WO2015095968A1 - Lipoic acid-resveratrol conjugates and uses thereof - Google Patents

Abstract

Described are covalent conjugates between lipoic acid (LA), or a derivative thereof, and resveratrol, or a derivative thereof. As an example, the covalent conjugate may be a compound of the formula: (I). Methods using the LA-resveratrol conjugates for the treatment of diseases, disorders, or conditions related to oxidative stress are also provided.

Description

LIPOIC ACID-RESVERATROL CONJUGATES AND USES THEREOF

FIELD OF INVENTION

The present invention relates generally to conjugates of lipoic acid and resveratrol, compositions comprising these compounds, and compounds that may be used as neuroprotectants, or in the prevention or treatment of diseases, disorders, or conditions related to oxidative stress, neurodegenerative diseases, stroke, ischemia, and/or reperfusion injury.

BACKGROUND

Worldwide mortality and disability rates due to cerebral ischemia (stroke) remain significant, and therapeutic options continue to be limited. Despite ongoing advances in understanding the causes of cerebral ischemia, the worldwide consequences of death and disability remain considerable and delivery of successful therapeutics remains challenging. Reactive oxygen species (ROS) and other free radicals generated during cerebral ischemia/reperfusion (I/R) are critical mediators of neuronal death (Bedard and Krause, 2007). Over the last decade since the introduction of rTPA for thrombolytic therapy, research into antioxidants for prevention of reperfusion injury to extend the window of opportunity for patient eligibility for this therapy has increased dramatically.

The application of combinatorial drug therapy in treating stroke has become increasingly attractive in recent years. As researchers uncover the complexity of disease progression following stroke which includes both immediate as well as delayed genetic components at multiple levels, it has become evident that multi-target drug therapy may hold more promise in the treatment and/or prevention of stroke than conventional single class drug regimens. In addition, there is evidence that some drug combinations display pharmacological potentiation (ie synergism) which optimistically translates into lower doses, fewer adverse side effects and an extended treatment window. Treatment outcomes for ischemic events involves the reestablishment of blood flow to compromised tissue, with the reintroduction of oxygen transiently adding to the injury with the generation of inflammatory mediators and toxic levels of oxidative free radicals (Ginberg, 2008) culminating in lipid peroxidation, protein synthesis arrest, and ultimately, cell death (Lip ton, 1999). Successful treatment options are therefore required to address several critical mediators of neuronal death simultaneously.

Resveratrol (3, 5, 4'-trihydroxystilbene) possesses multiple biological activities (de la Lastra and Villegas, 2005; Delmas et al., 2005), including being a potent antioxidant (Cadelario-Jalil et al., 2007) and anti-inflammatory (Kang et al., 2009) agent. These therapeutic uses of resveratrol have led researchers to further investigate its protective effects in several animal models of neurological diseases, particularly those with unknown etiology, or where inflammation and oxidative stress may play a role in the pathogenesis. Consequently, resveratrol has shown promise as a neuroprotectant in many animal models of cerebral ischemia through its ability to attenuate ischemia-induced cell death (Saleh et al., 2013; Connell et al., 2011; Tang et al., 2007; Jackman et al, 2009; Chen et al, 2009; Tang et al, 2008; Kelly et al, 2009; Zhao et al, 2010). Resveratrol possesses potent anti-oxidant and anti-inflammatory activities and have been shown to produce neuroprotection in several animal models of neurological diseases via complementary pathways (Yan et al., 2011; He et al., 2012).

Potential safety issues exist as high doses of resveratrol have been shown to cause renal toxicity (Crowell et al. 2004) and contribute to hepatic oxidative stress (Rocha et al., 2009). In the presence of peroxidase and/or transition metals, resveratrol may function as a pro-oxidant ultimately contributing to DNA damage and mitochondrial dysfunction (Galati et al., 2002; Ahmad et al., 2000). As well, resveratrol has been shown to inhibit cytochrome P450 enzyme CYP1A1 (Chun et al., 1999), an interference which may render other drugs in a patient's treatment plan ineffective at therapeutic doses.

Also a powerful antioxidant, lipoic acid (LA), for example a-lipoic acid, is characterized by high reactivity towards free radicals (Biewenga et al., 1997) and demonstrates potent neuroprotective effects in several animal models of stroke including models of reperfusion injury (I/R) (Connell et al., 2011b; Richard et al., 2011; Panigrahi et al., 1996; Wolz and Krieglstein, 1996; Clarke et al., 2001; Cao and Phillis, 1995). Further, the co-administration of LA with other compounds has been shown to enhance the protective effect of the drug in various animal models of pathology (Sola et al., 2005; Shotton et al., 2004; Mufherjee et al., 2011; Gonzalez-Perez et al., 2002; Garcia-Estrada et al., 2009). Research has shown that co -administration of non-protective (sub-threshold) doses of both LA and apocynin, an NADPH oxidase inhibitor, produced significant neuroprotection (Connell and Saleh, 2012).

Lipoic acid (LA) is a naturally occurring eight-carbon fatty acid that is synthesized by plants and animals, including humans. The natural configuration is "R", although the mixed RS (DL) lipoic acid is extensively used commercially.(Biewenga et al., 1997) It is chemically named 1,2- dithiolane-2-pentanoic acid (also referred to as thioctic acid). It is an important cofactor in the mitochondrial respiratory chain and serves as a cofactor for many enzyme reactions. (Constantinescu et al., 1995; Haramaki et al., 1997; Jones et al., 2002) It has also emerged as a potent antioxidant, anti-inflammatory, and a mitochondrial protective agent.(Packer et al., 1995)

Both the parent compounds as well as the dihydro form (both the sulfur atoms reduced to thiol functions) have been reported to have antioxidant properties. (Biewenga et al., 1997) LA has been reported to lower serum triglycerides, increase glucose uptake by cells, stimulate neurological function, decrease liver toxicity, increase levels of glutathione and ascorbic acid and decrease the expression of inflammatory molecules. (Morcos et al., 2005; Mari et al., 2009; Mijnhout et al., 2010; Ying et al., 2010) LA has also been shown to have a neuroprotective effect.(Connell et al., 2011; Richard et al., 2011) LA has a very short half-life in the bloodstream as it undergoes rapid metabolism in the liver. (Mignini et al., 2011) Dosing 3-4 times daily is necessary to accomplish reasonable blood levels. This rather limited bioavailability of LA could be extended by suitably incorporating chemical groups to attenuate the metabolic process in the liver.(Arner et al., 1996; Tiechert et al., 2003) Researchers have demonstrated the neuroprotective properties of LA administration in animal models of stroke including I/R (Connell et al., 2011; Richard et al., 2011; Panigrahi et al., 1996; Wolz and Krieglstein, 1996; Clarke et al., 2001; Cao and Phillis, 1995). The neuroprotective properties of a LA-apocynin co- drug following I/R injury have been demonstrated (Connell et al 2012; WO2013/071400).

Oxidative mechanisms are associated with central nervous system disorders such as stroke and dementia. Increased production of reactive oxygen species (ROS) has been implicated in various chronic diseases, including neurodegenerative diseases (Farooqui et al., 2000). Oxidative stress is implicated in endothelial dysfunction, inflammation, hypertrophy, apoptosis, fibrosis, angiogenesis, and rarefaction (Victor et al., 2009). There continues to be a need for additional neuroprotectants, and methods to treat oxidative stress, stroke, ischemia, and/or reperfusion injury.

SUMMARY OF THE INVENTION

An object of the invention is to provide covalent conjugates of lipoic acid and resveratrol.

In addition, an object of the invention is also to provide compounds that are useful, for example, for reducing or preventing cell damage caused by reactive oxygen species (ROS), or which can be used for treating diseases, disorders, or conditions related to oxidative stress.

Accordingly, there is provided herein a compound of Formula I:

(I) wherein

X, Y, and Z are each independently -OH, or represents a linkage to a moiety of Formula II, the linkage being in each instance, independently, an ester linkage, a thioester linkage, an amide linkage, an alkyl linkage, an -NH- or -N(alkyl)- linkage, an ether linkage, a thioether linkage, or a disulfide linkage; and at least one of X, Y, and Z is linked to the moiety of Formula II:

(Π), wherein n is 1, 2, 3, 4, 5 or 6; m is 0, 1 or 2; R¹ and R² are independently H, Ci_₆alkyl or C(0)Ci_₆-alkyl, or

R¹ and R² are absent and the two sulfur atoms are bonded together to form, together with the carbon atoms between them, a 4, 5 or 6 membered ring, or a pharmaceutically acceptable salt and/or solvate thereof.

In certain embodiments, at least one of X, Y, and Z may represent a linkage comprising an -0-, - 0-C(=0)-, -S-, -Ci-₆-alkyl-, or -NH- group. Thus, in further embodiments, the compound may be represented by Formula (la):

(la) wherein

R³, R⁴, and R⁵ are each independently H or a moiety of Formula Ila or Formula lib, and at least one of R³, R⁴, and R⁵ is a moiety of Formula Ila or Formula lib:

(Ila) wherein n, m, R¹ and R² are as defined above, or a pharmaceutically acceptable salt and/or solvate thereof.

In certain non-limiting embodiments of the above compound, n may be 3, 4 or 5, and in further embodiments, n is 4. In other non-limiting embodiments of the above compound, m may be 1 or 2, and in specific embodiments m is 2.

In yet further embodiments, R¹ and R² may independently be H,

or C(0)Ci_₄alkyl, and for example, may independently be H, CH₃ and C(0)CH₃. In further non-limiting embodiments, R¹ and R² are the same, and may both be hydrogen.

In addition, R¹ and R² may both be absent, and the two sulfur atoms bonded together to form, together with the carbon atoms between them, a 5 or 6 membered ring. In a non-limiting embodiment, the two sulfur atoms are bonded together to form a 5 membered ring.

In certain specific embodiments, the compound may have the formula:

(UPEI-201)

or a pharmaceutically acceptable salt thereof.

In further embodiments, the compound may have the formula:

(UPEI-200)

or a pharmaceutically acceptable salt thereof.

Also provided herein is a compound, or a pharmaceutically acceptable salt thereof, that is a covalent conjugate between 1-3 lipoic acids (LA), or a derivative thereof, and resveratrol, or a derivative thereof, wherein each conjugate linkage is independently an ester linkage, thioester linkage, amide linkage, alkyl linkage, -NH- or -N(alkyl)- linkage, ether linkage, thioether linkage, or disulfide linkage. For example, the conjugate linkage may comprise a -0-, -0-C(=0)- , -S-, -Ci-6-alkyl-, or -NH- group.

In a further embodiment, the Resveratrol derivative may be Pterostilbene, Oxyresveratrol, Piceatannol, Isorhapontin, 3,4'-5-Trihydroxystilbene-3-beta-D-glucopyranoside, Rhapontin, Polydatin, Deoxyrhapontin rhubarb root, Gnetifolin E, or Viniferin.

There is also provided a pharmaceutical composition comprising at least one compound as described above, and a pharmaceutically acceptable carrier.

In addition, a method is provided herein for preventing or treating a disease, disorder, or condition related to oxidative stress which comprises administering a compound or pharmaceutical composition as described above to a subject in need thereof.

For example, yet without wishing to be limiting, the disease, disorder or condition may be a cerebral disease, disorder, or condition, or a heart, kidney, liver, or skeletal muscle disease, disorder, or condition. In other non-limiting embodiments, the disease, disorder, or condition may include stroke, ischemia, reperfusion injury, neurodegenerative disease, inflammatory disease, neurovascular disorder, dementia, Multiple Sclerosis, Parkinson's disease, myocardial infarction, heart failure, renal failure, collagen vascular disease, metabolic disorder, cardiac disease, or combinations thereof.

According to the method described above, an anti-thrombolytic drug may also be provided for co-administration with the compound or pharmaceutical composition described above. For example, the anti-thrombolytic drug may be streptokinase, tPA (Tissue Plasminogen Activator), or rtPA (Recombinant Tissue Plasminogen Activator).

The above described compound and composition may also be used in methods for reducing or preventing cell damage caused by reactive oxygen species (ROS). In certain embodiments, the reducing or preventing cell damage may occur in vitro, or may occur in vivo.

Also provided herein is a process for preparing a covalent conjugate between lipoic acid (LA) and resveratrol of the formula:

(UPEI-201) (UPEI-200) The process comprises reacting lipoic acid (LA) of the formula:

with resveratrol of the formula:

in the presence of a coupling agent under conditions effective to form an ester linkage between the lipoic acid and the resveratrol moieties and form said LA-resveratrol conjugate, in particular, the ester linkage is formed between the oxygen of one or more carbonyl group of resveratrol and the carboxyl group of lipoic acid; and optionally purifying said compound.

In certain non-limiting embodiments of the process, the coupling agent may be a carbodiimide. For example, in embodiments wherein the compound is:

(UPEI-201) the coupling agent may be l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), and in embodiments wherein the compound is:

(UPEI-200) the coupling agent may be Ν,Ν'-dicyclohexylcarbodimide (DCC).

Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples are provided for illustration only, and that various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent from the following description in which reference is made to the following drawings:

FIGURE 1 : (A) Representative photomicrographs of TTC stained, 1 mm thick coronal slices illustrating the extent of the infarct within the prefrontal cortex following pretreatment (30 minutes prior to MCAO; i.v.) with either Vehicle (propylene glycol 4xl0^"3 % (v/v)) or resveratrol (2xl0^"7 and 2xl0^"3 mg/kg) following ischemia/reperfusion (I/R). (B) Bar graph summarizing the dose-response relationship between increasing doses of resveratrol and infarct size (mm³) calculated from TTC-stained, 1 mm thick coronal sections following I/R. Each bar represents the mean ± S.E.M. (n=5-6/group) and * indicates significance (p < 0.05) from the vehicle-treated control group;

FIGURE 2: Bar graph summarizing the effect of resveratrol injected 15 minutes into middle cerebral artery occlusion (15), or at 30 min intervals post-reperfusion. Each bar represents the mean ± S.E.M. (n=5-6/group) and * indicates significance (p < 0.05) from the pooled vehicle- treated (propylene glycol 4xl0^"3 % (v/v)) control group;

FIGURE 3: Bar graph summarizing the effect on infarct volume following the coadministration of a sub-threshold dose of lipoic acid (0.005 mg/kg) with increasing doses of resveratrol on infarct volume. Each bar represents the mean ± S.E.M. (n=5-6/group) and * indicates significance (p < 0.05) from the vehicle-treated (propylene glycol 4x10-3 % (v/v)) control group;

FIGURE 4: Bar graph summarizing the effect on infarct volume following the injection of the optimal combination of doses of lipoic acid and resveratrol 15 minutes during the occlusion (15 min), or immediately after reperfusion (30 min). Each bar represents the mean ± S.E.M. (n=5- 8/group) and * indicates significance (p < 0.05) from the vehicle-treated (propylene glycol 4xl0^"3 % (v/v)) control group;

FIGURE 5: Bar graph of the quantified cytoplasmic histone-associated-DNA fragmentation (an indicator of apoptotic cell death). Each bar represents the mean ± S.E.M. and * indicates significance (p < 0.05) from the vehicle-treated (propylene glycol 4xl0^"3 % (v/v)) control group;

FIGURE 6: (A) Bar graph illustrating the effect of UPEI-200 (3: 1 ratio of lipoic acid to resveratrol respectively) at increasing doses or a vehicle (propylene glycol 4xl0^"3 % (v/v)) injected 30 minutes prior to either ischemia/reperfusion (I/R; A) or permanent middle cerebral artery occlusion (6 hr MCAO; B). Each bar represents the mean ± S.E.M. (n=4-6/group);

FIGURE 7: (A) Bar graph illustrating the effect of UPEI-201 (1 : 1 ratio of lipoic acid to resveratrol respectively) at increasing doses or a vehicle (propylene glycol 4xl0^"3 % (v/v)) injected 30 minutes prior to either ischemia/reperfusion (I/R; A) or permanent middle cerebral artery occlusion (6 hr MCAO; B). Each bar represents the mean ± S.E.M. (n=5-7/group) and * indicates significance (p < 0.05) from the vehicle-treated control group;

FIGURE 8: (A) Bar graph illustrating the effect of UPEI-201 (1 : 1 ratio of lipoic acid to

6 3 resveratrol respectively) at a dose of 1x10^" mg/kg or a vehicle (propylene glycol 4x10^" % (v/v)) injected during the occlusion (15) or at 30 minute intervals immediately following reperfusion. Each bar represents the mean ± S.E.M. (n=5-7/group) and * indicates significance (p < 0.05) from the vehicle-treated control group;

FIGURE 9: Synthesis of UPEI-200 (A) and UPEI-201 (B).

DETAILED DESCRIPTION

Described herein are compounds that are covalent conjugates between lipoic acid (LA), or a derivative thereof, and resveratrol, or a derivative thereof. It will be understood by those of skill in the art that a wide variety of covalent linkages may be used to form covalent conjugates between LA and resveratrol. In some non-limiting embodiments, covalent linkages may include those comprising or consisting of an ester linkage, thioester linkage, amide linkage, alkyl linkage (such as Ci_₆alkyl or -CH2-), -NH- or -N(alkyl)- linkage, ether linkage, thioether linkage, disulfide linkage, biocleavable linkage, biocleavage-resistant linkage, or other appropriate linkage as will be known to one of skill in the art.

In certain embodiments, the covalent linkage may replace or modify a portion of the lipoic acid and/or resveratrol structure. For example, the linkage may be formed through an -OH portion of resveratrol and the -COOH portion of lipoic acid. Optionally, 1-3 lipoic acids may be linked to a single resveratrol. Where more than 1 lipoic acid is linked to a resveratrol, each lipoic acid may be linked by the same, or by a different, linkage type. In one embodiment, 1, 2, or 3 lipoic acids may each be linked to a resveratrol at a separate -OH group position on resveratrol. In embodiments, a -0-, -S-, -NH-, or -CH₂- group may link a -OH bearing carbon of resveratrol directly to the carbon of LA which normally bears the carboxylic acid functional group. Both the -OH function of resveratrol and the carboxlic acid -OH and (=0) groups of lipoic acid may be substituted out.

In some embodiments, a covalent linkage between lipoic acid (LA), or a derivative thereof, and resveratrol, or a derivative thereof, may be any biocleavage resistant linkage having a suitable metabolic profile such that the lipoic acid and resveratrol components of the conjugate remain linked in vitro or in vivo for at least a brief duration of time.

In a non-limiting embodiment, the covalent conjugate between LA and resveratrol may be a compound of Formula (I):

(I) wherein

X, Y, and Z are each independently -OH, or represents a linkage to a moiety of Formula II, the linkage being in each instance, independently, an ester linkage, a thioester linkage, an amide linkage, an alkyl linkage, an -NH- or -N(alkyl)- linkage, an ether linkage, a thioether linkage, or a disulfide linkage; and at least one of X, Y, and Z is linked to the moiety of Formula II:

(II) wherein n is 1, 2, 3, 4, 5 or 6; m is 0, 1 or 2;

R¹ and R² are independently selected from the group consisting of H, Ci_₆alkyl and C(0)Ci_₆ alkyl, or

R¹ and R² are absent and the two sulfur atoms are bonded together to form, together with the carbon atoms between them, a 4, 5 or 6 membered ring, or a pharmaceutically acceptable salt and/or solvate thereof.

In certain embodiments, at least one of X, Y, and Z may represent a linkage comprising an -0-, - 0-C(=0)-, -S-, -Ci-₆-alkyl-, or -NH- group. Thus, in further embodiments, the compound may be represented by Formula (la):

(la) wherein

R³, R⁴, and R⁵ are each independently H or a moiety of Formula Ila or Formula lib, and at least one of R³, R⁴, and R⁵ is a moiety of Formula Ila or Formula lib:

(Ila) wherein n, m, R¹ and R² are as defined above, or a pharmaceutically acceptable salt and/or solvate thereof.

In further non-limiting embodiments, the covalent conjugate between LA and resveratrol may be:

(UPEI-201)

or a pharmaceutically acceptable salt thereof.

In other non-limiting embodiments, the covalent conjugate between LA and resveratrol may be:

(UPEI-200)

or a pharmaceutically acceptable salt thereof.

In an embodiment, the LA-resveratrol conjugate may comprise a 1 :3 ratio, a 1 :2 ratio, or a 1 : 1 ratio of resveratrol (or derivative thereof) : Lipoic acid (or derivative thereof) moieties.

In further embodiments, the LA-resveratrol conjugate may be synthesized from resveratrol (or an appropriate derivative thereof) and lipoic acid (or an appropriate derivative thereof) using an appropriate coupling agent, as will be known to those of skill in the art. In an embodiment, the coupling agent may be a carbodiimide. In a further embodiment, the carbodiimide may be Ν,Ν'- dicyclohexylcarbodiimide (DCC), or l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI).

In an embodiment of the above-described process, wherein the compound may be:

(UPEI-201) or

(UPEI-200) the starting materials may be

(lipoic acid) and the ester linkage may be formed using a coupling agent. In some embodiments, the coupling agent may be DCC. In other embodiments, the coupling agent may be EDCI.

In certain embodiments, the compounds provided herein may be used as antioxidants.

In yet another non-limiting embodiment, compounds provided herein may be used for research or therapeutic applications. In still further embodiments, the research or therapeutic applications may involve oxidative stress and/or reactive oxygen species.

In certain non-limiting embodiments, the compounds provided herein may be used for the prevention or treatment of diseases, disorders, or conditions related to oxidative stress. In further embodiments, the diseases, disorders, or conditions may be stroke, ischemia, reperfusion injury, or combinations thereof. In still further embodiments, the diseases, disorders, or conditions may be cerebral diseases, disorders, or conditions.

In an embodiment, diseases, disorders, or conditions related to oxidative stress may include neurodegenerative disease, stroke, inflammatory disease, neurovascular disorder, dementia, Multiple Sclerosis, Parkinson's disease, myocardial infarction, heart failure, renal failure, collagen vascular disease, metabolic disorder, or cardiac disease.

In further embodiments, the diseases, disorders, or conditions related to oxidative stress, ischemia, reperfusion injury, or combinations thereof, may be cerebral, heart, kidney, liver, or skeletal muscle diseases, disorders, or conditions. In a further embodiment, the disease, disorder, or condition may be any ischemia/reperfusion injury. In another embodiment, the disease, disorder, or condition may be neurodegeneration and/or inflammation. In other non-limiting embodiments, the compounds and/or compositions provided herein may be used in combination with an anti-thrombolytic drug and/or a clot-busting drug. In an embodiment, the anti- thrombolytic drug may be tPA (Tissue Plasminogen Activator) or rtPA (Recombinant Tissue Plasminogen Activator). In some embodiments, the compounds and/or compositions provided herein may be used in combination with an anti-thrombolytic, for example, tPA or rtPA, for preventing and/or treating ischemia/reperfusion injury. In a further embodiment, the anti- thrombolytic may be streptokinase.

In another non-limiting embodiment, the compounds provided herein may be used for reducing/preventing cell damage caused by reactive oxygen species (ROS), either in vitro or in vivo.

Without wishing to be limited by theory, LA-resveratrol conjugates as provided herein may be considered co-drugs. In some embodiments, treatment with LA-resveratrol conjugates may produce neuroprotection while at least partially reducing at least one of resveratrol-induced renal toxicity, hepatic oxidative stress, DNA damage, mitochondrial dysfunction, or inhibition of cytochrome P450 enzyme (CYP1A1) which may be associated with a correspondingly effective dose of resveratrol alone.

In some embodiments, treatment with LA-resveratrol conjugates as provided herein may reduce infarct volume in ischemia/reperfusion injury. In additional embodiments, LA-resveratrol conjugates provided herein may be administered prior to an ischemia/reperfusion event, during a period of ischemia, during a period of reperfusion, following an ischemia/reperfusion event, or any combination thereof. As will be recognized by those of skill in the art, LA-resveratrol conjugates provided herein may be administered to a subject using any appropriate method known in the art. Methods of administration may include I.V. administration, subcutaneous injection, intraperitoneal injection, direct injection, or any other appropriate method of administration.

The dosages of the compound or composition provided herein may be formulated in a number of ways. For example, without wishing to be limiting, they may be formulated as an oral supplement, as a food/feed additive, or as pharmaceutical or nutraceutical compositions. The compounds or compositions described herein may also be formulated or combined with one or more acceptable additives, carriers or excipients suitable for preparation of the desired dosage form(s).

The compounds and compositions described herein can be employed in methods of treating or preventing diseases, disorders, or conditions related to oxidative stress. Such methods comprise administering the compounds or compositions provided herein to a subject in need thereof in an amount sufficient to ameliorate or prevent the diseases, disorders, or conditions related to oxidative stress, and/or to prevent or treat stroke, ischemia, reperfusion injury, or combinations thereof.

Therapeutic uses of the compounds and compositions described herein are also provided, whereby the compounds or compositions as described herein, or isomer, derivative, pharmaceutically acceptable salt or ester thereof, are used for treating or preventing diseases, disorders, or conditions related to oxidative stress, and/or to prevent or treat stroke, ischemia, reperfusion injury, or combinations thereof.

Specific examples of the diseases, disorders, or conditions related to oxidative stress include, but are not limited to, stroke, ischemia, reperfusion injury, or combinations thereof.

The compounds or compositions described herein are suitably formulated into one or more than one separate pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. Accordingly, the present application also includes a pharmaceutical composition comprising one or more compounds or compositions and a pharmaceutically acceptable carrier. The compounds or compositions may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. A compound may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington's Pharmaceutical Sciences (2000 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

The compounds or compositions may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or the compounds may be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly with the food of the diet. For oral therapeutic administration, the compound may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Oral dosage forms also include modified release, for example immediate release and timed-release, formulations. Examples of modified-re lease formulations include, for example, sustained-release (SR), extended-release (ER, XR, or XL), time-release or timed- release, controlled-release (CR), or continuous-release (CR or Contin), employed, for example, in the form of a coated tablet, an osmotic delivery device, a coated capsule, a microencapsulated microsphere, an agglomerated particle, e.g., as of molecular sieving type particles, or, a fine hollow permeable fiber bundle, or chopped hollow permeable fibers, agglomerated or held in a fibrous packet. In an embodiment, coatings that inhibit degradation of the compounds of the application by esterases, for example plasma esterases, are used in the oral administration forms. Timed-release compositions can be formulated, e.g. liposomes or those wherein the active compound is protected with differentially degradable coatings, such as by microencapsulation, multiple coatings, etc. Liposome delivery systems include, for example, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. It is also possible to freeze-dry the compounds or compositions and use the lyophilizates obtained, for example, for the preparation of products for injection.

Compounds or compositions may also be administered parenterally. Solutions of one or more compounds or compositions can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists.

Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively, the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as a fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer.

Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.

Compounds or compositions may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. Compounds or compositions may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, compounds or compsitions may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.

Compounds or compositions may be used alone or in combination with other known agents useful for treating diseases, disorders, or conditions related to oxidative stress and/or blood clotting. Compounds or compositions may also be used in combination with agents that inhibit esterases, such as plasma esterases. When used in combination with other agents useful in treating diseases, disorders, or conditions related to oxidative stress and/or blood clotting, it is an embodiment that the compounds or compositions are administered contemporaneously with those agents. As used herein, "contemporaneous administration" of two substances to a subject means providing each of the two substances so that they are both biologically active in the individual at the same time. The exact details of the administration will depend on the pharmacokinetics of the two substances in the presence of each other, and can include administering the two substances within a few hours of each other, or even administering one substance within 24 hours of administration of the other, if the pharmacokinetics are suitable. Design of suitable dosing regimens is routine for one skilled in the art. In particular embodiments, two substances will be administered substantially simultaneously, i.e., within minutes of each other, or in a single composition that contains both substances. In a further embodiment, a combination of agents may be administered to a subject in a non- contemporaneous fashion. In certain embodiments, the other known agents may be anti- thrombolyic drugs. In a further embodiment, the anti-thrombolytic drug may be rTPA.

The dosage of compounds can vary depending on many factors such as the pharmacodynamic properties of the compound, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the compound in the subject to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. Compounds of the application may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. As a representative example, oral dosages of one or more compounds of the application will range between about 1 mg per day to about 1000 mg per day for an adult, suitably about 1 mg per day to about 500 mg per day, more suitably about 1 mg per day to about 200 mg per day. In an embodiment of the application, compositions are formulated for oral administration and the compounds are suitably in the form of tablets containing 0.25, 0.5, 0.75, 1.0, 5.0, 10.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 75.0, 80.0, 90.0, 100.0, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg of active ingredient per tablet. Compounds of the application may be administered in a single daily dose or the total daily dose may be divided into two, three or four daily doses.

Treatment or prevention methods comprise administering to a subject or a cell, a therapeutically effective amount of the compounds or compositions, and optionally consists of a single administration, or alternatively comprises a series of administrations.

In an embodiment, the compounds may be administered to the subject in a series of administrations, for example about 1, 2, 3, 4, 5 or 6 times daily for 1 or more days either before or after the onset of the disease, disorder or condition. The length of the treatment period depends on a variety of factors, such as the cause of the disease, disorder or condition, severity of the disease, disorder or condition, the age of the subject, the concentration of the compounds, the activity of the compounds, and/or a combination thereof. It will also be appreciated that the effective dosage of the compound used for the treatment or prevention may increase or decrease over the course of a particular treatment or prevention regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compounds are administered to the subject in an amount and for a duration sufficient to treat the subject.

Definitions

Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the application herein described for which they are suitable as would be understood by a person skilled in the art.

Terms of degree such as "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least +5% of the modified term if this deviation would not negate the meaning of the word it modifies.

The term "derivative" as used herein refers to a compound that is derived from a parent compound by modification of one or more of the functional groups in the parent molecule. For example, a derivative of LA may be a reduced form (dithiol) of LA, or a reduced form in which the thiol groups are substituted with, for example, a C_1-6 alkyl group or a C_1-6 acyl group. Further, a derivative of resveratrol may be a compound such as pterostilbene, oxyresveratrol, piceatannol, isorhapontin, 3,4'-5-trihydroxystilbene-3-beta-D-glucopyranoside, rhapontin, Polydatin, deoxyrhapontin rhubarb root, gnetifolin E, amine derivatives of resveratrol, or viniferin.

The term "subject" as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans.

The term "pharmaceutically acceptable" means compatible with the treatment of subjects, in particular humans.

The term "pharmaceutically acceptable salt" means an acid addition salt which is suitable for, or compatible with, the treatment of patients.

The term "acid addition salt which is suitable for, or compatible with, the treatment of patients" as used herein means any non-toxic organic or inorganic salt of any basic compound. Basic compounds that form an acid addition salt include, for example, compounds comprising a thiol group. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, acid addition salts are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art.

The formation of a desired compound salt is achieved using standard techniques. For example, the basic compound is treated with an acid in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.

The term "solvate" as used herein means a compound or its pharmaceutically acceptable salt, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate". The formation of solvates will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.

In embodiments of the described invention, compounds and compositions described herein have at least one asymmetric center. These compounds exist as enantiomers. Where compounds possess more than one asymmetric center, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present application. It is to be further understood that while the stereochemistry of the compounds may be as shown in any given compound listed herein, such compounds may also contain certain amounts (e.g. less than 20%, suitably less than 10%, more suitably less than 5%) of compounds of the application having alternate stereochemistry. For example, compounds that are described or shown without any stereochemical designations are understood to be racemic mixtures. However, it is to be understood that all enantiomers and diastereomers are included within the scope of the present application, including mixtures thereof in any proportion.

In an embodiment, the resveratrol, or derivative thereof, moiety of compounds provided herein may by in a cis (Z) or a trans (E) isomeric configuration. The term "treating" or "treatment" as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "treatment" as used herein also include prophylactic treatment. For example, a subject can be treated to prevent onset or progression, or alternatively a subject in post-stroke or post- infarct can be treated with a compound or composition as described herein to reduce injury or prevent recurrence. Treatment methods comprise administering to a subject a therapeutically effective amount of the compounds described. As used herein, the term "effective amount" or "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired result. Effective amounts may vary according to factors such as the disease state, age, sex and/or weight of the subject. The amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of condition, disease or disorder, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

In some embodiments, treatment with the compounds or compositions provided herein may be long-term treatments for chronic conditions, or may be single dose treatments for acute conditions.

It will be appreciated that the embodiments described herein are for illustrative purposes, and not intended to be limiting in any way. The following examples are provided for the purposes of illustration, and may demonstrate embodiments of at least some of the compounds and uses provided herein.

EXAMPLE 1: USE OF LIPOIC ACID-RE SVERATROL CONJUGATES FOR NEUROPROTECTION IN ISCHEMIA/ REPERFUSION INJURY

In this example, 2 novel compounds combining resveratrol (3, 5, 4'-trihydroxystilbene), a naturally-occurring component of grapes, and a-lipoic acid (LA), a potent anti-oxidant found in common foods, were tested for neuroprotective effects in 2 animal models of ischemic stroke. Separately, resveratrol and LA possess potent anti-oxidant and anti-inflammatory activities and have been shown to produce neuroprotection in several animal models of neurological diseases via complementary pathways (Yan et al., 2011; He et al., 2012).

The current example investigated the potential for enhanced neuroprotective effects with LA by combining it with resveratrol in a rodent model of acute stroke and reperfusion injury (I/R) (Connell and Saleh, 2010). In addition, the effects of resveratrol and lipoic acid were compared to UPEI-200 and UPEI-201, two novel synthetic compounds linking resveratrol with LA (1 :3, 1 : 1 ratios, respectively), in both a transient occlusion-reperfusion model (tMCAO) as well as in a permanent occlusion model (pMCAO). Lastly, the feasibility of delayed treatment intervention was investigated for all drug combinations.

Methods

All experiments were carried out in accordance with the guidelines of the Canadian Council on Animal Care and were approved by the University of Prince Edward Island Animal Care Committee.

General surgical procedures for in vivo stroke surgery

All experiments were conducted on male Sprague-Dawley rats (250-350 g; Charles Rivers; Montreal, PQ, CAN). For all animals, food and tap water were available ad libitum. Rats were anaesthetized with sodium thiobutabarbital (Inactin; Sigma-Aldridge; St.Louis, MO, USA; 100 mg/kg; ip) and supplemented as needed. For intravenous administration of drugs, a polyethylene catheter (PE-10; Clay Adams, Parsippany, NJ, USA) was inserted into the right femoral vein. An endotracheal tube was inserted to facilitate breathing. Body temperature was monitored and maintained at 37 ± 1°C using a feedback system (Physitemp Instruments; Clifton, NJ, USA).

Transient and permanent middle cerebral artery occlusion (tMCAO and pMCAO)

The detailed methodology for transient occlusion of the middle cerebral artery was previously published (Connell and Saleh, 2010). Briefly, animals were placed in a David Kopf stereotaxic frame (Tujunga, CA, USA) and the right middle cerebral artery (MCA) approached through a rostra-caudal incision of the skin and frontalis muscle at the approximate level of bregma. Blood flow through the MCA was impeded by the placement of surgical suture behind the MCA at 3 designated positions along the exposed vessel for 30 minutes in the transient model (tMCAO), or left in place for a total of 6 hours in the permanent (pMCAO) model. The sutures were positioned so that the middle of each suture applied pressure to the underside of the MCA and impeded blood flow (ischemia) as previously confirmed using laser Doppler flowmetry (OxyFlo, Oxford-Optronix, Oxford, UK) (Connell and Saleh 2010). This 3-point placement of surgical sutures produced a highly reproducible and consistent focal ischemic lesion restricted to the prefrontal cerebral cortex. Blood flow in the tMCAO model was re-established (reperfusion) for an additional 5.5 hours following removal of the sutures.

Drug Preparation

Resveratrol (trans-3,5,4'-trihydroxy stilbene; Sigma Aldrich, St. Louis, MO, USA) stock solutions were prepared in 40% propylene glycol and diluted 10,000X in 0.9% saline The concentration of propylene glycol in each solution was 4xl0^"3 % (v/v). Lipoic acid (LA; Sigma- Aldridge; St. Louis, MO, USA; 0.005 mg/ml) was prepared in physiological saline (0.9%> sodium chloride) and the pH was adjusted to 7.0 - 7.4 with sodium hydroxide. The concentration of LA used was previously determined to be non-neuroprotective in our tMCAO model (Connell et al., 201 la,b). Appropriate vehicle solutions were prepared for each drug and dose.

Synthesis ofUPEI-200

The chemical synthesis of UPEI-200 (Figure 9 (A)) was performed as follows; resveratrol (0.01 M) was taken in a well-dried, 100-ml round-bottom flask followed by 0.05 M of LA, and 0.04 M of dimethylaminopyridine (DMAP) in 80 ml of anhydrous dichloromethane (CH₂CI₂). The 1- ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride (EDCI; 0.05 M) was added in small quantities over a period of 2hr. The entire reaction was performed under nitrogen atmosphere at room temperature. After stirring overnight, the crude mixture of compounds were quickly purified by pass through column using silica column chromatography after an aqueous work up. A pale yellow viscous liquid was again purified on chromatotran silica plate using 2mm pre- coated UV actove plate. Appropriate fractions were mixed and concentrated in rotary evaporator keeping the water bath temperature at 45°C. The final pure compound was obtained as a pale yellow viscous solid in very low yield and it was characterized by proton nuclear magnetic resonance spectroscopy and mass spectrometry. Yield, >5%. 1H NMR (600 MHz, DMSO-d6) δ 7.65 (2H), 7.35 - 7.20 (4H ,m), 7.15 (2H), 6.85 (1H)), 3.65 (3H,), 3.30 - 3.10 (6H, m), 2.60 - 2.40 (6H, m), , 1.90 - 1.652 (3H, m), 1.60 - 1.20 (12H, m), 1.70 - 1.85 (6H, dtd, ). High resolution mass (HRMS), Calculated : M+23. 815.1673, observed M+23, 815.1666.

Synthesis of UPEI-201

The chemical synthesis of UPEI-201 (Figure 9 (B)) was performed as follows: to a stirred solution of 1 mmol resveratrol in 20ml dimethylformamide (DMF) is added 10% mmol DMAP and 1 mmol lipoic acid. 1 mmol dicyclohexylcarbodimide (DCC) is added to the reaction mixture at 0 °C under N₂, which is then stirred for 5 min at 0°C and 3 h at 20°C. Precipitated urea is then filtered off and the filtrate evaporated down in vacuo. The residue is taken up in dichloromethane (CH₂CI₂) and, if necessary, filtered free of any further precipitated urea. The solvent is removed by evaporation and crude compound is purified on a silica column chromatography (Eluent, Hexanes:Ethylacetate (1 : 1; yellow oil), Yield; 57%. 1H NMR (300 MHz, Acetone) δ 7.40 (2H, d, J = 8.6 Hz), 7.05 - 6.79 (4H ,m), 6.52 (2H, d, J = 2.1 Hz), 6.25 (1H, t, J = 2.1 Hz), 3.58 (1H, tt, J = 12.7, 6.4 Hz), 3.24 - 3.04 (2H, m), 2.45 (1H, tt, J = 12.3, 6.2 Hz), 2.28 (1H, t, J = 7.2 Hz), 1.95 - 1.80 (1H, m), 1.79 - 1.52 (4H, m), 1.45 (2H, dtd, J = 11.1, 7.1, 4.1 Hz).

Effect of resveratrol on tMCAO model

In the first experiment, resveratrol (2 x 10-3 (n=5), 2 x 10-4 (n=5), 2 x 10-5 (n=6), 2 x 10-6 (n=5), 2 x 10-7 (n=5) mg/kg; 1 ml/kg; i.v.) or vehicle (propylene glycol; 4 x 10 -3 %> (v/v); 1 ml/kg; i.v.; n=5) was administered 30 minutes prior to the onset of MCAO. The sutures were left in place for 30 minutes, followed by 5.5 hours of reperfusion.

The feasibility of extended treatment options was investigated by administering the highest dose of resveratrol (2 x 10^"3 mg/kg; i.v.) or vehicle (propylene glycol; 4 x 10 ^"3 %> (v/v); 1 ml/kg; i.v.) at the following intervals during the I/R protocol; 15 minutes (n=5/group) following the onset of MCAO, and 0, 30, 60, 90 minutes (n=5,6,7,4 respectively) following the onset of reperfusion.

Co-administration of resveratrol and lipoic acid (tMCAO)

To examine neuroprotection following co-administration of various doses of resveratrol (2x10-5 (n=5), 2x10-6 (n=5), 2x10-7 (n=6), 2x10-8 (n=6), or 2x10-9 (n=6) mg/kg) with LA (0.005 mg/kg) on ischemia-reperfusion injury in our tMCAO model, resveratrol and LA were combined into a single solution and administered (1.0 ml/kg; iv) 30 minutes prior to MCAO. The MCA was occluded for 30 minutes followed by 5.5 hours of reperfusion.

Delayed treatment effects were also studied by co-injecting resveratrol (2 x 10-5 mg/kg) and LA (0.005 mg/kg; i.v) at the following intervals during the I/R protocol; 15 and 30 minutes (n=8 and 6) following the onset of MCAO, and 30, 60, 90 minutes (n=6,7,4 respectively) following the onset of reperfusion.

Co-administration of resveratrol and lipoic acid and permanent occlusion (pMCAO)

To determine if the co-administration of resveratrol and LA was neuroprotective on ischemia- induced cell death only, co-injection of resveratrol and LA (2 x 10-5 mg/kg and LA, 0.005 mg/kg; i.v.; n=4) or vehicle (propylene glycol; 4 x 10 ^"3 % (v/v); 1 ml/kg; i.v.; n=4) were made 30 minutes prior to pMCAO. The experiments were terminated at the end of 6 hours of occlusion with no reperfusion period.

UPEI-200 or 201 effects in tMCAO or pMCAO

The effects of UPEI-200 and UPEI-201 on infarct volume in both transient and permanent MCAO models were investigated. Dose-response curves were generated for both entities (n=4- 7/group). UPEI-201 was further studied for its effectiveness in delayed intervention by administering a neuroprotective dose (1x10-6 mg/kg) 15 and 30 minutes post-occlusion as well as 30 and 60 minutes into the 5.5 hr reperfusion period (n=4-7/group).

Histological Procedures At the end of each experiment, in which infarct volume was measured, animals were transcardially perfused with phosphate buffered saline (PBS; 0.1 M; 200 mis). The brains were removed and sliced into 1 mm coronal sections with the aid of a rat brain matrix (Harvard Apparatus; Holliston, MA, USA). Sections were incubated in a 2% solution of 2,3,5-triphenol tetrazolium chloride (TTC; Sigma- Aldrich; St. Louis; MO, USA) for 5 minutes. Infarct volumes were calculated with measurements taken from scanned digital images of each brain section. The infarct area for opposing views of each brain section was calculated using a computer- assisted imaging system (Scion Corporation; Frederick, MD, USA), averaged and multiplied by section thickness (1mm) to give a measure of infarct volume for each section. The sum total of the individual infarct volumes provided the infarct volume for each rat.

Co-administration of resveratrol - lipoic acid and apoptosis

In a separate set of experiments, the co-administration of resveratrol (2 x 10^"5 mg/kg) and LA (0.005 mg/kg; i.v.; n=4) or vehicle (propylene glycol; 4 x 10 ^"3 % (v/v); 1 ml/kg; i.v.; n=4) were made 30 minutes prior to tMCAO. The sutures were left in place for 30 minutes followed by 5.5 hours of reperfusion. Animals were transcardially perfused with 200 mL of 0.1M phosphate buffered saline (pH 7.4), the brains removed and the ipsilateral cerebral cortex isolated by careful dissection. A biopsy needle having an internal diameter of 8 mm was used to collect tissue from the region of infarct.

The tissue was weighed and homogenized (20% w/v) in ice cold PBS. The homogenate was centrifuged 12 000 X g for 15min at 4°C. Aliquots of the supernatant were stored at -80°C until assayed for protein. Apoptotic cell death was quantified using an ELISA based assay for determination of cytoplasmic histone-associated DNA fragments (Roche Diagnostics, Montreal, QC, CAN).

Statistical analysis

Data were analyzed using a statistical software package (SigmaStat and SigmaPlot; Jandel Scientific, Tujunga, CA). All data are presented as a mean ± standard error of the mean (S.E.M). Differences were considered statistically significant if p < 0.05 by an analysis of variance (ANOVA) followed by a Bonferroni post-hoc analysis. When only two groups were being compared the Student's t-test was used.

Results

Resveratrol and tMCAO

Pre-administration of resveratrol provided dose-dependent neuroprotection in our model of ischemia-reperfusion. This was evident by a reduction in mean infarct volume with increasing doses of resveratrol. A significant difference in infarct volume between resveratrol treated animals and vehicle treated controls was observed at the 2 highest doses tested (2 x 10^"3 and 2 x 10^"4 mg/kg; p<0.05; Figs.1 A and B).

Resveratrol or vehicle was injected (i.v) during MCAO or following the start of reperfusion (Figure 2). There were no significant differences in the mean infarct volumes when vehicle was injected during MCAO or at any time point during reperfusion (p>0.05), therefore, the vehicle data for all time points was pooled (n=29). However, all statistical comparisons were made between the infarct volumes measured following resveratrol and vehicle administration for each time point. When resveratrol treatment (2 x 10-3 mg/kg; iv) was delayed until 15 minutes into the ischemic period or 90 minutes into the reperfusion period (120 min post occlusion) there was no effect on infarct volume when compared to vehicle injected controls (p>0.05; Fig. 2). However, significant neuroprotection was observed when resveratrol (2 x 10-3 mg/kg) was administered at the start of the reperfusion period (30 minutes post-occlusion), or at 30 and 60 minutes into the reperfusion period (60 and 90 minutes post-occlusion; p<0.05 at each time point; Fig. 2).

Co-administration of resveratrol and lipoic acid

The combined pre-administration of resveratrol and LA 30 minutes prior to tMCAO produced a dose-dependent reduction in infarct volume compared to vehicle injected controls when measured following 5.5 hrs of reperfusion (Fig. 3). This effect was significant at the 2 highest doses of resveratrol (2xl0^"6 and 2xl0^"5 mg/kg; p<0.05; Fig. 3). Tissue sampled from the infarct region of rats injected with resveratrol (2 x 10^"5 mg/kg) and LA (0.005 mg/kg) 30 minutes prior to tMCAO displayed lower levels of cytoplasmic histone-associated-DNA fragmentation correlative with decreased apoptotic cell death (p<0.05; Fig. 5). Delaying treatment of resveratrol (2 x 10-5 mg/kg) and LA (0.005 mg/kg) until 15 minutes following the onset of tMCAO was neuroprotective however no significant effect was observed when the same combination of resveratrol and LA was injected immediately prior to suture removal and the onset of reperfusion (30 minutes post occlusion; Fig. 4)

Co-injection of resveratrol (2 x 10-5 mg/kg) and LA (0.005 mg/kg) 30 minutes prior to 6 hours of permanent MCAO did not result in significant neuroprotection (p>0.05; data not shown). The average infarct volumes following 6 hrs of permanent MCAO in the vehicle and resveratrol - LA treated groups were 25.3 ± 6 and 19.9 ± 5 mm³ respectively.

UPEI-200 and UPEI-201in tMCAO andpMCAO

UPEI 200 is a chemical construct composed of 3 LA moieties bonded to a single resveratrol molecule (3: 1). When administered 30 minutes prior to MCA occlusion in either tMCAO or pMCAO models, there was no significant neuroprotection observed at any of the doses tested (p>0.05; Fig. 6A, 6B).

Conversely, UPEI 201, which is composed of a single LA moiety bound to resveratrol (1 : 1), displayed potent neuroprotection when administered 30 minutes prior to MCA in tMCAO (Fig. 7 A; p<0.05). Delayed intervention with UPEI 201 (1 x 10^"6 mg/mg) was successful in reducing infarct volume when administered 15 minutes into the occlusion period (15 min; p<0.05, Fig. 8), but not when administered at the start or reperfusion or 30 minutes into the 5.5 hr reperfusion period (30, 60 min; Fig. 8).

Discussion

Dietary plant phenolics such as resveratrol are being widely used in supplement form to prevent and treat common health concerns. Potential safety issues exist as high doses of resveratrol have been shown to cause renal toxicity (Crowell et al. 2004) and contribute to hepatic oxidative stress (Rocha et al., 2009). In the presence of peroxidase and/or transition metals, resveratrol may function as a pro-oxidant ultimately contributing to DNA damage and mitochondrial dysfunction (Galati et al., 2002; Ahmad et al., 2000). As well, resveratrol has been shown to inhibit cytochrome P450 enzyme CYP1A1 (Chun et al., 1999), an interference which may render other drugs in a patient's treatment plan ineffective at therapeutic doses. Clearly, the health benefits of resveratrol are extensive and hence, finding ways to harness the potency of resveratrol in the absence of adverse side effects is desirable.

To this end, the inventors show in this study that resveratrol on its own produced dose-dependent neuroprotection against neuronal cell death in a rodent model of transient ischemia-reperfusion injury (Connell and Saleh, 2012). Combined injection of resveratrol with a non-neuroprotective dose of a-lipoic acid (Connell et al., 2011) prior to tMCAO produced neuroprotection at doses of resveratrol 100 fold less than when injected alone. By chemically bonding resveratrol to lipoic acid in a 1 : 1 ratio (UPEI-201), the inventors were able to show a further dose reduction (ten- fold lower) coincident with significant neuroprotection. These findings support the advantage of combination therapy in stroke treatment, while simultaneously demonstrating the utility of bioassay-guided optimization to achieve the ideal ratio of newly synthesized bioactive molecules.

Numerous studies have proven the efficacy of treatment with lipoic acid in disease states reflective of pro- and antioxidant imbalance such as diabetes, Alzheimer's disease, cancer and cerebrovascular disease. The chemical characteristics of LA, as well as its reduced form dihydrolipoic acid, qualify it as an effective scavenger of hydroxyl radicals, nitric oxide, peroxyl radicals and peroxinitrites, singlet oxygen species and hypochlorous acid (Gulcin, 2010). In a previous study in the inventors' laboratory, it was showed that LA pretreatment was effective as a neuroprotectant in reperfusion injury following tMCAO, but not after permanent occlusion (pMCAO) (Connell et al., 2011). In the present study, combination of LA with resveratrol also did not protect against neuronal death in a model of pMCAO. Prolonged ischemia is characterized by glutamate-induced neuronal toxicity ultimately leading to necrosis (for review, see Lipton, 1999). Generation of oxidative radicals is minimal owing to the lack of blood flow and dampening of mitochondrial activity, thereby rendering anti-oxidant therapy ineffectual. In contrast, anti-oxidants are highly effective in combating the oxidative stress generated during reperfusion injury which is demonstrated in the current study in our model of tMCAO. The reduction in infarct volume associated with resveratrol-LA treatment correlates with fewer necrotic cells at the ischemic core as evidenced with TTC staining, as well as reduced apoptotic cell death in the area of the penumbra as demonstrated by reduced oxidative DNA damage.

Other potential implications of combining resveratrol treatment with LA include their complementary participation in cell preserving pathways. For example, resveratrol and LA have both been shown to enhance aldehyde dehydrogenase-2-mediated detoxification of aldehydes in models of ethanol toxicity and ischemia-reperfusion injury respectively (Yan et al., 2011; He et al., 2012). Both compounds influence antioxidant status, in part through direct reduction of reactive oxygen species, but also as modulators of endogenous anti-oxidant systems. Resveratrol was shown to induce MnSOD activity in isolated rat liver mitochondria while LA inhibited glutathione peroxidase activity and induced mitochondrial uncoupling in the same model (Valdecantos et al., 2010). It is also noteworthy, that the LA/dihyrolipoate system is highly efficient in the reduction of the oxidized forms of anti-oxidants essentially aiding in their recycling allowing them to work more effectively without saturation (Smith et al, 2004). Its dual solubility in water and lipid allows LA to interact with antioxidants in extracellular (blood) as well as intracellular (both cytoplasmic and mitochondrial) compartments and to effectively cross the blood-brain barrier (Bilska and Wlodek, 2005).

To utilize the strategy of combinatorial therapy, the inventors created 2 new chemical entities, UPEI-200 and UPEI-201 and determined that a 1 : 1 ratio of resveratrol-LA moieties (UPEI-201) was preferred in providing neuroprotection following ischemia-reperfusion (tMCAO) but not during permanent ischemia (pMCAO). UPEI-201 effectively provided neuroprotection when injected 15 minutes into the period of occlusion but not when injected during reperfusion. With dosing in the nanomolar range providing significant neuroprotection in the model of transient ischemia, UPEI-201 is clearly a potent neuroprotectant against oxidative damage. Recent figures estimate medication non-compliance at around 50% at a cost of $300 hundred billion a year. Novel compounds such as UPEI-201 which aim to provide multi-level care in a single dose, may improve compliance and could make a significant contribution to global health initiatives in treating and/or preventing cerebrovascular disease.

UPEI-200 was less effective at providing neuroprotection in either tMCAO or pMCAO paradigms, possibly due to size of the molecule. The arrangement of 3 LA acid moieties connected to a single resveratrol molecule may have reduced the ability of UPEI-200 to cross lipid membranes (C₃₈H₄gC>6S6; Mass = 792.177; Mol wt = 793.174) thereby reducing absorption, as well as hindering diffusion across the blood-brain barrier. Stearic hindrance related to the arrangement of LA groups with resveratrol may also have interfered with ligand-receptor interactions required to reduce free radicals or mediate drug effects which are as of yet undetermined (Saleh et al., 2013).

In conclusion, the results presented above support the notion that combining antioxidants at subthreshold doses can produce equal or enhanced neuroprotective effects. In addition, creation of novel chemical entities via the synthetic bonding of these antioxidants can produce comparable effects to those observed by the co-administration of the 2 compounds, but at lower doses. The clear advantage to lowering the dose required to gain therapeutic effect is to minimize off-target effects on other organ systems which may lead to side effects as is seen in so many of the prescription drugs on the market today.

In summary, this example demonstrates the benefits of combinatorial anti-oxidant therapy in the treatment of ischemic stroke. Male Sprague-Dawley rats were anaesthetised and the middle cerebral artery (MCA) was occluded for 30 minutes followed by 5.5 hours of reperfusion. Pre treatment with resveratrol 30 minutes prior to MCA occlusion resulted in a significant, dose- dependent decrease in infarct volume (p<0.05) compared to vehicle-treated animals. Neuroprotection was also observed when resveratrol (2 x 10^"3 mg/kg; iv) was administered within 60 minutes following the return of blood flow (reperfusion). Pre treatment with non- neuroprotective doses of resveratrol (2x10^~6 mg/kg) and lipoic acid (LA; 0.005 mg/kg) in combination produced significant neuroprotection as well. This neuroprotection was also observed when resveratrol and LA were administered 15 minutes following the onset of MCA occlusion. Subsequently, the inventors synthetically combined resveratrol and LA in both a 1 :3 (UPEI-200) or 1 : 1 (UPEI-201) ratio, and screened these new chemical entities in the stroke model. UPEI-200 was less effective, while UPEI-201 demonstrated significant, dose-dependent neuroprotection.

Overall, these results demonstrate that combining subthreshold doses of resveratrol and LA prior to ischemia-reperfusion can provide significant neuroprotection likely resulting from concurrent effects on multiple pathways. The additional protection observed in the novel compound UPEI 201 may present opportunities for addressing ischemia-induced damage in patients presenting with transient ischemic episodes.

All documents cited are hereby incorporated herein by reference.

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Claims

WHAT IS CLAIMED IS:

1. A compound of Formula I:

(I) wherein

X, Y, and Z are each independently OH, or represents a linkage to a moiety of Formula II, said linkage in each instance being selected independently from an ester linkage, a thioester linkage, an amide linkage, an alkyl linkage, an -NH- or -N(alkyl)- linkage, an ether linkage, a thioether linkage, or a disulfide linkage; and at least one of X, Y, and Z is linked to said moiety of

Formula II:

(Π), wherein n is 1, 2, 3, 4, 5 or 6; m is 0, 1 or 2;

R¹ and R² are independently selected from the group consisting of H, Ci_₆alkyl and C(0)Ci_₆- alkyl, or

R¹ and R² are absent and the two sulfur atoms are bonded together to form, together with the carbon atoms between them, a 4, 5 or 6 membered ring, or a pharmaceutically acceptable salt and/or solvate thereof.

2. The compound of claim 1, wherein at least one of X, Y, and Z represents a linkage to said moiety of Formula II, said linkage comprising an -0-, -0-C(=0)-, -S-, -Ci_6-alkyl-, or -NH- group.

3. The compound of claim 1, wherein said compound is represented by Formula (la):

(la) wherein

R³, R⁴, and R⁵ are each independently H or a moiety of Formula Ila or Formula lib, and at least one of R³, R⁴, and R⁵ is a moiety of Formula Ila or Formula lib:

(Ila) (lib) wherein n, m, R¹ and R² are as defined in claim 1 , or a pharmaceutically acceptable salt and/or solvate thereof.

4. The compound of any one of claims 1-3, wherein n is 3, 4 or 5.

The compound any one of claims 1-3, wherein n is 4.

6. The compound of any one of claims 1-5, wherein m is 1 or 2.

7. The compound of claim 6, wherein m is 2.

8. The compound of any one of claims 1-7, wherein R¹ and R² are independently selected from the group consisting of H,

and C(0)ci-₄alkyl.

9. The compound of any one of claims 1-7, wherein R¹ and R² are independently selected from the group consisting of H, CH₃ and C(0)CH₃.

10. The compound of claim 8 or 9, wherein R¹ and R² are the same.

11. The compound of claim 10, wherein R¹ and R² are both H.

12. The compound of any one of claims 1-7, wherein R¹ and R² are absent and the two sulfur atoms are bonded together to form, together with the carbon atoms between them, a 5 or 6 membered ring.

13. The compound of claim 12, wherein R¹ and R² are absent and the two sulfur atoms are bonded together to form, together with the carbon atoms between them, a 5 membered ring.

14. The compound according to claim 1, wherein the compound has the formula

or a pharmaceutically acceptable salt thereof.

15. The compound according to claim 1, wherein the compound has the formula

or a pharmaceutically acceptable salt thereof.

16. A compound, or a pharmaceutically acceptable salt thereof, that is a covalent conjugate between 1-3 lipoic acids (LA), or a derivative thereof, and a resveratrol, or a derivative thereof, wherein each conjugate linkage is independently an ester linkage, thioester linkage, amide linkage, alkyl linkage, -NH- or -N(alkyl)- linkage, ether linkage, thioether linkage, or disulfide linkage.

17. The compound according to claim 16, wherein said conjugate linkage comprises a -0-, - 0-C(=0)-, -S-, -Ci_-6-alkyl-, or -NH- group.

18. A pharmaceutical composition comprising at least one compound according to any one of claims 1-17, and a pharmaceutically acceptable carrier.

19 A method of preventing or treating a disease, disorder, or condition related to oxidative stress comprising administering a compound according to any one of claims 1-17, or a composition according to claim 18, to a subject in need thereof.

20. Use of a compound according to any one of claims 1-17, or a composition according to claim 18, for the prevention or treatment of diseases, disorders, or conditions related to oxidative stress.

21. Use of a compound according to any one of claims 1-17, or a composition according to claim 18, in the manufacture of a medicament for the prevention or treatment of diseases, disorders, or conditions related to oxidative stress.

22. The method according to claim 19, or the use according to claim 20 or 21, wherein the disease, disorder, or condition is stroke, ischemia, reperfusion injury, neurodegenerative disease, stroke, inflammatory disease, neurovascular disorder, dementia, Multiple Sclerosis, Parkinson's disease, myocardial infarction, heart failure, renal failure, collagen vascular disease, metabolic disorder, cardiac disease, or combinations thereof.

23. The method according to claim 19 or 22, or the use according to any one of claims 20, 21, or 22, wherein the disease, disorder, condition is a cerebral disease, disorder, or condition.

24. The method according to claim 19 or 22, or the use according to any one of claims 20, 21, or 22, wherein the disease, disorder, condition is a heart, kidney, liver, or skeletal muscle disease, disorder, or condition.

25. The method according to claim 19 or 22, or the use according to any one of claims 20, 21, or 22, wherein an anti-thrombolytic drug is provided for co-administration with said compound or composition.

26. The method of claim 25, wherein the anti-thrombolytic drug is tPA (Tissue Plasminogen Activator), streptokinases, or rtPA (Recombinant Tissue Plasminogen Activator).

27. The use of a compound according to any one of claims 1-17, or a composition according to claim 18, for reducing or preventing cell damage caused by reactive oxygen species (ROS).

28. The use according to claim 27, wherein the reducing or preventing cell damage occurs in vitro. The use according to claim 27, wherein the reducing or preventing cell damage occurs in

A process for preparing a conjugate of lipioc acid and resveratrol of the formula:

the process comprising reacting lipoic acid of the formula:

with resveratrol of the formula:

in the presence of a coupling agent under conditions effective to form an ester linkage between the lipoic acid and the resveratrol moieties and form said conjugate of lipioc acid and resveratrol; and optionally purifying said compound. The process according to claim 30, wherein the coupling agent is a carbodiimide. The process according to claim 30 or 31, wherein the compound is:

and the coupling agent is l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI).

33. The process according to claim 30 or 31 , wherein the compound is:

coupling agent is Ν,Ν'-dicyclohexylcarbodimide (DCC).