HK1198123B - The use of oligonucleotide chelate complexes - Google Patents

Description

Use of oligonucleotide chelates

The application is a divisional application of PCT application No. 201180035946.6, named as 'oligonucleotide chelate', with the application date of 2011, 8, and 18, PCT/CA2011/000956 entering China national phase application.

Technical Field

The present invention relates to oligonucleotide chelates, compositions thereof, and methods of formulating Oligonucleotides (ONs) as chelates, as well as the use of these ON chelates for administration of ONs.

Background

Salts are ionic compounds produced by the interaction (neutralization) of an acid with a base. Salts are composed of cations and anions that interact to maintain an electrically neutral state. The anion may be inorganic (e.g. Cl)^-) Or organic, e.g. acetate (CH)₃COO^-). Aqueous solutions containing dissolved salts (electrolytes) are capable of conducting electricity due to the dissociated state of the anion and cation pairs in an aqueous environment. Oligonucleotides are polyanions and have previously been considered to behave only as salts, with their cationic counterparts present in solution in a dissociated state.

Administration of ONs to human patients is often accompanied by some broad side effects that are not related to the nucleotide sequences present. These include anticoagulation of Blood (increase in prothrombin time or PTT time) (Kandmimlla et al, 1998, bioorgan. Med. chem. Let.,8: 2103; Sheban et al, Blood,1998,92: 1617; Nicklin et al, 1197, Nucleotides & Nucleotides, 16: 1145; Kwoh,2008, Antisense Drug Tech. 2 nd edition, p.374) and injection site reactions or ISRs (sclerosis, inflammation, tenderness and pain) upon subcutaneous administration (Webb et al, 1997, Lancet,349: 9059; Schrieber et al, 2001, gastroenterol.,120: 1339; Seawell et al, 2002, J.rmacol. exp. rap. 1334; Kwoh,2008, Drug, 2 nd edition, La.375: 383; Lanceh et al, 2010, La < 8). Anticoagulation is thought to be mediated by non-sequence specific interactions with proteins of the coagulation cascade. Since ONs have been shown to have immunostimulatory properties (induced by Toll-like receptors or TLR-mediated cytokines), ISR is generally attributed to the need to administer high concentrations of ON in small volumes (typically 1cc) upon Subcutaneous (SC) injection, which is thought to cause local inflammation at the injection site.

With the advent of nucleic acid-based therapies in recent years, the growing number of ON-based compounds in clinical development has also increased. Historically most ON dosing regimens have employed multiple or single weekly doses that must be given parenterally because of the poor oral bioavailability of ONs. Because intravenous infusion of ONs is often dose and rate limited by reactivity (fever, chills, weakness) and will be a logical requirement in chronic dosage regimens, the recent clinical application of ONs has used the Subcutaneous (SC) route of administration. This causes minimal systemic administration side effects but is often accompanied by injection site reactions of varying severity (as described above), which also limits the administration achievable by this route of administration.

Therefore, it would be useful and desirable to provide ON formulations that would reduce reactivity during IV or SC route administration. Furthermore, while the anticoagulant effect of administration of ON is believed to be minimal, it is also useful to eliminate the side effects of ON to provide greater range of safety to human and non-human subjects.

Accordingly, there is a need to provide an improved ON formulation.

Disclosure of Invention

In accordance with the present description, there is now provided an oligonucleotide chelate complex comprising two or more oligonucleotides linked by a multivalent cation.

Further provided is an oligonucleotide formulation for subcutaneous administration comprising an oligonucleotide chelate complex as described herein.

Also disclosed is a pharmaceutical composition comprising an oligonucleotide chelate complex or oligonucleotide formulation described herein and a carrier.

Further disclosed is a method of reducing liver or kidney dysfunction associated with administration of an oligonucleotide to a subject, the method comprising the steps of: administering to the subject an oligonucleotide that is a chelate as described herein, an oligonucleotide formulation as disclosed herein, or a pharmaceutical composition as disclosed herein.

Disclosed herein are methods of suppressing, inhibiting, or reducing blood anticoagulation caused by ONs by administering the ONs as calcium chelates or other suitable ON metal chelates, the oligonucleotide formulations described herein, or the pharmaceutical compositions described herein.

Also disclosed herein are methods of improving the tolerability of any ON administered by IV infusion by preparing the ON as a calcium chelate complex or other suitable ON metal chelate complex, an oligonucleotide formulation described herein, or a pharmaceutical composition described herein.

Disclosed herein are methods for suppressing or reducing injection site reactions when administering an ON that is a calcium chelate complex or other suitable ON metal chelate complex, an oligonucleotide formulation described herein, or a pharmaceutical composition described herein. In particular, the ONs are administered subcutaneously.

Disclosed herein are methods of suppressing chelation of a metal with any ON administered by any route by providing the ON as a calcium chelate complex or other suitable ON metal chelate complex, an oligonucleotide formulation described herein, or a pharmaceutical composition described herein.

Disclosed herein are methods of reducing the serum half-life of any ON by administering the ON as a calcium chelate complex or other suitable ON metal chelate complex, an oligonucleotide formulation described herein, or a pharmaceutical composition described herein.

Disclosed herein are methods of reducing the interaction of serum proteins with any ON by administering the ON as a calcium chelate complex or other suitable metal chelate complex, an oligonucleotide formulation described herein, or a pharmaceutical composition described herein. In particular, the oligonucleotide chelate complex, formulation or composition is administered to a subject by IV infusion.

Also disclosed herein is an oligonucleotide formulation wherein the oligonucleotide is provided with a divalent metal cation source from any one of the following as an ON chelate complex when the oligonucleotide is used: calcium, magnesium, cobalt, iron, manganese, barium, nickel, copper and/or zinc. Thus described herein are oligonucleotide formulations comprising calcium; an oligonucleotide formulation comprising magnesium; an oligonucleotide formulation comprising cobalt; an oligonucleotide formulation comprising iron (2 +); an oligonucleotide formulation comprising manganese; an oligonucleotide formulation comprising copper; an oligonucleotide formulation comprising zinc.

Disclosed herein are methods for preparing ON metal chelates using any of the following metal cations, alone or in combination: calcium, magnesium, cobalt, iron, manganese, barium, nickel, copper and/or zinc.

Also disclosed is a method of preparing an oligonucleotide chelate complex as described herein, an oligonucleotide formulation as described herein or a pharmaceutical composition as described herein, said method comprising dissolving any sodium oligonucleotide salt in a pharmaceutically acceptable aqueous excipient and gradually adding a divalent metal salt solution to the dissolved oligonucleotide to maintain the solubility of the oligonucleotide chelate complex.

Disclosed herein is a method of chelating in a subject a divalent metal cation selected from the group consisting of: calcium, magnesium, cobalt, iron, manganese, barium, nickel, copper, zinc, cadmium, mercury, lead, beryllium, strontium, radium and/or any other metal, transition metal, post-transition metal, lanthanide or actinide capable of existing in a 2+ or 3+ charge state.

Also disclosed herein is a method of improving the stability of any ON in solution by preparing the ON as a calcium chelate complex or other suitable ON metal chelate complex, an oligonucleotide formulation described herein, or a pharmaceutical composition described herein. In particular, a method of stabilizing an oligonucleotide in an aqueous solution is disclosed.

It is contemplated herein that the multivalent cation is a divalent cation.

It is contemplated herein that the divalent cation is an alkaline earth metal in a 2+ charge state.

It is contemplated herein that the divalent cation is a transition or post-transition metal in a 2+ charge state.

It is contemplated herein that the divalent cation is a lanthanide metal in a 2+ charge state.

It is contemplated herein that the divalent cation is an actinide metal in a 2+ charge state.

The divalent cation may be, alone or in combination: calcium, magnesium, cobalt, iron, manganese, barium, nickel, copper and/or zinc.

In particular, the chelates described herein may comprise two or more different divalent metal cations.

In a further embodiment, the chelate complex comprises at least one double-stranded oligonucleotide.

In another embodiment, the chelate complex comprises at least one oligonucleotide having one phosphorothioate linkage.

The chelate complex may also comprise at least one fully phosphorothioated oligonucleotide.

The chelate complex may also comprise at least one oligonucleotide having a 2' modified ribose sugar.

The chelate complex may also comprise at least one oligonucleotide with each ribose 2' O-methylated.

In one embodiment, the chelate or formulation is suitable for subcutaneous administration, suitable for at least the following routes of administration: intraocular, oral, enteric, inhalation, cutaneous injection, intramuscular injection, intraperitoneal injection, intrathecal infusion, intratracheal, intravenous infusion, and topical administration. In particular, the inhalation administration may be an aerosol.

It is further contemplated that the oligonucleotide consists of SEQ ID NO 3 to 14.

It is further contemplated that the concentration of dissolved oligonucleotide prior to addition of the metal salt is 0.01-100 mg/ml.

In particular, the proportion of metal salt added to the dissolved oligonucleotide may be 0.1 to 40mg of divalent salt per 100mg of oligonucleotide.

It is further contemplated that the final concentration of the oligonucleotide is 0.1-100 mg/ml.

In further embodiments, the metal salt is at least one of: hydrochloride, gluconate, citrate, lactate, malate, aspartate, fumarate, ascorbate, benzoate, erythorbate, and propionate.

In another embodiment, the metal salt solution comprises at least one of: calcium, magnesium, cobalt, iron (2+), manganese, copper and/or zinc.

It is also contemplated herein that the chelate is a calcium chelate; a magnesium chelate; or mixed magnesium/calcium chelates.

Also provided is the use of a multivalent cation as described herein in the preparation of an oligonucleotide chelate complex.

Further provided is the use of an oligonucleotide chelate complex as described herein, an oligonucleotide formulation as described herein or a pharmaceutical composition as described herein for inhibiting or reducing anticoagulation of the administration of an oligonucleotide to a subject.

There is provided use of an oligonucleotide chelate complex as described herein, an oligonucleotide formulation as described herein or a pharmaceutical composition as described herein for inhibiting or reducing a subcutaneous injection site reaction in a subject administered said oligonucleotide subcutaneously.

Also provided is the use of an oligonucleotide chelate complex as described herein, an oligonucleotide formulation as described herein, or a pharmaceutical composition as described herein for improving tolerance of an oligonucleotide in a subject when administered by IV infusion.

Also provided is the use of an oligonucleotide chelate complex as described herein, an oligonucleotide formulation as described herein, or a pharmaceutical composition as described herein for reducing serum protein interactions of an oligonucleotide when administered to a subject by IV infusion.

Further provided is the use of an oligonucleotide chelate complex as described herein, an oligonucleotide formulation as described herein, or a pharmaceutical composition as described herein for reducing the serum half-life of an oligonucleotide in a subject.

Also provided is the use of an oligonucleotide chelate complex as described herein, an oligonucleotide formulation as described herein, or a pharmaceutical composition as described herein for reducing liver or kidney dysfunction associated with administration of the oligonucleotide in a subject.

Also provided is the use of an oligonucleotide chelate complex as described herein, an oligonucleotide formulation as described herein or a pharmaceutical composition as described herein for stabilizing an oligonucleotide in an aqueous solution.

The expression "anticoagulation" means inhibition of normal blood coagulation or clot formation.

The expression "chelation" means the masking or removal of a counter ion (negative or positive) from a free solution reaction by another molecule capable of binding to the counter ion to form a chelated complex.

The expression "divalent metal cation" refers to any metal cation naturally occurring in the 2+ charge state and including alkaline earth metals (group 2 elements according to IUPAC nomenclature), transition metals, post-transition metals, metalloids or lanthanides.

The expression "trivalent metal cation" refers to any metal cation that naturally exists in a 3+ charge state and includes transition metals, post-transition metals, metalloids, lanthanides or actinides.

Drawings

Reference will now be made to the accompanying drawings:

fig. 1 shows the general physiochemical characteristics of an ON. A) REP2006 and 21mer phosphorothioate ONs with defined sequences were co-separated by high performance liquid chromatography. B) Species in 21mer ONs were identified by mass spectrometry. C) Species in REP2006 ON were identified by mass spectrometry.

FIG. 2 shows the formation of ON-calcium chelates by fluorescently labeled degenerate phosphorothioate ONs, which are: A) various sizes of 6 mers (REP 2032-FL), 10 mers (REP 2003-FL), 20 mers (REP 2004-FL), 40 mers (REP 2006-FL) and B are selected using phosphorothioation (REP 2006-FL), phosphorothioation +2 'O methyl ribose (REP 2107-FL) or 2' O methyl ribose (REP 2086-FL) and different sequences (poly C-REP 2031-FL; 4) of the sequence of SEQ ID NO. The sequence-independent nature of ON chelate complex formation was demonstrated by using degenerate oligonucleotides, but was also demonstrated using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). ON-calcium chelate complex formation was demonstrated by binding increasing concentrations of ACS grade calcium chloride to FITC-labeled oligonucleotides in solution and monitoring oligonucleotide chelate complex formation by fluorescence polarization increase as described in example 1. Values represent mean +/-standard deviation of duplicate measurements.

FIG. 3 shows the formation of ON-magnesium chelates by fluorescently labeled degenerate phosphorothioate ONs, which are: A) various sizes of 6 mers (REP 2032-FL), 10 mers (REP 2003-FL), 20 mers (REP 2004-FL), 40 mers (REP 2006-FL) and B utilize the degenerate ON of phosphorothioation (REP 2006-FL), phosphorothioation +2 'O methyl ribose (REP 2107-FL) or 2' O methyl ribose (REP 2086-FL) and fluorescent labeling of different sequences (poly C-REP2031-FL, SEQ ID NO: 4). The sequence-independent nature of ON chelate complex formation was demonstrated by using degenerate oligonucleotides, but was also demonstrated using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). ON-magnesium chelate complex formation was demonstrated by binding increasing concentrations of ACS grade magnesium chloride to FITC-labeled oligonucleotides in solution and monitoring oligonucleotide chelate complex formation by fluorescence polarization increase as described in example 1. Values represent mean +/-standard deviation of duplicate measurements.

FIG. 4 shows the formation of ON-cobalt chelates by fluorescently labeled degenerate phosphorothioate ONs, which are: A) various sizes of 6 mers (REP 2032-FL), 10 mers (REP 2003-FL), 20 mers (REP 2004-FL), 40 mers (REP 2006-FL) and B utilize the degenerate ON of phosphorothioation (REP 2006-FL), phosphorothioation +2 'O methyl ribose (REP 2107-FL) or 2' O methyl ribose (REP 2086-FL) and fluorescent labeling of different sequences (poly C-REP2031-FL, SEQ ID NO: 4). The sequence-independent nature of ON chelate complex formation was demonstrated by using degenerate oligonucleotides, but was also demonstrated using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). Formation of ON-cobalt chelate was demonstrated by binding increasing concentrations of ACS grade cobalt chloride to FITC-labeled oligonucleotides in solution and monitoring formation of oligonucleotide chelate complexes by fluorescence polarization growth as described in example 1. Values represent mean +/-standard deviation of duplicate measurements.

FIG. 5 shows the formation of ON-iron chelate complexes by fluorescently labeled degenerate phosphorothioate ONs which are: A) various sizes of 6 mers (REP 2032-FL), 10 mers (REP 2003-FL), 20 mers (REP 2004-FL), 40 mers (REP 2006-FL) and B are selected using phosphorothioation (REP 2006-FL), phosphorothioation +2 'O methyl ribose (REP 2107-FL) or 2' O methyl ribose (REP 2086-FL) and different sequences (poly C-REP 2031-FL; 4) of the sequence of SEQ ID NO. The sequence-independent nature of ON chelate complex formation was demonstrated by using degenerate oligonucleotides, but was also demonstrated using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). ON-iron chelate complex formation was demonstrated by binding increasing concentrations of ACS-grade ferric chloride to FITC-labeled oligonucleotides in solution and monitoring oligonucleotide chelate complex formation by fluorescence polarization growth as described in example 1. Values represent mean +/-standard deviation of duplicate measurements.

FIG. 6 shows the formation of ON-manganese chelate complexes by fluorescently labeled degenerate phosphorothioate ONs which are: A) various sizes of 6 mers (REP 2032-FL), 10 mers (REP 2003-FL), 20 mers (REP 2004-FL), 40 mers (REP 2006-FL) and B are selected using phosphorothioation (REP 2006-FL), phosphorothioation +2 'O methyl ribose (REP 2107-FL) or 2' O methyl ribose (REP 2086-FL) and different sequences (poly C-REP 2031-FL; 4) of the sequence of SEQ ID NO. The sequence-independent nature of ON chelate complex formation was demonstrated by using degenerate oligonucleotides, but was also demonstrated using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). ON-manganese chelate formation was demonstrated by binding increasing concentrations of ACS grade manganese chloride to FITC-labeled oligonucleotides in solution and monitoring oligonucleotide chelate formation by fluorescence polarization growth as described in example 1. Values represent mean +/-standard deviation of duplicate measurements.

FIG. 7 shows the formation of ON-barium chelate complexes by fluorescently labeled degenerate phosphorothioate ONs which are: A) various sizes of 6 mers (REP 2032-FL), 10 mers (REP 2003-FL), 20 mers (REP 2004-FL), 40 mers (REP 2006-FL) and B are selected using phosphorothioation (REP 2006-FL), phosphorothioation +2 'O methyl ribose (REP 2107-FL) or 2' O methyl ribose (REP 2086-FL) and different sequences (poly C-REP 2031-FL; 4) of the sequence of SEQ ID NO. The sequence-independent nature of ON chelate complex formation was demonstrated by using degenerate oligonucleotides, but was also demonstrated using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). Formation of ON-barium chelate was demonstrated by binding increasing concentrations of ACS grade barium chloride to FITC-labeled oligonucleotide in solution and monitoring oligonucleotide chelate formation by fluorescence polarization increase as described in example 1. Values represent mean +/-standard deviation of duplicate measurements.

FIG. 8 shows the formation of ON-nickel chelates by fluorescently labeled degenerate phosphorothioate ONs, which are: A) various sizes of 6 mers (REP 2032-FL), 10 mers (REP 2003-FL), 20 mers (REP 2004-FL), 40 mers (REP 2006-FL) and B are selected using phosphorothioation (REP 2006-FL), phosphorothioation +2 'O methyl ribose (REP 2107-FL) or 2' O methyl ribose (RE P2086-FL) and different sequences (poly C-REP 2031-FL; 4) of the sequence of SEQ ID NO. The sequence-independent nature of ON chelate complex formation was demonstrated by using degenerate oligonucleotides, but was also demonstrated using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). ON-nickel chelate formation was demonstrated by binding increasing concentrations of ACS grade nickel chloride to FITC-labeled oligonucleotides in solution and monitoring oligonucleotide chelate formation by fluorescence polarization growth as described in example 1. Values represent mean +/-standard deviation of duplicate measurements.

FIG. 9 shows the formation of ON-copper chelates by fluorescently labeled degenerate phosphorothioate ONs: A) various sizes of 6 mers (REP 2032-FL), 10 mers (REP 2003-FL), 20 mers (REP 2004-FL), 40 mers (REP 2006-FL) and B are selected using phosphorothioation (REP 2006-FL), phosphorothioation +2 'O methyl ribose (REP 2107-FL) or 2' O methyl ribose (REP 2086-FL) and different sequences (poly C-REP 2031-FL; 4) of the sequence of SEQ ID NO. The sequence-independent nature of ON chelate complex formation was demonstrated by using degenerate oligonucleotides, but was also demonstrated using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). ON-copper chelate formation was demonstrated by binding increasing concentrations of ACS grade copper chloride to FITC-labeled oligonucleotides in solution and monitoring oligonucleotide chelate formation by fluorescence polarization increase as described in example 1. Values represent mean +/-standard deviation of duplicate measurements.

FIG. 10 shows the formation of ON-zinc chelates by fluorescently labeled degenerate phosphorothioate ONs, which are: A) various sizes of 6 mers (REP 2032-FL), 10 mers (REP 2003-FL), 20 mers (REP 2004-FL), 40 mers (REP 2006-FL) and B are selected using phosphorothioation (REP 2006-FL), phosphorothioation +2 'O methyl ribose (REP 2107-FL) or 2' O methyl ribose (REP 2086-FL) and different sequences (poly C-REP 2031-FL; 4) of the sequence of SEQ ID NO. The sequence-independent nature of ON chelate complex formation was demonstrated by using degenerate oligonucleotides, but was also demonstrated using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). ON-zinc chelate complex formation was demonstrated by binding increasing concentrations of ACS grade zinc chloride to FITC-labeled oligonucleotides in solution and monitoring oligonucleotide chelate complex formation by fluorescence polarization growth as described in example 1. Values represent mean +/-standard deviation of duplicate measurements.

FIG. 11 shows the formation of ON-cadmium chelates by fluorescently labeled degenerate phosphorothioate ONs: A) various sizes of 6 mers (REP 2032-FL), 10 mers (REP 2003-FL), 20 mers (REP 2004-FL), 40 mers (REP 2006-FL) and B are selected using phosphorothioation (REP 2006-FL), phosphorothioation +2 'O methyl ribose (REP 2107-FL) or 2' O methyl ribose (REP 2086-FL) and different sequences (poly C-REP 2031-FL; 4) of the sequence of SEQ ID NO. The sequence-independent nature of ON chelate complex formation was demonstrated by using degenerate oligonucleotides, but was also demonstrated using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). ON-cadmium chelate complex formation was demonstrated by binding increasing concentrations of ACS-grade cadmium chloride to FITC-labeled oligonucleotides in solution and monitoring oligonucleotide chelate complex formation by fluorescence polarization growth as described in example 1. Values represent mean +/-standard deviation of duplicate measurements.

FIG. 12 shows the formation of ON-mercury chelates by fluorescently labeled degenerate phosphorothioate ONs, which are: A) various sizes of 6 mers (REP 2032-FL), 10 mers (REP 2003-FL), 20 mers (REP 2004-FL), 40 mers (REP 2006-FL) and B are selected using phosphorothioation (REP 2006-FL), phosphorothioation +2 'O methyl ribose (REP 2107-FL) or 2' O methyl ribose (REP 2086-FL) and different sequences (poly C-REP 2031-FL; 4) of the sequence of SEQ ID NO. The sequence-independent nature of ON chelate complex formation was demonstrated by using degenerate oligonucleotides, but was also demonstrated using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). ON-mercury chelate formation was demonstrated by binding increasing concentrations of ACS-grade mercuric chloride to FITC-labeled oligonucleotides in solution and monitoring oligonucleotide chelate formation by fluorescence polarization growth as described in example 1. Values represent mean +/-standard deviation of duplicate measurements.

FIG. 13 shows the formation of ON-lead chelate complexes by fluorescently labeled degenerate phosphorothioate ONs which are: A) various sizes of 6 mers (REP 2032-FL), 10 mers (REP 2003-FL), 20 mers (REP 2004-FL), 40 mers (REP 2006-FL) and B are selected using phosphorothioation (REP 2006-FL), phosphorothioation +2 'O methyl ribose (REP 2107-FL) or 2' O methyl ribose (REP 2086-FL) and different sequences (poly C-REP 2031-FL; 4) of the sequence of SEQ ID NO. The sequence-independent nature of ON chelate complex formation was demonstrated by using degenerate oligonucleotides, but was also demonstrated using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). ON-lead chelate complex formation was demonstrated by binding increasing concentrations of ACS-grade lead chloride to FITC-labeled oligonucleotides in solution and monitoring oligonucleotide chelate complex formation by fluorescence polarization growth as described in example 1. Values represent mean +/-standard deviation of duplicate measurements.

Fig. 14A shows the general chemistry of ON independent of ON sequence. Regardless of the sequence, any ON is present as a polymer, which has both hydrophobic and hydrophilic activity. Thiophosphorylation (shown by the chemical structure in the figure) is used to increase the hydrophobicity of the ON polymer, but does not affect the hydrophilicity. Figure 14B conceptualizes the oligonucleotide chelating properties of divalent and trivalent metal cations. The metal cations (represented by the gray filled circles) are attached to the hydrophilic surface of the ON polymer by metal ionic bridges (represented by ovals) between two or three non-bridging oxygen or sulfur atoms in the phosphodiester linkage.

Fig. 15 shows a model of the solution behavior of ONs at different ON and divalent metal cation concentrations in the presence of divalent metal cations. A) Low divalent/trivalent metal cations, low ON concentrations produce dimers or low order ON chelates. B) Increasing the divalent/trivalent metal cation concentration results in more complete ON chelate complex formation in solution. C) Further increasing the ON concentration with increasing metal concentration in the presence of a divalent or trivalent metal can produce higher order ON chelate complexes. (A) All chelates of (a) to (C) are soluble in aqueous solution and thus maintain solubility due to their hydrophilic surface which is still exposed to an aqueous environment. D) At sufficient ON and metal concentrations, all hydrophilic surfaces are now confined within the ON chelate complex, leaving only hydrophobic surfaces exposed to an aqueous environment. This results in the precipitation of the ON chelate complex.

FIG. 16 shows the effect of solution behavior of fluorescence-ON chelate complexes ON fluorescence polarization. As the metal concentration increases, the size (and mass) of ON chelate complex formation also increases (see fig. 15) and thus tumbles more slowly in solution. This slower tumbling of the complex in solution results in an increase in fluorescence polarization and an increase in mP value.

Fig. 17 shows the formation of ON chelate complexes with calcium chloride or calcium sulfate as measured by fluorescence polarization. A) ON chelate complexes are formed with REP 2055-FL (SEQ ID NO:6) and REP2056-FL (SEQ ID NO: 7). B) ON chelate complexes were formed with REP2033-FL (SEQ ID NO:5) and REP 2029-FL (SEQ ID NO: 2). Values represent mean +/-standard deviation of duplicate measurements.

Fig. 18 shows the formation of ON chelate complexes with calcium chloride or calcium sulfate as measured by fluorescence polarization. A) No ON chelate complex was formed with REP 2028-FL and ON chelate complex was formed with REP2057-FL (SEQ ID NO: 8). B) ON chelate complexes are formed with REP 2120-FL and REP 2030-FL. Values represent mean +/-standard deviation of duplicate measurements.

Fig. 19 shows the formation of ON chelate complexes with calcium chloride or calcium sulfate as measured by fluorescence polarization. A) ON chelate complexes were formed with REP 2129-FL (SEQ ID NO:12) and REP 2126-FL (SEQ ID NO: 9). B) ON chelate complexes were formed with REP 2128-FL (SEQ ID NO:11) and REP 2127-FL (SEQ ID NO: 10). Values represent mean +/-standard deviation of duplicate measurements.

Fig. 20 shows the formation of ON chelate complexes with calcium chloride or calcium sulfate as measured by fluorescence polarization. A) ON chelate complexes are formed with REP 2139-FL (SEQ ID NO:13) and REP 2006-FL. B) ON chelate complexes are formed with REP 2045-FL and REP 2007-FL. Values represent mean +/-standard deviation of duplicate measurements.

Fig. 21 shows the formation of ON chelate complexes with magnesium chloride or magnesium sulfate as measured by fluorescence polarization. A) ON chelate complexes are formed with REP 2055-FL (SEQ ID NO:6) and REP2056-FL (SEQ ID NO: 7). B) ON chelate complexes were formed with REP2033-FL (SEQ ID NO:5) and REP 2029-FL (SEQ ID NO: 12). Values represent mean +/-standard deviation of duplicate measurements.

Fig. 22 shows the formation of ON chelate complexes with magnesium chloride or magnesium sulfate as measured by fluorescence polarization. A) No ON chelate was formed with REP 2028-FL (SEQ ID NO:11), and ON chelate was formed with REP2057-FL (SEQ ID NO: 8). B) ON chelate complexes were formed with REP 2120-FL and REP2030-FL (SEQ ID NO: 3). Values represent mean +/-standard deviation of duplicate measurements.

Fig. 23 shows the formation of ON chelate complexes with magnesium chloride or magnesium sulfate as measured by fluorescence polarization. A) ON chelate complexes were formed with REP 2129-FL (SEQ ID NO:12) and REP 2126-FL (SEQ ID NO: 9). B) ON chelate complexes were formed with REP 2128-FL (SEQ ID NO:11) and REP 2127-FL (SEQ ID NO: 10). Values represent mean +/-standard deviation of duplicate measurements.

Fig. 24 shows the formation of ON chelate complexes with magnesium chloride or magnesium sulfate as measured by fluorescence polarization. A) ON chelate complexes are formed with REP 2139-FL (SEQ ID NO:13) and REP 2006-FL. B) ON chelate complexes are formed with REP 2045-FL and REP 2007-FL. Values represent mean +/-standard deviation of duplicate measurements.

Figure 25 shows the formation of two different double-stranded ON chelates in the presence of calcium chloride or magnesium chloride, as measured by fluorescence polarization. Double-stranded ON is prepared by hybridization of REP 2055-FL (SEQ ID NO:6) and REP2033-FL (SEQ ID NO:5) and of REP2057-FL (SEQ ID NO:8) and REP2056-FL (SEQ ID NO: 7). Values represent the mean +/-standard deviation of triplicate measurements.

FIG. 26 shows the presence of divalent metal cations (Mg) only²⁺And Ca²⁺) In the presence of monovalent cations (Na) other than⁺、K⁺Or NH₄ ⁺) When present, different ON chelate complexes are formed. Values represent the mean +/-standard deviation of triplicate measurements.

Fig. 27 shows anticoagulation by adding ON of various concentrations of different sizes (REP 2004, REP 2006) and different chemical compositions (REP 2006, REP2107) to human blood. The sequence-independent manner of this interaction was demonstrated by using degenerate oligonucleotides (REP 2004, REP2006, REP2107), but also using sequence-specific oligonucleotides (REP 2031; SEQ ID NO: 4). Anticoagulation of blood in the presence of these compounds was monitored by measuring the prothrombin time (PTT) and comparing it to PTT in the presence of physiological saline in blood using accepted clinical laboratory test methods. The ratio of PTT in the presence and absence of drug yields a Normalized Ratio (NR). An NR of 1 indicates normal blood clotting activity and an NR greater than 1 indicates that blood clotting activity has diminished (anticoagulation).

FIG. 28 shows the addition of CaCl₂Inhibiting the anticoagulation of the oligonucleotide. REP2055 (having the sequence (AC)₂₀The 40mer thiophosphoric acid of (1); SEQ ID NO 6) was added to the blood at 2.5mM, which concentration induced significant blood anticoagulation. Reacting REP2055 with CaCl₂In combination and using accepted clinical laboratory test methods to determine each concentration of CaCl added₂The function of (1). Anticoagulation of blood in the presence of these compounds was monitored by measuring prothrombin time (PTT) and comparing it to PTT in the presence of physiological saline in blood using well-established test methods. The ratio of PTT in the presence and absence of drug yields a Normalized Ratio (NR). An NR of 1 indicates normal blood clotting activity and an NR greater than 1 indicates that blood clotting activity has diminished (anticoagulation).

Figure 29 shows calcium chelation of total serum calcium by chronic ON treatment in patients with chronic liver disease. Patients who did not receive the mineral supplement are shown in (a), while patients who received the supplement while undergoing ON treatment are shown in (B).

Detailed Description

Provided herein is evidence that ONs chelate various divalent metal cations, including calcium, magnesium, cobalt, iron, manganese, barium, nickel, copper, zinc, cadmium, mercury, and lead. It is further demonstrated that chelation of these divalent cations results in the formation of an ON chelate complex consisting of two or more ONs linked by a metal cation, and for example, but not limited to, an ON at a nucleotide length between 6 and 80, and occurs in the presence of a phosphodiester or phosphorothioate oligonucleotide. Chelation also occurs with oligonucleotides that contain 2' modifications or no modifications in the ribose. Furthermore, the chelation of the metal cation is independent of the sequence of the nucleotides present, but dependent on physiochemical characteristics common to all oligonucleotides (see FIG. 14A).

The discovery is presented herein that the presence of any simple divalent metal cation (such as, but not limited to, Ca)²⁺、Mg²⁺、Fe²⁺) The oligonucleotide in the aqueous solution of (a) is not present as a salt but as a chelated complex of the ON. These complexes consist of oligonucleotide dimers or higher order molecular organisations in which the ONs are linked in their phosphodiester backbone by divalent metal ion bridges (see figure 14B). At particular concentrations of ON and metal cations, these chelated complexes are stable and soluble in aqueous solution, and effectively mask any divalent cations in the ON chelate complex from solution interactions. This chelate formation is also possible with simple metal cations charged 3+ or more (as shown in fig. 14B). Thus the ON acts as a divalent cation chelator and does not form a salt with divalent cations.

Importantly, oligonucleotide chelates are formed without using monovalent cations such as Na⁺、K⁺Or NH₄ ⁺And therefore is unlikely to occur with any monovalent cation. Thus, the term "oligonucleotide salt" is expressly limited to only oligonucleotide salts having monovalent cations or cations that do not form chelates with oligonucleotides, and is incorrectly used to describe oligonucleotides in solution or powder form having divalent metal cations (or even trivalent metal cations).

The standard in the art clearly teaches the administration of ONs only as sodium salts. This is exemplified by the administration of a number of oligonucleotides as sodium salts in clinical trials, including Fominirisen (ISIS 2922), Mipomersen (ISIS 301012), Trecovirsen (GEM 91), Custirsen (OGX-011/ISIS 112989), Genasense (G3139), and Aprinocarsem (ISIS3531/LY 900003) (Geary et al, 2002, Clin. Pharmacokineticics, 41: 255-260; Yu et al, 2009, Clin. Pharmacetics, 48: 39-50; Sereni et al, 1999, J. Clin. Pharmacol.,39: 47-54; Chi et al, J. Nat. Inst.,97: 1287-1296; Marshall et al, 2004, Ann. Oncol.,15: 1274; Neosn. Oncol et al, 2005-12840: 12840).

Also provided herein is evidence that anticoagulation of blood by oligonucleotides results from chelation of calcium by oligonucleotides, as demonstrated by reversal of oligonucleotide-induced anticoagulation by the addition of calcium chloride to restore normal free calcium in blood.

Also provided herein is evidence that injection site reactions (cirrhosis, inflammation, tenderness and pain) observed with subcutaneous injections of oligonucleotides are due at least in part to the local chelation of calcium and possibly other divalent cations (e.g., magnesium) by the oligonucleotide at the injection site, as demonstrated by inhibition of Injection Site Reactions (ISRs) by injection of ONs prepared as calcium chelates.

Fluorescence polarization is a common method used to examine molecular interactions. In this technique, the bait (i.e., any ON) is labeled with a fluorescent label (e.g., FITC). In solution, the bait molecules tumble freely due to brownian motion, which results in a poorly polarized fluorescence emission when the bait is excited by light of the correct wavelength. Using a ligand of sufficient molecular weight (at least the same size as the bait), the interaction between the bait and the ligand introduces a substantial amount of inhibition of tumbling of the complex in solution. Due to the suppressed tumbling in this solution, the fluorescence emission becomes significantly polarized after excitation. Thus using this technique, interactions can be measured in solution without physical limitation to any binding partner. Fluorescence polarization is reported as dimensionless mP, which is proportional to the fraction of bait molecules bound in the reaction. For example, if a small fraction of the bait molecules are bound by a particular ligand, there will be little fluorescence polarization and thus a small mP value. At the other end of the spectrum, if a large proportion of the bait molecules are bound by a particular ligand (or with a higher concentration of ligand), there will be a large amount of fluorescence polarization and thus a large mP value. In this manner, binding isotherms for a particular bait-ligand interaction can be generated by varying the concentration of ligand in the presence of a fixed amount of fluorescently labeled bait.

Different fluorescently labeled ONs are employed herein to examine the formation of complexes thereof in the presence of monovalent and divalent cations. Although monitoring the formation of complexes by fluorescence polarization requires these ONs to be fluorescently labeled, attaching the label to the ON at the 3' end so as not to interfere with the nitrogenous base or phosphodiester backbone of the ON is problematic. The fluorescent tag is furthermore kept away from ON by a rigid 3-carbon linker to further exclude any perturbation of normal ON behavior in solution. The formation of any ON complex observed herein using fluorescence polarization with fluorescently labeled ON is therefore an accurate representation of the solution behavior of unlabeled ON (whether complexed or uncomplexed).

The term Oligonucleotide (ON) refers to an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) and/or analogues thereof. The term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring moieties that function similarly. Such modified or substituted oligonucleotides are generally preferred over their native forms due to their desirable properties, e.g., enhanced cellular uptake, enhanced affinity for the target nucleic acid, and enhanced stability in the presence of nucleases.

In the present application, the term "degenerate oligonucleotide" means a single-stranded oligonucleotide having one wobble (N) at each position, e.g. nnnnnnnn. Each base is synthesized as a wobble such that the ONs actually exist as a different randomly generated population of sequences of the same length and physiochemical properties. For example, for a 40 base length degenerate of ON, any particular sequence in a population would theoretically represent 1/4 in total⁴⁰Or 8.3X 10^-25. Considering 1 mole =6.022 × 10²³One molecule, and so far without the fact that degenerates have been synthesized in excess of 2 millimoles, any oligonucleotide with a particular sequence present is effectively present no more than 1 time in any preparation. The formation of any complex observed in such a formulation must therefore be due to the sequence-independent (or sequence-independent) physiochemical properties of the oligonucleotide, since any particular oligonucleotide (in preparation, it is) of defined sequenceUnique) cannot be expected to contribute to any activity from its particular nucleotide sequence.

As a further illustration of this concept, example 1 compares REP2006 (40mer ON with degenerate phosphorothioated sequence) with the characteristics of the 21mer of defined sequence by high performance liquid chromatography and mass spectrometry, and clearly shows that any ON with similar size and chemical modification (i.e. phosphorothioation) will have highly similar (if not identical) physiochemical properties that are not affected by the nucleotide sequence present.

The oligonucleotide may comprise various modifications, for example, stabilizing modifications, and thus may comprise at least one modification in the phosphodiester bond and/or in the sugar and/or in the base. For example, an oligonucleotide may include, but is not limited to, one or more phosphorothioate, phosphorodithioate, and/or methylphosphonate linkages. Different chemically compatible modified linkages can be combined, for example, where the synthesis conditions are chemically compatible modifications. While modified linkages are useful, oligonucleotides may include phosphodiester linkages in which the general physiochemical properties of the oligonucleotide polymer are not substantially affected. Additional useful modifications include, but are not limited to, modifications at the 2 '-position of the sugar, such as 2' -O alkyl modifications (e.g., 2 '-O-methyl modifications), 2' -amino modifications, 2 '-halo modifications (e.g., 2' -fluoro); acyclic nucleotide analogs. Other modifications are also known in the art and may be used, for example, to lock nucleotides. In particular, the entire oligonucleotide has modified linkages, e.g., phosphorothioate; having a 3 '-and/or 5' -cap; including a terminal 3 '-5' linkage; an oligonucleotide is or includes a concatemer consisting of two or more oligonucleotide sequences joined by a linking group.

Also provided is a pharmaceutical ON composition for preventing oligonucleotide-induced anticoagulation using a therapeutically effective amount of a pharmacologically acceptable oligonucleotide chelate complex as described herein, prepared using any of the following metal cations: calcium, magnesium, cobalt, manganese, iron, copper and/or zinc. The ON chelate complex may be prepared using two or more different cations as described above. In particular, the pharmaceutical composition is approved for administration to a human or non-human animal (e.g., a non-human primate).

Also provided is a pharmaceutical ON composition that prevents injection site reactions attendant to subcutaneous administration containing a therapeutically effective amount of a pharmacologically acceptable ON chelate complex as described herein prepared using any of the following metal cations: calcium, magnesium, cobalt, manganese, iron, copper and/or zinc. The ON chelate complex may also be prepared using two or more different cations as described above. In particular, the pharmaceutical composition is approved for administration to a human or non-human animal (e.g., a non-human primate).

Also provided is a pharmaceutical ON composition that improves the tolerability of IV infusions containing a therapeutically effective amount of a pharmacologically acceptable ON chelate complex prepared using any of the following metal cations: calcium, magnesium, cobalt, manganese, iron, copper and/or zinc. The ON chelate complex may also be prepared using two or more different cations as described above. In particular, the pharmaceutical composition is approved for administration to a human or non-human animal (e.g., a non-human primate).

Also provided is a pharmaceutical ON composition that prevents oligonucleotide-induced deficiency of calcium, magnesium, iron, manganese, copper or zinc using a therapeutically effective amount of a pharmacologically acceptable ON chelate complex prepared using any of the following metal cations: calcium, magnesium, cobalt, manganese, iron, copper and/or zinc. The ON chelate complex may also be prepared using two or more different cations as described above. In particular, the pharmaceutical composition is approved for administration to a human or non-human animal (e.g., a non-human primate).

Also provided is a pharmaceutical ON composition having improved storage stability, comprising a therapeutically effective amount of a pharmacologically acceptable ON chelate complex prepared using any of the following metal cations: calcium, magnesium, cobalt, manganese, iron, copper and/or zinc. The ON chelate complex may also be prepared using two or more different cations as described above. In particular, the pharmaceutical composition is approved for administration to a human or non-human animal (e.g., a non-human primate).

Also provided is a pharmaceutical ON composition having reduced serum half-life or reduced interaction with serum proteins, comprising a therapeutically effective amount of a pharmacologically acceptable ON chelate complex prepared using any of the following metal cations: calcium, magnesium, cobalt, manganese, iron, copper and/or zinc. The ON chelate complex may also be prepared using two or more different cations as described above. In particular, the pharmaceutical composition is approved for administration to a human or non-human animal (e.g., a non-human primate).

Furthermore, the above-mentioned composition may comprise physiologically and/or pharmaceutically acceptable carriers, adjuvants, vehicles and/or excipients. The characteristics of the carrier may depend on the route of administration. The term "pharmaceutically acceptable carrier, adjuvant, vehicle and/or excipient" refers to a carrier, adjuvant, vehicle and/or excipient that can be administered to a subject, incorporated into a composition of the invention, and does not destroy its pharmacological activity. Pharmaceutically acceptable carriers, adjuvants, vehicles and excipients that may be used in the pharmaceutical compositions described herein include, but are not limited to, the following: ion exchangers, aluminum oxide, aluminum stearate, lecithin, self-emulsifying drug delivery systems ("SEDDS"), surfactants used in pharmaceutical dosage forms (e.g., tween or other similar polymeric delivery matrices), serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium hydroxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat, cyclodextrins (e.g., alpha-, beta-, and gamma-cyclodextrins) or chemically modified derivatives (e.g., hydroxyalkylcyclodextrins, including 2-and 3-hydroxypropyl-beta-cyclodextrin) or other dissolved derivatives may also be used to enhance delivery of the compositions of the present invention.

The compositions described herein may contain other therapeutic agents as described below, and may be formulated, for example, according to techniques well known to those skilled in the art of pharmaceutical formulation, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the intended mode of administration (e.g., excipients, binders, preservatives, stabilizers, flavoring agents, and the like).

The compositions described herein may be administered by any suitable means, e.g., orally, e.g., in the form of tablets, capsules, granules, or powders; under the tongue; keeping in mouth; parenterally, e.g., by subcutaneous, intravenous, intramuscular, or intrasternal (intrastemal) injection or infusion techniques (e.g., as sterile injectable aqueous or nonaqueous solutions or suspensions); nasally, e.g. by inhalation spray; topical, e.g. in the form of creams or ointments; or rectally, e.g., in the form of suppositories; in dosage unit formulations containing a non-toxic, pharmaceutically acceptable vehicle or diluent. The compositions of the present invention may, for example, be administered in a form suitable for immediate release or extended release. Immediate or extended release may be achieved by use of a suitable pharmaceutical composition, or, particularly in the case of extended release, by use of a device (e.g. a subcutaneous implant or osmotic pump). Thus, the above compositions may be suitable for administration by any of the following routes: intraocular, oral, enteric, inhalation, cutaneous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrathecal injection or infusion, intratracheal, intravenous injection or infusion or topical administration.

Exemplary compositions for oral administration include suspensions which may contain, for example, microcrystalline cellulose for providing a plurality of alginic acid or sodium alginate as a suspending agent, methylcellulose as a thickening agent, and sweetening or flavoring agents such as those known in the art; and immediate release tablets may contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and/or lactose and/or other excipients, binders, fillers, disintegrants, diluents and lubricants such as those known in the art. The compositions of the present invention may also be delivered orally via sublingual and/or buccal administration. Molded, compressed or lyophilized tablets are exemplary forms that may be used. Exemplary compositions include those that formulate the compositions of the present invention with fast dissolving diluents such as mannitol, lactose, sucrose, and/or cyclodextrins. High molecular weight excipients such as cellulose (avicel) or polyethylene glycol (PEG) may also be included in such formulations. Such formulations may also include excipients to aid in mucosal adhesion, such as hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), sodium carboxymethyl cellulose (SCMC), maleic anhydride copolymers (e.g., Gantrez), and agents to control release, such as polyacrylic acid copolymers (e.g., Carbopol 934). Lubricants, glidants, flavoring agents, coloring agents and stabilizers may also be added for ease of processing and use.

An effective amount of a compound described herein can be determined by one of ordinary skill in the art and includes exemplary dosages for an adult human, i.e., about 0.1-500mg/kg body weight of active compound per day, which can be administered in a single or single divided dose, e.g., from 1 to 5 times per day. It will be understood that the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, the rate of excretion and clearance, drug combination and the severity of the particular condition. Preferred subjects for treatment include animals, more preferably mammals such as humans and domestic animals such as dogs, cats and the like, which are subject to angiogenesis-dependent or angiogenesis-related conditions.

The pharmaceutical composition may also contain other activity enhancing active factors and/or agents. Pharmaceutical compositions and formulations for administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders and aerosols. Conventional pharmaceutical carriers, aqueous solutions, powders or oily bases, thickeners and the like may be usedAs may be necessary or desirable. Other formulations include those in which the ONs are mixed with a local delivery agent (e.g., lipids, liposomes, fatty acids, fatty acid esters, steroids, chelators, and surfactants). Preferred lipids and liposomes include neutral (e.g., dioleoylphosphatidyDOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoylphetramethylaminopropyl DOTAP, dioleoylphosphatidylethanolamine DOTMA) and other delivery agents or molecules. The ONs may be encapsulated within liposomes or may form complexes, particularly cationic liposomes. Alternatively, the ON may be complexed to a lipid, in particular a cationic lipid. Preferred fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, oleic acid monoglyceride, dilaurin, 1-monocaprylic acid glyceride, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, or C_1-10Alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof.

The disclosure will be more readily understood by reference to the following examples.

Example I

Characterization of degenerate ONs

FIG. 1A details the separation by HPLC (using a hydrophobic column) of two oligonucleotide preparations injected simultaneously into the column. The first of these is called internal standard and is a 21mer phosphorothioate oligonucleotide with a specific defined sequence, the second is REP2006 (40mer degenerate phosphorothioate oligonucleotide). Separating these two species into distinct defined peaks based solely on their physiochemical properties (i.e. size and hydrophobicity); the nucleotide sequences present in each of these ONs had no significant effect ON their physiochemical properties, and thus ON their isolation. Thus, due to the difference in size of the two ON polymers alone, this internal standard elutes from the column as a well-defined peak with a smaller retention time compared to REP 2006. Note that the shoulders on either side of the REP2006 peak are due to the failure sequences typical in the generation of longer oligonucleotides. Despite the heterogeneous sequence nature of REP2006, it was resolved by HPLC to well-defined peaks similar as the 21mer specific sequence, which illustrates the common physiochemical properties of all species in REP2006 preparations, despite the presence of a very large number of different sequences. After HPLC separation of the REP2006 and 21-mer peaks, these can be subjected to Mass Spectrometry (MS) to identify the species present within these defined peaks (FIGS. 1B and 1C).

In FIG. 1B, 21 mers were resolved as a single species with MW 7402.6Da, consistent with this PS-ON with a defined sequence. However, MS analysis of REP2006 (fig. 1C) reveals that there are a very large number of species whose mass range has a nearly perfect normal distribution, consistent with its fully degenerate nature. This mass range is from C₄₀(minimum species) to A₄₀(largest species) and the prevalence of these species is very small with increasing number of species (peak intensity) because their mass is close to the center of the mass range. This is because an increasing number of different sequences will result in similar quality. The fact that all different ON species present in REP2006 have the same retention time ON a hydrophobic column during HPLC separation clearly demonstrates that all ONs of the same size and the same chemical modification (i.e. phosphorothioation) will have highly similar (if not identical) physiochemical properties and therefore, it can be considered that the sequence independent of the nucleotides present in a particular ON molecule is functionally similar for any application or property. Thus, any ON chelate complex formation observed with any particular degenerate ON (e.g. REP 2003, REP 2004) is unlikely to be dependent ON the sequence of the oligonucleotide present and must be dependent ON the conserved physiochemical properties of any ON.

Example II

Formation of chelates of ON with different divalent metal cations

The interaction of the oligonucleotide ammonium salts with various divalent metal cations was examined by Fluorescence Polarization (FP) as described above. During oligonucleotide synthesis, each oligonucleotide was conjugated to Fluorescein Isothiocyanate (FITC) at the 3' end through a rigid 3 carbon linker using well established reagents and synthesis protocols. These oligonucleotides are cleaved from the synthesis and leave as ammonium salts. The oligonucleotides used in this example are described in table 1.

TABLE 1

ON used in example 1

N = degenerate sequence (A, G, C or random combination of T)

PS = phosphorothioation of each bond

2 'O Me = 2' -O methylation per ribose

The 3 ' FITC labeled oligonucleotides used were REP 2032-FL (6mer phosphorothioated degenerate oligodeoxynucleotide), REP 2003-FL (10mer phosphorothioated degenerate oligodeoxynucleotide), REP 2004-FL (20mer phosphorothioated degenerate oligodeoxynucleotide), REP 2006-FL (40mer phosphorothioated degenerate oligodeoxynucleotide), REP2031-FL (40mer polycytidylic acid phosphorothioated oligodeoxynucleotide; SEQ ID NO:4), REP 2107-FL (40mer phosphorothioated degenerate oligonucleotide modified by 2 ' O methylation per ribose) and REP 2086-FL (40mer degenerate phosphodiester oligonucleotide modified by 2 ' O methylation per ribose). Each of these ONs was prepared as a 0.5mM stock solution in 1mM TRIS (pH 7.2). These stocks were used to prepare FP buffers (10 mM TRIS, 80mM NaCl, 1mM EDTA, 10 mM. beta. -mercaptoethanol, and 0.1% Tween)20) 3nM fluorescent ON solution. EDTA was present to remove any divalent metal present in the solution prior to FP measurement. Each of these buffer solutions also contained 80mM NaCl to assess ON complex formation in the presence of a molar excess of monovalent cations (this is reported as a 0mM metal chloride concentration in each of fig. 1-12). Various amounts of ACS grade divalent (2+) metal hydrochloride were added to each fluorescent ON in solution. These salts include calcium chloride, magnesium chloride, cobalt chloride, ferric chloride, manganese chloride, barium chloride, nickel chloride, copper chloride, zinc chloride, cadmium chloride, mercury chloride, and lead chloride. The formation of dimers or higher order ON chelate complexes is monitored by the increase in fluorescence polarization (quantified by dimensionless units "mP") so that the increased formation of ON complexes results in a greater change in mass. The slower tumbling of the generated ON chelates results in an increase in the polarization of the emitted fluorescence (see FIG. 16). The results of these tests are provided in fig. 2-13. In each case, a significant increase in fluorescence polarization was observed with all ONs in the presence of all divalent cations but not in the presence of a high molar excess of Na + (supplied as NaCl), indicating the formation of ON chelates only with divalent metal cations. These results are demonstrated as follows:

in the presence of 80mM NaCl ON does not show any detectable dimer or any other higher order ON complex formation.

In the presence of the following divalent metal cations present in a 2+ charge state, ONs form dimers and higher order complexes: calcium, magnesium, cobalt, iron, manganese, barium, nickel, copper, zinc, cadmium, mercury, and lead. The formation of these ON complexes involves the interaction of the ONs with these divalent metal cations.

The formation of ON complexes is unlikely due to hybridization between nitrogenous bases through traditional Watson-Crick interactions, resulting from the degenerate nature of the ON tested. In addition, REP2031 (SEQ ID NO:4) is unable to self-hybridize under the experimental conditions employed.

The formation of ON complexes is stable and soluble in aqueous solutions and, because these complexes appear to incorporate the divalent metal in question as part of the formed complex, these ON complexes have the effect of sequestering the divalent metal in question from the solution in which they are formed.

The chelation of these metals and the formation of ON chelates is independent of the particular nucleotide sequence, as evidenced by chelation observed with degenerate oligonucleotides, and likewise independent of nucleotide modifications involving phosphodiester linkages or modifications of the 2' ribose moiety.

Chelation of these metals occurs with oligonucleotides 6-40 nucleotides in length.

Chelation of these metals occurs in the presence or absence of phosphorothioate or 2' ribose modifications.

The extensive formation of ON chelate complexes with all ONs with a large number of divalent metal cations in this example also strongly suggests the following:

because divalent cations catalyze ON complex formation and monovalent cations cannot, and because ON complex formation does not occur by base hybridization as described above, ON chelate complex formation must involve some form of "metal ion bridge" between two ONs at a location where electrons capable of filling empty electron orbitals in the cation are readily shared. The most suitable position for "electron sharing" in the phosphodiester bond is the non-bridging oxygen (or sulfur in the case of phosphorothioation) (see FIG. 14B).

Double-stranded ONs, whether DNA or RNA, are expected to exhibit the same chelate formation and thus have the same tendency to chelate metal cations from solution.

These metal bridges must involve intermolecular interactions because intramolecular interactions do not result in any significant increase in fluorescence polarization (see fig. 15A-C and 16).

The soluble ON chelate complex is present at any concentration of ON and divalent metal cation that does not form a chelate precipitate (see fig. 14D).

The ONs cannot form salts with divalent metal cations and do not behave as salts in aqueous solutions. This is in contrast to monovalent cations (exemplified by sodium in this example, but also applies to other monovalent cations such as potassium, lithium or ammonium) which form salts with ONs and behave as salts (electrolytes) in solution. Furthermore, although all ONs used in this example were ammonium salts, in aqueous solution, the ammonium salt ions may dissociate from the ONs (as expected as a salt) and provide no inhibition of ON chelate complex formation from divalent cations. This is further enhanced by the observation that the additional monovalent salt (in this case 80mM NaCl) does not appear to interfere with the formation of ON chelate complexes with divalent cations.

The formation of ON chelate complexes can be expected to occur with any metal, transition metal, lanthanide or actinide in a 2+ charge state and may also occur with metal cations present in a 3+ or higher charge state (e.g., chromium).

ONs greater than 40 mers in length or carrying other modifications or having any particular defined nucleotide sequence are also contemplated to form ON chelates with divalent metal cations, provided they contain bonds capable of sharing electrons in the same manner as non-bridging oxygen (or sulfur) atoms in traditional phosphodiester linkages.

ON salts (e.g., sodium salts) may be used to sequester divalent metals in human or non-human patients such as, but not limited to: cadmium, mercury, lead. In addition, chelation of any one particular divalent metal cation (e.g., iron) by the oligonucleotide sodium salt can be suppressed by formulating the ON sodium salt as an ON chelate complex with another divalent metal cation (e.g., calcium).

Metal salts other than the hydrochloride salt may also allow the formation of ON chelate complexes. Taking calcium salts as an example, other calcium salts compatible with the formation of ON chelate complexes may include, but are not limited to, calcium gluconate, calcium citrate, calcium lactate, calcium malate, calcium aspartate, calcium fumarate, calcium ascorbate, calcium benzoate, calcium erythorbate, and/or calcium propionate.

Example III

Chelate complexes of ON with different calcium and magnesium salts

To further demonstrate the general nature of ON chelate complex formation and also the use of salts of different divalent metal cations in ON chelate complex formation, two different forms of calcium and magnesium salts were used to prepare different ON chelate complexes with different specific sequences of ON. The salts used are calcium chloride, calcium sulfate, magnesium chloride and magnesium sulfate. The ONs used are listed in table 2 below. The FP reaction conditions were the same as those in example 1 except that EDTA was omitted to demonstrate the formation of ON chelate complex in the absence of any EDTA-mediated action.

TABLE 2

ON used in example 2

N = degenerate sequence (A, G, C or random incorporation of T)

PS = phosphorothioation of each bond

2 'O Me = 2' -O methylation per ribose

NA = not applicable (sequence is degenerate)

The results of these experiments are shown in FIGS. 17-24, and demonstrate that all of the ONs tested are capable of forming ON complexes with different salts of calcium and magnesium. Two exceptions to the general observations are REP 2028-FL (40mer poly G, SEQ ID NO: 1; FIGS. 18A and 22A) and REP 2029-FL (40mer poly A, SEQ ID NO: 2; FIGS. 17B and 21B). Both of these ONs are polypurine and, particularly in the case of polyG, are known to form thermodynamically stable intramolecular interactions (known as "G-quatets" in the case of polyG structures), which result in the formation of compact intramolecular complexes of these ONs in which the phosphodiester backbone may partially fold or no longer be able to participate in solution interactions (like chelate formation). REP 2029-FL (SEQ ID NO:2) is able to weakly form chelates probably due to the weak "A-tetrad" interaction with this ON. Thus, not contemplated herein are ONs comprising only polyA and poly G and aptamers that form thermodynamically stable intramolecular interactions. An ON encompassed herein is not exclusively a poly a or polyG ON and/or heptamer (haptamer).

The results of example II show that different forms of magnesium and calcium salts can be used to prepare ON chelates and are further illustrated below:

all ONs in example ii (except REP 2006-FL) contain specific sequences, which do not contain any palindromic sequences capable of hairpin formation, and none of these sequences are self-complementary (self-complementary). Thus, any observed formation of ON chelate complexes is not attributable to hybridization events.

The poor ability of REP 2028-FL (SEQ ID NO: 1) and REP 2029-FL (SEQ ID NO:2) to form ON chelates accounts for the need for a loose phosphodiester backbone for ON chelate formation, again suggesting that a phosphodiester backbone is a chemical feature of an ON that is required to link two or more ONs to form an ON chelate.

Any ON containing a phosphodiester backbone is expected to be able to form chelates regardless of other existing modifications such as phosphorothioation, 2' ribose modification, or locked nucleic acid modification.

ON chelates can be formed with ONs up to 80mer in length, and ONs greater than 80mer also exhibit the same behavior in the presence of divalent metal cations.

Example IV

Formation of ON chelate complexes with double-stranded ON

Double-stranded oligonucleotides are formed from two single-stranded complementary oligonucleotides that hybridize to each other in aqueous solution via Watson-Crick interactions. Since double-stranded ONs still have a phosphodiester backbone exposed outside the DNA helix formed, they should be able to form chelates in the presence of divalent cations. To test this hypothesis, two different double-stranded DNA oligonucleotides were prepared by hybridizing REP 2055-FL (40mer poly AC; SEQ ID NO:6) and REP2033-FL (40mer poly TG; SEQ ID NO:5) and REP2057-FL (40mer poly AG; SEQ ID NO:8) and REP2056-FL (40mer poly TC; SEQ ID NO: 7). The resulting mass increase can be detected by an increase in fluorescence polarization relative to the single-stranded ONs used to prepare the complexes, as the ONs hybridize to give duplexes. Single-stranded ON (REP 2055-FL (SEQ ID NO:6), Rep2033-FL (SEQ ID NO:5), Rep2057-FL (SEQ ID NO:8) and REP2056-FL (SEQ ID NO:7) were each diluted to 20nmM in 1 xFP buffer hybridization of two complementary pairs as identified above was also performed in 1 xFP buffer (10 nM for each ON) and hybridization was confirmed by an increase in fluorescence polarization₂Or 100mM MgCl₂In (1). The formation of ON chelate complexes was monitored by a further increase in fluorescence polarization (see fig. 25). The results of this experiment confirm that two complementary pairs of ONs successfully hybridize to a double-stranded ON, as evidenced by an increase in fluorescence polarization. In addition, to these double-stranded ON adding CaCl₂Or MgCl₂Both lead to a further increase in fluorescence polarization, indicating that these double-stranded ONs can also form chelates in the presence of divalent metal cations. These results also strongly suggest that double-stranded ONs can form ON chelates with any divalent cations and are also expected to have the effect of masking the divalent cations from solution.

Example V

Multiple monovalent cations are not capable of forming chelates with ON

To more specifically demonstrate that the formation of ON chelate complexes cannot occur in the presence of monovalent cations and that divalent cations are particularly required for their formation, ON complex formation with many ON's was observed in FP buffers containing only a primary source of cations (see example 2). A 1 xfp buffer was prepared containing only one of the following salts: sodium chloride, potassium chloride, ammonium chloride, calcium chloride or magnesium chloride, all at a concentration of 80mM, so that the concentration of cations in the FP buffer is comparable. Fluorescently labeled ON as described in example 2 was diluted to 10nM in different FP buffers and ON chelate complex formation was monitored by fluorescence polarization (see fig. 26). In each ON case tested, only the cation Mg was used²⁺And Ca²⁺Without testing for any monovalent cation (Na)⁺、K⁺Or NH₄ ⁺) Chelate formation was observed. As observed in example III, REP 2029-FL (SEQ ID NO:2) and REP 2028-FL (SEQ ID NO: 1) showed only moderate or NO complex formation in the presence of calcium or magnesium, respectively. This further confirms that ONs can only form chelates with divalent cations, whereas ONs can only exist as salts with monovalent salts.

Example VI

Evaluation of Metal content in ON chelate complexes prepared in WFI

To demonstrate the widespread use of ON chelate preparations for all ONs, the ONs and²⁺metal chloride salts several different ON chelates were prepared as shown in table 3. All ONs used in these formulations are sodium salts that have been desalted to remove sodium from NaCl and leave only sodium in the final lyophilized ONs that is essential for ON salt formation. ON chelate complexes are prepared in water for injection (WFI) by first dissolving the prescribed amount of ON sodium salt to a concentration of 50mg/ml and adding the prescribed amount of divalent metal chloride salt to the ON solution. By inductively coupling plasmaBulk emission spectroscopy (ICP-OES) was used to analyze the sodium and related metals of the ON solution prior to divalent metal chloride addition/ON chelate formation. After ON chelate formation, the samples were desalted by ultrafiltration using a 5000MWCO regenerated cellulose filter. It has previously been demonstrated that the filter allows penetration of free salts but not of ONs or cations attached to the ONs. The retained solution (containing ON chelate) was analyzed for sodium and metal content by ICP-OES and for chlorine content by ion chromatography to confirm that the divalent metal present was chelated to the ON and not from the divalent metal salt present in the retained solution (table 4).

TABLE 3

Preparation of various ON chelate complexes

N = degenerate bases (A, G, T or random distribution of C)

PS = phosphorothioation of each bond

2 'O Me = 2' -O methylated ribose

5MeC = 5' methylcytidine

TABLE 4

Verification of Metal content in various ON chelate solutions

These results demonstrate the partial replacement of sodium by calcium, magnesium or iron (2+) ions in ONs varying in length from 20-60 mers, in sequence from fully degenerate ONs to three specific sequences (poly C, poly AC and poly AG) and in ONs with or without phosphorothioate modifications, with or without 2 'ribose modifications and with phosphorothioate and 2' ribose modifications. The ability to formulate 2 'O methyl ribose modified ONs into chelates is expected to extend equally to ONs containing any other 2' ribose modification such as, but not limited to, 2 'fluoro and 2' O methoxyethyl. In addition, they demonstrated that significant sodium displacement was achieved with minimal increase in divalent metal content, consistent with the structure of the ON chelate complex depicted in fig. 15. These results also illustrate the various combinations of calcium, magnesium, iron (2+) and mixed calcium/magnesium salt solutions that may be used to prepare ON chelates and show that any divalent metal salt solution or mixture of divalent metal salt solutions may be similarly used to prepare ON chelates.

Thus, ON chelates can be prepared from ONs that are fully or non-phosphorothioated, contain any number of phosphorothioated linkages, contain at least one 2 ' ribose modification, or are fully 2 ' ribose modified, or do not contain a2 ' ribose modification. The ON may be RNA or DNA or a mixture comprising RNA and DNA. Metal salts useful in preparing ON chelates include, but are not limited to, calcium salts, magnesium salts, iron salts, any other divalent metal salt.

Example VII

Preparation of ON chelate complexes stable in physiological saline

The widely conserved nature of the ON chelate complexes formed and prepared with different divalent metals has been demonstrated in examples II and III, examining the preparation of stable, soluble ON calcium chelate complexes in physiological saline, which is a more appropriate vehicle for administration of ON chelate complexes to subjects. For this experiment, a 200mg/ml solution of the sodium salt of REP2006 in physiological saline was used as the source of ON. The source of calcium was 10% CaCl in WFI₂Solution (100 mg/ml CaCl)₂). Various REP2006 calcium chelates (see table 5) were prepared in 1ml solutions at room temperature using different calcium and REP2006 concentrations according to the protocol of 1) adding REP2006 to a vial, 2) adding normal saline and mixing and 3) adding CaCl₂And mixed. Precipitation of these REP2006 calcium chelate solutions was observed within 36 days (see table 6).

TABLE 5

Conditions for preparing various REP2006 calcium chelates

TABLE 6

Formation of a precipitate in REP2006 calcium chelate at various ON and calcium concentrations

Clear, transparent solutions

+ very little white precipitate at the bottom of the vial

Good colloidal precipitation, translucency

Good colloidal precipitation of +++ and is opaque

The ++++ precipitates mainly at the bottom of the tube but there is residual colloid

The ++++ precipitates at the bottom of the tube and the remaining solution is clear and transparent

As evidenced by the more pronounced meniscus (meniscus) in the vial and in the viscous solution behavior upon gentle inversion of the vial, in all cases, the REP2006 calcium chelate solution exhibited a reduced surface tension and increased viscosity as compared to the REP2006 solution in the absence of calcium (at all concentrations tested). This solution behavior is consistent with the formation of a number of soluble multimeric complexes (chelates), as shown in FIGS. 15A-C. Even though a precipitate has formed in these vials, the remaining solution still exhibits this characteristic of increased surface tension and viscosity. This may be that the precipitate formed in these solutions has adopted a saturated (and insoluble) ON chelate structure as shown in FIG. 15D, where the remaining unprecipitated ON and calcium still form soluble chelates in solution. These behaviors are expected to generally represent the behavior of any ON chelate complex, regardless of ON length, chemical composition, structure (single or double stranded), or the presence of divalent metals. Furthermore, these experiments also demonstrate that while higher concentrations of ON and calcium may initially form fully soluble complexes, these may be dynamically unstable and slowly transition from soluble chelates (as shown in fig. 15A-C) to insoluble chelates (fig. 15D). It may therefore be desirable to prepare ON chelate complexes at, for example, the ON and metal concentrations shown in table 5, with the concentrations described in table 5 having soluble ON chelate complexes that are stable in solution. The optimal ON and metal concentrations that produce soluble ON chelates that remain soluble in solution over time can be varied from those of REP2006 and calcium shown in table 5 for different ON and metal combinations.

Examples vi and vii describe various combinations of ON and divalent metal salt concentrations in different excipients that can be used to prepare ON chelate complexes, and describe combinations of ON and divalent metal salts or mixtures of divalent metal salts that produce ON chelate complex solutions that precipitate rapidly in physiological saline, precipitate slowly, or remain fully soluble for extended periods of time. This is advantageous for using ON chelate complex solutions having any of these stability characteristics.

Thus, any ON salt (including but not limited to ON sodium or ON ammonium salts or mixed ON sodium/ammonium salts) may be used to prepare ON chelate complexes. Desirably, the ON salt is dissolved in an aqueous vehicle, including but not limited to water for injection or normal saline. The source of the divalent metal used to form the ON chelate may be a hydrochloride, sulfate, or any other pharmaceutically acceptable salt, including but not limited to gluconate, citrate, lactate, malate, aspartate, fumarate, ascorbate, benzoate, erythorbate, and/or propionate. The salt may comprise any of the following divalent metal cations: calcium, magnesium, iron (2+), manganese, copper and/or zinc. In addition, mixtures of more than one metal salt may be used. The metal salt may be used directly in powder form, but is preferably prepared as an aqueous solution in the same excipient, since the ON is dissolved therein. The metal salt can be prepared at any concentration up to the maximum limit of solubility of the metal salt in the excipient. The ON chelate complex is preferably prepared by slowly adding a metal salt solution to the ON solution with continuous mixing to prevent aggregation of the precipitate of the ON chelate complex during the addition of the salt solution. Depending ON the concentration of ON and metal salt used, the solution of ON chelate complex may slowly precipitate as ON chelate complex over time or remain completely soluble (see example VII).

Example VIII

Chelation of serum calcium by ON leads to anticoagulation of blood

Having previously described the anticoagulation of certain ONs but demonstrated in this disclosure the anticoagulation of ON chelates, non-FITC labeled ONs were prepared as highly pure sodium salts so as to be biocompatible. These ONs are REP 2004, REP2006, REP2107 and REP2031 (SEQ ID NO:4), which are unlabeled forms of those described in example 1. These various concentrations of ON (in 500. mu.l physiological saline) were added to 5mL of fresh human whole blood collected in a citrate tube. Thrombin time in the presence of these ON, a well-established measure of the coagulation status of blood, was assessed and compared to values using well-established clinical test methodsThe prothrombin time in the presence of the same volume of saline was compared. The relative effect on agglutination is expressed as a ratio (PPT)_{Oligo (A)}:PPT_{Physiological saline}) And is reported as Normalized Ratio (NR). NR of 1 indicates a normal coagulation state and NR greater than 1 indicates that blood is anticoagulated. The results of these tests are shown in fig. 27. For all ONs evaluated, there was a dose-dependent increase in anticoagulation.

REP2055 (SEQ ID NO:6) is a polypeptide having the sequence (AC)₂₀The 40mer phosphorothioation ON of (1). The effect of the addition of the sodium salt of 2.5mM REP2055 on the blood coagulation status was evaluated as described above. To determine whether anticoagulation is due to calcium sequestration from the blood, the effect of various amounts of calcium supplements (in the form of calcium chloride) on REP2055 (SEQ ID NO:6) -induced anticoagulation was observed. The results of this experiment are shown in fig. 28. As expected from the results in FIG. 27, 2.5mMREP 2055 (SEQ ID NO:6) induced significant anticoagulation of blood. This anticoagulation is effectively suppressed by calcium supplementation and can be completely reversed at calcium chloride concentrations greater than 2.25 mM. These results strongly suggest that anticoagulation by oligonucleotides is mediated by ON chelate complexes formed in the blood after administration of ON resulting in calcium chelation as described in the above examples. Furthermore, these results further identify a method of preventing anticoagulation of blood by ONs by eliminating chelation of ONs to be administered by preparing the ONs as calcium chelates. Elimination of calcium chelation may also be achieved with ON chelate complexes using another divalent metal salt including, but not limited to: magnesium, manganese, iron (2+), copper and/or zinc. These methods of suppressing oligonucleotide-mediated anticoagulation can be expected to be effective with oligonucleotides administered to human or non-human subjects intravenously or otherwise.

The results of this experiment also question the nature of the interaction of ON with serum proteins. The former hypothesis for the anticoagulant properties of ON was that ON interacts directly with proteins of the coagulation cascade, but we note that most of these proteins are calcium binding proteins or proteins involved in the calcium dependent coagulation cascade (Sheerhan and Lan, 1998, Blood, 92: 1617). The fact that blood anticoagulation by ON elimination by calcium addition and the fact that ON acts as a calcium chelator suggest the following:

● it is essential but not sufficient for anticoagulation to participate in the anticoagulation of the ON protein binding-removal of calcium from calcium dependent proteins by chelation by ON may be the mechanism by which ON exerts anticoagulation activity.

The interaction of the ● ON protein with components of the coagulation cascade may itself be calcium dependent.

Albumin, a major blood protein, also binds calcium and is part of the serum calcium regulation mechanism. Albumin is known to interact with ON and may play an important role in the circulating half-life of ON in the blood. From the results of the above anticoagulation experiments, it appears that most protein interactions in blood can be catalyzed to a large extent by the calcium-chelating function of ONs. Thus to reduce serum protein interactions, which may also have the effect of reducing the circulating half-life of the ON and improving its tolerability for parenteral administration, the ON may be administered to the subject as an ON chelate complex. These complexes can be prepared from calcium salts, but can also be prepared from other suitable metal salts to eliminate the tendency of ONs to chelate calcium when applied. The chelating activity of such complexes has been eliminated and would therefore be expected to have significantly reduced interactions with serum proteins. The advantages of such reduced serum protein interactions would be improved tolerance of the administered ONs (as chelates) and shorter half-lives of free ONs in the blood.

Example IX

Development and prevention of hypocalcemia in human subjects with chronic ON treatment

To further examine whether chelation of divalent metal cations by ONs described in the above examples was biologically relevant in human subjects, the effect of long-term administration of ONs ON serum calcium was examined in patients with chronic liver disease. These subjects are particularly suitable for testing the biological effects of ON sequestration (if any) as they have been shown to suffer from vitamin D deficiency, which is often accompanied by a disturbance in mineral metabolism and a reduction in bone mineral density (artemi et al, 2010, dig.dis.sci.,55:2624-2628 and George et al, 2009World J gasteroentol, 15: 3516-3522). Thus, if the chelation of long-term administration of ONs is altering the homeostasis of divalent metals (e.g., calcium) in human subjects, the altered effects will be more readily observed in these patients, as if such alterations occurred they would make it difficult to hinder any serum metal imbalance. Patients with chronic hepatitis B infection (diagnosed with chronic liver disease) were treated once a week with ON REP2055 (SEQ ID NO:6) in physiological saline (as GMP grade sodium salt) by slow IV infusion. Levels of total serum calcium were monitored in subjects by accepted clinical laboratory methods. The first two subjects receiving ON treatment developed significant hypocalcemia in 12 weeks of treatment (fig. 29A). This hypocalcemia varies in severity but persists during the subsequent 13 weeks of treatment. Mineral supplements (providing calcium, magnesium and zinc) were then administered to subjects receiving chronic ON treatment with REP2055 (SEQ ID NO:6) to counter chelation by ON treatment. Subjects receiving the mineral supplement at the time of ON treatment did not develop hypocalcemia (fig. 29B). These results demonstrate that the chelation activity of ONs does occur in human subjects. This can be observed directly with serum calcium, but can also occur with other biologically relevant divalent metal cations (such as magnesium, zinc and copper). In addition, metal deficiencies caused by ON chelation can be corrected by mineral supplements and can also be reduced by administering ON as a chelate.

Because ONs have been shown to chelate divalent metal cations in human subjects, administration of non-chelated ONs can be used to chelate harmful heavy metals in the subject, such as mercury, cadmium, lead, or even chromium (6 +). Such a method would involve the administration of a pharmaceutically acceptable salt of ON in a suitable excipient, preferably by intravenous administration but also by other parenteral routes, the ON being designed to lack sequence dependent function (such as, but not limited to, the specific sequence ON described in example iii). Patients with normal liver function would be expected to be able to block the calcium sequestration that would occur, however a mineral supplement (as in example ix) may be provided to ensure prevention of serum depletion of biologically important divalent cations. This unchelated ON will sequester heavy metals present in the blood, immediately reducing or eliminating the deleterious effects of these metals and likely also accelerating their elimination from the subject in question.

Since it has also been demonstrated that double stranded ONs can also form ON chelates (and thus also mask divalent metals), it is expected that administration of any double stranded nucleic acid (e.g., siRNA) would also be expected to have at least some chelation of a single stranded ON as described in the above examples. Thus, it is advantageous to prepare the double-stranded ONs as chelates prior to administration.

Example X

ON chelate complexes may suppress injection site reactions of subcutaneously administered oligonucleotides

Injection Site Reactions (ISRs) with subcutaneously administered oligonucleotides in human patients are common, even with highly modified oligonucleotides to reduce their immunostimulatory properties. Because subcutaneous administration involves injection of high concentrations of oligonucleotides (typically >100 mg/ml), chelation (most likely calcium but also other divalent metals such as magnesium) located around the injection site must be substantial and contribute to the often observed ISR. To test this hypothesis, a human patient was administered either REP2055 (SEQ ID NO:6) or REP2139 (SEQ ID NO:13, a REP2055 analog in which all ribose are 2 'O methylated and all cytosine bases are 5' methylated) by a subcutaneous route of administration. These two solutions were prepared aseptically as sodium salts or as calcium chelates in normal saline (see tables 7 and 8 and following the procedure in example viii). To control patient-to-patient variation, injection reactivity of both formulations of each ON in the same human subject was evaluated. The ISR of each injection site in the subject was monitored 12 hours after REP2055 (SEQ ID NO:6) administration and 72 hours after REP2139 (SEQ ID NO:13) administration. The results of this experiment are provided in tables 7 and 8.

TABLE 7

Prepared as a calcium chelate (1 cc bolus, 20mg CaCl)₂Per 100mg ON) suppression of injection site reaction of REP2055 (SEQ ID NO:6)

TABLE 8

By formulating into calcium chelate (2 cc bolus, 30mg CaCl)₂/100mg ON) inhibition of injection site reaction of REP2139 (SEQ ID NO:13)

These results demonstrate that administration of ONs as calcium chelates substantially reduces or eliminates the ISR of two different subcutaneously administered ONs. These results further demonstrate that calcium (and possibly other divalent metals) sequestration by ONs plays an important role in the manifestation of ISRs commonly associated with subcutaneous administration of ONs. Furthermore, these results identify a method of preventing ISR of any subcutaneously administered ONs by performing the administration of said ONs as chelates. In these examples, calcium was used as a vehicle for chelate formation, but reduction of ISR is also expected to occur with ON chelates that have been prepared with another suitable divalent metal other than calcium. Any ON metal chelate would be expected to have its propensity to chelate eliminated calcium, which would be a potential mechanism to improve SC tolerance of ONs administered as chelates. In light of the widely conserved nature of ON chelate complex formation disclosed in the examples herein, the ability of ON chelate complexes to suppress oligonucleotide-induced ISR is expected to be effective for any ON and or modification and or single or double stranded ON of any particular sequence. Using calcium as an exemplary divalent metal in this example, other calcium salts including, but not limited to, calcium gluconate, calcium citrate, calcium lactate, calcium malate, calcium aspartate, calcium fumarate, calcium ascorbate, calcium benzoate, calcium erythorbate, and/or calcium propionate may be used to prepare ON chelate complexes and are expected to produce ON chelate complexes with the same suppression of oligonucleotide-mediated ISR that accompanies subcutaneous administration. ON chelates may be prepared with other divalent metal cations such as, but not limited to: magnesium, manganese, iron, copper and zinc.

In the preparation of ON chelate complexes, it may be desirable to use other cations that are not divalent atoms, but which may similarly prevent chelation of the oligonucleotide. ON chelates prepared with these cations may also be used in formulations to suppress anticoagulation by ON or suppress injection site reactions associated with subcutaneous administration of ON or to prevent masking of biologically important divalent metals after ON administration. Such counterions can include, but are not limited to: 3+ or higher charge state or an atom of an organic cation.

In the preparation of ON chelate complexes, it is preferred to use mixtures of divalent cations (i.e., with calcium and magnesium salts) to prepare ON chelate complexes. Such mixed ON chelates are easier to prepare and have greater solubility than chelates prepared with a single divalent cation, and therefore would be better suited for high concentration applications.

Given the above examples using a variety of ON sequences, with a variety of modifications and in either single-stranded or double-stranded states and the use of a variety of divalent metals, the formation of ON chelate complexes, regardless of other modifications, can be considered a general feature of any and all ONs having a phosphodiester backbone (whether phosphorothioated or not). Thus, the formation of ON chelate complexes in the blood or subcutaneous space is a normal feature of any ON administration when the ON is administered as a salt (usually the sodium salt), which results in the masking of divalent metal cations, even though the secondary effects of such chelation may be asymptomatic in the particular population of subjects receiving the ON in question. Importantly, there are some examples of ON (PRO-051/GSK2402968-Goemans et al, 2011 New England J. Med.,364: 1513-. Even though these two ONs have different sequences and different 2' ribose modifications, they both have phosphodiester backbones (phosphorothioated in both cases) and can therefore form ON chelates and mask divalent metals from the local environment (in this case the subcutaneous space). Furthermore, even though ON chelation must occur, it has been shown that both ONs are capable of exerting their biological effects, and therefore ON chelates do not interfere with the biological activity of the ON in biological systems.

It is well accepted and well documented in the art that all phosphorothioated ONs (regardless of base sequence) generally achieve the highest drug concentrations in the kidney and liver. Historically, long-term administration of many different phosphorothioated ONs has been associated with mild hepatic or renal dysfunction. Although the causes of these dysfunctions have not been clearly elucidated, given the conserved chelation of ONs in general, it is likely that chelation of divalent metals in the liver and kidney with chronic ON administration is significant, since chelation activity may be most significant in these organs due to the presence of high concentrations of ON. Administration of ONs as chelates will not alter organ biodistribution (or affect bioactivity as shown above), but may help prevent metal deficiency in the liver and kidneys, which has an effect ON the normal function of these organs.

Whereas ON chelates lead to the formation of multimeric ON complexes in solution, these complexes may be much more resistant to nuclease degradation and possibly hydrolysis, and phosphorothioate ONs may also be more resistant to oxidation. Thus, storage of any ON as a chelate may greatly increase its stability in aqueous solution without significantly altering its biological activity when administered to a subject.

Claims (26)

1. Use of an oligonucleotide chelate complex comprising at least two oligonucleotides linked by a divalent cation through their phosphodiester bonds for the preparation of a medicament for suppressing or reducing anticoagulation of the oligonucleotide administration to a subject, for suppressing or reducing subcutaneous injection site reactions of the oligonucleotide administration to a subject, or for improving tolerance of the oligonucleotide when administered by IV infusion to a subject.

2. Use according to claim 1, wherein the chelate is a calcium chelate.

3. Use according to claim 1, wherein the chelate is a magnesium chelate.

4. Use according to claim 1 wherein the chelate is a mixed magnesium/calcium chelate.

5. The use of claim 1, wherein the chelate comprises at least one double-stranded oligonucleotide.

6. The use of claim 1, wherein the chelate complex comprises at least one oligonucleotide having at least one phosphorothioate linkage.

7. Use according to claim 1, wherein the chelate complex comprises at least one fully phosphorothioated oligonucleotide.

8. The use of claim 1, wherein said chelate complex comprises at least one oligonucleotide having one 2' modified ribose.

9. Use according to claim 1, wherein the chelate complex comprises at least one oligonucleotide in which each ribose is 2' O-methylated.

10. The use of claim 1, wherein said chelate complex comprises at least one oligonucleotide consisting of seq id NOs 3 to 14.

11. Use according to claim 1, wherein the chelate complex comprises at least one oligonucleotide comprising at least one 5-methylcytosine.

12. The use of claim 1, wherein said chelate complex comprises at least one oligonucleotide consisting of SEQ ID No. 6 comprising at least one 5-methylcytosine.

13. The use according to claim 1, wherein said chelate complex comprises at least one oligonucleotide consisting of SEQ ID NOs 3-12 wherein each cytosine is a 5-methylcytosine.

14. The use of claim 1, wherein the chelate further comprises a pharmaceutically acceptable excipient.

15. Use of a divalent cation in the manufacture of a medicament for suppressing or reducing anticoagulation of the administration of an oligonucleotide to a subject, for suppressing or reducing a subcutaneous injection site reaction of the administration of an oligonucleotide to a subject, said medicament formulated for subcutaneous administration to a subject, wherein said medicament comprises an oligonucleotide chelate complex comprising at least two oligonucleotides linked by divalent cations through their phosphodiester bonds.

16. Use of a divalent magnesium cation in the manufacture of a medicament for inhibiting or reducing anticoagulation by administration of an oligonucleotide to a subject, or for improving tolerance of an oligonucleotide when administered by IV infusion to a subject, said medicament formulated for intravenous administration to a subject, wherein said medicament comprises an oligonucleotide chelate complex comprising at least two oligonucleotides linked by divalent magnesium cations through their phosphodiester bonds.

17. Use of a divalent magnesium and calcium cation in the manufacture of a medicament for inhibiting or reducing anticoagulation or for improving tolerance of an oligonucleotide administered to a subject when administered by IV infusion, the medicament formulated for intravenous administration to a subject, wherein the medicament comprises an oligonucleotide chelate complex comprising at least two oligonucleotides linked by divalent magnesium and calcium cations through their phosphodiester bonds.

18. Use of a divalent zinc cation in the manufacture of a medicament for inhibiting or reducing anticoagulation by administration of an oligonucleotide to a subject, or for improving tolerance of an oligonucleotide when administered by IV infusion to a subject, said medicament formulated for intravenous administration to a subject, wherein said medicament comprises an oligonucleotide chelate complex comprising at least two oligonucleotides linked by a divalent zinc cation through their phosphodiester bond.

19. Use of two or more different divalent cations in the manufacture of a medicament for inhibiting or reducing anticoagulation of administration of an oligonucleotide to a subject, or for improving tolerance of an oligonucleotide when administered by IV infusion to a subject, the medicament formulated for intravenous administration to a subject, wherein the medicament comprises an oligonucleotide chelate complex comprising at least two oligonucleotides linked by two or more divalent cations through their phosphodiester bonds.

20. Use of a divalent cation in the manufacture of a medicament for suppressing or reducing anticoagulation of the administration of an oligonucleotide to a subject, for suppressing or reducing subcutaneous injection site reactions of the administration of an oligonucleotide to a subject, wherein said medicament comprises an oligonucleotide chelate complex comprising at least two oligonucleotides linked by divalent magnesium cations through their phosphodiester bonds and wherein said chelate complex comprises at least one double-stranded oligonucleotide.

21. Use of a divalent cation in the manufacture of a medicament for suppressing or reducing anticoagulation of the administration of an oligonucleotide to a subject, for suppressing or reducing subcutaneous injection site reactions of the administration of an oligonucleotide to a subject, wherein said medicament comprises an oligonucleotide chelate complex comprising at least two oligonucleotides linked by divalent cations through their phosphodiester bonds and wherein said chelate complex comprises at least one oligonucleotide having one 2' modified ribose.

22. Use of a divalent cation in the manufacture of a medicament for suppressing or reducing anticoagulation of the administration of an oligonucleotide to a subject, for suppressing or reducing subcutaneous injection site reactions of the administration of an oligonucleotide to a subject, wherein said medicament comprises an oligonucleotide chelate complex comprising at least two oligonucleotides linked by divalent cations through their phosphodiester bonds and wherein said chelate complex comprises at least one oligonucleotide with each ribose 2' O-methylated.

23. Use of a divalent cation in the manufacture of a medicament for suppressing or reducing anticoagulation of the administration of an oligonucleotide to a subject, for suppressing or reducing subcutaneous injection site reactions of the administration of an oligonucleotide to a subject, wherein said medicament comprises an oligonucleotide chelate complex comprising at least two oligonucleotides linked by divalent cations through their phosphodiester bonds and wherein said chelate complex comprises at least one oligonucleotide consisting of SEQ ID NOs 3 to 14.

24. Use of a divalent cation in the manufacture of a medicament for suppressing or reducing anticoagulation of the administration of an oligonucleotide to a subject, for suppressing or reducing subcutaneous injection site reactions of the administration of an oligonucleotide to a subject, wherein said medicament comprises an oligonucleotide chelate complex comprising at least two oligonucleotides linked by divalent cations through their phosphodiester bonds and wherein said chelate complex comprises at least one oligonucleotide comprising at least one 5-methylcytosine.

25. Use of a divalent cation in the manufacture of a medicament for suppressing or reducing anticoagulation of the administration of an oligonucleotide to a subject, for suppressing or reducing subcutaneous injection site reactions of the administration of an oligonucleotide to a subject, wherein said medicament comprises an oligonucleotide chelate complex comprising at least two oligonucleotides linked by divalent cations through their phosphodiester bonds and wherein said chelate complex comprises at least one oligonucleotide consisting of SEQ ID NO:6 further comprising at least one 5-methylcytosine.

26. Use of a divalent cation in the manufacture of a medicament for suppressing or reducing anticoagulation of oligonucleotide administration to a subject, for suppressing or reducing subcutaneous injection site reactions of oligonucleotide administration to a subject, wherein said medicament comprises an oligonucleotide chelate complex comprising at least two oligonucleotides linked by divalent cations through their phosphodiester bonds and wherein said chelate complex comprises at least one oligonucleotide consisting of SEQ ID NOs 3-12 wherein each cytosine is 5-methylcytosine.