Valproic Acid Derivative Compounds BACKGROUND OF THE INVENTION

Epilepsy is a neurological disease that is characterized by paroxysmal transient disturbances of the electrical activity of the brain. Epileptic seizures may be partial or focal seizures that are restricted to a particular locus within the brain, or generalized seizures which can result in abnormal activity throughout the brain [1]. Epilepsy is the most frequent neurodegenerative disease after stroke. It afflicts more than 2 million Americans and 50 million people worldwide [2]. Temporal lobe epilepsy (TLE), where seizures originate from the temporal lobe of the brain [3], is among the most frequent types of drug-resistant epilepsy. Individuals affected with TLE typically have an initial precipitating injury (IPI) such as the status epilepticus (SE), head trauma, encephalitis or childhood febrile seizures [4-6]. There is usually a latent period of several years between this injury and the emergence of the chronic TLE characterized by spontaneous recurrent motor seizures (SRMS) originating from temporal lobe foci, and learning and memory impairments [7, 8]. Human studies suggested that the hippocampal sclerosis was likely cause to initiate or contribute to the generation of most TLEs [9]. The standard treatment with appropriate antiepileptic drugs (AEDs) after an IPI or the SE appeared to be useful as an anti-seizure strategy. Valproic acid (VPA) and its sodium salt (sodium valproate) are among the most prescribed antiepileptic drugs. Among the conventional drugs, early administration of VPA after the SE appeared promising for preventing chronic epilepsy. But, based on currently available studies, it appears that the efficacy of prolonged administration of VPA after the onset of the SE for preventing chronic epilepsy development varies depending on the model employed [10]. These studies evaluated treatment with valproic acid for a relatively short duration, 28 days to 1 month, and it is possible that longer exposure to the drug may result in a different outcome. Also valproic acid was administered in these studies by intra-peritoneal (IP) injection in rats two to three times a day to maintain blood levels of drug above the therapeutic level, which gave rise to considerable fluctuation in blood levels [11, 12]. Valproic acid was also administered at a dose which gave rise to unexpected serious adverse effects in animals, e.g. sedation and ataxia, shortly after SE, which made it less suitable for prolonged treatment [12]. This invention provides a pro-drug of valproic acid, valproic acid conjugated to dextran, which will release the active drug slowly in vivo by hydrolysis and hence, prolong its effect. Dextrans are glucose polymers which have been used for more than 50 years as plasma volume expanders. Dextrans contain a large number of hydroxyl groups which can be easily conjugated to valproic acid by either direct attachment or, through a linker [13-15]. Hence an intra- cerebroventricular (ICV) injection of pro-drug of VPA will require lower amount of conjugate to achieve therapeutic level in cerebrospinal fluid (CSF), minimizing fluctuation in therapeutic level, will have minimum blood level of active drug and will have less incidence of adverse effects. Also, this prolonged release formulation will avoid multiple daily administrations and was very suitable for preventing

development of chronic epilepsy after an initial precipitating injury (IPI) such as status epilepticus (SE). This invention provides for delivering the dextran valeric acid conjugate to the relevant area of the brain, and provides a slow release of the active principle through chemical hydrolysis.

SUMMARY OF THE INVENTION

One aspect is a compound of formula (I) or salt thereof, solvate or hydrate thereof:

wherein L is a bond; -O-alkyl-; -O-alkyl-C(O)-; -O-alkyl-O-C(O)-; -O-alkoxy-, -O- alkoxy-C(O)-; or -O-alkoxy-O-C(O)-; and DEX is a dextran; wherein in each alkyl group one or more -CH ₂ - groups can optionally be replaced with an -O- group.

One aspect is a compound of formula (I) or salt thereof, solvate or hydrate thereof:

wherein L is a bond, -O-alkyl-, -O-alkyl-C(O)-; or -O-alkyl-O-C(O)-; and DEX is a dextran.

Other embodiments are those compounds wherein:

L is a bond;

the dextran is a l,000Da to 150,000Da dextran;

the dextran is a l,000Da to 100,000Da dextran;

the dextran is a 10,000Da to 150,000Da dextran;

the dextran is a 10,000Da to 100,000Da dextran;

wherein the dextran is a 70,000Da dextran;

wherein the dextran is a 10,000Da dextran or

wherein the dextran is a l,000Da dextran.

Another aspect is a composition comprising a compound of formula (I) or salt thereof, solvate or hydrate thereof:

wherein L is a bond; -O-alkyl-; -O-alkyl-C(O)-; -O-alkyl-O-C(O)-; -O-alkoxy-, -O- alkoxy-C(O)-; or -O-alkoxy-O-C(O)-; and DEX is a dextran; wherein in each alkyl group one or more -CH ₂ - groups can optionally be replaced with an -O- group.

Another aspect is a composition comprising a compound of formula (I) or salt thereof, solvate or hydrate thereof:

wherein L is a bond, -O-alkyl-, -O-alkyl-C(O)-; or -O-alkyl-O-C(O)-; and DEX is a dextran. Other embodiments are those compounds wherein:

L is a bond;

the dextran is a l,000Da to 150,000Da dextran;

the dextran is a l.OOODa to 100,000Da dextran;

the dextran is a 10,000Da to 150,000Da dextran;

the dextran is a 10,000Da to 100,000Da dextran;

wherein the dextran is a 70,000Da dextran;

wherein the dextran is a 10,000Da dextran;

wherein the dextran is a l,000Da dextran;

wherein degree of substitution (mg of valproic acid /100 mg of valproic acid:dextran conjugate) is between 0.10 and 0.90 mole fraction;

wherein degree of substitution (mg of valproic acid /100 mg of valproic acid:dextran conjugate) is between 0.10 and 0.50 mole fraction;

wherein degree of substitution is 0.42 mole fraction.

wherein degree of substitution is 0.23 mole fraction.

In one aspect, the invention provides a method of treating a subject suffering from or susceptible to brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof. The method includes administering to a subject in need thereof a therapeutically effective amount of a valproic acid:dextran compound (e.g., a compound herein).

In another embodiment, the invention provides a method of treating a subject having brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof. The method includes administering to a subject identified as in need thereof a therapeutically effective amount of a valproic acid dextran compound or composition thereof.

In another aspect, the invention provides a method of treating a subject suffering from or susceptible to brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof, comprising administering to the subject an effective amount of a valproic acid:dextran compound (e.g., a compound herein) or

composition thereof, such that the subject is treated.

In another aspect, the invention provides a method of treating a subject suffering from or susceptible to a disorder comprising administering to subject in need thereof a therapeutically effective amount of a compound herein or composition thereof.

In another aspect, the invention provides a packaged composition including a therapeutically effective amount of a valproic acid:dextran compound and a pharmaceutically acceptable carrier or diluent. The composition may be formulated for treating a subject suffering from or susceptible to brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof, and packaged with instructions to treat a subject suffering from or susceptible to brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof.

In one aspect, the invention provides a kit for treating brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof in a subject is provided and includes a compound herein, a pharmaceutically acceptable esters, salts, and prodrugs thereof, and instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described below with reference to the following non-limiting examples and with reference to the following figures, in which:

FIG 1. depicts a first order plot of hydrolysis of VPA-D 10 conjugate in phosphate buffer.

FIG 2. depicts a zero order plot of hydrolysis of VPA-D 10 conjugate in phosphate buffer.

FIG 3. depicts a first order plot of hydrolysis of VPA-D 10 conjugate in NaHC0 ₃ /Na ₂ C0 ₃ buffer.

FIG 4. depicts a zero order plot of hydrolysis of VPA-D 10 conjugate in NaHC0 ₃ /Na ₂ C0 ₃ buffer.

FIG 5. depicts a first order plot of hydrolysis of VPA-D70 conjugate in phosphate buffer. FIG 6. depicts a zero order plot of hydrolysis of VPA-D70 conjugate in phosphate buffer.

FIG 7. depicts a first order plot of hydrolysis of VPA-D70 conjugate in NaHC0 ₃ /Na ₂ C0 ₃ buffer.

FIG 8. depicts a zero order plot of hydrolysis of VPA-D70 conjugate in

NaHC0 ₃ /Na ₂ C0 ₃ buffer.

FIG 9. depicts hydrolysis of conjugate VPA-TEG-D1 in different media.

FIG 10. depicts hydrolysis of conjugate VPA-PEG-D1 in different media.

DETAILED DESCRIPTION OF THE INVENTION

1. DEFINITIONS

Before further description of the present invention, and in order that the invention may be more readily understood, certain terms are first defined and collected here for convenience.

The term "administration" or "administering" includes routes of introducing the compound of the invention(s) to a subject to perform their intended function. Examples of routes of administration that may be used include injection

(subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal), oral, inhalation, rectal and transdermal. The pharmaceutical preparations may be given by forms suitable for each administration route. For example, these preparations are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred. The injection can be bolus or can be continuous infusion. Depending on the route of administration, the compound of the invention can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally effect its ability to perform its intended function. The compound of the invention can be administered alone, or in conjunction with either another agent as described above or with a pharmaceutically- acceptable carrier, or both. The compound of the invention can be administered prior to the administration of the other agent, simultaneously with the agent, or after the administration of the agent. Furthermore, the compound of the invention can also be administered in a pro-drug form which is converted into its active metabolite, or more active metabolite in vivo.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term heteroalkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C ₃ -C ₃₀ for branched chain), preferably 26 or fewer, and more preferably 20 or fewer, and still more preferably 4 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 3, 4, 5, 6 or 7 carbons in the ring structure.

Moreover, the term alkyl as used throughout the specification and sentences is intended to include both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or hetero aromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. Cycloalkyls can be further substituted, e.g., with the substituents described above. An "alkylaryl" moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). The term "alkyl" also includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively. Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six, and still more preferably from one to four carbon atoms in its backbone structure, which may be straight or branched-chain. Examples of lower alkyl groups include methyl, ethyl, n-propyl, i-propyl, tert-butyl, hexyl, heptyl, octyl and so forth. In preferred embodiment, the term "lower alkyl" includes a straight chain alkyl having 4 or fewer carbon atoms in its backbone, e.g., C1-C4 alkyl.

The term "alkoxy" refers to an -O-alkyl radical, wherein the alkyl group may optionally have one or more additional -O- atoms in place of any -CH ₂ - group in the alkyl chain.

The terms "alkoxyalkyl," "polyaminoalkyl" and "thioalkoxyalkyl" refer to alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or sulfur atoms.

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively. For example, the invention contemplates cyano and propargyl groups.

The term "aryl" as used herein, refers to the radical of aryl groups, including 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like. Those aryl groups having

heteroatoms in the ring structure may also be referred to as "aryl heterocycles,"

"heteroaryls" or "heteroaromatics." The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).

The term "associating with" refers to a condition of proximity between a chemical entity or compound, or portions thereof, and a binding pocket or binding site on a protein. The association may be non-covalent (wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions) or it may be covalent.

The term "binding pocket", as used herein, refers to a region of a molecule or molecular complex, that, as a result of its shape, favorably associates with another chemical entity or compound.

The language "biological activities" of a compound of the invention includes all activities elicited by compound of the inventions in a responsive cell. It includes genomic and non-genomic activities elicited by these compounds.

"Biological composition" or "biological sample" refers to a composition containing or derived from cells or biopolymers. Cell-containing compositions include, for example, mammalian blood, red cell concentrates, platelet concentrates, leukocyte concentrates, blood cell proteins, blood plasma, platelet-rich plasma, a plasma concentrate, a precipitate from any fractionation of the plasma, a supernatant from any fractionation of the plasma, blood plasma protein fractions, purified or partially purified blood proteins or other components, serum, semen, mammalian colostrum, milk, saliva, placental extracts, a cryoprecipitate, a cryo supernatant, a cell lysate, mammalian cell culture or culture medium, products of fermentation, ascites fluid, proteins induced in blood cells, and products produced in cell culture by normal or transformed cells (e.g., via recombinant DNA or monoclonal antibody technology). Biological compositions can be cell-free. In a preferred embodiment, a suitable biological composition or biological sample is a red blood cell suspension. In some embodiments, the blood cell suspension includes mammalian blood cells. Preferably, the blood cells are obtained from a human, a non-human primate, a dog, a cat, a horse, a cow, a goat, a sheep or a pig. The term "chiral" refers to molecules which have the property of non- superimposability of the mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner.

The term "diastereomers" refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not mirror images of one another.

The term "effective amount" includes an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., sufficient to treat a treating brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof. An effective amount of compound of the invention may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the compound of the invention to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (e.g., side effects) of the compound of the invention are outweighed by the therapeutically beneficial effects.

A therapeutically effective amount of compound of the invention (i.e., an effective dosage) may range from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a compound of the invention can include a single treatment or, preferably, can include a series of treatments. In one example, a subject is treated with a compound of the invention in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of a compound of the invention used for treatment may increase or decrease over the course of a particular treatment. The term "enantiomers" refers to two stereoisomers of a compound which are non-superimposable mirror images of one another. An equimolar mixture of two enantiomers is called a "racemic mixture" or a "racemate."

The term "haloalkyl" is intended to include alkyl groups as defined above that are mono-, di- or polysubstituted by halogen, e.g., fluoromethyl and trifluoromethyl.

The term "halogen" designates -F, -CI, -Br or -I.

The term "hydroxyl" means -OH.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.

The term "homeostasis" is art-recognized to mean maintenance of static, or constant, conditions in an internal environment.

The language "improved biological properties" refers to any activity inherent in a compound of the invention that enhances its effectiveness in vivo. In a preferred embodiment, this term refers to any qualitative or quantitative improved therapeutic property of a compound of the invention, such as reduced toxicity.

The term "optionally substituted" is intended to encompass groups that are unsubstituted or are substituted by other than hydrogen at one or more available positions, typically 1, 2, 3, 4 or 5 positions, by one or more suitable groups (which may be the same or different). Such optional substituents include, for example, hydroxy, halogen, cyano, nitro, CrCgalkyl, C ₂ -C8 alkenyl, C ₂ -Cgalkynyl, C - Cgalkoxy, C ₂ -Cgalkyl ether, C3-Cgalkanone, CrCgalkylthio, amino, mono- or di-(Cl- Cgalkyl)amino, haloCrCgalkyl, haloCrCgalkoxy, CrCgalkanoyl, C ₂ -Cgalkanoyloxy, Ci-Cgalkoxycarbonyl, -COOH, -CONH ₂ , mono- or di-(Ci -Cgalkyl)aminocarbonyl, - S0 ₂ NH ₂ , and/or mono or as well as carbocyclic and heterocyclic groups. Optional substitution is also indicated by the phrase "substituted with from 0 to X substituents," where X is the maximum number of possible substituents. Certain optionally substituted groups are substituted with from 0 to 2, 3 or 4 independently selected substituents (i.e., are unsubstituted or substituted with up to the recited maximum number of substituents). The term "isomers" or "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical

administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

The terms "polycyclyl" or "polycyclic radical" refer to the radical of two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl,

alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term "prodrug" or "pro-drug" includes compounds with moieties that can be metabolized in vivo. Generally, the prodrugs are metabolized in vivo by esterases or by other mechanisms to active drugs. Examples of prodrugs and their uses are well known in the art (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19). The prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Hydroxyl groups can be converted into esters via treatment with a carboxylic acid. Examples of prodrug moieties include substituted and unsubstituted, branch or unbranched lower alkyl ester moieties, (e.g., propionoic acid esters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g. , acetyloxymethyl ester), acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g. , with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkyl amides, and hydroxy amides. Preferred prodrug moieties are propionoic acid esters and acyl esters. Prodrugs which are converted to active forms through other mechanisms in vivo are also included.

The language "a prophylactically effective amount" of a compound refers to an amount of a compound of the invention any formula herein or otherwise described herein which is effective, upon single or multiple dose administration to the patient, in preventing or treating disease, e.g., treating brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof.

The language "reduced toxicity" is intended to include a reduction in any undesired side effect elicited by a compound of the invention when administered in vivo.

The term "sulfhydryl" or "thiol" means -SH.

The term "subject" includes organisms which are capable of suffering from a cell a disease or disorder or who could otherwise benefit from the administration of a compound of the invention of the invention, such as human and non-human animals. Preferred humans include human patients suffering from or prone to suffering from brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof or associated state, as described herein. The term "non-human animals" of the invention includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non-mammals, such as non-human primates, sheep, dogs, cats, cows, chickens, amphibians, reptiles, etc.

The term "susceptible to epilepsy, seizures or ocular diseases, disorders or symptoms thereof is meant to include subjects at risk of developing epilepsy, seizures or ocular diseases, disorders or symptoms thereof, or subjects having a family or medical history of treating brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof, and the like.

The phrases "systemic administration," "administered systemically",

"peripheral administration" and "administered peripherally" as used herein mean the administration of a compound of the invention(s), drug or other material, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

The language "therapeutically effective amount" of a compound of the invention of the invention refers to an amount of an agent which is effective, upon single or multiple dose administration to the patient, in treating brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof.

With respect to the nomenclature of a chiral center, terms "d" and "1" configuration are as defined by the IUPAC Recommendations. As to the use of the terms, diastereomer, racemate, epimer and enantiomer will be used in their normal context to describe the stereochemistry of preparations.

2. COMPOUNDS OF THE INVENTION

In one aspect, the invention provides compounds that are dextran derivatives of valproic acid (also known as 2-propylpentanoic acid), including those delineated in any of the formulae herein. The compounds are also depicted as those of formula I:

wherein L is a bond, -O-alkyl-, -, -O-alkyl-C(O)-; or -O-alkyl-O-C(O)-; and DEX is a dextran of molecular weight between about lkD and 150 kD, in an alternate embodiment between about lOkD and 150kD.

In another depiction the valproic acid:dextran conjugate is of the formula below where the conjugate is a dextran of molecular weight between lkD and 150 kD, in an alternate embodiment between about lOkD and 150kD that is attached via the 4-position oxygen (glucose numbering) Valproic acid-Dextran conjugate wherein the darker squiggly bonds indicate attachment to the dextran chain of the appropriate molecular weight range (e.g., between lkD and 150kD; lOkD and

150kD).

Certain preferred compounds include compounds specifically delineated herein:

Valproic acid dextran conjugate (lOkD MW) [VPA-D-10];

Valproic acid dextran conjugate (70kD MW) [VPA-D-70];

Valproic acid -triethylene glycol-dextran conjugate (lkD MW) [VPA-TEG-D-1];

Valproic acid -pentaethylene glycol-dextran conjugate (lkD MW) [VPA-PEG-D- i];

The invention also relates to the pharmaceutically acceptable salts and esters of the above-mentioned compounds.

Naturally occurring or synthetic isomers can be separated in several ways known in the art. Methods for separating a racemic mixture of two enantiomers include chromatography using a chiral stationary phase (see, e.g., "Chiral Liquid Chromatography," W.J. Lough, Ed. Chapman and Hall, New York (1989)).

Enantiomers can also be separated by classical resolution techniques. For example, formation of diastereomeric salts and fractional crystallization can be used to separate enantiomers. For the separation of enantiomers of carboxylic acids, the

diastereomeric salts can be formed by addition of enantiomerically pure chiral bases such as brucine, quinine, ephedrine, strychnine, and the like. Alternatively, diastereomeric esters can be formed with enantiomerically pure chiral alcohols such as menthol, followed by separation of the diastereomeric esters and hydrolysis to yield the free, enantiomerically enriched carboxylic acid. For separation of the optical isomers of amino compounds, addition of chiral carboxylic or sulfonic acids, such as camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid can result in formation of the diastereomeric salts.

Another object of the present invention is the use of a compound as described herein (e.g., of any formulae herein) in the manufacture of a medicament for use in the treatment of a disorder or disease (e.g., brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof). Another object of the present invention is the use of a compound as described herein (e.g., of any formulae herein) for use in the treatment of a disorder or disease (e.g., brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof).

Another aspect is a compound made by any of the processes described herein

(e.g., in the examples herein).

3. USES OF THE COMPOUNDS OF THE INVENTION

The compounds delineated herein are useful in methods for modulating disease and disorders and symptoms thereof, particularly those where valproic acid is indicated. Such conditions include for example, brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof.

In one embodiment, the invention provides a method of treating brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof in a subject comprising administering to the subject identified as in need thereof a compound that is any compound delineated herein, or composition thereof.

In certain embodiments, the methods of the invention include administering to a subject a therapeutically effective amount of a compound of the invention in combination with another pharmaceutically active compound. Examples of pharmaceutically active compounds include compounds known to treat brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof, e.g., brain trauma agent, antiepileptic agent, antiseizure agent, ocular disease agent. Other pharmaceutically active compounds that may be used can be found in Harrison's Principles of Internal Medicine, Thirteenth Edition, Eds. T.R. Harrison et al.

McGraw-Hill N.Y., NY; and the Physicians Desk Reference 50th Edition 1997, Oradell New Jersey, Medical Economics Co., the complete contents of which are expressly incorporated herein by reference. The compound of the invention and the pharmaceutically active compound may be administered to the subject in the same pharmaceutical composition or in different pharmaceutical compositions (at the same time or at different times).

Determination of a therapeutically effective amount or a prophylactically effective amount of the compound of the invention of the invention, can be readily made by the physician or veterinarian (the "attending clinician"), as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. The dosages may be varied depending upon the

requirements of the patient in the judgment of the attending clinician; the severity of the condition being treated and the particular compound being employed. In determining the therapeutically effective amount or dose, and the prophylactically effective amount or dose, a number of factors are considered by the attending clinician, including, but not limited to: the specific brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof involved; pharmacodynamic characteristics of the particular agent and its mode and route of administration; the desired time course of treatment; the species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment (i.e., the interaction of the compound of the invention with other co-administered therapeutics); and other relevant circumstances.

Treatment can be initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage may be increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. A therapeutically effective amount and a prophylactically effective amount of a compound of the invention of the invention is expected to vary from about 0.1 milligram per kilogram of body weight per day (mg/kg/day) to about 100 mg/kg/day.

Compounds determined to be effective for the prevention or treatment of brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof in animals, e.g., mammals, primates, dogs, chickens, and rodents, may also be useful in treatment of in humans. Those skilled in the art of treating brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof in humans will know, based upon the data obtained in animal studies, the dosage and route of administration of the compound to humans. In general, the dosage and route of administration in humans is expected to be similar to that in animals.

The identification of those patients who are in need of prophylactic treatment for brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof is well within the ability and knowledge of one skilled in the art. Certain of the methods for identification of patients which are at risk of developing brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof which can be treated by the subject method are appreciated in the medical arts, such as family history, and the presence of risk factors associated with the development of that disease state in the subject patient, or actual trauma or observation of symptoms. A clinician skilled in the art can readily identify such candidate patients, by the use of, for example, clinical tests, physical examination and medical/family history.

A method of assessing the efficacy of a treatment in a subject includes determining the pre-treatment extent of brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof by methods well known in the art and then administering a therapeutically effective amount valproic acid dextran compound (e.g., those described herein) according to the invention to the subject. After an appropriate period of time after the administration of the compound (e.g., 1 day, 1 week, 2 weeks, one month, six months), the extent of the brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof is determined again. The modulation (e.g., decrease) of the extent or invasiveness of the brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof indicates efficacy of the treatment. The extent or invasiveness of the brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof may be determined periodically throughout treatment. For example, the extent or invasiveness of the brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof may be checked every few hours, days or weeks to assess the further efficacy of the treatment. A decrease in extent or invasiveness of the brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof indicates that the treatment is efficacious. The method described may be used to screen or select patients that may benefit from treatment with a compound herein.

As used herein, "obtaining a biological sample from a subject," includes obtaining a sample for use in the methods described herein. A biological sample is described above.

In another aspect, a compound of the invention is packaged in a

therapeutically effective amount with a pharmaceutically acceptable carrier or diluent. The composition may be formulated for treating a subject suffering from or susceptible to brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof, and packaged with instructions to treat a subject suffering from or susceptible to brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof.

In another aspect, methods of inhibiting a brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof in a subject include administering an effective amount of a compound of the invention (i.e., a compound described herein) to the subject. The administration may be by any route of administering known in the pharmaceutical arts. The subject may have brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof, may be at risk of developing brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof, or may need prophylactic treatment prior to anticipated or unanticipated exposure to one or more conditions capable of increasing susceptibility to a brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof.

In one aspect, a method of monitoring the progress of a subject being treated with a compound herein includes determining the pre-treatment status of the brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof, administering a therapeutically effective amount of a compound herein to the subject, and determining the status of the brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof after an initial period of treatment with the compound, wherein the modulation of the status indicates efficacy of the treatment.

The subject may be at risk of brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof , may be exhibiting symptoms of brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof, may be susceptible to brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof and/or may have been diagnosed with brain trauma, epilepsy, seizures or ocular diseases, disorders or symptoms thereof.

If the modulation of the status indicates that the subject may have a favorable clinical response to the treatment, the subject may be treated with the compound. For example, the subject can be administered therapeutically effective dose or doses of the compound.

Kits of the invention include kits for treating brain trauma, epilepsy, seizures and ocular diseases, disorders or symptoms thereof in a subject. The kit may include a compound of the invention, for example, a compound described herein,

pharmaceutically acceptable esters, salts, and prodrugs thereof, and instructions for use. The instructions for use may include information on dosage, method of delivery, storage of the kit, etc. The kits may also include, reagents, for example, test compounds, buffers, media (e.g., cell growth media), cells, etc. Test compounds may include known compounds or newly discovered compounds, for example, combinatorial libraries of compounds. One or more of the kits of the invention may be packaged together, for example, a kit for assessing the efficacy of a treatment for brain trauma, epilepsy, seizures and ocular diseases, disorders or symptoms thereof, or may be packaged with a kit for monitoring the progress of a subject being treated for brain trauma, epilepsy, seizures and ocular diseases, disorders or symptoms thereof according to the invention.

4. PHARMACEUTICAL COMPOSITIONS

The invention also provides a pharmaceutical composition, comprising an effective amount of a compound of the and a pharmaceutically acceptable carrier. In a further embodiment, the effective amount is effective to treat a disease, disorder, or symptom thereof as described previously.

In an embodiment, the compound of the invention is administered to the subject using a pharmaceutically- acceptable formulation, e.g., a pharmaceutically- acceptable formulation that provides sustained delivery of the compound of the invention to a subject for at least 12 hours, 24 hours, 36 hours, 48 hours, one week, two weeks, three weeks, or four weeks after the pharmaceutically-acceptable formulation is administered to the subject.

In certain embodiments, these pharmaceutical compositions are suitable for topical or oral administration to a subject. In other embodiments, as described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.

The phrase "pharmaceutically acceptable" refers to those compound of the inventions of the present invention, compositions containing such compounds, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically-acceptable carrier" includes pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically- acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Compositions containing a compound of the invention(s) include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, more preferably from about 10 per cent to about 30 per cent.

Methods of preparing these compositions include the step of bringing into association a compound of the invention(s) with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Compositions of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the invention(s) as an active ingredient. A compound may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically- acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight

polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or

preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compound of the invention(s) include pharmaceutically- acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compound of the invention(s) may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Pharmaceutical compositions of the invention for rectal or vaginal

administration may be presented as a suppository, which may be prepared by mixing one or more compound of the invention(s) with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent. Compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a compound of the invention(s) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound of the invention(s) may be mixed under sterile conditions with a pharmaceutically- acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to compound of the invention(s) of the present invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of the

invention(s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

The compound of the invention(s) can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound. Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically-acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (T weens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the invention(s) to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the active ingredient across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active ingredient in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of the invention.

Pharmaceutical compositions of the invention suitable for parenteral administration comprise one or more compound of the invention(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers, which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of compound of the invention(s) in biodegradable polymers such as polylactide- polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

When the compound of the invention(s) are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically- acceptable carrier.

Regardless of the route of administration selected, the compound of the invention(s), which may be used in a suitable hydrated form, and/or the

pharmaceutical compositions of the present invention, are formulated into

pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of the invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. An exemplary dose range is from 0.1 to 10 mg per day.

A preferred dose of the compound of the invention for the present invention is the maximum that a patient can tolerate and not develop serious side effects.

Preferably, the compound of the invention of the present invention is administered at a concentration of about 0.001 mg to about 100 mg per kilogram of body weight, about 0.001 - about 10 mg/kg or about 0.001 mg - about 100 mg/kg of body weight. Ranges intermediate to the above-recited values are also intended to be part of the invention.

EXAMPLES

The invention is further illustrated by the following examples which are intended to illustrate but not limit the scope of the invention. Definitions of variables in the structures in schemes herein are commensurate with those of corresponding positions in the formulae delineated herein.

EXAMPLE 1

Synthesis of valproic acid-dextran conjugate

To synthesize the conjugate, 964 μΐ of valproic acid (VPA) (6 mmol) was added to 10 ml of dried DMSO. After that, 1.07 g of carbonyl diimidazole (CDI) (6.6 mmol) was added to this solution slowly in portions while stirring on a magnetic stirrer. This mixture was stirred continuously for 24 hours on top of Argon gas. Then, 3.0 g of Dextran (MW 10 kD, or 70 kD) was added to 30 ml of anhydrous DMSO in a separate conical flask and the mixture was heated at 70°C for 30 minutes, while stirring on a magnetic stirrer, to help dissolve dextran in DMSO. After the addition of 6 ml of triethylamine (TEA), the solution was stirred for 10 minutes. Approximately

20 g of dried molecular sieves (4 A) was added to this solution and the solution was allowed to stand for 24 hours. After 24 hours, the two solutions was added into a spinner flask under the positive pressure of Argon gas, and about 8 g of dried molecular sieves was added to this solution. The reaction mixture was left for 24 hours at 70°C under continuous stirring. Finally, conjugates was precipitated by the addition of different organic solvents. After precipitation, the supernatant was discarded and the conjugates was dissolved in DMSO and again precipitated with the addition of organic solvent. In the next step the supernatant was discarded again and the conjugates was dispersed in methanol. Finally, the conjugates was dried in a rota- vap by removing the excess solvent [16-19].

Scheme 1:

EXAMPLE 2

Determination of the degree of substitution.

To determine the drug content in mg per 100 mg of the dextran conjugate, 10-15 mg of the VPA-dextran conjugate was dissolved in 10 ml of NaOH solution (0.5 N). The solution was kept in the refrigerator for complete hydrolysis and samples was collected at different time points for the next 48 hours. For the sample collection, 500 μΐ of the solution was collected into 1 ml HPLC vials and 500 μΐ of HCl solution (0.5 N) was added to the vials to neutralize the base [17]. The experiment was done in triplicate. The released VPA was analyzed by HPLC [20] .

EXAMPLE 3

Characterization of valproic acid-dextran conjugate by IR spectroscopy.

Infrared (IR) Characterization

FT-IR Spectrum of VPA

FT-IR Spectrum of VPA-D10

FT-IR Spectrum of VPA-D70

The FTIR spectra from the VPA-dextran conjugates was recorded on a Thermo Nicolet NEXUS 670 FTIR. All samples was measured, using KBr pellets made with the different conjugates. The FT-IR spectrum of valproic acid showed a strong absorption band at 1706 cm ^"1 attributable to the C=0 group stretching. Valproic acid- dextran conjugate showed the shift of peak of C=0 stretching from 1706 cm ^"1 to 1733 cm ^"1 indicating the formation of ester bond between valproic acid and dextran. EXAMPLE 4

Characterization of valproic acid-dextran conjugate by NMR spectroscopy.

NMR Characterization

^-NMR spectra was obtained from a Bruker, Avance II 600 Spectrometer with 5 mm TXI CryoProbe sample head, operating at 600 MHz. The sample tube size is 5 mm with a sample concentrations of 50 mg/0.5 ml for valproic acid, 10 mg/0.5 ml for the dextran 10 000, 7 mg/0.5 ml for the dextran 70 000 as control substances, 16 mg/0.5 ml for conjugate D-70-II and 20 mg/0.5 ml for conjugate D-10-II. DMSO-d ₆ was used as a solvent. The samples was measured at 40°C.

The ^-NMR spectrum showed a distinctive chemical shift between δ= 3.1 and 5.0 ppm, these broad bands belonged to the protons of dextran, especially the bands at δ= 3.5 and 4.7, which belonged to the protons linked to the dextran [14]. The bands for the protons of valproic acid could be found between δ= 0.8 and 2.3. There was also one band around δ= 12 for the organic acid group, which was very small in case of the NMR chromatograms of the conjugates. The band at δ= 2.5 was present in all chromatograms and represented part of the DMSO used as solvent which was not deuterated completely (e.g. DMSO-ds). Due to the broad bands for the conjugates and the fact that there were the bands both for the valproic acid and for the dextran, present in the spectra provided strong indication for the formation of the conjugate between valproic acid and dextran.

1H NMR Spectrum of VPA-D-10 (B) compared to those of Dextran (A) and VPA

(C)

1H NMR Spectrum of VPA-D-70 (B) compared to those of Dextran (A) and VPA

(C)

EXAMPLE 5

Determination of aqueous solubility of valproic acid-dextran conjugate. To determine aqueous solubility of the conjugate at 25 °C, approximately 50 mg of conjugate was added to 1.5 ml of double distilled water. The mixture will then be sonicated for 60 minutes to dissolve as much conjugate as possible and afterwards it was centrifuged for 10 minutes at 13,200 rpm to precipitate the un-dissolved conjugate. Then, 1ml of the supernatant was added to 10 ml of NaOH solution (0.5 N), to perform complete hydrolysis. At different time points, 500 μΐ of each sample was collected into 1 ml HPLC vials and 500 μΐ of HCl solution (0.5 N) was added to each vial to neutralize the base [17]. The experiment was done in triplicate. The samples thus prepared was stored in the refrigerator before being analyzed by HPLC.

EXAMPLE 6

Study in vitro hydrolysis of valproic acid-dextran conjugate.

A portion of conjugate was dissolved in 50 ml of buffer solutions (0.1 M). Three different buffer solutions was used for 3 different pH- Citric Acid/Na ₂ HP0 ₄ buffer (pH 5.0), Phosphate buffer (pH 7.5) and NaHC0 ₃ /Na ₂ C0 ₃ buffer (pH 10.22) The hydrolysis study was carried out at 37 °C and at 47 °C in an incubator under continuous stirring of the samples. Samples of 1 ml was collected at predetermined time intervals and 1 ml of buffer solution was added to the samples to keep the volume constant [15, 17, 21]. The experiment was done in triplicate. All samples was kept in the freezer at -20 °C before being analyzed by HPLC. See FIGs. 1-8.

The conjugate showed negligible, or no hydrolysis in acidic medium (p 5.0).

There was a slow rate of hydrolysis of conjugate in phosphate buffer (p 7.5) and much faster hydrolysis in basic bicarbonate buffer (p 10.22). From the plots, it was evident that data for the hydrolysis of both conjugates in phosphate buffer (P 7.5) and at the temperatures of 37 °C and 47 °C could be adequately described by both zero- and first-order models with R values of 0.99. Cartensen [22] showed that if degradation was less than 15%, it would be difficult to distinguish a first-order reaction from a zero-order reaction. But when percent of hydrolysis of both conjugates was higher in case of bicarbonate buffer (P 10.22) at both temperatures; first-order reaction model gave better fit to the data compared to zero-order model. So, it could be concluded that hydrolysis of conjugates followed first-order reaction kinetics in bicarbonate buffer at 37 °C and 47 °C and it could be assumed that hydrolysis of conjugates followed first order reaction kinetics in phosphate buffer at both temperatures. Estimated observed rate constants (from the slopes of the plots) and calculated t for the hydrolysis of the conjugates have been presented in the following table:

EXAMPLE 7

Synthesis of valproic acid-spacer-dextran conjugate and study in vitro hydrolysis of the conjugate in the presence of esterase enzyme.

The primary objective of this aim is to synthesize valproic acid-dextran conjugate having a spacer in between to make the bond formed between VPA and linker more accessible by the esterase enzyme and to study in vitro hydrolysis of the conjugate in the presence of esterase enzyme. Also, study of in vitro hydrolysis of the conjugate formed by direct attachment of VPA to dextran in the presence of esterase enzyme to determine the nature and extent of the effect of enzyme on the rate of hydrolysis of this type of conjugate is performed. [23-25]. Macromolecular pro-drugs synthesized by direct attachment of drug to the dextran appeared to be stable towards enzymatic cleavage by various esterase enzymes. The bonds formed between drug and dextran seemed to be inaccessible by the active sites of the enzymes due to the bulky nature of the dextran molecule. Spacer in between drug and dextran molecule might make the bond between VPA and linker more accessible by the esterase enzyme and help accelerate hydrolysis of the conjugate. Both the affinity and reactivity of the substrates with respect to horse liver carboxylesterase increased as either chain (acyl or alkyl) was lengthened to about C4 to C6. Further increase in the alkyl chain length resulted in a decrease in both the affinity and reactivity, suggesting that the alkyl binding site was only large enough to accommodate a butyl to hexyl chain [26]. Based on this, 1,6-hexanediol can be used as a spacer in between VPA and dextran.

Synthesis of valproic acid-spacer-dextran conjugate.

Scheme 2

To valproic acid- 1,6-hexanediol conjugate, carbonyl diimidazole (CD I) is added followed by solvent THF to dissolve the mixture. The reaction is continued for about 16 hours to complete the activation of the conjugate. The solvent is evaporated in a rota-vap to precipitate activated valproic acid-linker conjugate. This product is used without further purification. After that, dextran is dissolved in DMSO followed by addition of dimethyl aminopyridine (DMAP) to this solution. This solution is added to the activated valproic acid-linker conjugate and the reaction is allowed to continue at room temperature for 4 days. At the end, reaction is stopped by adding concentrated HC1 to neutralize DMAP and imidazole [27]. Finally, ethyl acetate is added to the reaction medium to precipitate the conjugate.

EXAMPLE 8

Study in vitro hydrolysis of valproic acid-linker-dextran conjugate in the presence of esterase enzyme

Hydrolysis of valproic acid-dextran conjugate (with and without the linker in between) is studied in the presence of porcine liver esterase. Porcine liver esterase is used for the study, because of its stability, low cost and the ability to hydrolyze a wide range of substrates without the need for co-enzymes. A 0.001 M stock solution of both conjugates is prepared in de-ionized double-distilled water. At the beginning of each experiment, the stock solution is diluted with 0.15 M phosphate-buffered saline

(PBS) at p 7.4 and 1 ml of a 10 U/ml esterase solution is added to it. The solutions are then diluted to one of five final concentrations by the addition of PBS: 1, 50, 100, 150 and 200 μΜ [21]. At each concentration, controls are prepared by repeating the dilutions described above without the addition of the esterase solution. The solutions are placed in an incubator at 37 °C and are agitated by magnetic stirring bars throughout the experiment. Over a period of 30 minutes to 48 hours, aliquots of 500 μL· are removed periodically and added to Eppendorf vials containing an equal volume of acetonitrile to quench enzyme-catalyzed reaction. The samples are centrifuged at 10,500g for 10 minutes and after that 500 μΐ _^ aliquots of the supernatant are removed and added to glass auto-sampler vials and stored at -20 °C prior to analysis by HPLC [28]. Stability of porcine liver esterase- Esterase from porcine liver is available as lyophilized powder (Sigma- Aldrich) and should be stored at -20 °C for maximum stability. All samples with enzymes are prepared on the day of experiment and stored at temperatures of 0-4 °C (on ice-bath) prior to the experiment. The samples are allowed to equilibrate in screw-capped bottles for one hour at 37 °C, before the addition of pro-drug solution. All enzyme preparations are kept for no more than 50 hours [29] . However, the suitable conditions for carrying out the experiment (for optimal activity) and for storage (for stability) of the enzyme should be reevaluated based on the supplier's specification.

EXAMPLE 9

Protocol for the synthesis of VPA-linker-Dextran conjugate: Two conjugates of valproic acid were synthesized according to the following protocol. Two different linkers were used for the two conjugates. These are- triethylene glycol (TEG) and pentaethylene glycol (PEG). We used dextrans with molecular weights about 1 KDa.

Synthesis of VPA-linker conjugate

1) Add 6.1962 ml (46.43 mmol) of triethylene glycol to 20 ml dried acetonitrile in a conical flask and dissolve it with the help of magnetic stirrer, (sol- A)

2) Take 20 ml dried dicholoromethane (DCM) (dried over molecular sieves) in a 125 ml conical flask and add sol-A slowly to DCM in this flask while shaking. (sol-B)

3) Now, add 1848 μΐ _^ of valproic acid (1664 mg, or, 11.6 mmol), 432.2 mg of DMAP, dimethyl aminopyridine (3.49mmol) and purge with argon gas.

Dissolve 2631.4 mg of DCC, dicyclohexyl carbodiimide (12.76 mmol) in 10 ml of dried DCM and add this solution dropwise for 5 minutes. Purge with argon gas. After that continue the reaction at room temperature for 24 hours

Extraction

1) There should be precipitated urea in the reaction mixture. Filter the urea from the reaction mixture by using conventional paper filter.

2) Evaporate the reaction mixture in a rota-vap under high vacuum at 55 °C for about 15-20 minutes and concentrate to a small amount of oily liquid (about 5 ml). Do not dry to total dryness. 3) Add about 10 ml of dried DCM to dissolve the oily liquid and transfer it into a separatory funnel.

4) Add 10 ml of 5% NaHS0 ₄ in water to the funnel and shake it vigorously for 5 to 10 minutes to have DMAP dissolved in the aqueous layer. Drain the upper aqueous layer in a conical flask and collect it.

5) Repeat the wash (10 ml) with 5% NaHS0 ₄ in water 1 time to ensure complete removal of DMAP from the reaction mixture.

6) Now add 10 ml of 5% NaHC0 ₃ solution in water to the funnel and also shake it vigorously for 5 to 10 minutes to remove excess acid from the reaction mixture. Drain the upper aqueous layer in a conical flask.

7) Repeat the wash (10 ml) with 5% NaHC0 ₃ solution 1 time for the

completeness.

8) During the wash periods, some conjugates might be precipitated out of the organic phase. Carry it over until wash period ends.

9) Collect the organic phase (with some precipitated conjugates) in a conical flask and add 80 ml of DCM to dissolve the precipitated conjugate completely. Then, add 5.31 g of dry MgS0 ₄ to remove water from the organic solvent. Leave it for 30 minutes.

Filter the salt out completely with a conventional paper filter and transfer the organic liquid in a rota-vap and evaporate under high vacuum at 50 °C to complete dryness. The mixture was dried for 3 hours. Product yield: 2.48 mg (8.99 mmol). You have to calculate mmol of the conjugate by dividing the weight of conjugate in mg by 276

Synthesis of VPA-linker-Dextran conjugate

1) Dissolve the conjugate (2.48 mg) in 60 ml of THF in an argon atmosphere in a conical flask and add 1.602 g (8.99 mmol of VPA-linker conjugate *1.1) of CDI. The clear solution should result. Close the flask with a bottom and parafilm.

2) The solution will be stirred for 16 hours at room temperature.

3) Evaporate the solvent in a rota-vap (50°C) under reduced pressure yielding a viscous oil (VPA-linkerCI). This product will be used without further purification. 4) Dissolve 1.5098 g of freeze-dried Dextran (mw 10k) in 20 ml of dried DMSO in a conical flask in an argon atmosphere. 3 g of molecular sieves (4A) will be dried for one hour and the added to the Dextran solution.

6.03 g of molecular sieves (3A) will be dried for around 1 hour. The "reaction flask" has been dried before with the "hairdryer". Then the Dextran-DMSO solution will be transferred in this flask followed by the VPA-linker conjugate (activated with CDI) (before dissolve with 15 ml DMSO). At last 1.6461 g (8.99 mmol of VPA-linker conjugate* 1.5) of DMAP will be added to the "reaction flask". All the procedure will be done in an argon atmosphere. The flask will be placed on the magnetic stirrer and heated at 50°C.

5) The solution will be stirred at 50°C for 3 to 4 days.

Extraction

1) Now add EtOAc to the reaction medium to to precipitate the conjugate.

Discard the clear supernatant and add DMSO to dissolve the conjugate (use homogenizer, if needed). Again, add EtOAc to this solution to precipitate the conjugate and discrad the clear supernatant.

1 ml of the conjugate solution and 5 ml of ethyl acetate will be transferred in a centrifugal tube to make sure that conjugate is precipitated. If conjugate precipitates, then around 6 ml of conjugate solution will be transferred in 4 centrifugal tubes and around 30 ml of ethyl acetate will be added to each of them. Then the mixtures will be centrifuged at 2500 rpm for 5 minutes. After that the supernatant will be discarded and the conjugate will be dissolved in 6 ml DMSO (each tube) with the homogenizer.

30 ml of ethyl acetate will be added to precipitate the conjugate again and the mixtures will be centrifuged at 2500 rpm for 5 minutes. The supernatant will be discarded and the conjugate will be dissolved in 6 ml of DMSO (each tube) with the homogenizer.

Again 30 ml of ethyl acetate will be added to precipitate the conjugate and the mixtures will be centrifuged at 2500 rpm for 5 minutes. The supernatant will be discarded again.

Now, add 10 ml MeOH to one tube and disperse the conjugate in MeOH. Then transfer it to another one and the same will be done. If needed, a few ml of MeOH will be added to wash the tubes. Then dry the conjugate in a rota-vap for about 3 hours to remove MeOH and water. cheme 4

1H-NMR spectra of VPA (A), VPA-TEG (B), Dextran (C) and VPA-TEG-DEX

1H-NMR spectra of VPA (A), VPA-PEG (B), Dextran (C) and VPA-PEG-DEX

(D)

EXAMPLE 10

Determination of drug content in coniugates (degree of substitution)

1) 10.0 mis of NaOH 0.5 N are added to 10.0 -15.0 mg of conjugate and vortex the mixture for 10 to 20 minutes to dissolve the conjugate. The experiment will be done in triplicate.

Conjugate- 1 - VPA-triethylene glycol-dextran (lk)

Conjugate-2 - VPA-pentaethylene glycol-dextran (lk)

Conj-1-1 : 16.1 mg Conj-1-2: 11.7 mg Conj-1-3: 19.6 mg

Conj-2-1: 15.3 mgConj-2-2: 22.6 mg Conj-2-3: 19.2 mg

2) The conjugate solutions are left in the refrigerator (4 °C) for a total of 46 hours.

3) 500 μΐ from each solution will be collected in 1 ml-HPLC vials and 500 μΐ of

HCL 0.5 N will be added to each vial to neutralize the base. The collected samples will then be analyzed for drug content by HPLC analysis.

EXAMPLE 11

Protocol for enzymatic hydrolysis study of the coniugates VPA-linker-Dl Enzyme solution-I: Take 7 ml of IX PBS solution (p ^w 7.4, 0.01 M) in a small glass vial at room temperature. Then, weigh about 17 mg (approx.

17 units/mg) of esterase enzyme and add it to the buffer solution in glass vial (approx. 40 units/ml).

Hydrolysis study in IX PBS solution (pH 7.4) with Porcine Liver Esterase

(coniugate-1. VPA-PEG-D1)

Take 200 ml of IX PBS solution (p ^H 7.4, 0.01 M) in a conical flask and keep it in the incubator to preheat the solution at 37 °C. Then, weigh 50 mg of conjugates in glass vials and add to it 9 ml of preheated PBS solution and shake the vials to dissolve the conjugate in PBS solution. Place the glass vials in the incubator at 37 °C and keep the magnetic stirrer running at 100 rpm. After that, add 1 ml of enzyme solution (approx. 40 U/ml) to the glass vial. Mark the time of addition of the enzyme solution. This will serve as zero- time for the hydrolysis study. This experiment will be done in triplicate.

Hydrolysis study in IX PBS solution (pH 7.4) with Rat Brain Homogenate

(coniugate-1, VPA-PEG-D1)

Weigh 50 mg of conjugates in glass vials and add to it 9.5 ml of preheated PBS solution and shake the vials to dissolve the conjugate in PBS solution. Place the glass vials in the incubator at 37 °C and keep the magnetic stirrer running at 100 rpm. After that, add 0.5 ml of rat brain homogenate to the glass vial. Mark the time of addition of the enzyme solution. This will serve as zero-time for the hydrolysis study. This experiment will be done in triplicate. Negative control -1 (IX PBS + Coniugate-1) (no enzyme solution)

The vial without the addition of enzyme solution will serve as negative control. Weigh 50 mg of conjugates in glass vials and add to it

10 ml of preheated PBS solution and shake the vials to dissolve the conjugate in PBS solution. Place the glass vials in the incubator at 37 °C and keep the magnetic stirrer running at 100 rpm. This experiment will be done in triplicate.

Negative control -2 (IX PBS + PLE) (no conjugate)

No conjugate will be added in this control. Take 9 ml of preheated phosphate buffer solution (p 7.4, 0.01 M) in glass vials and add 1 ml of enzyme solution (approx. 40 U/ml) to the glass vial. This experiment will be done in triplicate. Sample will be collected for one-time point at the end of the experiment.

Negative control -3 (IX PBS + RBH) (no conjugate)

No conjugate will be added in this control. Take 9.5 ml of preheated phosphate buffer solution (p 7.4, 0.01 M) in glass vials and add 0.5 ml of rat brain homogenate to the glass vial. This experiment will be done in triplicate. Sample will be collected for one-time point at the end of the experiment. Sample collection-

Sample collection from the vials- 500 μΐ of the reaction solution from each glass vial will be removed periodically and collected into eppendorf tubes already containing 500 μΐ of acetonitrile. The, centrifuge these tubes at 13,200 rpm for 5 minutes. After that 800 μΐ of supernatant will be collected in 1 ml HPLC glass vials and kept in the refrigerator before being analyzed by HPLC.

Add 500 μΐ of PBS solution after each sample collection to each vial to replenish the volume.

Time intervals for collection of sample-4 min, 8 min, 12 min, 16 min, 20 min, 24min.

Hydrolysis results for the VPA-TEG-D1 conjugate. (See, FIG 9. )

Hydrolysis results for the VPA-PEG-D1 conjugate. (See, FIG 10. )

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The disclosures of each and every patent, patent application and publication cited herein are hereby incorporated herein by reference in their entirety.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other

embodiments or portions thereof. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Although the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The claims are intended to be construed to include all such embodiments and equivalent variations.