Patent Application
20130281504
The present invention relates generally to the fields of chemistry, pharmacology and medicine and more particularly to the treatment of neurodegenerative diseases, deterring or reducing neuronal damage following ischemic/hypoxic/anoxic events and treatment of other conditions wherein microglia-mediated neurotoxicity occurs.
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Microglia are non-neural, interstitial cells of mesodermal origin that form part of the supporting structure of the central nervous system in humans and other mammals. Microglia are tissue resident macrophages of the brain. Microglia come in various forms and may have slender branched processes. They are migratory and, when activated (usually by some instigating stimulus), can act as phagocytes, which engulf and remover nervous tissue waste products.
In various neurodegenerative diseases, damage to nerve cells is believed to occur, at least in part, due to activation of microglia by some instigating stimulus (an “activator”). For example, in Alzheimer's disease (AD), amyloid plaques accumulate between nerve cells (neurons) in the brain. Amyloid is a term, which broadly refers to protein fragments that the body produces normally. Beta amyloid (Aβ) is a protein fragment that comes from an amyloid precursor protein. In healthy brains, these Aβ protein fragments are broken down and eliminated. However, in AD, the Aβ protein fragments aggregate to form hard, insoluble plaques. Aggregated forms of Aβ as well as soluble precursor forms called oligomeric Aβ act as microglial activators. The activated microglia have a beneficial effect of phagocytiozing Aβ deposits, but they also have deleterious neuron-damaging effects, such as direct microglial neuron killing and by causing production of neurotoxic nitric oxide (NO) and inflammatory cytokines.
Microglia also play a roll in causing brain damage following hypoxic or anoxic insults to the brain. Hypoxic or anoxic brain insults may occur due to various causes, including but not limited to ischemic or hemorrhagic strokes, cardiac arrest and resuscitation, carbon monoxide poisoning, trauma, asphyxiation, strangulation, drowning, hemorrhagic shock, inhalant substance abuse (“huffing”), brain edema, iatrogenic disruption of cerebral circulation during surgery or other medical procedures, etc.
Inhibition of the intermediate-conductance calcium-activated potassium channel KCa3.1 in microglial cells is viewed as a potential therapeutic approach for reducing microglia-mediated neurotoxicity. However, it is desirable for therapies aimed at microglia-mediated neurotoxicity to meet the following goals:
In accordance with the present invention, there is provided a method for deterring microglia-mediated neurotoxicity in a human or non-human animal subject, said method comprising the step of inhibiting or blocking the intermediate-conductance calcium-activated potassium channel KCa3.1 in microglia. The inhibition or blocking of the KCa1.3 channels may be accomplished by administering to the subject a therapeutically effective amount of a KCa3.1 inhibiting substance, non-limiting examples of which are described in U.S. Pat. No. 7,235,577. One such substance comprises 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34).
Further in accordance with the invention, in some embodiments, the inhibition or blockade of the intermediate-conductance calcium-activated potassium channel KCa3.1 may be carried out in a manner that reduces neurotoxic effects of the microglia without preventing beneficial effects of the microglia.
Still further in accordance with the invention, the method may be carried out to deter or slow neuron damage in subjects who suffer from a neurodegenerative disease. Some such subjects may have Aβ deposits (such as those suffering from Alzeheimer's Disease or who are in the process of developing Alzeheimer's Disease) and the inhibition or blockade of the intermediate conductance calcium-activated potassium channel KCa3.1 may be carried out in a manner that reduces at least one neurotoxic effect of microglia (e.g., microglia-mediated neuronal killing, microglial production of NO and/or microglial cytokine production) while not preventing microglia from phagocytosing Aβ deposits.
Still further in accordance with the invention, in some embodiments, the method will be carried out to reduce neural damage in subjects who have suffered or are suffering an ischemic, anoxic or hypoxic condition, event or insult, such as those who suffer a) ischemic stroke, b) hemorrhagic stroke, c) cardiac arrest and resuscitation, d) carbon monoxide poisoning, e) trauma, f) asphyxiation, g) strangulation, h) drowning, i) hemorrhagic shock, j) inhalant substance abuse or huffing, k) brain edema and l) iatrogenic disruption of cerebral circulation during a surgery or other medical procedure.
Still further aspects and details of the present invention will be understood upon reading of the detailed description and examples set forth herebelow.
The following detailed description and the accompanying figures to which it refers are intended to describe some, but not necessarily all, examples or embodiments of the invention. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The contents of this detailed description and the accompanying drawings do not limit the scope of the invention in any way.
To examine microglia activators that are pathologically relevant to the early stage of AD development, Applicants investigated the ability of low levels of Aβ oligomers (AβO) to activate microglia and discovered, in the course of such work, that AβO can activate microglia at concentrations in the range of 5-50 nM. Such low concentrations of AβO are usually not sufficient to cause direct neurotoxicity. This effect follows a bell-shaped dose-response curve and tapers off at concentrations above 100 nM. Furthermore, Applicants have determined that AβO-activated microglia release soluble neurotoxic substances that cause neuronal damage, and this mode of microglia activation and neurotoxicity is dependent on microglial KCa3.1. This indicates that neuroinflammation mediated by microglia is a significant contributor to early AD pathogenesis when Aβ starts to accumulate in brain prior to fAβ deposition.
Chemicals—
Lipopolysaccharides (LPS), Congo red (CR), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), polyinosinic acid (poly I), [3,8-diamino-5-(3-(diethylmethylamino)propyl)-6-phenyl phenanthridinium diiodide (PI), apamin, and doxycycline were purchased from Sigma (St. Louis, Mo.). The CD40 ligand, CD 154, was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.). The macrophage colony stimulatory factor (MCSF) was purchased from R&D Systems. The KCa3.1 inhibitor TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) was synthesized as described in expressly incorporated U.S. Pat. Nos. 6,803,375 and 7,235,577.
Preparation of Aβ Solutions—
AβO solutions as well as the unaggregated and fibrillary Aβ were prepared as described. Our preparation of AβO follows the procedure described by Lambert et al.; (1998) Proc Natl Acad Sci USA 95, 6448-6453, except that the Aβ42 peptide was diluted with Opti-MEM culture medium instead of the F12 medium originally described, before incubation at 4° C. for 24 h to generate oligomers. This preparation of AβO has been extensively characterized in our laboratory. To ensure consistency of quality, a random sample from each batch was chosen and imaged using electron microscopy and atomic force microscopy to characterize the size and shape of the aggregates. The biological activities of each batch were confirmed by determining AβO's neurotoxic activity, synaptic binding activity and ability to rapidly induce exocytosis of MTT formazan.
Soluble AβO from Human Brain Tissue—
Hippocampal tissue samples were obtained postmortem from three AD subjects and two cognitively and pathologically normal control subjects. A11 subjects had comparable postmortem intervals averaging 5.5 h. Soluble extracts from brain tissues were prepared as described by Lacor, P. N., et al.: (2004) J Neurosci 24, 10191-10200. Molecular weight fractionation of oligomeric species was performed using Centricon YM-100 and YM-10 concentrators (Millipore, Bedford, Mass., USA). The relative abundance of AβO in the resulting solutions was determined by Western blots using the 6E10 antibody and dot blots using the A11 antibody. While the AD samples contained various amounts of AβO, the two control samples showed no detectable AβO on Western blots and almost background levels on dot blots.
To ensure accurate measurement of the total quantity of Aβ in the extract, Applicants used two Aβ ELISA kits purchased from IBL America (Minneapolis, Minn.) and Wako Chemicals USA (Richmond, Va.), respectively. The procedures were conducted according to manufacturer's protocols.
Primary Cultures—
Primary microglia cultures derived from newborn C57BL/6J mice were prepared from mixed glia cultures with the “shaking off” method described by Maezawa, I., et al.; (2006) FASEB J 20, 797-799. Cultures were ≧99% pure for microglia as demonstrated by anti-IBA1 immunostaining. To obtain conditioned medium (CM) for treating neurons, microglia were first cultured in 24-well culture plates at a density of 1.3×105 cells/cm2 for 24 hrs in DMEM with 10% fetal bovine serum (DMEM10). Cultures were washed extensively and changed to the Neurobasal medium with B27 supplement (NB/B27, Invitrogen) without serum and cultured for another 24 hrs. The NB/B27-based microglia CMs were collected, briefly centrifuged, and used immediately or frozen for future uses.
Hippocampal neuronal cultures were prepared from newborn wild type C57BL/6J mice according to the method of Xiang et al (26). Neurons were cultured in NB/B27 at a density of 2.5×105 cells/well in 12-well plates or 8×105 cells/well in 6-well plates for at least 14 days before they were treated with microglia CM.
Hippocampal slice cultures (400 μm-thick) were prepared from 7-day-old C57BL/6J mice as previously described (25) and cultured for 10 days in vitro before use. Neuronal damage in the slices was monitored by PI uptake following Bernardino et al (27). PI itself is not toxic to neurons (27). Briefly, hippocampal slices were pretreated with or without doxycycline (20 μM) or TRAM-34 (1 μM) for 1 hr and then treated with medium containing PI (2 μM) and AβO of indicated concentration, with or without doxycycline or TRAM-34. After 24 hrs, the slices were observed under a Nikon Eclipse E600 microscope and the red (630 nm) fluorescence emitted by PI taken up by damaged cells was photographed by a digital camera (SPOT RTke, SPOT Diagnostics, Sterling Heights, Mich.) with fixed exposure time.
BrdU Incorporation Assay—
Microglia were plated onto 48-well culture plates at a density of 1×105 cells/well in DMEM10 and incubated for 24 hrs. Cells were washed with serum-free Opti-MEM medium three times and treated with indicated concentrations of AβO in Opti-MEM. After 5 hr incubation, 10 μM BrdU was added and allowed to be incorporated into DNA of proliferating cells during an additional 16 hr of incubation. Cells with positive BrdU incorporation were determined by an immunocytochemical stain for BrdU using an anti-BrdU antibody conjugated with Alexa594 (Invitrogen) following the manufacturer's protocol (Chemicon), and counted.
NFκB Assay—
The detection of cells with active NFκB was performed according to the method described by Franciosi, S., et al.; (2006) J Neurosci 26, 11652-11664. Briefly, microglia were plated as described above and treated with activators for 2 hrs. Cells were fixed and stained with anti-NF-κBp65 antibody (1:250, Chemicon) and with the nuclear dye DAPI.
Assays for Neuronal Viability—
Hippocampal neurons were prepared as described above and were plated onto 96-well plate at a density of 6×104 cells/well and cultured for 14 days. CM from microglia cultures was added onto neurons with indicated dilutions, and cultures were incubated for 24 hrs. Neuronal viability was evaluated by the MTT assay and the LDH release assay as previously described by Maezawa, I., et al.; (2006) J Neurochem 98, 57-67 and Maezawa, I., et al.; (2006) FASEB J 20, 797-799.
Immunofluorescence Staining and Quantification—
For immunofluorescent staining of neurons, cultures were fixed in 4% paraformaldehyde and stained with anti-PSD95 (1:200, Cell Signaling), anti-MAP2 (1:500, Chemicon) and anti-acetylated α-tubulin (1:250, Zymed). For immunofluorescent staining of microglia, cultures or hippocampal slices were fixed in 4% paraformaldehyde and stained with anti-IBA1 (1:500, Wako Chemicals USA), anti-SRA (E-20, 1:200, Santa Cruz Biotechnology), and anti-CD11b (1:200, AbD Serotec USA, Raleigh, N.C.). The fixed cells or hippocampal slices were incubated with antibodies for overnight at 4° C. followed by secondary Alexa488 conjugated anti-mouse or Alexa568 conjugated anti-rabbit antibody (1:700, Molecular probes). Immunostained images were observed under a Nikon Eclipse E600 microscope and photographed by a digital camera (SPOT RTke, SPOT Diagnostics, Sterling Heights, Mich.).
For quantification of the PSD95-immunoreactive puncta along the dendrites, photomicrographs of PSD95 immunostained cultures were randomly taken from each culture condition. The images were transformed to 8 bit gray scale and analyzed with the Image J program. The number of puncta in each photomicrograph was counted and normalized by dendritic length. The photography and analysis were conducted in an investigator-blinded manner
Western Blot Analysis—
To obtain lysates, cells or tissues were washed with ice-cold PBS and incubated with a buffer containing 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 2% SDS, proteinase inhibitor cocktail (Sigma), and phosphatase inhibitor cocktail (Sigma). Lysates were briefly sonncated and cleared by centrifugation at 50,000 rpm for 10 min. Equivalent amounts of protein were analyzed by Tris/HCL gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes and probed with antibodies. Visualization was performed using enhanced chemiluminescence (ECL, Amersham Pharmacia, Piscataway, N.J.).
The following primary antibodies (dilutions) were used: anti-p38MAPK (1:1000, Cell Signaling Technology, Boston, Mass.), anti-phospho-p38MAPK (1:1000, Cell Signaling Technology), anti-Synaptophysin (1:1000, Abcam), anti-PSD95 (1:1000, cell signaling), anti-GRIP 1 (1:1000, UpState), anti-MAP2 (1:1000, Chemicon), anti-acetylated α-tubulin (1:2000, Zymed), and anti-β-actin (1:3000, Sigma). Secondary antibodies were HRP-conjugated anti-rabbit, anti-goat, or anti-mouse antibody (1:3000 Amersham).
Assay for Nitric Oxide (NO)—
Conditioned medium collected from microglia cultures (1×105/0.75 cm2) and hippocampal slices treated with Aβ for 24 hrs were analyzed by the Nitric Oxide Quantization Kit according to the protocol of the manufacturer (Active Motif, Carlsbad, Calif.). Data were normalized to the amount of total protein.
Patch-Clamp Experiments—
Microglia, that were “floating off” from their feeding astrocyte layer, were harvested with a pipette, washed, attached to poly-L-lysine coated cover-slips, and then studied in the whole-cell mode of the patch-clamp technique with an EPC-10 HEKA amplifier. The pipette solution contained 145 mM K+ aspartate, 2 mM MgCl2, 10 mM HEPES, 10 mM K2EGTA and 8.5 mM CaCl2 (1 μM free Ca2+), pH 7.2, 290 mOsm. To reduce chloride “leak” currents, we used a Na+ aspartate external solution containing 160 mM Na+ aspartate, 4.5 mM KCl, 2 CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4, 300 mOsm. K+ currents were elicited with voltage ramps from −120 to 40 mV of 200 ms duration applied every 10 s. Whole-cell KCa3.1 conductances were calculated from the slope of the TRAM-34 sensitive KCa current between −80 mV and −75 mV where KCa3.1 currents are not “contaminated” by Kv1.3 (which activates at voltages above −40 mV) or inward-rectifier K+ currents (which activate a voltages more negative than −80 mV). Cell capacitance, a direct measurement of cell surface area, was continuously monitored during recordings. KCa3.1 current density was determined by dividing the TRAM-34-sensitive slope conductance by the cell capacitance.
Statistical Analysis—
Statistical analyses were performed using SigmaPlot 11 software (Systat Software, Inc). Analysis of variance (ANOVA) was used to compare quantitative values from cultures across groups. Tukey's studentized range test was used to adjust for multiple comparisons in post-hoc pairwise tests.
AβO at Low Nanomolar Concentrations Stimulates Microglia into a Distinct Activation Phenotype.
Our AβO preparations affected viability of cultured hippocampal neurons at concentrations above 100 nM. The same preparations induced proliferation of cultured microglia, starting at 5 nM (22.5 ng/ml) and maximizing at around 50 nM (225 ng/ml), as seen in
At 24 hr and 48 hr post-stimulation, cell counts in cultures treated with 20 nM AβO were 184±8.59% (mean±S.E, n=6, p<0.001, see
As shown in
To determine if AβO derived from human brain would also promote microglia proliferation, we obtained soluble extracts from hippocampi of AD and age-matched non-demented control subjects and prepared the fractions containing AβO but excluding unaggregated Aβ or large aggregates. This preparation of human AβO has been well characterized in previous studies. The quantities of Aβ in these extracts were determined by sandwich ELISA. Because ELISA may underestimate the total quantity of Aβ when in oligomeric forms, we first deaggregated Aβ in the extract with guanidine HCl as previously described. In addition, Applicants used two different commercial ELISA kits and obtained consistent Aβ concentrations. Surprisingly, soluble extracts containing AβO from all three AD brains consistently stimulated microglia proliferation at sub- to low nanomolar concentrations (˜0.11 to 1.24 nM Aβ42) (
As shown in
As mentioned above, microglia activation is often accompanied by increased release of nitric oxide (NO), synthesized by the inducible NO synthase (iNOS). However, Applicants have determined that AβO treatment significantly increased NO generation as evaluated by measuring the concentration of nitrite, its stable metabolite, released into the medium. After normalization to total amount of cellular protein, the data indicate a ˜80% increase of NO release per cell; therefore this increase can not be explained solely by AβO-induced cell proliferation (
AβO-Stimulated Microglia Activation Depends on SRA and can be Blocked by Doxycycline.
To explore the molecular mechanisms and potential pharmacological blockage of AβO-induced microglia activation, Applicants tested several known inhibitors of microglia activation. It has previously been reported that scavenger receptors, in particular SRA, participate in the binding of fAβ. Because AβO treatment increased microglial expression of SRA (
Doxycycline, a second generation tetracycline, has been shown to provide neuroprotection in various models of neuronal injuries by inhibiting microglia activation. Applicants found that doxycycline was able to inhibit AβO-induced microglia activation, including NFκB activation and NO generation (
Regulation of AβO-Stimulated Microglia Activation by KCa3.1.
By modulating Ca2+ signaling, the calcium-activated potassium channel KCa3.1 has been reported to be involved in microglia proliferation, oxidative burst, NO production and microglia mediated neuronal killing in rat microglia. To determine if mouse microglia also express functional KCa3.1 channels, Applicants studied their K+ channel expression in the whole-cell configuration of the patch-clamp technique. Similar to previous electrophysiological studies of mouse microglia cultured under various conditions, Applicants observed KCa, KV and Kir currents. The KV currents exhibited the typical biophysical properties of Kv1.3. KCa currents became visible when microglia were dialyzed through the patch-pipette with 1 μM of free Ca2+ (
Using TRAM-34 as a test compound and pharmacological tool, Applicants probed the role of KCa3.1 in AβO-induced microglia activation and neurotoxicity. TRAM-34 alone did not affect microglia viability, as judged by cell count and NO generation (
Aβ Cause Indirect Neuronal Damage Via Microglia.
Although AβO induced microglia activation at concentrations lower than those required for direct neurotoxicity, Applicants asked if this activation would result in indirect neuronal damage. To test this possibility, Applicants activated cultured microglia using sub-neurotoxic concentrations of AβO, and transferred the microglia-conditioned medium (AβO-CM) to cultured hippocampal neurons. As controls, Applicants also treated neurons with CM derived from microglia mock-treated with solvent (Con-CM), and medium with Aβ but without being conditioned by microglia.
Because dendritic and synaptic damage likely play a more important role than neuronal loss in early stages of AD, Applicants asked if AβO could enhance the ability of microglia to damage dendrites and synapses. Applicants found that neurons treated with AβO-CM showed much more robust signs of dendritic damage than those treated with Con-CM, as demonstrated by immunofluorescent stains with two dendritic markers, acetylated tubulin (Ac-TN) and microtubule associated protein 2 (MAP2) (
To confirm that the indirect neurotoxicity was a consequence of microglia activation by AβO, Applicants tested if blocking AβO-induced microglia activation by doxycycline or TRAM-34 also blocks the indirect neurotoxicity. CM from AβO-treated microglia co-incubated with doxycycline or TRAM-34 did not alter the dendritic morphology (
AβO Induces Neurotoxicity Via Activating Microglia in Organotypic Hippocampal Slices.
Applicants further tested the AβO effect on microglia using organotypic hippocampal slice cultures, which would better reflect the conditions in the brain in turns of microglial density and their interaction with astroglia and neurons. Treating hippocampal slices with 5-50 nM Aβ caused a significant increase in cells immunoreactive for SRA and CD11b, markers for activated microglia (
NO is the Major Mediator of 40-Induced Microglial Neurotoxicity.
NO was found to be a major inflammatory mediator released by AβO-stimulated microglia, Applicants tested whether NO mediates neurotoxicity. Applicants used two selective inhibitors of iNOS, N-iminoethyl-Llysine (L-NIL) (57) and N-[(3-aminomethyl)benzyl]acetamidine (1400 W) and found that both compounds significantly blocked the increased NO release induced by AβO (
These effects were unlikely to be caused by direct actions of the compounds on neurons because neurons do not express KCa3.1 as discussed above, and doxycycline was shown to provide neuroprotection by regulating microglia, but not via neuronal mechanisms. Although a direct anti-amyloidogenic property of doxycyline was previously shown, the above-described experimental results do not indicate any direct effect on AβO because there was no effect of doxycycline on the morphology, size, or neurotoxic activities of AβO. Therefore, in the more physiological hippocampal slice cultures, low nanomolar AβO caused indirect neurotoxicity by activating microglia.
The experimental data summarized above show that Aβ is able to activate microglia at concentrations at least 10-fold lower than those used to induce direct neurotoxicity. Low nanomolar AβO activates microglia to release soluble neurotoxic factors and thus indirectly damages the integrity of neurons and synapses. AβO stimulates a unique neuroinflammatory pattern with increased NO generation but without the production of a regular panel of inflammatory mediators such as prostaglandin E2, glutamate, and the cytokines TNF-α, IL1-β, and IL6. These observations were reproduced using the more physiological hippocampal slices in addition to dissociated microglia cultures. Neurotoxicity was ameliorated by a well-studied inhibitor of microglia activation, doxycycline, and by the KCa3.1 blocker TRAM-34. Therefore, our results support the possibility that the early neurotoxicity seen during the initial buildup of AβO might be mediated, at least in part, by microglia.
Soluble AβO extracted from AD hippocampi were about 50 times more potent than synthetic AβO in activating microglia, further suggesting a role of AβO in activating microglia. The reason for the higher potency of brain-derived AβO is not known, but is possibly due to the presence of co-fractionated costimulators or in vivo modifications of brain-derived Aβ that are not present in synthetic Aβ peptides.
The ability of fAβ to activate microglia is generally low or absent when fAβ is the only stimulant; activation requires micromolar concentrations (2-100 μM) of fAβ and enhancement by costimulators such as γ-interferon, lipopolysaccharides, advanced glycation endproducts, complement factors, and MCSF. A few reports have showed that unaggregated forms of Aβ at micromolar concentrations were able to activate microglia. Freshly solubilized, non-aggregated Aβ-42 at 500 nM has been reported by others to activate microglia in the presence of CD40 ligand. It has also been reported that activation of microglia by variably prepared ADO, in concentrations ranging from 2 to 50 μM, was equally or less potent than fAβ in activating microglia. In contrast to previous reports, Applicants used AβO at concentrations about 1,000 times lower and were able to show AβO's significantly higher potency than that of fAβ or unaggregatd Aβ in activating microglia. Because this activity has a bell-shaped concentration-response curve, which maximizes at 20-50 nM Aβ, it was not recognized in prior studies using μM or high nM Aβ.
Applicants' data indicate that detrimental effects of AβO upon synapses were ameliorated by inhibitors of microglia activation, and therefore support an alternative, microglia-mediated mechanism of synaptic dysfunction. These data further show that AβO at low nM concentrations activates microglia and causes reduced levels of critical dendritic and postsynaptic proteins in both dissociated neuronal cultures and hippocampal slices. Applicants also found a pattern of preferential postsysynaptic damage mediated by AβO-activated microglia similar to those found in Tg2576 transgenic mice and in human AD cortex, suggesting a pathological relevance.
It has been determined that microglia processes make regular direct contact with synapses and that prolonged contact increases the turnover of synapses. Therefore Applicants hypothesize that synaptic AβO might attract microglia by a potent chemotactic effect and promote a synapse-centered neuroinflammatory reaction to damages synapses. Supporting this notion, a previous study showed that the inhibition of NMDA receptor-dependent long term potentiation by soluble Ap (500 nM, which might contain oligomers) can be prevented by minocycline, a microglia activation inhibitor in the same class as doxycycline, and iNOS inhibition to reduce NO production from microglia. NO is the only AβO-promoted neuroinflammatory mediator we have so far uncovered.
TRAM-34 is a small molecule that selectively blocks the intermediate-conductance calcium-activated potassium channel KCa3.1. The data described herein provide the first evidence that a specific K+ channel regulates Aβ-induced microglia activation and neurotoxicity. KCa3.1 regulates Ca2+-signaling by maintaining a negative membrane potential through K+ efflux, thus facilitating Ca2+ entry through CRAC (calcium-release activated Ca2+ channel), a channel responsible for the store-operated Ca2+ entry required for microglia activation. The anti-inflammatory and neuroprotective properties of KCa3.1 blockers have been shown in models of traumatic brain injury, multiple sclerosis, and retinal ganglion cell degeneration after optic nerve transection. In addition, TRAM-34, although inhibiting microglia-mediated neurotoxicity, does not affect the beneficial activities of microglia such as migration and phagocytosis. Accordingly, the present invention provides compositions and methods by which KCa3.1 blockers, targeting microglia selectively because of the microglia-restrictive cellular expression of KCa3.1 in the CNS, provide a novel anti-inflammatory effects in subjects suffering from or in the process of developing AD. Thus, inhibition or blockade of KCa3.1 constitutes a useful therapeutic method for reducing the detrimental effects of microglia-mediated neurotoxicity, such as in Alzheimer's disease, while preserving beneficial microglial effects.
Further in accordance with the present invention, inhibition of microglial activity by KCa3.I blockade in AD can preserve the beneficial functions of microglia such as phagocytosis of amyloid-beta deposits while inhibiting their deterimenatal effects like microglia mediated neuronal killing and the production on NO and inflammatory cytokines. This is additionally evidenced by the data shown graphically in
Microglia and brain infiltrating macrophages significantly contribute to the secondary inflammatory damage in the wake of ischemic stroke. The following is an example, which demonstrates that inhibition of KCa3.1 (IKCal/KCNN4) reduces microglia and macrophage activation.
Using an HPLC/MS assay, Applicants first confirmed that our small molecule KCa3.1 blocker TRAM-34 effectively penetrates into the brain and achieves micromolar plasma and brain concentrations following intraperitoneal (i.p.) injection. Applicants then subjected male Wistar rats to 90 min of middle cerebral artery occlusion (MCAO) and administered either vehicle or TRAM-34 (10 or 40 mg/kg i.p. twice daily) for 7 days starting 12 h after reperfusion. Both compound doses reduced infarct area by ˜50% as determined by H&E staining on day-7 and the higher dose also significantly improved neurological deficit. Significant reductions in ED1+ activated microglia and TUNEL-positive neurons were observed as well as increases in NeuN+ neurons in the infarcted hemisphere. These findings suggest that KCa3.1 blockade constitutes an attractive approach for the treatment of ischemic stroke because it is still effective when initiated 12 hours after the insult.
In addition to directly causing neuronal damage, focal ischemic stroke elicits a strong and long-lasting inflammatory response. Activated by multiple stimuli, which include hypoxia, neuronal debris, ATP and glutamate, microglia retract their branched processes, round up and transform into “reactive” microglia. Partial breakdown of the blood-brain barrier additionally promotes the infiltration of macrophages, neutrophils and activated T cells from the blood. In both rodent models of cerebral ischemia and in histopathological studies on human postmortem brain sections activated microglia/macrophages are abundant in the infarcted area and the peri-infarct zone 18-96 hours after an ischemic insult, and are still present in chronic cystic stages months after a stroke. More recen PET imaging in ischemic stroke patients demonstrated microglia activation in the peri-infarct zone on a slightly more delayed time scale: starting at 72 hours and lasting for at least 4 weeks. While microglia can of course exert neuroprotective functions by releasing neurotrophic growth factors such as brain-derived neuroprotective factor (BDNF) or phagocytosing debris and potentially even invading neutrophils, activated microglia/macrophages are also the main source of inflammatory cytokines such as IL-1β and TNF-α, reactive oxygen species, nitric oxide and cyclooxygenase-2 reaction products.
TRAM-34 blocks the KCa3.1 channel with an IC50 of 20 nM and exhibits 200-1500 fold selectivity over other IC channels. KCa3.1 is expressed on proliferating fibroblasts, dedifferentiated vascular smooth muscle cells, and on immune cells including microglia and macrophages, activated CCR7+ T cells and IgD B cells. In all these cells KCa3.1 is part of signaling cascades that involve relatively global and prolonged calcium rises during cellular proliferation, cytokine secretion and volume regulation. KCa3.1 channels are voltage-independent and only require a small increase in intracellular calcium to open and then maintain a negative membrane potential through IC efflux. KCa3.1 channels thus provide the driving force for store-operated inward-rectifier calcium channels like CRAC (calcium-release activated Ca2+ channel) or transient receptor potential channels like TRPC1.
Particularly in microglia, KCa3.1 has been shown to be involved in respiratory bursting, migration, proliferation and LPS or amyloid-β oligomer induced nitric oxide production as well as in microglia-mediated neuronal killing in cultures and organotypic hippocampal slices, suggesting that KCa3.1 suppression might be useful for reducing microglia activity in stroke, traumatic brain injury, multiple sclerosis and Alzheimer's disease. It has previously been reported that intraocular injection of the KCa3.1 blocker TRAM-34 reduced retinal ganglion cell degeneration after optic nerve transection in rats and that TRAM-34 treats experimental autoimmune encephalomyelitis (EAE) in mice. In the nerve transection study KCa3.1 blockade did not prevent microglia from aligning with damaged axons and from phagocytosing damaged neurons but increased the number of surviving retinal ganglion cells presumably by reducing the production and/or secretion of neurotoxic molecules in the retina. In addition, it has been reported that two structurally different KCa3.1 inhibitors, a triarylmethane and a cyclohexadiene, reduced infarct volume and brain edema following traumatic brain injury caused by acute subdural haematoma in rats.
Applicants have studied whether KCa3.1 blockade such as by TRAM-34, which inhibits LPS-stimulated p38 mitogen-activated protein kinase (MAPK) activation but not nuclear-factor κB (NF-κB) activation in microglia, might preferentially target microglia activities that are involved in neuronal killing without affecting beneficial functions such as scavenging of debris.
MCAO with 7 Days of Reperfusion
Adult male Wistar rats weighing 160-180 g were purchased from Charles River (Wilmington, Mass.), acclimatized to the new vivarium for 5-7 days and used for the surgery when they weighed 200-230 g. Rats were anesthetized using box induction with 5% isoflurane and then maintained on 0.5%-1.5% isoflurane in medical grade oxygen via a facemask. In order to assure consistent reduction of cerebral blood flow (CBF) throughout the procedure, we affixed a small hand-made adaptor for the Laser Doppler probe (Moor Instruments, Wilmington, Del.) to the surface of the skull. The center of the adapter was 5 mm lateral to the central fusion line and 2.5 mm posterior to bregma. Instant adhesive and dental cement were applied to the base and around the edges of the small plastic adapter to hold the Doppler probe. The adapter with the attached probe remained in place throughout the MCAO surgery to confirm continuous occlusion and later the establishment of reperfusion. Focal cerebral ischemia was then induced by occlusion of the left middle cerebral artery (MCA). The left common carotid artery was surgically exposed, the external carotid artery was ligated distally from the common carotid artery, and a silicone rubber coated nylon monofilament with a tip diameter of 0.43±0.02 mm (Doccol Corp., Redlands, Calif.) was inserted into the external carotid artery and advanced into the internal carotid artery to block the origin of the middle cerebral artery (when maximum CBF reduction observed). The filament was kept in place for 90 min and then withdrawn and removed from the blood vessel to restore blood supply. Rats received TRAM-34 at 10 mg/kg, 40 mg/kg or vehicle (Miglyol 812 neutral oil at 1 μl/g) twice daily i.p. for 7 days starting 12 hour after reperfusion. Neurological deficits were scored according to a 4-score test and a tactile and/or proprioceptive limb-placing test as follows: 1) 4-score test (higher score for more severe neurological deficits): 0=no apparent deficit; 1=contralateral forelimb is consistently flexed during suspension by holding the tail; 2=decreasing grip ability on the contralateral forelimb while tail pulled; 3=spontaneous movement in all directions but circling to contralateral side when pulled by the tail; 4=spontaneous contralateral circling or depressed level of consciousness. 2) 14-score limb placing test (lower score for more severe neurological deficits): Proprioception, forward extension, lateral abduction, and adduction were tested with vision or tactile stimuli. For visual limb placing, rats were held and slowly moved forward or lateral toward the top of a table. Normal rats placed both forepaws on the tabletop. Tactile forward and lateral limb placing were tested by lightly contacting the table edge with the dorsal or lateral surface of a rat's paw while avoiding whisker contact and covering the eyes to avoid vision. For proprioceptive hind limb placing, each rat was pushed along the edge of an elevated platform in order to test proprioceptive hind limb adduction. The paw was pulled down and away from the platform edge, and the ability to retrieve and place the paw on the table surface upon sudden release was assessed. For each test, limb placing scores were 0=no placing; 1=incomplete and/or delayed (>2 seconds) placing; or 2=immediate and complete placing. For each body side, the maximum summed visual limb placing score was 4 and the maximum summed tactile and proprioceptive limb placing score, including the platform test, was 10.
TRAM-34 was synthesized in our laboratory as previously described and its chemical identity and purity checked by 1H-NMR and HPLC/MS. For intravenous application TRAM-34 was dissolved at 5 mg/ml in a mixture of 25% Cremophor®EL (Sigma-Aldrich, St. Louis, Mo.) and 75% PBS and then injected at 10 mg/kg into the tail vein of male Wistar rats. At various time points following the injection approximately 100-200 μl of blood were collected from a tail nick into EDTA blood sample collection tubes. For simultaneous determinations of plasma and brain concentrations TRAM-34 was dissolved in Miglyol 812 neutral oil (caprylic/capric triglyceride; Tradename Neobee M5®, Spectrum Chemicals, Gardena, Calif.) at 10 or 40 mg/ml and injected i.p. at 10 or 40 mg/kg. Blood samples were taken by cardiac puncture under deep isoflurane anesthesia. The right atrium was then cut open and 20 mL of saline slowly injected into the left ventricle to flush the blood out of the circulation. The rats were then sacrificed and brains removed. Plasma was separated by centrifugation and samples stored at −80° C. pending analysis. Plasma and homogenized brain samples were purified using C18 solid phase extraction (SPE) cartridges. Elutioned fractions corresponding to TRAM-34 were dried under nitrogen and reconstituted in acetonitrile. LC/MS analysis was performed with a Hewlett-Packard 1100 series HPLC stack equipped with a Merck KGaA RT 250-4 LiChrosorb RP-18 column interfaced to a Finnigan LCQ Classic MS. The mobile phase consisted of acetonitrile and water, both containing 0.2% formic acid. With a flow rate of 1.0 ml per min the gradient was ramped from 20/80 to 70/30 in 5 min, then to 80/20 over 11 min, to 5/95 till 16.5 min and finally back to 80/20 till 38 min. With the column temperature maintained at 30° C., TRAM-34 eluted at 14.4 min and was detected by a variable wavelength detector (VWD) set to 190 nm and the MS in series. Using electrospray ionization/ion trap MS (capillary temperature 270° C., capillary voltage 1V, tube lens offset −15 V, positive ion mode) TRAM-34 was quantified by its base peak of 277 m/z (2-chlorotrityl fragment) and concentrations calculated with a 5-point calibration curve from 25 nM to 2.5 Concentrations above 2.5 μM were quantified by their UV absorption at 190 nm. The related compound TRAM-46 (base peak of 261 m/z, 2-fluorotrityl fragment) was used as an internal standard.
The percentage of plasma protein binding for TRAM-34 was determined by ultrafiltration. Rat plasma was spiked with 50 and 100 μM TRAM-34 in 1% DMSO and the sample loaded onto a Microcon YM-100 Centrifugal Filter (Millipore Corporation, Bedford, Mass.) and centrifuged at 14000 rpm for 15 min at RT. The centrifugate (=free TRAM-34) was directly analyzed for TRAM-34 by HPLC-MS. The retentate was collected by inverting the filter into an Eppendorf tube and spinning at 14000 rpm for 15 min. The retentate then underwent sample preparation as per the above-described procedure for determining total TRAM-34 concentration in plasma. The plasma protein binding of TRAM-34 was found to be 98±0.5% (n=3) and the unbound (=free) fraction 2.0±0.4%.
Rats were euthanized with an overdose of isoflurane. Blood samples for determination of electrolytes, pH, pCO2, glucose and hemoglobin (I-STAT; Abbott, Princeton, N.J.) were drawn from the vena cava and brains quickly removed and sectioned into eight 2-mm thick slices starting from the frontal pole. Slices were then fixed in 10% buffer formalin embedded in paraffin and sectioned at 5 Sections were stained with hematoxylin & eosin and scanned. The resulting jpg images were analyzed in Adobe Photoshop CS3 for infarct area using the Magnetic Lasso tool to outline the area and the Histogram tool to determine the number of pixels in the respective area. Percent infarct for each slice was calculated as: (pixels in ipsilateral side/pixels in whole control hemisphere)×100. Percentage of total infarct area of whole hemisphere was calculated as: (summation of pixels in infarct from 8 slices/summation of pixels in whole control hemisphere from 8 slices)×100. The degree of brain shrinkage was calculated from the same data.
Sections were dewaxed with xylene, rehydrated through an alcohol gradient, and heated with 10 mM Na citrate (pH 6) in a microwave for 15 min to retrieve antigenic determinants After treatment with 1% H2O2 to inactivate endogenous peroxidase activity and blocking with 5% goat serum in PBS, the sections were incubated overnight at 4° C. with the primary antibody in PBS/2% goat serum. The following primary antibodies were used: KCa3.1 (1:500; AV35098, Sigma), CD68 (ED1, 1:1000; Serotec Raleigh, N.C.), and NeuN (1:1000; A60, Millipore, Calif.). The polyclonal anti-KCa3.1 antibody, which recognizes human, rat and mouse KCa3.1 was tested for specificity with spleen and vascular sections from KCa3.1-wild-type and KCa3.1−/− mice. Bound primary antibodies were detected with a biotinylated donkey anti-mouse IgG secondary antibody for CD68 and NeuN, or with a biotinylated goat anti-rabbit IgG secondary antibody (both 1:500, Jackson ImmunoResearch, West Grove, Pa.) for KCa3.1 followed by a horseradish peroxidase-conjugated avidin complex (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, Calif.). Peroxidase activity was visualized with 3,3′-diaminobenzidine (DAB Substrate Kit for Peroxidase, Vector Laboratories). Sections were counterstained with hematoxylin (Fisher, Pittsburg, Pa.), dehydrated and mounted with Permount (Fisher, Pittsburg, Pa.). AβOptosis was assessed with the AβOpTag Peroxidase in Situ AβOptosis detection kit (Millipore, Billerica, Mass.) according to the manufacturer's protocol.
ED1 stains CD68, a lysosomal membrane protein, which is mainly found in phagocytosing macrophages and reactive microglia. At 1:1000 the antibody produced no stain on resting microglia. Infiltration of ED1+ cells was evaluated. Sections stained for ED1 were photographed and the resulting photos composited into whole-slide images with Photoshop. The Magnetic Lasso tool was used to outline hemisphere borders, brown pixels were selected with the magic wand tool and the number of brown pixels determined with the Histogram tool. The results are reported as brown=ED1+ positive pixels per one millimeter square area (pixels/mm2). NeuN is a DNA-binding, neuron-specific protein present in neuronal nuclei, perikarya and some proximal neuronal processes. Strong nuclear staining suggests proper nuclear regulatory protein function representative of a healthy neuron. Sections stained for NeuN were photographed and the resulting photos composited into whole-slide images with Photoshop. NeuN and TUNEL positive cells in the infracted hemisphere were counted with the Photoshop CS3 extended count tool.
Statistical analyses of infarct area, neurological deficit scoring and IHC were performed with one-way analysis of variance (ANOVA; Origin software) followed by post-hoc pair-wise comparison of the different groups using Tukey's method, also referred to as honestly significant difference test, as recommended by Schlattmann and Dirnagle for MCAO studies. P<0.05 was used as the level of significance. *=P<0.05, **=P<0.01, ***=P<0.001. A11 data with the exception of the pharmacokinetic data in
MCAO with 7 Days of Reperfusion Induces Substantial Activation of Microglia/Macrophages Expressing KCa3.1
In determining the optimal length of reperfusion for this experiment, it was noted that 48 hours of reperfusion only induced mild to moderate inflammation as measured by the number of ED1+ (=CD68+) activated microglia/macrophages (data not shown), while seven days of reperfusion resulted in a dramatic increase in ED1+ cells in the infarcted brain areas as shown in
Staining of serial sections with a polyclonal anti-KCa3.1 antibody, which did not produce any stain on lymphoid and vascular tissues from KCa3.1−/− mice, revealed strong KCa3.1 expression on cells with the round or “ruffled” shape characteristic of reactive microglia/macrophages in the infarcted areas. While the two stains clearly were on the same cells, they did not strictly co-localize because CD68 is a lysosomal protein, whereas the KCa3.1 channel is expressed on the plasma membrane and traffics through the ER. As seen on the microvessel in the right panels of
To reduce brain inflammation the KCa3.1 blocker TRAM-34 should ideally reach pharmacologically active concentrations in the brain. In order to address this question and to determine TRAM-34's pharmacokinetics in rats we established an HPLC/MS assay to measure TRAM-34 concentrations in plasma and tissue. Following intravenous administration at 10 mg/kg, total TRAM-34 plasma concentrations fell from a peak of 40 μM at 8 min after application to 250 nM at 24 hours. This decay in plasma levels was best fitted tri-exponentially reflecting a 3-compartment model with rapid distribution from blood into tissue followed by elimination and slow repartitioning from body fat acting as a deep compartment back into plasma (
Taken together these results demonstrate that TRAM-34 has reasonably good pharmacokinetics in rats and effectively reaches the brain even when the blood-brain barrier is intact (Cbrain/Cplasma=1.2). Based on its plasma half-life of 2 hours and its fast availability following intraperitoneal application we decided to administer TRAM-34 twice daily intraperitoneally at 12 hour intervals.
KCa3.1 Blockade with TRAM-34 Reduces Infarction and Microglia Activation in MCAO with 7 Days of Reperfusion when Treatment is Started 2 Hours after Reperfusion
In preliminary experiments we induced relatively mild infarcts by reducing cerebral blood flow by only 50% (control: 51.2±8.2% flux reduction, mean±SD, n=10; TRAM-34: 49.0±7.4% flux reduction, n=11) and then administering TRAM-34 at 10 mg/kg twice daily for 7 days starting 2 h after reperfusion. Under these conditions we found a reduction in mean infarct area from 18.8±3.5% of the ipsolateral triphenyltetrazolium chloride positive hemisphere area (n=10) in controls to 6.6±1.9% in TRAM-34 treated animals (n=11; mean±SEM, P=0.007). As shown in
KCa3.1 Blockade with TRAM-34 Reduces Infarction in MCAO with 7 Days of Reperfusion when Treatment is Started 12 Hours after Reperfusion
The above-described results suggest that KCa3.1 blockade can indeed reduce infarction and microglia activation. In view of this, Applicants perfoiined a second set of MCAO experiments where cerebral blood flow was reduced more severely and evaluated hemotoxylin & eosin (H&E) defined lesion area since this method is known to be more accurate for aged infarcts than TTC. However, in order to be able to prepare undamaged μm thin sections from the often brittle and delicate aged infarcts, the 2-mm coronal slices were fixed in 10% formalin for 1 day and the resulting paraffin blocks trimmed until several undamaged section could be obtained from each slice. In order to better simulate possible treatment conditions in the clinic we also delayed the start of the TRAM-34 treatment until 12 hours after reperfusion.
As in our previous experiments, male Wistar rats were subjected to 90 min of MCAO with 7 days of reperfusion and then treated with either TRAM-34 at 10 mg/kg or 40 mg/kg or vehicle twice daily starting 12 hour after successful reperfusion. Cerebral blood flow reduction was 67.3±9.6% in the controls (n=8), 71.4±5.8% in the high-dose TRAM-34 group (n=8) and 67.9±5.9% in the low-dose TRAM-34 group (n=6; values are means±SD), which also depicts a representative example of a laser Doppler recording during MCAO surgery and reperfusion.
Table 1, above, shows that there were no differences with respect to plasma concentrations of Na+, K+, Cl−, HCO3−, glucose, blood urea nitrogen, hemoglobin, pH, PCO2 or hematocrit in venous blood samples taken after the surgery and at the time of sacrifice between animals subjected to 90 min of MCAO and treated with either vehicle or TRAM-34. (Please note that PCO2 directly after surgery was elevated in all groups due to the respiratory depression from ˜2 h of isoflurane anesthesia). We also measured TRAM-34 concentration at the time of sacrifice, which was 12 hours after the last application, and found an average plasma concentration of 91±46 nM (n=6) for the 10 mg/kg group and 662±416 nM (n=7; mean±SD) for the 40 mg/kg group, while no TRAM-34 was detectable in vehicle treated animals. Treatment with TRAM-34 resulted in a significant reduction in H&E defined lesion area with the mean infarct size (
KCa3.1 Blockade with TRAM-34 Reduces Neurological Deficit, Microglia Activation and Neuronal Death
Using both a 4-score neurological evaluation scale shown to correlate well with infarct sizes in the frontoparietal cortex and a 14-score tactile and proprioceptive limb-placing test rats were evaluated for neurological deficit 12 h after MCAO and then every 24 h for 7 days. The combination of both tests was chosen since filament MCAO in rats not only induces infarction in the major MCA territory, the lateral and parietal cortex, but also in the underlying striatum. Rats subjected to MCAO exhibited an average deficit score of 3 in the 4-score system (
In order to determine if the delayed TRAM-34 application (12 hours after MCAO) also reduced microglia/macrophage activation similar to what we had previously seen when treatment was started 2 hours after reperfusion, we stained sections from the center of the infarct in the 8 and 10-mm slices from all animals in the vehicle, low-dose and high-dose TRAM-34 group for ED1+ microglia and determined the ED1+ area according to the pixel based method by Lehr et al. While the delayed administration of 10 mg/kg TRAM-34 did not result in a significant reduction in microglia activation, the higher TRAM-34 dose of 40 mg/kg reduced the ED1+ area from 3770.6±594.2 pixels/mm2 to 1632.6±363.75 pixels/mm2 (P=0.03) in the infracted hemisphere (
The calcium-activated K+ channel KCa3.1 plays an important role in several microglia functions such as respiratory burst, migration and microglia-mediated neuronal killing in vitro and in vivo. TRAM-34 is lipophilic and effectively crosses the blood-brain barrier. Based on the data provided in this example, KCa3.1 has a neuroprotective effect and reduces infarct in the rat ischemic stroke model tested. It is reasonable to conclude from these data that TRAM 34, when administered in doses effective to inhibit the calcium-activated K+ channel KCa3.1 in microglial cells will deter neuronal damage following ischemic, anoxic or hypoxic brain insult.
It is to be appreciated that the invention has been described hereabove with reference to certain examples or embodiments of the invention but that various additions, deletions, alterations and modifications may be made to those examples and embodiments without departing from the intended spirit and scope of the invention. For example, any element or attribute of one embodiment or example may be incorporated into or used with another embodiment or example, unless otherwise specified of if to do so would render the embodiment or example unsuitable for its intended use. Also, where the steps of a method or process have been described or listed in a particular order, the order of such steps may be changed unless otherwise specified or unless doing so would render the method or process unworkable for its intended purpose. A11 reasonable additions, deletions, modifications and alterations are to be considered equivalents of the described examples and embodiments and are to be included within the scope of the following claims.
This patent application claims priority to U.S. Provisional Patent Application No. 61/359,318, filed Jun. 28, 2010 and is a continuation in part of copending U.S. patent application Ser. No. 11/805,763 filed May 25, 2007, which is a division of U.S. patent application Ser. No. 10/402,532 filed Mar. 28, 2003 and issued as U.S. Pat. No. 7,235,577 on Jun. 26, 2007, which is a division of U.S. patent application Ser. No. 09/479,375, filed Jan. 6, 2000 and issued as U.S. Pat. No. 6,803,375 on Oct. 12, 2004, the entire disclosure of each such application and patent being expressly incorporated herein by reference.
This invention was made with United States Government support under Grant Nos. AG025500 and AG031362 awarded by the National Institutes of Health. The United States Government may have certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/42243 | 6/28/2011 | WO | 00 | 7/8/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61359318 | Jun 2010 | US |
