WO2013044176A2 - Methods and compositions for the treatment of ischemic stroke - Google Patents

Methods and compositions for the treatment of ischemic stroke Download PDF

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WO2013044176A2
WO2013044176A2 PCT/US2012/056760 US2012056760W WO2013044176A2 WO 2013044176 A2 WO2013044176 A2 WO 2013044176A2 US 2012056760 W US2012056760 W US 2012056760W WO 2013044176 A2 WO2013044176 A2 WO 2013044176A2
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treatment
dha
npdl
alkyl
cycloalkyl
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WO2013044176A3 (en
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Nicolas G. Bazan
Nicos A. Petasis
Charles N. Serhan
Ludmila Belayev
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University Of Southern California
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/60—Salicylic acid; Derivatives thereof
    • A61K31/612—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid
    • A61K31/616—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid by carboxylic acids, e.g. acetylsalicylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C59/40—Unsaturated compounds
    • C07C59/42—Unsaturated compounds containing hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
    • C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • This invention relates to compounds, methods and compositions for the treatment of ischemic stroke and related conditions.
  • Stroke is the third leading cause of death and the leading cause of long term disability in developed countries. Each year, over 15 million people worldwide suffer a stroke, and among these 5 million die and another 5 million are permanently disabled (World Health Report - 2007, from the World Health Organization). Despite progress made in understanding the pathophysiology of stroke, today the only efficacious treatment approved for ischemic stroke is thrombolysis. Unfortunately, due to its narrow therapeutic window and complexity of administration, only 3 to 5% of patients fully benefit from this therapy. Therefore, the development of an effective method for the treatment for stroke remains an unmet therapeutic need.
  • Stroke is a sudden interruption in the blood supply to the brain, most commonly caused by a blockage or occlusion of blood vessels involved in blood supply to the brain. Ischemic stroke is most often caused by a thromboembolic lesion obstructing the middle cerebral artery. Although recanalization by thrombolytic therapy is a modifiable predictor of clinical outcome, only a small number of these patients are amenable to this treatment.
  • Acute ischemic stroke produces a brain lesion composed of a severely injured core and a peripheral area of hypoperfusion (penumbra), where tissue is damaged but potentially salvageable.
  • the penumbra has a limited life span and undergoes irreversible damage within a few hours unless reperfusion is initiated.
  • a major goal in the management of ischemic stroke is the rescue of the penumbra (Dirnagl et al, 2009; Lo et al, 1996; Moskowitz et al, 2010).
  • the complex cascade trigerred by ischemic stroke includes as prevalent events neurovascular and neuroinflammatory signaling that in turn converges to induce brain damage and apoptotic cell death.
  • neurovascular unit leads to rapid neutrophil (PMN) infiltration that participates in the initial brain inflammatory response and also in innate immunity and host defense. Moreover, excessive accumulation of PMN leads to amplification of the inflammatory response and brain damage. Therefore modulation of neutrophil infiltration is key in brain ischemia reperfusion damage, and anti-inflammatory agents are needed to control excess neutrophil responses that lead to sustain inflammation and in turn to severe brain damage (Marcheselli et al, 2003).
  • PMN rapid neutrophil
  • aspirin is unique in that it jump-starts resolution. This is mainly due to the effect of aspirin on COX-2. Aspirin acetylates COX-1 within the enzyme's catalytic region preventing thus the alignment of the substrate arachidonic acid for oxygenation. As a consequence, the formation of prostaglandin endoperoxide intermediate (PGG2) and the biosynthesis of thromboxanes and prostaglandins is blocked. Since the catalytic site of COX-2 is larger than that of COX-1, its acetylation by aspirin does not lead to its complete inhibition.
  • COX-2 is abundantly constitutively expressed in the central nervous system where is involved both in normal function as well as in pathological conditions.
  • DHA docosahexaenoic acid
  • this DHA-derived mediator was initially termed neuroprotectin Dl (10R,17S- dihydroxy-docosa-4Z,7Z,llE,13E,15Z,19Z-hexaenoic acid) (Bazan et al, 2010; Mukherjee et al, 2004; Stark et al, 2011).
  • neuroprotectin Dl 10R,17S- dihydroxy-docosa-4Z,7Z,llE,13E,15Z,19Z-hexaenoic acid
  • NPD1/PD1 10J2,17S-dihydroxy-docosa- 4Z,7Z,llE,13E,15Z,19Z-hexanenoic acid
  • Aspirin-triggered DHA-derived mediators were identified in self-limited resolving murine exudates as well as the brain (Marcheselli et al, 2003;
  • AT-NPD 1 The structural identification of AT-NPD 1 and the related experimental findings led to the present invention for the treatment if ischemic stroke.
  • the present invention is based on the unexpected findings detailed herein, that the neuroprotective effects of this class of compounds for the effective treatment of ischemic stroke depend significantly on the detailed stereochemistry and carboxylate structure of these compounds. In particular, it was found that ester derivatives can be more effective than the
  • substitution patterns, and methods of use of these agents for an effective treatment of ischemic stroke were also identified.
  • This invention provides compounds, methods and compositions for the effective treatment of ischemic stroke based on our findings on the beneficial role of a DHA pathway involving neuronal COX- 2, and the identification of suitable compounds, stereochemical patterns, methods, compositions and treatment methods involving DHA, a combination of DHA and aspirin, or a neuroprotective 10,17-dihydroxyl derivative of DHA, including NPD1 and AT- NPD1 and their alkynyl derivatives.
  • This invention provides compounds, methods and compositions for the treatment of ischemic stroke, comprising the timely administration of docosahexaenoic acid (DHA), a combination of DHA and aspirin, or a neuroprotective 10,17-dihydroxyl derivative of DHA.
  • DHA docosahexaenoic acid
  • a combination of DHA and aspirin or a neuroprotective 10,17-dihydroxyl derivative of DHA.
  • the invention provides treatment methods within up to 6 hours following focal ischemia, by administering a composition comprising DHA, a combination of DHA and aspirin, or a neuroprotective 10,17- dihydroxyl derivative of DHA.
  • compositions include pharmaceutically acceptable salts, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids
  • compositions for local or systemic delivery comprising of DHA, a combination of DHA and aspirin, or a neuroprotective 10,17-dihydroxyl derivative of DHA selected from the list comprising: NPDl, AT-NPDl, or an R/S stereochemical isomer of NPDl and AT-NPDl, either as the free carboxylic acid, a carboxyl derivative or a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.
  • preferred compounds, methods and compositions include an active neuroprotective compound or a compound that can be converted in vivo to an active neuroprotective compound via known processes including the action of esterases, and have the general formula:
  • Ri and R2 are independently selected from the group consisting of H and COR3 (acyl), where R3 is alkyl, cycloalkyl, aryl or heteroaryl, and
  • R is H, alkyl, cycloalkyl, aryl or heteroaryl
  • Figure 1 shows the experimental design, chemical structures and behavioral assessment of novel aspirin-triggered Neuroprotectin Dl (AT- NPD1) in focal cerebral ischemia in rats,
  • AT- NPD1 novel aspirin-triggered Neuroprotectin Dl
  • Figure 2 shows that AT-NPDl reduces lesion volumes, brain edema and improves water mobility. Cortical, subcortical and total lesion areas and volumes, computed from T2WI were s reduced by iv injection of either AT- NPDl sodium salt or methyl ester ( AT-NPD1ME) on day 7. (d)
  • T2-DWI from saline, AT-NPDl and AT-NPDl-ME treated rats T2 hyperintensites were observed in the cortex and striatum of saline-treated rat, consistent with edema formation.
  • AT-NPDl-treated animal has smaller lesion size, visible only in small portion of cortex and subcortical area.
  • AT-NPDl-ME treated rat has a lesion located only in a small portion of the striatum
  • (e) Brain edema and (f) ADC maps were computed and generated. On both the T2 and ADC maps, the ROIs were manually drawn in cortical and subcortical areas (see brain diagram for ROI: C -Cortex, S- subcortex).
  • T2 values are elevated in saline-treated group, AT-NPDl significantly decreases brain edema and ADC in the cortex, (g) 3D lesion volumes were computed from T2WI.
  • Saline -treated rats showed large cortical and subcortical lesion volumes. Lesion volume was reduced in rats treated with AT-NPDl and was mostly localized in the small cortical and subcortical areas.
  • AT-NPDl-ME treatment dramatically reduced lesion volume, which was mostly localized in the subcortical areas.
  • FIG. 3 shows that DTI metrics demonstrate that AT-NPDl-ME preserves white matter
  • T2WI T2 weighted
  • RA relative anisotropy
  • FIG. 4 shows that AT-NPDl reduces brain damage
  • rat treated with AT-NPDl-ME has small infarction only in subcortical area, (b-d) Cortical, subcortical and total infarct areas measured at 9 coronal levels and integrated infarct volumes in rats with 2-h MCAo and on day 7 of survival. Treatment with both AT-NPDl and AT-NPDl-ME
  • Figure 5 shows that AT-NPDl attenuates cellular damage after focal cerebral ischemia, (a) Computer- generated MosaiX processed images of SMI- 71 (positive vessels), GFAP (positive astrocytes), ED-1 (positive
  • microglia/microphages and GFAP/ED-1 double staining from saline, AT-NPDl and AT-NPD1-ME rats at a magnification lOx. All treatments were
  • SMI positive vessels Numbers of SMI positive vessels, GFAP positive astrocytes, ED-1 positive microglia cells presented in Panels b- d.
  • Coronal brain diagram (bregma +1.2 mm) showing locations of regions for SMI-71, GFAP, ED-1 counts in cortex (a, b and c) and subcortex (S).
  • AT-NPDl and AT-NPDl -ME increased SMI-71 positive vessels and GFAP positive astrocytes and decreased ED-1 microglia/microphages cell count.
  • Figure 6 shows the endogenous biosynthesis of NPDl and of AT-NPDl as well of markers of COX-2 activity in the ipsilateral penumbra after MCAo after iv injection of DHA and aspirin, a) NPDl and AT-NPDl, shown by a representative multiple reaction monitoring (MRM) chromatogram, and b) typical LC-MS/MS spectrum of endogenous AT-NPDl. c) NPDl and AT-NPDl authentic synthetic standards shown by a MRM chromatogram. d) 17R-HDHA and 17S-HDHA, stable derivatives of the short-lived precursors of AT-NPDl and NPDl, respectively, are depicted by a representative selective reaction monitoring (SRM) chromatogram.
  • SRM selective reaction monitoring
  • Rats were perfused with ice cold saline at 3 days after the onset of stroke.
  • the brain tissue was rapidly collected and immediately frozen in liquid nitrogen, then homogenized in cold methanol. Purification was performed by solid-phase extraction technique. In short, samples pre-equilibrated at pH 4.0 were loaded onto C18 columns (Varian) and eluted with 10 ml methanol. Samples were concentrated by a nitrogen stream evaporator, and analyzed by a TSQ Quantum Ultra UPLC-MS MS triple stage tandem mass spectrometer (Thermo).
  • Figure 7 shows a therapeutic window study: a Neurological score
  • Saline-treated rat shows large cortical and subcortical infarction.
  • rats treated with DHA at 3, 4 and 5 h show less extensive damage, mostly in the subcortical area.
  • DHA-treated rat at 6 h shows infarct involving cortical and subcortical regions, f Computer-generated MosaiX processed images of GFAP (green), ED-1 (red) and GFAP/ED-1 double staining (overlay) on day 7 after 2 h of MCAo at a magnification lOx. Treatment with DHA or saline was given at 3 h after onset of stroke.
  • Figure 8 shows a) Coronal brain diagram showing locations of regions for cell counts in cortex (1, 2 and 3) and striatum (S).
  • b-d Number of GFAP positive astrocytes, ED-1 positive microglia cells and NeuN positive neurons on day 7 after 2 h of MCAo.
  • DHA or saline was given at 3 h after onset of stroke.
  • DHA treatment decreased ED-1, increased NeuN and GFAP positive cell counts.
  • Data are mean ⁇ SEM. *, significantly different from saline (P ⁇ 0.05; repeated-measures ANOVA followed by Bonferroni tests).
  • Figure 9 shows that DHA significantly increased AKT Phosphorylation at 4hr when given as a Treatment after MCAo:
  • A Coronal brain diagram showing locations of regions for western blot analysis. Individual regions of the penumbra, adjacent regions, and all penumbral regions pooled were analyzed for (B) p473 AKT and (C) p308 AKT using western blot analysis.
  • D D
  • Figure 10 shows that aged rats have a reduced capacity to synthesize
  • NPD1 after MCAo This deficiency is overcome with DHA Treatment:
  • (B) Young and aged rats underwent 2hr of MCAo, were treated with either saline or DHA, and NPDl content was analyzed at 4hr (N 4). * and ** indicate significant difference between groups (p ⁇ 0.05;
  • alkyl herein used means Ci -Cio straight or branched chain alkyl, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, n-pentyl, i-pentyl, neo-pentyl, tert-pentyl, and the like.
  • cycloalkyl 11 herein means an alkyl having a C3-C8 aliphatic ring and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.
  • the alkyl or cycloalkyl may be optionally substituted.
  • Substituents for optionally substituted alkyl or optionall substituted cycloalkyl are hydroxy, alkoxy (e.g., methoxy and ethoxy), halogen (e.g., fluoro, chloro, bromo, and iodo), carboxy, alkoxycarbonyl, nitro, cyano, and substituted or unsubstituted amino, phenyl, benzyloxy, and the like. These substituents are able to bind them at one or more of any possible ositions of the alkyl or cycloalkyl.
  • aryl used means monocyclic or condensed ring aromatic hydrocarbons, preferably a phenyl.
  • heteroaryl herein used means an aromatic heterocyclic group, preferably a 5 to 6 membered aromatic heterocyclic group, which contains one or more hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur atoms in the ring.
  • aryl and heteroaryl groups may be optionally substituted on the aromatic ring.
  • Substituents for the aromatic ring of an optionally substituted aryl" and optionally substituted heteroaryl are hydroxy, alkoxy, halogen (e.g., fluoro, chloro, bromo, and iodo), carboxy, alkoxycarbonyl (e.g.,
  • substituents are able to bind to it at one or more of any possible position aromatic ring.
  • acyl refers to a -COR group, including for example
  • alkylcarbonyl cycloalkylcarbonyl, arylcarbonyl, or heteroarylcarbonyls, all of which may be optionally substituted.
  • the compounds of the invention means compounds disclosed herein. Particular compounds of the invention are compounds of Formulas I-XIX (set forth below) and pharmaceutically acceptable salts, hydrates, enantiomers, diastereomer, racemates or mixtures of stereoisomers thereof.
  • the compounds of the invention may be identified herein by their chemical structure and/or chemical name and/or formula number and/or assigned name. Where a compound is referred to by both a chemical structure and a chemical name or formula number or assigned name, and there is a conflict between for instance, between the chemical structure and the chemical name, the chemical structure is to be accorded more weight.
  • focal cerebral ischemia occurs when a blood clot has occluded a cerebral vessel. Focal ischemia reduces blood flow to a specific brain region, increasing the risk of cell death to that particular area. It can be either caused by thrombosis or embolism.
  • This invention provides compounds, methods and compositions for the treatment of ischemic stroke, comprising the administration of docosahexaenoic acid (DHA), a combination of DHA and aspirin, or a neuroprotective 10,17-dihydroxyl derivative of DHA.
  • DHA docosahexaenoic acid
  • the administration should be timely given to a subject in need thereof.
  • the treatment should be administered timely, generally within up to about 6 hours, but preferably less than 6 hours, following focal ischemia.
  • the method includes administering an effective amount of a composition comprising DHA or a neuroprotective 10, 17-dihydroxyl derivative of DHA to a patient in need thereof.
  • One aspect of the present invention is directed to neuroprotective compounds that are suitable for the treatment of ischemic stroke.
  • Certain preferred embodiments of the neuroprotective compounds are neuroprotectin Dl (NPD1), aspirin-triggerred neuroprotectin Dl (AT-NPDl) or an R/S stereochemical isomer of NPD1 and AT-NPDl, either as the free carboxylic acid, or preferably as a carboxy ester or other carboxyl derivative or a pharmaceutically acceptable salt.
  • Another aspect of the present invention is directed to compounds that are active neuroprotective compounds or compounds that can be converted in vivo to active neuroprotective compounds via known processes including the action of esterases and have the Formula I and II:
  • Ri and R2 are independently selected from the group consisting of H and COR3 (acyl), where R3 is alkyl, cycloalkyl, aryl or heteroaryl, and R is selected from the group consisting of H, alkyl, cycloalkyl, aryl and heteroaryl,
  • Another aspect of the present invention is directed to neuroprotective compounds having Formulas III, IV, V and VI with the stereoisomers structure:
  • Rj and R2 are independently selected from the group consisting of H and COR3 (acyl), where R3 is selected from the group consisting of alkyl, cycloalkyl, aryl and heteroaryl, and
  • R is selected from the group consisting of H, alkyl, cycloalkyl, aryl and heteroaryl,
  • AT-NPDl Formula VII (below, referred to as "AT-NPDl"), the sodium salt of AT-NPDl of Formula VIII, the methyl ester of AT-NPDl of Formula IX or the carboxy ester derivative of Formula X
  • R is selected from the group consisting of alkyl, cycloalkyl, aryl or heteroaryl,
  • Another preferred embodiment of the present invention includes compounds of Formula XI (below, referred to as "NPDl”) or the carboxy ester derivativ of Formula XII:
  • R is selected from the group consisting of methyl, alkyl, cycloalkyl, aryl and heteroaryl,
  • Another preferred embodiment of the present invention includes compounds of Formula XIII (below, referred to as "10-Epi-NPDl”) or the carboxy ester derivative of Formula XIV:
  • R is methyl, alkyl, cycloalkyl, aryl or heteroaryl, or a salt.
  • Another aspect of the invention is directed to neuroprotective
  • Ri and R2 are independently selected from the group consisting of H and COR3 (acyl), where R3 is alkyl, cycloalkyl, aryl or heteroaryl, and
  • R is selected from the group consisting of H, alkyl, cycloalkyl, aryl and heteroaryl, or a pharmaceutically acceptable salt thereof.
  • pharmaceutically acceptable refers to a component that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention.
  • pharmaceutically acceptable counterion is an ionic portion of a salt that is not toxic when released from the salt upon administration to a recipient.
  • salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid affording a physiologically acceptable anion.
  • Alkali metal e.g., sodium, potassium or lithium
  • alkaline earth metal e.g., calcium
  • the provided compounds can be prepared in a stereochemically pure form by using reactions and processes according to Petasis et al., 2012.
  • the preferred compound of Formula IX is
  • Step 1 palladium-catalyzed Sonogashira coupling of compounds of Formula XX and XXI, for example by using Pd(PPhs)4 catalyst, Cul, 25 °C benzene
  • R4 is a protective group such as a trialkylsilyl group
  • Step 2 removal of protective group using known methods. For example if R 4 is a trialkylsilyl group it can be removed with tetrabutyl ammonium fluoride.
  • Precursor intermediate of Formula XX can be prepared from
  • the compounds of the present invention can be formulated as
  • compositions The pharmaceutical compositions of the present invention generally comprise a compound according to the present invention dissolved or dispersed in carrier.
  • the pharmaceutical compositions, and thus the compounds they include, can be administered to a subject in need thereof, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, or subcutaneous routes.
  • compositions, and thus the compounds they include may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be
  • the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
  • the active compound may also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile -filtered solutions.
  • Useful dosages can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
  • the amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
  • Another aspect of the present invention is directed to a method for the treatment of ischemic stroke, comprising the timely administration of a provided neuroprotective compound.
  • the invention provides treatment methods within up to 6h following focal ischemia, by administering a composition comprising a provided neuroprotective compound.
  • Another aspect of the present invention is directed to a method for the treatment of ischemic stroke, comprising the timely administration of a provided neuroprotective compound as a complex with albumin.
  • the method comprises administering an effective amount of the compound as a complex with albumin to a patient in need thereof.
  • the preferred timely administration for effective treatment is within 6 hours from the initiation of an ischemic event.
  • the method generally comprises administering an effect amount of the compound to a subject in need thereof.
  • Another aspect of the present invention is directed to a method for the treatment of ischemic stroke, comprising the timely administration of docosahexaenoic acid.
  • the method comprises administering an effective amount of docosahexaenoic acid to a subject in need thereof.
  • the preferred timely administration for effective treatment is within 6 hours from the initiation of an ischemic event.
  • Another aspect of the present invention is directed to a method for the treatment of ischemic stroke, comprising the timely administration of a combination of docosahexaenoic acid and
  • the method comprises administering an effective amount of docosahexaenoic acid and aspirin to a subject in need thereof.
  • the preferred timely administration for effective treatment is within 6 hours from the initiation of an ischemic event.
  • Another aspect of the present invention is directed to a method of administration for the provided compositions, comprising the timely systemic administration via intravenous injection or via an oral formulation.
  • the preferred timely administration for effective treatment is within 6 hours from the initiation of an ischemic event.
  • an “effective amount” of the composition is an amount sufficient to carry out a specifically stated purpose.
  • An “effective amount” may be determined empirically and in a routine manners in relation to the stated purpose. Generally, the amount should be effective to produce at least one of reduced cortical, subcortical and total infarct volumes relative to untreated controls. Alternatively, the effective amount may be effecto to produce a smaller lesion size, either in aa portion of cortex or the subcortical areas relative to untreated controls.
  • the combination method may include at least one of the following: (1) administering a combination of DHA, aspirin and at least one compound according to Formulas I to XIX; (2) administering a combination of aspirin and at least one compound according to Formulas I to XIX.
  • the provided combination method comprises a combination treatment comprising a method according to the present invention in combination with other existing methods of treatment for ischemic stroke, including the administration of thrombolytic agents such as the protein tissue-plasminogen activator (t-PA) and surgical methods known in the art.
  • thrombolytic agents such as the protein tissue-plasminogen activator (t-PA) and surgical methods known in the art.
  • Another aspect of the present invention is directed to a non-invasive method for monitoring the progression of the treatment with the provided methods and compositions comprising the use of 3D images obtained via magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • AT-NPDl in either sodium salt or as methyl ester, was systemically injected (iv), after one hour of reperfusion following two hours of middle cerebral artery occlusion (MCAo). Then neurological functions were assessed up to 7 days latter. At time MRI, histology and immunostaining were performed to precisely define the size and features of the ischemia-reperfusion induced lesions.
  • Focal ischemic stroke leads to impaired sensorimotor and cognitive function, and 70% to 80% of patients display hemiparesis immediately after stroke.
  • Functional deficits in rodents due to MCAo a model of focal ischemic stroke, resemble this outcome including sensorimotor dysfunctional deficit.
  • Animals were treated with either the sodium salt of AT-NPDl or the methyl ester form, AT-NPD1-ME, at 3h after onset of stroke ( Fig l,a). All rats developed a high-grade neurological deficit when examined at 60 min of MCAo (Fig. Id). Vehicle -treated animals exhibited severe behavioral impairments throughout the 7-day survival period.
  • Example 2 volumes, brain edema and improves water mobility.
  • Cerebral ischemia induces edema within minutes of the insult, and is a leading cause of morbidity and death after stroke.
  • MRI is a highly sensitive tool for the detection of changes in water diffusion that characterize acute ischemic stroke and also to define potential effects of experimental therapies in patients and animals. Imaging the penumbra is a new strategy for detecting tissue at risk (Donnan et al, 2009). Up to 44% of patients may still have penumbral tissue 18 hours after stroke (Ebinger et al, 2009).
  • MRI apparent diffusion coefficient
  • 3D images were obtained to delineate the volumetric development of ischemic injury. Analysis included extraction of lesion, non-lesioned brain, and total brain volumes. Region of interest (ROI) was drawn semi-automatically on each slice and manually checked (see brain diagrams in Fig 2a, b, c). Treatment with both AT-NPDl and AT-NPD1-ME significantly reduced cortical (by 65 and 82%), subcortical (by 40 and 76%) and total infarct volumes (by 59 and 80%; respectively), computed from T2WI on day 7 (Fig. 2a-c). AT-NPDl-treated group has smaller lesion size, visible only in a small portion of cortex and subcortical areas.
  • AT-NPDl-ME treated rats have a lesion located only in a small portion of the striatum (Fig. 2d). Both AT-NPDl decreases brain edema and water mobility (ADC) in the cortex (Fig. 2e-f). 3D lesion volumes presented in Fig. 2g. Saline -treated rats showed large cortical and subcortical lesion volumes. In contrast, lesion volume was dramatically reduced by both AT-NPDl treatments (Fig. 2g).
  • RA l/fractional anisotropy
  • AT-NPDl attenuates brain damage.
  • the histology of focal cerebral ischemia consists of infarction and a variable rim of selective neuronal necrosis, a "histologic penumbra" (Auer, 1998).
  • the area peripheral to the lesion is characterized by eosinophilic neurons. Without reperfusion, the penumbra progressively deteriorates over a few hours, becoming an extension of the ischemic core.
  • a purpose of the present study was to ask if the novel AT-NPDl molecules would rescue the penumbra from transforming away from a core-like section of the ischemic insult. All treatments were administered at 3 h after onset of stroke. Histology of the brain of saline-treated animals followed by 7 days survival showed large zones of infarction involving the frontoparietal neocortex and underlying
  • Fig. 4a caudoputamen
  • Figure 5 illustrates further assessment of AT-NPDls on cerebral parenchyma changes after acute ischemic stroke because cerebral ischemia initiates a complex cascade of cellular, molecular and metabolic events that lead to irreversible brain damage.
  • the neurovascular lesions in necrotic area worsen, finally being disintegrated (Mogoanta et al, 2010).
  • Dead and injured tissue are scavenged by activated resident microglia and/or macrophages that invaded the injured tissue from the blood stream (Graber et al, 2002).
  • Surviving astrocytes and activated microglia in the penumbra may facilitate restoring neuronal integrity by producing growth factors, cytokines, and extracellular matrix molecules, involved in repair and regenerative neuronal mechanisms (Panickar and Norenberg, 2005).
  • Macrophages, astrocytes and blood vessels involved in cerebral infarction were studied by immunohistochemistry on day 7.
  • Controls exhibited pannecrotic lesions involving cortical and subcortical regions of the right hemisphere, characterized microscopically by loss of neuronal, glial, vascular elements and massive ED-l-positive microglia/macrophage infiltration (Fig. 5a).
  • AT-NPDl attenuated damage as well as decreased ED-l-positive microglia/macrophages and increased blood vessels and GFAP-positive reactive astrocytes (Fig.
  • FIG. 7a day 1 (DHA: 3 h, 7.7 ⁇ 0.2; 4 h, 6.9 ⁇ 0.7; 5 h, 7.2 ⁇ 0.5; and 6 h, 8.3 ⁇ 0.2 vs. Saline: 9.8 ⁇ 0.2; p ⁇ 0.01), day 2 (DHA: 3 h, 7.7 ⁇ 0.2; 5 h, 6.5 ⁇ 0.8; and 6 h, 7.8 ⁇ 0.3 vs. Saline: 9.8 ⁇ 0.2; pO.01), day 3 (DHA: 3 h, 7.1 ⁇ 0.6; 5 h, 6.0 ⁇ 1.0; and 6 h, 8.2 ⁇ 0.4 vs.
  • saline -treated rats exhibited a consistent pannecrotic lesion involving both cortical and subcortical (mainly striatal) regions of the right hemisphere, characterized microscopically by destruction of neuronal, glial, and vascular elements.
  • infarct size was dramatically reduced in rats treated with DHA at 3, 4, and 5 h after onset of ischemia, but not in 6-h group (Fig. 7e).
  • Saline-treated rats showed extensive neuronal loss, GFAP-positive reactive astrocytes outlining the lesion territory, and massive ED-l-positive microglia/macrophage infiltration (Fig. 7f, top).
  • FIG. 7f presents GFAP-, ED-1-, and NeuN-positive cell counts.
  • DHA treatment significantly decreased ED-1 (p ⁇ 0.01) and increased NeuN (p ⁇ 0.01) and GFAP- positive (p ⁇ 0.005) cell counts.
  • Dawley rats were anesthetized with isofiurane/nitrous oxide and mechanically ventilated. Rectal and cranial temperatures were regulated at 36-37.5°C. Rats received 2hr MCAo by retrograde insertion of an intraluminal suture. Animals were treated with DHA (5mg/kg, IV), NPD1 (300 fg/kg), or vehicle (5ml/kg isotonic saline, IV) 3hr after MCAo onset. Behavioral function was evaluated at 60min after occlusion onset and on days 1, 2, 3, and 7 on a grading scale of 0-12 (O ⁇ normal and 12 ⁇ maximal deficit).
  • DHA or NPDl treatment resulted in improved neurobehavioral outcome on all days with a 36% improvement in DHA-treated animals and 38% improvement in NPDl treated animals by day 7 when compared to vehicle-treated animals.
  • NPDl content was analyzed 4hr after MCAo, aged rats had a 55% reduction in NPDl synthesis when compared to young rats but this deficit was overcome with DHA treatment.
  • the protein phosphorylation state of aged rats at 4hr was analyzed, we found that aged rats did not show a treatment effect on the phosphorylation of p308 AKT or pGSK.
  • pS6 expression was increased with DHA (62% increase) or NPDl (280% increase) when compared to vehicle.
  • DHA modulates protein signaling in the penumbra and is
  • DHA upregulated activation of AKT at 4hr versus vehicle (4hr DHA: 36% increase in p473 AKT; 79% increase in p308 AKT) and when compared to DHA-treated animals at 24hr (4hr DHA: 114% increase in p473 AKT; 98% increase in p308 AKT).
  • DHA also increased pS6 at 4hr by 160% and pGSK at 24hr by 61% as compared to vehicle -treated animals.
  • Auer RN "Histopathology of Cerebral Ischemia.” Cerebrovascular Disease: Pathophysiology, Diagnosis and Management. Vol. 1. Blackwell Science. 1998; 90-101.
  • Bazan NG Bazan NG. Cell survival matters: docosahexaenoic acid signaling, neuroprotection and photoreceptors. Trends Neurosci. 2006; 29:263-71.
  • Bazan NG Neuroprotectin Dl-mediated anti-inflammatory and survival signaling in stroke, retinal degenerations, and Alzheimer's disease. J Lipid Res. 2009; 50 Suppl: S400-5.
  • Inducible cycloxygenase may have anti-inflammatory
  • Dl a docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress. Proc Natl Acad Sci USA. 2004; 101, 8491-8496.
  • Moussignac RL Resolvins: a family of bioactive products of omega- 3 fatty acid transformation circuits initiated by aspirin treatment that counter pro- inflammation signals. J Exp Med. 2002; 196:1025-1037.
  • Docosahexaenoic acid prevents white matter damage after spinal cord injury. J Neurotrauma. 2010; 27:1769-1780.

Abstract

This invention provides compounds, methods and compositions for the treatment of ischemic stroke, comprising the timely administration of doeosahexaenoic acid (DHA), a combination of DHA and aspirin, or a neuroprotective 10,17-dihydroxyl derivative of DHA. In particular, the invention provides treatment methods within up to 6h following focal ischemia, by administering a composition comprising DHA, a combination of DHA and aspirin, or a neuroprotective 10,17-dihydroxyl derivative of DHA. The invention also provides a non-invasive method for monitoring the progression of the treatment.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT
OF ISCHEMIC STROKE
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 61/538,504 filed September 23, 2011.
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
This invention was made with government support under National Institutes of Health Grant No. RC2AT005909. The government has certain rights in the invention.
FIELD OF THE INVENTION
This invention relates to compounds, methods and compositions for the treatment of ischemic stroke and related conditions.
BACKGROUND OF THE INVENTION
Stroke is the third leading cause of death and the leading cause of long term disability in developed countries. Each year, over 15 million people worldwide suffer a stroke, and among these 5 million die and another 5 million are permanently disabled (World Health Report - 2007, from the World Health Organization). Despite progress made in understanding the pathophysiology of stroke, today the only efficacious treatment approved for ischemic stroke is thrombolysis. Unfortunately, due to its narrow therapeutic window and complexity of administration, only 3 to 5% of patients fully benefit from this therapy. Therefore, the development of an effective method for the treatment for stroke remains an unmet therapeutic need.
Stroke is a sudden interruption in the blood supply to the brain, most commonly caused by a blockage or occlusion of blood vessels involved in blood supply to the brain. Ischemic stroke is most often caused by a thromboembolic lesion obstructing the middle cerebral artery. Although recanalization by thrombolytic therapy is a modifiable predictor of clinical outcome, only a small number of these patients are amenable to this treatment.
Acute ischemic stroke produces a brain lesion composed of a severely injured core and a peripheral area of hypoperfusion (penumbra), where tissue is damaged but potentially salvageable. The penumbra has a limited life span and undergoes irreversible damage within a few hours unless reperfusion is initiated. A major goal in the management of ischemic stroke is the rescue of the penumbra (Dirnagl et al, 2009; Lo et al, 1996; Moskowitz et al, 2010).
The complex cascade trigerred by ischemic stroke includes as prevalent events neurovascular and neuroinflammatory signaling that in turn converges to induce brain damage and apoptotic cell death. The damage to the
neurovascular unit leads to rapid neutrophil (PMN) infiltration that participates in the initial brain inflammatory response and also in innate immunity and host defense. Moreover, excessive accumulation of PMN leads to amplification of the inflammatory response and brain damage. Therefore modulation of neutrophil infiltration is key in brain ischemia reperfusion damage, and anti-inflammatory agents are needed to control excess neutrophil responses that lead to sustain inflammation and in turn to severe brain damage (Marcheselli et al, 2003).
Unlike several current anti-inflammatory agents, which delay
inflammatory resolution and can be toxic (Gilroy et al, 1999; Schwab et al, 2007), aspirin is unique in that it jump-starts resolution. This is mainly due to the effect of aspirin on COX-2. Aspirin acetylates COX-1 within the enzyme's catalytic region preventing thus the alignment of the substrate arachidonic acid for oxygenation. As a consequence, the formation of prostaglandin endoperoxide intermediate (PGG2) and the biosynthesis of thromboxanes and prostaglandins is blocked. Since the catalytic site of COX-2 is larger than that of COX-1, its acetylation by aspirin does not lead to its complete inhibition. Even though acetylated COX-2 does not generate PGG2, this aspirin-modified enzyme is able to form the lipoxygenase-like 15R-HETE (Claria and Serhan, 1995; Rowlinson et al, 2000), which is converted to the potent aspirin- triggered lipoxins (Chiang et al, 2004). COX-2 is abundantly constitutively expressed in the central nervous system where is involved both in normal function as well as in pathological conditions.
CNS is enriched and avidly retains the omega-3 fatty acid family member docosahexaenoic acid (DHA) (Bazan, 2006). DHA is an essential fatty acid and the precursor of several potent metabolites including a 10,17 docosatriene that during brain ischemia reperfusion acts as a potent regulator of PMN infiltration, reducing stroke-mediated brain damage (Marcheselli et al, 2003, Serhan et al, 2002). Given its potent bioactivity in retina and brain, this DHA-derived mediator was initially termed neuroprotectin Dl (10R,17S- dihydroxy-docosa-4Z,7Z,llE,13E,15Z,19Z-hexaenoic acid) (Bazan et al, 2010; Mukherjee et al, 2004; Stark et al, 2011). These studies explained that the unexpected early increase in unesterified DHA pool in brain ischemia is the initiation of a pathway for the biosynthesis of NPD1. Because this mediator also displays activities in the immune, cardiovascular and renal systems, the name protectin Dl (PDl) was also used for it's non-neuronal specific activities. The complete stereochemistry of NPD1/PD1 (10J2,17S-dihydroxy-docosa- 4Z,7Z,llE,13E,15Z,19Z-hexanenoic acid) was established (Serhan et al, 2006, Bazan et al, 2010).
Aspirin-triggered DHA-derived mediators were identified in self-limited resolving murine exudates as well as the brain (Marcheselli et al, 2003;
Serhan et al, 2002). The complete stereochemistry of the aspirin-triggered NPD1/PD1 pathway from murine exudates and human PMN displays potent protective bioactions comparable to NPD1/PD1 in vitro and in vivo, reducing both PMN infiltration and enhancing the removal of apoptotic PMN by macrophages were recently reported (Serhan et al, 2011).
Recently, we unambiguously established by chirality of carbon- 10 and carbon-17 hydroxyl groups and the geometry of the conjugated triene unit that is essential for the potent bioactivity of aspirin-triggered neuroprotectin Dl, AT-NPD 1/PD 1, (10R, 17R-dihydroxy-docosa-4Z,7Z, 1 IE, 13E, 15Z, 19Z-hexaenoic acid)a novel aspirin-triggered omega-3 fatty acid lipid mediator matching with materials prepared by total organic synthesis (Serhan et al, 2011).
Without being limited to theory, the postulated biosynthetic conversion of DHA in the presence of aspirin to AT-NPDl/PDl, abbreviated herein as AT- NPDl, involves the acetylation of the COX-2 enzyme by aspirin, and is shown in the following Scheme 1:
Figure imgf000005_0001
AT-NPD1 or AT-NPD1/PD1
Scheme 1
The structural identification of AT-NPD 1 and the related experimental findings led to the present invention for the treatment if ischemic stroke.
Several reports show that DHA is protective in ischemia (Bazan, 2009; Moskowitz et al, 2010) and in spinal cord injury (Ward et al. 2010). The potential neuroprotective effects of DHA and a DHA/albumin complex in focal cerebral ischemia have been previously investigated (Belayev et al, 2005, 2009). However, the mechanism/s and bioactive mediator/s involved - and particularly whether or not these mechanisms/mediators target the salvageable area of the stroke penumbra - have not yet been defined.
Despite these earlier studies, effective methods and compositions for the systemic administration of DHA or related DHA-derived neuroprotective agents on the ischemic brain penumbra have not yet been developed. Given the complex nature of the neuroinflammatory cascade involved in ischemic stroke, and the narrow therapeutic window for effective treatment, it is essential to identify specific agents and specific methods and compositions for systemic treatment that are suitable for sustainable neuroprotection and effective rescue of the penumbra with concomitant neurobehavioral recovery.
The present invention is based on the unexpected findings detailed herein, that the neuroprotective effects of this class of compounds for the effective treatment of ischemic stroke depend significantly on the detailed stereochemistry and carboxylate structure of these compounds. In particular, it was found that ester derivatives can be more effective than the
corresponding acids or salts, presumably because they have a longer half-life and are converted to their active forms upon hydrolysis with esterase enzymes. Additional unexpected findings include the stereochemical isomers,
substitution patterns, and methods of use of these agents for an effective treatment of ischemic stroke. Suitable methods of administration and monitoring within the critical time window that improves the efficacy of this method were also identified.
This invention provides compounds, methods and compositions for the effective treatment of ischemic stroke based on our findings on the beneficial role of a DHA pathway involving neuronal COX- 2, and the identification of suitable compounds, stereochemical patterns, methods, compositions and treatment methods involving DHA, a combination of DHA and aspirin, or a neuroprotective 10,17-dihydroxyl derivative of DHA, including NPD1 and AT- NPD1 and their alkynyl derivatives. BRIEF SUMMARY OF THE INVENTION
This invention provides compounds, methods and compositions for the treatment of ischemic stroke, comprising the timely administration of docosahexaenoic acid (DHA), a combination of DHA and aspirin, or a neuroprotective 10,17-dihydroxyl derivative of DHA.
In particular, the invention provides treatment methods within up to 6 hours following focal ischemia, by administering a composition comprising DHA, a combination of DHA and aspirin, or a neuroprotective 10,17- dihydroxyl derivative of DHA.
Preferred methods and compositions include pharmaceutical
compositions for local or systemic delivery comprising of DHA, a combination of DHA and aspirin, or a neuroprotective 10,17-dihydroxyl derivative of DHA selected from the list comprising: NPDl, AT-NPDl, or an R/S stereochemical isomer of NPDl and AT-NPDl, either as the free carboxylic acid, a carboxyl derivative or a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.
In particular, preferred compounds, methods and compositions include an active neuroprotective compound or a compound that can be converted in vivo to an active neuroprotective compound via known processes including the action of esterases, and have the general formula:
Figure imgf000007_0001
wherein:
Ri and R2 are independently selected from the group consisting of H and COR3 (acyl), where R3 is alkyl, cycloalkyl, aryl or heteroaryl, and
R is H, alkyl, cycloalkyl, aryl or heteroaryl,
or a harmaceutically acceptable salt thereof. BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the experimental design, chemical structures and behavioral assessment of novel aspirin-triggered Neuroprotectin Dl (AT- NPD1) in focal cerebral ischemia in rats, (a) Experimental design; (b) AT- NPDl (C22H3iNaO4i10R, 17R-llE,13E,15Z-diHDHA sodium salt) and AT- NPDl-ME (C23H3404, 10R, 17R-llE,13E,15Z-diHDHA methyl ester), (c) Total neurological score (normal score= score=12) in rats during MCAo
(lh) and at various survival times after MCAo. All iv treatments were at 3 h after onset of ischemia. Both AT-NPDls improved the behavioral score at 24, 48, 72 h and 7 days compared to saline group. (d~g) Time course of recovery of postural reflex, tactile and proprioceptive contralateral forelimb reactions (normal score = 0, maximal deficit = 2) following MCAo in rats. Bar graphs show improvement of postural reflex and placing reactions in both AT-NPD1- treated rats vs. saline-treated group. Behavioral data are means ± SEM; n=6-9 per group. *} significantly different from saline-treated group ( <0.05;
repeated- measures ANOVA followed by Bonferroni tests)
Figure 2 shows that AT-NPDl reduces lesion volumes, brain edema and improves water mobility. Cortical, subcortical and total lesion areas and volumes, computed from T2WI were s reduced by iv injection of either AT- NPDl sodium salt or methyl ester ( AT-NPD1ME) on day 7. (d)
Representative T2-DWI from saline, AT-NPDl and AT-NPDl-ME treated rats. T2 hyperintensites were observed in the cortex and striatum of saline-treated rat, consistent with edema formation. AT-NPDl-treated animal has smaller lesion size, visible only in small portion of cortex and subcortical area. In contrast, AT-NPDl-ME treated rat has a lesion located only in a small portion of the striatum, (e) Brain edema and (f) ADC maps were computed and generated. On both the T2 and ADC maps, the ROIs were manually drawn in cortical and subcortical areas (see brain diagram for ROI: C -Cortex, S- subcortex). T2 values are elevated in saline-treated group, AT-NPDl significantly decreases brain edema and ADC in the cortex, (g) 3D lesion volumes were computed from T2WI. Saline -treated rats showed large cortical and subcortical lesion volumes. Lesion volume was reduced in rats treated with AT-NPDl and was mostly localized in the small cortical and subcortical areas. By contrast, AT-NPDl-ME treatment dramatically reduced lesion volume, which was mostly localized in the subcortical areas.
Figure 3 shows that DTI metrics demonstrate that AT-NPDl-ME preserves white matter A) Representative T2 weighted (T2WI) and relative anisotropy (RA) images reveal preservation of tissue within the corpus callosum (CC). Saline treated animals exhibited large infarct regions on T2WI (*) with a decreased lesion (*) volume in the AT-NPDl treatment group. No lesion was visible in most of the AT-NPDl-ME treated animals. RA maps demonstrate reduced values in the both the saline- and AT-NPDl treated animals. In contrast, RA values returned to normative values after treatment with AT-NPDl-ME. White box on T2WI is the expanded RA region. Arrows demarcate the CC. B) Quantitative assessment of RA values demonstrates a significant difference in RA between saline and AT-NPDl-ME treated animals (* p<0.05) on the side of the MCAO lesion (right). No differences were evident on the contralateral side.
Figure 4 shows that AT-NPDl reduces brain damage, (a)
Representative histology from Nissl stained paraffin-embedded brain sections at coronal level (bregma +1.2 mm) from rats treated with saline, AT-NPDl and AT-NPDl-ME at 7 days after MCAo. A well- demarcated infarct involving cortical and subcortical regions is present in rat treated with saline. Note smaller area of cortical and subcortical infarction in rats treated with AT- NPDl. In contrast, rat treated with AT-NPDl-ME has small infarction only in subcortical area, (b-d) Cortical, subcortical and total infarct areas measured at 9 coronal levels and integrated infarct volumes in rats with 2-h MCAo and on day 7 of survival. Treatment with both AT-NPDl and AT-NPDl-ME
significantly reduced cortical, subcortical and total infarct areas and volumes on day 7. Results are presented as mean ± SEM; n=6-9 per group.
^Significantly different from corresponding vehicle group (p <0.05).
Figure 5 shows that AT-NPDl attenuates cellular damage after focal cerebral ischemia, (a) Computer- generated MosaiX processed images of SMI- 71 (positive vessels), GFAP (positive astrocytes), ED-1 (positive
microglia/microphages) and GFAP/ED-1 double staining from saline, AT-NPDl and AT-NPD1-ME rats at a magnification lOx. All treatments were
administered at 3 h after onset of stroke. Numbers of SMI positive vessels, GFAP positive astrocytes, ED-1 positive microglia cells presented in Panels b- d. Coronal brain diagram (bregma +1.2 mm) showing locations of regions for SMI-71, GFAP, ED-1 counts in cortex (a, b and c) and subcortex (S). AT-NPDl and AT-NPDl -ME increased SMI-71 positive vessels and GFAP positive astrocytes and decreased ED-1 microglia/microphages cell count. Data are mean values ± SEM; n=6-9 per group *, significantly different from saline (P<0.05; repeated-measures ANOVA followed by Bonferroni tests).
Figure 6 shows the endogenous biosynthesis of NPDl and of AT-NPDl as well of markers of COX-2 activity in the ipsilateral penumbra after MCAo after iv injection of DHA and aspirin, a) NPDl and AT-NPDl, shown by a representative multiple reaction monitoring (MRM) chromatogram, and b) typical LC-MS/MS spectrum of endogenous AT-NPDl. c) NPDl and AT-NPDl authentic synthetic standards shown by a MRM chromatogram. d) 17R-HDHA and 17S-HDHA, stable derivatives of the short-lived precursors of AT-NPDl and NPDl, respectively, are depicted by a representative selective reaction monitoring (SRM) chromatogram. The structure and mechanism for the generation of diagnostic mass spectrometric fragment ion for each compound are illustrated as the insert in each panel. Rats were perfused with ice cold saline at 3 days after the onset of stroke. The brain tissue was rapidly collected and immediately frozen in liquid nitrogen, then homogenized in cold methanol. Purification was performed by solid-phase extraction technique. In short, samples pre-equilibrated at pH 4.0 were loaded onto C18 columns (Varian) and eluted with 10 ml methanol. Samples were concentrated by a nitrogen stream evaporator, and analyzed by a TSQ Quantum Ultra UPLC-MS MS triple stage tandem mass spectrometer (Thermo).
Figure 7 shows a therapeutic window study: a Neurological score
(normal score^O; maximum scores 12) was improved after DHA administration when administered 3, 4 and 5 h after onset of stroke. Cortical (b), subcortical (c) and total corrected infarct volumes (d) on day 7. DHA reduced cortical and total infarct volumes when administered 3, 4 and 5 h after stroke. Data are means ± SEM. *, significantly different from saline (P<0.05; repeated- measures ANOVA followed by Bonferroni tests), e Computer-generated
MosaiX processed images of Nissl stained paraffin-embedded brain sections from rats treated with saline or DHA at 3, 4, 5 and 6 h after the onset of ischemia. Saline-treated rat shows large cortical and subcortical infarction. In contrast, rats treated with DHA at 3, 4 and 5 h show less extensive damage, mostly in the subcortical area. DHA-treated rat at 6 h shows infarct involving cortical and subcortical regions, f Computer-generated MosaiX processed images of GFAP (green), ED-1 (red) and GFAP/ED-1 double staining (overlay) on day 7 after 2 h of MCAo at a magnification lOx. Treatment with DHA or saline was given at 3 h after onset of stroke.
Figure 8 shows a) Coronal brain diagram showing locations of regions for cell counts in cortex (1, 2 and 3) and striatum (S). b-d) Number of GFAP positive astrocytes, ED-1 positive microglia cells and NeuN positive neurons on day 7 after 2 h of MCAo. DHA or saline was given at 3 h after onset of stroke. DHA treatment decreased ED-1, increased NeuN and GFAP positive cell counts. Data are mean ±SEM. *, significantly different from saline (P <0.05; repeated-measures ANOVA followed by Bonferroni tests).
Figure 9 shows that DHA significantly increased AKT Phosphorylation at 4hr when given as a Treatment after MCAo: (A) Coronal brain diagram showing locations of regions for western blot analysis. Individual regions of the penumbra, adjacent regions, and all penumbral regions pooled were analyzed for (B) p473 AKT and (C) p308 AKT using western blot analysis. (D)
Representative Western Blots of A T trials. Data are mean ± SEM. * and § indicate significant difference (p<0.05; repeated measures ANOVA followed by Bonferroni tests).
Figure 10 shows that aged rats have a reduced capacity to synthesize
NPD1 after MCAo. This deficiency is overcome with DHA Treatment: (A) Behavioral outcomes over 7 days. DHA (5mg kg) or NPD1 (30C^g/kg) was neuroprotective when administered 3hr after stroke onset (IV) in aged animals on days 1, 2, 3, & 7. (B) Young and aged rats underwent 2hr of MCAo, were treated with either saline or DHA, and NPDl content was analyzed at 4hr (N=4). * and ** indicate significant difference between groups (p<0.05;
repeated measures ANOVA followed by Bonferroni tests).
DETAILED DESCRIPTION OF THE INVENTION
The term "alkyl" herein used means Ci -Cio straight or branched chain alkyl, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, n-pentyl, i-pentyl, neo-pentyl, tert-pentyl, and the like.
The term " cycloalkyl11 herein means an alkyl having a C3-C8 aliphatic ring and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.
The alkyl or cycloalkyl may be optionally substituted. Substituents for optionally substituted alkyl or optionall substituted cycloalkyl are hydroxy, alkoxy (e.g., methoxy and ethoxy), halogen (e.g., fluoro, chloro, bromo, and iodo), carboxy, alkoxycarbonyl, nitro, cyano, and substituted or unsubstituted amino, phenyl, benzyloxy, and the like. These substituents are able to bind them at one or more of any possible ositions of the alkyl or cycloalkyl.
The term "aryl" used means monocyclic or condensed ring aromatic hydrocarbons, preferably a phenyl.
The term "heteroaryl" herein used means an aromatic heterocyclic group, preferably a 5 to 6 membered aromatic heterocyclic group, which contains one or more hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur atoms in the ring.
The aryl and heteroaryl groups may be optionally substituted on the aromatic ring. Substituents for the aromatic ring of an optionally substituted aryl" and optionally substituted heteroaryl are hydroxy, alkoxy, halogen (e.g., fluoro, chloro, bromo, and iodo), carboxy, alkoxycarbonyl (e.g.,
methoxycarbonyl and ethoxycarbonyl), nitro, cyano, alkyl, optionally
substituted alky, cycloalkyl and optionally substituted alkyl. These
substituents are able to bind to it at one or more of any possible position aromatic ring.
The term "acyl" refers to a -COR group, including for example
alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, or heteroarylcarbonyls, all of which may be optionally substituted.
The compounds of the invention means compounds disclosed herein. Particular compounds of the invention are compounds of Formulas I-XIX (set forth below) and pharmaceutically acceptable salts, hydrates, enantiomers, diastereomer, racemates or mixtures of stereoisomers thereof. The compounds of the invention may be identified herein by their chemical structure and/or chemical name and/or formula number and/or assigned name. Where a compound is referred to by both a chemical structure and a chemical name or formula number or assigned name, and there is a conflict between for instance, between the chemical structure and the chemical name, the chemical structure is to be accorded more weight.
Ischemic stroke or focal cerebral ischemia is referred to synonymously as focal ischemia. As used herein, occurs when a blood clot has occluded a cerebral vessel. Focal ischemia reduces blood flow to a specific brain region, increasing the risk of cell death to that particular area. It can be either caused by thrombosis or embolism.
This invention provides compounds, methods and compositions for the treatment of ischemic stroke, comprising the administration of docosahexaenoic acid (DHA), a combination of DHA and aspirin, or a neuroprotective 10,17-dihydroxyl derivative of DHA. The administration should be timely given to a subject in need thereof. In particular, the treatment should be administered timely, generally within up to about 6 hours, but preferably less than 6 hours, following focal ischemia. The method includes administering an effective amount of a composition comprising DHA or a neuroprotective 10, 17-dihydroxyl derivative of DHA to a patient in need thereof.
One aspect of the present invention is directed to neuroprotective compounds that are suitable for the treatment of ischemic stroke. Certain preferred embodiments of the neuroprotective compounds are neuroprotectin Dl (NPD1), aspirin-triggerred neuroprotectin Dl (AT-NPDl) or an R/S stereochemical isomer of NPD1 and AT-NPDl, either as the free carboxylic acid, or preferably as a carboxy ester or other carboxyl derivative or a pharmaceutically acceptable salt.
Another aspect of the present invention is directed to compounds that are active neuroprotective compounds or compounds that can be converted in vivo to active neuroprotective compounds via known processes including the action of esterases and have the Formula I and II:
Figure imgf000014_0001
Formula I Formula II wherein:
Ri and R2 are independently selected from the group consisting of H and COR3 (acyl), where R3 is alkyl, cycloalkyl, aryl or heteroaryl, and R is selected from the group consisting of H, alkyl, cycloalkyl, aryl and heteroaryl,
or a pharmaceutically acceptable salt thereof.
Another aspect of the present invention is directed to neuroprotective compounds having Formulas III, IV, V and VI with the stereoisomers structure:
Figure imgf000015_0001
Formula V Formula VI
wherein:
Rj and R2 are independently selected from the group consisting of H and COR3 (acyl), where R3 is selected from the group consisting of alkyl, cycloalkyl, aryl and heteroaryl, and
R is selected from the group consisting of H, alkyl, cycloalkyl, aryl and heteroaryl,
or a pharmaceutically acceptable salt thereof.
A preferred embodiment of the present invention includes compounds of
Formula VII (below, referred to as "AT-NPDl"), the sodium salt of AT-NPDl of Formula VIII, the methyl ester of AT-NPDl of Formula IX or the carboxy ester derivative of Formula X
Figure imgf000016_0001
Formula VII, "AT-NPDl" Formula VIII, "AT-NPD1 Na salt"
Figure imgf000016_0002
Formula D "AT-NPD 1 methyl ester" Fo mula X
wherein R is selected from the group consisting of alkyl, cycloalkyl, aryl or heteroaryl,
or a pharmaceutically acceptable salt thereof.
Another preferred embodiment of the present invention includes compounds of Formula XI (below, referred to as "NPDl") or the carboxy ester derivativ of Formula XII:
Figure imgf000016_0003
Formula XI, "NPDl" Formula XII
wherein R is selected from the group consisting of methyl, alkyl, cycloalkyl, aryl and heteroaryl,
or a pharmaceutically acceptable salt.
Another preferred embodiment of the present invention includes compounds of Formula XIII (below, referred to as "10-Epi-NPDl") or the carboxy ester derivative of Formula XIV:
Figure imgf000017_0001
wherein R is methyl, alkyl, cycloalkyl, aryl or heteroaryl, or a salt.
Another aspect of the invention is directed to neuroprotective
compounds of Formulas XV-XVI with stereoisomer ic structure comprising:
Figure imgf000017_0002
Formula XV Formula XVI
Figure imgf000017_0003
Formula XVII Fomula XVIII wherein:
Ri and R2 are independently selected from the group consisting of H and COR3 (acyl), where R3 is alkyl, cycloalkyl, aryl or heteroaryl, and
R is selected from the group consisting of H, alkyl, cycloalkyl, aryl and heteroaryl, or a pharmaceutically acceptable salt thereof.
The term "pharmaceutically acceptable," as used herein, refers to a component that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. A "pharmaceutically acceptable salt" means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention. A
"pharmaceutically acceptable counterion" is an ionic portion of a salt that is not toxic when released from the salt upon administration to a recipient.
Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be made.
The provided compounds can be prepared in a stereochemically pure form by using reactions and processes according to Petasis et al., 2012. In one method, the preferred compound of Formula IX:
Figure imgf000018_0001
Formula IX
can be prepared via the use of Zn(Cu/Ag), MeOH/H20 or via selective Lindlar hydrogenation of the provided acetylenic compound of Formula XIX:
Figure imgf000019_0001
Formula XIX
which can be prepared in two steps:
Step 1: palladium-catalyzed Sonogashira coupling of compounds of Formula XX and XXI, for example by using Pd(PPhs)4 catalyst, Cul, 25 °C benzene
Figure imgf000019_0002
Formula XX Formula XXI
where R4 is a protective group such as a trialkylsilyl group
Step 2: removal of protective group using known methods. For example if R4 is a trialkylsilyl group it can be removed with tetrabutyl ammonium fluoride.
Precursor intermediate of Formula XX can be prepared from
stereoehemically pure starting materials using methods known in the art, according to the following Scheme 2;
Figure imgf000020_0001
Formula XX
Scheme 2. Synthesis of intermediate of Formula XX: (a) nBuLi, BF3 ' OEt2, -78 °C, tetrahydrofuran; (b) tBuPh2Si-Cl, imidazole, dimethylamino pyridine, 25 °C, CH2CI2; (c) camphorsulfoinic acid, 25 °C, CH2Cl2 MeOH; (d) E /Lindlar catalyst, quinoline, 25 °C, EtOAc; (e) dimethyl sulfoxide, (COCl)2 Et3N, -78 °C, CH2C12; (f) PPhs, CBr4, 0 °C, CHsCla; (g) BuLi, Et20.
Precursor intermediate of formula XXI can be prepared from
stereochemically pure starting materials using methods known in the art, according to the following Scheme 3:
Figure imgf000021_0001
8 10 11 12
Figure imgf000021_0002
Formula XXI
Scheme 3: Synthesis of intermediate of Formula XXI: (a) ra-BuLi, BF3* Et20, -78 °C, tetrahydrofuran; (b) tBuPh2Si -CI, imidazole, dimethylamino pyridine, 25 °C, CH2CI2; (c) camphorsulfoinic acid, 25 °C, CH2Cl2 MeOH; (d) NBS, PPh3) 0 °C, CH2CI2; (e) tBuMe2SiOTf, lutidine, 0 °C, CH2CI2; (f) Cul, Nal, K2C03, 25 °C, dimethylformamide; (g) camphorsulfoinic acid, 25 °C, CH2Cl MeOH; (h) H2/Lindlar catalyst, quinoline, 25 °C, EtOAc; (i) dimethyl sulfoxide, (COCl)2f Et3N, -78 °C, CH2CI2; (j) Ph3P=CHCHO, PhMe, reflux, 2h; (k) CHI3, CrCl2, 0 °C, tetrahydrofuran.
The compounds of the present invention can be formulated as
pharmaceutical compositions. The pharmaceutical compositions of the present invention generally comprise a compound according to the present invention dissolved or dispersed in carrier. The pharmaceutical compositions, and thus the compounds they include, can be administered to a subject in need thereof, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, or subcutaneous routes.
The compositions, and thus the compounds they include, may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be
compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile -filtered solutions.
Useful dosages can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art. The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
Another aspect of the present invention is directed to a method for the treatment of ischemic stroke, comprising the timely administration of a provided neuroprotective compound. In particular, the invention provides treatment methods within up to 6h following focal ischemia, by administering a composition comprising a provided neuroprotective compound.
Another aspect of the present invention is directed to a method for the treatment of ischemic stroke, comprising the timely administration of a provided neuroprotective compound as a complex with albumin. The method comprises administering an effective amount of the compound as a complex with albumin to a patient in need thereof. In particular, the preferred timely administration for effective treatment is within 6 hours from the initiation of an ischemic event. The method generally comprises administering an effect amount of the compound to a subject in need thereof.
Another aspect of the present invention is directed to a method for the treatment of ischemic stroke, comprising the timely administration of docosahexaenoic acid. The method comprises administering an effective amount of docosahexaenoic acid to a subject in need thereof. In particular, the preferred timely administration for effective treatment is within 6 hours from the initiation of an ischemic event.
Another aspect of the present invention is directed to a method for the treatment of ischemic stroke, comprising the timely administration of a combination of docosahexaenoic acid and The method comprises administering an effective amount of docosahexaenoic acid and aspirin to a subject in need thereof. In particular, the preferred timely administration for effective treatment is within 6 hours from the initiation of an ischemic event.
Another aspect of the present invention is directed to a method of administration for the provided compositions, comprising the timely systemic administration via intravenous injection or via an oral formulation. The preferred timely administration for effective treatment is within 6 hours from the initiation of an ischemic event.
An "effective amount" of the composition is an amount sufficient to carry out a specifically stated purpose. An "effective amount" may be determined empirically and in a routine manners in relation to the stated purpose. Generally, the amount should be effective to produce at least one of reduced cortical, subcortical and total infarct volumes relative to untreated controls. Alternatively, the effective amount may be effecto to produce a smaller lesion size, either in aa portion of cortex or the subcortical areas relative to untreated controls.
Another aspect of the present invention is directed to a combination method for the treatment of ischemic stroke, comprising a combination of two or more of the methods provided herein. For instance, the combination method may include at least one of the following: (1) administering a combination of DHA, aspirin and at least one compound according to Formulas I to XIX; (2) administering a combination of aspirin and at least one compound according to Formulas I to XIX. In another aspect, the provided combination method comprises a combination treatment comprising a method according to the present invention in combination with other existing methods of treatment for ischemic stroke, including the administration of thrombolytic agents such as the protein tissue-plasminogen activator (t-PA) and surgical methods known in the art.
Another aspect of the present invention is directed to a non-invasive method for monitoring the progression of the treatment with the provided methods and compositions comprising the use of 3D images obtained via magnetic resonance imaging (MRI).
Some representative details of one or more embodiments of the invention are exemplified in the following examples. These examples are provided in order to demonstrate and further illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. EXAMPLES
Example 1
improves neurological outcome after focal
cerebral ischemia.
AT-NPDl, in either sodium salt or as methyl ester, was systemically injected (iv), after one hour of reperfusion following two hours of middle cerebral artery occlusion (MCAo). Then neurological functions were assessed up to 7 days latter. At time MRI, histology and immunostaining were performed to precisely define the size and features of the ischemia-reperfusion induced lesions.
Focal ischemic stroke leads to impaired sensorimotor and cognitive function, and 70% to 80% of patients display hemiparesis immediately after stroke. Functional deficits in rodents due to MCAo, a model of focal ischemic stroke, resemble this outcome including sensorimotor dysfunctional deficit. Animals were treated with either the sodium salt of AT-NPDl or the methyl ester form, AT-NPD1-ME, at 3h after onset of stroke ( Fig l,a). All rats developed a high-grade neurological deficit when examined at 60 min of MCAo (Fig. Id). Vehicle -treated animals exhibited severe behavioral impairments throughout the 7-day survival period. In contrast, both AT-NPDl significantly improved the total neurological score and the time course of recovery of postural reflex, tactile and proprioceptive contralateral forelimb reactions at 24, 48, 72 h and 7 days (Fig. ld-h). Example 2: volumes, brain edema and improves water mobility.
Cerebral ischemia induces edema within minutes of the insult, and is a leading cause of morbidity and death after stroke. MRI is a highly sensitive tool for the detection of changes in water diffusion that characterize acute ischemic stroke and also to define potential effects of experimental therapies in patients and animals. Imaging the penumbra is a new strategy for detecting tissue at risk (Donnan et al, 2009). Up to 44% of patients may still have penumbral tissue 18 hours after stroke (Ebinger et al, 2009). Here we employed MRI to define AT-NPDl intervention in a relevant model of ischemic stroke. Ex vivo MRI was conducted on day 7. T2 and apparent diffusion coefficient (ADC) maps were computed from T2-weighted imaging (T2WI). 3D images were obtained to delineate the volumetric development of ischemic injury. Analysis included extraction of lesion, non-lesioned brain, and total brain volumes. Region of interest (ROI) was drawn semi-automatically on each slice and manually checked (see brain diagrams in Fig 2a, b, c). Treatment with both AT-NPDl and AT-NPD1-ME significantly reduced cortical (by 65 and 82%), subcortical (by 40 and 76%) and total infarct volumes (by 59 and 80%; respectively), computed from T2WI on day 7 (Fig. 2a-c). AT-NPDl-treated group has smaller lesion size, visible only in a small portion of cortex and subcortical areas. In contrast, AT-NPDl-ME treated rats have a lesion located only in a small portion of the striatum (Fig. 2d). Both AT-NPDl decreases brain edema and water mobility (ADC) in the cortex (Fig. 2e-f). 3D lesion volumes presented in Fig. 2g. Saline -treated rats showed large cortical and subcortical lesion volumes. In contrast, lesion volume was dramatically reduced by both AT-NPDl treatments (Fig. 2g).
Example 3
White matter reorganization enhanced by AT-NPDl-ME
in a rat model of focal ischemia.
Here we quantitatively investigated the effect of AT-NPDl treatments on white matter reorganization using ex vivo MRI measurement of diffusion tensor imaging (DTI). Ex vivo DTI is equivalent to in vivo diffusion imaging (Sun et al, 2003). Measures of axial (axonal), radial (myelin) and mean
(apparent diffusion coefficient, ADC) diffusivity were obtained and no significant differences were observed in these DTI measures of white matter integrity. Diffusion anisotropy, as measured by relative a isotropy (RA)is sensitive to alterations in white matter fiber integrity (Le Bihan et al, 2001) and has been successfully used to detect subtle abnormalities in a variety of diseases that involve disruption of white matter fibers, including trauma and stroke (RA=l/fractional anisotropy (FA)). In human and in experimental models of stroke, the change of diffusion anisotropy within the ischemic area discloses the degree of structural damage within the white matter and provides indices of functional potential (Li et al, 2009).
We demonstrate here that the RA within corpus callosum (CC) of saline treated animals was decreased and that treatment with AT-NPDl-ME results in a return to normative RA values (Sun et al, 2003) (Fig. 3). A significant difference (p<0.05) in RA was found between saline and AT-NPDl- ME treated animals. AT-NPDl treated animals show a non-statistically significant trend for protection. However, treatment with AT-NPDl-ME after stroke significantly enhances and protects white matter, which is associated with improved neurological function. Presumably, the ester form of AT-NPDl has increased half life resulting in sustained bioactivity needed for the protection of edema resolution.
Example 4
AT-NPDl attenuates brain damage.
Neuroprotection against 2h of MCAo was evaluated and the actions of AT-NPDl on penumbral survival was assessed by histology. The histology of focal cerebral ischemia consists of infarction and a variable rim of selective neuronal necrosis, a "histologic penumbra" (Auer, 1998). The area peripheral to the lesion , the penumbra, is characterized by eosinophilic neurons. Without reperfusion, the penumbra progressively deteriorates over a few hours, becoming an extension of the ischemic core. A purpose of the present study was to ask if the novel AT-NPDl molecules would rescue the penumbra from transforming away from a core-like section of the ischemic insult. All treatments were administered at 3 h after onset of stroke. Histology of the brain of saline-treated animals followed by 7 days survival showed large zones of infarction involving the frontoparietal neocortex and underlying
caudoputamen (Fig. 4a). These infarcted regions showed pancellular necrosis as well as denser areas of eosinophilic, shrunken neurons along the infarct margin. By contrast, AT-NP l treated rats showed markedly smaller cortical infarcts and reduced zones of basal- anglionic infarct as well (Fig.4a). The maximal protection was detected in the cortex (represent penumbral area) (by 76 and 96%) and also in subcortical area (by 61 and 70%) (Fig. 4b-c).
Treatment with either AT-NPDl or its carboxy methyl ester AT-NPD1-ME, reduced total (cortical + subcortical) infarct volumes (by 69 and 84%;
respectively) and total infarct areas at multiple coronal levels (Fig 4d).
Figure 5 illustrates further assessment of AT-NPDls on cerebral parenchyma changes after acute ischemic stroke because cerebral ischemia initiates a complex cascade of cellular, molecular and metabolic events that lead to irreversible brain damage. As the infarct progresses, the neurovascular lesions in necrotic area worsen, finally being disintegrated (Mogoanta et al, 2010). Dead and injured tissue are scavenged by activated resident microglia and/or macrophages that invaded the injured tissue from the blood stream (Graber et al, 2002). Surviving astrocytes and activated microglia in the penumbra may facilitate restoring neuronal integrity by producing growth factors, cytokines, and extracellular matrix molecules, involved in repair and regenerative neuronal mechanisms (Panickar and Norenberg, 2005).
Macrophages, astrocytes and blood vessels involved in cerebral infarction were studied by immunohistochemistry on day 7. Controls exhibited pannecrotic lesions involving cortical and subcortical regions of the right hemisphere, characterized microscopically by loss of neuronal, glial, vascular elements and massive ED-l-positive microglia/macrophage infiltration (Fig. 5a). In contrast, AT-NPDl attenuated damage as well as decreased ED-l-positive microglia/macrophages and increased blood vessels and GFAP-positive reactive astrocytes (Fig. 5b) As a result of AT-NPDl treatment, blood vessel density increased in the penumbra, with the parallel formation of a denser GFAP-rich scar tissue. Additionally, the generation of new blood vessels facilitates neurogenesis and synaptogenesis, which in turn contributes to repair and improved functional recovery.
Example 5
Mediator lipidomics demostrate endogenous biosynthesis of AT-NPDl in the ipsilateral-side after stroke.
We found that NPDl and AT-NPDl are formed in the ipsilateral side after MCAo by matching the LC-MS/MS chromato rams and spectrum to authentical synthetic compunds (Fig. 6, a-c). The MRM chromatogram at m/z 359 > (m/z 153, 206) showed that more AT-NPDl was generated in penumbra regions after MCAo upon iv injection of DBA and aspirin (Fig.6 a,b). The
MS/MS ions m/z 359[M-H]-, 341[M-H-H20]-, 323[M-H-2H20]-, 315[M-H-C02]- , 297[M-H-H20-C02]-, 261, 243 [261-H20]-, 217[261-C02]-; 206, and 153 were consistent with one carboxyl, two hydroxyls, and the molecular weight (M) of AT-NPDl and NPDl, which was 360, as well as the positions of the hydroxyl groups (Fig. 6b). The markers for the biosynthetic pathways of AT-NPDl and NPDl, 17R-HDHA and 17S-HDHA as shown by chiral-LC SRM chromatogram at m/z 343 > 245 respectively were found (Fig. 6,d).
Example 6
Therapeutic window Study
Neurological deficits were reduced by DHA, even when treatment was initiated as late as 5 h after MCAo onset (Fig. 7a: day 1 (DHA: 3 h, 7.7±0.2; 4 h, 6.9±0.7; 5 h, 7.2±0.5; and 6 h, 8.3±0.2 vs. Saline: 9.8±0.2; p<0.01), day 2 (DHA: 3 h, 7.7±0.2; 5 h, 6.5±0.8; and 6 h, 7.8±0.3 vs. Saline: 9.8±0.2; pO.01), day 3 (DHA: 3 h, 7.1±0.6; 5 h, 6.0±1.0; and 6 h, 8.2±0.4 vs. Saline: 9.7±0.2; p<0.003) and day 7 (DHA: 3 h, 6.0±0.6; 4 h, 5.7±1,0; 5 h, 5.5±0.9 vs. Saline; 9.0±0.3; p<0.0004). DKA- mediated protection was extensive in the frontal- parietal cortex (tissue salvage, 49% (p<0.02), 77% (p<0.002), and 71%
(p<0.004), respectively for 3 h, 4 h and 5 h; Fig. 7b) and across multiple coronal levels. DHA administration did not affect the subcortical infarct, except when it was infused at 4 h (p<0.01; Fig. 7c). Total infarct volume, corrected for brain swelling, was reduced by 40% (p<0.02) when DHA was administered at 3 h, by 66% (pO.001) at 4 h, and by 59% (p<0.003) at 5 h (Fig. 7d). The brains of saline -treated rats exhibited a consistent pannecrotic lesion involving both cortical and subcortical (mainly striatal) regions of the right hemisphere, characterized microscopically by destruction of neuronal, glial, and vascular elements. In contrast, infarct size was dramatically reduced in rats treated with DHA at 3, 4, and 5 h after onset of ischemia, but not in 6-h group (Fig. 7e). Saline-treated rats showed extensive neuronal loss, GFAP-positive reactive astrocytes outlining the lesion territory, and massive ED-l-positive microglia/macrophage infiltration (Fig. 7f, top). In contrast, DHA attenuated damage as well as decreased ED-l-positive microglia/macrophages and increased GFAP-positive reactive astrocytes (Fig. 7f, bottom). Figure 8 presents GFAP-, ED-1-, and NeuN-positive cell counts. DHA treatment significantly decreased ED-1 (p<0.01) and increased NeuN (p<0.01) and GFAP- positive (p<0.005) cell counts.
Example 7
Behavioral outcome, lipidoraics and western blot analysis study.
Young (287-385g} 3-4mos) or aged (515-630g, 15-17mos) male Sprague-
Dawley rats were anesthetized with isofiurane/nitrous oxide and mechanically ventilated. Rectal and cranial temperatures were regulated at 36-37.5°C. Rats received 2hr MCAo by retrograde insertion of an intraluminal suture. Animals were treated with DHA (5mg/kg, IV), NPD1 (300 fg/kg), or vehicle (5ml/kg isotonic saline, IV) 3hr after MCAo onset. Behavioral function was evaluated at 60min after occlusion onset and on days 1, 2, 3, and 7 on a grading scale of 0-12 (O^normal and 12~maximal deficit). In the behavioral outcome study, behavioral outcome was tested at 60 min (during MCAo) and then on days 1, 2, 3 and 7 after MCAo. In the lipidomic study, NPDl content was quantified at 4hr in young rats treated with vehicle and aged rats treated with vehicle or DHA. In the proteomic study, DHA, NPDl, or vehicle was administered 3hr after MCAo onset (n=4) and western blot analysis of AKT signaling proteins was conducted 4hr after reperfusion.Results: Rectal and cranial (temporalis muscle) temperatures, arterial blood gases, hematocrit, and plasma glucose in young and aged animals showed no significant differences among groups. DHA or NPDl treatment resulted in improved neurobehavioral outcome on all days with a 36% improvement in DHA-treated animals and 38% improvement in NPDl treated animals by day 7 when compared to vehicle-treated animals. When NPDl content was analyzed 4hr after MCAo, aged rats had a 55% reduction in NPDl synthesis when compared to young rats but this deficit was overcome with DHA treatment. When the protein phosphorylation state of aged rats at 4hr was analyzed, we found that aged rats did not show a treatment effect on the phosphorylation of p308 AKT or pGSK. However, pS6 expression was increased with DHA (62% increase) or NPDl (280% increase) when compared to vehicle. Conclusions: Behavior, lipidomics, and western blot analysis confirm that aged animals have a reduced capacity to synthesize NPD and that this deficit is overcome with DHA therapy. Furthermore, treatment with NPDl results in increased protein activation and marked neurobehavioral recovery after MCAo in aged animals and should be
investigated as a potential therapy after stroke in the clinical setting (Figure 9).
Example 8
DHA modulates protein signaling in the penumbra and is
neuroprotective in experimental stroke. Male Sprague-Dawley rats (287-385g) were anesthetized with
isoflurane/nitrous oxide and mechanically ventilated. Rectal and cranial temperatures were regulated at 36-37.5°C. Rats received 2hr MCAo by retrograde insertion of an intraluminal suture. Animals were treated with DHA (5mg/kg, IV) or vehicle (5ml/kg isotonic saline, IV) 3hr after MCAo onset. Behavioral function was evaluated at 60min after occlusion onset and at 24hr on a grading scale of 0-12 (0=normal and 12=maximal deficit). In the histopathology study, GFAP (reactive astrocytes), ED-1 (activated
microglia/microphages), and NeuN (neurons) were analyzed in the infarct core and penumbra at 4hr and 24hr. In the proteomic study, western
blot/immunohistochemical analysis of pAKT, pGSK, and pS6 (proteins of the AKT signaling pathway) was conducted in penumbra regions at 4hr and 24hr. Results: DHA treatment significantly improved behavioral scores compared to vehicle at 24hrs (DHA: 6.75±0.8 vs. Vehicle: 9.8810.3; p<0.01). DHA
significantly reduced microglia infiltration by 85% and increased astrocytosis by 119% when compared to vehicle at 24hr. DHA upregulated activation of AKT at 4hr versus vehicle (4hr DHA: 36% increase in p473 AKT; 79% increase in p308 AKT) and when compared to DHA-treated animals at 24hr (4hr DHA: 114% increase in p473 AKT; 98% increase in p308 AKT). DHA also increased pS6 at 4hr by 160% and pGSK at 24hr by 61% as compared to vehicle -treated animals. The findings from this work support further investigation into DHA's potential to reduce the burden of stroke damage in a clinical setting. These studies on behavior, histopathology, and proteomics confirm marked
neuroprotective changes within 24hrs of DHA treatment after focal cerebral ischemia (Figure 11).
While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.
All references cited herein or appearing under the heading
"REFERENCES," including but not limited to any patents, patent
applications, and non-patent literature, are hereby incorporated by reference herein in their entirety.
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Lo EH, Rogowska J, Bogorodzki P, Trocha M, Matsumoto K, Saffran B, Wolf GL. Temporal correlation analysis of penumbral dynamics in focal cerebral ischemia. J Cereb Blood Flow Metab. 1996; 16:60-8. Marcheselli VL, Hong S, Lukiw WJ, Tian XH, Gronert K, Musto A, Hardy M, Gimenez JM, Chiang N, Serhan CN, Bazan NG. Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and proinflammatory gene expression. J Biol Chem. 2003; 278:43807-17.
Mogoanta L, Pirici D, Pop OT, Bal§eanu AT, Rolea E, Dahnovici RM.
Study of vascular microdensity in areas of cerebral ischemia on experimental model. Rom J Morphol Embryol. 2010; 51:725-731.
Moskowitz MA, Lo EH, Iadecola C. The science of stroke: mechanisms in search of treatments. Neuron. 2010; 67:181-98.
Mukherjee PK, Marcheselli VL, Serhan CN, Bazan NG. Neuroprotectin
Dl: a docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress. Proc Natl Acad Sci USA. 2004; 101, 8491-8496.
Panickar KS, Norenberg MD. Astrocytes in cerebral ischemic injury; morphological and general considerations. Glia. 2005; 50:287-298.
Petasis, NA, Yang R, Winkler JW, Zhu M, Uddin J, Bazan NG, Serhan CN StereocontroUed total synthesis of Neuroprotectin Dl/Protectin Dl and its aspirin-triggered stereoisomer, Tetrahedron Letters, 2012; 53:1695-1698.
Rowlinson SW, Crews BC, Goodwin DC, Schneider C, Gierse JK, Marnett LJ. Spatial requirements for 15-(R)-hydroxy-5Z,8Z,llZ,13E- eicosatetraenoic acid synthesis within the cyclooxygenase active site of murine COX-2. J Biol Chem. 2000; 275, 6586-6591.
Schwab JM, Chiang N, Arita M, Serhan, C.N. Resolvin El and protectin Dl activate inflammation-resolution programmes. Nature. 2007; 447, 869-874.
Serhan CN, Hong S, Gronert K, Colgan SP, Devchand PR, Mirick G,
Moussignac RL. Resolvins: a family of bioactive products of omega- 3 fatty acid transformation circuits initiated by aspirin treatment that counter pro- inflammation signals. J Exp Med. 2002; 196:1025-1037.
Serhan CN, Gotlinger K, Hong S, Lu Y, Siegelman J, Baer T, Yang R, Colgan SP, Petasis NA. Anti-inflammatory actions of neuroprotectin Dl/protectin Dl and its natural stereoisomers: assignments of dihydroxy- containing docosatrienes. J Immunol. 2006; 176:1848-1859.
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Claims

WHAT IS CLAIMED IS: as
Figure imgf000038_0001
wherein:
Ri and R2 are independently selected from the group consisting of H and COR3 (acyl), where R3 is selected from the group consisting of alkyl, cycloalkyl, aryl or heteroaryl; and
R is H, alkyl, cycloalkyl, aryl or heteroaryl,
or a pharmaceutically acceptable salt thereof.
2. The compound according to claim 1, having formula selected from the grou consisting of:
Figure imgf000038_0002
wherein:
Ri and R2 are independently selected from the group consisting of H and COR3 (acyl), where R3 is alkyl, cycloalkyl, aryl or heteroaryl, and
R is H, alkyl, cycloalkyl, aryl or heteroaryl, a pharmaceutically acceptable salt thereof.
The compound accordin to claim 1 having the formula:
Figure imgf000039_0001
wherein R is selected from the group consisting of H, methyl, alkyl, cycloalkyl, aryl and heteroaryl,
or a pharmaceutically acceptable salt thereof.
4. The compound of Claim 1 havin the formula:
Figure imgf000039_0002
wherein R is selected from the group consisting of H, methyl, alkyl, cycloalkyl, aryl and heteroaryl,
or a pharmaceutically acceptable salt thereof.
5. The compo the formula:
Figure imgf000039_0003
wherein R is selected from the group consisting of H, methyl, alkyl, cycloalkyl, aryl or heteroaryl,
or a pharmaceutically acceptable salt thereof.
Figure imgf000040_0001
wherein:
Ri and a are independently selected from the group consisting of H and COR3 (acyl), where R3 is alkyl, cycloalkyl, aryl or heteroaryl, and
R is Hf alkyl, cycloalkyl, aryl or heteroaryl,
or a pharmaceutically acceptable salt thereof.
7. A method for the treatment of ischemic stroke, comprising administering to a subject in need thereof an effective amount of at least one compound according to any of claims 1 to 6.
8. The method according to claim 6, wherein the adminsteration is within up to 6 hours following focal ischemia.
9. A method for the treatment of ischemic stroke, comprising admmistermg to a subject in need thereof at least one compound according to claims 1 to 6 as a complex with albumin.
10. A method for the treatment of ischemic stroke, comprising administering to a patent in need thereof an effective amount of
docosahexaenoic acid. 11. A method for the treatment of ischemic stroke, comprising
administering to a patient in need thereof a combination of docosahexaenoic acid and aspirin.
12. A pharmaceutical composition for the treatment of ischemic stroke, comprising a compound according to any of claims 1 to 6 dissolved or dispersed in a pharmaceutically acceptable carrier.
13. A method of treatment for ischemic stroke, comprising
administering the pharmaceutical compositions according to claim 11 to a subject in need thereof, where the administration comprises one of intrave ous injection and an oral formulation.
14. The method according to claim 13, wherein the treatment is within 6 hours from the initiation of an ischemic event.
15. A non-invasive method for monitoring the progression of the treatment comprising 3D images obtained via magnetic resonance imaging (MM). 16. A method for treatment of ischemic stroke comprising one of: (1) administering a combination of DHA, aspirin and at least one compound according to Formulas I to XIX and (2) administering a combination of aspirin and at least one compound according to Formulas I to XIX.
17. The method according to any of claims 7-11 and 13-16, further comprising a step of either administering a thrombolytic agents such as the protein tissue-plasminogen activator (t-PA) and/or a surgical procedure for treatment of ischemic stroke.
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