WO2004078204A1 - Marqueurs de la permeabilite de la barriere sanguine et leurs methodes d'utilisation - Google Patents

Marqueurs de la permeabilite de la barriere sanguine et leurs methodes d'utilisation Download PDF

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WO2004078204A1
WO2004078204A1 PCT/US2003/018780 US0318780W WO2004078204A1 WO 2004078204 A1 WO2004078204 A1 WO 2004078204A1 US 0318780 W US0318780 W US 0318780W WO 2004078204 A1 WO2004078204 A1 WO 2004078204A1
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ttr
blood
bbb
protein
looβ
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PCT/US2003/018780
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Damir Janigro
Nicola Marchi
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The Cleveland Clinic Foundation
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Priority to EP03816193A priority patent/EP1551450A4/fr
Publication of WO2004078204A1 publication Critical patent/WO2004078204A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the blood brain barrier and blood cerebrospinal fluid barriers prevent many compounds in the blood stream from entering the tissues and fluids of the brain and central nervous system (“CMS"). It is generally recognized that nature provides these barriers to ensure a toxin free environment for neurologic function.
  • the blood brain barrier (“BBB”) is of great importance for the maintenance of a constant environment for optimal neurological function. Most metabolic substrates (i.e. sugars and amino acids) are hydrophilic, and traverse these barriers only by specific carrier-mediated transport systems. Other molecules traverse the barriers more freely.
  • BBB blood-brain barrier
  • Macromolecules such as polypeptides/protein, can cross an endothelial cell barrier primarily in three ways: between the cells through cell-cell junctions (paracellular pathway), through the EC, via pores (fused vesicles), or transcellularly via shuttling specific vesicles and receptors. Electron microscopic evidence suggests that macromolecules are shuttled across the endothelial barrier via vesicles.
  • CSF cerebrospinal fluid
  • Samples of CSF can be intra surgically taken from the ventricles or from the sub-arachnoid space in the brain.
  • An obvious limitation of intrathecal detection of blood brain-barrier intactness resides in the fact that sampling of CSF is invasive, and that the sample itself may be contaminated by the procedure.
  • a gradient in protein content exists from the brain to the lumbar cord.
  • concentrations of protein in segments distal to the site of CSF reduction are known to be much higher. It appears that, at least in part, the increased protein in the lumbar compartment of the CSF is due to a combination of protein secreted by parenchymal cells plus a small amount of protein leakage across the blood brain-barrier.
  • the present invention is directed to a peripheral marker, used alone or in combination with other markers, which is indicative of the permeability of the blood brain barrier (“BBB”) or blood cerebrospinal fluid barrier (“BCSFB”). and the blood cerebrospinal fluid barrier as BCSFB.
  • BBB blood brain barrier
  • BCSFB blood cerebrospinal fluid barrier
  • the term blood barrier may be used herein to refer to and include both BBB and BCSFB.
  • the present invention is based on monitoring or measuring a marker or markers of BBB and/or BCSFB permeability, particularly transthyretin TTR alone or in combination with other markers of neuronal disease or damage (e.g. S-lOO ⁇ ).
  • S-lOO ⁇ is primarily synthesized in the brain by the end feet process of the astrocytes and is quickly released from the brain in the blood when the BBB is disrupted.
  • S-lOO ⁇ has also been found in other tissues but at lower concentrations. Although the appearance of S-lOO ⁇ in plasma correlates well with BBB openings, S-lOO ⁇ has been shown to increase in plasma, CSF or both as a consequence of other pathologies not limited to the CNS.
  • S-lOO ⁇ may detect brain damage or indicate advanced metastasis in melanoma patients.
  • Transthyretin is a reliable marker of permeability of the BCSFB.
  • levels of TTR are determined and compared with a control sample (e.g., levels found in a normal population); changes above these baseline values being indicative of blood brain barrier dysfunction or permeability. Both the amount and form of the TTR protein may be used. TTR protein may also be observed as an indicator of brain damage, along with other markers of neuronal distress.
  • the embodiments of the present invention provide: methods; compositions; and kits for the diagnostic and prognostic evaluation of permeability of the BCSFB and/or the BBB. Additionally, certain embodiments of the present invention may be utilized to identify compromise of the blood-CSF barrier rather than the blood brain barrier and vice- versa. These embodiments are particularly useful in the identification of subjects possessing a predisposition to passing or preventing agents from passing the BCSFB or BBB, and for monitoring patients undergoing treatment involving the integrity of the BCSFB or BBB, based on the detection of increased levels of TTR protein expression in a blood derived sample of subjects alone or in combination with other neuronal markers.
  • the present invention provides for a method for diagnosis of blood barrier permeability by means of detecting levels of TTR protein in a sample of biological fluid, preferably a blood sample.
  • the method for diagnosis can include detecting the levels of markers of neuronal distress, suitable markers being, for example, NSE and GFAP, and albumin.
  • suitable markers being, for example, NSE and GFAP, and albumin.
  • comparisons can be made with BBB function vis-a-vis the levels of S-lOO ⁇ in the sample.
  • the level of TTR protein detected can be used as a measure of BCSFB permeability.
  • the TTR protein is preferably detected using an immunoassay, such as an immunoprecipitation assay, but can also be detected by bi- dimensional gels or other means of detecting TTR.
  • Another embodiment of the present invention provides for detecting onset of neuronal distress in a patient by identifying elevated levels of TTR protein in the biological fluid, such as blood or serum. Additionally, the method for detection can include detecting the levels of markers of neuronal distress, combined with alternative methods to measure BBB function (S-lOO ⁇ ) alone or in conjunction with suitable markers of neuronal distress including NSE, GFAP and MBP.
  • BBB function S-lOO ⁇
  • Another embodiment of the present invention provides for a method of treating a patient in need thereof with a therapeutic agent.
  • the method includes the steps of administering an agent which opens the BBB or BCSFB and verifying elevated levels of TTR protein in the blood (alone or in combination with other suitable markers).
  • the therapeutic agents that may be employed include chemotherapeutics, pharmaceuticals, neuropharmaceuticals, potential neuropharmaceuticals, and other neurologically active agents.
  • Another embodiment of the present invention comprises assays developed to detect the level of TTR proteins in a subject's serum sample.
  • assays include immunoassays wherein the TTR proteins are detected by their interaction with anti-TTR specific antibodies.
  • TTR antibodies or fragments of antibodies may be used to quantitatively detect the presence of TTR proteins in a serum sample.
  • the embodiments may also involve the use of the TTR protein antigens in immunoassays designed to detect the presence of serum autoantibodies to the TTR protein antigens.
  • immunoassays can be utilized to determine the permeability of the BCSFB and BBB.
  • the monitoring of TTR levels can be used prognostically to stage progression of the disease or the treatment.
  • kits for diagnosis and prognosis of blood barrier integrity in a subject includes a component for detecting a peripheral marker of blood barrier integrity.
  • the component is utilized for the purpose of detecting the presence of TTR protein in a blood sample, where elevated levels of TTR protein is indicative of blood barrier opening.
  • the kit may also be designed to localize the area of permeability or rule out disruption of a particular portion of the blood barrier (e.g. BBB disruption).
  • kits which will be conveniently used in clinical settings, to diagnose or monitor patients with impaired BBB or BCSFB integrity.
  • the kits will also be utilized to monitor the efficacy of compounds used for treatment of diseases associated with BBB or BCSFB integrity or lack thereof.
  • Figure 1 is a diagrammatic representation of the different distribution between S-lOO ⁇ and TTR in the brain.
  • Figure 2 is a 2D gel electrophonesis of human blood illustrating the detection of penetrating BBB markers.
  • Figure 3 illustrates a time course of serum protein changes after BBB disruption.
  • Figure 4 illustrates the immunological of protein changes induced by BBB disruption.
  • Figure 5 illustrates non-SDS separation of pre-BBBD and post-BBBD samples.
  • sample it is meant a volume of fluid or tissue, such as blood or CSF, but preferably blood which is obtained at one point in time.
  • a sample can be as little as 2.5 mL (or less) taken from the subject.
  • all the markers can be measured with one assay device or by using a separate assay device for each marker, in which case aliquots of the same fluid sample can be used or different fluid samples can be used.
  • the analyses should be carried out within some short time frame after the sample is taken, e.g., within about one-half hour, so the data can be used to prescribe treatment as quickly as possible.
  • above normal and above threshold are used herein to refer to a level of TTR that is greater than the level of TTR observed in normal individuals in a given environment, that is, individuals who are not undergoing an event, i.e. an opening of a blood barrier. These terms contemplate a level that is significantly above the normal level found in individuals.
  • significantly refers to statistical significance.
  • the assay method by which the analysis for the TTR protein is carried out must be sufficiently sensitive to be able to detect the level of the marker which is present over the concentration range of interest and also must be highly specific. Ranges of TTR should be detectable from about 0.001 mg/L to about 100 mg/L in blood.
  • the assay devices used according to the invention can be arranged to provide a semi-quantitative or a quantitative result.
  • the term "semi-quantitative" is meant to reflect the ability to discriminate between a level which is above the elevated marker protein value, and a level which is not above that threshold.
  • the statistical methods used herein generally present data as means ⁇ SEM ANONA was needed to determine significance. Origin 7.0 (Microcal) was used for statistical analysis.
  • the barrier that separates the blood from the cerebral interstitial fluid is defined as the BBB, while the one that separates the blood and cerebrospinal fluid (BCSFB) discontinues the circulation between the blood and cerebrospinal fluid.
  • BBB is made of endothelial cells
  • the blood-to-CSF barrier consists of epithelial cells.
  • both CNS barriers are impermeant to macromolecules, thus hampering passage of proteins from the blood to the brain/CSF and vice versa.
  • CSF protein content is significantly lower than plasma.
  • plasma proteins leak into the CSF.
  • BBB openings may have both therapeutic and etiologic significance, since a correlation between severity of symptoms and BBB function has been suggested and given the fact that promising therapies based on brain-derived proteins have failed largely because of poor penetration of these protein compounds across the BBB.
  • Protein profiles from blood samples obtained from patients undergoing iatrogenic BBB disruption (BBBD) with intrarterial hyperosmotic mannitol were compared with pre-BBB opening serum.
  • BBBD iatrogenic BBB disruption
  • TTR transthyretin
  • TTR Protein gel electrophoresis and immunodetection confirmed that TTR was indeed extravasated in its monomeric form when CNS barriers were breached.
  • CSF cerebrospinal fluid
  • the Cleveland Clinic Brain Tumor Institute provides a treatment called blood- brain barrier disruption for primary central nervous system lymphomas, primitive neuroectodermal tumors, some gliomas, CNS germinoma and some metastatic brain tumors (such as breast, small cell lung or germ cell). All procedures were performed after informed consent was obtained using protocols approved by the Cleveland Clinic Foundation Institutional Review Board. In this protocol, intrarterial mannitol (1.4 M) is administered via a carotid or vertebral artery, and BBBD is confirmed by CT immediately after chemotherapy.
  • Sucrose gradient separation was performed to divide proteins by molecular weight. 10 ml of discontinuous 10%-25%-40% gradient and 200 ⁇ l of sample (15 ⁇ of serum, 125 ⁇ l of gradient buffer) were used. The upper fraction was collected after 16 hours of centrifugation at 4°C (225,0000 x g). The low molecular weight fraction was filtered with a 3 kDa molecular weight cut-off (Amicon Centricon YM 3000) for 6 hours (5800 x g) to remove sucrose. Both SDS and NO-SDS-PAGE were used. Non-SDS-PAGE samples were analyzed in non-denaturing condition.
  • TTR protein was identified by Western blotting techniques. Serum samples were obtained from the BBBD procedures and protein were probed overnight at 4°C with primary TTR rabbit anti-human antibody (1:1000; Dako). Protein concentration was estimated according to the Bradford assay method. Relative expressions of proteins were determined by densitometric analysis using Scion Image Software. This approach was used to quantify the TTR and haptoglobin data shown in Figure 3. Radial Immunodiffusion (RID) was used to quantitatively determine TTR in serum. Prefabricated immunodiffusion plates were purchased from Kent Laboratories, Inc. (Bellingham, WA). Experiments were performed as recommended by the vendor.
  • RID Radial Immunodiffusion
  • LC-MS liquid chromatography-mass spectroscopy
  • Finnigan LCQ-Deca ion trap mass spectrometer system with a Protana microelectrospray ion source interfaced to a self -packed 10 cm x 75 um inner diameter Phenomenex Jupiter C18 reversed-phase capillary chromatography column was used.
  • Data were analyzed by using all collision-induced dissociation spectra collected in the experiment to search the National Center for Biotechnology Information non-redundant database with the search program TurboSequest. All matching spectra were verified by manual interpretation. The interpretation process was also aided by additional searches using the programs Mascot and Fasta, performed as needed.
  • Transthyretin represents a disproportionate fraction (25%) of CSF protein, prompting the suggestion that it is either selectively transported across the blood-CSF barrier or synthesized de novo within the CNS. It has been demonstrated that the latter is the case and that the epithelial cells of the choroid plexus are the site of synthesis in both rats and humans.
  • TTR shows high-affinity binding to plasma retinol-binding protein and is involved in the transport of tyrosine in to the brain. TTR variants have been implicated in a variety of human disorders, including Alzheimer's dementia.
  • TTR is usually present in its tetrameric form and originates from liver secretion. About 40% of plasma TTR circulates in a tight protein-protein complex with the plasma retinol-binding protein (RBP). TTR is synthesized by choroid plexus epithelial cells, and subsequently released into the CSF. Plasma TTR is present in the homotetramer form, while its CSF form is primarily monomeric. In contrast to the BBB marker S- lOO ⁇ , TTR is not expressed by perivascular astrocytes or any other cells in the brain parenchyma.
  • RBP plasma retinol-binding protein
  • FIG. 1 illustrates different distribution between S-lOO ⁇ and TTR in the brain.
  • S-lOO ⁇ is synthesized primarily by the astrocytes surrounding the BBB
  • TTR is synthesized by the choroids plexuses and is found in the ventruicular CSF. This topographic segretation may explain the different roles of these markers.
  • Transthyretin is a candidate marker for blood-to- CSF barrier dysfunction, in a manner similar to S-lOO ⁇ in its relationship to the BBB proper.
  • Transthyretin has broad utility in the management or diagnosis of disorders such as hydrocephalus, meningitis, and other cerebrovascular disorders.
  • the BBB is a continuous, tight-junctioned, endothelial cell layer.
  • the endothelial layer actually consists of two separate cell membranes, one on the inside of the vessels (luminal) and one on the outside (abluminal) separated by 300-500 nm of thick cytoplasm.
  • the endothelial cells are only one part of a "BBB complex", which consists of astrocytes, pericytes, microglia, and neurons. All of these cell types play a role in the induction and maintenance of the specialized BBB endothelium.
  • the microvascular endothelium shares a common basement membrane with astrocytes and pericytes. Beyond the basement membrane in parenchymal vessels of the brain lies a close investment of end feet from neuroglial cells, predominantly astrocytes. Astrocytes and their processes invest more than 90% of endothelial capillaries and their end feet are projected tightly around the endothelial cells. Therefore, the glial end feet are a natural candidate to mediate a communication link between neurons and capillaries. Recent evidence has shown that shear stress promotes the expression of numerous genes involved in various aspects of endothelial cell function.
  • the BBB is not a static organ, and numerous factors affect BBB permeability.
  • Majno and Palade showed that after exposure of tissue to histamine, carbon particles injected into the blood compartment entered the parenchyma (tissue) selectively via post capillary venule endothelial cells ("ECs"). Moreover, gaps were occasionally seen.
  • Majno et al observed that the nucleus of these ECs had a wrinkled appearance and postulated that contraction of the EC was the basis for the increased extravasation of macromolecules. This insightful observation is supported by more recent findings and constitutes the rationale for "osmotic opening" of the BBB.
  • the BBB is of great importance for the maintenance of a constant environment for optimal CNS function.
  • the macromolecules may be taken up, transported through the endothelial cells, and ultimately released on the abluminal side of the vessel (brain parenchyma).
  • the "blood brain barrier organ” is constituted not only by endothelial cells, but glial end feet as well.
  • Current understanding of the mechanism of neuro-immunological interactions of specialized cells with the blood brain barrier has also suggested involvement of both perivascular pericytes and microglia as active components of the blood brain barrier. Under neuro-pathological conditions, both perivascular astrocytes and blood brain barrier endothelial cells undergo significant changes.
  • the cerebral endothelium plays an active part in the disease process with the BBB becoming disrupted, or modified, in such a way there is a dramatic increase in vascular permeability.
  • BBB dysfunction may be a cause or consequence of a particular disease process.
  • Diseases in which increased BBB permeability have been reported include neoplasia, ischemia, hypertension, dementia, epilepsy, infection, multiple sclerosis, and trauma.
  • TTR appears to be being released from the cerebrospinal fluid may explain why its appearance in plasma delayed compared to other markers directly available in the perivascular space. This allows for an embodiment of the present invention, wherein TTR is used to measure BBB permeability or BCSFB permeability. The late appearance would appear to be particularly useful in detecting or diagnosing BBB permeability if this marker is used in conjunction with other fast-released protein such as S- lOO ⁇ .
  • TTR In its tetrameric form, TTR is in plasma at levels much higher than CSF. According to the considerations listed above, one would expect TTR to behave, in the presence of a leaky BBB as albumin, thus promptly moving from the blood to the brain compartment.
  • the CSF contains high levels of the monomeric, low molecular weight form of TTR. It is this form that preferably allows peripheral determinations of BBB intactness in the serum. It may also be possible to detect differential amounts of the tetramer in the blood.
  • a peripheral marker of blood barrier permeability should include most of the following properties: plasma levels in control subjects must be exceedingly low or undetectable; similarly, under normal conditions CSF levels must be constant or, ideally, low; significant increases of the marker's concentrations must occur at early stages of neuronal distress; CSF changes must be reflected by comparable changes in plasma levels.
  • TTR monomer would appear to be a suitable peripheral marker in reflecting BBB permeability and/or neuronal damage with regard to those characteristics. Blood samples from three patients affected by primary brain lymphoma who underwent monthly hemispheric BBBD by intrarterial infusion of 1.4 M mannitol before receiving intrarterial methotrexate were tested.
  • Blood samples (29 total) were obtained at four times for each hemispheric disruption: after anesthesia induction, 45 seconds after mannitol infusion, approximately 45 seconds after methotrexate infusion, and during recovery in the neurointensive care unit (4-5 hours after the procedure).
  • Two-dimensional (“2-D”) electrophoresis was used to compare human serum samples obtained from BBBD protocols to detect changes in protein content before and after opening the blood-brain barrier.
  • Figure 2 shows the results of a typical experiment. Care was taken to ensure that an equal amount of protein was loaded on each gel. A quantitative analysis was performed to confirm that gels prepared with pre-BBBD and post-BBBD were comparable. To this end, a comparison spot corresponding to haptoglobin (18 kDa, pi 5.4-6), was used as internal control.
  • FIG. 2 illustrates detection of putative BBB markers by 2D gel electrophoresis of human blood proteins before and after osmotic opening of the BBB by intra-arterial mannitol.
  • the samples used for loading were taken before mannitol injection and after chemotherapy.
  • the time of the injection of the osmotic agent and the introduction of the chemotherapic agent (methotrexate) are shown in the timeline.
  • Protein signals that remained unchanged are indicated by arrows.
  • the region in which significant changes were observed is boxed by a dashed line. Note the appearance of a distinct spot after BBB disruption.
  • FIG. 3 illustrates time course of serum protein changes after BBB disruption. S-lOO ⁇ , TTR, haptoglobin, and NSE were measured at the time indicated by the inset. Note that S-lOO ⁇ and TTR increased significantly after BBB opening but with different kinetics. NSE and haptoglobin-1 (Hapto- 1) remained unchanged throughout the procedure. The mean ⁇ 5D of three experiments is shown; *p ⁇ 0.05.
  • TTR and haptoglobin-1 are expressed as percentage change of spot intensity
  • NSE and S-lOO ⁇ were measured by immunodetection techniques, and the values are expressed in nannograms per milliliter.
  • the values for S-lOO ⁇ were scaled for clarity (lOOx). The asterisks indicate the actual time points at which the samples used for the gels shown were taken. This protein was subsequently identified as transthyretin. Previous results demonstrated that opening of the BBB by osmotic means causes a reproducible increase in serum levels of S-lOO ⁇ . In contrast, levels of the putative marker of neuronal damage, neuron-specific enolase (NSE) remained unchanged.
  • NSE neuron-specific enolase
  • TTR time-dependent appearance of TTR was compared with changes of NSE and S-lOO ⁇ during the same procedures.
  • BBBD caused S-lOO ⁇ and TTR changes characterized by distinct time dependency, whereas the internal controls haptoglobin and NSE remained essentially unaffected, on average. Note, however, that while S-lOO ⁇ increased significantly immediately (40 seconds) after mannitol injection and BBB disruption, TTR levels were elevated only after a longer delay.
  • Protein identification was carried out by LC-MS microelectrospry MS. The region of interest was cut out from the gel and digested overnight with trypsin. The digest was analyzed by mass spectroscopy to determine peptide molecular weight and amino acid sequence. An additional spot (haptoglobin) was also processed to standardize the procedure for each individual gel.
  • Transthyretin is the major protein product of the choroids plexuses and represents 20% of total amount of protein in CSF.
  • TTR is present in a homotetrameric form with specific binding to several other proteins.
  • TTR CSF is predominantly represented as a monomer, with accumulation of the tetrameric protein in epithelial cells of the choroid plexus.
  • FIG. 4 illustrates immunological analysis of protein changes induced by BBB disruption. Top panel, denaturated proteins were run in parallel with purified TTR (left lane). Western blot analysis revealed a significant increase of immunosignal for both low molecular weight isoforms. Quantitative analysis was performed on the same samples by RED (bottom panels); note the progressive increase of the immunoprecipitation signal surrounding the sample port (see Materials and Methods for details). The numeric values represent TTR levels extrapolated from these measurements and are expressed as micrograms per milliliter.
  • TTR immunoreactivity consistent with increased monomeric and dimeric TTR levels after BBBD.
  • a commercially available TTR tetramer 55 kDa was used as reference and loaded in the gel after processing under identical conditions. After denaturation, both dimeric and monomeric bands were identified by comparison with molecular weight standards.
  • RID quantitative radial immuno-diffusion
  • FIG. 5 illustrates non-SDS separation of pre- BBBD and post-BBBD samples.
  • Low molecular weight proteins obtained after separation with a sucrose gradient (cutoff 50 kDa) were loaded on a non-denaturating gel. Note the appearance of at 15 kDa molecular weight band after BBB disruption. Also note that this band corresponds to the monomeric form of standard TTR loaded on a separate gel. Purified TTR was again loaded as reference.
  • Transthyretin particularly the monomeric form of transthyretin is a new marker of barrier integrity and affords proteomic strategies with clinical procedures where the BBB is disrupted to allow penetration of chemotherapic agents to treat brain tumors. Both mass spectroscopy and immunoblotting confirmed that TTR is increased early (minutes) after BBB disruption.
  • S-lOO ⁇ increases in serum may also reflect peripheral neoplasms (e.g., melanoma or schwannoma), or other conditions where the BBB may be intact and neuronal damage absent.
  • peripheral neoplasms e.g., melanoma or schwannoma
  • a broad proteomic analysis strategy was utilized to reveal other peripheral markers of BBB (dys)function.
  • Plasma electrophoresis has been used to diagnose human diseases. Conversely, CSF protein analysis has been instrumental in understanding CNS disorders. In particular, the presence of abnormal levels of plasma protein in CSF has been interpreted as sign of BBB failure. The same interpretation may be applied to modern contrast agent-based radiological investigations, where variously labeled plasma proteins are detected in the CNS when the BBB is breached.
  • the present invention allows for a substantially non-invasive determination of blood brain barrier permeability by peripheral detection of TTR. Due to its localization in the cerebrospinal fluid, TTR may, in addition to detect blood-brain barrier opening also correlate with failure of the blood-to-cerebrospinal fluid barrier or be independent of the blood-brain barrier opening. It is expected that TTR will be released very close in time to the opening of the blood brain barrier but that its appearance will lag behind that of other markers present in the extravascular space (S-lOO ⁇ ). Thus upon detection of TTR, other markers of brain damage may be utilized to determine the onset of neuronal distress or damage. TTR and S- lOO ⁇ may be used to quantify the extent of blood-brain barrier damage and to confirm or rule out opening of the blood-to-cerebrospinal fluid interface.
  • One particular useful aspect of the present invention is in chronic neurological disease.
  • chronic neurological diseases where the BBB opens before the disease becomes, pathology, becomes apparent.
  • An example may be Alzheimer's disease, multiple sclerosis, or other diseases where it is believed that the BBB opens before the symptoms occur.
  • TTR would be elevated in the plasma before markers of neuronal damage become elevated. If routine measurements or samples are taken, a peripheral increase in TTR should precede an increase in the other markers.
  • the present invention encompasses a method for diagnosis and prognosis of a subject's BBB, comprising: contacting a serum sample derived from a subject with a sample containing TTR protein under conditions such that a specific antigen-antibody binding can occur; and detecting the presence of TTR present in the subject's serum, wherein the presence of immunospecific binding indicates level or degree to which the BBB is open.
  • the TTR protein is utilized to screen a subject's serum for the presence of TTR by means of sensitive and rapid immunoadsorbent assays or by other procedures.
  • the present invention also provides for kits for carrying out the above described methods. The methods can be performed, for example, by utilizing prepackaged diagnostic kits comprising at least a reagent for detecting TTR protein such as an anti- TTR antibody.
  • the diagnostic kits may comprise an TTR peptide for detection of TTR autoantibodies in a subject derived sample.
  • TTR protein levels in serum or body fluids can be used for the early diagnosis of diseases associated with an open BBB, such as neurological disorders.
  • monitoring of TTR protein levels can be used prognostically to stage the progression of the disease and to evaluate the efficacy of compounds in penetrating the BBB.
  • TTR proteins in a body fluid from a subject can be accomplished by any of a number of methods.
  • Preferred diagnostic methods for the detection of TTR proteins in the serum of a patient can involve, for example, immunoassays wherein TTR proteins are detected by their interaction with an TTR specific antibody.
  • Antibodies useful in the present invention can be used to quantitatively or qualitatively detect the presence of TTR peptides.
  • reagents other than antibodies, such as, for example, polypeptides that bind specifically to TTR proteins can be used in assays to detect the level of TTR protein expression.
  • Immunoassays to be used in the practice of the invention include but are not limited to assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),"sandwich” immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Filtering of protein to isolate the monomeric form are necessary step prior to immunological detection by the aforementioned means.
  • a biological sample which may contain TTR proteins, such as serum or other biological fluids in which secreted proteins can localize is obtained from a subject suspected of having a particular breach of the BBB or BCSFB a patient in which it is desirable to open the BBB.
  • Immunoassays for detecting expression of TTR protein typically comprise contacting the biological sample, such as a serum sample derived from a subject, with an anti-TTR antibody under conditions such that specific antigen-antibody binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody.
  • such binding of antibody for example, can be used to detect the presence and increased expression of TTR proteins wherein the detection of increased expression of TTR proteins is an indication of a diseased condition.
  • the levels of TTR protein in a serum sample are compared to norms established for normal individuals and for subjects at a variety of stages of BBB integrity or opening.
  • the biological sample such as a serum sample is brought in contact with a solid phase support or carrier, such as nitrocellulose, for the purpose of immobilizing any proteins present in the sample.
  • a solid phase support or carrier such as nitrocellulose
  • the support is then washed with suitable buffers followed by treatment with detectably labeled TTR specific antibody.
  • the solid phase support is then washed with the buffer a second time to remove unbound antibody.
  • the amount of bound antibody on the solid support is then determined according to well known methods.
  • TTR antibodies can be detectably labeled is by linking the antibody to an enzyme, such as for use in an enzyme immunoassay (EIA) (Noller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2: 1- 7, Microbiological Associates Quarterly Publication, Walkersville, MD; Noller, A., et al., 1978, J. Clin. Pathol. 31: 507-520; Butler, J. E., 1981, Meth. Enzymol. 73: 482-523).
  • EIA enzyme immunoassay
  • the enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric, or by visual means.
  • Enzymes that can be used to detectable label the antibody include, but are not limited to, horseradish peroxidase and alkaline phosphatase. The detection can also be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme.
  • Detection of TTR antibodies may also be accomplished using a variety of other methods. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect TTR protein expression through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986).
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • the antibody may also be labeled with a fluorescent compound.
  • fluorescent labeling compounds are fluorescein isothiocyanate rhodamine, phycoerythrin and fluorescamine.
  • a bioluminescent compound may be used to label the TTR antibody. The presence of a bioluminescence protein is determined by detecting the presence of luminescence. Important bioluminescence compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • TTR proteins in biological samples can be analyzed by two-dimensional gel electrophoresis.
  • Methods of two-dimensional electrophoresis are known to those skilled in the art.
  • Biological samples such as serum samples, are loaded onto electrophoretic gels for isoelectric focusing separation in the first dimension which separates proteins based on charge.
  • a number of first-dimension gel preparations may be utilized including tube gels for carrier ampholytes-based separations or gels strips for immobilized gradients based separations.
  • proteins are transferred onto the second dimension gel, following an equilibration procedure and separated using SDS PAGE which separates the proteins based on molecular weight.
  • the proteins are transferred from the two dimensional gels onto membranes commonly used for Western blotting.
  • the techniques of Western blotting and subsequent visualization of proteins are also well known in the art (Sambrook et al, "Molecular Cloning, A Laboratory Manual", 2 nd Edition, Volume 3,1989, Cold Spring Harbor).
  • the standard procedures may be used, or the procedures may be modified as known in the art for identification of proteins of particular types, such as highly basic or acidic, or lipid soluble, etc. (See for example, Ausubel, et at., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. ⁇ .).
  • Antibodies that bind to the TTR proteins are utilized in an incubation step, as in the procedure of Western blot analysis.
  • a second antibody specific for the first antibody is utilized in the procedure of Western blot analysis to visualize proteins that reacted with the first antibody.
  • the immunoassays can be conducted in a variety of ways. For example, one method to conduct such assays involves anchoring of TTR protein onto a solid support and detecting anti-TTR antibodies specifically bound thereto.
  • the TTR proteins to be utilized in the assays of the invention can be prepared via recombinant DNA techniques well known in the art. For example, in instances where the nucleotide sequence of a DNA encoding an TTR protein is available, the DNA can be genetically engineered into an appropriate expression vector for large scale preparation of TTR protein. It may be advantageous to engineer fusion proteins that can facilitate labeling, immobilization or detection of the TTR protein. See, for example, the techniques described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N. Y.
  • the TTR protein may be purified from natural sources, e. g., purified from cells, using protein separation techniques well known in the art. Such purification techniques may include, but are not limited to molecular sieve chromatography and/or ion exchange chromatography. In practice, microtitre plates are conveniently utilized as the solid support for the TTR proteins. The surfaces may be prepared in advance and stored.
  • a kit according to the invention comprises components for detecting and/or measuring human IgG antibodies directed toward TTR antigen.
  • the antibodies are detected and/or measured by enzyme linked immunoabsorbent assay (ELISA)
  • such components may comprise target antigen, in the form of at least one and preferably a plurality of different TTR antigens or epitopes thereof, linked to a solid phase, and a means for detecting a human antibody bound to target antigen.
  • Such means for detection may be, for example, an antibody directed toward the constant region of human IgG (e. g., rabbit anti-human IgG antibody), which may itself be detectably labeled (e. g., with a radioactive, fluorescent, colorimetric or enzyme label), or which may be detected by a labeled secondary antibody (e. g., goat anti-rabbit antibody).
  • a kit according to the invention may comprise components which detect and/or measure TTR antigens in the biological sample of a subject.
  • TTR proteins are detected and/or measured by enzyme linked immunoabsorbent assay (ELISA)
  • ELISA enzyme linked immunoabsorbent assay
  • such components may comprise an antibody directed to epitopes of the TTR proteins which can be used to detect and/or quantitate the level of TTR expression in the biological sample.
  • the antibody itself may be detectably labeled with a radioactive, flourescent, colorimetric or enzyme label.
  • the kit may contain a labeled secondary antibody.
  • neuropharmacologic agents include, by way of non- limiting example, any of neuropharmacologic agents, neuroactive peptides (e.g., hormones, gastrointestinal peptides, angiotensin, sleep peptides, etc.), proteins (e.g, calcium binding proteins), enzymes (e.g., cholineacetyltransferase, glutamic acid decarboxylase, etc.), gene therapy agents, neuroprotective or growth factors, biogenic amines (e.g., dopamine, GABA), trophic factors to brain or spinal transplants, immunoreactive proteins (e.g, antibodies to neurons, myelin, antireceptor antibodies), receptor binding proteins (e.g., opiate receptors), radio
  • neuroactive peptides e.g., hormones, gastrointestinal peptides, angiotensin, sleep peptides, etc.
  • proteins e.g, calcium binding proteins
  • enzymes e.g., cholineacetyltransfera
  • disorders include tumors, cancer, degenerative disorders, sensory and motor abnormalities, seizure, infection, immunologic disorder, mental disorder, behavioral disorder, and localized CNS disease, among others.
  • the invention provides methods for modification of neurologic and neurologically-related activity (e.g., behavioral activity, memory-related activity, and sexual activity, among others) by such methods.
  • neurologic and neurologically-related activity e.g., behavioral activity, memory-related activity, and sexual activity, among others.
  • the invention provides methods for modification of neurologic and neurologically-related activity (e.g., behavioral activity, memory-related activity, and sexual activity, among others) by such methods.
  • neurologic and neurologically-related activity e.g., behavioral activity, memory-related activity, and sexual activity, among others.

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Abstract

La présente invention concerne globalement un marqueur périphérique ou des marqueurs de l'intégrité de la barrière sanguine et leurs méthodes d'utilisation pour le diagnostic, le pronostic et le traitement de diverses maladies. Ces marqueurs périphériques sont particulièrement utiles pour le diagnostic différentiel d'états pathologiques. Les modes de réalisation préférés de la présente invention concernent des méthodes, des compositions, des nécessaires et des dosages permettant de déterminer l'intégrité ou la perméabilité d'une barrière sang-LCR ou d'une barrière sang-cerveau. Les divers modes de réalisation de la présente invention peuvent être utilisés, d'une part, pour identifier des sujets présentant un risque de développer une maladie associée à une perméabilité trop grande de la barrière sang-cerveau, et, d'autre part, pour fournir un aperçu de la capacité d'un ou plusieurs agent à traverser la barrière sang-cerveau. Des modes de réalisation de la présente invention consistent de préférence à utiliser des prélèvements sanguins d'un sujet pour déterminer l'occurrence et le taux de protéines circulant dans le sang indiquant la perméabilité ou l'intégrité de la barrière sang-cerveau. Les modes de réalisation de la présente invention concernent également des méthodes de dosage destinées au diagnostic, au pronostic, à la mesure de la sensibilité ou du degré de perméabilité ou de pénétration de la barrière sang-cerveau, consistant à détecter la présence de transthyrétine sérique soit directement soit à l'aide d'anticorps.
PCT/US2003/018780 2002-06-12 2003-06-12 Marqueurs de la permeabilite de la barriere sanguine et leurs methodes d'utilisation WO2004078204A1 (fr)

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WO2010030203A1 (fr) * 2008-09-09 2010-03-18 Biocodex - Incubação De Empresas De Ciências Da Vida, S.A. Anticorps monoclonal dirigé contre la transthyrétine humaine amyloïdogène et des formes modifiées de celle-ci et son utilisation dans la détection et le traitement de la fap et de pathologies présentant une ttr modifiée
US9810698B2 (en) 2004-04-15 2017-11-07 University Of Florida Research Foundation, Incorporated Neural proteins as biomarkers for nervous system injury and other neural disorders
US10041959B2 (en) 2009-09-14 2018-08-07 Banyan Biomarkers, Inc. Micro-RNA, autoantibody and protein markers for diagnosis of neuronal injury
US10794918B2 (en) 2003-09-24 2020-10-06 Matthias Sitzer Use of GFAP for identification of intracerebral hemorrhage
US11994522B2 (en) 2008-08-11 2024-05-28 Banyan Biomarkers, Inc. Biomarker detection process and assay of neurological condition
US12077601B2 (en) 2016-10-28 2024-09-03 Banyan Biomarkers, Inc. Antibodies to ubiquitin C-terminal hydrolase L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) and related methods

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10794918B2 (en) 2003-09-24 2020-10-06 Matthias Sitzer Use of GFAP for identification of intracerebral hemorrhage
US9810698B2 (en) 2004-04-15 2017-11-07 University Of Florida Research Foundation, Incorporated Neural proteins as biomarkers for nervous system injury and other neural disorders
US10330689B2 (en) 2004-04-15 2019-06-25 University Of Florida Research Foundation, Inc. Neural proteins as biomarkers for nervous system injury and other neural disorders
US11221342B2 (en) 2004-04-15 2022-01-11 University Of Florida Research Foundation, Inc. Neural proteins as biomarkers for nervous system injury and other neural disorders
US11994522B2 (en) 2008-08-11 2024-05-28 Banyan Biomarkers, Inc. Biomarker detection process and assay of neurological condition
WO2010030203A1 (fr) * 2008-09-09 2010-03-18 Biocodex - Incubação De Empresas De Ciências Da Vida, S.A. Anticorps monoclonal dirigé contre la transthyrétine humaine amyloïdogène et des formes modifiées de celle-ci et son utilisation dans la détection et le traitement de la fap et de pathologies présentant une ttr modifiée
US10041959B2 (en) 2009-09-14 2018-08-07 Banyan Biomarkers, Inc. Micro-RNA, autoantibody and protein markers for diagnosis of neuronal injury
US12077601B2 (en) 2016-10-28 2024-09-03 Banyan Biomarkers, Inc. Antibodies to ubiquitin C-terminal hydrolase L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) and related methods

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