This includes the diagnosis and treatment of disturbances in the blood-brain/retina barrier, brain (including the eye) disorders, as well as peripheral vascular disorders. Additionally, the present invention further relates to the use of anti-LPSS polypeptide antibodies or ligands as diagnostic probes, as blood-brain barrier targeting agents or as therapeutic agents as well as the use of ligands or modulators of expression, activation or bioactivity of LPSS polypeptides as diagnostic probes, therapeutic agents or drug delivery enhancers.
2. Description of the Background Art
In order to function properly, neurons require a tightly regulated extracellular milieu. This essential, well-defined microenvironment is locally maintained by nursing brain cells called astrocytes (or astroglia). To cope with the considerable and variable dissimilarity between the composition of the blood and the extracellular compartment of the brain, the central nervous system (CNS) is also shielded from the general blood circulation by a number of blood-CNS barriers, i.e. the blood-brain barrier, blood-cerebral spinal fluid (CSF) barrier, pial vessel-CSF barrier, the ependyma and glia limitans, and also the blood-retina barrier, blood-nerve barrier, blood-spinal cord barrier. The blood-brain barrier (BBB) is considered as the most important blood-CNS barrier, because it covers a 1000 times larger surface area when compared to the other blood-CNS barriers. The BBB is characterized by a unique tight endothelial cell layer that covers capillary blood vessels in the CNS. Again, astrocytes are the principal inducers of BBB properties in these endothelial cells, by projecting ‘glial foot’ on the capillaries.
In particular, the BBB regulates the trafficking of ions (Na+, K+, Ca2+), water, nutrients, metabolites, neurotransmitters (glutamic acid, tryptophan), plasma proteins (albumin, fibrinogen, immunoglobulins), cells from the immune system and also xenobiotics (drugs) in and out of the brain. The capillary endothelium in the brain has special properties when compared to peripheral capillaries. It has narrow tight-junctions, no fenestrae, low pinocytotic activity and a continuous basement membrane. The narrow tight-junctions result in a high electrical resistance of 1500-2000 Ohm·cm2. In addition, the endothelial cells have a negative surface charge that repulses negatively charged compounds. They have many mitochondria and enzymes to break down compounds and various selective transport systems to actively transport nutrients and other compounds into and out of the brain. Under healthy conditions, the BBB not only regulates the entry of drugs or endogenous compounds into the brain, but also cellular infiltration is lower compared to peripheral organs. The normal endothelial cell layer provides a thrombo-resistant surface that prevents platelet and leukocyte adhesion and activation of any coagulation system. The highly specialized brain microvascular endothelial cells form a tight barrier which isolates the brain from immune surveillance, and allow only a few mononuclear cells (such as activated T-cells) to migrate into the CNS. The low expression of major histocompatibility complex antigens, the low number of antigen-presenting cells in the healthy CNS, and the fact that the CNS is not properly drained by a fully developed lymphatic vasculature, make the brain an “immuno-secluded” site.
The present understanding of the anatomical basis of the BBB is that it functions as a dynamically regulated organ, influenced by peripheral (e.g., cortisol, adrenaline) and local (e.g., cytokines, chemokines) hormones. In addition to astrocytes, several other cells like pericytes, neurons and cells of the immune system, influence its properties. Next to that, the endothelium is involved in other processes like coagulation, control of vasotonus, antigen-presentation and the control of the basement membrane by e.g., growth factors. Particularly, under pathological conditions like brain and cerebral inflammation, angiogenesis in brain tumors, the activated endothelium plays an important role.
In general, the BBB can be regarded as an organ that serves to protect the homeostasis of the brain. Not surprisingly, dysfunction of the BBB plays a central role in the vast majority of brain disorders. Some examples are:
Healing a dysfunctional BBB thus opens new avenues for the treatment of brain disorders. Brain disorders are the principal cause of morbidity and disabilities in the western world. The identification and characterization of novel LPSS polypeptides, whose gene expression is modulated in brain microvascular endothelial cells undergoing early dynamic inflammation-induced changes in blood-brain bather functionality, will prove useful to meet these needs.
In addition to the desirable drugs with a BBB-healing capacity for the treatment of brain disorders, a proper functioning BBB is also essential to block or reduce the entry into the brain of lymphocytes, which mediate an immune response. The same holds for the entry into the brain of metastatic cancer cells. The identification and characterization of novel LPSS polypeptides, whose gene expression is modulated in brain microvascular endothelial cells undergoing early dynamic inflammation-induced changes in blood-brain barrier functionality, will prove useful to meet these needs.
The BBB, however, also limits the delivery of xenobiotics (such as drugs and diagnostic agents) to the brain, which complicates classical drug therapy (i.e. targeted against neurons) of brain disorders. It is therefore also desirable to either manipulate the permeability of the BBB in order to deliver blood-borne, membrane-impermeant drugs to the brain by reversibly opening the BBB, or to selectively target drugs to the brain via endogenous BBB transport systems. The same holds for drug delivery across the blood-testis barrier and the blood-placenta barrier. The identification and characterization of novel LPSS polypeptides, whose gene expression is modulated in brain microvascular endothelial cells undergoing early dynamic inflammation-induced changes in blood-brain barrier functionality, will also prove useful to meet these needs.
It is also desirable to manipulate BBB properties in microvessels of other organs than the brain or eye affected in vascular disorders. Introducing BBB properties in peripheral microvessels will be beneficial in conditions involving (micro)angiopathies, pathological angiogenesis, failure of blood-testis barrier or blood-placenta barrier, and conditions such as pulmonary edema, shock caused by bacterial endotoxins, hyperfibrinolysis and anaphylactic shock. The identification and characterization of novel LPSS polypeptides, whose gene expression is modulated in brain microvascular endothelial cells undergoing early dynamic inflammation-induced changes in blood-brain barrier functionality, will also prove useful to meet these needs.
It has been known for a long time that brain astrocytes induce BBB properties in brain capillary endothelial cells (BCEC) by the projection perivascular end feet (Arthur et al., 1987, Brain Res. 433: 155-159; Janzer and Raff, 1987, Nature 325: 253-257). It is also known for a long time that this induction is brought about by soluble factor(s), since astrocyte conditioned medium (ACM) can reproduce some of the inductive effects (Tio et al., 1990, Eur. J. Morphol. 28(2-4): 289-300). Several candidate molecules have been identified, capable of mimicking aspects of ACM-mediated barrier induction in BCEC; these include TGFβ, GDNF, bFGF, IL-6 and steroids. Others have found that the factor is not a protein or peptide and that it contains an iron-nitric oxide adduct (Federici et al., 1995, J. Neurochem. 64(3): 1008-1015; Regina et al., 2001, Biochim Biophys. Acta 1540(3): 233-242). So one can conclude that despite the effort of several research groups, the responsible astrocyte-derived factor has not been identified yet.
In previous experiments, we found that primary isolated BCEC, exposed to ACM, retained many of the essential BBB properties in culture (Gaillard et al., 2001, Eur J Pharm Sci. 12:215-22). By introducing primary cultured brain astrocytes at the bottom of the cell culture well, the transendothelial electrical resistance (TEER) across BCEC monolayers cultured on filter inserts above was increased to about 150% of BCEC monolayers cultured in ACM alone. Moreover, when culturing the astrocytes on the bottom side of the filter insert, thus in close proximity of the BCEC, TEER multiplied by a factor 3-8. In addition, the paracellular transport of sodium fluorescein (FLU, mol. weight 376 Da) and FITC-labeled dextran (FD4, MW 4 kDa) decreased to about 50% of BCEC cultured in ACM alone. In conclusion, the proximity of astrocytes from the BCEC determined the magnitude of the effect, although they did not make physical contact with the BCEC (Gaillard et al., supra).
TEER is a sensitive measure to quantify the permeability of small ions through the tight junctions between BCEC. TEER thus represents the functionality of tight junctions, which are considered the major hallmark of the BBB. The absolute value of TEER is mainly dependent on the amount and complexity of tight junctions between the cells. Likewise, this is also the limiting factor for the paracellular transport of large and hydrophilic compounds.
In additional studies, we found that astrocytes cultured on the bottom side of the filter insert:
(1) maintained (or re-induced) the expression of P-glycoprotein (Pgp, a drug efflux pump involved in multidrug resistance) on BCEC after the first passage (Gaillard et al., 2000, Pharm. Res. 17:1198-1205);
(2) decreased the sensitivity for vinblastine induced BBB disruption (a Pgp functionality assay) (Gaillard et al., 2000, supra);
(3) induced active transport of Pgp substrates from the basolateral (CNS) side to the apical (blood) side of a filter, while this was not observed in BCEC monolayers, despite the fact that Pgp was expressed in BCEC monolayers (Gaillard et al., 2000, supra);
(4) mediated a protective response to LPS-induced BCEC disruption (Gaillard, P., 2000a, Ph.D. Thesis, Leiden University, p 81-97). None of these effects were induced by ACM alone. Apparently, the physical and proximate presence of astrocytes on the bottom side of the filter inserts is superior in inducing BBB properties in BCEC when compared to ACM alone.
There exists thus a need for additional products, methods and assays that provide a means to control BBB properties or identify and modulate cellular responses to early dynamic inflammation induced changes in BBB functionality and tissue response to such changes. Such products, methods and assays will provide benefit in numerous medical conditions and procedures, such as those mentioned above.
An “alteration of the activity or steady state level of a polypeptide” herein means any detectable change in the biological activity exerted by the polypeptide or in the steady state level of the protein as compared the normal activity or steady-state in a healthy individual.
An “agonist” is herein defined as any molecule that mimics a biological activity, preferably the biological activity of a polypeptide, a receptor or its ligand. An antagonist is any molecule that partially or fully blocks, inhibits or neutralizes such a biological activity.
The term “treatment” of vascular disorders refers to, inter alia, reducing or alleviating one or more symptoms in an individual, preventing one or more symptoms from worsening or progressing, promoting recovery or improving prognosis, and/or preventing disease in an individual who is free therefrom as well as slowing or reducing progression of existing disease. For a given individual, improvement in a symptom, its worsening, regression, or progression may be determined by an objective or subjective measure. Efficacy of treatment may be measured as an improvement in morbidity or mortality (e.g., lengthening of survival curve, for a selected population).
Increased permeability of the endothelial/vascular barrier makes it more leaky (i.e., less tight, more permeable). Decreased permeability of the endothelial/vascular barrier makes it more tight (i.e., less leaky, less permeable). Treating a vascular disorders thus means decreasing vascular permeability, whereas increasing drug delivery thus requires increased vascular permeability. LPSS polypeptides of the invention which are upregulated in BCEC from BCEC-astrocyte cocultures are involved in increased vascular permeability. LPSS polypeptides of the invention which are downregulated in BCEC from BCEC-astrocyte cocultures are involved in increased vascular permeability. LPSS polypeptides of the invention which are differentially up- or downregulated between BCEC monolayers and BCEC-astrocyte cocultures are involved in the ability to recover from the inflammatory stimulus (
In a first aspect the invention relates to a method for modulating the permeability of endothelial cells. The method comprises altering in the endothelial cells the activity or the steady-state level of a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NOs:1-25. Sequence identities or similarities are herein defined as described below.
The endothelial cells are preferably vascular endothelial cells, more preferably microvascular endothelial cells. Most preferably the endothelial cells are microvascular endothelial cells that constitute or are part of one of the blood-central nervous system (CNS) bathers, such as the blood-brain bather, blood-retina barrier, blood-nerve barrier, blood-spinal cord barrier, of which brain microvascular endothelial cells are most preferred.
Such endothelial barrier cells may be characterized in situ, ex situ (i.e., in isolated capillaries) or in vitro by e.g., specific endothelial cell markers, specific barrier markers, but also by barrier functional assays. More specifically, endothelial cells may be characterized by their morphology in situ, i.e. a tube-like structure of with a diameter of about 10-20 micrometers, formed by single (or no more than three) continuously connected endothelial cells, surrounded by a continuous basal lamina, in which perivascular pericytes reside and astrocyte endfeet are projected upon. Both in and ex situ, as well as in vitro, barrier-like endothelial cells are between 1 and 5 micrometers thick, have many mitochondria, are connected by tight junctions, have no intercellular clefts, no fenestrations and very few pinocytotic vesicles, as can be observed by e.g., electron microscopy. In vitro, capillary structures may be characterized by their morphology in culture i.e., a tube-like structure with a diameter of about 10-20 micrometers, between 50 and 200 micrometers long. In vitro, endothelial cells may be characterized by their morphology in culture i.e., cobblestone shape (when growing in a cluster, e.g., out from a capillary) and spindle shape (when confluent), with a centered oval nucleus, as can be observed by e.g., phase-contrast microscopy. They may also be characterized by using a panel of general endothelial cell specific markers and functions e.g., expression of endothelial specific cluster of differentiation (CD) antigens (VCAM (CD106), CD31, EN-4, ICAMs, E-Selectin, PECAM, RBA), cadherins, integrins, actin, vimentin, factor VIII related antigen (vWF), collagen I and IV, fibronectin, matrix metalloproteinases, tissue inhibitor of metalloproteinases; non-thrombogenicity; low leukocyte adherence; release of vasoactive compounds (nitric oxide, endothelin-1 and prostacyclins); uptake of DiI-labeled-acetylated low density lipoprotein (DiI-Ac-LDL); lectin binding; presence of angiotensin converting enzyme, alkaline phosphatase, monoamine oxidase and anionic sites. In addition, typical barrier markers and functions may be used, like visualization of tight junctions or tight junction-related proteins (ZO-1) and restricted paracellular transport of reference compounds (such as e.g., Evans blue (binds to albumin), mannitol, sucrose, fluorescein, dextrans, albumin, AIB); absence of vesicular transport; absence of non-barrier markers like PAL-E; expression of gamma-glutamyl-transpeptidase (γ-GTP); expression and functionality of P-glycoprotein (Pgp), multi-drug resistance proteins 1-7, glucose transporters, nucleoside transporters, organic anion transporters, large and neutral amino acid transporters; transferrin receptors, insulin-growth factor receptors, scavenger receptors; marginal F-actin localization and expression of many mitochondria, although none of these are specific for endothelial cells. (Functional) expression of these markers may be determined by e.g., molecular biological, biochemical, (immuno)-histo(cyto)chemical techniques as well as by functional assays using known substrates, ligands and/or inhibitors. These markers have been described and reviewed in international scientific journals (de Boer et al., 1999, Eur J Pharm Sci. 8):1-4; Hofman et al., 2001, Invest Ophthalmol Vis Sci. 42:895-901; Schlingemann et al., 1997, Ophthalmic Res. 29:130-8; Schlingemann et al., 1999, Diabetologia. 42:596-602; Vorbrodt et al., 1986, Brain Res. 394:69-79; Dai et al., 2002, Brain Res. 954:311-316).
The permeability of the endothelial cells is herein understood to mean the measure of the ease with which a compound (may that be ions (e.g., Na+, K+, Ca2+), water, nutrients (e.g., glucose, amino acids), metabolites, neurotransmitters (e.g., glutamic acid, tryptophan), hormones, peptides, plasma proteins (e.g., albumin, fibrinogen, immunoglobulins, cytokines, growth factors), cells and xenobiotics (e.g., drugs, diagnostic markers)) can diffuse across, or be (actively) transported into or across, an endothelial cell layer in the luminal to abluminal direction or visa versa. Changes in permeability of the endothelial cells can also be the result of endothelial biotransformation of a given compound (may that be nutrients (e.g., glucose, amino acids), metabolites, neurotransmitters (e.g., glutamic acid, tryptophan), hormones, peptides, plasma proteins (e.g., albumin, fibrinogen, immunoglobulins, cytokines, growth factors), cells and xenobiotics (e.g., drugs, diagnostic markers)). The modulation of the permeability includes both increases and decreases in permeability. The permeability may conveniently be determined in vitro by determining the transendothelial electrical resistance (TEER) as described in the Examples. TEER is a sensitive measure to quantify the permeability of ions through the tight junctions between cells. In the method of the invention, a modulation of the permeability of the endothelial cells preferably is a modulation that results in a change of the TEER of at least 20, 50, 100, 300 or 1000% (Gaillard et al., 2000b, Eur J Pharm Sci. 12:95-102). Other methods for determining the permeability include e.g., the demonstration of changes in (functional) expression of the endothelial/barrier markers described above involved in permeability control by e.g., molecular biological, biochemical, (immuno)-histo(cyto)chemical techniques or by functional assays using known substrates, ligands and/or inhibitors of transporter systems. More specifically, changes in permeability may be demonstrated by loss of tight junction expression, appearance of intercellular clefts, fenestrations and/or number and localization of pinocytotic vesicles, as can be observed by e.g., electron microscopy (Hofman et al., supra). Changes in expression levels of the endothelial cell markers involved in endothelial permeability like ZO-1, PAL-E, RBA, F-actin, factor VIII related antigen (vWF), γ-GTP, Pgp, glucose transporters, PECAM, integrins, cadherin-5, transferrin receptors, lectin-binding sites or alkaline phosphatase, are all indicative for changes in endothelial permeability (Gaillard et al., 2001, supra; de Boer et al., 1999, supra; Schlingemann et al., 1997, supra; Schlingemann et al., 1999, supra; Tio et al., 1990, supra; Vorbrodt et al., 1986, supra; Dai et al., 2002, supra). Functional assays for restricted paracellular transport of reference compounds (e.g., mannitol, sucrose, fluorescein, dextrans, albumin), polar and active and inhibitable (with e.g., verapamil, PSC-833, temperature) transport of Pgp-substrates (rhodamine 123, vinblastine, etc.) or transferrin across endothelial cell layers are indicative for changes in endothelial permeability (Gaillard et al., 2000, 2001, supra).
In vivo the permeability of the endothelial cells may be determined by the demonstration of changes in (functional) expression of the endothelial/barrier markers that are involved in permeability control as described above for the in vitro situation (by e.g., molecular biological, biochemical, (immuno)-histo(cyto)chemical techniques or by functional assays using known substrates, ligands and/or inhibitors of transporter systems). In addition, extravasation of endogenous (e.g., fibrinogen, IgG) or (fluorescence- or radiolabeled) exogenous (e.g., Evans blue (binds to albumin), mannitol, sucrose, fluorescein, dextrans, albumin, AIB) reference compounds may be determined by (immuno)-histo(cyto)chemical techniques or by several in vivo sampling methods, like brain uptake index (BUI, Oldendorf, 1970 Brain Res. 24:372-6), brain efflux index (BEI, Kakee et al., 1996 J Pharmacol Exp Therap. 277:1550-9), in situ perfusion (Takasato et al., 1984 Am J. Physiol. 247(3 Pt 2):H484-93), single or multiple pass brain perfusion (Brodie et al., 1960, J Pharmacol Exp Ther. 130:519-28), CSF sampling (unit impulse response, van Bree et al., 1989. J. Pharmacokin. Biopharm. 17:441-62), positron emission tomography (PET, Hendrikse et al., 1998, Br J. Pharmacol. 124:1413-18), magnetic resonance techniques (MRI, MRS, Jenkins et al., 1999, Ann NY Acad. Sci. 893:214-42), quantitative autoradiography (QAR, Smith, 1989 In Implications of the Blood-Brain Barrier and its Manipulation, vol. 1: Basic science aspects. New York: Plenum Publ. Corp., E. A. Neuwelt, (ed.) pp 85-118), and intracerebral microdialysis (de Lange et al., 2000, Adv Drug Deliv Rev. 45: 125-48).
In the method of the invention, preferably the activity or steady-state level of the LPSS polypeptide may be altered at the level of the polypeptide itself, e.g., by providing the LPSS polypeptide to the endothelial cells from an exogenous source, or by adding an antagonist or inhibitor of the LPSS polypeptide to the endothelial cells, such as e.g., an antibody against the LPSS polypeptide. For provision of the LPSS polypeptide from an exogenous source the LPSS polypeptide may conveniently be produced by expression of a nucleic acid encoding the LPSS polypeptide in suitable host cells as described below. An antibody against the LPSS polypeptide may be obtained as described below.
Alternatively, the activity or steady-state level of the LPSS polypeptide may be altered by regulating the expression level of a nucleotide sequence encoding the polypeptide. Preferably, the expression level of a nucleotide sequence is regulated in the endothelial cells. The expression level of the LPSS polypeptide may be up-regulated by introduction of an expression vector into the endothelial cells, whereby the expression vector comprises a nucleotide sequence encoding the LPSS polypeptide, and whereby the nucleotide sequence is under control of a promoter capable of driving expression of the nucleotide sequence in the endothelial cells. The expression level of the LPSS polypeptide may also be up-regulated by introduction of an expression vector into the endothelial cells, whereby the expression vector comprises a nucleotide sequence encoding a factor capable of trans-activation of the endogenous nucleotide sequence encoding the LPSS polypeptide.
Alternatively, the expression level of the LPSS polypeptide may be down regulated by providing an antisense molecule to the cells, whereby the antisense molecule is capable of inhibiting the expression of the nucleotide sequence encoding the LPSS polypeptide. The antisense molecule may be provided as such or it may be provided by introducing an expression vector into the endothelial cells, whereby the expression vector comprises an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding the LPSS polypeptide, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in the endothelial cells. The expression level of the LPSS polypeptide may also be down-regulated by introducing an expression vector into the endothelial cells, whereby the expression vector comprises a nucleotide sequence encoding a factor capable of trans-repression of the endogenous nucleotide sequence encoding the LPSS polypeptide.
Generally, the activity or steady state level of LPSS polypeptides may thus be modified by:
1. Increasing gene expression, e.g., by providing:
Thus, in the method of the invention, the permeability of the endothelial cells is preferably decreased by increasing the activity or the steady-state level of a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:2, 3, 4, 6, 7, 8, 9, 10, 11, 19, 20, 23, 24 and 25. More preferably, the permeability is decreased by increasing the activity or the steady-state level of a LPSS polypeptide selected from the groups consisting of downregulated secreted factors (SEQ ID NOs: 2, 3, 4 and 23), downregulated signal transduction pathways (SEQ ID NO:6, 7, 8, 9, 10 and 11), differentially upregulated signal transduction pathways (SEQ ID NO:19), downregulated receptors and adhesion molecules (SEQ ID NO:20), and differentially upregulated metabolic enzymes (SEQ ID NOs:23-25). The activity or the steady-state level of the LPSS polypeptide may be increased by any of the means described above, e.g., by introducing an expression vector into the endothelial cells, whereby the expression vector comprises a nucleotide sequence encoding the LPSS polypeptide, and whereby the nucleotide sequence is under control of a promoter capable of driving expression of the nucleotide sequence in the endothelial cells.
Alternatively, in the method of the invention, the permeability of the endothelial cells may be decreased by decreasing the activity or the steady-state level of a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:1, 5, 12, 13, 14, 15, 16, 17, 18, 21 and 22. More preferably, the permeability is decreased by decreasing the activity or the steady-state level of a LPSS polypeptide selected from the groups consisting of upregulated secreted factors (SEQ ID NOs:1, 13, 14 and 22), upregulated signal transduction pathways (SEQ ID NOs:5, 12, 15, 16 and 17), differentially downregulated signal transduction pathways (SEQ ID NO:18), and upregulated receptors and adhesion molecules (SEQ ID NOs:21 and 22). The activity or the steady-state level of the LPSS polypeptide may be decreased by any of the means described above, e.g., the activity or the steady-state level of the LPSS polypeptide may be decreased by introducing an expression vector into the endothelial cells, whereby the expression vector comprises an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding the LPSS polypeptide, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in the endothelial cells.
In the method of the invention, the permeability of the endothelial cells may be increased by increasing the activity or the steady-state level of a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:1, 5, 12, 13, 14, 15, 16, 17, 18, 21 and 22. More preferably, the permeability is decreased by decreasing the activity or the steady-state level of a LPSS polypeptide selected from the groups consisting of upregulated secreted factors (SEQ ID NOs:1, 13, 14 and 22), upregulated signal transduction pathways (SEQ ID NOs:5, 12, 15, 16 and 17), differentially downregulated signal transduction pathways (SEQ ID NO:18), and upregulated receptors and adhesion molecules (SEQ ID NOs:21 and 22). The activity or the steady-state level of the LPSS polypeptide may be increased by any of the means described above, e.g., by introducing an expression vector into the endothelial cells, whereby the expression vector comprises a nucleotide sequence encoding the LPSS polypeptide, and whereby the nucleotide sequence is under control of a promoter capable of driving expression of the nucleotide sequence in the endothelial cells.
Alternatively, in the method of the invention, the permeability of the endothelial cells may be increased by decreasing the activity or the steady-state level of a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:2, 3, 4, 6, 7, 8, 9, 10, 11, 19, 20, 23, 24 and 25. More preferably, the permeability is decreased by increasing the activity or the steady-state level of a LPSS polypeptide selected from the groups consisting of downregulated secreted factors (SEQ ID NOs:2, 3, 4 and 23), downregulated signal transduction pathways (SEQ ID NO:6, 7, 8, 9, 10 and 11), differentially upregulated signal transduction pathways (SEQ ID NO:19), downregulated receptors and adhesion molecules (SEQ ID NO:20), and differentially upregulated metabolic enzymes (SEQ ID NOs:23-25). The activity or the steady-state level of the LPSS polypeptide may be decreased by any of the means described above, e.g., the activity or the steady-state level of the LPSS polypeptide may be decreased by introducing an expression vector into the endothelial cells, whereby the expression vector comprises an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding the LPSS polypeptide, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in the endothelial cells.
In another aspect, the invention relates to a method for treating or preventing a microvascular permeability-modifying disorder in a subject. The method comprises pharmacologically altering the activity or the steady-state level in the subject's microvascular endothelial cells, of a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NOs:1-25. Preferably, the alteration is sufficient to reduce the symptoms of the microvascular permeability-modifying disorder. The method preferably comprises administering to the subject in a therapeutically effective amount, a pharmaceutical composition comprising a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NOs:1-25, or a nucleic acid molecule comprising a nucleotide sequence encoding the LPSS polypeptide or another entity that is effective in modifying the activity or steady state level of a LPSS polypeptide as listed herein above. Preferably, in the method the LPSS polypeptide is a polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:2, 3, 4, 6, 7, 8, 9, 10, 11, 19, 20, 23, 24 and 25. More preferably, the permeability is decreased by increasing the activity or the steady-state level of a LPSS polypeptide selected from the groups consisting of downregulated secreted factors (SEQ ID NOs:2, 3, 4 and 23), downregulated signal transduction pathways (SEQ ID NO:6, 7, 8, 9, 10 and 11), differentially upregulated signal transduction pathways (SEQ ID NO:19), downregulated receptors and adhesion molecules (SEQ ID NO:20), and differentially upregulated metabolic enzymes (SEQ ID NOs:23-25). The nucleic acid molecule preferably is a gene therapy vector, in which the nucleotide sequence is under control of a promoter capable of driving expression of the nucleotide sequence in endothelial cells, preferably microvascular endothelial cells.
Alternatively, the method of treatment is a method comprising the step of administering to the subject in a therapeutically effective amount, a pharmaceutical composition comprising an antagonist of a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:1, 5, 12, 13, 14, 15, 16, 17, 18, 21 and 22, whereby preferably the antagonist is an antibody against the LPSS polypeptide. More preferably the amino acid sequence is selected from the groups consisting of upregulated secreted factors (SEQ ID NOs:1, 13, 14 and 22), upregulated signal transduction pathways (SEQ ID NOs:5, 12, 15, 16 and 17), differentially downregulated signal transduction pathways (SEQ ID NO:18), and upregulated receptors and adhesion molecules (SEQ ID NOs:21 and 22). The same effect may be achieved in a method comprising the step of administering to the subject in a therapeutically effective amount, a pharmaceutical composition comprising a gene therapy vector. The gene therapy vector preferably comprises an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:1, 5, 12, 13, 14, 15, 16, 17, 18, 21 and 22, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in endothelial cells, preferably microvascular endothelial cells. More preferably the amino acid sequence is selected from the groups consisting of upregulated secreted factors (SEQ ID NOs:1, 13, 14 and 22), upregulated signal transduction pathways (SEQ ID NOs:5, 12, 15, 16 and 17), differentially down-regulated signal transduction pathways (SEQ ID NO:18), and upregulated receptors and adhesion molecules (SEQ ID NOs:21 and 22).
In the method of treatment of the invention, the microvascular permeability disorder preferably is selected from the group consisting of neurodegenerative disorders, such as cerebrovascular accidents (CVA), Alzheimer's disease (AD), vascular-related dementia, Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), Parkinson's disease (PD), brain trauma, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Huntington's chorea; peripheral disorders with a CNS component, such as septic shock, hepatic encephalopathy, (diabetic) hypertension, diabetic microangiopathy, sleeping sickness, Whipple disease, Duchenne muscular dystrophy (DMD), aspartylglucosaminuria, cholesterol ester storage disease, Wolman disease, cystinosis, Danon disease, Fabry disease, Farber lipogranulomatosis, Farber disease, fucosidosis, galactosialidosis types I/II, Gaucher disease types I/II/III, Gaucher disease, globoid cell leukodystrophy, Krabbe disease, glycogen storage disease II, Pompe disease, GM1-gangliosidosis types 1/11/11I, GM2-gangliosidosis type I, Tay Sachs disease, GM2-gangliosidosis type II, Sandhoff disease, GM2-gangliosidosis, α-mannosidosis types 1/11, mannosidosis, metachromatic leukodystrophy, mucolipidosis type I, sialidosis types 1/11 mucolipidosis types 11/III 1-cell disease, mucolipidosis type IIIC pseudo-Hurler polydystrophy, mucopolysaccharidosis type I, mucopolysaccharidosis type II, Hunter syndrome, mucopolysaccharidosis type IIIA, Sanfilippo syndrome, mucopolysaccharidosis type IIIB, mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID, mucopolysaccharidosis type IVA, Morquio syndrome, mucopolysaccharidosis type IVB Morquio syndrome, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, Sly syndrome, mucopolysaccharidosis type IX, multiple sulfatase deficiency, neuronal ceroid lipofuscinosis, CLN1 Batten disease, Niemann-Pick disease, Niemann-Pick disease types A/B, C1, or C2, pycnodysostosis, Schindler disease, Schindler disease types VII, and sialic acid storage disease, pre-eclampsia; neuropsychiatric disorders, such as depression, autism, anxiety attention deficit hyperactivity disorder (ADHD), neuropsychiatric systemic lupus erythematosus, bipolar disorder, schizophrenia and other psychoses; other CNS disorders, such as brain tumors, epilepsy, migraine, narcolepsy, insomnia, chronic fatigue syndrome, mountain sickness, encephalitis, meningitis, AIDS-related dementia; and angiogenesis-related disorders, such as vascular tumors, proliferative vitreoretinopathy, rheumatoid arthritis, Crohn's disease, atherosclerosis, ovarian hyperstimulation, psoriasis, endometriosis associated with neovascularization, restenosis subsequent to balloon angioplasty, scar tissue overproduction, peripheral vascular disease, hypertension, inflammatory vasculitides, Reynaud's disease, Reynaud's phenomenon, aneurysms, arterial restenosis, thrombophlebitis, lymphangitis, lymphedema, wound healing and tissue repair, ischemia reperfusion injury, angina, myocardial infarctions, chronic heart conditions, heart failure such as congestive heart failure, age-related macular degeneration, and osteoporosis.
In a further aspect, the invention relates to a method for reversibly increasing the microvascular permeability in a subject. This method comprises the step of administering to the subject, a pharmaceutical composition comprising a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NOs:1-25, or a nucleic acid molecule comprising a nucleotide sequence encoding the LPSS polypeptide, or another entity that is effective in modifying the activity or steady state level of a LPSS polypeptide as listed herein above, in an amount effective to increase the microvascular permeability. Preferably, the LPSS polypeptide is a polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:1, 5, 12, 13, 14, 15, 16, 17, 18, 21 and 22. More preferably the amino acid sequence is selected from the groups consisting of upregulated secreted factors (SEQ ID NOs:1, 13, 14 and 22), upregulated signal transduction pathways (SEQ ID NOs:5, 12, 15, 16 and 17), differentially downregulated signal transduction pathways (SEQ ID NO:18), and upregulated receptors and adhesion molecules (SEQ ID NOs:21 and 22). Preferably the nucleic acid molecule is a gene therapy vector, in which the nucleotide sequence is under control of a promoter capable of driving expression of the nucleotide sequence in endothelial cells, preferably microvascular endothelial cells. In this method, the reversibility of the increase in permeability of the microvascular permeability is preferably achieved by using a gene therapy vector that is capable of only transient expression of the nucleotide sequence (see below), and/or, the promoter capable of driving expression of the nucleotide sequence in endothelial cells, preferably is an inducible promoter. More preferably, the inducible promoter is a promoter that may be induced by the administration of small organic or inorganic compounds (see below).
Alternatively, method for reversibly increasing the microvascular permeability in a subject may also be a method comprising the step of administering to the subject in a therapeutically effective amount, a pharmaceutical composition comprising an antagonist of a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:2, 3, 4, 6, 7, 8, 9, 10, 11, 19, 20, 23, 24 and 25, whereby the antagonist preferably is an antibody against the LPSS polypeptide. More preferably, the amino acid sequence is selected from the groups consisting of downregulated secreted factors (SEQ ID NOs:2, 3, 4 and 23), downregulated signal transduction pathways (SEQ ID NO:6, 7, 8, 9, 10 and 11), differentially upregulated signal transduction pathways (SEQ ID NO:19), downregulated receptors and adhesion molecules (SEQ ID NO:20), and differentially upregulated metabolic enzymes (SEQ ID NOs:23-25). Similarly, the method may also comprise the step of administering to the subject in a therapeutically effective amount, a pharmaceutical composition comprising a gene therapy vector, whereby the gene therapy vector comprises an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:2, 3, 4, 6, 7, 8, 9, 10, 11, 19, 20, 23, 24 and 25, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in endothelial cells, preferably microvascular endothelial cells. More preferably, the amino acid sequence is selected from the groups consisting of downregulated secreted factors (SEQ ID NOs:2, 3, 4 and 23), downregulated signal transduction pathways (SEQ ID NOs:6, 7, 8, 9, 10 and 11), differentially upregulated signal transduction pathways (SEQ ID NO:19), down-regulated receptors and adhesion molecules (SEQ ID NO:20), and differentially upregulated metabolic enzymes (SEQ ID NOs:23-25).
The methods for reversibly increasing the microvascular permeability in a subject may advantageously be applied when one wants to deliver blood-borne, membrane-impermeant drugs to the brain. The drug may be any pharmaceutically, veterinarily or diagnostically useful compound or composition of compounds, which is normally impermeant to the blood-brain or other physiological barrier or at least insufficiently permeant. The pharmacological nature of the drug is otherwise unimportant. The invention is therefore useful in the delivery of a wide range of drugs across physiological barriers such as the blood-brain barrier. However, it is anticipated that among the primary candidates for delivery by means of this aspect of the invention will be: anti-tumor compounds, such as methotrexate, adriamycin, cisplatin and the other antineoplastic agents or cytotoxic drugs that are defined herein below (see e.g., pages 24-27); growth factors, such as NGF, RDNF and CNTF, which are used to treat neurodegenerative disease; imaging agents, especially those that are antibody based; and neurotransmitter antagonists or agonists which do not penetrate the blood-brain barrier (such as certain NMDA receptor blockers)
For most countries other than the USA, in a further aspect the invention relates to various uses of the compounds of the invention for the manufacture of a medicament for treating or preventing a microvascular permeability-modifying disorder. For example, in one such aspect the invention relates to the use of a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NOs:1-25, or a nucleic acid molecule comprising a nucleotide sequence encoding the LPSS polypeptide, or another entity that is effective in modifying the activity or steady state level of a LPSS polypeptide as listed herein above, for the manufacture of a composition for treating or preventing a microvascular permeability-modifying disorder. Preferably, the LPSS polypeptide is a polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:2, 3, 4, 6, 7, 8, 9, 10, 11, 19, 20, 23, 24 and 25. More preferably, the amino acid sequence is selected from the groups consisting of downregulated secreted factors (SEQ ID NOs:2, 3, 4 and 23), downregulated signal transduction pathways (SEQ ID NO:6, 7, 8, 9, 10 and 11), differentially upregulated signal transduction pathways (SEQ ID NO:19), down-regulated receptors and adhesion molecules (SEQ ID NO:20), and differentially upregulated metabolic enzymes (SEQ ID NOs:23-25). The nucleic acid molecule preferably is a gene therapy vector comprising the nucleotide sequence, whereby the nucleotide sequence is under control of a promoter capable of driving expression of the nucleotide sequence in endothelial cells. An antagonist of a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:1, 5, 12, 13, 14, 15, 16, 17, 18, 21 and 22, may also be used for the manufacture of a composition for treating or preventing a microvascular permeability-modifying disorder, whereby the antagonist preferably is an antibody against the LPSS polypeptide. More preferably the amino acid sequence is selected from the groups consisting of upregulated secreted factors (SEQ ID NOs:1, 13, 14 and 22), upregulated signal transduction pathways (SEQ ID NOs:5, 12, 15, 16 and 17), differentially downregulated signal transduction pathways (SEQ ID NO:18), and upregulated receptors and adhesion molecules (SEQ ID NOs:21 and 22).
Alternatively, a gene therapy vector comprising an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:1, 5, 12, 13, 14, 15, 16, 17, 18, 21 and 22, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in endothelial cells, preferably microvascular endothelial cells, may be used for the manufacture of a composition for treating or preventing a microvascular permeability-modifying disorder. More preferably the amino acid sequence is selected from the groups consisting of upregulated secreted factors (SEQ ID NOs:1, 13, 14 and 22), upregulated signal transduction pathways (SEQ ID NOs:5, 12, 15, 16 and 17), differentially downregulated signal transduction pathways (SEQ ID NO:18), and upregulated receptors and adhesion molecules (SEQ ID NOs:21 and 22).
In the above uses of the compounds of the invention for the manufacture of a medicament for treating a microvascular permeability-modifying disorder, the disorder preferably is a microvascular permeability-modifying disorder as described above.
Similarly, for most countries other than the USA, in a yet further aspect the invention relates to various uses of the compounds of the invention for the manufacture of a medicament or composition for reversibly increasing the microvascular permeability in a subject. Preferably the compound is a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NOs:1-25, or a nucleic acid molecule comprising a nucleotide sequence encoding the LPSS polypeptide or another entity that is effective in modifying the activity or steady state level of a LPSS polypeptide as listed herein above. Preferably, the LPSS polypeptide is a polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:1, 5, 12, 13, 14, 15, 16, 17, 18, 21 and 22. More preferably the amino acid sequence is selected from the groups consisting of upregulated secreted factors (SEQ ID NOs:1, 13, 14 and 22), upregulated signal transduction pathways (SEQ ID NOs:5, 12, 15, 16 and 17), differentially downregulated signal transduction pathways (SEQ ID NO:18), and upregulated receptors and adhesion molecules (SEQ ID NOs:21 and 22). The nucleic acid molecule preferably is a gene therapy vector, in which the nucleotide sequence encoding the LPSS polypeptide is under control of a promoter capable of driving expression of the nucleotide sequence in endothelial cells, preferably microvascular endothelial cells.
Alternatively, an antagonist of a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:2, 3, 4, 6, 7, 8, 9, 10, 11, 19, 20, 23, 24 and 25, may be used for the manufacture of a composition for reversibly increasing the microvascular permeability in a subject, whereby preferably the antagonist is an antibody against the LPSS polypeptide. More preferably, the amino acid sequence is selected from the groups consisting of downregulated secreted factors (SEQ ID NOs:2, 3, 4 and 23), downregulated signal transduction pathways (SEQ ID NO:6, 7, 8, 9, 10 and 11), differentially upregulated signal transduction pathways (SEQ ID NO:19), downregulated receptors and adhesion molecules (SEQ ID NO:20), and differentially upregulated metabolic enzymes (SEQ ID NOs:23-25). Or a gene therapy vector comprising an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NOs:2, 3, 4, 6, 7, 8, 9, 10, 11, 19, 20, 23, 24 and 25, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in endothelial cells, preferably microvascular endothelial cells, may be used for the manufacture of a composition for reversibly increasing the microvascular permeability in a subject. More preferably, the amino acid sequence is selected from the groups consisting of downregulated secreted factors (SEQ ID NOs:2, 3, 4 and 23), downregulated signal transduction pathways (SEQ ID NO:6, 7, 8, 9, 10 and 11), differentially upregulated signal transduction pathways (SEQ ID NO:19), downregulated receptors and adhesion molecules (SEQ ID NO:20), and differentially upregulated metabolic enzymes (SEQ ID NOs:23-25). Preferably the gene therapy vector is a vector for transient expression (see below) and/or the promoter preferably is an inducible promoter. More preferably, the inducible promoter is a promoter that may be induced by the administration of small organic or inorganic compounds (see below).
In yet another aspect, the invention involves a method of treating or diagnosing a CNS or microvascular disorder by administering a therapeutic or diagnostic agent, e.g., a neuroactive agent, by targeting that agent, or its pharmaceutically acceptable carrier, to a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NOs:1-25, to a patient suffering from or at risk for developing the CNS or microvascular disorder. Preferably, the neuroactive agent or its carrier is targeted to an upregulated LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NOs:1, 5, 12, 13, 14, 15, 16, 17, 18, 19, 21-25. More preferably, the amino acid sequence is selected from the groups consisting of the amino acid sequences of upregulated secreted factors (SEQ ID NOs:1, 13, 14 and 22), upregulated signal transduction pathways (SEQ ID NOs:5, 12, 15, 16, 17 and 19), upregulated receptors and adhesion molecules (SEQ ID NOs:21 and 22), and upregulated metabolic enzymes (SEQ ID NOs:23-25). Still more preferably an upregulated LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NOs:21 or 22.
The targeting agent can be an antibody to a LPSS polypeptide, a protein, a peptide, a LPSS polypeptide agonist, a LPSS polypeptide antagonist, a peptidomimetic, a small molecule, or another compound that specifically binds to a LPSS polypeptide. As used herein, the term “specific binding” means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity, for example, a peptide of similar size that lacks a specific binding sequence. Specific binding is present if the molecule has measurably higher affinity for the LPSS polypeptide than the control molecule. Specificity of binding can be determined, for example, by competition with a control molecule that is known to bind to a target. The term “specific binding,” as used herein, includes both low and high affinity specific binding. Specific binding can be exhibited, e.g., by a low affinity targeting agent having a Kd of at least about 10−4 M. E.g., if a LPSS polypeptide has more than one binding site for a targeting agent, a targeting agent having low affinity can be useful for targeting the microvascular endothelium. Specific binding also can be exhibited by a high affinity targeting agents, e.g., a targeting agent having a Kd of at least about of 10−7M, at least about 10−8M, at least about 10−9M, at least about 10−10M, or can have a Kd of at least about 10−11M or 10−12M or greater. Both low and high affinity-targeting agents are useful for targeting the microvascular endothelium.
The targeting agent is preferably conjugated to the therapeutic agent or its pharmaceutically acceptable carrier. A “conjugate” is herein defined as consisting of two entities that are covalently coupled together. In the context of the present invention the first entity will usually be a targeting agent as herein defined above, whereas the second entity may be a therapeutic or diagnostic moiety, such as a molecule or structure, for use in the treatment or diagnosis of a CNS or microvascular disorder. Such therapeutic or diagnostic moieties may e.g., be anti-tumor compounds, such as antineoplastic agents or cytotoxic drugs, such as alkylating agents, e.g., Mechlorethamine hydrochloride (Nitrogen Mustard, Mustargen, HN2), Cyclophosphamide (Cytovan®, Endoxana®), Ifosfamide (IFEX®), Chlorambucil (Leukeran®), Melphalan (Phenylalanine Mustard, L-sarcolysin, Alkeran®, L-PAM), Busulfan (Myleran®), Thiotepa (Triethylenethiophosphoramide), Carmustine (BiCNU, BCNU), Lomustine (CeeNU, CCNU), Streptozocin (Zanosar®) and the like; plant alkaloids, e.g., Vincristine (Oncovin®), Vinblastine (Velban®, Velbe®), Paclitaxel (Taxol®), and the like; antimetabolites, e.g., methotrexate (MTX), Mercaptopurine (Purinethol®, 6-MP), Thioguanine (6-TG), Fluorouracil (5-FU), Cytarabine (Cytosar-U®®, Ara-C), Azacitidine (Mylosar®, 5-AZA) and the like; antibiotics, e.g., Dactinomycin (Actinomycin D, Cosmegen®), Doxorubicin (Adriamycin®), Daunorubicin (Daunomycin®, Cerubidine®), Idarubicin (Idamycin®), Bleomycin (Blenoxane®), Picamycin (Mithramycin®, Mithracin®), Mitomycin (Mutamycin®) and the like, and other anticellular proliferative agents, e.g., Hydroxyurea (Hydrea®), Procarbazine (Mutalane®), Dacarbazine (DTIC-Dome®), cisplatin (Platinol®), Carboplatin (Paraplatin®), Asparaginase (Elspar®), Etoposide (VePesid®, VP-16-213), Amsarcrine (AMSA, m-AMSA), Mitotane (Lysodren®), Mitoxantrone (Novatrone®), and the like; gefitinib (ZD1839 or Iressa®) and imatinib mesylate (Gleevec® or Glivec®); anti-cancer biopharmaceutical drugs including antibodies (Rituxan® or rituximab; Herceptin® or trastuzumab; Zevalin® or ibritumomab tiuxetan (radiolabeled); Erbitux® or cetuximab; Avastin™ or bevacizumab or rhuMAb-VEGF) and cytokines (Intron® or α-interferon; Proleukin® IL-2 or aldesleukin) to treat primary brain tumors or brain metastasis of somatic tumors; anti-inflammatory drugs including antibodies (Enbrel® or etanercept; Remicade® or infliximab; Simulect® or basiliximab; Zenapax® or daclizumab; Kineret® or anakinra; Xolair® or omalizumab; Humira® or adalimumab; Antegren® or natalizumab; RhuFab™ or ranibizumab; Raptiva™ or efalizumab) and cytokines such as interferon-α, interferon-beta (Avonex® or interferon beta-1a; Betaseron®/Betaferon® or interferon β-1b; Rebif® or interferon-beta-1a), interferon-γ, interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), TNF, granulocyte macrophage colony stimulating factor (GM-CSF: Leukine® or sargramostim), granulocyte colony stimulating factor (G-CSF: Neupogen® or filgrastim), macrophage colony stimulating factor (M-CSF), platelet-derived growth factor (PDGF); to treat e.g., neuroinflammation related to neurodegenerative disorders; neurotrophic factors (e.g., NGF or nerve growth factor; BDNF or brain-derived neurotrophic factor; NT3 or neurotrophin-3; NT4 or neurotrophin-4; NT5 or neurotrophin-5; RDGF or retina-derived growth factor; CNTF or ciliary neurotrophic factor; activin; bFGF or basic fibroblast growth factor; aFGF or acidic fibroblast growth factor; GDNF or glial cell line-derived neurotrophic factor or neublastin or artemin or enovin, presephin, neurturin; CTGF or connective tissue growth factor; EGF or epithelial growth factor); erythropoietins (EPO) (Procrit®/Eprex® or erythropoietin alfa; Epogen® or erythropoietin; NeoRecormon® or erythropoietin β; Aranesp® or darbepoietin alfa); growth hormone or somatotropin (Humatrope®; Protropin®/Nutropin®; Serostim®; Saizen®); anti-NogoA Mab (IN-1); NogoA antagonist of Nogo66 inhibitor (NEP1-40), to treat e.g., neurodegenerative disorders; enzymes (e.g., Cerezyme® or glucocerebrosidase; Aldurazyme™ or laronidase; Aryplase™ or arylsulfatase B; 12S or iduronate-2-sulfatase; α-L-iduronidase; N-acetylgalactosamine 4-sulfatase; phenylase; aspartylglucosaminidase; acid lipase; cysteine transporter; Lamp-2; a galactosidase A; acid ceramidase; α-L-fucosidase; ss-hexosaminidase A; GM2-activator deficiency; α-D-mannosidase; ss-D-mannosidase; arylsulfatase A; saposin B; neuraminidase; α-N-acetylglucosaminidase phosphotransferase; phosphotransferase 7-subunit; heparan-N-sulfatase; α-N-acetylglucosaminidase; acetyl-CoA: N-acetyltransferase; N-acetylglucosamine 6-sulfatase; galactose 6-sulfatase; 0-galactosidase; hyaluronoglucosaminidase; multiple sulfatases; palmitoyl protein thioesterase; tripeptidyl peptidase I; acid sphingomyelinase; cholesterol trafficking; cathepsin K; α-galactosidase B; sialic acid transporter; SOD or Cu/Zn superoxide dismutase) to treat e.g., (neurological symptoms related to) lysosomal storage diseases or other neurodegenerative disorders; brain-acting hormones and neurotransmitters such as somatostatin, oxytocin, vasopressin, guaranine, VIP, adrenocorticotropic hormone (ACTH), cholecystokinin (CCK), substance-P, bombesin, motilin, glicentin, glucagon, glucagon-like peptide (GLP-1); and neuropeptides and derivatives thereof such as peptide YY (PYY), neuropeptide Y (NPY), pancreatic polypeptide (PP), neurokinin A, neurokinin B, endorphin, enkephalin, neurotensin, neuromedin K, neuromedin L, calcitonin related peptide (CGRP), endothelin, ANP (“atrial natriuretic peptide”), BNP (“brain natriuretic peptide”), CNP (C-type natriuretic peptide”), and PACAP (“pituitary adenylate cyclase activating peptide”); imaging agents, especially those that are antibody based; neurotransmitter antagonists or agonists which do not penetrate the blood-brain barrier (such as certain NMDA receptor blockers); antibiotics, such as: aminoglycosides, e.g., amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin, fortimicin, gentamicin, isepamicin, kanamycin, micronomcin, neomycin, netilmicin, paromycin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin, trospectomycin; amphenicols, e.g., azidamfenicol, chloramphenicol, florfenicol, and theimaphenicol; ansamycins, e.g., rifamide, rifampin, rifamycin, rifapentine, rifaximin; beta.-lactams, e.g., carbacephems, carbapenems, cephalosporins, cehpamycins, monobactams, oxaphems, penicillins; lincosamides, e.g., clinamycin, lincomycin; macrolides, e.g., clarithromycin, dirthromycin, erythromycin, etc.; polypeptides, e.g., amphomycin, bacitracin, capreomycin, etc.; tetracyclines, e.g., apicycline, chlortetracycline, clomocycline, etc.; synthetic antibacterial agents, such as 2,4diaminopyrimidines, nitrofurans, quinolones and analogs thereof, sulfonamides, sulfones; antifungal agents, such as: polyenes, e.g., amphotericin B, candicidin, dermostatin, filipin, fungichromin, hachimycin, hamycin, lucensomycin, mepartricin, natamycin, nystatin, pecilocin, perimycin; synthetic antifungals, such as allylamines, e.g., butenafine, naftifine, terbinafine; imidazoles, e.g., bifonazole, butoconazole, chlordantoin, chlormidazole, etc., thiocarbamates, e.g., tolciclate, triazole, e.g., fluconazole, itraconazole, terconazole; anthelmintics, such as: arecoline, aspidin, aspidinol, dichlorophene, embelin, kosin, napthalene, niclosamide, pelletierine, quinacrine, alantolactone, amocarzine, amoscanate, ascaridole, bephenium, bitoscanate, carbon tetrachloride, carvacrol, cyclobendazole, diethylcarbamazine, etc.; antimalarials, such as: acedapsone, amodiaquin, arteether, artemether, artemisinin, artesunate, atovaquone, bebeerine, berberine, chirata, chlorguanide, chloroquine, chlorprogaunil, cinchona, cinchonidine, cinchonine, cycloguanil, gentiopicrin, halofantrine, hydroxychloroquine, mefloquine hydrochloride, 3-methylarsacetin, pamaquine, plasmocid, primaquine, pyrimethamine, quinacrine, quinine, quinine, quinocide, quinine, dibasic sodium arsenate; antiprotozoal agents, such as: acranil, timidazole, ipronidazole, ethylstibamine, pentamidine, acetarsone, aminitrozole, anisomycin, nifuratel, timidazole, benzidazole, suramin, and the like; genes (including expression vectors and/or promoters, preferably the GFAP- and/or γ-GTP promoters) encoding for polypeptides (preferably for Neprilysin and the proteins, peptides, enzymes, cytokines, interleukins, hormones and growth factors described herein above) or antisense DNA for polypeptides; and antisense probes (nucleic acids or peptide nucleic acids). In addition to direct conjugation between the therapeutic or diagnostic moieties and the targeting agent, such therapeutic or diagnostic moieties may be encapsulated within nanocontainers, such as nanoparticles, liposomes or nanogels, where the targeting agent is preferably covalently coupled to such a nanocontainer. Such conjugation to the nanocontainer may be either directly or via any of the well-known polymeric conjugation agents such as sphingomyelin, polyethylene glycol (PEG) or other organic polymers, and either with a single targeting agent or in combination with any of the well-known blood-brain barrier targeting moieties against the insulin, transferrin, IGF, leptin, LRP (1B) or LDL receptor on the blood-brain barrier and brain cell membrane. Details of producing such pharmaceutical compositions comprising targeted (PEG) liposomes are described in U.S. Pat. No. 6,372,250.
A large variety of methods for conjugation of targeting agents with therapeutic or diagnostic moieties are known in the art. Such methods are e.g., described in Hermanson (1996, Bioconjugate Techniques, Academic Press; U.S. Pat. No. 6,180,084 and U.S. Pat. No. 6,264,914, and include, e.g., methods used to link haptens to carriers proteins as routinely used in applied immunology (see Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). It is recognized that, in some cases, a targeting agent or a therapeutic or diagnostic moiety may lose efficacy or functionality upon conjugation depending, e.g., on the conjugation procedure or the chemical group utilized therein. However, given the large variety of methods for conjugation the skilled person is able to find a conjugation method that does not or least affects the efficacy or functionality of the entities to be conjugated.
Suitable methods for conjugation of a targeting agent with a therapeutic or diagnostic moiety include e.g., carbodiimide conjugation (Bauminger and Wilchek, 1980, Meth. Enzymol. 70: 151-9). Alternatively, a moiety can be coupled to a targeting agent as described by Nagy et al., Proc. Natl. Acad. Sci. USA 93:7269-73 (1996); and Nagy et al., Proc. Natl. Acad. Sci. USA 95:1794-99 (1998), each of which is incorporated herein by reference. Another method for conjugating that may suitable be used are e.g., sodium periodate oxidation followed by reductive alkylation of appropriate reactants and glutaraldehyde crosslinking.
A particularly advantageous method of conjugation may be applied when both the targeting agent and the therapeutic moiety are (poly)peptides. In such instances the two entities may be synthesized as a single (poly)peptide chain comprising the amino acid sequences of both the targeting agent and the therapeutic peptide. When the sum of the amino acid sequences of the targeting agent and the therapeutic peptide does not exceed 50, 80 or 100 amino acids the conjugate may be synthesized by solid phase peptide synthesis as herein described above. Alternatively, when the sum of the amino acid sequences is larger the single (poly)peptide chain comprising the targeting agent and the therapeutic peptide may be produced by recombinant expression techniques as outlined herein below. In such instances e.g., the two nucleic acid sequences encoding each the targeting agent and the therapeutic peptide may be operably linked in frame to form a single open reading frame. The nucleic acid sequence containing the single open reading frame may then be inserted in a suitable expression vector for expression in a suitable host from which the conjugate may then be recovered and optionally further purified as herein described below. In these methods, targeting peptides may be placed on either or both ends of the therapeutic peptide, or may be inserted within the amino acid sequence of the therapeutic peptide in one or more positions that do not disturb the function or efficacy of the respective peptides. Using routine methods the skilled person can establish the optimal position of the targeting peptide(s) with respect to the therapeutic peptide.
In a further aspect the invention relates to methods for diagnosing the status of the microvascular permeability in a subject. Such a method preferably comprises the steps of: (a) determining the expression level of a nucleic acid sequence encoding a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NOs:1-25 in the subject's microvascular endothelium; and, (b) comparing the expression level of the nucleic acid sequence with a reference value for expression level of the nucleic acid sequence, the reference value preferably being the average value for the expression level in the microvascular endothelium of healthy individuals. The expression level of the nucleic acid sequence may be determined indirectly by quantifying the amount of the LPSS polypeptide encoded by the nucleic acid sequence. In a preferred method, the expression level of more than one nucleic acid sequences are compared. When more than one nucleic acid sequence is analyzed this may conveniently be done using microarrays comprising complementary nucleic acids as described below and in the Examples. The expression level may be determined ex vivo in a sample obtained from the subject. The method preferably is a method for diagnosing a microvascular permeability disorder or for diagnosing a susceptibility to a microvascular permeability disorder, whereby the microvascular permeability may be as described above. The method may also be used to assess the efficacy of a treatment for restoration of the microvascular permeability.
In yet another aspect the invention relates to methods for identification of substances capable of modulating the permeability of microvascular endothelial cells. The method preferably comprises the steps of: (a) providing a test cell population capable of expressing one or more nucleic acid sequences encoding a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NOs:1-25; (b) contacting the test cell population with a composition comprising a substance to be tested; (c) determining the expression level of a nucleic acid sequence encoding a LPSS polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NOs:1-25, in the test cell population contacted with the substance; (d) comparing the expression of the nucleic acid sequence with the expression level of the nucleic acid sequence in a test cell population that is not contacted with the substance; and, (e) identifying a substance that produces a difference n expression level of the nucleic acid sequence, between the test cell population that is contacted with the substance and the test cell population that is not contacted with the substance. In the method the expression level of the nucleic acid sequence may be determined indirectly by quantifying the amount of the LPSS polypeptide encoded by the nucleic acid sequence. The expression level of more than one nucleic acid sequence may be compared. In a preferred method, the test cell population comprises endothelial cells, preferably vascular endothelial cells, more preferably microvascular endothelial cells, most preferably brain microvascular endothelial cells. The cells in the test cell population are preferably mammalian cells, preferably human cells. Preferably in the method, the test cell population that is contacted with the substance and the test cell population that is not contacted with the substance are derived from one cell population, preferably from one cell line, more preferably from one cell. In a further preferred method, the test cell population is co-cultured with a helper cell population, whereby the test cell population is cultured on one side of the filter and the helper cell population is cultured on the other side of the filter, and whereby the helper cell population preferably comprises astrocytes.
We disclose herein specifically differentially expressed polypeptides that are involved in decreased vascular permeability. We therefore refer to these polypeptides as “lipopolysaccharide-sensitive” polypeptides or LPSS polypeptides. LPSS polypeptides are involved in several different types of mechanisms involved in the control of blood-brain barrier functionality. These include secreted factors, signal transduction pathways, receptors and adhesion molecules, and metabolic enzymes. LPSS polypeptides and these mechanisms are discussed in greater detail below. If known or applicable, for each LPSS polypeptide the following information is given:
Extracellularly secreted or operational LPSS polypeptides (like hormones, enzymes, growth factors, cytokines, chemokines, binding proteins, etc.) are preferably used for specific modulation or monitoring of the permeability of the blood-brain barrier in the embodiments of the present invention. We have identified several of such novel specifically differentially expressed polypeptides, including pre-B-cell colony-enhancing factor, bone morphogenic protein 4, latent transforming growth factor beta binding protein 2, tumor necrosis factor α-induced protein 6, heparin-binding epidermal growth factor-like growth factor (diphtheria toxin receptor) and phospholipase A2, group VII. These are discussed in greater detail below, except for diphtheria toxin receptor (SEQ ID NO:22) and phospholipase A2, group VII (SEQ ID NO:23), which are discussed in different sections (Receptors and adhesion molecules and Metabolic enzymes, respectively).
The PBEF gene (SEQ ID NO:1; LPSS01), encoding pre-B-cell colony-enhancing factor, is upregulated in BCEC after an exposure for 2 hours to LPS in both BCEC monolayers and BCEC-astrocyte cocultures (Table 1 and Table 2). Upregulated LPSS polypeptides are involved in increased vascular permeability. Pre-B-cell colony-enhancing factor is a cytokine that acts on early B-lineage precursor cells. It increases the pre-B-cell colony formation activity of stem cell factor (MGF) and interleukin 7 (IL7). The surprising finding that the PBEF gene, or pre-B-cell colony-enhancing factor, was modified by LPS in the cells that constitute the blood-brain barrier has not been reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality. Therefore, any agent that changes the biological activity of the PBEF gene product (pre-B-cell colony-enhancing factor) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. Pre-B-cell colony-enhancing factor activity may be conveniently decreased by antisense inhibition of the PBEF gene, while pre-B-cell colony-enhancing factor activity may be conveniently increased by introduction of the PBEF gene into the cell or by exposure of the endothelial cells to exogenous pre-B-cell colony-enhancing factor. In addition, Ognjanovic et al. (2001, J Mol. Endocrinol. 26: 107-17) developed useful antibodies against pre-B-cell colony-enhancing factor, which may be used for diagnostic or treatment purposes. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. For this purpose both nucleic acids complementary to the PBEF gene as well as antibodies against the PBEF protein may be applied.
The BMP4 gene (SEQ ID NOs:2, 3 and 4; LPSS02), encoding bone morphogenic protein 4, is downregulated in BCEC after an exposure for 2 hours to LPS in BCEC-astrocyte cocultures (Table 1 and Table 2). Downregulated LPSS polypeptides are involved in increased vascular permeability. Bone morphogenic protein 4 (or bone morphogenetic protein 2B (BMP2B or BMP2B1), or ZYME) is a member of the bone morphogenetic protein family which is part of the transforming growth factor-beta superfamily. The superfamily includes large families of growth and differentiation factors. Bone morphogenetic proteins were originally identified by an ability of demineralized bone extract to induce endochondral osteogenesis in vivo in an extraskeletal site. This particular family member plays an important role in the onset of endochondral bone formation in humans, and a reduction in expression has been associated with a variety of bone diseases, including the heritable disorder Fibrodysplasia Ossificans Progressiva. Alternative splicing in the 5′ untranslated region of this gene has been described and three variants are described, all encoding an identical protein. The surprising finding that the expression of the BMP4 gene, or bone morphogenic protein 4, was modified by LPS in the cells that constitute the blood-brain barrier has not been reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality. Therefore, any agent that changes the biological activity of the BMP4 gene product (bone morphogenic protein 4) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. Bone morphogenic protein 4 activity may be conveniently decreased by antisense inhibition of the BMP4 gene or by exposure of the endothelial cells to the bone morphogenic protein 4 inhibitors noggin or chordin, while bone morphogenic protein 4 activity may be conveniently increased by introduction of the BMP4 gene into the cell or by exposure of the endothelial cells to exogenous bone morphogenic protein 4. In addition, R&D Systems Europe Ltd., UK offers useful antibodies against bone morphogenic protein 4, which may be used for diagnostic or treatment purposes. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. For this purpose both nucleic acids complementary to the BMP4 gene as well as antibodies against the BMP4 protein may be applied.
The LTBP2 gene (SEQ ID NO:13; LPSS06), encoding latent transforming growth factor beta binding protein 2, is upregulated in BCEC after an exposure for 2 hours to LPS in BCEC-astrocyte cocultures (Table 1 and Table 2). Upregulated LPSS polypeptides are involved in increased vascular permeability. Latent transforming growth factor beta binding protein 2 (formerly known as LTBP3) is involved in binding of tgf-beta in the extracellular-matrix. It serves as an important mechanism to regulate tgf-beta function. Mutations in LTBP2 have been identified in two instances of atypical Marfan Syndrome. The surprising finding that the expression of the LTBP2 gene, or latent transforming growth factor beta binding protein 2, was modified by LPS in the cells that constitute the blood-brain barrier has not been reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality. Therefore, any agent that changes the biological activity of the LTBP2 gene product (latent transforming growth factor beta binding protein 2) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. Latent transforming growth factor beta binding protein 2 activity may be conveniently decreased by antisense inhibition of the LTBP2 gene, while latent transforming growth factor beta binding protein 2 activity may be conveniently increased by introduction of the LTBP2 gene into the cell or by exposure of the endothelial cells to exogenous latent transforming growth factor beta binding protein 2. In addition, Elastin Products Company, Inc. (Missouri, USA) offers useful antibodies against latent transforming growth factor beta binding protein 2, which may be used for diagnostic or treatment purposes. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. For this purpose both nucleic acids complementary to the LTBP2 gene as well as antibodies against the LTBP2 protein may be applied.
The TNFAIP6 gene (SEQ ID NO:14; LPS S07), encoding tumor necrosis factor α-induced protein 6, is upregulated in BCEC after an exposure for 2 hours to LPS in both BCEC monolayers and BCEC-astrocyte cocultures (Table 1 and Table 2). Upregulated LPSS polypeptides are involved in increased vascular permeability. Tumor necrosis factor α-induced protein 6 (or tumor necrosis factor-stimulated gene-6 protein or TSG6, or hyaluronate-binding protein, or tumor necrosis factor-inducible protein 6, or tumor necrosis factor α-inducible protein 6) is a secretory protein that contains a hyaluronan-binding domain, and thus is a member of the hyaluronan-binding protein family. The hyaluronan-binding domain is known to be involved in extracellular matrix stability and cell migration. This protein has been shown to form a stable complex with inter-α-inhibitor (I α I), and thus enhance the serine protease inhibitory activity of I α I, which is important in the protease network associated with inflammation. The expression of this gene can be induced by tumor necrosis factor α, interleukin-1 and LPS in normal fibroblasts, peripheral blood mononuclear cells, synovial cells, and chondrocytes. The expression can also be induced by mechanical stimuli in vascular smooth muscle cells, and is found to be correlated with proteoglycan synthesis and aggregation. TNFAIP6 is similar to the adhesion receptor CD44. The surprising finding that the TNFAIP6 gene, or tumor necrosis factor α-induced protein 6, was modified by LPS in the cells that constitute the blood-brain barrier has not been reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality. Therefore, any agent that changes the biological activity of the TNFAIP6 gene product (tumor necrosis factor α-induced protein 6) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. Tumor necrosis factor α-induced protein 6 activity may be conveniently decreased by antisense inhibition of the TNFAIP6 gene or by the use of antibodies against the TNFAIP6 protein, while tumor necrosis factor α-induced protein 6 activity may be conveniently increased by introduction of the TNFAIP6 gene into the cell or by exposure of the endothelial cells to exogenous tumor necrosis factor α-induced protein 6. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. For this purpose both nucleic acids complementary to the TNFAIP6 gene as well as antibodies against the TNFAIP6 protein may be applied.
Polypeptides involved in intracellular signal transduction pathways are preferably used to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. We have identified several novel of such specifically differentially expressed polypeptides, including retinoblastoma-binding protein 6, calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma, macrophage myristoylated alanine-rich C kinase substrate, GTP-binding protein RHO6, phosducin isoform phosducin-like protein/orphan 1, calreticulin precursor and a G-protein-coupled receptor induced protein. These are discussed in greater detail below.
The RBBP6 gene (SEQ ID NO:5; LPSS03), encoding retinoblastoma-binding protein 6, is upregulated in BCEC after an exposure for 2 hours to LPS in BCEC-astrocyte cocultures (Table 1 and Table 2). Upregulated LPSS polypeptides are involved in increased vascular permeability. Retinoblastoma-binding protein 6 (or RBQ-1 or DKFZp761B2423) is a ubiquitously expressed nuclear protein. It is found among several proteins that bind directly to retinoblastoma protein which regulates cell proliferation. It interacts preferentially with under-phosphorylated retinoblastoma protein. The surprising finding that the expression of the RBBP6 gene, or retinoblastoma-binding protein 6, was modified by LPS in the cells that constitute the blood-brain barrier has not been reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality. Therefore, any agent that changes the biological activity of the RBBP6 gene product (retinoblastoma-binding protein 6) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. Retinoblastoma-binding protein 6 activity may be conveniently decreased by antisense inhibition of the RBBP6 gene or by the use of antibodies against the RBBP6 protein, while retinoblastoma-binding protein 6 activity may be conveniently increased by introduction of the RBBP6 gene into the cell or by exposure of the endothelial cells to exogenous retinoblastoma-binding protein 6. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. For this purpose both nucleic acids complementary to the RBBP6 gene as well as antibodies against the RBBP6 protein may be applied.
The CAMK2G gene (SEQ ID NOs:6, 7, 8, 9, 10 and 11; LPSS04), encoding calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma, is downregulated in BCEC after an exposure for 2 hours to LPS in BCEC-astrocyte cocultures (Table 1 and Table 2). Downregulated LPSS polypeptides are involved in increased vascular permeability. Calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma (or CAMK, CAMKG, CAMK-II, MGC26678) belongs to the Serine/Threonine protein kinase family, and to the Ca(2+)/calmodulin-dependent protein kinase subfamily. Calcium signaling is crucial for several aspects of plasticity at glutamatergic synapses. In mammalian cells the enzyme is composed of four different chains: α, beta, gamma, and delta. The product of this gene is a gamma chain. Six alternatively spliced variants that encode six different isoforms have been characterized to date. Additional alternative splice variants that encode different isoforms have been described, but their full-length nature has not been determined. The surprising finding that the expression of the CAMK2G gene, or calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma, was modified by LPS in the cells that constitute the blood-brain barrier has not been reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality. Therefore, any agent that changes the biological activity of the CAMK2G gene product (calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. Calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma activity may be conveniently decreased by antisense inhibition of the CAMK2G gene or by the use of antibodies against the CAMK2G protein, while calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma activity may be conveniently increased by introduction of the CAMK2G gene into the cell or by exposure of the endothelial cells to exogenous calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. For this purpose both nucleic acids complementary to the CAMK2G gene as well as antibodies against the CAMK2G protein may be applied. In addition, Bui et al. (2000, Cell 100(4): 457-67) generated transgenic mice expressing CaMKIIγB* (T287D), a partially calcium-independent mutant of CaMKIIγB, which may be useful to specifically investigate the blood-brain barrier in the embodiments of the present invention.
The MACMARCKS gene (SEQ ID NO:12; LPSS05), encoding macrophage myristoylated alanine-rich C kinase substrate, is upregulated in BCEC after an exposure for 2 hours to LPS in both BCEC monolayers and BCEC-astrocyte cocultures (Table 1 and Table 2). Upregulated LPSS polypeptides are involved in increased vascular permeability. Macrophage myristoylated alanine-rich C kinase substrate (also known as F52, or MARCKS-like protein or MLP or MLP1, or MARCKS-related protein, MRP) functions in coupling the calmodulin signal transduction and protein kinase C systems. It is involved in central nervous system development. The surprising finding that the MACMARCKS gene, or macrophage myristoylated alanine-rich C kinase substrate, was modified by LPS in the cells that constitute the blood-brain barrier has not been reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality. Therefore, any agent that changes the biological activity of the MACMARCKS gene product (macrophage myristoylated alanine-rich C kinase substrate) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. Macrophage myristoylated alanine-rich C kinase substrate activity may be conveniently decreased by antisense inhibition of the MACMARCKS gene or by antibodies against the MACMARCKS protein, while macrophage myristoylated alanine-rich C kinase substrate activity may be conveniently increased by introduction of the MACMARCKS gene into the cell or by exposure of the endothelial cells to exogenous macrophage myristoylated alanine-rich C kinase substrate. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. For this purpose both nucleic acids complementary to the MACMARCKS gene as well as antibodies against the MACMARCKS protein may be applied. In addition, Wu et al. (1996, Proc Natl Acad Sci USA, 93(5): 2110-2115) generated F52-deficient mice, which may be useful to specifically investigate the blood-brain barrier in the embodiments of the present invention.
The RHO6 gene (SEQ ID NO:15; LPSS08), encoding GTP-binding protein RHO6, is upregulated in BCEC after an exposure for 2 hours to LPS in both BCEC monolayers and BCEC-astrocyte cocultures (Table 1 and Table 2). Upregulated LPSS polypeptides are involved in increased vascular permeability. GTP-binding protein RHO6 (or round1, RND1) is involved in the regulation of the actin cytoskeleton and cell adhesion. RHO6 is highly similar to ARHE (or RND3, or Rho8, or RhoE). The surprising finding that the RHO6 gene, or GTP-binding protein RHO6, was modified by LPS in the cells that constitute the blood-brain bather has not been reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality. Therefore, any agent that changes the biological activity of the RHO6 gene product (GTP-binding protein RHO6) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. GTP-binding protein RHO6 activity may be conveniently decreased by antisense inhibition of the RHO6 gene or by antibodies against the RHO6 protein, while GTP-binding protein RHO6 activity may be conveniently increased by introduction of the RHO6 gene into the cell or by exposure of the endothelial cells to exogenous GTP-binding protein RHO6. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. For this purpose both nucleic acids complementary to the RHO6 gene as well as antibodies against the RHO6 protein may be applied.
The PDC gene (SEQ ID NOs:16 and 17; LPSS09), encoding phosducin isoform phosducin-like protein/orphan 1, is upregulated in BCEC after an exposure for 2 hours to LPS in BCEC-astrocyte cocultures (Table 1 and Table 2). Upregulated LPSS polypeptides are involved in increased vascular permeability. Phosducin (also known as phosducin-like protein or PhLP1, or phosducin, pineal gland, or G beta gamma binding protein, or 33 kDA phototransducing protein, or PHD, or MEKA) is located in the outer and inner segments of the rod cells in the retina. Phosducin may participate in the regulation of visual phototransduction or in the integration of photoreceptor metabolism. Phosducin modulates the phototransduction cascade by interacting with the beta and gamma subunits of the retinal G-protein transducin. Two alternatively spliced transcript variants have been described. One of the isoforms encoded by the variants, the phosducin-like orphan protein, does not bind the G protein. The phosducin protein and its isoform are also present in other tissues where they may participate in signal transduction pathways. The gene encoding this protein is a potential candidate gene for retinitis pigmentosa and Usher syndrome type II. The surprising finding that the expression of the PDC gene, or phosducin isoform phosducin-like protein/orphan 1, was modified by LPS in the cells that constitute the blood-brain barrier has not been reported earlier and offers new opportunities to modify or monitor blood-brain bather functionality. Therefore, any agent that changes the biological activity of the PDC gene product (phosducin isoform phosducin-like protein/orphan 1) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. Phosducin isoform phosducin-like protein/orphan 1 activity may be conveniently decreased by antisense inhibition of the PDC gene or by using antibodies against the PDC protein, while phosducin isoform phosducin-like protein/orphan 1 activity may be conveniently increased by introduction of the PDC gene into the cell or by exposure of the endothelial cells to exogenous phosducin isoform phosducin-like protein/orphan 1. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. For this purpose both nucleic acids complementary to the PDC gene as well as antibodies against the PDC protein may be applied.
The CALR gene (SEQ ID NO:18; LPSS10), encoding calreticulin precursor, is differentially downregulated in BCEC after an exposure for 2 hours to LPS in BCEC-astrocyte cocultures (Table 1 and Table 2). Differentially downregulated LPSS polypeptides between BCEC monolayers and BCEC-astrocyte cocultures are involved in the ability to recover from the LPS stimulation (
The C8FW gene (SEQ ID NO:19; LPSS11), encoding G-protein-coupled receptor induced protein, is differentially upregulated in BCEC after an exposure for 2 hours to LPS in BCEC-astrocyte cocultures (Table 1 and Table 2). Differentially upregulated LPSS polypeptides between BCEC monolayers and BCEC-astrocyte cocultures are involved in the ability to recover from the LPS stimulation (
Polypeptides functioning as membrane (signaling or internalizing) receptors or (signaling) adhesion molecules are preferably used to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. We have identified several novel of such specifically differentially expressed polypeptides, including chemokine (C—X—C motif) receptor 4, growth hormone receptor and diphtheria toxin receptor (heparin-binding epidermal growth factor-like growth factor). These are discussed in greater detail below.
The CXCR4 gene (SEQ ID NO:20; LPSS12), encoding chemokine (C—X—C motif) receptor 4, is downregulated in BCEC after an exposure for 2 hours to LPS in BCEC-astrocyte cocultures (Table 1 and Table 2). Downregulated LPSS polypeptides are involved in increased vascular permeability. Chemokine receptor 4 is a G protein-coupled receptor that binds CXC cytokines. It mediates intracellular calcium flux. Chemokine receptor 4 is involved in activation of MAPK, apoptosis, chemotaxis, histogenesis and organogenesis, immune response, inflammatory response, invasive growth, neurogenesis, response to viruses and virulence (it is a coreceptor for HIV-1 entry in cells). Depending on what properties were being studied, this protein has been called neuropeptide Y receptor Y3 (NPY3R); fusin; leukocyte-derived 7-transmembrane-domain receptor (LESTR); seven-transmembrane-segment receptor, spleen; lipopolysaccharide (LPS)-associated protein 3 (LAPS), among various designations (like HM89, NPYR, HSY3RR, NPYY3R, D2S201E). The surprising finding that the expression of the CXCR4 gene, or chemokine receptor 4, was modified by LPS in the cells that constitute the blood-brain barrier has not been reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality. Therefore, any agent that changes the biological activity of the CXCR4 gene product (chemokine receptor 4) is useful to specifically modulate the permeability of the blood-brain bather in the embodiments of the present invention. Chemokine receptor 4 activity may be conveniently decreased by antisense inhibition of the CXCR4 gene or by chemokine receptor 4 antagonists including antibodies against the CXCR4 protein, while chemokine receptor 4 activity may be conveniently increased by introduction of the CXCR4 gene into the cell or by exposure of the endothelial cells to exogenous chemokine receptor 4 agonists. To date, the following CXCR4 antagonists have been described: peptidic compounds (T22, T134, T140, ALX40-4C, CGP64222), bicyclam derivatives (AMD3100), neutralizing antibodies (12G5, 44717-111), and natural antagonists (HIV-1 tat protein) (Sachpatzidis et al., 2003, J Biol Chem 278: 896-907; De Clercq et al., 2001, Antivir Chem Chemother 12 Suppl 1:19-31; Tamamura et al., 1998, Biochem Biophys Res Commun 253:877-82). To date, the following CXCR4 agonists have been described: peptidic compounds (RSVM, ASLW), and natural agonists (stromal cell-derived factor 1a and beta (CXCL12) (Sachpatzidis et al., supra). In addition, R&D Systems Europe Ltd., UK offers recombinant human and mouse CXCL12 and useful antibodies against chemokine receptor 4 and its ligand CXCL12, which may be used for diagnostic or treatment purposes. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. For this purpose both nucleic acids complementary to the CXCR4 gene as well as antibodies against the CXCR4 protein may be applied.
The GHR gene (SEQ ID NO:21; LPSS13), encoding growth hormone receptor, is upregulated in BCEC after an exposure for 2 hours to LPS in BCEC-astrocyte cocultures (Table 1 and Table 2). Upregulated LPSS polypeptides are involved in increased vascular permeability. Biologically active growth hormone binds its transmembrane receptor (GHR), which dimerizes to activate an intracellular signal transduction pathway leading to synthesis and secretion of insulin-like growth factor I (IGF1). In plasma, IGF1 binds to the soluble IGF1 receptor (IGF1R). At target cells, this complex activates signal-transduction pathways that result in the mitogenic and anabolic responses that lead to growth. GHR is also known as growth hormone binding protein (GHBP), which is derived from the extracellular hormone-binding region of the GHR and GHBP remains bound to growth hormone in the circulation, and serves to stabilize growth hormone in the circulation. The surprising finding that the expression of the GHR gene, or growth hormone receptor, was modified by LPS in the cells that constitute the blood-brain bather has not been reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality. Therefore, any agent that changes the biological activity of the GHR gene product (growth hormone receptor) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. Growth hormone receptor activity may be conveniently decreased by antisense inhibition of the GHR gene, by growth hormone receptor antagonists (including high concentrations of growth hormone, which then become antagonistic) or by antibodies against the GHR protein, while growth hormone receptor activity may be conveniently increased by introduction of the GHR gene into the cell or by exposure of the endothelial cells to exogenous growth hormone receptor agonists (like growth hormone). Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. For this purpose both nucleic acids complementary to the GHR gene as well as antibodies against the GHR protein may be applied.
The DTR (or HEGFL) gene (SEQ ID NO:22; LPSS14), encoding diphtheria toxin receptor (or heparin-binding epidermal growth factor-like growth factor), is upregulated in BCEC after an exposure for 2 hours to LPS in BCEC-astrocyte cocultures (Table 1 and Table 2). Upregulated LPSS polypeptides are involved in increased vascular permeability.
Diphtheria toxin receptor (or known as HB-EGF, or heparin-binding EGF-like growth factor precursor, or diphtheria toxin sensitivity, DTS) is the receptor for diphtheria toxin (DT), a potent exotoxin produced by lysogenized strains of Corynebacterium diphtheriae. DT, a 58 kDa protein, is a multifunctional protein that kills susceptible mammalian cells. It is composed of two disulfide-linked protein fragments, both of which are required for the intoxication process. The A-fragment catalyzes the ADP-ribosylation of eukaryotic elongation factor 2, thereby inhibiting protein synthesis. The B-fragment is responsible for binding of the toxin to cells and is essential for facilitating the entry of the A-fragment into the cytosol. The existence of specific cell-surface DT receptors was first demonstrated in 1973, and it is now known that DT enters susceptible mammalian cells via receptor-mediated endocytosis. The initial step involves the binding of DT to the DTR, followed by internalization of the toxin:receptor complexes into coated pits and translocation of the A-fragment into the cytosol. In fact, after DT toxin binds to its cellular receptor, it is endocytosed, and while in this endocytic vesicle, it is exposed to an acidic pH environment. The acidic pH induces a structural change in the toxin molecule which provides the driving force for membrane insertion and translocation to the cytosol. Not all mammalian cells are equally sensitive to DT. For example, monkey kidney cells such as Vero cells, are highly sensitive, whereas e.g., human, rabbit, guinea pig and hamster cells are moderately sensitive and mouse and rat cells are resistant. Also chicken cells are sensitive to DT. As exemplified in example 2, DT is toxic to bovine BCEC in (sub)nanomolar range, in a concentration -and time dependent manner, after apical and basolateral exposure.
HB-EGF was originally identified in 1990 as a macrophage-secreted heparin binding growth factor. Like other members of the EGF family, HB-EGF exerts its biological effects by binding to the erb class of EGF receptor (EGF-R) molecules. HB-EGF activates two EGF receptor subtypes, HER1 and HER4 and binds to cell surface HSPG. However, unlike most members of the EGF family including EGF, HB-EGF binds heparin with a high affinity. Heparin appears to potentiate binding of HB-EGF to the signal-transducing EGF-R, and may also modulate the biologic effects of the growth factor on target cells, including cellular migration and proliferation. HB-EGF is mitogenic for fibroblasts, smooth muscle cells and epithelial cells, but not for endothelial cells. In addition, HB-EGF is produced by epithelial cells and acts as an autocrine growth factor for these cells. It is a heat-resistant, cationic protein, with a molecular weight of approximately 22 kDa that elutes from heparin-affinity chromatography columns with 1.0 M NaCl. HB-EGF gene expression is highly upregulated in response to for example oxidative, ischemic, osmotic (high glucose, or hyperosmolarity), electrical and mechanical (shear) stresses and after exposure to cytokines (TNF-α, IL-1beta, TGF-α), LPS, growth factors (EGF, HB-EGF, amphiregulin, bFGF, PDGF), lyso-Phosphatidylcholine, mercuric chloride, phorbol ester, Ca-ionophore, serum, thrombin, endothelin-1, angiotensin II, lipoprotein, platelet activation factor (PAF), α-adrenergic agonists, and transcription factors such as MyoD, Raf, v-Ha-ras. Soluble mature HB-EGF is proteolytically processed from a larger membrane-anchored precursor by matrix metalloproteinases (MMP's, in particular MMP-3) and ADAM's (a disintegrin and metalloprotease family, including ADAM9, ADAM10/Kuzbanian, ADAM12/meltrin-α, and ADAM17/TACE (TNF-α converting enzyme)). This process is called ectodomain shedding and is induced or upregulated by UV-light, IL-1beta, anisomycin, sorbitol, LPS, hydrogen peroxide, phenylephrine, endothelin-1, angiotensin II, insulin-like growth factor-1,12-O-tetradecanoylphorbol-13-acetate (TPA), via activation of protein kinase C delta and subsequent binding to the cytoplasmic domain of ADAM9/MDC9/meltrin-gamma, or by lysophosphatidic acid (LPA), via Ras-Raf-MEK and small GTPase Rac signalling pathways, or by a stress- and inflammatory cytokine-induced p38 MAPK-mediated pathway (Takenobu et al., 2003, J. Biol. Chem. 278: 17255-62; Umata et al., 2001, J. Biol. Chem. 276: 30475-82; Asakura et al., 2002, Nature Med, 8: 35-40). Pro-HB-EGF shedding is inhibited by MMP inhibitors like the hydroxamic acid-based KB-R8301, general MMP inhibitors (including TIMP's) and BB-94 (batimastat) and ADAM12 inhibitor KB-R7785 and the ADAM10 inhibitors XL784 and XL081, or analogues thereof. TPA-induced shedding is inhibited by the PKC inhibitor Ro-31-8220, LPA-induced shedding by the MEK antagonist PD98059, and p38 MAPK-mediated shedding by SB203580 (Takenobu et al., 2003, supra). Still, after the process of proteolytic cleavage from the membrane, a considerable amount of HB-EGF precursor remains uncleaved on the cell surface (Nakamura et al., 1995, J. Cell Biol. 129:1691-1705).
HB-EGF has been implicated as a participant in a variety of normal physiological processes such as blastocyst implantation and wound healing, and in pathological processes such as tumor growth, SMC hyperplasia and atherosclerosis. HB-EGF gene expression has been demonstrated in a variety of tissues, including vascular endothelial and smooth muscle cells, inflammatory cells (mostly NK-cells), skeletal and heart muscle, kidney mesangial cells, keratinocytes, small intestine, brain (neurons and glial cells), whole joints, trophoblasts, blastocysts, ovary and uterus, placenta, skin, lymph node, bladder and tumor cells (including glioma).
HB-EGF precursor has recently been found to function as the receptor for diphtheria toxin (Naglich et al., 1992, Cell 69: 1051-61). Although HB-EGF precursor is expressed in species including human, monkey, rat and mouse with a similar tissue distribution, rat and mice are resistant to DT because of an amino acid substitution in the sequence that is specifically recognized by DT (the receptor-binding domain for DT on HB-EGF) in humans and monkeys, that reduces binding of DT to rodent HB-EGF (Mitamura et al., 1995, J. Biol. Chem. 270:1015-19). Recently, AA141 (Glu141) has been shown to be a critical residue for DT-binding and toxin sensitivity (Hooper and Eidels, 1996, Biochem. Biophys. Res. Commun. 220: 675-680). Later, Mitamura et al., 1997, J. Biol. Chem. 272: 27084-90, discovered two additional critical residues for DT-binding and toxin sensitivity (AA115 (Phe115) and AA127 (Leu127)). Table 3 shows the sequence of the DT-receptor binding domain (AA106-147) of HB-EGF across different DT-insensitive (mouse, rat) and DT-sensitive (chinese hamster, rabbit, pig, monkey, human and chicken) species.
CIHGECRYLQE
RTPSCKCLPGYHG
RCHGLT
CIHGECRYLKELRIPSCHCLPGYHG
RCHGLT
Residues that differ from human are depicted in italic, and the residues depicted in bold are critical residues for DT-binding and toxin sensitivity (AA115 (Phe115), AA127 (Leu127) and AA141 (Glu141))
Heparin, heparan sulfate and heparan sulfate proteoglycans (HSPG), and other associate proteins like CD9/DRAP27 and α3-beta1-integrin, modulate DTR function by increasing DTR affinity for its ligands (as well as for HB-EGF binding to its receptor (Shishido et al., 1995, J. Biol. Chem. 270:29578-85)). Anti-CD9/DRAP27 monoclonal antibodies (IgG1: ALB-6 and TP82, and IgG2a: BU16 and 007, and MAB1206), inhibit the binding and toxicity of DT to human cells (Nakamura et al., 1995, supra; Mitamura et al., 1992, J. Cell Biol. 118: 1389-99; Iwamoto et al., 1991, J. Biol. Chem. 266: 20463-9).
The finding that the DTR gene (or diphtheria toxin receptor (or heparin-binding epidermal growth factor-like growth factor), was expressed on the cells that constitute the blood-brain barrier (as exemplified in examples 1 and 2) and that the biological activity of DTR was modified by disease stimuli (as exemplified for LPS in examples 1 and 3), heparin binding (as exemplified for exposure to exogenous heparin in example 3), antagonists (as exemplified for CRM197 (a competitive antagonist of DT) in example 2 and soluble HB-EGF (a non-competitive antagonist of DT) in examples 2 and 4), inhibitors of ectodomain shedding (as exemplified by exposure to the matrix metalloproteinase BB-94 (or batimastat) in example 3), or combinations thereof (as exemplified in example 3), offers new opportunities to specifically target drugs to and across the blood-brain barrier and/or to an intracellular compartment, particularly the lysosome. Any agent that changes the biological activity of the DTR gene product (i.e., heparin-binding epidermal growth factor-like growth factor) is useful to specifically modulate the targeting capacity to the blood-brain barrier in the embodiments of the present invention. Any ligand that specifically binds to (the receptor-binding domain of) DTR (like (parts of) DT, (parts of) the B-fragment of DT, (parts of) CRM197 (as exemplified in example 4), or any other ligand) is useful to target drugs to the blood-brain barrier in the embodiments of the present invention.
The general concept of the use of protein toxins (or non-toxic derivatives thereof) as carriers for e.g., peptides and proteins, across membranes and into the cytosol is not new (see for references on this subject the recent review of Sandvig et al. (2002, Annu. Rev. Cell Dev. Biol. 18: 1-24). DT, after binding to its receptor HB-EGF, is internalized by a process called receptor-mediated endocytosis. Receptor-mediated endo-/transcytosis is a well-known safe and effective cargo-carrying transport mechanism for the selective targeting of drugs to the brain (Pardridge, 2002, Nat. Rev. Drug Discov., 1:131-9). However, the use of specific ligands for DTR to carry drugs directly across the blood-brain barrier into the CNS by a mechanism involving receptor-mediated endo-/transcytosis, as has been described for e.g., the transferrin receptor, has never been appreciated earlier. In fact, only the non-toxic C fragment of the tetanus toxin protein (TTC or Tet451), and the non-toxic derivative of the tetanus toxin protein (Glu234 substitution by Ala), have been exploited to carry drugs into the CNS, however by a clearly distinctive mechanism of action (as compared to receptor-mediated endo-/transcytosis at the blood-brain bather), involving uptake into peripheral nerve endings followed by retrograde axonal transport to their cell body and trans-synaptic transfer to central neurons (described in U.S. Pat. No. 5,780,024 and in the references cited therein, and Li et al., 2001, J. Biol. Chem. 276: 31394-401). Even though constitutive and disease-induced HB-EGF expression in neurons, glial cells and blood vessels in the brain of rats was already described earlier by Mishima et al., 1996, Neurosci. Lett. 213: 153-6; Nakagawa et al., 1998, Dev. Brain Res. 108: 263-72; Hayase et al., 1998, Brain Res. 784:163-78; and Tanaka et al., 1999, Brain Res. 827:130-8. None of these authors appreciated the opportunity for the targeted delivery of drugs coupled to ligands of the DTR to (intracellular compartments in) these cells in the brain. This omission is best explained by the fact that rodent HB-EGF is not a receptor for DT. In fact, in rodents the permeability of DT across the microvasculature of brain tumors is equal to or less than the permeability of other large proteins (Wrobel et al., 1990, J. Neurosurg. 72:946-50), even though it could effectively kill tumor cells within the brain after passive diffusion (Arguello et al., 1996. Blood 87:4325-32). Therefore these studies only reported on the auto- and juxtacrine growth -and adhesion factor properties of HB-EGF. Even more surprisingly, neither renowned CNS drug delivery experts on receptor-mediated endo-/transcytosis (including Pardridge and Rapoport who published numerous scientific and review papers/books, patents and patent applications on the subject), nor the authors of the most recently published U.S. patent application “Covalent conjugates of biologically-active compounds with amino acids and amino acid derivatives for targeting to physiologically-protected sites,” Yatvin et al. (US2003-0087803), in which a comprehensive overview of the available technologies for drug delivery to the brain is given and which is included herein as reference, have appreciated the opportunity for the targeted delivery of drugs coupled to ligands of the DTR to (intracellular compartments in) cells in the brain.
The fact that DT is very toxic to susceptible mammalian cells makes drug targeting to the DTR with its natural ligand DT not preferable. However, the identification of non-toxic parts of DT, including (parts of) the B-fragment of DT, or (parts of) CRM197 (a non-toxic mutant protein of DT), have opened the door to the safe and effective targeting of drugs to the brain via the DTR. CRM197 is most preferred because polysaccharides conjugated to CRM197 have already been safely applied for human use to millions of people (babies, toddlers, adolescents and adults) in vaccination programs (e.g., Haemophilus influenzae type b oligosaccharide CRM197 conjugate vaccine (HibTiter™); Pneumococcal 7-valent Conjugate Vaccine (Prevnar™); meningococcal C oligosaccharide conjugate vaccines (Menjugate™ and Meningtec™)). From these indications it is known that the CRM197 protein is a safe and effective T-cell dependent carrier for saccharides. Likewise, CRM66 (cross-reactive 66 kDa protein) is an inactive mutant form of Pseudomonas aeruginosa exotoxin A, which specifically binds to the low density lipoprotein (LDL) receptor-related protein (LRP) and LRP 1B, and may be exploited to deliver drugs across the blood-brain barrier in a similar fashion as has been described for p97 (melanotransferrin) by Demeule et al. (2002, J. Neurochem. 83:924-33), which binds to the same receptor (WO03/009815).
CRM197 is produced by Corynebacterium diphtheriae infected by the nontoxigenic phage (beta197tox−) created by nitrosoguanidine mutagenesis of the toxigenic corynephage(β) (Uchida, et al. 1971, Nature New Biol. 233:8-11). The CRM197 protein has the same molecular weight as the diphtheria toxin but differs therefrom by a single base change (guanine to adenine) in the structural gene. This single base change causes an amino acid substitution (AA52: glutamic acid for glycine) in the mature protein and eliminates the toxic properties of diphtheria toxin.
Carrier proteins in vaccines were discovered based on the following concept. Vaccination with full (but inactivated) viruses or bacteria is effective but has many disadvantages and side-effects. For this reason, vaccination protocols with only the immunogenic parts (or mimics) of the viruses and bacteria (e.g., (poly)saccharides or capsular proteins) have been developed. However, such vaccines are least effective in the segment of the population most at risk for infections: B cell immunocompromised individuals, the elderly and infants younger than two years old who depend on T cell responses for immune protection. Since such vaccines are poor inducers of T-cell responses, conversion of the immunogenic parts of the viruses and bacteria into immunogens capable of inducing T cell responses is the key to producing adequate protection in this target population. It was discovered that linking the immunogenic parts of the viruses and bacteria to a suitable protein carrier, such as keyhole limpet hemocyanin (KLH), tetanus toxoid (TT), diphtheria toxoid (formalinized DT), bovine serum albumin (BSA), or human serum albumin (HSA), produced such an immunogen, by an nonspecific mechanism of action. In fact, the mechanism of action for diphtheria toxoid-conjugates was herein not linked to the effects mediated by binding to DTR on lymphocytes. To circumvent any remaining toxicity of diphtheria toxoid-conjugates, the diphtheria toxoid was later replaced by the non-toxic mutant of DT, CRM197. The notion that the carrier proteins are used on the basis of a nonspecific mechanism of action is further endorsed by the fact that the efficacy of CRM197-conjugate vaccines is routinely tested in DT-insensitive mice. Gupta et al. (1997, Vaccine 15:1341-3) showed that large differences in immunogenicity of CRM197-conjugated vaccines can be demonstrated between DT-insensitive mice and DT-sensitive guinea pigs, indicating that CRM197-conjugated vaccines do indeed make use of specific DTR-mediated cellular uptake in DT-sensitive species.
Neutralizing antibodies against (receptor-binding domain of) DT may be present in serum of the recipient because of earlier exposure to, or vaccination against Corynebacterium diphtheriae, (part of) DT (B-fragment) or (part of) CRM197 (or any other (part of) non-toxic DT) which may prevent the specific binding of drugs conjugated to CRM197 (or any other compound that binds specifically to the DT-binding domain) on the DTR, thereby reducing the overall efficacy of the drug delivery system. Such neutralizing antibodies are preferably inactivated prior to the application of the drug delivery system by exposure of the recipient to an effective, minimal amount of free CRM197, or any other compound that binds specifically to the DT-binding domain on the neutralizing antibody (like (part of) DT (B-fragment) or (part of) CRM197 fragments of CRM197, small molecules, peptides, mimetics, anti-idiotypic antibodies, etc.).
In addition, the surprising finding that the expression of the DTR gene, or diphtheria toxin receptor (or heparin-binding epidermal growth factor-like growth factor), was modified by LPS in the cells that constitute the blood-brain bather offers new opportunities to modify or monitor blood-brain barrier functionality. In addition, any agent that changes the biological activity of the DTR gene product (i.e., heparin-binding epidermal growth factor-like growth factor) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention. Diphtheria toxin receptor (heparin-binding epidermal growth factor-like growth factor) activity may be conveniently decreased by antisense inhibition of the DTR gene or by heparin-binding epidermal growth factor-like growth factor antagonists or by antibodies against the DTR protein, while diphtheria toxin receptor (heparin-binding epidermal growth factor-like growth factor) activity may be conveniently increased by introduction of the DTR gene into the cell or by exposure of the endothelial cells to exogenous heparin-binding epidermal growth factor-like growth factor. In addition, R&D Systems Europe Ltd., UK offers recombinant human HB-EGF and useful antibodies against HB-EGF, which may be used for diagnostic or treatment purposes. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. For this purpose both nucleic acids complementary to the HB-EGF gene as well as antibodies against the HB-EGF protein may be applied.
To greatly enhance the ease of experimentation and availability of relevant animal disease models in order to specifically study the permeability of the blood-brain barrier in the embodiments of the present invention, it is highly preferred that a human-like HB-EGF transgenic or knock-in (KI) mice is generated. Human-like HB-EGF transgenic mice can be genetically engineered by introducing the human DTR gene (encoding HB-EGF) under the control of a constitutively activated (e.g., tumor) promoter or a tissue specific promoter, preferably the GFAP and/or gamma-GTP promoters to obtain brain and/or cerebrovascular expression of the gene. Most preferred, however, is the introduction by homologous recombination in ES cells of a genetically engineered Hegfl gene (encoding the mouse HB-EGF gene under its endogenous promoter(s)) such that the exons 2 and 3 will contain the human sequences for the receptor-binding domain for diphtheria toxin, preferably containing positive and negative selection marker sequences. The coding sequences for the receptor-binding domain for diphtheria toxin are located at the end of exon 2 and the beginning of exon 3. To this end, mouse genomic DNA clones are derived from PAC libraries, preferably the pPAC4 library (129/SvevTACfBr strain). In the targeting vector the original second and third exon are replaced by the human specific sequences for the receptor-binding domain for diphtheria toxin by genetic engineering, creating a diphtheria toxin sensitive receptor-binding domain. Within intron 2 or downstream of exon 3, a PGK-driven neo cassette flanked by LoxP sites are present. Embryonic stem cells (E14) are electroporated and clones selected for homologous recombination by Southern blot analysis using external probes. The presence of the human sequences for the receptor-binding domain for diphtheria toxin are tested by PCR using human specific primers, preferably in addition to subsequent digestion with restriction enzyme, as well as by sequencing analysis of exons 2 and 3. Targeted ES cells are injected into blastocysts to create chimeric animals. F1 agouti progeny are genotyped for transmission of the mutant allele, generating transgenic line human-like HB-EGF+Neo. Heterozygous human-like HB-EGF+Neo mice are bred with mice of the EIIA-Cre strain (Lakso et al., 1996, Proc. Natl. Acad. Sci. USA. 93:5860-65) to remove the neo cassette. By these means, germline transmission is obtained and transgenic line human-like HB-EGF KI is established. Mice are further bred with C57BL/6J for five generations. Homozygous human-like HB-EGF KI and wt littermates are used for further analysis (˜97% C57BL/6J background). Then, in order to grow a xenogeneic tumor implant in the human-like HB-EGF KI mice, a number of immunodeficient mice are available for use in the invention. These mice, include but are not limited to, nude mice, scid mice and mice deficient in the rag-1 and rag-2 genes. Other animals with diverse types of immunodeficiency as a result of mutations of certain genetic loci can be found in the website immunology.tch.harvard.edu. These mice are crossed with the aforementioned human-like HB-EGF KI mice to produce progenies that are deficient in immune function, but do express the human-like HB-EGF. In addition, no immunogenic response (leading to e.g., neutralizing antibodies) against exogenous carrier proteins, like CRM197, is expected in these mice. Further, the aforementioned human-like HB-EGF KI (original and/or immunodeficient) mice is crossed with any of the knockout of transgenic/KI mice described in the art serving as disease models to produce progenies that both have the disease phenotype and are sensitive to exogenous carrier proteins, like CRM197. Likewise, transgenic rats and pigs can also be generated accordingly.
“Sequence identity” is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. “Similarity” between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A M, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D W, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A M et al., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M et al., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math. 48:1073 (1988).
Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g., the GCG program package (Devereux, J., et al., Nucl Acids Res 12:387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S F et al., J. Mol. Biol. 215:403-10 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S F, et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, et al., 1990, supra). The well-known Smith Waterman algorithm may also be used to determine identity.
Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-53 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-19 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the “Ogap” program from Genetics Computer Group, in Madison, Wis. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).
Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, supra); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group. Given above are the default parameters for nucleic acid comparisons.
Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called “conservative” amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gln or his; Asp to glu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; H is to asn or gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.
Polypeptides for use in the present invention can be prepared using recombinant techniques, in which a nucleotide sequence encoding the polypeptide of interest is expressed in suitable host cells. The present invention thus also concerns the use of a vector comprising a nucleic acid molecule or nucleotide sequence as defined above. Preferably the vector is a replicative vector comprising on origin of replication (or autonomously replication sequence) that ensures multiplication of the vector in a suitable host for the vector. Alternatively the vector is capable of integrating into the host cell's genome, e.g., through homologous recombination or otherwise. A particularly preferred vector is an expression vector wherein a nucleotide sequence encoding a polypeptide as defined above, is operably linked to a promoter capable of directing expression of the coding sequence in a host cell for the vector.
As used herein, the term “promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active under most physiological and developmental conditions. An “inducible” promoter is a promoter that is regulated depending on physiological or developmental conditions. A “tissue specific” promoter is only active in specific types of differentiated cells/tissues.
Expression vectors allow the LPSS polypeptides as defined above to be prepared using recombinant techniques in which a nucleotide sequence encoding the LPSS polypeptide of interest is expressed in suitable cells, e.g., cultured cells or cells of a multicellular organism, such as described in Ausubel et al., “Current Protocols in Molecular Biology”, Greene Publishing and Wiley-Interscience, New York (1987) and in Sambrook and Russell (2001, supra); both of which are incorporated herein by reference in their entirety. Also see, Kunkel, 1985, Proc. Natl. Acad. Sci. USA 82:488 (describing site directed mutagenesis) and Roberts et al., 1987, Nature 328:731-4 or Wells, J A et al., 1985, Gene 34:315 (describing cassette mutagenesis).
Typically, nucleic acids encoding the desired polypeptides are used in expression vectors. The phrase “expression vector” generally refers to nucleotide sequences that are capable of effecting expression of a gene in hosts compatible with such sequences. These expression vectors typically include at least suitable promoter sequences and optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression can also be used as described herein. DNA encoding a polypeptide is incorporated into DNA constructs capable of introduction into and expression in an in vitro cell culture. Specifically, DNA constructs are suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or can be introduced into a cultured mammalian, plant, insect, e.g., Sf9, yeast, fungi or other eukaryotic cell lines.
DNA constructs prepared for introduction into a particular host typically include a replication system recognized by the host, the intended DNA segment encoding the desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment. A DNA segment is “operably linked” when it is placed into a functional relationship with another DNA segment. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. Generally, DNA sequences that are operably linked are contiguous, and, in the case of a signal sequence, both contiguous and in reading phase. However, enhancers need not be contiguous with the coding sequences whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof.
The selection of an appropriate promoter sequence generally depends upon the host cell selected for the expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art (see, e.g., Sambrook and Russell, 2001, supra). The transcriptional regulatory sequences typically include a heterologous enhancer or promoter that is recognized by the host. The selection of an appropriate promoter depends upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g., Sambrook and Russell, 2001, supra). Expression vectors include the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment can be employed. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Russell (supra) and in Metzger et al. (1988) Nature 334: 31-6. For example, suitable expression vectors can be expressed in, yeast, e.g., S. cerevisiae, e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells and bacterial cells, e.g., E. coli. The host cells may thus be prokaryotic or eukaryotic host cells. The host cell may be a host cell that is suitable for culture in liquid or on solid media. The host cells are used in a method for producing a LPSS polypeptide as defined above. The method comprises the step of culturing a host cell under conditions conducive to the expression of the polypeptide. Optionally the method may comprise recovery the polypeptide. The polypeptide may e.g., be recovered from the culture medium by standard protein purification techniques, including a variety of chromatography methods known in the art per se.
Alternatively, the host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal, preferably a non-human animal. A transgenic plant comprises in at least a part of its cells a vector as defined above. Methods for generating transgenic plants are e.g., described in U.S. Pat. No. 6,359,196 and in the references cited therein. Such transgenic plants may be used in a method for producing a LPSS polypeptide as defined above, the method comprising the step of recovering a part of a transgenic plant comprising in its cells the vector or a part of a descendant of such transgenic plant, whereby the plant part contains the polypeptide, and, optionally recovery of the polypeptide from the plant part. Such methods are also described in U.S. Pat. No. 6,359,196 and in the references cited therein. Similarly, the transgenic animal comprises in its somatic and germ cells a vector as defined above. The transgenic animal preferably is a non-human animal. Methods for generating transgenic animals are e.g., described in WO 01/57079 and in the references cited therein. Such transgenic animals may be used in a method for producing a LPSS polypeptide as defined above, the method comprising the step of recovering a body fluid from a transgenic animal comprising the vector or a female descendant thereof, wherein the body fluid contains the polypeptide, and, optionally recovery of the polypeptide from the body fluid. Such methods are also described in WO 01/57079 and in the references cited therein. The body fluid containing the polypeptide preferably is blood or more preferably milk.
Another method for preparing polypeptides is to employ an in vitro transcription/translation system. DNA encoding a polypeptide is cloned into an expression vector as described supra. The expression vector is then transcribed and translated in vitro. The translation product can be used directly or first purified. Polypeptides resulting from in vitro translation typically do not contain the post-translation modifications present on polypeptides synthesized in vivo, although due to the inherent presence of microsomes some post-translational modification may occur. Methods for synthesis of polypeptides by in vitro translation are described by, for example, Berger & Kimmel, Meth Enzymol. 152, Guide to Molecular Cloning Techniques, Academic Press, San Diego, Calif., 1987.
Some aspects of the invention concern the use of expression vectors comprising the nucleotide sequences as defined above, wherein the vector is a vector that is suitable for gene therapy. Vectors that are suitable for gene therapy are described in Anderson 1998, Nature 392: 25-30; Walther and Stein, 2000, Drugs 60: 249-71; Kay et al., 2001, Nat. Med. 7: 33-40; Russell, 2000, J. Gen. Virol. 81: 2573-2604; Amado and Chen, 1999, Science 285: 674-6; Federico, 1999, Curr. Opin. Biotechnol. 10: 448-53; Vigna and Naldini, 2000, J. Gene Med. 2: 308-16; Marin et al., 1997, Mol. Med. Today 3: 396-403; Peng and Russell, 1999, Curr. Opin. Biotechnol. 10: 454-7; Sommerfelt, 1999, J. Gen. Virol. 80: 3049-64; Reiser, 2000, Gene Ther. 7: 910-3; and references cited therein.
Particularly suitable gene therapy vectors include Adenoviral and Adeno-associated virus (AAV) vectors. These vectors infect a wide number of dividing and non-dividing cell types. In addition adenoviral vectors are capable of high levels of transgene expression. However, because of the episomal nature of the adenoviral and AAV vectors after cell entry, these viral vectors are most suited for therapeutic applications requiring only transient expression of the transgene (Russell, 2000, supra) as indicated above. Preferred adenoviral vectors are modified to reduce the host response as reviewed by Russell (supra).
Generally, gene therapy vectors will be as the expression vectors described above in the sense that they comprise the nucleotide sequence encoding the LPSS polypeptide to be expressed, whereby the nucleotide sequence is operably linked to the appropriate regulatory sequences as indicated above. Such regulatory sequence will at least comprise a promoter sequence. Suitable promoters for expression of the nucleotide sequence encoding the polypeptide from gene therapy vectors include e.g., cytomegalovirus (CMV) intermediate early promoter, viral long terminal repeat promoters (LTRs), such as those from murine moloney leukemia virus (MMLV) Rous sarcoma virus, or HTLV-1, the simian virus 40 (SV40) early promoter and the herpes simplex virus thymidine kinase promoter.
Several inducible promoter systems have been described that may be induced by the administration of small organic or inorganic compounds. Such inducible promoters include those controlled by heavy metals, such as the metallothionine promoter (Brinster et al. 1982 Nature 296: 39-42; Mayo et al. 1982 Cell 29: 99-108), RU-486 (a progesterone antagonist) (Wang et al. 1994 Proc. Natl. Acad. Sci. USA 91: 8180-4), steroids (Mader and White, 1993 Proc. Natl. Acad. Sci. USA 90: 5603-7), tetracycline (Gossen and Bujard 1992 Proc. Natl. Acad. Sci. USA 89: 5547-51; U.S. Pat. No. 5,464,758; Furth et al. 1994 Proc. Natl. Acad. Sci. USA 91: 9302-6; Howe et al. 1995 J. Biol. Chem. 270: 14168-74; Resnitzky et al. 1994 Mol. Cell. Biol. 14: 1669-79; Shockett et al. 1995 Proc. Natl. Acad. Sci. USA 92: 6522-6) and the tTAER system that is based on the multi-chimeric transactivator composed of a tetR polypeptide, as activation domain of VP16, and a ligand binding domain of an estrogen receptor (Yee et al., 2002, U.S. Pat. No. 6,432,705).
The gene therapy vector may optionally comprise a second or one or more further nucleotide sequence coding for a second or further protein. The second or further protein may be a (selectable) marker protein that allows for the identification, selection and/or screening for cells containing the expression construct. Suitable marker proteins for this purpose are e.g., the fluorescent protein GFP, and the selectable marker genes HSV thymidine kinase (for selection on HAT medium), bacterial hygromycin B phosphotransferase (for selection on hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection on G418), and dihydrofolate reductase (DHFR) (for selection on methotrexate), CD20, the low affinity nerve growth factor gene. Sources for obtaining these marker genes and methods for their use are provided in Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory Press, New York.
Alternatively, the second or further nucleotide sequence may encode a protein that provides for fail-safe mechanism that allows to cure a subject from the transgenic cells, if deemed necessary. Such a nucleotide sequence, often referred to as a suicide gene, encodes a protein that is capable of converting a prodrug into a toxic substance that is capable of killing the transgenic cells in which the protein is expressed. Suitable examples of such suicide genes include e.g., the E. coli cytosine deaminase gene or one of the thymidine kinase genes from Herpes Simplex Virus, Cytomegalovirus and Varicella-Zoster virus, in which case ganciclovir may be used as prodrug to kill the IL-10 transgenic cells in the subject (see e.g., Clair et al., 1987, Antimicrob. Agents Chemother. 31: 844-9).
The gene therapy vectors are preferably formulated in a pharmaceutical composition comprising a suitable pharmaceutical carrier as defined below.
Some aspects of the invention concern the use of an antibody or antibody-fragment that specifically binds to a LPSS polypeptide of the invention as defined above. Methods for generating antibodies or antibody-fragments that specifically bind to a given polypeptide are described in e.g., Harlow and Lane (1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and WO 91/19818; WO 91/18989; WO 92/01047; WO 92/06204; WO 92/18619; and U.S. Pat. No. 6,420,113 and references cited therein. The term “specific binding,” as used herein, includes both low and high affinity specific binding. Specific binding can be exhibited, e.g., by a low affinity antibody or antibody-fragment having a Kd of at least about 10−4 M. Specific binding also can be exhibited by a high affinity antibody or antibody-fragment, for example, an antibody or antibody-fragment having a Kd of at least about of 10−7 M, at least about 10−8 M, at least about 10−9 M, at least about 10−10 M, or can have a Kd of at least about 10−11 M or 10−12 M or greater.
Peptide-like molecules (referred to as peptidomimetics) or non-peptide molecules that specifically bind to a LPSS polypeptide or a LPSS polypeptide receptor and that may be applied in any of the methods of the invention as defined herein may be identified using methods known in the art per se, as e.g., described in detail in U.S. Pat. No. 6,180,084 which incorporated herein by reference. Such methods include e.g., screening libraries of peptidomimetics, peptides, DNA or cDNA expression libraries, combinatorial chemistry and, particularly useful, phage display libraries. These libraries may be screened for agonists and antagonists of LPSS polypeptides or receptors thereof by contacting the libraries with substantially purified LPSS polypeptides, LPSS polypeptide receptors, fragments thereof or structural analogues thereof.
The invention further relates to a pharmaceutical preparation comprising as active ingredient a LPSS polypeptide, an antibody or a gene therapy vector as defined above. The composition preferably at least comprises a pharmaceutically acceptable carrier in addition to the active ingredient.
In some methods, the polypeptide or antibody of the invention as purified from mammalian, insect or microbial cell cultures, from milk of transgenic mammals or other source is administered in purified form together with a pharmaceutical carrier as a pharmaceutical composition. Methods of producing pharmaceutical compositions comprising polypeptides are described in U.S. Pat. Nos. 5,789,543 and 6,207,718. The preferred form depends on the intended mode of administration and therapeutic application.
The pharmaceutical carrier can be any compatible, non-toxic substance suitable to deliver the polypeptides, antibodies or gene therapy vectors to the patient. Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier. Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like, may also be incorporated into the pharmaceutical compositions.
The concentration of the LPSS polypeptides or antibodies of the invention in the pharmaceutical composition can vary widely, i.e., from less than about 0.1% by weight, usually being at least about 1% by weight to as much as 20% by weight or more.
For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
The LPSS polypeptides, antibodies or gene therapy vectors are preferably administered parentally. The polypeptide, antibody or vector for preparations for parental administration must be sterile. Sterilization is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The parental route for administration of the LPSS polypeptide, antibody or vector is in accord with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, intracranial, intrathecal, transdermal, nasal, buccal, rectal, or vaginal routes. The polypeptide, antibody or vector is administered continuously by infusion or by bolus injection. A typical composition for intravenous infusion could be made up to contain 10 to 50 ml of sterile 0.9% NaCl or 5% glucose optionally supplemented with a 20% albumin solution and 1 to 50 μg of the LPSS polypeptide, antibody or vector. A typical pharmaceutical composition for intramuscular injection would be made up to contain, for example, 1-10 ml of sterile buffered water and 1 to 100 μg of the LPSS polypeptide, antibody or vector of the invention. Methods for preparing parenterally administrable compositions are well known in the art and described in more detail in various sources, including, for example, Remington's Pharmaceutical Science (15th ed., Mack Publishing, Easton, Pa., 1980) (incorporated by reference in its entirety for all purposes).
For therapeutic applications, the pharmaceutical compositions are administered to a patient suffering from a microvascular permeability disorder in an amount sufficient to reduce the severity of symptoms and/or prevent or arrest further development of symptoms. An amount adequate to accomplish this is defined as a “therapeutically-” or “prophylactically-effective dose”. Such effective dosages will depend on the severity of the condition and on the general state of the patient's health. In general, a therapeutically- or prophylactically-effective dose preferably is a dose, which restores the microvascular permeability to the average levels found in normal unaffected healthy individuals.
In the present methods, the LPSS polypeptide or antibody is usually administered at a dosage of about 1 μg/kg patient body weight or more per week to a patient. Often dosages are greater than 10 μg/kg per week. Dosage regimes can range from 10 μg/kg per week to at least 1 mg/kg per week. Typically dosage regimes are 10 μg/kg per week, 20 μg/kg per week, 30 μg/kg per week, 40 μg/kg week, 60 μg/kg week, 80 μg/kg per week and 120 μg/kg per week. In preferred regimes 10 μg/kg, 20 μg/kg or 40 μg/kg is administered once, twice or three times weekly. Treatment is preferably administered by parenteral route.
Another aspect of the invention relates to microarrays (or other high throughput screening devices) comprising the nucleic acids, polypeptides or antibodies as defined above. A microarray is a solid support or carrier containing one or more immobilized nucleic acid or polypeptide fragments for analyzing nucleic acid or amino acid sequences or mixtures thereof (see e.g., WO 97/27317, WO 97/22720, WO 97/43450, EP 0 799 897, EP 0 785 280, WO 97/31256, WO 97/27317, WO 98/08083 and Zhu and Snyder, 2001, Curr. Opin. Chem. Biol. 5: 40-45). Microarrays comprising the nucleic acids may be applied e.g., in methods for analyzing genotypes or expression patterns as indicated above. Microarrays comprising polypeptides may be used for detection of suitable candidates of substrates, ligands or other molecules interacting with the polypeptides. Microarrays comprising antibodies may be used for in methods for analyzing expression patterns of the polypeptides as indicated above.
1.1.1 Isolation of bovine brain capillaries
Brain capillaries were isolated from bovine (calf) brain, obtained at the slaughterhouse from freshly killed animals. The brain was transported to the laboratory in ice-cold phosphate buffered saline (LPSS, 1.1 mM KH2PO4, 5.6 mM Na2HPO4 and 150 mM NaCl, pH 7.4). Meninges and white matter were removed and gray matter was collected in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% (v/v) heat inactivated (30 min at 56° C.) fetal calf serum (DMEM+S). The DMEM, formulated with high D-glucose (4.5 g/l), NaHCO3 (3.7 g/l) and HEPES (25 mM), contained extra MEM non-essential amino acids, L-glutamine (2 mM), streptomycin sulfate (0.1 g/l) and penicillin G sodium (100000 U/1). Blood vessel fragments were prepared by manual homogenization using a Wheaton homogenizer and subsequently trapped on 150 μm nylon meshes. The blood vessels were digested in collagenase CLS3 (210 U/ml), trypsin TRL (91 U/ml) and DNAse I (170 U/ml, final concentrations) in DMEM+S for 1 hour at 37° C. and subsequently filtered through a 200 μm nylon mesh. The brain capillary fraction was resuspended in freeze mix (fetal calf serum (FCS) with 10% (v/v) DMSO) and stored at −80° C.
1.1.2 Isolation of Astrocytes
Astrocytes were isolated from newborn Wistar rat pups (Harlan B. V., Zeist, The Netherlands). Isolated cortices were fragmented and incubated with 0.016% (w/v) trypsin-EDTA (final concentration) in DMEM (fully HEPES buffered (50 mM), without NaHCO3) in a shaking water bath (80 rpm, 30 minutes) at 37° C. The suspension was filtered through a 120 and 45 μm nylon mesh, respectively. The cell-suspension was cultured for 3 days in DMEM+S in 250 ml plastic tissue culture flasks (Greiner B. V., Alphen a/d Rijn, The Netherlands) in a humidified incubator (Napco Scientific Company, Tualatin, Oreg., USA) at 37° C. in a mixture of air with 10% CO2. Thereafter, the medium was refreshed every other day. After 7 days of culturing, other cells than astrocytes were removed by shaking the cultures in a shaking water bath (80 rpm) overnight at room temperature. Two days later the cultures were passaged with 0.05% (w/v) trypsin-EDTA in a split ratio of 1:3 to poly-D-lysine coated flasks (overnight stirred 10 μg/ml poly-D-lysine solution, air dried and washed with LPSS (3 times)). When confluent, astrocyte conditioned medium was collected, every other day, for 2-4 weeks, sterile filtered and stored at −20° C. For co-culture purpose, 2 weeks old cultures were passaged and stored in freeze mix in liquid nitrogen.
1.1.3 Differential Seeding of Brain Capillaries and Culture of BCEC
Brain capillaries were seeded in collagen (human placenta type IV, 10 μg/ml solution in 0.1% (v/v) acetic acid for 2 hours and washed with LPSS 3 times) and human plasma fibronectin (10 μg/ml solution in LPSS, 30 minutes) coated 250 ml plastic tissue culture flasks and allowed to adhere for 4 hours in the incubator. Thereafter, the culture medium was replaced with growth medium (DMEM+S with 50% (v/v) astrocyte conditioned medium, supplemented with 125 μg/ml heparin) and the outgrowing cells, predominately BCEC and some pericytes, were cultured at 37° C., 10% CO2.
1.1.4 Preparation of the In Vitro BBB on Filters
The in vitro BBB model was prepared on collagen coated (as above) Transwell polycarbonate filters (surface area: 0.33 cm2, pore-size: 0.4 μm, Corning Costar, Cambridge, Mass., USA). At about 70% confluence (day 4 or 5 after seeding of the brain capillaries), BCEC were passaged with trypsin-EDTA for endothelial cells (500 BAEE units porcine trypsin and 180 μg EDTA per ml) for approximately 1 minute, leaving the majority of pericytes still adhered to the substratum. BCEC and astrocyte co-cultures were prepared with astrocytes seeded on the bottom of the filter at a density of 45000 astrocytes per filter. Astrocytes were allowed to adhere to the bottom of the filter for 8 minutes, 2 or 3 days before BCEC were passaged. BCEC were seeded at a density of 30000 BCEC per filter. BCEC+astrocyte co-cultures were cultured to tight monolayers in DMEM+S supplemented with 125 μg/ml heparin for the first 2 days and in DMEM+S for the last 2 days. BCEC monolayers were cultured accordingly, but with 50% (v/v) astrocyte conditioned medium added to the culture medium.
1.2.1 Isolation of total RNA
In case of BCEC+astrocyte co-cultures, astrocytes were removed prior to BCEC RNA isolation by scraping the basolateral side of the Transwell filters. Total RNA was isolated from BCEC (containing <5% pericytes (Gaillard et al., 2001, supra)) using the RNeasy mini kit (Qiagen, Hilden, Germany). For this, the cell-culture medium was removed and replaced by 40 μl of lysis buffer per Transwell filter. Subsequently, lysates were resuspended and collected from multiple (12-18) Transwell filters, whereupon the manufacturer's recommended procedures for the isolation of total RNA from animal cells was followed. QIA shredders were used to homogenize cell lysates. When necessary, total RNA was concentrated with sodium acetate and ethanol.
1.2.2 Preparation of Labeled RNA
Subsequent protocols for the preparation of biotinylated cRNA from total RNA for Affymetrix GeneChip® gene expression analysis were performed according to the manufacturer's recommendations, as described in the Affymetrix GeneChip® Expression Analysis Manual (Affymetrix, Santa Clara, Calif., USA). Briefly, 6-16 μg of total RNA per sample was used for double-stranded cDNA synthesis using the Gibco BRL Superscript Choice System (Life Technologies, Rockville, Md., USA). A T7-dT24 primer and Superscript II reverse transcriptase (Life Technologies, Rockville, Md., USA) were utilized for first-strand synthesis. The second-strand synthesis involved E. coli DNA polymerase I (Life Technologies, Rockville, Md., USA). The double-stranded cDNA was then purified using phenol/chloroform extraction (utilizing phase lock gels (Eppendorf AG, Hamburg, Germany)) followed by precipitation with ammonium acetate and ethanol. Biotinylated cRNA was synthesized by in vitro transcription from cDNA using the BioArray HighYield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, N.Y., USA), by incubation at 37° C. for 5 hours. The labeled cRNA was then purified using the RNA cleanup protocol of the RNeasy mini kit (Qiagen, Hilden, Germany). Subsequently, 15-20 μg of labeled cRNA was fragmented by heating at 94° C. for 35 minutes in fragmentation buffer (40 mM Tris-acetate (pH 8.1), 125 mM KOAc, 30 mM MgOAc).
1.2.3 GeneChip® Hybridization
Labeled and fragmented cRNA was hybridized to the HG-U95Av2 and HG-U133A array (Affymetrix, Santa Clara, Calif., USA) under conditions recommended by the manufacturer. cRNA was first hybridized to a Test2 Chip (Affymetrix), to ensure the quality of the preparation. In brief, cRNA was diluted in hybridization mix (1×MES hybridization buffer, 100 μg/ml herring sperm, 50 μg/ml acetylated BSA, control oligonucleotide B2 and eukaryotic hybridization controls) denatured and then hybridized for 16 hours at 45° C. at 60 rpm. Following hybridization, arrays were washed and stained with streptavidin-phycoerythrin using the Affymetrix Genechip® Fluidics Station 400. Fluorescent signals on the arrays were measured using the Hewlett-Packard Affymetrix GeneArray® scanner.
Identification of “Lipopolysaccharide-Sensitive” Genes, Differentially Expressed in and Between BCEC-ACM Monolayers and BCEC-Astrocyte Cocultures
In earlier experiments (as detailed in Gaillard (2000a, supra), which is included as a reference), we found that astrocytes and inflammatory processes (mimicked by lipopolysaccharide, LPS) display opposing effects in our dynamic co-culture model of the BBB. Briefly, astrocytes increase barrier functionality, whereas LPS decreases it. Moreover, astrocytes bring about a recovery process from LPS which was not observed without the physical presence of astrocytes (i.e., in BCEC-ACM monolayers). Finally, this recovery process was dependent on protein synthesis, which indicates that specific gene transcription is involved. In
For the identification of the involved LPSS genes, and their involvement in the recovery process, four different cell culture conditions for our BCEC cultured from primary isolated brain capillaries from calf brain were used (as detailed in Gaillard et al., 2001, supra, which is included as a reference and briefly herein in “1.1 Cell Culture”): 1) BCEC monolayers on filter inserts in 50% ACM (
After 2 hours of exposure to LPS, BBB functionality was assessed by TEER across the filters using an electrical resistance system (ERS) with a current-passing and voltage-measuring electrode (Millicell-ERS, Millipore Corporation, Bedford, Mass., USA). TEER (Ohm·cm2) was calculated from the displayed electrical resistance on the readout screen by subtraction of the electrical resistance of a collagen coated filter without cells and a correction for filter surface area. TEER across collagen coated filters with only astrocytes on the bottom was close to zero (Gaillard et al., 2001, supra).
The average TEER across BCEC-ACM monolayers was 29.3±2.1 Ohm·cm2 (mean±standard error, n=12) before exposure to LPS and decreased to 21.2±4.2 Ohm·cm2 (mean±standard error, n=18) after 2 h of LPS exposure. See
The average TEER across BCEC-Astrocytes cocultures was 149.8±5.4 Ohm·cm2 (mean±standard error, n=18) before exposure to LPS and decreased to 65.5±2.1 Ohm·cm2 (mean±standard error, n=18) after 2 h of LPS exposure. See
For all experimental conditions (BCEC-ACM monolayers±LPS and BCEC-Astrocytes cocultures±LPS) the RNA isolation, labeling of cRNA and hybridization protocol was performed in triplo and all samples were analyzed on both the HG-U95Av2 and HG-U133A arrays. Affymetrix Microarray Suite 5.0 and Affymetrix Data Mining Tool 2.0 were used for primary analysis of the acquired intensity data. Microsoft Excel (Microsoft, USA) was used for further analysis. Global scaling, where the data of each chip is scaled to a user-defined target intensity, was performed to make experiments comparable. Genes that were designated as “absent” by Affymetrix Microarray Suite 5.0 in all samples were eliminated from further analysis. When applicable, only the genes that were designated as “present” or as “marginally present” in all three “triplo” samples were included for further analysis. Mann-Whitney tests were performed to identify genes that were statistically significant differentially expressed (between control BCEC-ACM monolayers and LPS-treated BCEC-ACM monolayers; between control BCEC-Astrocytes cocultures and LPS-treated BCEC-Astrocytes cocultures; between LPS-treated BCEC-ACM monolayers and LPS-treated BCEC-Astrocytes cocultures). Differences were considered to be statistically significant when p-value was <0.05. Furthermore, for the LPS effect between BCEC-ACM monolayers and BCEC-Astrocytes cocultures, fold changes were calculated based on the average intensity value (only changes of 2 fold or more were considered biologically relevant).
The identified genes are designated herein as “lipopolysaccharide-sensitive (LPSS)” genes, and coded accordingly (LPSS01-LPSS25) and presented in Table 1. Included are also the SEQ ID NOs, LPS effect (up, down, differentially expressed (dif+ or dif−)), the accession codes for reference in publicly accessible databases (RefSeq), the gene symbol, and a description (title or gene name) for the LPSS gene. For each identified LPSS gene, the specific result is presented in Table 2.
For the characterization of LPSS14 (DTR) on the blood-brain barrier, BCEC cultured from primary isolated brain capillaries from calf brain were used as monolayers on filter inserts with primary isolated newborn rat brain astrocytes cultured on the bottom side of the filter insert (
Every hour after exposure to DT, BBB functionality was assessed by TEER across the filters using an electrical resistance system (ERS) with a current-passing and voltage-measuring electrode (Millicell-ERS, Millipore Corporation, Bedford, Mass., USA). TEER (Ohm·cm2) was calculated from the displayed electrical resistance on the readout screen by subtraction of the electrical resistance of a collagen coated filter without cells and a correction for filter surface area. TEER across collagen coated filters with only astrocytes on the bottom was close to zero (Gaillard et al., 2001, supra). Effects on TEER were normalized for control treated filters and represented as such.
After apical exposure to 1 ng/ml up to 10 microgram/ml of DT, the TEER across BCEC-Astrocytes cocultures decreased in a concentration- and time dependent manner, while concentrations as low as 1 ng/ml were toxic after an overnight incubation period (
After basolateral exposure to 25 ng/ml up to 1000 ng/ml of DT, the TEER across BCEC-Astrocytes cocultures decreased in a concentration- and time dependent manner, while when compared to equimolar apical concentrations or amounts of DT these effects occurred about 1 hour earlier (
After apical exposure to 100 ng/ml DT which was preincubated with soluble HB-EGF, the toxic effect of DT on BCEC-Astrocytes cocultures decreased in a concentration dependent manner (
After BCEC were preincubated with CRM197, the toxic effect after apical exposure to 100 ng/ml DT on BCEC-Astrocytes cocultures decreased (
For the modulation of the biological activity of LPSS14 (DTR) on the blood-brain barrier, BCEC cultured from primary isolated brain capillaries from calf brain were used as monolayers on filter inserts with primary isolated newborn rat brain astrocytes cultured on the bottom side of the filter insert (
Every hour after exposure to DT, BBB functionality was assessed by TEER across the filters using an electrical resistance system (ERS) with a current-passing and voltage-measuring electrode (Millicell-ERS, Millipore Corporation, Bedford, Mass., USA). TEER (Ohm·cm2) was calculated from the displayed electrical resistance on the readout screen by subtraction of the electrical resistance of a collagen coated filter without cells and a correction for filter surface area. TEER across collagen coated filters with only astrocytes on the bottom was close to zero (Gaillard et al., 2001, supra). Effects on TEER were normalized for control treated filters and represented as such.
After BCEC were preincubated with heparin, the toxic effect after apical exposure to 100 ng/ml DT on BCEC-Astrocytes cocultures increased in a way that the effect took place about 1 hour earlier then untreated controls (
After BCEC were preincubated with LPS or BB94, the toxic effect after apical exposure to 100 ng/ml DT on BCEC-Astrocytes cocultures moderately increased in a way that the effect took place faster then in untreated controls (
For the assessment of the blood-brain barrier drug targeting ability via LPSS14 (DTR), BCEC cultured from primary isolated brain capillaries from calf brain were used as monolayers in 96-wells plates (as detailed in Gaillard et al., 2001, supra, which is included as a reference and briefly herein in “1.1 Cell Culture”). We used: 1) proteins (CRM197, BSA and holo-transferrin (TrF)) conjugated to horseradish peroxidase (HRP, a 40 kDa enzyme) in a 10:1 weight/weight ratio (
Proteins were conjugated to HRP by means of a HRP conjugation kit according to the manufacturers instructions (a Diagnostic International, San Antonio, Tex., USA). In addition, conjugated proteins were further purified on a HiPrep 16/60 column packed with Sephacryl S-200 HR matrix (Amersham Biosciences, UK).
Liposomes (100 nm) were prepared essentially according to Mastrobattista et al. (1999, Biochim. Biophys. Acta. 1419: 353-63) and consisted of EPC-35 and cholesterol in a 2:1 ratio, with 2.5% PEG2000-DSPE and 2.5% PEG2000-maleimide-PE, conjugated to about 3-60 CRM197 proteins per liposome. Briefly, after evaporation of organic solvents the lipid film was resuspended in HBS pH 6.5 containing 0.3 mg HRP per μmol PL and liposomes were extruded 3-5 times through a series of filters (200-50 nm). CRM197 was modified with a thiol group using SATA according to Bloemen et al. (1995, FEBS Lett. 357:140-4). CRM197 and SATA (1:8 molar ratio) were incubated for 1 h at room temperature under constant shaking. Free SATA was removed by centrifugation over 30 kDa cut-off filters (Vivaspin). Directly before coupling to PEG2000-maleimide-PE, the thiol group was activated (deprotected) by incubation with 0.1 M hydroxylamine (pH 7.4) for 45 min at room temperature. The amount and stability of the thiol groups was determined with Ellman's reagent (Ellman, 1959, Arch. Biochem. Biophys. 82:70-7). HRP-preloaded liposomes were coated with CRM197-conjugated PEG2000 according to the post-insertion method (Iden et al., 2001, Biochim Biophys Acta. 1513:207-16). Briefly, micelles of 2.5% CRM197-conjugated PEG2000-maleimide-PE and 2.5% PEG2000-DSPE were transferred into preformed HRP-loaded liposomes during a 2 h incubation at 40° C., before separation on a Sephadex CL4B column, followed by concentration using ultracentrifugation (60.000 g, 30 min, 10° C.). For specific experiments (as indicated for each example), CRM197 was replaced by BSA undergoing the herein above described protocol, serving as control liposomes. After preparation the phospholipid content was determined according to Fiske and Subarrow, and the protein content was determined with the Biorad protein assay (modification of Bradford). The CRM197-coated PEG-liposomes contained 3.3 proteins per liposome, where the BSA-coated PEG-liposomes contained 26.9 proteins per liposome. Furthermore size (112 nm for the CRM197-coated PEG-liposomes and 104 nm for the BSA-coated PEG-liposomes) and polydispersity (0.21 for the CRM197-coated PEG-liposomes and 0.08 for the BSA-coated PEG-liposomes) were determined by dynamic light scattering with a Malvern 4700 system (Malvern Ltd. Malvern, UK). The zeta potential (−18.6±0.7 for the CRM197-coated PEG-liposomes and −25.2±11.2 for the BSA-coated PEG-liposomes) was determined with a Malvern 3000 HSa Zetasizer (Malvern Ltd. Malvern, UK).
HRP activity of conjugated proteins, the HRP content of the liposomes and in cell lysate samples was detected using a standard colorimetric assay with the appropriate calibration curves. Cells and liposomes were lysed (after thorough washing of the cells with cold PBS) by 40 microliters of an aqueous solution of 0.1% Na-deoxycholate.
After BCEC were incubated with HRP-conjugated proteins corresponding to a concentration of 5 microgram/ml of un-conjugated HRP, the CRM197-HRP conjugate was preferably taken up by the BCEC when compared to BSA- and transferrin-HRP conjugates (
After BCEC were incubated with CRM197-HRP-conjugate (corresponding to a concentration of 5 microgram/ml of un-conjugated HRP) which was preincubated with 10 microgram/ml soluble HB-EGF, the specific uptake of the CRM197-HRP conjugate was completely inhibited, as compared to the a-specific uptake of BSA-HRP-conjugate (
After BCEC were incubated with HRP-loaded CRM197-coated PEG-liposomes corresponding to a concentration of 5 microgram/ml of free HRP, the 37° C. HRP-loaded CRM197-coated PEG-liposomes were actively taken up by the BCEC when compared to the uptake at 4° C. (
For the assessment of the blood-brain barrier drug targeting ability via LPSS14 (DTR) by transcytosis, BCEC cultured from primary isolated brain capillaries from calf brain were used as monolayers on filter inserts with primary isolated newborn rat brain astrocytes cultured on the bottom side of the filter insert (
BBB functionality was assessed by TEER across the filters using an electrical resistance system (ERS) with a current-passing and voltage-measuring electrode (Millicell-ERS, Millipore Corporation, Bedford, Mass., USA). TEER (Ohm·cm2) was calculated from the displayed electrical resistance on the readout screen by subtraction of the electrical resistance of a collagen coated filter without cells and a correction for filter surface area. TEER across collagen coated filters with only astrocytes on the bottom was close to zero (Gaillard et al., 2001, supra).
Proteins were conjugated to HRP by means of a HRP conjugation kit according to the manufacturers instructions (a Diagnostic International, San Antonio, Tex., USA). In addition, conjugated proteins were further purified on a HiPrep 16/60 column packed with Sephacryl S-200 HR matrix (Amersham Biosciences, UK).
Liposomes (100 nm) were prepared as described above. HRP conjugated to CRM197 or BSA, or HRP-loaded PEG-liposomes conjugated to either CRM197 or BSA, were added to the apical side of the filter insert and the filter was directly transferred into a fresh well containing warm (or cold for the HRP-conjugated proteins) 250 microliter hepes buffered DMEM+S. Every hour, up to 4 hours in total, this procedure was repeated in order to prevent possible re-endocytosis of HRP-conjugated proteins or HRP-loaded PEG-liposomes conjugated to either CRM197 or BSA by the abluminal side of the BCEC. Cumulated HRP activity of transcytosed HRP into the basolateral compartment was detected using a standard colorimetric assay with the appropriate calibration curves.
The average TEER across BCEC-Astrocytes cocultures increased from 149.8±5.4 Ohm·cm2 (mean±standard error, n=18) to 834±77 Ohm·cm2 (mean±standard error, n=24) after treatment with 8-4-CPT-cAMP and RO-20-1724. No difference in DT sensitivity was observed between cells un-treated and cell treated as such (data not shown).
After BCEC were incubated with HRP-conjugated proteins corresponding to a concentration of 5 microgram/ml of un-conjugated HRP, the CRM197-HRP conjugate was preferably transcytosed across the BCEC when compared to BSA-HRP conjugates (
After BCEC were incubated with HRP-loaded PEG-liposomes conjugated to either CRM197 or BSA corresponding to a concentration of 5 microgram/ml of free HRP, the CRM197-coated PEG-liposomes were preferably transcytosed across the BCEC when compared to the BSA-coated PEG-liposomes (
Brain uptake of CRM197 or holo-transferrin (TrF) conjugated to HRP (2:1 weight/weight ratio) was determined 1.5 h after an intracarotid bolus injection of the conjugates (corresponding to a concentration of 500 microgram/ml in 0.5 ml saline of un-conjugated HRP), and compared to an equal concentration of free HRP, in young male Guinea pigs (Dunkin-Hartley HsdPoc:HD, 250-300 g). The proteins were conjugated to HRP by means of a HRP conjugation kit according to the manufacturers instructions (α Diagnostic International, San Antonio, Tex., USA). In addition, conjugated proteins were further purified on a HiPrep 16/60 column packed with Sephacryl S-200 HR matrix (Amersham Biosciences, UK). Briefly, the animals were anesthetized with isoflurane inhalation (4% induction, 1-1.5% maintenance) in an air/oxygen mixture (2:1). A cannula was placed in the carotid artery for blood sample collection and drug administration. At 1.5 h after injection of the proteins, animals were deeply anesthetized with 4% isoflurane (1-2 min) and subsequently the whole animal (including the brain) was perfused with saline via the cardiac aorta (<5 min), to clear the blood vessels from blood. Directly after, the animal was decapitated and the brain was removed from the skull for further analysis. Only brains that were cleared from all blood (based on a visual inspection of the brain) were used for further analysis.
The cortex of perfused brains (and one non-perfused control brain) was dissected and weighted. Directly after, the cortex fragments were homogenized and one half volume of the homogenate was filtrated through an 120 micrometer nylon mesh. Both the filtrate (containing the vascular structures) and the eluent (containing the brain parenchymal cells), in addition to the full homogenate, were used to analyze the HRP activity of transcytosed HRP into the three brain cortex samples (i.e., full homogenates (designated “homogenate” in
Central cross-sections (ear to ear) of about 0.5 cm of the perfused brains (and one non-perfused control brain (i.e., no injections of HRP or HRP-conjugates)) were dissected and were directly snap-frozen in isopentane and stored till use at minus 80° C. The tissue sections were cut into 14 micrometer cryosections on a cryostat. Some sections were air fixed and HRP (or endogenous peroxidases in case of the non-perfused control brain) activity was stained directly by TMB (peroxidase substrate kit TMB, Vector Laboratories) for 30 min, washed in demi water for 5 min, and dehydrated in a series of ethanol and xylene (90% ethanol 2×1 min; 100% ethanol 2×1 min; xylene 2×1 min) and finally embedded in Entellan (Merck). Other sections were fixed in 4% paraformaldehyde (15 min), and CRM197 distribution in the brain was stained by immunohistochemistry for diphtheria toxin by mouse-anti-diphtheria toxin 1:10 (OBT0746, ImmunologicalsDirect.com), and a secondary HRP-goat-anti-mouse antibody 1:250 (Jackson Immunoresearch). This primary antibody was able to selectively stain CRM197 as well as CRM197-HRP conjugates in dot-blot pilot experiments, both on pure proteins samples and in the CRM197-HRP conjugated homogenate samples (data not shown). Endogenous peroxidases were blocked (20 min in PBS with 0.3% H2O2en 0.1% NaN3), and non-specific staining was prevented by 5% normal goat serum. HRP activity of the secondary antibody was stained by TMB (peroxidase substrate kit TMB, Vector Laboratories) for 10 min, washed in demi water for 5 min, and dehydrated in a series of ethanol and xylene (90% ethanol 2×1 min; 100% ethanol 2×1 min; xylene 2×1 min) and finally embedded in Entellan (Merck). No counterstaining was performed.
1.5 hours after guinea pigs were injected with HRP-conjugated CRM197 and TrF (both corresponding to a concentration of 500 microgram/ml of un-conjugated HRP), as well as with an equal concentration of free HRP, only HRP activity in all three brain cortex homogenate samples (i.e., full homogenates (designated “homogenate” in
After the cryosections of the non-perfused control brain was directly stained for endogenous peroxidase activity by TMB a distinct and strong staining pattern characteristic for blood vessels was observed throughout the whole section. A typical example for this pattern is shown in
Cryosections in which the CRM197 distribution in the brain was stained by immunohistochemistry for diphtheria toxin by mouse-anti-diphtheria toxin showed a faint homogeneously distributed pattern throughout the whole section (
When all exemplary methods are considered and combined with the available prior art regarding the DTR as is included herein, the following mechanism of action for the delivery of drugs into and across the blood-brain bather is proposed: Following specific binding of the B domain of the drug- or liposome-conjugated carrier protein (e.g., CRM197) to the diphtheria toxin receptor, the carrier protein/drug complex is endocytosed. Due to a pH shift-induced change in conformation in the carrier protein, the T domain of the carrier protein/drug complex is inserted into the membrane of the endosome, followed by subsequent translocation of the A domain (including the drug complex) into the cytosol. Thereafter, drug and carrier protein (cleaved of still conjugated) are transported across the blood-brain barrier. Since a part of the endosomes are likely to end up in lysosomes this mechanism of action is also useful as a means of delivering therapeutic agents to the lysosome. In this instance, conjugates with enzymes (e.g., an enzyme deficient in a patient with a lysosomal storage disease) are particularly of interest.
| Number | Date | Country | Kind |
|---|---|---|---|
| 03075390.9 | Feb 2003 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| 60446270 | Feb 2003 | US | |
| 60491522 | Aug 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10544991 | Aug 2005 | US |
| Child | 13188458 | US |
