FIELD OF THE INVENTION

The present invention relates to water soluble compositions comprising lipophilic diesters of l,2-bis(2-aminophenoxy)ethane-N,N,N' ,N' -tetraacetic acid denoted herein as B APTA-DE, or salts thereof, non-covalently associated with organic amine compounds, the organic amine preferably comprising an amino acid. The invention further relates to pharmaceutical compositions comprising said water soluble compositions and their use in the treatment of conditions or diseases related to abnormal levels of divalent metal ions, wherein said treatment minimizes undesired side effects associated with administration of B APTA-DE sodium salts.

BACKGROUND OF THE INVENTION

Perturbed balance of divalent metal ions, such as zinc, calcium, copper or iron, is thought to be a contributory factor to cell damage or cell death following cell insult by ischemic or hypoxic events, such as stroke, brain and spinal cord trauma, myocardial ischemia or birth asphyxia, or acute and recurrent pancreatitis; as well as long-lasting or chronic conditions or diseases involving metal dyshomeostasis, such as Alzheimer's disease, Parkinson's disease and Lewy Body dementia; diseases of protein misfolding and Prion protein related disorders; atherosclerosis and chronic pancreatitis. A potential way to address this phenomenon is by using ion chelation in order to restore ionic balance. The therapeutic use of divalent metal ion chelators is, however, problematic due either to the non-selective nature of these compounds or to the perturbation of normal ionic balance that they may cause, leading to a high incidence of cardio- and hepatotoxic effects. One way to overcome this drawback is by restricting the action of such agents to the cell and intracellular membranes, which is the primary site of cellular damage. US Patent No. 6,458,837 to some of the inventors of the present invention discloses stable lipophilic diesters of chelating agents, BAPTA chelators in particular, designed such that metal ion chelation is significantly increased in a lipophilic milieu. Without being limited to theory or mechanism of action it is assumed that in a living organism chelation occurs preferably in the vicinity of the cell membrane. Sodium salts of these lipophilic diesters were found useful in the treatment of a condition or disease related to an excess of divalent metal ions, and in particular for the treatment of a condition or disease related to elevated levels of intracellular calcium and/or zinc ions, such as in brain or cardiac ischemia, stroke, epilepsy, Alzheimer's disease, Parkinson's disease or Lewy Body dementia, acute, recurrent or chronic pancreatitis, or cardiac arrhythmia.

One of these lipophilic diester salts, the disodium salt of l,2-bis(2-aminophenoxy)ethane-

Ν,Ν,Ν'Ν'-tetraacetic acid, N,N-di(octyloxyethyl ester), also called DP-b99, was found to be a promising drug-candidate for the treatment of ischemic stroke. The disodium salt of DP-b99 is a lipophilic, cell permeable disodium salt derivative of BAPTA-DE characterized by its preferential binding activity for zinc, calcium, copper as well as other divalent cations in cellular hydrophobic milieus, such as cell membranes. In vitro, DP-b99 was shown to protect primary cortical neurons from cell death induced by either deprivation of oxygen and glucose or hydrogen peroxide (H2O2), with protection conferred when DP-b99 is added to cells up to 4 hours after the addition of H2O2. DP-b99 was also shown to attenuate basal activity of MMP-9 as well as TNFa induced MMP-9 activation in glia and C6 cells (US Patent No. 7,799,831). In a rodent focal ischemia model, DP-b99 attenuates the extent of cerebral infarction, even when treatment is administered 6 hours following the onset of ischemic insult. In a range of ischemic animal models DP-b99 was shown to significantly reduce neuron-specific enolase (NSE) levels at 24 and 72 h following ischemia. Without being limited by theory or mechanism of action, it is hypothesized that DP-b99 is capable of restoring zinc and calcium homeostasis and thus moderate the cascade of events leading to cell death as a result of ischemic damage.

Multiple clinical studies of DP-b99 in healthy volunteers and patients with acute ischemic stroke indicated that DP-b99 disodium salt may be safely administered intravenously as a single dose of up to 3 mg/kg and as lmg/kg/day over 4-6 days while displaying acceptable systemic tolerability. The most prevalent study drug-related adverse event (AE) was injection site reaction (mostly mild phlebitis). Currently, the risk of venous inflammation (e.g. phlebitis) precludes DP-b99 disodium salt administration via central vein infusion.

In order to reduce the risk of venous inflammation, efforts are currently made to avoid repeated use of the same infusion site location during the 4-day treatment. Cannulation of peripheral veins is a painful procedure and repeated re-cannulation is distressing for patients and may lead to future venous access difficulties, as well as to rapid exhaustion of available sites. The current recommended practice of establishing a new peripheral venous cannulation every infusion day is particularly challenging in the treatment of elderly patients who often have a limited number of peripheral venous access points.

Lipophilic diesters of l,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid are very weak electrolytes, for which solubility is highly dependent on the degree of ionization (dissociation) since the affinity of the ionized species for water is markedly higher than that of the un-ionized species. The degree of dissociation is in turn dependent on the pKa of the weak electrolyte and the pH of the solution into which it is dissolved. Simplistically, at pH values above the pKa of a weak acid, the solubility increases significantly as a result of acid dissociation and at pH values below the pKa it decreases, as the acid is present in the de-ionized state thereof.

One way to increase solubility of a weak acid is to form a salt of said acid. However, the solubility of salts formed with a variety of different counter-ions can vary significantly. For example, development of phlebitis following the infusion of sodium salt of DP-b99 may be attributed to rapid dissociation of the sodium salts at pH close to physiological level and irritation of tissues surrounding the infusion site by the poorly-soluble lipophilic diester. Therefore, alternative salts of DP-b99 were suggested to overcome the side effects associated with administration of the sodium salt.

CN Patent Application Publication No. 102079717 is directed to mono- and di-arginine salts of DP-b99, having excellent water solubility and stability and decreased adverse reactions, such as vascular stimulation and muscular stimulation, when injected, as compared to the sodium salt of DP-b99.

CN Patent Application Publication No. 102079715 is directed to mono- and di-lysine salts of DP-b99, having excellent water solubility and stability and decreased adverse reactions, such as vascular stimulation and muscular stimulation, when injected.

However, even mono- and di-arginine and lysine salts of DP-b99 do not eliminate completely the inflammation risks associated with intravenous administration of DP-b99. Orally- administrable compositions can eliminate said risk, where oral administration is applicable. It is apparent, however, that the degree of ionization (and therefore solubility) of an orally administered weak acid is likely to be dictated by the pH of the gastrointestinal (GI) fluids rather than the intrinsic pH attained by the dissolution and dissociation of the salt of a weak acid in water.

There thus remains an unmet need for compositions comprising lipophilic diesters of BAPTA-DE chelators and in particular l,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, N,N-di(octyloxyethyl ester), which are soluble in aqueous solutions at weak basic pH for parenteral and in acidic pH for oral drug administration, thereby being capable of effectively treating or preventing metal ion-associated disorders, while minimizing the side effects associated with administration of BAPTA-DE sodium salts.

SUMMARY OF THE INVENTION

The present invention provides water soluble, pharmaceutically acceptable non-covalent mixtures comprising lipophilic diesters of l,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-DE) or salts thereof, non-covalently associated with organic amine compounds. In particular, the present invention relates to pharmaceutical compositions comprising organic amines non-covalently associated with l,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, N,N- di(octyloxyethyl ester) designated throughout the specification as DP-b99, or l,2-bis(2- aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, N,N-di(dodecyloxyethyl ester) designated throughout the specification as DP-460 and their salts, which are effective in the treatment of metal ion-associated disorders, such as stroke, brain and spinal cord trauma, myocardial ischemia or birth asphyxia, acute, acute high risk, recurrent and chronic pancreatitis, Alzheimer's disease, Parkinson's disease and Lewy Body dementia; diseases of protein misfolding and Prion protein related disorders and atherosclerosis.

The term "non-covalent mixture", as used herein, refers to any physical combination of at least two discrete chemical entities, such as, for example, a physical combination of a lipophilic diester of 1,2- bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or salt thereof and an organic amine, preferably comprising a free amino acid, wherein the organic amine is present in a molar excess of greater than 2 moles of the organic amine to 1 mole of the lipophilic diester. According to some embodiments, the discrete chemical entities are present in the non-covalent mixture in non-covalent association with each other. Therefore, the terms "compositions comprising a lipophilic diester in non-covalent association with an organic amine or a free amino acid" and "non-covalent mixtures of a lipophilic diester and an organic amine or a free amino acid" can be used interchangeably.

According to some embodiments, the term "non-covalent mixture" refers to any pharmaceutical composition having a BAPTA-DE or salt thereof, in non-covalent association with an organic amine, and in particular a free amino acid, wherein the organic amine is present in a molar excess of greater than 2 moles of the organic amine to 1 mole of the lipophilic diester.

Arginine and lysine salts of lipophilic esters of 1 ,2-bis(2-aminophenoxy)ethane- Ν,Ν,Ν',Ν'-tetraacetic acid were reported to be more water soluble than the sodium salt thereof. However, the inventors found that even said arginine and lysine salts, which are soluble at physiological pH, precipitate at a pH of below 6. The present invention is based in part on the unexpected finding that compositions comprising the lipophilic esters of l,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or salts thereof non-covalently associated with organic amines, wherein the organic amine is present in a molar excess of greater than two moles to one mole of the lipophilic diester are significantly more soluble than the sodium, arginine and lysine salts of DP-b99. In particular, the solubility of DP-b99 non-covalently associated with arginine and lysine, wherein the stoichiometric ratio between the lipophilic diester and the free amino acid was 1:6, was found to be higher both in HC1 solutions and in plasma as compared to the solubility of sodium, mono-arginine, di-arginine and di-lysine salts of DP-b99 in the same media. An additional beneficial feature of the compositions of the present invention is that such compositions can be formed from the less water-soluble precursors (such as, for example the sodium salt of the lipophilic diester) at the patient's bedside.

Thus, according to a first aspect, the present invention relates to pharmaceutical compositions comprising a lipophilic diester of a chelating agent or salt thereof, of the general Formula I:

CH ₂ COOM MOOCCH ₂

ROOCCH ₂ N C ₆ H4 OCH ₂ CH ₂ 0 N CH ₂ COOR

Formula 1 in non-covalent association with a free amino acid, wherein a stoichiometric ratio of the free amino acid and the lipophilic diester in the pharmaceutical composition is at least 3: 1, wherein M denotes a hydrogen ion and/or a physiologically acceptable cation, and wherein R is selected from the group consisting of CnEbn+i (n=l-10), C _n H2n+i(OCH2CH2) _m (n=l-20, m=l-6), (C _n H2n+i)2N(CH2)m (n=l-6, m=l-6) and substituted or unsubstituted ArC¾; the substituents on the aromatic rings being in the ortho position.

According to some embodiments, the free amino acid is selected from the group consisting of naturally occurring amino acids, synthetic amino acids and combinations thereof. According to some currently preferred embodiments, the free amino acid is naturally occurring. According to some embodiments, the free amino acid has an amino group side chain. According to some embodiments, the free amino acid has a guanidino group side chain. According to some currently preferred embodiments, the free amino acid is selected from the group consisting of arginine, lysine and combinations thereof. According to particular embodiments, the free amino acid is arginine. According to further particular embodiments, the free amino acid is lysine.

According to some embodiments, the free amino acid displays cell protective activity, such as neuroprotective activity. In this case and according to some further embodiments, a combined cell protective activity of both the BAPTA diesters (BAPTA-DE) and the free amino acid may occur in vivo. The combined cell protective activity according to some embodiments may be additive or synergistic.

According to other embodiments, the free amino acid is devoid of biological activity.

According to some embodiments, there are provided the pharmaceutical compositions, wherein the lipophilic diester is in a form of a salt. The lipophilic diester salt can be any pharmaceutically acceptable salt of the lipophilic diester of general Formula I. According to some embodiments, the salt of the lipophilic diester comprises one physiologically acceptable cation and one mono-anion of the lipophilic diester. In certain such embodiments, one M denotes a hydrogen ion and another M denotes a physiologically acceptable cation. In other embodiments, the salt of the lipophilic diester comprises two physiologically acceptable cations and one dianion of the lipophilic diester. In certain embodiments, each M denotes a physiologically acceptable cation.

According to some embodiments, the physiologically acceptable cation is selected from inorganic or organic cations. According to some embodiments, the inorganic cation is a monovalent metal cation selected from the group consisting of a sodium cation (Na ⁺ ), lithium cation (Li ⁺ ), and potassium cation (K ⁺ ). Each possibility represents a separate embodiment of the invention. According to certain embodiments, the monovalent metal cation is a sodium cation. According to further embodiments, the salt of the lipophilic diester of the general Formula I is a disodium salt. According to some embodiments, the organic cation is an organic amine cation. In certain such embodiments, the lipophilic diester salt is an organic amine salt. According to some embodiments, the lipophilic diester salt comprises one or two organic amine cations. According to some embodiments, the organic amine cation is derived from organic non-toxic molecules selected from the group consisting of lysine, arginine, aminoguanidine (pimagedine), trimetazidine, taurine, choline, histamine, tromethamine and meglumine. According to further embodiments, the salt of the lipophilic diester of the general Formula I is selected from the group consisting of a lysine, arginine, aminoguanidine (pimagedine), trimetazidine, taurine, choline, histamine, tromethamine and meglumine salt. According to some currently preferred embodiments, the organic amine cation is derived from an amino acid. According to some embodiments, the amino acid from which the organic amino cation is the same as the amino acid of the composition, being in non-covalent association with the lipophilic diester of Formula I. According to other embodiments, said amino acid is different from the amino acid of the non- covalent mixture. According to certain embodiments, the organic cation comprises arginine or lysine cations. According to further embodiments, the salt of the lipophilic diester of the general Formula I is selected from the group consisting of mono-arginine, di-arginine, mono-lysine and di-lysine salts. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the stoichiometric ratio between the free amino acid and the lipophilic diester of the general Formula I is above 3: 1. In these embodiments, M denotes a hydrogen ion. In some embodiments, said stoichiometric ratio is 4: 1. In certain embodiments, said stoichiometric ratio is 5: 1. In some exemplary embodiments, said stoichiometric ratio is 6: 1. According to some embodiments, said stoichiometric ratio is above 6: 1.

According to some embodiments, the free amino acid and the lipophilic diester or salt thereof, of the general Formula I form a non-covalent complex. According to further embodiments, the free amino acid is associated with the lipophilic diester or salt thereof in the non-covalent complex via electrostatic bonds (i.e., hydrogen bonds), van der Waals forces, or hydrophobic-hydrophilic interactions. Each possibility represents a separate embodiment of the invention. According to some embodiments, the complexes are formed at a particular stoichiometric ratio between the free amino acid and the lipophilic diester. According to some embodiments, said stoichiometric ratio is 6: 1.

According to some embodiments, R of the lipophilic diester is selected from the group consisting of: C ₂ H ₅ , C3H7, i- C3H7, C4H9, C ₇ H ₁₅ , C ₈ H ₁₇ , CH ₂ C ₆ H ₅ , CH3OCH2CH2, C2H5OCH2CH2, C3H7OCH2CH2, C4H9OCH2CH2, C ₇ H ₁₅ OCH2CH2, C ₈ H ₁₇ OCH2CH2, C10H21OCH2CH2, C12H25OCH2CH2, C16H33OCH2CH2, C18H37OCH2CH2, CH ₃ (OCH ₂ CH2)2, C2H ₅ (OCH ₂ CH2)2, C ₄ H ₉ (OCH2CH2) ₂ , C ₆ H ₁₃ (OCH2CH2)2, C ₇ H ₁₅ (OCH2CH2) ₂ ,

C ₈ H ₁₇ (OCH2CH2) ₂ , CioH2i(OCH ₂ CH2)2, CH ₃ (OCH ₂ CH2)3, (CH ₃ )2NCH ₂ CH2, C ₇ H ₁₅ (OCH ₂ CH2)3.

According to some embodiments, R is selected from the group consisting of: C2H5, C3H-7, C4H9, C ₇ H ₁₅ , C ₈ H ₁₇ , C ₈ H ₁₇ OCH ₂ CH2, C10H21OCH2CH2, C12H25OCH2CH2, C16H33OCH2CH2, Ci ₈ H ₃₇ OCH ₂ CH2, C ₈ H ₁₇ (OCH ₂ CH2) ₂ , CioH2i(OCH ₂ CH2)2.

According to certain preferred embodiments, R is C ₈ Hn or CsHnOCthCth. According to some exemplary embodiments, R is C8H1-7OCH2CH2. In other embodiments, R is

According to some embodiments, the present invention relates to the pharmaceutical compositions, wherein R is CsHnOCthCth (DP-b99) and the free amino acid comprises arginine, lysine or combinations thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, said salt is a mono- or di-arginine salt of DP- b99. In other embodiments, said salt is a di-sodium salt of DP-b99. According to some embodiments, the present invention relates to the non-covalent mixtures of the free amino acids with lipophilic diesters or salts thereof of the general Formula I, wherein R is C ₁ 2H25OCH2CH2 (DP-460) and the free amino acid comprises arginine, lysine or combinations thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, said salt is a mono- or di-arginine salt of DP-460. In other embodiments, said salt is a di-sodium salt of DP-460.

In further embodiments, the pharmaceutical composition comprises a lipophilic diester or salt thereof of the general Formula I, wherein R is CsHnOCFhCFh and the stoichiometric ratio between the free amino acid and the lipophilic diester is 6:1. In yet further embodiments, the pharmaceutical composition comprises a lipophilic diester of the general Formula I, wherein R is C ₁ 2H25OCH2CH2 and the stoichiometric ratio between the free amino acid and the lipophilic diester is 6: 1. In certain such embodiments, the organic amine comprises arginine, lysine or combinations thereof.

The pharmaceutical compositions comprising lipophilic diesters or their salts, of the general Formula I, in non-covalent association with free amino acids, according to the principles of the present invention, display several advantages in comparison with the sodium or organic amine salts of said diesters alone. According to some embodiments, the pharmaceutical compositions of the invention are soluble in aqueous solutions having a physiological pH. In particular embodiments, the compositions are soluble at a pH in the range of 7.35 - 7.45, while the sodium salt solutions tend to precipitate at pH lower than 7.5. According to further embodiments, the solubility of the pharmaceutical compositions of the invention at a physiological pH, and in particular in the range of 7.1 - 7.4, is higher than solubility of the organic amine salts of said lipophilic diesters.

According to further embodiments, the pharmaceutical compositions according to the principles of the present invention further comprise a pharmaceutically acceptable diluent or carrier. The pharmaceutical compositions may be in liquid or solid dosage forms and may be administered via a parenteral, enteral or topical route. Each possibility represents a separate embodiment of the invention. According to currently preferred embodiments, the pharmaceutical compositions of the invention are suitable for parenteral administration. According to further embodiments, the parenteral administration is into central vein. According to some embodiments, the parenteral route includes intravenous, intra-arterial and intracerebral routes of administration. The intracerebral route may include epidural or intracerebroventricular administration. According to other embodiments, the pharmaceutical compositions of the invention are suitable for enteral administration. The currently preferred enteral administration route includes an oral administration. The orally administrable compositions may comprise an enteric coating.

The pharmaceutical compositions, according to the principles of the present invention are useful in the treatment or prevention of metal ion-associated disorders for example, disorders associated with abnormal levels of manganese, magnesium, copper, iron, cadmium, cobalt, and in particular calcium and zinc ions. The use of the pharmaceutical compositions of the current invention allows minimizing the occurrence of undesired side effects associated with the administration of the salts of the lipophilic diesters. Thus, in some embodiments, the pharmaceutical compositions according to the principles of the present invention are for use in the treatment or prevention of a disease or disorder related to an imbalance of divalent metal ions According to some embodiments, said disease or disorder is related to an elevated level of intracellular Ca ⁺⁺ and/or Zn ⁺⁺ ions.

The divalent metal ions can be selected from the group consisting of Ca ⁺⁺ , Zn ⁺⁺ , Cd ⁺⁺ ,

Co ⁺⁺ , Cu ⁺⁺ , Fe ⁺⁺⁺ , Mg ⁺⁺ , and Mn ⁺⁺ , ions. In some embodiments, the imbalance comprises elevated level of intracellular divalent metal ions. In the currently preferred embodiments, the disease or disorder is related to an elevated level of intracellular Ca ⁺⁺ and/or Zn ⁺⁺ ions. In some embodiments, undesirable side effects associated with the use of the pharmaceutical compositions of the present invention or the pharmaceutical compositions comprising same, are reduced relative to the side effects associated with the use of the salts of the diesters of the general Formula I. According to further embodiments, the undesirable side effects are reduced as compared to the use of sodium salts of the lipophilic diesters. According to yet further embodiments, the undesirable side effects are reduced as compared to the use of organic amine salts of the lipophilic diesters. According to some embodiments, the disease or disorder related to an imbalance of intracellular Ca ⁺⁺ and/or Zn ⁺⁺ ions, is selected from the group consisting of brain and cardiac ischemia, stroke, brain and spinal cord trauma, myocardial infarction, epilepsy, Alzheimer's disease, Parkinson's disease, Lewy body dementia, acute inflammation, prostatic hypertrophy, muscular spasm, arterial hypertension, asthma, irritable bowel syndrome, acute, recurrent and chronic pancreatitis. Each possibility represents a separate embodiment of the invention. According to particular embodiments, said disease or disorder is a brain stroke. According to some embodiments, the stroke is an acute ischemic stroke. According to some further embodiments, the stroke is a hemispheric acute ischemic stroke. According to further particular embodiments, said disease or disorder is pancreatitis. According to some embodiments, the pancreatitis is an acute pancreatitis. According to some embodiments the undesirable side effect is phlebitis.

In another aspect, the present invention provides a method for treating a disease or disorder related to an imbalance of divalent metal ions, comprising administering to an individual in need of such treatment a pharmaceutical composition, according to the principles of the present invention. In yet another aspect, the present invention provides a method for preventing or reducing brain damage due to a stroke, comprising administering to a patient in need of such treatment a pharmaceutical composition, according to the principles of the present invention. In still another aspect, the present invention provides a method for preventing or reducing cellular damage due to pancreatitis, comprising administering to a patient in need of such treatment a pharmaceutical composition, according to the principles of the present invention.

According to some embodiments the pharmaceutical composition is administered by an injection or infusion. According to some embodiments, the pharmaceutical compositions of the present invention are intravenously administered to a stroke patient in from 1 to 7, preferably 2 to 4, more preferably 4 separate intravenous infusions. Each possibility represents a separate embodiment of the invention. According to some embodiments, the dosage regimen given in separate intravenous infusions per day is from about 0.1 to about 3 mg of the lipophilic diester or salt thereof of the general Formula I to kg of body weight per infusion. According to further embodiments, the total daily dosage regimen is from about 0.3 to about 9 mg of the lipophilic diester or salt thereof of the general Formula I to kg of body weight. The methods of the invention comprise administration of at least the first intravenous infusion within 12 hours, preferably within 9 hours after the onset of the insult. The term "onset of an insult" is defined as the time that the subject was last seen in a normal state, or bedtime for unwitnessed insults occurring during sleep. According to currently preferred embodiments, the pharmaceutical compositions are intravenously administered in up to 3 daily intravenous infusions for up to 10 days. According to further embodiments, the individual dose comprises 3mg/kg/infusion and the daily dosage is up to about 9 mg/kg of body weight, wherein the first dose is administered within 9 hours after the onset of the insult.

According to some embodiments, the pharmaceutical compositions of the present invention are intravenously administered to a pancreatitis patient in from 1 to 10, preferably 4 to 8, separate intravenous infusions. According to some embodiments, the dosage regimen given in separate intravenous infusions per day is from about 0.1 to about 3 mg of the lipophilic diester or salt thereof of the general Formula I to kg of body weight per infusion. According to further embodiments, the total daily dosage regimen is from about 0.3 to about 9 mg of the lipophilic diester or salt thereof of the general Formula I to kg of body weight. The methods of the invention comprise administration of at least the first intravenous infusion within 72 hours, preferably within 48 hours of acute pancreatitis symptoms onset. According to currently preferred embodiments, the pharmaceutical compositions are intravenously administered in up to 3 daily intravenous infusions for up to 10 days. According to further embodiments, the individual dose comprises 3mg/kg/infusion and the daily dosage is up to about 9 mg/kg of body weight, wherein the first dose is administered within 48 hours after the acute pancreatitis symptoms onset.

The low tendency of the pharmaceutical compositions of the invention to cause venous inflammation and in particular phlebitis enables according to some embodiments their introduction into a blood vessel over extended periods of time. According to particular embodiments, their low tendency to cause venous inflammation enables their administration into a single blood vessel through the course of the treatment avoiding the need to change the administration site on a daily basis during the course of the treatment. According to some embodiments, the administration site is a peripheral vein. According to some currently preferred embodiments, the pharmaceutical compositions of the invention are administered into a central vein.

The pharmaceutical compositions comprising are preferably administered by intravenous infusion in the form of a 0.1- 3 mg/ml solution in sterile physiological saline. According to some embodiments, the pharmaceutical compositions may be diluted prior to injection or infusion in pharmaceutically acceptable solutions.

According to some embodiments, administration refers to chronic administration. In certain embodiments, the pharmaceutical compositions of the present invention are administered via an enteral route. In currently preferred embodiments, the enteral route includes oral administration. The pharmaceutical compositions of the present invention can be administered to stroke patients and/or pancreatitis patients. Each possibility represents a separate embodiment of the invention. In particular embodiments, the pancreatitis patient is a chronic pancreatitis patient. The pharmaceutical compositions of the present invention can be orally-administered at a dose of about 100-500 mgs/patient/day.

According to an additional aspect, the present invention provides a non-covalent mixture of a free amino acid and a lipophilic diester of the general Formula I, for use in the preparation of a medicament for the treatment of a disease or disorder related to an imbalance of divalent metal ions. In accordance with another aspect of the invention, there is provided a kit for the treatment or prevention of a disease or disorder related to an imbalance of divalent metal ions, the kit comprising, in separate containers, a lipophilic diester or salt thereof, of the general Formula I, wherein M denotes a hydrogen ion and/or a physiologically acceptable cation, and wherein R is selected from the group consisting of CnEhn+i (n=l-10), C _n H2n+i(OCH2CH2) _m (n=l-20, m=l-6), (C _n H2n+i)2N(CH2)m (n=l-6, m=l-6) and substituted or unsubstituted ArCFb; the substituents on the aromatic rings being in the ortho position, and a pharmaceutically acceptable diluent or carrier comprising a free amino acid, wherein the molar ratio between the free amino acid and the lipophilic diester or the salt is at least 1: 1. According to some embodiments, each M denotes a physiologically acceptable cation comprising an organic amine cation. In particular embodiments, said organic amine cation is derived from arginine or lysine. According to further embodiments, the molar ratio between the free amino acid and the lipophilic diester or the salt is above 1 : 1.

According to further embodiments, said molar ratio is at least 2: 1. According to yet further embodiments, one M denotes a physiologically acceptable cation comprising an organic amine cation and one M denotes a hydrogen ion. In particular embodiments, said organic amine cation is derived from arginine or lysine.

According to further embodiments, said molar ratio is at least 3: 1. According to yet further embodiments, each M denotes a physiologically acceptable cation comprising a monovalent metal cation. According to particular embodiments, said monovalent metal cation is a sodium cation. According to alternative embodiments, each M denotes a hydrogen ion.

According to the currently preferred embodiments, the free amino acid comprises arginine, lysine or combinations thereof. The free amino acid can be present in the kit container in a form of a cation, its conjugated salt (e.g. chloride salt, bromide salt, hydrochloride salt, hydrobromide salt) and/or its free base.

According to some embodiments, the kit is suitable for the preparation of the pharmaceutical composition comprising the lipophilic diester or salt thereof, of the general Formula I, in non-covalent association with a free amino acid. According to further embodiments, the kit is suitable for the preparation of said pharmaceutical composition at the patient's bedside. According to some embodiments, the pharmaceutical composition is formed within about 10 minutes to about 1 hour after the lipophilic diester or salt thereof salt is mixed with the diluent or carrier, comprising the free amino acid.

These and other features and advantages of the present invention will become more readily understood and appreciated from the detailed description of the invention that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be understood and appreciated more fully from the detailed description below, in conjunction with the drawings, in which:

Figure 1: shows UV absorbance of the titration assays of DP-b99-Arg salt with arginine as a function of moles of arginine in the mixture at 260 nm.

Figures 2A-2F: show the DSC curves of pure components and the prepared DP-b99 arginine salts and non-covalent mixtures: DSC curve of pure L-arginine (Figure 2A); DSC curve of DP- b99 acid (Figure 2B); DSC curve of DP-b99 di-L-arginine salt (Figure 2C); and DSC curve of DP-b99-L-arginine non-covalent mixture (Figure 2D) DSC curve of DP-b99 di-L-lysine salt (Figure 2E) and DSC curve of DP-b99-L-lysine non-covalent mixture (Figure 2F).

Figure 3A-3C: show an image of the DP-b99 mono-L-arginine salt (Figure 3A), DP-b99 di-L- arginine salt (Figure 3B) and DP-b99-L-arginine non-covalent mixture (Figure 3C) solutions in plasma after 2 hr.

Figures 4A-4D: show absorbance measured at 230 (diamond), 260 (square), 280 (triangle) and 320 (cross) nm for up to 5 hours of incubation in water of: DP-b99-disodium salt (Figure 4A); DP-b99-mono-L-arginine salt (Figure 4B); DP-b99-di-L-arginine salt (Figure 4C); and DP-b99- L-arginine non-covalent mixture (Figure 4D).

Figures 5A-5C: show solubility of DP-b99 non-covalent mixture and salts measured by UV absorption spectroscopy at 260 nm of di-Sodium salt (diamond), DP-b99-mono-L-arginine salt (square), DP-b99-di-L-arginine salt (triangle) and DP-b99-L-arginine non-covalent mixture (cross) in phosphate buffer in pH=7.1 (Figure 5A); 7.2 (Figure 5B); and 7.3 (Figure 5C).

Figure 6: shows a considerable decrease in LDH release following exposure to DP-b99 di-L- arginine or di-L-lysine non-covalent mixtures in comparison with the corresponding salts. DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, there are provided stable, water soluble compositions comprising lipophilic diesters of l,2-bis(2-aminophenoxy)ethane-N,N,N',N' -tetraacetic acid (BAPTA-DE) or their salts non-covalently associated with organic amine compounds, and in particular pharmaceutically acceptable non-covalent mixtures of 1 ,2-bis(2-aminophenoxy)ethane- Ν,Ν,Ν'Ν'-tetraacetic acid-N,N-di(2-octyloxyethyl ester) and of 1 ,2-bis(2-aminophenoxy)ethane- Ν,Ν,Ν'Ν'-tetraacetic acid-N,N-di(2-dodexyloxyethyl ester) or their salts, with amino acids, which are effective in the treatment of metal ion-associated disorders, such as stroke, while the risk of developing side effects associated with their administration are substantially lower than that of said lipophilic diester salts alone.

The term "stable" denotes any composition which possess stability sufficient to allow manufacture and which maintains the integrity of the composition for a sufficient period of time to be useful for the purposes detailed herein (e.g., administration to a subject, preferably intravenously). According to some embodiments, a stable non-covalent mixture is one that is not substantially altered when kept at a temperature of 25°C or less, in the absence of moisture or other chemically reactive conditions, for at least a week.

The compositions provided according to embodiments of the invention are stable and water soluble non-covalent mixtures of diesters of chelating agents of divalent metal ions with a physiologically acceptable organic amine. Preferably, said organic amine is an amino acid. The divalent metal ions include, but are not limited to, manganese, magnesium, copper, cobalt, and cadmium, more preferably zinc and ferrous ions, and most preferably calcium ions.

The term "chelating agent" as used herein denotes any molecule or anion capable of chelating divalent metal ions as known in the art.

According to one aspect of the invention, there are provided compositions comprising a lipophilic diester or salt thereof of the general Formula I:

CH ₂ COOM MOOCCH ₂ ROOCCH ₂ N C ₆ H4 OCH ₂ CH ₂ 0 C ₆ H4 N CH ₂ COOR

Formula 1 in non-covalent association with a physiologically acceptable organic amine, wherein a stoichiometric ratio of the organic amine and the lipophilic diester in the composition is at least 3: 1, wherein M denotes a hydrogen ion and/or a physiologically acceptable cation, and wherein R is selected from the group consisting of CnEfen ₊ i (n=l-10), C _n H2n ₊ i(OCH2CH2)m (n=l-20, m=l-6), (C _n H2n ₊ i)2N(CH2)m (n=l-6, m=l-6) and substituted or unsubstituted; the substituents on the aromatic rings being in the ortho position.

The term "substituted", as used herein refers to the replacement of hydrogen atoms of the aryl of ArC¾ with other functional groups. Such other functional groups include, but are not limited to, amino, hydroxyl, alkyl and derivatives thereof, carboxylic acids and derivatives thereof, and the like.

The term "non-covalent mixture", as used herein, refers to any physical combination of at least two discrete chemical entities. The term "non-covalent mixture" refers, in some embodiments, to an intimate interaction of the at least two chemical entities. The intimate interaction is of a non- covalent type. According to some embodiments, the at least two discrete chemical entities are present in the non-covalent mixture in a non-covalent association. The terms "non-covalent mixture" and "non-covalent association" are used herein to describe the interactions between the lipophilic diester of the general Formula I and the physiologically acceptable organic amines, in particular an amino acid, and can be used interchangeably.

According to particular embodiments, the term "non-covalent mixture" refers to any pharmaceutical composition having a lipophilic diester of the general Formula I or salt thereof, in non-covalent association with a physiologically acceptable organic amine, wherein the organic amine is present in a molar excess relative to the lipophilic diester, and wherein the molar excess is greater than 2 moles of the organic amine to 1 mole of the lipophilic diester. According to the currently preferred embodiments, the organic amine and the lipophilic diester are present in a stoichiometric ratio of at least 3: 1. According to further currently preferred embodiments, the term "non-covalent mixture" refers to any pharmaceutical composition having a lipophilic diester of the general Formula I or salt thereof, in non-covalent association with an amino acid.

According to some embodiments, the pharmaceutically acceptable organic amine is an organic compound displaying cell protective activity, such as neuroprotective activity. In this case and according to some further embodiments, a combined cell protective activity of both the BAPTA diesters (BAPTA-DE) and the organic amine may occur in vivo. The combined cell protective activity according to some embodiments may be additive or synergistic.

The term "cell protective activity" as used herein, refers to the ability to prevent, reduce, arrest or ameliorate a cellular insult.

The term "cellular insult" as used herein, refers to an organ or cellular damage, including, but not limited to a degeneration of nervous tissue, pancreatic tissue or acinar cells damage.

The term "neuroprotective activity" as used herein, refers to the effect of reducing, arresting or ameliorating an insult to a nervous tissue, and/or protecting, resuscitating, or reviving insulted nervous tissue.

The term "tissue insult" refers to any damaging process that affects the cells or tissue in a living body, caused by any negative impact on the organ, such as but not limited to, traumatic blow or force, loss or decrease of oxygen, loss or decrease of blood flow, radiation, infection (viral or bacterial), a toxin, a toxic drug, iatrogenic, thermal or chemical insult, and includes without limitation chronic or acute pathological states such as metabolic, excitotoxic, ischemic, hypoxic, diseases in particular of the nervous system including neurodegenerative diseases or psychotic disease or gastrointestinal system disease, such as pancreatitis.

According to other embodiments, the physiologically acceptable organic amine is devoid of biological activity.

According to some embodiments, the physiologically acceptable organic amines of the non-covalent mixtures of the invention are selected from organic non-toxic molecules. The term "organic amine", as used herein, refers in some embodiments to primary amines, secondary amines, tertiary amines or quaternary ammonium compounds. The term "quaternary ammonium", refers in some embodiments, to the positively charged form of said organic amines.

The term "organic amine", as used herein, refers in some embodiments to non-limiting examples of: organic amino cations of primary amines (R'Ntb ^"1" , such as methylammonium, ethylammonium and hydrazinium); organic amine cations derived from secondary amines (R'2NH2 ⁺ , such as dimethylammonium, diethylammonium, ethylmethylammonium and diethanolammonium); organic amine tertiary amines (RVNH ^"1" , such as trimethylammonium and triethylammonium); quaternary ammonium cations (R N ^"1" , such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, trimethyl ammonium compounds, glycine betaine, homarine, trigonelline, carnitine, gama-butyrobetaine, didecyldimethylammonium and pentamethylhydrazinium); and guanidinium cations ([CHeN3] ⁺ and derivatives thereof (for example (S)-2-ammonium-5-guanidinium valerate). According to embodiments of the invention R' can independently be any of alkyl-, alkenyl-, alkynyl-, substituted or unsubstituted aryl- and substituted or unsubstituted arylalkyl. The choice of the preferred R' residue or the specific organic amine that are appropriate for any given composition is dependent on the intended therapeutic use of the non-covalent mixture, and may be optimized by the artisan in accordance with the principles of the invention.

The organic amines according to some embodiments of the invention, can be cyclic such as for example piperazinium and derivatives thereof (non- limiting examples of piperazinium derivative cations include ranolazinium, trimetazidinium, alnespironium, amoxapinium, befuralinium, binospironium, buspironium, flesinoxaium, fluphenazinium, gepironium, ipsapironium, nefazodonium, piberalinium, 1-benzylpiperazinium, 1 ,4-dibenzylpiperazinium, 4- methyl- 1 -benzylpiperazinium, 3 ,4-methylenedioxy- 1 -benzylpiperazinium, 4- methoxyphenylpiperazinium, 4-fluorophenylpiperazinium, 1 -(2,3,4- trimethoxybenzyl)piperazinium, tandospironium, trazodonium, vilazodonium, and zalospironium).

The organic amines according to some embodiments of the invention comprise or can be derived from any organic base, preferably an organic base that contains at least one nitrogen atom, which can be easily protonated, such as amines and nitrogen-containing heterocyclic compounds. Non limiting examples include an amino acid (lysine, arginine, histidine and the like), substituted or unsubstituted imidazoles, substituted or unsubstituted pyridines and substituted and unsubstituted benzimidazoles. According to some currently preferred embodiments, the organic amine comprises amino acids. According to particular embodiments, the amino acid is a free amino acid. The term "free amino acid", as used herein, refers to an amino acid which is not a part of a peptide or a protein.

As used herein, the term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; and other unusual amino acids including, but not limited to, 2- aminoadipic acid and hydroxy lysine. Furthermore, the term "amino acid" includes both L- and D-amino acids. The amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. When there is no indication, either the L- or D- isomers of the amino acids may be used.

According to some currently preferred embodiments, the amino acid is naturally occurring. According to some embodiments, the amino acids suitable for the composition of the present invention include an amino side chain. According to some embodiments, the suitable amino acids include an alkaline side chain.

In some embodiments of the invention, the pharmaceutically acceptable organic amine is in a form of a cation, its conjugated salt (e.g. chloride salt, bromide salt, hydrochloride salt, hydrobromide salt) and/or its free base. In further embodiments, the amino acid is in a form of a cation, its conjugated salt (e.g. chloride salt, bromide salt, hydrochloride salt, hydrobromide salt) and/or its free base.

According to some embodiments, said pharmaceutically acceptable organic amine or amino acid in a form of a cation, its conjugated salt and/or its free base, is an organic compound displaying cell protective activity, and in particular neuroprotective activity, by itself. For example L-lysine monohydrochloride was shown to have neuroprotective effect of on acute iterative anoxia (Hong-Ping G and Bao-Shan KU. (1999) Life Sci. 65(2):PL19-25) and reduce the effects of cerebral ischemic insults while inhibiting glutamate-induced neuronal activity (Kondoh T, et al., (2010) Front Integr Neurosci. 4: 18). Taurine has been implicated in a wide array of physiological phenomena including inhibitory neurotransmission, long-term potentiation in the striatum/hippocampus, membrane stabilization, feedback inhibition of neutrophil/macrophage respiratory burst, adipose tissue regulation and possible prevention of obesity, calcium homeostasis, recovery from osmotic shock, protection against glutamate excitotoxicity and prevention of epileptic seizures.

According to other embodiments, the pharmaceutically acceptable organic amine or amino acid in a form of a cation, its conjugated salt and/or its free base, is physiologically inert by itself.

Persons skilled in the art will appreciate in what manner the concept of the invention may be applied to conditions and diseases which are related to abnormal levels of divalent metal ions, particularly zinc and calcium ions, so that the compositions according to the invention will comprise a stable and water soluble non-covalent mixture of an organic amine with a diester of an active compound which is a metal ion-chelator but which will possess optimized pharmacological activity.

According to some embodiments, the pharmaceutically acceptable organic amines of the invention are derived from organic non-toxic bases or salts selected from the group consisting of arginine, lysine, aminoguanidine (pimagedine), trimetazidine, taurine sulfonamide, choline and histamine. According to further embodiments, the amino acids of the invention are selected from the group consisting of L-arginine, L-lysine, D-arginine, D-lysine and combinations thereof. According to some currently preferred embodiments, the amino acids are selected from L-arginine, L-lysine and combinations thereof.

According to some embodiments, there are provided non-covalent mixtures of the lipophilic diester of the general Formula I with organic amines, or, preferably, amino acids, wherein the lipophilic diester is in a form of salt.

The lipophilic diester salt can be any pharmaceutically acceptable salt of the lipophilic diester of general Formula I. According to some embodiments, the salt of the lipophilic diester comprises one physiologically acceptable cation and one mono-anion of the lipophilic diester. In these embodiments, M denotes a hydrogen ion and a physiologically acceptable cation. In other embodiments, the salt of the lipophilic diester comprises two physiologically acceptable cations and one dianion of the lipophilic diester. In these embodiments, M denotes a physiologically acceptable cation.

According to some embodiments, the physiologically acceptable cation is selected from inorganic or organic cations. According to some embodiments, the inorganic cation is a monovalent metal cation selected from the group consisting of a sodium cation (Na ⁺ ), lithium cation (Li ⁺ ), and potassium cation (K ⁺ ). Each possibility represents a separate embodiment of the invention. According to certain embodiments, the monovalent metal cation is a sodium cation. According to further embodiments, the salt of the lipophilic diester of the general Formula I is a disodium salt. According to some embodiments, the inorganic cation is ammonium (NH/t ⁺ ). According to some embodiments, the organic cation is an organic amine cation. According to some embodiments, the lipophilic diester salt comprises one or two organic amine cations. According to further embodiments the organic amine cation comprises organic amine cations derived from organic non-toxic molecules, including, without limitation, primary amines (R'NH3 ⁺ ); secondary amines (R'2NH2 ⁺ ); and tertiary amines (RVNH ^"1" ), or quaternary ammonium cations having the structure R N ^"1" . According to some embodiments, the organic amine cation is derived from organic non-toxic molecules selected from the group consisting of lysine, arginine, aminoguanidine (pimagedine), trimetazidine, taurine, choline, histamine, tromethamine and meglumine. According to some currently preferred embodiments, the organic amine cation is derived from an amino acid. According to some embodiments, the amino acid from which the organic amino cation is the same as the amino acid of the composition, being in a non-covalent association with the lipophilic diester of Formula I. According to other embodiments, said amino acid is different from the amino acid of the non-covalent mixture. According to certain embodiments, the organic cation comprises arginine or lysine cations. According to further embodiments, the salt of the lipophilic diester of the general Formula I is selected from the group consisting of mono-arginine, di-arginine, mono-lysine and di-lysine salts. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the stoichiometric ratio between the physiologically acceptable organic amine and the lipophilic diester of the general Formula I is above 3: 1. In some embodiments, said stoichiometric ratio is 4:1. In certain embodiments, said stoichiometric ratio is 5: 1. In some exemplary embodiments, said stoichiometric ratio is 6: 1. According to some embodiments, said stoichiometric ratio is above 6:1.

The choice of the particular stoichiometric ratio between the organic amine or an amino acid and the lipophilic diester or salt thereof is determined partly by the desired non-covalent mixture characteristics, such as, but not limited to water solubility at various pH conditions and stability thereof.

According to some embodiments, the stoichiometric ratio between the amino acid and the lipophilic diester of the general Formula I is above 3:1. In some exemplary embodiments, said stoichiometric ratio is 4:1. In certain embodiments, said stoichiometric ratio is 5:1. In further exemplary embodiments, said stoichiometric ratio is 6: 1. According to some embodiments, said stoichiometric ratio is above 6: 1.

According to some embodiments, the stoichiometric ratio between the amino acid and the lipophilic diester salt of the general Formula I is at least 3: 1. In certain such embodiments, the lipophilic diester salt comprises two physiologically acceptable cations, comprising an inorganic cation and a dianion of the lipophilic diester. In these embodiments, each M denotes a physiologically acceptable cation, comprising an inorganic cation. According to some embodiments, the inorganic cation comprises a monovalent metal cation. According to particular embodiments, the monovalent metal cation is a sodium cation and the salt of the lipophilic diester of the general Formula I is a disodium salt. According to other embodiments, the lipophilic diester salt comprises two physiologically acceptable cations, comprising an organic cation and a dianion of the lipophilic diester. In these embodiments, each M denotes a physiologically acceptable cation, comprising an organic cation. Said organic cation can be an organic amine cation, preferably an amino acid cation. In certain such embodiments, the amino acid cation of the lipophilic diester salt is different from the amino acid of the non-covalent mixture. In some embodiments, said stoichiometric ratio is above 3: 1. In some embodiments, said stoichiometric ratio is 4: 1. In certain embodiments, said stoichiometric ratio is 5: 1. In further embodiments, said stoichiometric ratio is 6:1. According to some embodiments, said stoichiometric ratio is above 6: 1.

According to some embodiments, the stoichiometric ratio between the amino acid and the lipophilic diester salt of the general Formula I is at least 2: 1. In certain such embodiments, the lipophilic diester salt comprises one physiologically acceptable cation, comprising an organic cation and one mono-anion of the lipophilic diester. In these embodiments, one M denotes a hydrogen ion and another M denotes a physiologically acceptable cation, comprising an organic cation. Said organic cation can be an organic amine cation, preferably an amino acid cation. Preferably, said amino acid cation of the lipophilic diester salt is the same as the amino acid of the non-covalent mixture. In some embodiments, said stoichiometric ratio is above 2:1. In some embodiments, said stoichiometric ratio is 3:1. In certain embodiments, said stoichiometric ratio is 4:1. In certain embodiments, said stoichiometric ratio is 5:1. In further embodiments, said stoichiometric ratio is above 5: 1.

According to some embodiments, the stoichiometric ratio between the amino acid and the lipophilic diester salt of the general Formula I is at least 1: 1. In certain such embodiments, the lipophilic diester salt comprises two physiologically acceptable cations, comprising an organic cation and one di-anion of the lipophilic diester. In these embodiments, one M denotes a hydrogen ion and another M denotes a physiologically acceptable cation, comprising an organic cation. Said organic cation can be an organic amine cation, preferably an amino acid cation. In certain such embodiments, the amino acid of the lipophilic diester salt is the same as the amino acid of the non-covalent mixture. In some embodiments, said stoichiometric ratio is above 1 : 1. In some embodiments, said stoichiometric ratio is 2:1. In certain embodiments, said stoichiometric ratio is 3: 1. In certain embodiments, said stoichiometric ratio is 4: 1. In further embodiments, said stoichiometric ratio is above 4: 1.

According to some embodiments, there are provided non-covalent mixtures of the lipophilic diesters of the general Formula I with organic amines, or, preferably, amino acids, wherein the lipophilic diester is in a form of a free acid.

According to some embodiments, the physiologically acceptable amine or, preferably, an amino acid and the lipophilic diester or salt thereof, of the general Formula I form a non-covalent complex. According to further embodiments, the amino acid is associated with the lipophilic diester or salt thereof in the non-covalent complex via electrostatic bonds (i.e., hydrogen bonds), van der Waals forces, or hydrophobic-hydrophilic interactions. Each possibility represents a separate embodiment of the invention. According to some embodiments, the complexes are formed at a particular stoichiometric ratio between the amino acid and the lipophilic diester. According to some embodiments, said stoichiometric ratio is 6: 1. According to some embodiments, the complex is characterized by interaction between the lipophilic diester or salt thereof of the general Formula I and the organic amine, or, preferably, an amino acid, which leads to elimination of endothermic peaks corresponding to the lipophilic diester salt, appearance of new peaks, and/or changes in peak shape, peak temperature/melting point and/or relative peak area. Each possibility represents a separate embodiment of the invention According to some embodiments, the complex is characterized by deviation of at least one melting temperature onset as compared to the corresponding melting temperature onset of the lipophilic diester salt of at least about 3%. According to some embodiments, the melting temperature onset deviation is about 5%, 7.5%, 10%, 12.5%, 15 %, 20%, 25% or even 30%. Each possibility represents a separate embodiment of the invention.

In some embodiments, the present invention relates to pharmaceutically acceptable organic amine non-covalent complexes of lipophilic diesters of the general Formula I, comprising six organic amine ligands and one molecule of the lipophilic diester. In other embodiments, the non-covalent complex comprises at least four organic amine ligands and a lipophilic diester salt. In particular embodiments, the non-covalent complex comprises four organic amine molecules and one lipophilic diester salt. In other particular embodiments, the non-covalent complex comprises three organic amine molecules and one lipophilic diester salt. In yet other particular embodiments, the non-covalent complex comprises two organic amine molecules and one lipophilic diester salt. The organic amino cations of the lipophilic diester salt and the organic amine compound ligands may be same or different. In some embodiments, the organic amine compound ligands are same as the organic amine cations of the salt.

According to some embodiments, R of the lipophilic diester is selected from the group consisting of: C ₂ H ₅ , C3H7 , 1-C3H7 , C4H9 , C ₇ H ₁₅ , C ₈ H ₁₇ , CH ₂ C ₆ H ₅ , CH3OCH2CH2 , C2H5OCH2CH2 , C3H7OCH2CH2 , C4H9OCH2CH2 , C7H15OCH2CH2 , C8H17OCH2CH2 , C10H21OCH2CH2 , C12H25OCH2CH2 , C16H33OCH2CH2 , C18H37OCH2CH2 ,CH ₃ (OCH ₂ CH2)2 , C2H ₅ (OCH ₂ CH2)2 , C ₄ H ₉ (OCH2CH2) ₂ , C ₇ H ₁₅ (OCH2CH2) ₂ , C ₈ Hi ₇ (OCH2CH2) ₂ , CioH2i(OCH ₂ CH2)2 , CH ₃ (OCH ₂ CH2)3 , (CH ₃ )2NCH ₂ CH2, C ₇ H ₁₅ (OCH ₂ CH2)3 .

According to some embodiments, R is selected from the group consisting of: C2H5, C3H7, C4H9, C7H15, CsHn, C8H17OCH2CH2, C10H21OCH2CH2, C12H25OCH2CH2, C16H33OCH2CH2, C18H37OCH2CH2, C ₈ Hi ₇ (OCH2CH2) ₂ , CioH2i(OCH ₂ CH2) ₂ . According to certain preferred embodiments, R is CsHn or CsHnOCthCtb. According to some exemplary embodiments, R is C8H17OCH2CH2. According to additional embodiments, R is C12H25OCH2CH2.

According to some embodiments, the present invention relates to non-covalent mixtures of lipophilic diesters or salts thereof, of the general Formula I, with an organic amine, wherein R is C8H ₁ 7OCH2CH2 and the organic amine is derived from organic non-toxic bases or salts selected from the group consisting of arginine, lysine, aminoguanidine (pimagedine), trimetazidine, taurine sulfonamide, choline and histamine. Each possibility represents a separate embodiment of the invention. According to further embodiments, the present invention relates to non-covalent mixtures of lipophilic diesters or salts thereof, of the general Formula I, with an amino acid, wherein R is CsHnOCFhCFb and the amino acid is selected from the group consisting of arginine, lysine, and combinations thereof. Each possibility represents a separate embodiment of the invention. According to other embodiments, the present invention relates to non-covalent mixtures of lipophilic diesters or salts thereof, of the general Formula I, with organic amines, wherein R is C12H25OCH2CH2 and the organic amine is derived from organic non-toxic bases or salts selected from the group consisting of the organic amine salts of arginine, lysine, aminoguanidine (pimagedine), trimetazidine, taurine sulfonamide, choline and histamine. Each possibility represents a separate embodiment of the invention. According to further embodiments, the present invention relates to non-covalent mixtures of lipophilic diesters or salts thereof, of the general Formula I, with an amino acid, wherein R is C12H25OCH2CH2 and the amino acid is selected from the group consisting of arginine, lysine, and combinations thereof.

According to further embodiments, the present invention relates to non-covalent mixtures of lipophilic diesters or salts thereof, of the general Formula I, with amino acids, wherein R is C8H ₁ 7OCH2CH2, the amino acid is selected from the group consisting of arginine, lysine, and combinations thereof and the stoichiometric ratio between the amino acid and the lipophilic diester is 6: 1. According to further embodiments, the present invention relates to non-covalent mixtures of lipophilic diesters or salts thereof, of the general Formula I, with amino acids, wherein R is C12H25OCH2CH2, the amino acid is selected from the group consisting of arginine, lysine, and combinations thereof and the stoichiometric ratio between the amino acid and the lipophilic diester is 6:1.

The water-soluble non-covalent mixtures of organic amines, or, preferably, amino acids with the lipophilic diesters or salts thereof, of the general Formula I, according to the principles of the current invention, display several advantages in comparison with the sodium or organic amine salts of said diesters, which are not present in combination with said organic amine or amino acid. According to some embodiments, the organic amine-lipophilic diester non-covalent mixtures of the present invention are soluble in aqueous solutions at room temperature and at a pH lower than 8.0, even more preferably at a pH lower than 7.5.

According to some embodiments, the aqueous solutions of the non-covalent mixtures of the invention are stable at a physiological pH and in particular in the range of 7.35 - 7.45, while the sodium salts of DP-b99 tend to precipitate in aqueous solutions having a pH lower than 7.5. Obtaining a water soluble DP-b99 composition which is stable at physiological pH is thus of a great advantage.

In further embodiments, the non-covalent mixtures of the present invention are soluble in aqueous solutions at room temperature and at a pH between 7.0-7.45. In yet further embodiments, the solubility of the non-covalent mixtures of the present invention at a physiological pH, and in particular in the range of 7.0-7.4, is higher than the solubility of the di- sodium salts of the lipophilic diesters of the general Formula I. In still further embodiments, the solubility of the non-covalent mixtures of the present invention at a physiological pH, and in particular in the range of 7.1-7.4, is higher than the solubility of the organic amine salts of lipophilic diesters of the general Formula I. In some embodiments, said non-covalent mixtures and said salt comprise the same organic amines. In certain embodiments, the solubility of the non-covalent mixture of the lipophilic diester or salt thereof, of the general Formula I, with arginine or lysine in aqueous solutions at room temperature and at a pH between 7.1-7.4 is higher than that of the corresponding mono-arginine, di-arginine and di-lysine salts. In additional embodiments, the solubility of the non-covalent mixture in aqueous solutions at room temperature and at a pH as low as 7.0 is higher than that of the corresponding mono-arginine salt.

In further embodiments, the non-covalent mixtures of the present invention are soluble in a hydrochloric solution (HC1) having a molar concentration of about 0.7 mM, preferably in an HC1 solution of about 7 mM, more preferably in an HC1 solution of about 21 mM HC1. Each possibility represents a separate embodiment of the invention. In yet further embodiments, the solubility of the non-covalent mixtures of the present invention in hydrochloric solutions having a molar concentration of about 5 mM to about 25 mM is higher than that of the di-sodium salts of the lipophilic diesters of the general Formula I. In further embodiments, the solubility of the non-covalent mixtures of the present invention in hydrochloric solutions having a molar concentration of about 5 mM to about 25 mM is higher than that of the organic amine salts of the lipophilic diesters of the general Formula I. In some embodiments, said non-covalent mixtures and said salt comprise the same organic amino cations. In certain embodiments, the solubility of the non-covalent mixture of the lipophilic diester or salt thereof, of the general Formula I with arginine or lysine in HC1 solutions having a molar concentration of about 5 mM to about 25 mM is higher than that of the corresponding mono-arginine, di-arginine and di-lysine salts.

According to further embodiments, the non-covalent mixtures of the present invention are soluble in human plasma. Human plasma is characterized by a pH of about 7.3. In certain embodiments, the non-covalent mixtures are soluble in human plasma for at least two hours, preferably for at least 5 hours, more preferably for at least 10 hours, even more preferably for at least 15 hours. Each possibility represents a separate embodiment of the invention. According to yet further embodiments, the solubility of the non-covalent mixtures of the present invention in human plasma is higher than that of the di-sodium salts of the lipophilic diesters of the general Formula I. According to the preferred embodiments, the solubility of the non-covalent mixtures of the present invention in human plasma is higher than that of the organic amine salts of the lipophilic diesters of the general Formula I. In some embodiments, said non-covalent mixture and said salt comprise the same organic amino cations. In certain embodiments, the solubility of the non-covalent mixture of the lipophilic diester or salt thereof, of Formula I, with arginine in human plasma is higher than that of the corresponding mono-arginine or di-arginine salts. In certain embodiments the solubility of the non-covalent mixture of the lipophilic diester or salt thereof, of the general Formula I with lysine in human plasma is higher than that of the corresponding di-lysine salt. According to the further embodiments, the solubility of the non- covalent mixtures of the present invention incubated for at least two hours in human plasma is higher than that of the disodium and organic amine salts of the lipophilic diesters of general Formula I.

In accordance with another aspect of the invention, there are provided pharmaceutical compositions comprising the composition comprising an organic amine non-covalently associated with a lipophilic diester or salt thereof, of the general Formula I, and a pharmaceutically acceptable diluent or carrier. According to some embodiments, the pharmaceutical composition comprises the non-covalent mixture according to the principles of the present invention, wherein R is C8H ₁ 7OCH2CH2 and the organic amine compound is derived from organic non-toxic molecules selected from the group consisting of arginine, lysine, aminoguanidine (pimagedine), trimetazidine, taurine sulfonamide, choline, tromethamine and histamine. Each possibility represents a separate embodiment of the invention. According to other embodiments, the pharmaceutical composition comprises a lipophilic diester of Formula I, wherein R is C12H25OCH2CH2 and the organic amine compound is derived from organic non-toxic molecules selected from the group consisting of arginine, lysine, aminoguanidine (pimagedine), trimetazidine, taurine sulfonamide, choline and histamine. Each possibility represents a separate embodiment of the invention. According to the currently preferred embodiments, the organic amine comprises an amino acid. In preferred embodiments, the organic amine compound comprises arginine, lysine or combinations thereof. Each possibility represents a separate embodiment of the invention. Pharmaceutical compositions comprising as an active ingredient the non-covalent mixtures according to the principles of the present invention and will contain in addition any pharmaceutically acceptable diluents or carriers. By "pharmaceutically acceptable carrier" is meant a component such as a carrier, vehicle, adjuvant, diluent, excipient, and the like of a composition that is compatible with the particular pharmacological agent and other optional ingredients of the subject pharmacological agent compositions in that a pharmaceutically acceptable carrier may be combined with the pharmacological agent without eliminating the biological or therapeutically effective activity of the pharmacological agent, and is suitable for use in subjects as provided herein without undue adverse side effects or mortality (such as toxicity, irritation and allergic response). Side effects are "undue" when their risk outweighs the benefit provided by the pharmacological agent.

The pharmaceutical compositions according to some embodiments of the invention may be in liquid or solid dosage forms, and may be formulated into any suitable formulations including but not limited to solutions, suspensions, aerosols, micelles, emulsions, microemulsions, tablets, and the like, as will be required for the appropriate route of administration. Any suitable route of administration is encompassed by the invention including, but not limited to, oral, intravenous, intra-arterial, intracerebral, epidural, intracerebroventricular, intramuscular, subcutaneous, inhalation, intranasal, rectal, topical or other known routes. According to some embodiments, the pharmaceutical compositions of the invention are administered enterally or parenerally. According to currently preferred embodiments, the pharmaceutical compositions are administered intravenously by infusion selected from central vein infusion and peripheral vein infusion. According to some embodiments, the pharmaceutical compositions are administered via central vein infusion. According to other embodiments, the pharmaceutical compositions are administered intra-arterially or intracelebrally. Each possibility represents a separate embodiment of the invention. According to alternative embodiments, the pharmaceutical compositions of the invention are administered orally. The orally administrable compositions may comprise an enteric coating.

The choice of carrier is determined partly by the particular active ingredient, as well as by the particular method used to administer the composition. Suitable formulations for parenteral administration preferably include aqueous solutions of the active compounds. Non-limiting examples of pharmaceutically acceptable carriers suitable for parenteral administration include, but are not limited to, any of the standard pharmaceutical carriers such as water, saline solutions (e.g. lactated Ringer's solution, acetated Ringer's solution, phosphate buffered saline solutions etc.).

Pharmaceutical compositions formulated for parenteral administration may be present in unit dose or multiple dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, e. g., water, for injections immediately prior to use.

In another aspect, the present invention relates to the non-covalent mixtures of organic amines, or, preferably, amino acids, with lipophilic diesters or salts thereof, of the general Formula I, and pharmaceutical compositions comprising same, which are useful in the treatment or prevention of metal ion-associated disorders for example, disorders associated with abnormal levels of manganese, magnesium, copper, iron, cadmium, cobalt, and in particular calcium and zinc ions. The administration of the non-covalent mixtures and pharmaceutical compositions comprising said non-covalent mixtures, according to the principles of the current invention allows minimizing the occurrence of undesired side effects associated with the administration of sodium or organic amine salts of the lipophilic diesters. Each possibility represents a separate embodiment of the invention.

In yet another aspect, the present invention provides a method for treating or preventing a disease or disorder related to an imbalance (e.g. excess or dyshomeostasis) of divalent metal ions, for example, disorders associated with abnormal levels of manganese, magnesium, copper, iron, cadmium, cobalt, lead, strontium and in particular calcium and zinc ions, comprising administering to an individual in need of such treatment a therapeutically effective amount of a stable, water-soluble non-covalent mixture of a lipophilic diester or salt thereof, of the general Formula I, with organic amines, preferably comprising amino acids. In some embodiments, the risk of developing undesirable side effects associated with the administration of the non-covalent mixtures of the present invention are reduced relative to the risk of developing side effects associated with the administration of the salts of the diesters of Formula I alone. In some embodiments, the risk of developing said undesirable side effects are reduced as compared to the administration of the sodium salts of the lipophilic diesters of Formula I. In other embodiments, the risk of developing said undesirable side effects are reduced as compared to the administration of the organic amine salts of the lipophilic diesters of Formula I. In further embodiments, the risk of developing said undesirable side effects are reduced as compared to the administration of the arginine salts of the diesters of Formula I. The arginine salt may be a mono- or a di-arginine salt. Each possibility represents a separate embodiment of the invention. In yet further embodiments, the risk of developing said undesirable side effects are reduced as compared to the administration of the lysine salts of the diesters of Formula I. The lysine salt may be a mono- or a di-lysine salt. Each possibility represents a separate embodiment of the invention. According to some embodiments, the methods of the invention comprise administration of a therapeutically effective amount of the non-covalent mixtures of the invention. According to some embodiments, the administration refers to an injection or infusion. According to other embodiments, the administration refers to an oral administration. According to some embodiments, the disease or disorder related to an imbalance of intracellular Ca ⁺⁺ and/or Zn ⁺⁺ ions, is selected from brain and cardiac ischemia, stroke, brain and spinal cord trauma, myocardial infarction, epilepsy, Alzheimer's disease, Parkinson's disease, acute inflammation, urinary incontinence, prostatic hypertrophy, muscular spasm, Lewy body dementia, acute inflammation, prostatic hypertrophy, muscular spasm, arterial hypertension, asthma, irritable bowel syndrome and pancreatitis. Each possibility represents a separate embodiment of the invention. According to particular embodiments, the present invention provides a method for the treatment of stroke. According to some embodiments, the stroke is an acute ischemic stroke. According to some further embodiments, the stroke is a hemispheric acute ischemic stroke. According to further particular embodiments, the present invention provides a method for the treatment of pancreatitis. According to some embodiments, pancreatitis includes acute, recurrent or chronic pancreatitis. According to particular embodiments, pancreatitis is an acute pancreatitis. According to some embodiments the undesirable side effect is phlebitis.

Phlebitis refers to the clinical finding of pain, tenderness, swelling, erythema, warmth and palpable cord-like veins due to inflammation (Fernandez L. Superficial phlebitis. In: Up-To- Date [online] 2004; 12.2:4. Available at: www.uptodate.com). Phlebitis is one of the most common complications of peripheral intravenous catheter use, which may occur in up to 75% of hospitalized patients (Sutariya B and Berk W. Vascular access. In: Tintinally J, Kelen G, Stapczynski S, eds. Emergency Medicine. 5th ed. New York: McGraw-Hill, 2000: 103-4).

According to some embodiments, the risk of developing phlebitis after the administration of the non-covalent mixtures of the organic amines with the lipophilic diesters or salts thereof, of general Formula I is reduced by at least 20% compared to the risk of developing phlebitis after the administration of the sodium salts of the diesters of general Formula I. Alternatively, the risk of developing phlebitis is reduced by at least 30% , 40%, 50%, 60%, 70%, or even 80% compared to the risk of developing phlebitis after the administration of the sodium salts of the diesters of general Formula I. Each possibility represents a separate embodiment of the invention.

According to other embodiments, the risk of developing phlebitis after the administration of the non-covalent mixtures of the organic amines with the lipophilic diesters or salts thereof, of the general Formula I is reduced by at least 20% compared to the risk of developing phlebitis after the administration of the organic amine salts of the diesters of general Formula I. Alternatively, the risk of developing phlebitis is reduced by at least 30% , 40%, or even 50% compared to the risk of developing phlebitis after the administration of the organic amine salts of the diesters of general Formula I. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the present invention provides methods for preventing or reducing brain damage due to a stroke comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine with a lipophilic diester or salt thereof, of the general Formula I. In some embodiments, the risk of developing undesirable side effects associated with the administration of said non-covalent mixture are reduced relative to the risk of developing side effects associated with the administration of the salts of the diesters of the general Formula I. In some embodiments, the risk of developing said undesirable side effects are reduced as compared to the administration of the sodium salts of the diesters of the general Formula I. In other embodiments, the risk of developing said undesirable side effects are reduced as compared to the administration of the organic amine salts of the diesters of the general Formula I. According to some embodiments, the undesirable side effect is phlebitis. According to particular embodiments, the present invention provides methods for preventing or reducing brain damage due to a stroke comprising administering to a patient a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine, with a lipophilic diester or salt thereof, of the general Formula I, wherein R is C8H ₁ 7OCH2CH2. According to other particular embodiments, the present invention provides methods for preventing or reducing brain damage due to a stroke comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non- covalent mixture of an organic amine with a lipophilic diester or salt thereof, of the general Formula I, wherein R is C ₁ 2H25OCH2CH2. According to further particular embodiments, the present invention provides methods for preventing or reducing brain damage due to a stroke comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine with a lipophilic diester or salt thereof, of the general Formula I, wherein R is C8H ₁ 7OCH2CH2 and the organic amines are derived from organic non-toxic molecules selected from the group consisting of arginine, lysine, aminoguanidine (pimagedine), trimetazidine, taurine sulfonamide, choline and histamine. Each possibility represents a separate embodiment of the invention. According to yet further particular embodiments, the present invention provides methods for preventing or reducing brain damage due to a stroke comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine with a lipophilic diester or salt thereof, of the general Formula I, wherein R is C12H25OCH2CH2 and the organic amines are derived from organic non-toxic molecules selected from the group consisting of arginine, lysine, aminoguanidine (pimagedine), trimetazidine, taurine sulfonamide, choline and histamine. Each possibility represents a separate embodiment of the invention. According to the currently preferred embodiments, the present invention provides methods for preventing or reducing brain damage due to a stroke comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an amino acid with the lipophilic diester or salt thereof, of the general Formula I, wherein R is C8H17OCH2CH2 or C12H25OCH2CH2. Each possibility represents a separate embodiment of the invention. Said amino acid preferably comprises arginine, lysine or a combination thereof. According to further embodiments, the stoichiometric ratio between the organic amine and the lipophilic diester is 6:1 or 4:1. According to further embodiments, the stoichiometric ratio between the amino acid and the lipophilic diester is 6:1.

According to some embodiments, the present invention provides methods for preventing or reducing neurological deficits due to a stroke comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine with a lipophilic diester or salt thereof, of the general Formula I. In some embodiments, the risk of developing undesirable side effects associated with the administration of said non-covalent mixture are reduced relative to the risk of developing side effects associated with the administration of the salts of the diesters of the general Formula I. In some embodiments, the risk of developing said undesirable side effects are reduced as compared to the administration of the sodium salts of the diesters of the general Formula I. In other embodiments, the risk of developing said undesirable side effects are reduced as compared to the administration of the organic amine salts of the diesters of the general Formula I. According to some embodiments, the undesirable side effect is phlebitis. According to particular embodiments, the present invention provides methods for preventing or reducing neurological deficits due to a stroke comprising administering to a patient a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine, with a lipophilic diester or salt thereof, of the general Formula I, wherein R is CsHnOCFhCFh. According to other particular embodiments, the present invention provides methods for preventing or reducing neurological deficits due to a stroke comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine with a lipophilic diester or salt thereof, of the general Formula I, wherein R is C ₁ 2H25OCH2CH2. According to further particular embodiments, the present invention provides methods for preventing or reducing neurological deficits due to a stroke comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine with a lipophilic diester or salt thereof, of the general Formula I, wherein R is CsHnOCFbCFb and the organic amines are derived from organic non-toxic molecules selected from the group consisting of arginine, lysine, aminoguanidine (pimagedine), trimetazidine, taurine sulfonamide, choline and histamine. Each possibility represents a separate embodiment of the invention. According to yet further particular embodiments, the present invention provides methods for preventing or reducing neurological deficits due to a stroke comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine with a lipophilic diester or salt thereof, of the general Formula I, wherein R is C ₁ 2H25OCH2CH2 and the organic amines are derived from organic non-toxic molecules selected from the group consisting of arginine, lysine, aminoguanidine (pimagedine), trimetazidine, taurine sulfonamide, choline and histamine. Each possibility represents a separate embodiment of the invention. According to the currently preferred embodiments, the present invention provides methods for preventing or reducing neurological deficits due to a stroke comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an amino acid with the lipophilic diester or salt thereof, of the general Formula I, wherein R is CsHnOCFhCFh or C12H25OCH2CH2. Each possibility represents a separate embodiment of the invention. Said amino acid preferably comprises arginine, lysine or a combination thereof. According to further embodiments, the stoichiometric ratio between the organic amine and the lipophilic diester is 6: 1 or 4: 1. According to further embodiments, the stoichiometric ratio between the amino acid and the lipophilic diester is 6: 1.

The term "brain damage" as used herein refers to neuronal and/or glial injury or neuronal or glial cell death whereas the term "neurological deficits" refer to a functional abnormalities, disabilities or pathological conditions of apparent neurological origin.

According to some embodiments, the present invention provides methods for preventing or reducing cellular damage due to pancreatitis comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine with a lipophilic diester or salt thereof, of the general Formula I. In some embodiments, the risk of developing undesirable side effects associated with the administration of said non-covalent mixture are reduced relative to the risk of developing side effects associated with the administration of the salts of the diesters of the general Formula I. In some embodiments, the risk of developing said undesirable side effects are reduced as compared to the administration of the sodium salts of the diesters of the general Formula I. In other embodiments, the risk of developing said undesirable side effects are reduced as compared to the administration of the organic amine salts of the diesters of the general Formula I. According to some embodiments, the undesirable side effect is phlebitis. According to particular embodiments, the present invention provides methods for preventing or reducing cellular damage due to pancreatitis comprising administering to a patient a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine, with a lipophilic diester or salt thereof, of the general Formula I, wherein R is CsHnOCFhCFb. According to other particular embodiments, the present invention provides methods for preventing or reducing cellular damage due to pancreatitis comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine with a lipophilic diester or salt thereof, of the general Formula I, wherein R is C ₁ 2H25OCH2CH2. According to further particular embodiments, the present invention provides methods for preventing or reducing cellular damage due to pancreatitis comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine with a lipophilic diester or salt thereof, of the general Formula I, wherein R is CsHnOCFbCFb and the organic amines are derived from organic non-toxic molecules selected from the group consisting of arginine, lysine, aminoguanidine (pimagedine), trimetazidine, taurine sulfonamide, choline and histamine. Each possibility represents a separate embodiment of the invention. According to yet further particular embodiments, the present invention provides methods for preventing or reducing cellular damage due to pancreatitis comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non-covalent mixture of an organic amine with a lipophilic diester or salt thereof, of the general Formula I, wherein R is C12H25OCH2CH2 and the organic amines are derived from organic non-toxic molecules selected from the group consisting of arginine, lysine, aminoguanidine (pimagedine), trimetazidine, taurine sulfonamide, choline and histamine. Each possibility represents a separate embodiment of the invention. According to the currently preferred embodiments, the present invention provides methods for preventing or reducing cellular damage due to pancreatitis comprising administering to a patient in need of such treatment a therapeutically effective amount of a stable water-soluble non- covalent mixture of an amino acid with the lipophilic diester or salt thereof, of the general Formula I, wherein R is CsHnOCFhCFh or C12H25OCH2CH2. Each possibility represents a separate embodiment of the invention. Said amino acid preferably comprises arginine, lysine or a combination thereof. According to further embodiments, the stoichiometric ratio between the organic amine and the lipophilic diester is 6: 1 or 4: 1. According to further embodiments, the stoichiometric ratio between the amino acid and the lipophilic diester is 6: 1.

The therapeutically effective amount of the pharmaceutical compositions comprising the non-covalent mixtures of the present invention is to produce the desired protective effect. According to some embodiments, administration refers to an injection or infusion. According to some embodiments, the compositions of the present invention are intravenously administered to a stroke patient in from 1 to 7, preferably 2 to 4, more preferably 4 separate intravenous infusions, the total dosage regimen given in one intravenous infusion being from 1.0 mg/kg to about 3.0 mg/kg of body weight. According to some embodiments, the total daily dosage regimen is from about 0.3 to about 9 mg/kg. The methods of the invention comprise administration of at least the first intravenous infusion within 12 hours, preferably within 9 hours after the onset of the insult. The term "onset of an insult" is defined as the time that the subject was last seen in a normal state, or bedtime for unwitnessed insults occurring during sleep. According to currently preferred embodiments, the pharmaceutical compositions are intravenously administered up to 3mg/kg/infusion, three times per day, for up to 10 consecutive days, the daily dosage being up to about 9 mg/kg of body weight, wherein the first dose is administered within 9 hours after the onset of the insult.

According to some embodiments, the compositions of the present invention are intravenously administered to a pancreatitis patient in from 1 to 10, preferably 4 to 8, separate intravenous infusions, the total dosage regimen given in one intravenous infusion being from 1.0 mg/kg to about 3.0 mg/kg of body weight. According to some embodiments, the total daily dosage regimen is from about 0.3 to about 9 mg/kg. The methods of the invention comprise administration of at least the first intravenous infusion within 72 hours, preferably within 48 hours of acute pancreatitis symptoms onset. According to currently preferred embodiments, the pharmaceutical compositions are intravenously administered up to 3mg/kg/infusion, three times per day, for up to 10 consecutive days, the daily dosage being up to about 9 mg/kg of body weight, wherein the first dose is administered within 48 hours after the acute pancreatitis symptoms onset.

The term "cellular damage" as used herein refers to changes within the pancreatic acinar cells which are common in acute pancreatitis. Key amongst these changes is premature intracellular activation of digestive enzymes. This is also accompanied by the appearance of cytosolic vacuoles, co-localization of digestive and lysosomal enzymes, activation of NF-κΒ, and release of proinflammatory cytokines. Although the exact mechanism responsible for enzyme activation is unknown, it is clear that all of these changes are triggered by an abnormal, sustained rise in cytosolic calcium concentration, which is itself dependent both on release of calcium from endoplasmic reticulum stores and uptake from the extracellular milieu. Activated enzymes are directly damaging to the acinar cell themselves, but recruitment of circulating neutrophils leads to further cellular damage. Cytokines and neutrophil activation are also responsible for the systemic inflammatory response typically seen in severe acute pancreatitis.

The low tendency of the non-covalent mixtures of the present invention to cause venous inflammation and in particular phlebitis enables according to some embodiments their introduction into a blood vessel over extended periods of time. According to particular embodiments, their low tendency to cause venous inflammation enables their administration into a single blood vessel through the course of the treatment avoiding the need to change the administration site on a daily basis during the course of the treatment. According to some embodiments, the administration site is a peripheral vein. According to some currently preferred embodiments, the compositions of the invention are administered into a central vein.

The pharmaceutical compositions comprising the non-covalent mixtures of the invention are preferably administered by intravenous infusion in the form of a 0.1- 3 mg/ml solution in sterile physiological saline. According to some embodiments, the non-covalent mixtures of the present invention may be diluted prior to injection or infusion in pharmaceutically acceptable solutions. Organic amine compounds may be further added to the solution while keeping the pH and osmolarity of the solution within the physiological range. In the preferred embodiments, the cation of said organic amine is similar to the organic amine contained in the non-covalent mixture to be injected.

According to some embodiments, administration refers to chronic administration. In certain embodiments, the non-covalent mixtures and the pharmaceutical compositions of the present invention are administered orally. The non-covalent mixtures and the pharmaceutical compositions of the present invention can be administered to stroke patients and/or pancreatitis patients. Each possibility represents a separate embodiment of the invention. In particular embodiments, the pancreatitis patient is a chronic pancreatitis patient. The pharmaceutical compositions of the present invention can be orally-administered at a dose of about 100-500 mgs/patient/day.

According to another aspect, there is provided a kit for the treatment or prevention of a disease or disorder related to an imbalance of divalent metal ions, the kit comprising, in separate containers, a lipophilic diester or salt thereof, of the general Formula I, wherein M denotes a hydrogen ion and/or a physiologically acceptable cation, and wherein R is selected from the group consisting of C _n H _2n+ i (n=l-10), CnH2n ₊ i(OCH ₂ CH2) _m (n=l-20, m=l-6), (CnH2n ₊ i)2N(CH ₂ )m (n=l-6, m=l-6) and substituted or unsubstituted ArCth; the substituents on the aromatic rings being in the ortho position, and a pharmaceutically acceptable diluent or carrier comprising a physiologically acceptable organic amine, wherein the molar ratio between the organic amine and the lipophilic diester or salt thereof is at least 1 : 1. According to the preferred embodiments, said organic amine comprises a free amino acid. The free amino acid can be preferably selected from the group consisting of arginine, lysine and combinations thereof. According to some embodiments, the pharmaceutically acceptable organic amine or amino acid is in a form of a cation, its conjugated salt and/or its free base. The kit for the treatment or prevention of a disease or disorder related to an imbalance of divalent metal ions, can be used for the preparation of the non-covalent mixture of the present invention, by combining the containers of said kit. The kit can thus advantageously be used for the preparation of the highly water-soluble non-covalent mixture or the pharmaceutical composition comprising said mixture, from a less water-soluble lipophilic diester or salt thereof. According to certain embodiments, the kit is used for the preparation of the non-covalent mixture of the present invention or the pharmaceutical composition comprising said mixture, at the patient's bedside. According to some embodiments, the non-covalent mixture the pharmaceutical composition comprising said mixture can be prepared by combining the containers of the kit in less than about 4 hours, preferably less than about 3 hours, more preferably less than about 2 hours, even more preferably less than about 1 hour, most preferably less than about 30 minutes. Each possibility represents a separate embodiment of the invention. Without wishing to being bound by theory, the choice of the particular molar ratio between the organic amine or a free amino acid comprised in the pharmaceutically acceptable carrier or diluent, and the lipophilic diester or salt thereof can be determined partly by the desired non-covalent mixture characteristics, such as, but not limited to water solubility of the non-covalent mixture at various pH conditions and stability thereof.

According to some embodiments, the kit comprises a lipophilic diester or salt thereof, salt of the general Formula I, wherein each M denotes a physiologically acceptable cation, comprising an organic amine cation. According to some embodiments, the organic amine cation is derived from organic non-toxic molecules selected from the group consisting of lysine, arginine, aminoguanidine (pimagedine), trimetazidine, taurine, choline, histamine, tromethamine and meglumine. According to some embodiments, the organic amine cation is similar to the organic amine cation comprised in the pharmaceutically acceptable diluent or carrier. In certain such embodiments, the molar ratio between the organic amine and the lipophilic diester salt is at least 1 :1. The molar ratio can further be selected to be 2:1, 3: 1, or 4: 1. According to some embodiments, the molar ratio is above 4: 1. According to the currently preferred embodiments, the physiologically acceptable cation, comprising an organic amine cation, comprises an amino acid cation. Said amino acid cation can be preferably selected from the group consisting of arginine, lysine and combinations thereof. According to some embodiments, the kit comprises a lipophilic diester or salt thereof, of the general Formula I, wherein one M denotes a physiologically acceptable cation, comprising an organic amine cation and another M denotes a hydrogen ion. According to some embodiments, the organic amine cation is derived from organic non-toxic molecules selected from the group consisting of lysine, arginine, aminoguanidine (pimagedine), trimetazidine, taurine, choline, histamine, tromethamine and meglumine According to some embodiments, the organic amine cation is similar to the organic amine cation comprised in the pharmaceutically acceptable diluent or carrier. In certain such embodiments, the molar ratio between the organic amine and the lipophilic diester salt is at least 2:1. The molar ratio can further be selected to be 3: 1, 4:1 or 5: 1. According to some embodiments, the molar ratio is above 5: 1. According to the currently preferred embodiments, the physiologically acceptable cation, comprising an organic amine cation, comprises an amino acid cation. Said amino acid cation can be preferably selected from the group consisting of arginine, lysine and combinations thereof.

According to some embodiments, the kit comprises a lipophilic diester or salt thereof, of the general Formula I, wherein each M denotes a physiologically acceptable cation, comprising an organic amine cation or a monovalent metal cation. Each possibility represents a separate embodiment of the invention. According to some embodiments, the organic amine cation is derived from organic non-toxic molecules selected from the group consisting of lysine, arginine, aminoguanidine (pimagedine), trimetazidine, taurine, choline, histamine, tromethamine and meglumine. According to some embodiments, the organic amine cation is different from the organic amine cation comprised in the pharmaceutically acceptable diluent or carrier. According to some embodiments, the monovalent metal cation selected from the group consisting of a sodium cation (Na ⁺ ), lithium cation (Li ⁺ ), and potassium cation (K ⁺ ). Each possibility represents a separate embodiment of the invention. According to certain embodiments, the monovalent metal cation is a sodium cation. In certain such embodiments, the molar ratio between the organic amine and the lipophilic diester salt is at least 3: 1. The molar ratio can further be selected to be 4: 1, 5: 1, or 6: 1. According to some embodiments, the molar ratio is above 6: 1. According to the currently preferred embodiments, the physiologically acceptable cation, comprising an organic amine cation, comprises an amino acid cation. Said amino acid cation can be selected from the group consisting of arginine, lysine and combinations thereof. According to some embodiments, the kit comprises a lipophilic diester or salt thereof, of the general Formula I, wherein each M denotes a hydrogen ion. In certain such embodiments, the molar ratio between the organic amine and the lipophilic diester is at least 3: 1. The molar ratio can further be selected to be 4: 1 , 5: 1, or 6: 1. According to some embodiments, the molar ratio is above 6: 1.

As used herein, the term "about", when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +/-10%, more preferably +1-5%, even more preferably +/-1 , and still more preferably +/-0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

The examples hereinbelow are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art may readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

EXAMPLE 1: Synthesis of l,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid- N,N'-di[(2-octyloxy)ethyl ester] -2,6-diammonium hexaate non-covalent mixture

Method 1 (starting from a diester precursor):

BAPTA diesters (e.g. alkyl or aryl diester of BAPTA and monoalkyl ethers of mono-, di- and triethylene glycol diesters of BAPTA diesters, were prepared as described in the International Patent Publication No. WO 99/16741.

1 g (1.27 mmol) l,2-bis(2-aminophenoxy)ethane-N,N,N' ,N'-tetraacetic acid-N,N'-di[(2- octyloxy) ethyl ester] was added to the solution of 1.1 g (7.62 mmol) 2,6-diaminohexanoic acid (L-lysine) in 30 ml water. The reaction mixture was stirred at room temperature until a clear solution was obtained. The final pH of reaction was 9.8. The clear solution was lyophilized. Yield was 2.18 g (white solid powder).

Method 2 (starting from an organic amine salt precursor):

Lysine salt of BAPTA diesters was prepared as follows: a suspension of 1,2-bis (2- aminophenoxy)ethane-N,N,N',N' -tetraacetic acid-N,N'-di[(2-octyloxy)ethyl ester] (5g, 6.3 mmol) in water (100 ml) was titrated at room temperature under Ar with water solution (40 ml) of 2,6- diaminohexanoic acid monohydrate (lysine) (2.28 g, 13.9 mmol). The final pH of reaction solution was 9.8. The solution obtained was filtered through an analytical filter (Acrodisk 0.45 μπι) and lyophilized. Yield was 6.26 (white solid, 92%).

A solution of 0.70 g (4.79 mmol) lysine was added to a solution of 1.09 g (1 mmol) 1,2- bis(2-aminophenoxy)ethane-N,N,N' ,N'-tetraacetic acid-N,N'-di[(2-octyloxy) ethyl ester]-di- lysine salt in 40 ml water. The reaction mixture was stirred for 5 hours at room temperature. The obtained clear solution was lyophilized.

Method 3 (starting from a sodium salt precursor):

A solution of 1.75 g (12 mmol) lysine was added to a solution of 0.83 g (1 mmol) 1 ,2- bis(2-aminophenoxy)ethane-N,N,N' ,N'-tetraacetic acid-N,N'-di[(2-octyloxy) ethyl ester]-di- sodium salt in 40 ml water. The reaction mixture was stirred at room temperature until a clear solution was obtained. This solution was lyophilized.

EXAMPLE 2: Synthesis of l,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid- N,N'-di[(2-octyloxy)ethyl ester]-(S)-2-ammonium-5-guanidinium valerate non-covalent mixture

Method 1 (starting from a diester precursor):

BAPTA diesters (e.g. alkyl or aryl diester of BAPTA and monoalkyl ethers of mono-, di- and triethylene glycol diesters of BAPTA diesters, were prepared as described in the International Patent Publication No. WO 99/16741.

A suspension of 1 g (1.27 mmol) 1 ,2-bis (2-aminophenoxy)ethane-N,N,N' ,N'-tetraacetic acid-N,N'-di[(2-octyloxy)ethyl ester] was added to the solution of (5)-2-amino-5- guanidinopentanoic acid (L-arginine) (1.33 g, 7.62 mmol). The reaction mixture was stirred at room temperature until a clear solution was obtained. The final pH of reaction was 10. The clear solution was lyophilized. Yield was 1.92 g (white solid powder).

Method 2 (starting from an organic amine salt precursor):

Arginine salt of BAPTA diesters was prepared as follows: A suspension of 1,2-bis (2- aminophenoxy)ethane-N,N,N',N' -tetraacetic acid-N,N'-di[(2-octyloxy)ethyl ester] (5 g, 6.3 mmol) in water (100 ml) was titrated at room temperature under Ar with an aqueous solution (40 ml) of (S)-2- Amino-5-guanidinopentanoic acid (L-arginine) (2.28g, 13.1mmol). The final pH of reaction solution was 9.8. The solution obtained was filtered through analytical filter (Acrodisk 0.45 μηι) and lyophilized. Yield was 6.5g (white solid, 90%).

A solution of 0.83 g (4.79 mmol) arginine was added to a solution of 1.14 g (1 mmol) l,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-N,N'-di[(2-octyloxy) ethyl ester]-di- arginine salt in 40 ml water. The reaction mixture was stirred for 5 hours at room temperature. The obtained clear solution was lyophilized.

Method 3 (starting from a sodium salt precursor):

A solution of 2.09 g (12 mmol) arginine was added to a solution of 0.83 g (1

mmol) l,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-N,N'-di[(2-octyloxy) ethyl ester] -di-sodium salt in 40 ml water. The reaction mixture was stirred at room temperature until a clear solution was obtained. The solution was lyophilized.

EXAMPLE 3: Synthesis of 1,2-Bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid- N,N'-di[(2-octyloxy)ethyl ester] - [N-(2-hydroxyethyl)-N,N,N-trimethyl- ammonium] non- covalent mixture

(CH ₃ ) ₃ NCH ₂ CH ₂ OH

N-(2-hydroxyethyl)-N,N,N-trimethyl- ammonium (choline cation)

A suspension of 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-N,N'-di[(2- octyloxy)ethyl ester] is mixed at room temperature with water solution of N-(2-hydroxyethyl)- Ν,Ν,Ν-trimethylammonium hydroxide under argon, until a clear solution is obtained. The obtained solution is lyophilized.

EXAMPLE 4: Synthesis of 1,2-Bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid- N,N'-di[(2-octyloxy)ethyl ester] - 2-(4-imidazolyl)ethylammonium non-covalent mixture

2-(4-imidazolyl)ethylammonium (histamine cation) A suspension of 1 ,2-bis (2-aminophenoxy)ethane-N,N,N' ,N'-tetraacetic acid-N,N'-di[(2- octyloxy)ethyl ester] in water (60ml) is mixed at room temperature with an aqueous solution of 2-(lH-imidazol-4-yl) ethanamine (histamine), until a clear solution is obtained. The obtained solution is lyophilized.

EXAMPLE 5: Synthesis of 1,2-Bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid- N,N'-di[(2-octyloxy)ethyl ester] - [2-(l-hydroxy-2-methylprop-2-yl) ammonium] non- covalent mixture

2-(l-hydroxy-2-methylprop-2-yl) ammonium

A suspension of 1 ,2-bis (2-aminophenoxy)ethane-N,N,N' ,N'-tetraacetic acid-N,N'-di[(2- octyloxy)ethyl ester] in water is mixed at room temperature with an aqueous solution of 2-(l- hydroxy-2-methylprop-2-yl) amine, until a clear solution is obtained. The solution is lyophilized.

EXAMPLE 6: Synthesis of 1,2-Bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid- N,N'-di[(2-octyloxy)ethyl] - aminoguanidinium non-covalent mixtures

H ₂ N-C ⁺ -NH-N H ₂

NH ₂ aminoguanidinium (pimagedine cation)

A suspension of 1 ,2-bis (2-aminophenoxy)ethane-N,N,N' ,N'-tetraacetic acid-N,N'-di[(2- octyloxy)ethyl ester] in water is mixed at room temperature with an aqueous solution of aminoguanidine (pimagedine), until a clear solution is obtained. The solution is lyophilized.

EXAMPLE 7: Synthesis of 1,2-Bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid- N,N'-di[(2-octyloxy)ethyl] - [l-(2,3,4-trimethoxybenzyl)piperazinium] non-covalent mixtures l-(2,3,4-trimethoxybenzyl)piperazinium (tri

A suspension of 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-N,N'-di[(2- octyloxy)ethyl ester] in water (100 ml) is mixed at room temperature with an aqueous solution of l-(2,3,4-trimethoxybenzyl)piperazine (trimetazidine) free base, until a clear solution is obtained. The solution is lyophilized.

EXAMPLE 8: Synthesis of 1,2-Bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid- N,N'-di[(2-octyloxy)ethyl] - 2-ammoniumethanesulfonamide non-covalent mixtures

2-aminoethansulfonamide (taurine sulfonamide cation)

A suspension of 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-N,N'-di[(2- octyloxy)ethyl ester] in water is mixed at room temperature with an aqueous solution of 2- aminoethansulfonamide (taurine sulfonamide), until a clear solution is obtained. The solution is lyophilized.

EXAMPLE 9: Stoichiometry of the DP-b99-arginine and DP-b99-lysine non-covalent mixtures

UV absorption

The stoichiometric ratios between arginine and the lipophilic diester and between lysine and the lipophilic diester, which form non-covalent complexes were evaluated by UV absorption. DP-b99-di-arginine salt was reacted with the excess of arginine and lysine (in separate experiments) and absorbance of the obtained clear solution was measured. Upon the formation of a stable non-covalent complex absorbance of the non-covalent complex is not affected by addition of arginine or lysine cations. Table 1 represents mixtures of the DP-b99 salt mixtures with excess arginine, which were prepared and which UV absorption was assessed.

DP-b99-di-arginine salt concentration was chosen to be 0.25 mmol to obtain absorption of 2.5 AU at 260 nm. 0.25 mmol solution of DP-b99-di-arginine salt means that 0.2 ml of the reaction mixture contains 50 nmol DP-b99-di-arginine salt. To obtain 5 mM solution of DP-b99- di-arginine salt, 5.7 mg of the salt was dissolved in 1 ml water. To prepare 0.01 mM arginine, 1.74 mg arginine was dissolved in 1 ml water.

TABLE 1: DP-b99 salt and arginine mixtures tested by UV absorbance

Figure 1 shows UV absorbance of the tested DP-b99-di-arginine mixtures as a function of moles of arginine in the mixture at 260 nm. The absorbance curve reached steady state when the ratio between arginine and the salt is equal to 4:1, when the ratio between the total arginine cations and DP-b99 in the non-covalent complex is 6: 1. It can be thus concluded that the stable non-covalent complex is formed between the lipophilic diester and arginine, wherein the stoichiometric ratio between arginine and the lipophilic diester is 6:1.

EXAMPLE 10: The stoichiometric ratio between arginine and the lipophilic diester and between lysine and the lipophilic diester in the non-covalent complexes as further assessed by LiChroult RP-18 elution column.

LiChrolut RP-18 column was used for determination of stoichiometry of the non-covalent complexes. Contents of DP-b99 component in the complex was determined by spectrophotometry. Contents of L-arginine and L-lysine in the complex was determined using method described M.A.A.Al-Bayati et al., Modification of Spectroscopic Method for Determination of L-Arginine in Different Tissue extracts, Pak. J.Chem., 2(1) 1-5 (2012).

The method includes the following stages: a) loading; b) washing with water (until OD=0); c) washing with 30% ethanol (until OD=0); d) elution of a complex with 100% ethanol.

TABLE 2: Quantitative determination of DP-b99-acid in eluate.

Eluate of the b99-di-L-arginine salt -L-arginine complex contains 0.4 μπιοΐ b99 acid and 2.63 μπιοΐ L-arginine. The ratio b99 acid:L-arginine= 1 :6.6.

Eluate of the b99-di-L- lysine salt -L-lysine complex contains 0.054 μπιοΐ b99 acid and

0.32 μπιοΐ L-lysine. The ratio b99 acid:L-lysine= 1 :6.

EXAMPLE 11: Physicochemical characterization of DP-b99-Arginine and DP-b99-Lysine non-covalent mixtures.

DP-b99-L-arginine and DP-b99-L-lysine non-covalent mixtures prepared as described above were lyophilized. The obtained dried final residue (non-covalent mixture) was further characterized by means of: Differential Scanning Calorimetry (DSC), Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance ( ^J H-NMR and ¹³ C-NMR).

Characterization of DP-b99-Arginine and DP-b99-Lysine non-covalent mixtures

DSC analysis

The DSC analysis of DP-b99 acid, L-arginine, the DP-b99 di-arginine salt (molar ratio

1 :2), the DP-b99-arginine non-covalent mixture (molar ratio 1 :6 in the preparation solution), the DP-b99-di-lysine salt (molar ratio 1 :2), the DP-b99-lysine non-covalent mixture (molar ratio 1 :6 in the preparation solution) were carried out using Polymer Laboratories PL-DSC (RSI Orchestrator software). Samples of approximately 2-3 mg were weighed in aluminum pans. The samples were heated from 25°C to 300°C at a ramp rate of 5°C/min under N2 purge.

In DSC, formation of a non-covalent mixture characterized by an interaction of DP-b99 and the amino acid, is evidenced. Said interaction between DP-b99 and the amino acid leading to elimination of endothermic peaks corresponding to precursors, appearance of new peaks, changes in peak shape, peak temperature/melting point and/or relative peak area (as can be seen in Table 3) can be attributed to the formation of a non-covalent complex, between the lipophilic diester and the amino acid.

TABLE 3: Temperature onset of melting point (°C)

The DSC curves of pure components, salts and the non-covalent mixtures are illustrated in

Figures 2A-2F.

FTIR analysis

FTIR spectra of DP-b99 acid, L-arginine, the DP-b99-di-arginine salt (molar ratio 1 :2) and the DP-b99-arginine non-covalent mixture were obtained using a Thermo Scientific Nicolette IS5 equipped with ID5 ATR accessory (Omnic software). A spectrum was collected for each sample within the wave number region 4000-400 cm ¹ . The spectra were analyzed for the absence or shift in the wave numbers of the characteristic peaks.

NMR spectroscopic analysis

¾-NMR and ¹³ C-NMR spectra for DP-b99 sodium salt, DP-b99 arginine salt and DP-b99- arginine non-covalent mixture were recorded at 25°C on a 400 MHz Bruker instrument, using D2O for sample solubilization.

EXAMPLE 12: Water solubility of DP-b99-Arginine and DP-b99-Lysine non-covalent mixtures.

Solubility of the DP-b99-Arginine and Lysine non-covalent mixtures was evaluated in acidic media and in human plasma. The solubility of the non-covalent mixture was compared to the solubility of DP-b99 disodium salt and the corresponding DP-b99 di-arginine and di-lysine and mono-arginine salts. Solubility in acidic media

Solubility in acidic media was evaluated by visually testing solutions of DP-b99 non- covalent mixture and salts dissolved containing 1 mmole, 10 mmole and 30 mmole HCl. 1.5 ml (20 mg/ml) of DP-b99 non-covalent mixture and salts solutions were added to 3.5 ml 1 mmol, 10 mmol and 30 mmol HCl. Resulting concentration of HCl in the obtained solutions was 0.7 mM, 7mM and 21 mM respectively (Tables 4a and 4b).

It can be seen from Tables 4a and 4b that solubility of the arginine and lysine non-covalent mixtures with DP-b99 in solutions containing hydrochloric acid, particularly at high concentrations thereof, was higher than solubility of DP-b99 sodium, arginine and lysine salts.

TABLE 4a: Visual evaluation of solubility of arginine-DP-b99 non-covalent mixture and DP-b99 salts in HCl aqueous solutions

* - immediately

** - pH-meter

*** - pH Test Strips 4.5-10.0 TABLE 4b: Visual evaluation of solubility of lysine-DP-b99 non-covalent mixture and DP-b99 salts in HC1 aqueous solutions

* - immediately

** - pH-meter

*** - pH Test Strips 4.5-10.0

Solubility in human plasma

Solubility in human plasma was evaluated by visually examining solutions of DP-b99 arginine and lysine non-covalent mixtures in plasma as a function of time. 1 ml (20 mg/ml) of DP-b99 non-covalent mixture and salts solutions were added to 3ml of human plasma (centrifuged and filtered).

The results of the plasma solubility experiments are summarized in Tables 5a and 5b.

TABLE 5a: Visual evaluation of solubility of DP-b99-Arginine non-covalent mixtures and DP-b99 salts in human plasma.

* - immediately TABLE 5b: Visual evaluation of solubility of DP-b99-Lysine non-covalent mixtures and DP-b99 salts in human plasma.

- immediately

Figures 3A-3C represent images of the DP-b99 non-covalent mixture and salts solutions in plasma after 2 hrs.

Solubility of DP-b99 non-covalent mixtures and corresponding salts in plasma has been further evaluated by UV absorption spectroscopy. Figure 4A-4D show absorbance of the DP- b99-disodium salt, DP-b99-mono-arginine salt, DP-b99-di-arginine salt and DP-b99-arginine non-covalent mixture, measured at 230, 260, 280 and 320 nm for up to 5 hours of incubation in human plasma.

It can be concluded that solubility of the DP-b99 non-covalent mixture in human plasma, particularly when incubated for long periods of time, is higher than solubility of DP-b99 sodium, arginine and lysine salts.

Solubility in buffered solution

Solubility of DP-b99 non-covalent mixture and salts in phosphate buffer has been evaluated by UV absorption spectroscopy. Figures 5A-5C show absorbance of the DP-b99- disodium salt, DP-b99-mono-arginine salt, DP-b99-di-arginine salt and DP-b99-arginine non- covalent mixture, measured at 260 in pH=7.1, 7.2 and 7.3 phosphate buffers respectively.

It can be concluded that solubility of the DP-b99 non-covalent mixture in the neutral and physiological pH is higher than solubility of DP-b99 sodium and arginine salts.

EXAMPLE 13: Lactate Dehydrogenase (LDH) release test.

Differences in the LDH concentrations were measured using cytotoxicity detection kit (Roche). Briefly, human neuroblastoma SK-N-SH cells were treated with 10 μπιοΐ DP-b99 di- arginine or DP-b99 di-lysine salts and DP-b99 arginine non-covalent mixture with the stoichiometric ratio of 6: 1 (arginine :DP-b99) or DP-b99 lysine non-covalent mixture with the stoichiometric ratio of 6: 1 (lysine :DP-b99) for 20 min. For the analysis 100 μΐ supernatant were extracted from each well and placed in separate 96-well plate. 100 μΐ of catalyst solution were added to each well and incubated at 25°C for 30 min. Absorbance was measured at 490 nm using microplate reader. Total cellular LDH was determined using total cell lysate following 2% Triton X-100. The assay medium served as a low control and its absorbance was subtracted from all absorbance measurement to estimate the cytotoxicity (%).

The equation used was (OD _tes t -ODi _ow controi)/( ODhigh control -ODi _ow controi)x 100.

Figure 6 shows reduced cell toxicity when SK-N-SH cells are exposed to the non-covalent mixtures, as compared to the salts.

While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, rather the scope, spirit and concept of the invention will be more readily understood by reference to the claims which follow.