Patent Application
20140086827
Disruption, particularly uncontrolled disruption, of the blood brain barrier (BBB) is generally believed to be harmful in most circumstances as it can cause influx of potentially dangerous pathogens and inflammatory molecules. However, normal BBB can restrict the entry of potential therapeutic agents into the brain. Therefore, an increased understanding of the molecular mechanisms of the BBB which include the identification of markers of BBB disruption, will provide new avenues of therapeutic intervention for brain disorders.
Described herein are studies of S100B, a marker of blood brain-barrier disruption. Experiments described herein further elucidate properties of this marker. There are several proteins in CFS that, when present in serum, are indicators of disruption of the BBB. S100B has been the most useful so far because its levels are normally very low or undetectable in blood (e.g., see U.S. Pat. No. 6,884,591 and U.S. Pat. No. 7,144,708 which are incorporated herein by reference in their entirety). However, several confounding factors interpreting this test have been pointed out and these are addressed herein. The invention described herein is based, in part, on the fact that S100B is present in brain or CFS in different configurations, namely as a monomer, or a dimer, the latter being either heterodimer or homodimer (S100AB or S100BB). Shown herein is that an indicator of blood brain-barrier permeability is the homodimer S100B-S100B.
Current means of detection of S100B at the clinical level do not distinguish between monomeric or dimeric form. This said, there was a commercially available ELISA kit which enabled the detection of any of the above species (Canag).
As shown herein, S100B-S100B dimmer is superior to S100B detection in serum as an indicator of blood brain-barrier permeability. In particular, the dimer provides increased sensitivity and specificity for testing blood brain-barrier permeability (see Pham, N., et al., PLoS ONE, 5(9):e12691 (2010) which is incorporated herein by reference). With the exception of the defunct Canag Ab, commercially available and ELISA or automated kits, all based on the detection of a combination of dimer and monomer forms, can be used.
Accordingly, in one aspect, the invention is directed to a method of assessing blood brain barrier permeability in an individual comprising selectively or specifically detecting a level of S100BB homodimer in a sample of the individual, and comparing the level of S100BB homodimer in the sample to a level of S100B a control. An elevated level of S100BB homodimer in the sample compared to the level of S100BB homodimer in the control, is indicative of blood brain barrier permeability in the individual.
In another aspect, the invention is directed to a method for delivering an agent for delivery to the brain of an individual in need thereof comprising introducing a first agent that opens the blood brain barrier into the individual. The level of S100BB homodimer in a sample of the individual is selectively determined, wherein an elevated level of S100BB homodimer in the sample compared to the level of S100BB homodimer in the control, indicates that the blood brain barrier of the individual is permeable to the agent for delivery to the brain. The agent for delivery to the brain is then introduced to the individual when the blood brain barrier of the individual is permeable, thereby delivering the agent for delivery to the brain of the individual.
In another aspect, the invention is directed to a method of detecting whether a cancer has metastasized to a cancer patient's brain in a patient that has, or is at risk of having, metastasis, comprising detecting a level of S100B in a sample of the cancer patient using a first immunoassay, and detecting a level of S100B in a sample using an immunoassay that differs from the first immunoassay (a second immunoassay; e.g., an immunoassay that is performed at the same time as, or subsequent to, the first immunoassay). The level of S100B in the first and second immunoassays are compared to the level of S100B in a control, wherein if the level of S100B in the first immunoassay and the level of S100B in the second immunoassay are the same as, or lower than the level of S100B in the control then, the metastasis has not spread to the cancer patient's brain.
In yet another aspect, the invention is directed to a method of determining the effectiveness of a treatment for a neurological disorder wherein blood-brain barrier permeability is present in an individual in need thereof comprising detecting a level of S100B in a sample of the individual undergoing the treatment, and comparing the level of S100B in the sample to the level of S100B in a sample from the individual obtained prior to treatment. Decreased levels of S100B in the sample compared to the level of S100B in the sample from the individual obtained prior to treatment indicate that the treatment for a neurological disorder wherein blood-brain barrier permeability is present is effective in the individual.
In another aspect, the invention is directed to a method of determining the effectiveness of a treatment for seizures triggered by blood brain barrier damage in an individual in need thereof comprising detecting a level of S100B in a sample of the individual undergoing the treatment, and comparing the level of S100B in the sample to the level of S100B in a sample from the individual obtained prior to treatment. Decreased levels of S100B in the sample compared to the level of S100B in the sample from the individual obtained prior to treatment indicate that the treatment is effective to treat the seizures in the individual.
In another aspect, the invention is directed to a method of determining the effectiveness of a hypothermia treatment in an individual in need thereof comprising detecting a level of S100B in a sample of the individual undergoing the hypothermia treatment, and comparing the level of S100B in the sample to the level of S100B in a sample from the individual obtained prior to hypothermia treatment. Decreased levels of S100B in the sample compared to the level of S100B in the sample from the individual obtained prior to treatment indicate that the hypothermia treatment is effective to treat the individual.
In another aspect, the invention is directed to a method of detecting a positive outcome for a newborn that has undergone asphyxia during birth comprising detecting a level of S100B in a sample of the newborn at birth, detecting at least one level of S100B in one or more samples of the newborn after birth, and comparing the at least one level of S100B in the sample after birth to the level of S100B in the sample at birth. A decreased level of S100B in the sample after birth compared to the level of S100B at birth indicate a positive outcome for the newborn.
In another aspect, the invention is directed to a method of detecting a sub-concussion in an individual in need thereof comprising detecting a level of S100B in a sample of the individual, and comparing the level of S100B in the sample to a level of S100B a control, wherein elevated levels of S100B in the sample compared to the level of S100B in the control indicate that the individual has a sub-concussion.
In another aspect, the invention is directed to a method of detecting a history of blood brain barrier disruption in an individual in need thereof comprising detecting auto-antibodies directed against S100B in a sample of the individual, wherein the presence of auto-antibodies directed against S100B in the sample indicates that the individual has a history of blood brain barrier disruption.
S100B, established as prevalent protein of the central nervous system, is a peripheral biomarker for blood-brain barrier disruption and often also a marker of brain injury. However, reports of extracranial sources of S100B, especially from adipose tissue, may confound its interpretation in the clinical setting. Described herein is the characterization of the tissue specificity of S100B and the assessment of how extracranial sources of S100B affect serum levels. Specifically, as described herein, the extracranial sources of S100B were determined by analyzing nine different types of human tissues by ELISA and Western blot. In addition, brain and adipose tissue were further analyzed by mass spectrometry. A study of 200 subjects was undertaken to determine the relationship between body mass index (BMI) and S100B serum levels. The levels of S100B homo- and heterodimers in serum were measured quantitatively after blood-brain barrier disruption. Analysis of human tissues by ELISA and Western blot revealed variable levels of S100B expression. By ELISA, brain tissue expressed the highest S100B levels. Similarly, Western blot measurements revealed that brain tissue expressed high levels of S100B but comparable levels were found in skeletal muscle. Mass spectrometry of brain and adipose tissue confirmed the presence of S100B but also revealed the presence of S100A1. The analysis of 200 subjects revealed no statistically significant relationship between BMI and S100B levels. Moreover, shown herein is that the main species of S100B released from the brain was the B-B homodimer. The results show that extracranial sources of S100B do not affect serum levels. Thus, the diagnostic value of S100B and its negative predictive value in neurological diseases in intact subjects (without traumatic brain or bodily injury from accident or surgery) are not compromised in the clinical setting.
Also described herein is the investigation of the targeting pro-inflammatory events to reduce seizures and the ability to evaluate the efficacy of such treatments using S100B. Experimentally, antagonism of inflammatory processes and of blood-brain barrier (BBB) damage has been demonstrated to be beneficial in reducing status epilepticus (SE). Clinically, a role of inflammation in the pathophysiology of drug resistant epilepsies is suspected. However, the use anti-inflammatory drug such as glucocorticosteroids (GCs) is limited to selected pediatric epileptic syndromes and spasms. Lack of animal data may be one of the reasons for the limited use of GCs in epilepsy. The effect of the CG dexamethasone in reducing the onset and the severity of pilocarpine SE in rats was evaluated. BBB integrity was assessed by measuring serum S100β and Evans Blue brain extravasation. Electrophysiological monitoring and hematologic measurements (WBCs and IL-1β) were performed. The effect of add on dexamethasone treatment on a population of pediatric patients affected by drug resistant epilepsy was reviewed. Subjects affected by West, Landau-Kleffner or Lennox-Gastaut syndromes and Rasmussen encephalitis, known to respond to GCs or adrenocorticotropic hormone (ACTH), were excluded. The effect of two additional GCs, methylprednisolone and hydrocortisone, was also reviewed in this population. When dexamethasone treatment preceded exposure to the convulsive agent pilocarpine, the number of rats developing status epilepticus (SE) was reduced. When SE developed, the time-to-onset was significantly delayed compared to pilocarpine alone and mortality associated with pilocarpine-SE was abolished. Dexamethasone significantly protected the BBB from damage. The clinical study included pediatric drug resistant epileptic subjects receiving add on GC treatments. Decreased seizure frequency (≧50%) or interruption of status epilepticus was observed in the majority of the subjects, regardless of the underlying pathology. The experimental results point to a seizure-reducing effect of dexamethasone. The mechanism encompasses improvement of BBB integrity. The results also indicate that add on GCs could be of efficacy in controlling pediatric drug resistant seizures (Marchi, N., et al., PLoS ONE, 6(3):e18200 (2011) which is incorporated herein by reference).
In addition to seizures, S100B can be used as a prognostic indicator for a variety of disorders associated with blood brain barrier dysfunction and the treatment thereof.
Finally, also shown herein is that autoantibodies directed against S100B, S100BB, S100AB or a combination thereof can be detected in samples form individuals with a history of blood brain barrier disruption.
Accordingly, in one aspect, the invention is directed to a method of assessing blood brain barrier permeability in an individual comprising selectively or specifically detecting a level of S100BB homodimer in a sample of the individual, and comparing the level of S100BB homodimer in the sample to a level of S100B a control. An elevated level of S100BB homodimer in the sample compared to the level of S100BB homodimer in the control, is indicative of blood brain barrier permeability in the individual.
The blood brain barrier is a naturally occurring barrier created by the modification of brain capillaries (as by reduction in fenestration and formation of tight cell-to-cell contacts) that prevents many substances from leaving the blood and crossing the capillary walls into the brain tissues. “S100B” is a prevalent protein of the central nervous system which is used as a peripheral biomarker for blood-brain barrier disruption and as a marker of brain injury. S100B can occur in a monomeric form, referred to herein as S100B or S100B monomer; a homodimeric form referred to herein as S100BB homodimer, S100BB, B-B homodimer, or S100B-B; or a heterodimeric form referred to herein as S100AB heterodimer, S100AB, A-B heterodimer, or S100A-B. Thus, depending on the context, the term “S100B” can be used to refer to the S100B monomer, the S100B homodimer, the S100B heterodimer, or the combined total of S100B monomer, S100BB homodimer and S100AB heterodimer (total S100B).
In the methods described herein a (one or more) level of S100B is measured. Unless otherwise specified, the level of S100B measured can be the level of S100B monomer, S100BB homodimer, S100AB, total S100B or a combination thereof.
In some embodiments of the methods described herein, one or more of the S100B proteins is selectively measured. Selective detection of one or more S100B proteins refers to the ability to detect one or more S100B proteins in a sample to the exclusion of other forms of one or more S100B proteins and other molecules in a sample. Thus, for example, selective detection of S100BB homodimer refers to the ability to detect the S100BB heterodimer in a sample to the exclusion of other forms of the S100B protein (e.g., to the exclusion of S100B monomer, S100AB heterodimer) and other molecules (e.g., protein) in a sample.
As will be appreciated by those of skill in the art, the methods described herein can further comprise detecting one or more levels of one or more markers of neuronal distress. Examples of markers of neuronal distress include Ubiquitin C-terminal hydrolase 1, NSE, GFAP, tau protein, beta trace protein, cystatin C.
The method of assessing blood brain barrier permeability in an individual can further comprising detecting whether auto-antibodies directed against S100B, S100BB, S100AB or a combination thereof are present in a sample (e.g., the same sample used to detect S100BB homodimer, or a different sample) of the individual.
The detection of the S100B protein (e.g., S100BB homodimer) and/or autoantibodies directed against S100B, S100BB, S100AB or a combination thereof can be detected at the same time or at different times and at one or more time periods as determined necessary by one of skill in the art. Thus, in the methods described herein, the S100BB homodimer and/or autoantibodies directed against S100B, S100BB, S100AB or a combination thereof can be detected in a single sample or in multiple samples (samplings) and/or over a period of time, as needed.
In another aspect, the invention is directed to a method for delivering an agent for delivery to the brain of an individual in need thereof comprising introducing an agent (a first agent) or condition that opens (transiently opens) the blood brain barrier into the individual. The level of S100BB homodimer in a sample of the individual is selectively determined, wherein an elevated level of S100BB homodimer in the sample compared to the level of S100BB homodimer in the control, indicates that the blood brain barrier of the individual is permeable to the agent for delivery to the brain. The agent for delivery to the brain (a second agent) is then introduced to the individual when the blood brain barrier of the individual is permeable, thereby delivering the agent for delivery to the brain of the individual.
Agents which cause the blood brain barrier to open are known to those of skill in the art. Examples of such agents include hyperosmolar osmotic agents such as mannitol (intraarterial injection), bradikinin and its analog RPM-7 (B2 receptor agonist), and alkilglycerola (Stamataovic, S., et al., Current Neuropharmacology, 6:179-192 (2008)). In some instances the blood brain barrier of the individual will be open due to conditions to which the individual is exposed or is undergoing, such as inflammation or exposure of BBB to radiotherapy (20-30 Gy) (Stamataovic, S., et al., Current Neuropharmacology, 6:179-192 (2008)).
As will be appreciated by those of skill in the art, a variety of agents can be delivered to the brain using the methods described herein. Examples of agents include a contrast agent, a neuropharmacologic agent, a neuroactive peptides, a protein, an enzyme, a gene therapy agent, a neuroprotective factor, a growth factor, a biogenic amine, a trophic factor to any of brain and spinal transplants, an immunoreactive proteins, a receptor binding protein, a radioactive agent, an antibody, a cytotoxin or a combination thereof.
Methods for delivering the agent that is to be delivered to the brain are also apparent to those of skill in the art. In a particular aspect, the agent for delivery to the brain is introduced into the individual's bloodstream in a vicinity of the individual's brain. For example, the agent can be delivered via intra-carotid and/or intranasal injection.
In another aspect, the invention is directed to a method of detecting whether a cancer has metastasized to a cancer patient's brain in a patient that has, or is at risk of having, metastasis, comprising detecting a level of S100B in a sample of the cancer patient using a first immunoassay, and detecting a level of S100B in a sample using an immunoassay that differs from the first immunoassay (a second immunoassay; e.g., an immunoassay that is performed at the same time as, or subsequent to, the first immunoassay). The level of S100B in the first and second immunoassays are compared to the level of S100B in a control, wherein if the level of S100B in the first immunoassay and the level of S100B in the second immunoassay are the same as, or lower than the level of S100B in the control then, the metastasis has not spread to the cancer patient's brain. In one aspect, the level of S100B monomer, S100BB homodimer, S100AB or total S100B or a combination thereof is detected. In another aspect, the level of S100BB homodimer is selectively detected.
In yet another aspect, the invention is directed to a method of determining the effectiveness of a treatment for a neurological disorder wherein blood-brain barrier permeability is present in an individual in need thereof comprising detecting a level of S100B in a sample of the individual undergoing the treatment, and comparing the level of S100B in the sample to the level of S100B in a sample from the individual obtained prior to treatment. Decreased levels of S100B in the sample compared to the level of S100B in the sample from the individual obtained prior to treatment indicate that the treatment for a neurological disorder wherein blood-brain barrier permeability is present is effective in the individual. In one aspect, the level of S100B monomer, S100BB homodimer, S100AB or total S100B or a combination thereof is detected. In another aspect, the level of S100BB homodimer is selectively detected. Examples of a neurological disorder wherein blood-brain barrier permeability is present includes multiple sclerosis, tumors, psychiatric disorders and the like.
In another aspect, the invention is directed to a method of determining the effectiveness of a treatment for seizures triggered by blood brain barrier damage in an individual in need thereof comprising detecting a level of S100B in a sample of the individual undergoing the treatment, and comparing the level of S100B in the sample to the level of S100B in a sample from the individual obtained prior to treatment. Decreased levels of S100B in the sample compared to the level of S100B in the sample from the individual obtained prior to treatment indicate that the treatment is effective to treat the seizures in the individual. In one aspect, the level of S100B monomer, S100BB homodimer, S100AB or total S100B or a combination thereof is detected. In another aspect, the level of S100B13 homodimer is selectively detected.
In another aspect, the invention is directed to a method of determining the effectiveness of a hypothermia treatment in an individual in need thereof. As will be appreciated by those of skill in the art, hypothermia is administered to treat a variety of reasons such as to treat ischemic-hemorrhagic stroke, to mitigate seizures or during a surgical cardiac procedure in the individual. The method comprises detecting a level of S100B′ in a sample of the individual undergoing the hypothermia treatment, and comparing the level of S100B in the sample to the level of S100B in a sample from the individual obtained prior to hypothermia treatment. Decreased levels of S100B in the sample compared to the level of S100B in the sample from the individual obtained prior to treatment indicate that the hypothermia treatment is effective to treat the individual. In one aspect, the level of S100B monomer, S100BB homodimer, S100AB or total S100B or a combination thereof is detected. In another aspect, the level of S100BB homodimer is selectively detected.
In another aspect, the invention is directed to a method of detecting a positive outcome for a newborn that has undergone asphyxia during birth comprising detecting a level of S100B in a sample of the newborn at birth, detecting at least one level of S100B in one or more samples of the newborn after birth, and comparing the at least one level of S100B in the sample after birth to the level of S100B in the sample at birth. A decreased level of S100B in the sample after birth compared to the level of S100B at birth indicate a positive outcome for the newborn. In one aspect, the level of S100B monomer, S100BB homodimer, S100AB or total S100B or a combination thereof is detected. In another aspect, the level of S100BB homodimer is selectively detected.
In another aspect, the invention is directed to a method of detecting a sub-concussion in an individual in need thereof comprising detecting a level of S100B in a sample of the individual, and comparing the level of S100B in the sample to a level of S100B a control, wherein elevated levels of S100B in the sample compared to the level of S100B in the control indicate that the individual has a sub-concussion. In one aspect, the level of S100B monomer, S100BB homodimer, S100AB or total S100B or a combination thereof is detected. In another aspect, the level of S100BB homodimer is selectively detected. In yet another aspect, the individual has had one or more concussions, sub-concussions, seizures or a combination thereof.
In another aspect, the invention is directed to a method of detecting a history of blood brain barrier disruption in an individual in need thereof comprising detecting auto-antibodies directed against S100B in a sample of the individual, wherein the presence of auto-antibodies directed against S100B in the sample indicates that the individual has a history of blood brain barrier disruption. In one aspect, the auto-antibodies are directed against S100B monomer, S100BB heterodimer, S100AB heterodimer or a combination thereof. The method can further comprise detecting a level of S100B in a sample of the individual wherein elevated levels of S100B in the sample compared to the level of S100B in the control further indicates that the individual has a history of blood brain barrier disruption. In one aspect, the level of S100B monomer, S100BB homodimer, S100AB or total S100B or a combination thereof is detected. In another aspect, the level of S100BB homodimer is selectively detected. In particular aspects, the individual has ongoing blood brain barrier disruption, has an increased risk for degenerative brain disease, or has had one or more concussion, sub-concussions, seizures or a combination thereof.
In a particular embodiment, the invention is directed to a method of detecting a history of blood brain barrier disruption in an individual in need thereof comprising detecting S100B and auto-antibodies directed against S100B in a (one or more) sample of the individual, wherein the presence of S100B and auto-antibodies directed against S100B in the sample indicates that the individual has a history of blood brain barrier disruption. In one aspect, the level of S100B monomer, S100BB homodimer, S100AB or total S100B or a combination thereof is detected. In another aspect, the level of S100BB homodimer is selectively detected. In yet another aspect, the auto-antibodies are directed against S100B monomer, S100BB heterodimer, S100AB heterodimer or a combination thereof. In particular aspects, the individual has ongoing blood brain barrier disruption, has an increased risk for degenerative brain disease, or has had one or more concussion, sub-concussions, seizures or a combination thereof.
The methods described herein can further comprise obtaining a sample from the individual (e.g., prior to treatment). The methods can also comprise contacting the sample with an agent that detects S100B (e.g., S100B monomer, S100BB homodimer, S100AB or total S100B or a combination thereof) or auto-antibodies directed against S100B), thereby producing a combination or mixture. The combination can be maintained under conditions which allow detection of the S100B or auto-antibodies directed against S100B.
As described herein, in some aspects of the invention, decreased levels of S100B indicate the effectiveness of a treatment. In a particular embodiment, a decrease of about 10%, 20%, 30%, 40% or 50% in the level of S100B (e.g., S100B monomer, S100BB homodimer, S100AB or total S100B or a combination thereof) detected in the sample is indicative of the effectiveness of a treatment.
Detection of the one or more forms of S100B can be performed using a variety of methods known to those of skill in the art. The S100B (e.g., S100B monomer, S100BB homodimer, S100AB or total S100B or a combination thereof) molecules can be detected alone or in a complex with another molecule. In one aspect, the S100B is detected using mass spectrometry or a proteomic test based on immunodetection. In other aspects, the S100B is detected using an immunoassay such as an immunoprecipitation assay. In particular aspects, a sample obtained from an individual can be contacted with an agent that captures (e.g., binds to) one or more forms of S100B (e, g, such an agent can be added to a sample obtained from an individual), thereby faulting a combination or mixture. The combination or mixture can be maintained under conditions in which the agent captures the one or more forms of S100B in the sample, thereby forming a complex between the one or more forms of the S100B and the capture agent. In a specific embodiment, the capture agent is an antibody or antigen binding fragment thereof that specifically binds to, or has a binding affinity for, one or more forms of S100B (e.g., an antibody that specifically binds to one or more forms of S100B monomer, S100BB homodimer, S100AB heterodimer or a combination thereof).
As used herein, the terms “specific”, “selective”, “specifically”, “selectively” when referring to a capture agent such as an antibody-antigen interaction, is used to indicate that the capture agent (e.g., antibody) can selectively bind to one or more forms of S100B. In particular aspects, the capture agent is an antibody. In one embodiment, the antibody selectively binds to all or a portion of S100B monomer. In another embodiment, the antibody selectively binds to all or a portion of S100BB homodimer. In yet another embodiment, the antibody specifically binds to all or a portion of S100AB heterodimer.
An antibody that is specific for one or more forms of S100B is a molecule that selectively binds to one or more forms of S100B (e.g., selectively binds to S100BB homodimer) but does not substantially bind to other forms of S100B (e.g., S100B monomer, S100AB heterodimer) or other molecules in a sample, e.g., in a biological sample that contains one or more forms of S100B. The term “antibody,” as used herein, refers to an immunoglobulin or a part thereof (e.g., an antigen binding fragment thereof), and encompasses any polypeptide comprising an antigen-binding site regardless of the source, method of production, and other characteristics. The term includes but is not limited to polyclonal, monoclonal, monospecific, polyspecific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, conjugated and CDR-grafted antibodies. The term “antigen-binding site” refers to the part of an antibody molecule that comprises the area specifically binding to or complementary to, a part or all of an antigen. An antigen-binding site may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). An antigen-binding site may be provided by one or more antibody variable domains (e.g., an Fd antibody fragment consisting of a VH domain, an Fv antibody fragment consisting of a VH domain and a VL domain, or an scFv antibody fragment consisting of a VH domain and a VL domain joined by a linker). For example, the term “anti-S100B monomer antibody,” “antibody against S100B monomer,” refers to any antibody that specifically binds to at least one epitope of S100B monomer; the term “anti-S100BB homodimer antibody,” “antibody against S100BB homodimer,” refers to any antibody that specifically binds to at least one epitope of S100BB homodimer; and the term “anti-S100AB heterodimer antibody,” “antibody against S100AB heterodimer,” refers to any antibody that specifically binds to at least one epitope of S100AB heterodimer.
The various antibodies and portions thereof can be produced using known techniques (Kohler and Milstein, Nature 256:495-497 (1975); Current Protocols in Immunology, Coligan et al., (eds.) John Wiley & Sons, Inc., New York, N.Y. (1994); Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen et al., European Patent No. 0 451 216 B1; and Padlan, E. A. et al., EP 0 519 596 A1; Newman, R. et al., BioTechnology, 10: 1455-1460 (1992); Ladner et al., U.S. Pat. No. 4,946,778; Bird, R. E. et al., Science, 242: 423-426 (1988)).
As will also be appreciated by those of skill in the art, a variety of methods can be used to detect auto-antibodies directed against S100B. Examples of such assays include an enzyme-linked immunosorbent assay (ELISA) and immunofluoresence (e.g., indirect immunofluorescence).
As described herein, in the methods the amount of S100B and/or autoantibodies directed against S100B in the individual (e.g., a sample of the individual) can be compared to the amount of S100B and/or autoantibodies directed against S100B in a suitable control. As will be appreciated by one of skill in the art, there are a variety of suitable controls that can be used in the methods described herein. For example, the control can be a sample from an individual that does not have a permeable BBB, a BBB disruption, a brain disorder and/or a brain trauma.
As will also be appreciated by those of skill in the art, any suitable biological sample can be used in the methods described herein. Examples of biological samples include urine, blood, serum, spinal fluid (e.g. cerebral spinal fluid), lymph, and tissue. The sample can be obtained from the individual and/or analyzed for the presence of S100B and/or autoantibodies directed against S100B using known methods.
The amount of S1000B and/or autoantibodies directed against S100B in the sample can be compared to the amount of S100B or autoantibodies directed against S100B in a suitable control. Suitable controls include a previous reading of S100B and/or autoantibodies directed against S100B in the individual prior to BBB permeability or disruption (e.g., a reading obtained from the same individual prior to metastasis of a cancer to the brain, an epileptic seizure, a brain trauma, a brain disorder, a sub-concussion, a concussion), readings (from one or more individuals with normal BBB, and of a known standard.
As used herein, the term “individual” refers to an “animal” which includes mammals, as well as other animals, vertebrate and invertebrate (e.g., birds, fish, reptiles, insects (e.g., Drosophila species), mollusks (e.g., Aplysia). In a particular embodiment, the animal is a mammal. The terms “mammal” and “mammalian”, as used herein, refer to any vertebrate animal, including monotremes, marsupials and placental, that suckle their young and either give birth to living young (eutharian or placental mammals) or are egg-laying (metatharian or nonplacental mammals). Examples of mammalian species include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs), ruminents (e.g., cows, pigs, horses) felines and canines.
+Originally Sangtec Medical, Sweden
++Now Fujirebio Diagnostics
All studies were performed in accordance with the Declaration of Helsinki and written approval by the IRB Committee at the Cleveland Clinic. All samples (blood and tissue) were obtained by written informed consent and given a coded identifier to retain subject anonymity. Tissue samples were obtained as surplus tissue from various medical procedures conducted at the Cleveland Clinic, generously provided by the department of Pathology. For the purpose of this study we analyzed 200 subjects, consisting of 155 patients and 45 controls (Table 1). Note that the patient population comprised of individuals with a broad spectrum of ages, races, and pathologies, which ranges from psychiatric disorders to brain tumors. A large number of volunteers without any pathology or psychiatric condition, as detected by a psychiatrist delivering a brief psychiatric exam, were also recruited. Samples were analyzed by different techniques, the details of which are presented in the Methods Section and below. Tissue protein contents was analyzed by antibody-dependent assays or by mass spectrometry (MS). When possible, two antibodies were used for the detection of different antigen regions in the S100B protein. BMI (body mass index) was calculated based on the weight (in kilograms) divided by the square of the patient's height (in meters) taken from the medical record.
The Cleveland Clinic Brain Tumor Institute provides a treatment called blood-brain barrier disruption for primary CNS lymphomas. All procedures were performed after informed consent was obtained using protocols approved by the Cleveland Clinic Foundation IRB. In this protocol, intra-arterial mannitol (1.4 M) is administered via a carotid or vertebral artery, and BBB disruption was confirmed by contrast CT immediately after chemotherapy. The details are described elsewhere [Marchi, N., et al., Epilepsia, 48:732-742 (2007)].
Proteins were extracted from various tissues using the Millipore Total Protein Extraction Kit (Chemicon subsidiary, Temecula, Calif.). Briefly, tissues were weighed, chopped into small pieces, and kept on dry ice. Then 1×TM buffer [13 mL of HEPES (pH 7.9), MgCl2, KCl, EDTA, sucrose, glycerol, sodium deoxycholate, NP-40, sodium orthovanadate] and a protease inhibitor cocktail was added to each tissues at 2.5 mL per gram of tissue and put on ice for 5 minutes. The tissue was homogenized for 20 seconds and then put on dry ice for 15 seconds. This cycle was repeated 3 times. The homogenized tissues were rotated at 4° C. for 20 minutes and centrifuged at 11,000 rpm at 4° C. for 20 minutes. The supernatant was collected and stored at −80° C. until analyzed further.
Protein concentration was determined by the Bradford assay method (Bio-Rad, Hercules, Calif.). Total proteins (50 μg/lane) were separated on 10-20% polyacrylamide gels with SDS-PAGE at 80 V and transferred onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, Mass.) by electroblotting at 100 V of constant voltage for 1 hour. After blocking with TBST and milk (Tris-buffered saline, 0.05% milk powder, and 0.05% Tween 20) for at least 2 hours, the membrane was probed overnight at 4° C. either with the Sangtec-Diasorin or OriGene S100B primary antibody (1:1000). The OriGene monoclonal antibody was made by immunizing against a synthetic peptide corresponding to residues on the C-terminus of human S100B. The polyclonal Sangtec antibody was raised against the whole human protein. These antibodies were selected because they target different regions of the S100B protein (see legend of
Western blots were scanned on a scanner interfaced with a PC using HP Precision Scan Pro 3.02 analysis software (Hewlett-Packard Co., Palo Alto, Calif.). The scanned grayscale images were saved in an uncompressed TIFF format and further analyzed using software specifically designed for measuring grayscale image density, developed by Nonlinear USA, Inc. (Durham, N.C.) Phoretix™ ID (Version 2003.01).
The colorimetric immunosorbent assay, Sangtec® 100 ELISA, by DiaSorin, Inc. (Stillwater, Minn.) was used to quantify S100B. The limit of detection is 0.03 ng/mL. The Canag/Fujirebio system by Fujirebio Diagnostics, Inc. (Tokyo, Japan) was also used to measure various mono- and hetero-dimers of S100B. The Elecsys system (Roche Diagnostics, Indianapolis, Ind.) was used as well for the purpose of measuring S100B.
A LC-MS system Finnigan LCQ-Deca ion trap mass spectrometer system with a Protana microelectrospray ion source interfaced to a self-packed 10 cm×75 um id Phenomenex Jupiter C18 reversed-phase capillary chromatography column was used. Data were analyzed by using all CID spectra collected in the experiment to search the National Center of Biotechnology Information (NCBI) non-redundant database with the search program TurboSequest. All matching spectra were verified by manual interpretation. The interpretation process was also aided by using the programs Mascot and Fasta to perform additional searches, as needed.
Data are presented as mean±standard deviation (SD). JMP® 8.0 (Cary, N.C.) was used for statistical analysis. Correlation plots were produced using statistical software by the Origin Lab Corporation (versions 7.0 and higher, Northampton, Mass.) to calculate correlation coefficients (R) and 95% confidence limits. Significant difference or correlation was assessed by P-values of <0.05, calculated using the Student's t-statistic.
A study of whether tissue sources, outside the brain, contribute significantly to serum levels of S100B is described herein. In this study, the expression of S100B in human tissue was characterized by Western blot using two different antibodies and this data was corroborated with mass spectrometry. How expression in these tissues affects the current clinical methods for detection of S100 was also assessed using the LIAISON® S100 or LIAMAT systems (Sangtec-Diasorin), and the Elecsys system (Roche Diagnostics). 200 subjects were also analyzed to determine if adiposity or fat S100B affected serum levels.
S100B protein content was first analyzed in a variety of tissue samples (Table 2). Western blot analysis revealed that, regardless of the antibody used, S100B is found in tissues other than brain. The results in
The two antibodies gave comparable but not identical results. While both revealed a strong signal from brain tissue, muscle and fat also exhibited strong levels of expression. The data were quantified to construct the bar graph shown in
MS analysis confirmed that the brain tissue signal, consistent by molecular weight with S100B, was indeed this protein (Table 3). When the same lanes were isolated from fat samples, S100B expression was similarly found. Fat tissue was chosen because it was most consistently reported in the literature to contribute to elevated serum S100B levels [Anderson, R E, et al., Ann Thorac Surg, 71:1512-1517 (2001); Steiner, J., et al., Psychoneuroendocrinology, 35:321-324 (2010)]. In addition to S100B, MS analysis also revealed the presence of another protein of the S100 family, namely S100A1 [Donato, R., et al., Int J Biochem Cell Biol, 33:637-668 (2001)].
Results with a Clinically Relevant Platform
Most of the clinical results dealing with the utilization of S100B as a predictor of neurological disorders were obtained with one of several immunoassays that are commercially available (for a complete recent summary see Goncalves C A, et al., Cllin Biochem, 41:755-763 (2008)).
S100B was then measured by ELISA (Sangtec-LIAISON;
In addition to this commercially available immunoassay, previous literature dealing with clinical samples has often used an automated version of the same test [Goncalves C A, et al., Cllin Biochem, 41:755-763 (2008)]. The results of a direct comparison between the automated and manual test are shown in
The literature dealing with the detection of S100B as an indicator of neurological dysfunction or BBB leakage was profoundly affected by the discovery of Marchi et al. (Epilepsia, 48:732-742 (2007)), who demonstrated that upon iatrogenic BBB disruption (BBBD) S100B levels were elevated minutes after the procedure itself, which prompted characterization of S100B as an indicator of BBB damage rather than a protein related to neuronal cell death or other types of brain injuries. However, recent findings by others have shed doubt on the utility of this approach, primarily because of extracranial sources of S100B. The data herein, in fact, show that this is indeed the case and that tissue other than brain expresses this protein. However, it is also known that S100B may be detected in its monomeric or dimeric form. In addition, S100B may form a homo- or hetero-dimer with its companion S100A1.
The molecular nature of the S100B extravasating from the human brain under conditions of iatrogenic BBB disruption was investigated (
The results presented so far unveiled a complex scenario where several different molecular species act, upon BBBD, to modify serum values of S100B. Whether the S100B values in serum of patients or controls across the broadest published population of subjects was investigated. The results are summarized in
Shown herein is that in spite of robust expression by extracranial sources, changes in serum levels are primarily dictated by extravasation across the disrupted BBB. In addition, the main molecular species of total S100B related to blood-brain barrier disruption is the S100B homodimer.
The study described herein compares and cross-validates various approaches to the detection of S100B. In addition, a vast array of subjects and tissues have been studied. Western blot analysis revealed that, regardless of the antibody used, S100B is found in tissues other than brain. The results show, surprisingly, a poor correlation between different antibodies. While cross-reactivity of antibodies is well known and widely accepted, it was nevertheless surprising that the rank order of expression depended on the antibodies used. In addition, testing by ELISA showed a different profile, where expression by extracranial sources was less prominent. However, the gel-based approach was sensitive for S100B which was detected by MS. The co-expression of S100A1 was expected, based on results by others. Although discussion of the possibilities of why such varying expressions of S100B protein between the platforms of Western blot and ELISA was observed is very noteworthy. These findings may have implications beyond recent S100B research efforts, inasmuch as most of the current knowledge on protein function is based on antibody detection of levels by gel electrophoresis. The use of antibody-independent detection therefore is advisable.
Different levels of S100B expression using two different antibodies, and poor qualitative and quantitative correlation were found. Similar results were obtained in rodent tissue with other commercially available antibodies.
A BMI calculation was used to assess individuals' relative body fat. Recent studies on nutrition and metabolism have validated techniques such as ultrasound, air displacement plethysmography and bioelectrical impedance to be superior to BMI for accurately measuring body fat. These techniques were not readily available nor could they be easily implemented and therefore the BMI calculation was utilized not only out of practicality, but also out of the widespread use of it in other S100B studies.
Demonstrated herein is that the clinical detection of S100B is more reproducible and robust. In addition, the type of instrument, or the platform used did not alter the results, nor did it affect the predictive value of the test. The clinical tests all measure total S100B, regardless of it monomeric or dimeric state, nor do they consider whether S100B is bound to S100B or S100A1. Preliminary results with tests detecting only S100B-S100B dimers have demonstrated, as expected, that the ceiling for “normal values” is significantly lower than the published “0.1 ng/ml” dogma [1], [6], [10], [14], [19], [24], [40], [44]-[46] (Kanner A. A., et al. (2003) Cancer 97: 2806-2813; Rothermundt M., et al. (2004) Int Rev Neurobiol 59: 445-470; Biberthaler P., et al. (2001) World J Surg 25: 93-97; Fazio V., et al. (2004) Ann Thorac Surg 78: 46-52; Anderson R. E., et al. (2001) Ann Thorac Surg 71: 1512-1517; Vogelbaum M. A., et al. (2005) Cancer 104: 817-824; Mussack T., et al. (2000) Acta Neurochir Suppl 76: 393-396; Anderson R. E., et al. (2001) Neurosurgery 48: 1255-1258; Jonsson H., et al. (2000) J Cardiothorac Vase Anesth 14: 698-701; Raabe A., et al. (2003) Restor Neurol Neurosci 21: 159-169).
Confirmed herein is that S100B was not exclusively produced by CNS cells. It was found that muscle and fat were chief extracranial expressors, which is consistent with the literature [Anderson R. E., et al. (2001) Ann Thorac Surg 71: 1512-1517]. However, the results are in sharp contrast with the findings linking serum S100B to BMI [Steiner J., et al. (2010) Psychoneuroendocrinology 35: 321-324]. These results used the same system used by us (LIAMAT), but their sample size was significantly smaller. In addition, no cross-validation with other detection systems was used. The range of BMI values was larger, the ages broader, and the racial samples were balanced to reflect the general US population. Diseased patients' samples were also included to add to the clinical significance of the findings. No correlation between S100B and weight or height was found, but, as expected, a correlation with age was found [Portela L. V., et al. (2002) Clin Chem 48: 950-952]. When the values were restricted within a given category (e.g., pediatric controls in Table 1), no correlation between BMI and S100B was found. Why these results are in sharp contrast with Steiner et al. [Steiner J., et al, (2010) Psychoneuroendocrinology 35: 321-324] remains at present unknown.
The results show that extracranial sources of S100B do not significantly affect serum levels. Thus, the reported low sensitivity and positive predictive value (relative to the reported strong specificity and negative predictive value) for S100B [Vogelbaum M. A., et al. (2005) Cancer 104: 817-824] is not apparently due to extracranial release of the protein.
There are two possible explanations that may account for the discrepancy between previous studies documenting elevated S100B levels from extracranial sources and the present findings. There have been recent reports that serum S100B levels are positively correlated with body mass index without evidence of traumatic brain injuries. Interestingly, obesity has been hypothesized to be a state of heightened systemic oxidative stress and inflammatory response, which is mechanistically linked to other co-morbid conditions such as hypertension and small vessel disease. Therefore, it is not clear whether obesity itself or obesity-associated comorbidities contribute to a rise in serum S100B in the previous studies. In this study, the tissue specific expression of S100B in addition to serum S100B was measured, which represents a collective source from multiple disease processes. The study showed that an increase in fat mass might not in isolation be a major contributor to elevated S100B levels. Rather, obesity-related diseases are likely contributors. For example, small vessel ischemic disease associated with obesity is a source of serum S100B [Vogelbaum M. A., et al. (2005) Cancer 104: 817-824; Mazzone P. J., et al. (2009) PLoS One 4: e7242].
Prior studies have shown that cardiothoracic surgeries resulted in higher serum levels of S100B. However, Fazio et al. demonstrated that S100B antibodies from certain ELISA kits might cross-react with other proteins found in serum [Fazio V., et al. (2004) Ann Thorac Surg 78: 46-52]. It is difficult to characterize biomarkers in serum because of the wide range of protein concentrations and predominance of 10 to 20 proteins (albumin, immunoglobulins, etc.) that overwhelm the less abundant signals. This indicates that in other studies yet unknown cross-reactants were artificially increasing the apparent S100B levels measured in serum.
S100B homodimer is expressed in human brain astrocytes. The use of the Proximity Ligation Assay™ (PLA) (Olink Bioscience, Sweden) revealed that S100BB, the homodimer of S100BB, is the astrocytic and brain form of S100B (see
In the experiments shown in
S100B tests used in combination have an improved negative predictive value and are thus less likely to reveal false negative patients. The test was used to analyze serum samples taken from patients diagnosed with systemic lung cancer (small cell and non-small cell carcinoma) who were at risk of brain metastases or with ongoing metastatic disease. The negative predictive value refers to contrast enhanced MRI scans used to visualize brain masses. NPV and PPV are negative and positive predictive value respectively. Specificity (negative predictive value) shows the proportion of negatives which are correctly identified. There was a synergistic benefit when S100B total by Diasorin and S100B Total by Canag in ruling out the presence of brain metastases diagnosed in this population by MRI. Blood was drawn at first diagnosis of systemic lung cancer; within a few days, the MRI scan was performed. Gadolinium was used as a contrast agent to reveal by MRI blood-brain barrier leakage due to tumor invasion in the brain. See
Rats were housed in a controlled environment (21±1° C.; humidity 60%; lights on 08:00 AM-8:00 PM; food and water available ad libitum). Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996). Cleveland Clinic IACUC approved the protocol number 08491 for the performance of the presented experiments.
Rats (male Sprague-Dawley 225-250 g) were injected with methylscopolamine (0.5 mg/kg, i.p., Sigma-Aldrich) and 30 minutes after with pilocarpine (340 mg/Kg, Sigma-Aldrich). Data obtained from a total of 45 rats was analyzed (see also
The number of rats used, timing of drug treatments, blood drawings-analysis and animal sacrifice are indicated in
Stereotactic electrode implantation was performed in rats under isofluorane anaesthesia, using the Kopf stereotactic frame. Approximately, half of the rats used were implanted. Four stainless steel screws (MX-0090-2, Small Parts Inc., Miami, Fla.) were placed bilaterally on the dura mater of the fronto-parietal cortex. A prefabricated Pinnacle pre-amplifier was connected to the screws. The system has three bio-potential channels—2 EEG and 1 EMG. Prefabricated head implants (Pinnacle Inc., USA) ensured accurate electrode positioning and reliable, robust contacts. Cable artifacts are eliminated by pre-amplification of the EEG and EMG waveforms at the animal's head. EEG data were sampled a rate of 200 Hz. All data are transferred via a USB connection to a PC. Rats were left unrestrained for 2 weeks to recovery from surgery before EEG recordings were performed. Each rat was kept in a separate cage under 12-hours dark-light cycles with free access to food and water. Origin Microcal 7.0 and Diadem (National Instruments) was used in conjunction to the acquisition system for data analysis (e.g., time joint frequency analysis).
The schedule of blood sampling is illustrated in
BBB status was assessed using two independent modalities: serum S100β and brain Evan's Blue extravasation [Marchi N., et al, (2009) Neurobiol Dis 33: 171-181; Kanner A. A., et al. (2003) Cancer 97: 2806-2813]. Blood samples were obtained from the tail vein and brains were collected after sacrifice (control, dexamethasone-treated or at onset of SE, n=5 rats/group). S100β: blood samples were collected at the times indicated in
The study was conducted according to the Declaration of Helsinki Criteria and to the procedures for compassionate drug administration approved by the Ethic Committees of Carlo Besta Institute (Milan, Italy). Oral informed consent from was obtained from parents by specialized neurologists. Administration of GCs was considered compassion care since all patients had intractable life threatening seizures. The clinical data of 43 patients (Table 4) of the Carlo Besta Neurological Institute (Milan, Italy) was reviewed. All the patients had a known history of intractable seizures and antiepileptic treatment with conventional AEDs was administered (Table 4). Patients with a history of epileptic syndromes known to respond to steroids (i.e., West, Lennox-Gastaut, Landau-Kleffner and Rasmussen encephalitis) as well as patients affected by documented inflammatory brain disease were excluded. All the subjects received steroid treatment on an in-patient basis.
Different treatments were employed in different patients: ACTH, dexamethasone, methylprednisolone, and hydrocortisone. Details on glucocorticoids (GCs) or ATCH dosage are summarized in Table 4. Patients received GCs or ACTH therapy because of one of the following: 1) >50% increase in seizure frequency; 2) development of non-convulsive status epilepticus; 3) presence of epilepsia partialis continua (EPC). Before GCs or ATCH treatment, patients were evaluated by a team of neurologists, physiologists, and underwent EEG and routine laboratory examinations. Antiepileptic treatments were maintained during the steroid course (Table 4).
Steroidal treatment was, when successful, repeated in case of seizure recurrence. For this reason, the number of treatments reported is greater than the number of patients (treatments=92, see Table 4). GCs or ATCH therapy was considered successful when: 1) seizure frequency decreased by 50%; 2) status epilepticus was stopped; 3) or when epilepsia partialis continua (EPC) was stopped/reduced enough to allow voluntary movement in the affected body district. Duration of treatment was limited to acute dosing, which was repeated when the initial response was beneficial. The effects of length of treatments and efficacy/toxicity of chronic use are not presented herein, since this study design only addressed a proof of principle use of steroids in pediatric epilepsy.
Spike detection, spike area and instantaneous frequency calculation were performed using pClamp 9.2. Statistics were performed with aid of Origin 7.0 (Microcal) and Jump 7.0; data were considered to be significantly different when p<0.05 (by ANOVA or paired t-test for multiple comparisons). Normal distribution of data was evaluated with Wilk-Shapiro routine. Mosaic plots were graphed with Jump 7.0 and transferred to CorelDraw as metafiles. The Diadem (National Instruments) package was used to construct time-frequency plots. Fisher exact test was used (Jump 7.0) to evaluate the significance of probability of SE and incidence of mortality between groups of animals.
Described herein is the evaluation of the effect of anti-inflammatory agents in experimental seizures, and the efficacy of gluco-corticosteroids. The efficacy of anti-inflammatory treatments was evaluated in drug resistant pediatric epilepsies, excluding those conditions already known to benefit from steroidal treatment, i.e. West, Landau-Kleffner, Lennox-Gastaut syndromes and Rasmussen's encephalitis [Grosso S., et al. (2008) Epilepsy Res 81: 80-85; Sevilla-Castillo R. A., et al. (2009) Neuropediatrics 40: 265-268; Verhelst H., et al. (2005) Seizure 14: 412-421]. The response to gluco-corticosteroids, or ACTH was analyzed in a pediatric population and the results were used to develop a hypothesis that also takes into account data obtained from animal experiments where rats were exposed to convulsive doses of the cholinergic agonist pilocarpine.
The justification for extrapolating data obtained from pilocarpine-induced SE to drug resistant epilepsy may be considered inappropriate and one should ideally compare human data to pilocarpine-treated chronic rats who do not respond to AED. Thus, two points of asymmetry can be found in the current study, one related to chronicity of seizures in humans vs. acute nature of BBB disruption-induced seizures, as well as the issue of human epileptic vs. normal brain induced to seize. In fact, to segregate and study drug resistant rats would constitute the best animal correlate of human multiple drug resistance to antiepileptic drugs. However, recent experimental findings suggested that correlates of acute seizures (e.g., as triggered by iatrogenic BBB disruption or pilocarpine) are not dissimilar from chronic seizures. For instance, seizures acutely induced by intraarterial mannitol have EEG features similar to pilocarpine seizure and the histological and immunohistochemical tracts of acute seizures (e.g., cerebrovascular damage) are similar to the ones observed in the chronic epileptic human brain [Marchi N., et al. (2010) Blood-brain barrier damage, but not parenchymal white blood cells, is a hallmark of seizure activity. Brain Res.].
Whether dexamethasone prevents the onset of pilocarpine-induced seizures in rats was evaluated, and seizure-induced mortality was quantified. Experimental details are shown in
Time-joint frequency analysis was performed to examine changes not immediately apparent by EEG inspections. Note that the early burst clusters (single asterisks in
The integrity of the BBB in animals treated with pilocarpine and in those pre-treated with dexamethasone was compared (
Circulating white blood cells (WBCs) were analyzed to determine the level of T-lymphocyte activation after pilocarpine or after dexamethasone followed by pilocarpine. The most important effect of dexamethasone was a drastic reduction in the number of circulating T-cells (CD3+,
The efficacy of add-on glucocorticosteroids (GCs) or ACTH treatment in patients affected by drug-resistant epileptic seizures was investigated (see Table 4). Inclusion criteria are described in the Methods. For the whole study (
The results have shown that dexamethasone reduces the number of rats experiencing status epilepticus (SE) and abolishes mortality. The mechanism by which dexamethasone lessens pilocarpine seizure burden encompasses improved BBB function. This was shown by analysis of dye and marker extravasation in treated vs. untreated animals. The efficacy of add-on gluco-corticosteroids in a population of pediatric drug resistant patients excluding those syndromes known to be responsive to GCs and ACTH (L-G, L-K, West or Rasmussen's) was also studied. The effect was beneficial regardless of the pathology and epileptic syndrome. A selected case where a decrease in FLAIR signal was associated with seizure reduction was also observed. Previous studies have shown that FLAIR hyperintense regions or regions of gadolinium enhancement correspond to sites of BBB leakage [Cornford E. M. (1999) Adv Neurol 79: 845-862; van Vliet E. A., et al. (2007) Brain 130: 521-534; Amato C., et al. (2001) Eur J Radiol 38: 50-5; Huang C. C., et al. (1995) Eur Neurol 35: 199-205; Lansberg M. G., et al. (1999) Neurology 52: 1021-1027; Alvarez V., et al. (2010) Epilepsy Behav 17: 302-303; Ivens S., et al. (2010) J Neurol 257: 615-620].
The findings are presented herein in a format where clinical data are presented together with animal results. This is appropriate because: 1) there is a recognized urgency to provide rapid therapeutic advancement by comparing clinical and laboratory results (Kwan P., et al. (2009) Definition of drug resistant epilepsy: Consensus proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies. Epilepsia; Stefan H., Lopes et al. (2006) Acta Neurol Scand 113: 139-155; Elkassabany N. M., et al. (2008) J Neurosurg Anesthesiol 20: 45-4) the anecdotal use of corticosteroids in clinical epilepsy has recently expanded (see below), but its full potential for widespread use is limited by the lack of scientific validation of their use. The study described herein compared the efficacy of glucocorticosteroids and ATCH: 1) in patients across a wide spectrum of epileptic etiologies and syndromes, 2) with the exclusion of those syndromes known to be steroid responsive (i.e. L-G, L-K, West or Rasmussen's). The efficacy of steroids in patients were also compared with the effects observed in the pilocarpine model. It has been previously demonstrated that direct inhibition of leukocyte-mediated blood-brain barrier disruption, comparable to the effects of GCs, prevents SE in this model [Fabene P. F., et al. (2008) Nat Med 14: 1377-1383; Marchi N., et al. (2009) Neurobiol Dis 33: 171-181].
These patients failed at least three AED medications and were exposed to all traditional anti-epileptic care that benefits most of their drug-respondent counterparts. Thus, while use of a placebo arm is mandatory to evaluate once and for all whether anti-inflammatory therapy is a valid AED alternative, the studies herein have shown their utility at least as an add-on maneuver.
Animal data were obtained exclusively from adult rats; this does not appear to be a crucial limitation since patient age ranged from a few months to adolescence and no differences in GCs or ACTH effect were observed with regards to age. Finally, patients received a combination of GCs and anti-epileptic drugs (Table 4) while rodents did not.
There are several lines of evidence suggesting that the blood-brain barrier could be a valid adjunctive target for anti-epileptic drug therapy. Animal studies that have shown that breaching the BBB is a reliable mean towards decreased seizure threshold [van Vliet E. A., et al. (2007) Brain 130: 521-534] or seizure development [Marchi N., et al. (2007) Epilepsia 48: 732-742; Seiffert E., et al. (2004) J Neurosci 24: 7829-7836]. More importantly, these animal studies have been supported by concurrent clinical data showing that blood-brain barrier disruption (BBBD) causes seizures in human subjects [Marchi N., et al, (2007) Epilepsia 48: 732-742] and that BBBD has a causative link with post-traumatic epilepsy [Korn A., et al. (2005) J Clin Neurophysiol 22: 1-9]. It has been previously demonstrated that prevention of BBB failure reduces seizure onset [Marchi N., et al. (2009) Neurobiol Dis 33: 171-181]. The data presented herein further support a pharmacological approach aimed to prevent BBB failure or restore BBB integrity.
There are important clinical correlations resulting from the findings. First and foremost is the fact that the efficacy of corticosteroids in pediatric epilepsy is not limited to epileptic encephalopathies, such as infantile spasms and Rasmussen encephalitis [Vezzani A., et al. (2005) Epilepsia 46: 1724-1743; Granata T. (2003) Rasmussen's syndrome. Neurol Sci 24: Suppl 4S239-S243; Granata T., et al. (2003) Neurology 60: 422-425]. Rather, the effects seemed to be pronounced in focal epilepsy, including those due to focal dysplastic lesions (
The results of the experimental study herein support the hypothesis that inflammatory mechanisms and BBB damage contribute to seizure generation and severity [Ravizza T., et al, (2008) Neurobiol Dis 29: 142-160; Vezzani A., et al. (2010) Curr Opin Investig Drugs 11: 43-50]. The pattern of WBC activation was confirmed in the experimental model used [Marchi N., et al. (2009) Neurobiol Dis 33: 171-181]. Interestingly, in rats pre-treated with dexamethasone, a decrease in SE severity (
The efficacy of corticosteroids observed in patients supports the hypothesis that seizures of different etiologies (including those due to congenital malformations or acquired brain damage) may be aggravated by inflammatory mechanisms and BBB disruption. The hypothesis linking BBB damage to seizures is in agreement with histological studies that have shown BBB dysfunction in human epileptic tissue and with MRI studies showing changes corresponding to the location of EEG activity in patients with partial status or focal epilepsy [van Vliet E. A., et al. (2007) Brain 130: 521-534; Amato C., et al, (2001) Eur J Radiol 38: 50-54; Lansberg M. G., et al. (1999) Neurology 52: 1021-1027; Paladin F., et al. (1998) Ital J Neurol Sci 19: 217-220; Paladin F., et al. (1999) Ital J Neurol Sci 20: 237-242; Tan K. M., et al. (2008) Epilepsy. Res 82: 190-193; Alvarez V., et al. (2010) Epilepsy Behav 17: 302-303; Vezzani A., et al. (2005) Epilepsia 46: 1724-1743]. The clinical study design herein did not include the systematic evaluation of MRI before and after treatments, nevertheless the decrease in FLAIR hyperintensity concurrent with seizure reduction after steroid treatment (
Pilocarpine has been historically used to model human disease and several features of this model have common traits with human epilepsy [Leite J. P., et al. (1995) Epilepsy Res 20: 93-104; Leite J. P., et al. (2002) Epilepsy Res 50: 93-103]. For example, it was shown that pilocarpine seizures cause cerebrovascular changes which are consistent with those seen on MRI of patients [Leite J. P., et al. (2002) Epilepsy Res 50: 93-103]. These consist of homeostatic failure leading, in patients, to MRI hyperintensity and in animals to increased extravasation of intravascular (Evans blue) or cerebral-to-serum (S100β) indicators (
If BBB leakage is the main initiator of seizures, and if BBB repair is protective against neurological disease, which are the mechanisms involved? The experimental evidence provided herein, together with previous findings [Vezzani A., et al. (2005) Epilepsia 46: 1724-1743; Marchi N., et al. (2007) Epilepsia 48: 1934-1946: Fabene P. F., et al. (2008) Nat Med 14: 1377-1383], point to an inflammatory-induced damage of the BBB. Corticosteroids have a profound effect on human and rodent BBB permeability [Vezzani A., et al. (2005) Epilepsia 46: 1724-1743; Hermsen C. C., et al. (1998) J Infect Dis 178: 1225-1227; Betz A. L., et al. (1990) Stroke 21: 1199-1204; Cucullo L., et al. (2004) Brain Res 997: 147-151; Gutin P. H. (1975) Semin Oncol 2: 49-56; Rook G. A. (1999) Baillieres Best Pract Res Clin Endocrinol Metab 13: 567-581]. Moreover, corticosteroids have an impact on the number of circulating T-cells. Downstream signaling by IL1-β appears to be a common thread in pilocarpine-induced seizures [Vezzani A., et al. (2005) Epilepsia 46: 1724-1743; Marchi N., et al. (2009) Neurobiol Dis 33: 171-181; Riazi K., et al. (2010) Epilepsy Res 89: 34-42]. It therefore seems reasonable to assume that the immune system acts in concert to produce BBB leakage and seizures, while the counteraction of corticosteroids has the opposite effect [Riazi K., et al. (2010) Epilepsy Res 89: 34-42]. These results further indicate that seizures and inflammation belong to the same chapter of neuro-immunology, as also shown recently [Maroso M., et al. (2010) Nat Med 16: 413-419].
An important correlate of the findings herein is the fact that animal data strongly indicate that anti-inflammatory treatments have a pronounced effect on survival, which in the pilocarpine model is usually achieved only by stopping SE with diazepam or barbiturates [Blair R. E., et al. (2009) Neurosci Left 453: 233-237]. This likely bears significant relevance for the treatment of catastrophic epilepsies in pediatric or adult settings.
It is important to note that a non-inflammatory mechanism for steroidal action exists, namely the modulation of GABA receptors [Rogawski M. A., et al. (2002) Int Rev Neurobiol 49: 199-219]. This is a possible interpretation of results but is unlikely given that a number of corticosteroids, ACTH, or IL1-RA exerted the same effects. While the results cannot fully confirm or rule out a specific neuronal action of steroids, the fact that the response spanned across a wide range of anti-inflammatory molecules makes this unlikely.
Finally, while steroidal treatment showed promise in the study described herein, the fact remains that the use of potent and potentially harmful anti-inflammatory drugs is not a viable long-term option. The treatment, when successful, had to be repeated once the efficacy waned. This is likely due to traditional, concurrent etiologic mechanisms including cortical dysplasia, other brain lesions, etc. The proposed scenario thus indicates that: 1) reduced BBB function on an abnormal cortical/hippocampal background facilitates seizures; 2) the lesional tissue itself promotes BBB dysfunction, cooperating towards a further decrease of the threshold for seizures; 3) steroids “repair” the BBB while having no impact on circuital and structural abnormalities. This reduces seizure probability; and 4) the anti-inflammatory efficacy decreases over time. This vicious cycle, to be interrupted requires simultaneous targeting of neurons and endothelial cells. In multiple drug resistant patients, AEDs are obviously not sufficient to reduce hypersynchronous firing: a new therapy combing anti-inflammatory potency with neuronal targeting may be the necessary and winning combination.
The results herein indicate the potential application of GCs in treating drug resistant seizures and support further studies assessing the effect of GCs in experimental chronic seizures.
The experiment described herein measured S100B (total, S100AB, S100BB by Canag ELISA after subarachnoid hemorrhage (SAH). The antibodies used were Canag Prod. No. 708-10 (total S100B), 706-10 (heterodimer) and 701-10 (homodimer). In addition, total S100B was measured by an independent method (Diasorin, shown as S100B*). The data in
Hypothermia is used to reduce blood-brain barrier damage (and development of brain damage) in patients suffering from an ischemic-hemorrhagic stroke. Note that regardless of the test used, there was a significant difference between hypothermia (HT) or normothermia (NT) patients. Hypothermia was induced using surface cooling in patients with severe acute ischemic stroke (part of a multicenter study “Cooling for Acute Ischemic Brain Damage, COOL AID I”). Patients were cooled for periods 12-72 hours, with a mean duration of hypothermia of 47.4±20.4 hours (including a rewarming period of 22.6±15.6 hours). Blood samples were drawn at 72 hours post-procedure. Serum was separated by centrifugation (2000 RPM, 10′) and stored at −80 C. Samples were then analyzed by ELISA and processed as recommended by the vendor.
As shown in
In this example, it is shown that anti-S100B auto-antibodies can be detected in serum of patients with a history of blood-brain barrier disruption. Results in
To test the hypothesis that increased serum S100B due to BBBD are sufficient to trigger an autoimmune response, AS100BAb in patients undergoing periodic, repeated, osmotic BBBD was measured. In these patients, and in analogous animal models, proportionality exists between serum S100B and extent of BBB disruption. Data show that BBBD triggers a time-dependent increase in autoimmune load and that the process itself peaks at approximately 6-8 months.
The time of autoantibody persistence after a minor traumatic brain injury was also measured. The data in
The significance of these finding is that measuring autoantibodies against S100B is a tool to quantify or detect post-traumatic events.
The data in
Provided below is an examples of methods that can be employed to detect autoantibodies against S100B in a sample such as human serum
1) Coated wells with 100 μL of human S-100β protein Catalog number-559291, EMD chemicals (1 μg/ml). Incubated overnight at 4° C.
2) Washed 3× with PBS.
3) Added 100 μL/well of 1% BSA as blocking solution; incubate at RT (room temperature) for 2 hrs
4) Aspirated & washed wells 3× with 200 μL of PBS containing 0.05% Tween 20/wash
5) Added 100 μL of serially diluted standards (positive ctrls), or serum samples (1:1000 and 1:5000) from patients to the 96 well Nunc Maxisorp plate. Incubate 1 hr at RT.
6) Aspirated & washed wells 3× with 200 μL of PBS containing 0.05% Tween 20/wash.
7) Added 200 μL of HRP (horseradish-peroxidase) Goat Anti-mouse IgG to positive controls and 200 μL of HRP conj. Goat anti-human IgG for serum samples. Incubated for 1 hr. at RT.
8) Aspirated & washed the wells, 3× with 200 μL of PBS containing 0.05% Tween 20/wash
9) Added 100 μL of OPD solution and incubate for 20-30 minutes at RT
10) Stopped the reaction by adding 100 μL of 2.5 M Sulfuric acid and read the plate using an ELISA plate reader @ 490 mu.
PB, VPA, TPM,
PB, VPA, TPM,
LVT, PHT, MDZ,
PB, VPA, TPM,
LVT, PHT, MDZ,
CZP
CBZ, TPM, PB,
CBZ, PB, VPA,
PB, PHT, LTG,
Clo, CBZ, VPA,
PB, PHT, LTG,
Clo, CBZ, VPA,
TPM, Pir, CZP,
PB, TPM, GVG,
PB, TPM, GVG,
Clo, MDZ, PHT,
TPM, VPA, PHT,
CBZ, GVG, LTG,
Clo, CZP, LVT,
PB, VPA, PHT,
VPA, CZP, Clo,
VPA, CZP, Clo,
GVG, LTG, Clo,
TPM, LTG
LTG, MSM
VPA, ESM, LVT,
VPA, ESM, LVT,
VPA, ESM, LVT,
Clo, TPM, BR,
VPA, ESM, LVT,
VPA, GVG, CBZ,
VPA, GVG, CBZ,
PB
SUL, PHT,
VPA, ESM, LTG
VPA, CBZ, PRM,
GVG, PHT, PB,
PRM, Clo, CBZ,
PHT, CBZ, Clo,
LTG, GVG, PRM,
Clo, ESM, LTG,
CBZ, TPM, PB,
MDZ
FBM, ESM, LVT,
FBM, ESM, LVT,
FBM, ESM, LVT,
FBM, ESM, LVT,
FBM, ESM, LVT,
VPA, Clo, ESM,
VPA, ESM
VPA, CBZ, CNZ
VPA, CBZ, CNZ
VPA, CBZ, CNZ
VPA, CBZ, CNZ
VPA, CBZ, CNZ
VPA, CBZ, CNZ
VPA, CBZ, CNZ
VPA, CBZ, CNZ
LTG, CBZ, VPA,
LTG, CBZ, VPA,
VPA, Clo, LVT,
LTG
VPA, ESM, Clo,
PB, VPA, Clo,
PB, VPA, Clo,
VPA, ESM, LTG,
TPM,
VPA, ESM, Clo,
VPA, ESM, Clo,
VPA, ESM, Clo,
VPA, CBZ, LTG,
VPA, CBZ, LTG,
LTG, VPA
VPA, Clo, TPM,
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 61/483,974, filed on May 9, 2011 and U.S. Provisional Application No. 61/522,557 file on Aug. 11, 2011. The entire teachings of the above applications are incorporated herein by reference.
This invention was made with government support under RO1 NS43284, RO1 NS38195, and R21 HD057256 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/037170 | 5/9/2012 | WO | 00 | 11/8/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61483974 | May 2011 | US | |
| 61522557 | Aug 2011 | US |
