CA2746981A1 - Methods for inhibition of apolipoprotein h - Google Patents

Abstract

The present disclosure relates to methods for the inhibition of apolipoprotein H (apoH) and uses for the treatment of various diseases. In particular, the ability of amphipathic, nucleic-acid based polymers to bind to and inhibit the activity of apoH are disclosed herein. This inhibition is not dependent on the sequence of the nucleic acid, but rather on the presence of phosphorothioation or other sulfur modifications to increase hydrophobicity along the length of the polymer, thus rendering the oligonucleotide polymer more amphipathic.

Description

METHODS FOR INHIBITION OF APOLIPOPROTEIN H
TECHNICAL FIELD
[0001] The present invention relates to compositions and methods for the inhibition of apolipoprotein H (apoH) including using oligonucleotides whose apoH interaction is not depedent on the sequence of nucleic acids present and comprising phosphorothioation or other sulfur modifications.

BACKGROUND ART

[0002] ApoH (also known as (32-glycoprotein I) is comprised of 345 amino acids (as predicted by analysis of the open reading frame or "ORF") which includes 19 hydrophobic signal peptide residues not present in the mature protein, which is 326 amino acids (Lozier et al., 1984, Proc. Nat. Acad. Sci.
81:
3640). ApoH contains 5 homologous segments of approximately 60 amino acids each (GP-1 domains) which are related to domains commonly found in several complement control proteins (Kristensen et al., 1987, Federation Proceedings 46: 2463). The human apoH gene is located on chromosome 17q23-24 and is expressed primarily in the liver (Steinkasserer et al., 1991, Biochemistry Journal 277: 387). The overall tertiary structure of apoH is thought to be conserved amongst mammalian species including humans (Kato et al., 1991, Biochemistry 30: 11687; Aoyama et al., 1989, Nucleic Acids Res. 17: 6401; Nonaka et al., 1992, Genomics 13: 1082; Sellar et al., 1993, Biochem. Biophys. Res. Comm.
191: 1288).

[0003] ApoH is a multifunctional apolipoprotein known to play a role in many areas of human physiology which include lipoprotein metabolism (Kamboh et al., 1991, Advances in Lipid Res. 1: 9), coagulation (Roubey et al., 1994, Blood 82: 2854) and the production of anti-phospholipid antibodies (Schousboe et al., 1985, Blood 66: 1086). ApoH is a component of HDL, VLDL and chylomicrons and may play a role in the metabolism of triglyceride-rich lipoproteins. In the blood, apoH inhibits serotonin release from platelets and prevents subsequent waves of ADP-induced platelet aggregation (Nimpf et al., 1987, Atherosclerosis 63: 109). It is also involved in the binding of negatively charged compounds in the agglutination process and acts to block contact-mediated agglutination in the intrinsic blood coagulation pathway (Schousboe et al., 1985, Blood 66:
1086). ApoH complexed with anionic phospholipids is known to be the antigen involved in the production of anti-phospholipid antibodies (aPA) in patients with systemic lupus erythematosus (SLE) and primary antiphospolipid syndrome (APS) (McNeil et al., 1999, Proc. Natl. Acad. Sci. 87: 4120; Galli et al., 1990, The Lancet 335: 1544; and Jones et al., 1992, J. Rheumatology 19: 1397).
ApoH has also been indirectly linked to hepatitis B virus (HBV) infection by virtue of its identification as a potential receptor for HBV in hepatocytes (Neurath et al., 1994, Virology 204: 475; Stefas et al., 2001, Hepatology 33:
207) and its association with the hepatitis B surface antigen (HBsAg) (Gao et al., 2003, World J. Gastroenterology 9: 2114). It is possible that the concomitant association of apoH with HBsAg and anionic phospholipids (Mehdi et al., 2008, Biochem. Biophys. Acta. 1782: 163) indicates that this apolipoprotein may play an important role in facilitating the release of hepatitis B subviral particles (which lack a viral capsid) by rendering HBsAg/anionic phospholipid complexes inside cells competent for secretion by hepatocytes in the absence of a capsid. The secretion of subviral particles by infected hepatocytes is the mechanism by which HBV infection results in the development of massive concentrations of HBsAg in the blood which in turn is thought to inhibit immune function and allow the maintenance of chronic HBV infection.

[0004] SLE and APS are chronic inflammatory disorders which result from chronic dysfunction of the immune system. Inflammation in SLE is known to affect the connective tissues and is associated with the generation of antibodies directed against many host cellular components. This disease is more prevalent among women than men (5:1) and typically becomes most symptomatic between the ages of 15 and 45. The prevalence of this disease is approximately 50-75 per 100,000 individuals. The frequency of detection of aPA in patients with SLE varies between 20 and 60%. While only a third of patients with aPA
will have clinical manifestation of the disease, these manifestations can include thrombotic complications affecting the cerebral, pulmonary, retinal and placental vessels. Due to the critical nature of these disorders, it is important to provide new methods for their therapeutic intervention.

[0005] Hepatitis B virus (HBV) is an enveloped virus of the family hepanadnaviridae. This virus is the cause of the largest known pandemic viral infection, having affected more than 2 billion people worldwide and left more than 400 million of these individuals with chronic liver infection (Kowdley et al., 2004, J. Clin. Gastroenterology 38(10 suppl): S132). Annually, there are approximately 4 million new acute cases of hepatitis caused by HBV and many million deaths attributable to the effects of HBV infection (World Health Organization, 1996, State of the world's vaccines and immunization. Geneva) which include active hepatitis, cirrhosis or primary liver cancer (Lavanchy et al., 2004, J Viral Hepatitis. 11: 97). To meet the need for treatment of this disease, several drugs have been approved for the treatment of HBV which include lamivudine, adefovir, entecavir, telbivudine, clevudine and others. However, while these drugs can suppress the replication of the virus in the liver, they usually do not achieve a sustained virological response (SVR) when treatment is halted, even after several years of continuous treatment (Yuen et al., 2004, J
Clin Microbiol 42: 4882; Manesis et al., 2007, Antivir Therapy 12: 73;
Brunetto et al., 2009, Hepatology 49: 1141; Heathcote et al., 2009, AASLD 2009, Boston, USA: Abstract 483; Wursthorn et al., 2009, AASLD 2009, Boston, USA: Abstract 487). As such, there is still a need for a more effective treatment for HBV.

[0006] It would thus be desirable to be provided with more effective treatment for HBV infection, SLE and APS.

SUMMARY

[0007] In accordance with the present disclosure there is now provided a composition for the inhibition of apoH comprising at least one phosphorothioate oligonucleotide, wherein the oligonucleotide interacts with apoH and wherein the interaction of the oligonucleotide is non-sequence dependent.

[0008] It is also provided a composition for the inhibition of apoH comprising at least one phosphorothioate oligonucleotide, wherein the oligonucleotide is comprised of alternating adenosine and cytosine.

[0009] Additionally, it is provided a composition for the inhibition of apoH
comprising at least one phosphorothioate oligonucleotide, wherein the oligonucleotide is comprised of alternating adenosine and guanosine.

[0010] It is also provided a composition for the inhibition of apoH comprising at least one oligonucleotide consisting of SEQ ID NO:3.

[0011] It is also provided a composition for the inhibition of apoH comprising at least one oligonucleotide, wherein the at least one oligonucleotide comprises any of the following modified bases: 4'thiouracil, 5' thiouracil, 5;
thiocytidine, 4'thiothymine or deoxy-4'thiouracil and 6'thioguanine.

[0012] In an embodiment, the at least one oligonucleotide is between 20 and 80 nucleotides in length, is oligonucleotide is phosphorothioated, contains at least one phosphodiester linkage, and/or contains at least one unmodified thymidine, uridine, adenosine, cytosine or guanosine base.

[0013] In another embodiment, the at least one phosphorothioate oligonucleotide is SEQ ID NO-1 or SEQ ID NO-2.

[0014] In another embodiment, the oligonucleotide has at least one 2' ribose modification, all ribose sugars 2' modified, has at least one 2' 0-methyl ribose modification, has all ribose sugars 2' 0-methyl modified, has at least one 2'-O
(2-methoxyethyl) ribose modification, has all ribose sugars 2'-O (2-methoxyethyl) modified, contains at least one locked nucleic acid, and/or contains at least one methylphosphonate.

[0015] In a further embodiment, the composition also comprises a carrier.

[0016] It is also provided a method of inhibiting the function of apoH in a patient comprising the step of administering to said patient the composition described herein.

[0017] In an embodiment, the patient is a human patient or a non-human animal patient.

[0018] In another embodiment, the method described herein is further treating at least one of systemic lupus erythematosus, anti-phospholipid syndrome and hepatitis B infection.

[0019] It is also provided the use of the composition described herein for the treatment of systemic lupus erythematosus, for the treatment of anti-phospholipid syndrome, and/or for the treatment of hepatitis B infection in a human patient or a non-human animal.

[0020] It is also provided the use of the composition described herein in the manufacture of a medicament for the treatment of systemic lupus erythematosus, in the manufacture of a medicament for the treatment of anti-phospholipid syndrome, and/or in the manufacture of a medicament for the treatment of hepatitis B infection in a human patient or a non-human animal.

[0021] It is also provided an antisense oligonucleotide complimentary to the mRNA of an isoform of apoH; a siRNA complimentary to the mRNA of an isoform of apoH; a morpholino antisense oligonucleotide complimentary to the mRNA of an isoform of apoH; and a miRNA that targets the mRNA of any isoform of apoH.

[0022] It is additionally provided an oligonucleotide aptamer optimized for interaction with apoH.

[0023] In an embodiment, the oligonucleotide comprises either D- or L-nucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Reference will now be made to the accompanying drawings, and in which:

[0025] Figs. 1A and B illustrate a graphic representation showing the non sequence-dependent and size dependent interaction of phosphorothioate oligonucleotides with apoH wherein FITC-labelled phosphorothioated oligonucleotide randomer baits of different sizes were mixed with various concentrations of purified human apoH in solution. Oligonucleotide-apoH
complex formation was monitored by measurement of fluorescence polarization as described in the methods.

[0026] Fig. 2 illustrates a graphic representation showing the effect of other sulfur modifications of oligonucleotides in their interaction with apoH. 40mer oligonucleotides containing only 4-thiodeoxyuridine were prepared with either phosphodiester or phosphorothioate linkages. These compounds were added to pre-formed FITC-REP 2006-apoH complexes. Interactions of these compounds with apoH were monitored by a reduction in fluorescence polarization, indicating that FITC-REP 2006 had been displaced from the apoH in solution by the compounds in question.

DETAILED DESCRIPTION

[0027] It is provided a family of compounds have been identified (amphipathic oligonucleotide-based polymers) which interact with apoH via non-sequence-dependent amphipathic interactions. The interaction is dependent on sulfur modifications of the oligonucleotide polymer such as phosphorothioation or sulfur modification to the nitrogenous bases and tolerates the addition of 2' modification of riboses. The strength of this interaction becomes longer as the size of the polymer grows so that oligonucleotides less than 20 nucleotides in length show minimal to moderate binding and oligonucleotides greater than 20 nucleotides show stronger binding.

[0028] It is also provided compounds and formulations for the inhibition of apoH function, methods for the inhibition of apoH function and or synthesis and methods for the treatment of systemic lupus erythematosus, primary antiphospolipid syndrome, and hepatitis B infection in human and non-human patients .

[0029] It is disclosed herein methods for the inhibition of apolipoprotein H
(apoH) and the potential uses for these methods in the treatment of various diseases. In particular, the ability of amphipathic, oligonucleotide based polymers to bind to and inhibit the activity of apoH are disclosed herein.
This inhibition is not dependent on the sequence of the nucleic acid, but rather on the presence of phosphorothioation or other sulfur modifications to increase hydrophobicity along the length of the polymer, thus rendering the oligonucleotide polymer more amphipathic. The use of these compounds has also identified a novel therapeutic application of apoH inhibition, namely that apoH inhibition is an effective method for the inhibition of hepatitis B
infection.
Further methods for the inhibition of apoH and their therapeutic applications are also disclosed.

[0030] The expression "inhibition of apoH" is intended to mean the inhibition of apoH function and/or synthesis.

[0031] Administration of REP 2055 (also identified as SEQ ID NO:1) to patients with chronic hepatitis B infection produced a rapid and potent suppression of subviral particle release which results in the rapid clearance of HBsAg from the blood of infected patients. HBsAg is known to effectively suppress the immune response to HBV infection and its removal from the blood also removes the suppression of immune function, allowing patients to regain immunological control over their infection. This immunological control persisted even after REP 2055 administration was halted. This provides for the first time evidence that modulation of apoH function is a highly effective method for the treatment of HBV and also strongly suggests that other methods which modulate the function of apoH, either by direct or indirect inhibition of apoH
function or direct or indirect modulation of apoH synthesis, will also be effective in the treatment of hepatitis B.

[0032] Accordingly, the primary object of the present disclosure was to identify a family of compounds (amphipathic oligonucleotide-based polymers) which can modulate apoH function by direct interaction with apoH and to define the parameters affecting apoH interaction of these compounds:

1. polymer size: oligonucleotides less than 20mer are suboptimal for apoH binding, oligonucleotides 20mer or greater are optimal for apoH
binding and binding is observed with oligonucleotides up to 80mer;

2. polymer chemistry: oligonucleotides must possess at least one sulfur based modification on each nucleotide along the entire polymer (eg phosphorothioation of the phosphodiester linkage or sulfurization of the nitrogenous base); 2' ribose modifications of oligonucleotides can be tolerated without substantially affecting the apoH interaction; and 3. in the context of phosphorothioated or otherwise sulfur modified oligonucleotides, the oligonucleotide-apoH interaction is not sequence dependent.

[0033] Fluorescence polarization is a common methodology used to examine intermolecular interactions. In this technique, the bait is labeled with a fluorescent tag (e.g. FITC or CY2). In solution, the bait molecule tumbles freely in solution due to Brownian motion which results is poor polarized fluorescence emission when the bait is subjected to excitation with the correct wavelength of light. With a ligand of sufficient molecular weight (at least the same size as the bait), the interaction between the bait and the ligand introduced a substantial inhibition of the tumbling of the complex in solution. As a result, fluorescence emission becomes significantly polarized upon excitation. Thus with this technique, interactions can be measured in solution with no physical constraints on either binding partner. Fluorescence polarization is reported as the dimensionless mP, which is directly proportional to the fraction of bound bait molecules in the reaction. For example, if a very small fraction of bait molecules were bound by a particular ligand, there would be very little fluorescence polarization and consequently small mP values. At the other end of the spectrum, if a large proportion of bait molecules were bound by a particular ligand (or with a higher concentration of ligand), there would be substantial fluorescence polarization and consequently large mP values. In this fashion, binding isotherms for particular bait-ligand interactions can be generated by varying concentrations of ligand in the presence of a fixed amount of fluorescently tagged bait.

[0034] It is also disclosed a method of inhibition of apoH function using a non-nucleic acid based polymer which mimics the chemical characteristics of amphipathic oligonucleotide based polymers.

[0035] Another object of the present disclosure is to provide for a method of inhibition of apoH function by a small molecule which targets apoH.

[0036] Another object of the present disclosure is to provide for a method if inhibition of apoH function by sequence-specific oligonucleotide aptamers (using D-nucleotides or L-nucleotides) or peptide aptamers which are optimized for specific interaction with apoH.

[0037] In the context of the present disclosure, an aptamer refers to a single stranded peptide or oligonucleotide which forms a specific tertiary structure based on its specific amino acid or nucleotide sequence. This tertiary structure acts in an antibody-like fashion to selectively bind to a particular protein.
The sequence of oligonucleotide aptamers is usually optimized through a process called systematic evolution of ligands by exponential enrichment (SELEX).

(0038] Yet another object of the present disclosure is to provide a method for inhibition of the synthesis of apoH by blocking the translation of apoH mRNA
using sequence-specific morpholino oligonucleotides directed against the start of translation or other appropriate region of the apoH mRNA.

[0039] Yet another object of the present disclosure is to provide a method for inhibition of the synthesis of apoH by catylizing the degradation of apoH mRNA
via a variety of sequence specific nucleic-acid based technologies, but not limited to:

1. an antisense oligonucleotide (DNA or RNA) complimentary to the apoH mRNA

2. a siRNA which is complimentary to the apoH mRNA; and 3. a miRNA which targets the 3' UTR of the apoH RNA.

[0040] Short interfering RNA (siRNA) is a double stranded RNA molecule typically 20-25 nucleotides in length. siRNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process. The specific sequence of the siRNA strands then guide the RISC complexes to complementary RNA molecules, where they cleave and destroy the target RNA.

[0041] Micro RNAs (miRNA) as short single stranded RNA molecule typically 22 nucleotides in length which, as part of the miRISC complex, target complimentary sequences in the 3' untranslated regions of mRNA, catalyzing the degradation of the target RNA.

[0042] It is provided a method of treatment of hepatitis B virus infection in humans using the methods of apoH inhibition described herein, excluding the use of amphipathic oligonucleotide-based compounds having a non-sequence dependent apoH interaction as previously described.

[0043] The term oligonucleotide (ON) refers to an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) and/or analogs thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0044] In the present application, the term "randomer oligonucleotide" is intended to mean a single stranded oligonucleotide having a wobble (N) at every position, such as NNNNNNNNNN. Each base is synthesized as a wobble such that this ON actually exists as a population of different randomly generated sequences of the same length and physiochemical properties. For example, for an ON randomer 40 bases in length, any particular sequence in the population would theoretically represent only 1/440 or 8.3 X 10"25 of the total fraction.
Given that 1 mole = 6.022X1023 molecules, and the fact that no synthesis of randomers has exceeded 2 mmoles to date, any oligonucleotide with a specific sequence present cannot exist more that once in any preparation. Thus any activity derived from such a preparation must be due to the non-sequence dependent physiochemical properties of oligonucleotides since any particular oligonucleotide of a defined sequence, being unique in the preparation, cannot be expected to contribute any activity derived from its specific nucleotide sequence. Of course, one skilled in the art applying the teaching of the present disclosure could also use ONs that have specific sequences but utilize the sequence independent activity discovered in the present invention.
Accordingly, with respect to the non-sequence dependent apoH binding activity of oligonucleotides described herein, the present disclosure is not to be restricted to non-sequence complementary ONs, but does note encompass only what has been disclosed in the prior art regarding sequence-specific antisense and RNAi (e.g., siRNA) and sequence-specific aptamer ONs to inhibit apoH synthesis and or function.

[0045] ApoH interacting oligonucleotides can include various modifications, e.g., stabilizing modifications, and thus can include at least one modification in the phosphodiester linkage and/or on the sugar, and/or on the base. For example, the oligonucleotide can include one or more phosphorothioate linkages, phosphorodithioate linkages, and/or methylphosphonate linkages.
Different chemically compatible modified linkages can be combined, e.g., modifications where the synthesis conditions are chemically compatible. While modified linkages are useful, the oligonucleotides can include phosphodiester linkages where the general physiochemical properties of the oligonucleotide polymer are not substantially affected. Additional useful modifications include, without restriction, modifications at the 2'-position of the sugar, such as 2'-O-alkyl modifications such as 2'-O-methyl modifications, 2'-amino modifications, 2'-halo modifications such as 2'-fluoro; acyclic nucleotide analogs. Other modifications are also known in the art and can be used such as locked nucleic acids. In particular, the oligonucleotide has modified linkages throughout, e.g., phosphorothioate; has a 3'- and/or 5'-cap; includes a terminal 3'-5' linkage;
the oligonucleotide is or includes a concatemer consisting of two or more oligonucleotide sequences joined by a linker(s).

[0046] It is also provided a pharmaceutical composition inhibiting apoH
function that includes a therapeutically effective amount of a pharmacologically acceptable, small molecule, oligonucleotide or mixture of oligonucleotides as described herein or a non-oligonucleotide based polymer which either interact with apoH or otherwise modulate its synthesis and or activity. In particular, the oligonucleotide or a combination or mixture of oligonucleotides is as specified above for individual oligonucleotides or combinations or mixtures of oligonucleotides. In particular, the pharmaceutical compositions are approved for administration to a human, or a non-human animal such as a non-human primate.

[0047] In particular, the pharmaceutical composition is adapted for the treatment, control, or prevention of a disease which involves apoH function and is adapted for delivery by intraocular administration, oral ingestion, enteric administration, inhalation, cutaneous, subcutaneous, intramuscular, intraperitoneal, intrathecal, intratracheal, or intravenous injection, or topical administration. In particular embodiments, the composition includes a delivery system, e.g., targeted to specific cells or tissues; or a liposomal formulation.

[0048] Furthermore, the composition may include physiologically and/or pharmaceutically acceptable carriers. The characteristics of the carrier may depend on the route of administration. The pharmaceutical composition may also contain other active factors and/or agents which enhance activity.
Pharmaceutical compositions and formulations for administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Other formulations include those in which the ONs are in mixed with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP, dioleoylphosphatidyl ethanolamine DOTMA) and other delivering agents or molecules. ONs may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, ONs may be complexed to lipids, in particular to cationic lipids. 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, monoolein, dilaurin, glyceryl 1 -monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1_10 alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.

[0049] For oligonucleotides, useful examples of pharmaceutically acceptable salts include but are not limited to salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and salts formed from elemental anions such as chlorine, bromine, and iodine.

[0050] The present disclosure will be more readily understood by referring to the following examples.

EXAMPLE I

Stability of oligonucleotides with various modifications [0051] Both phosphorothioation and 2'O methyl ribose modifications have long been known to improve the resistance of oligonucleotides to nuclease mediated degradation. Oligonucleotides were subjected to nuclease mediated degradation under physiological conditions using two exemplary endonucleases (phosphodiesterase II and S1 nuclease) and two exemplary exonucleases (Bal 31 [5' and 3' ends] and Exo 1 [5' ends only]). The oligonucleotides used and their descriptions can be found in Table 1 below. Degradation of oligonucleotides after 4 hours of incubation in the presence of different nucleases was monitored by ethidium bromide visualization of oligonucleotides and degradation products after separation on agarose gels. Table 1 summarizes the results of these experiments.

Table 1 Nuclease stability of various modified oligonucleotides Nuclease Compound Description Sequence (5' - 3') Phospho-diesterase S1 Nuclease Bal 31 Exo I

REP2004 20mer NNNNNNNNNNNNNNNNNNNN +++ - ++++ ++++
randomer, PS
REP2006 40mer NNNNNNNNNNNNNNNNNNNN ++ ++++ ++++
randomer, PS NNNNNNNNNNNNNNNNNNNN
40mer NNNNNNNNNNNNNNNNNNNN
REP2086 randomer, 2-0- NNNNNNNNNNNNNNNNNNNN ++++ ++++ - ++++
Me 40mer NNNNNNNNNNNNNNNNNNNN
REP2107 randomer, PS, NNNNNNNNNNNNNNNNNNNN ++++ ++++ ++++ ++++
2-0-Me 40mer poly 4-REP2115 thio SEQ ID NO:3 - - - -deoxyuridine 40mer poly 4-REP2116 thin SEQ ID NO:3 - - ++++ ++++
deoxyuridine, PS
= completely degraded, ++++ = completely intact; PS = all linkages phosphorothioated; 2-0-Me = all riboses 2'O-methylated [0052] These experiments demonstrated that phosphorothioation gave moderate to complete protection from all nucleases except S1 nuclease. The 2' 0-methyl modification gave complete protection from all nucleases except Bal 31 and REP 2107, which combines both modifications, was completely resistance to all nucleases. REP 2115, having no stabilization modifications, was not resistant to any of the nucleases tested.

EXAMPLE II

Interaction of phosphorothioated oligonucleotides with apoH is non-sequence dependent and size dependent and not related to oligonucleotide stability [0053] The interaction of various oligonucleotides with purified human apolipoprotein H was examined by fluorescence polarization (FP). During oligonucleotide synthesis, each oligonucleotide was conjugated to fluorescein isothiocyanate (FITC) by a rigid 3 carbon using well established reagents and synthesis protocols. Then apoH in increasing concentrations was added to each oligonucleotide bait (at a fixed concentration of 3nM) in order to generate binding isotherms. All reactions were done in solution under physiological salt and pH conditions. Fluorescence polarization was measured using a Tecan Ultra fluorescence plate reader. To examine if the oligonucleotide interaction with apoH was non-sequence dependent, all oligonucleotides were prepared as randomers so that no sequence dependent activity could be present. The size dependent oligonucleotide interaction was examined using phosphorothioated randomers of different lengths: timer (Rep 2032; NNNNNN), 1Omer (REP 2003;
NNNNNNNNNN), 20mer (REP2004), 40mer (REP 2006) and 80 mer (REP
2007; sequence being 80 X N). To examine the dependence of oligonucleotide stability on apoH interactions, 40mer randomers with different degrees of stabilization were assessed: phosphorothioated (REP 2006), 2' 0-methylation of ribose (REP 2086) and both phosphorothioation and 2' 0-methylation (REP
2107). Results can be seen in Figs. 1A and 1 B.

[0054] The first observation in Fig. 1A is that a significant interaction with apoH (significant increase in fluorescence polarization) is seen only with phosphorothioated randomers 20mer in size and larger. This demonstrates that there is an oligonucleotide interaction with apoH that is size dependent.
Moreover, the fact that all oligonucleotides in this experiment that interacted with apoH were randomers also demonstrates that this interaction is not dependent on a specific oligonucleotide sequence and therefore must be dependent on the physiochemical properties present in phosphorothioated oligonucleotides.

[0055] In Fig. 1 B, a similar experimental setup as for Fig. 1A showed that no binding was detected with REP 2086 (which has comparable or better stability compared to REP 2006) and moderate binding was observed with REP 2107 (which was the most stable of all nucleotides tested). These results demonstrate that there is no relationship between the stabilizing effects of phosphorothioation or 2' 0- methylation and the non-sequence dependent apoH
interaction with oligonucleotides. These results also demonstrate that the phosphorothioate modification of oligonucleotides is required for oligonucleotide interaction because of the increased hydrophobicity it confers to oligonucleotides.

[0056] Since the apoH interaction is a function of the non-sequence dependent physiochemical properties of phosphorothioated oligonucleotides, it is expected that the apoH interaction will occur even in the presence of one or more phosphodiester linkages, provided that proportion of phosphorothioated linkages is sufficient to provide a suitable increase in hydrophobicity to drive the apoH interaction.

EXAMPLE III
Competing the REP 2006:apoH interaction with phosphorothioated and phosphodiester oligonucleotides composed 4-thiodeoxyuridine [0057] To examine if sulfur modifications could be used at other locations on the oligonucleotide so that apoH interaction would occur, a competition experiment was conducted. In this experiment, 3nM of FITC labeled REP 2006 was complexed with apoH at a final concentration of 0.025ug/ui in solution. To this complex was then added the following unlabelled oligonucleotides: REP
2006 (as a control), REP 2115 (poly 4'thiodeoxyuracil; SEQ ID NO.-3) and REP
2116 (a phosphorothioated version of REP 2115). In this experiment, the strength of the interaction between a particular oligonucleotide was monitored by its ability to displace the pre-complexed FITC-labelled REP 2006, thus resulting in a drop of fluorescence polarization in the reaction. The results of these experiments are found in Fig. 2. In the absence of any competitor, the FITC-REP 2006/apoH mixture exhibited strong fluorescence polarization, indicating that REP 2006 was complexed with apoH (as had been previously shown in Fig. 1). Unlabelled REP 2006 was able to significantly compete away the bound REP 2006 off apoH, as demonstrated by a drop in fluorescence polarization. REP 2115 showed an almost identical ability to displace FITC-REP
2006 from apoH compared to REP 2006 itself. REP 2116 showed a much stronger ability to displace bound REP 2006 from apoH than either of the other two compounds, exhibiting strong competition at lower concentrations and also an ability to compete away almost all the bound REP 2006 from apoH. This indicated that REP 2116 has a much stronger binding affinity to apoH than REP
2006 or REP 2115.

[0058] These results demonstrate that sulfur modifications placed at other sites on the oligonucleotide can also render it competent to bind to apoH.
Combining two different sulfur modifications (phosphorothioation and 4'thio modification of deoxyuridine) gave a pronounced increase in the binding affinity of oligonucleotides. Interestingly, REP 2115 exhibited comparable apoH binding behavior to REP 2006, even though this compound has no nuclease stability at all, further demonstrating that oligonucleotide stability has no bearing on its ability to interact with apoH.

[0059] Therefore, the sulfur modification may be at any location in order to confer the necessary chemical properties required for the sequence-independent interaction of oligonucleotides with apoH. Such sulfur modifications may include without restriction mono and diphosphorothioation of the phosphodiester linkage, 4' or 5' thiolation of the uracil moiety, 5' thiolation of the cytidine moiety, 2' or 4' thiolation of the thymine moiety, 6' thiolation of the guanine moiety, sulfur modifications to any other nucleobase moiety and sulfur modification to the ribose moiety of any nucleotide. Moreover, ONs can have more than one sulfur substitution on each nucleotide, which has now been shown to increase the apoH binding activity. Finally, any single or multiple sulfur substitution may be combined with other modifications known to improve properties of ONs. These modifications include without restriction: any 2' ribose modification including 2'-0 methyl, 2'-fluorine, 2'-FANA, 2'-O (2-methoxyethyl), locked nucleic acids, methylphosphonates and morpholino nucleic acids. These latter modifications may have a positive effect on therapeutic activity, even if they do not positively impact apoH interaction (e.g. REP 2107) because they will provide a much greater stability of the compound in human tissues.

[0060] Since the apoH interaction is a function of the non-sequence dependent physiochemical properties of oligonucleotides containing sulfur modifications, it is expected that the apoH interaction will occur even in the presence of one or more unmodified nucleotides, provided that proportion of sulfur-modified nucleotides is sufficient to provide a suitable increase in hydrophobicity to drive the apoH interaction.

EXAMPLE IV
REP 2055 mediated apoH inhibition results in inhibition of HBV subviral particle release and seroclearance of HBsAg [0061] The role of apoH in HBV infection is unclear, but since it has been demonstrated that apoH may be a cellular receptor for HBV and that apoH also interacts with HBsAg, the effects of apoH inhibition as an antiviral methodology was examined in human patients with chronic hepatitis B treated with REP
2055. REP 2055 is a 40mer phosphorothioate oligonucleotide with the sequence (AC)20. This compound is identical to REP 2006 in its physiochemical characteristics (and therefore apoH interaction) but whose sequence is optimized for minimal toxicological impact in patients. Patients with established chronic hepatitis B infection were subjected to REP 2055 treatment and the antiviral effects of REP 2055 treated were monitored by assessing serum concentrations of HBsAg (by ELISA) as well as HBV virus titers (by quantitative PCR) and are seen in Table 2 Table 2 Effects of REP 2055 treatment on HBsAg and HBV titers in human patients Serum HBsA Serum HBV titer (co ies/ml) Ultimate serum Patient REP 2055 At point of HBV titer Pretreatment treatment Pretreatment HBsAg outcome seroclearance I positive undetectable 2.1x106 3.7x105 Undetectablea 2 positive undetectable 1.3x10' 3.9x106 Undetectableb 3 positive undetectable 1.3x10 1.1x105 3 log drop from baseline' a 7 weeks after HBsAg seroclearance b 20 weeks after HBsAg seroclearance c 37 weeks after HBsAg seroclearance [0062] REP 2055 treatment rapidly cleared HBsAg from the serum but had minimal effect on the production of infectious virus. These results are consistent with the hypothesis that apoH is essential for the secretion of subviral particles containing HBsAg and no capsid but not for infectious HBV virions.
Additionally, 2 of the three patients in Table 2 when on to clear their serum of infectious HBV
virus, consistent with the notion that removal of HBsAg allows restoration of immune function and with it the host's ability to clear the infection from the liver.
Most importantly, these results validate for the first time the effectiveness of targeting apoH as a method for treating hepatitis B infection in human patients.
EXAMPLE V
Inhibition of apoH by antisense oligonucleotides [0063] Antisense oligonucleotides are typically comprised of DNA or RNA
and are typically in the size range of 18-20 nucleotides but can be longer.
Antisense oligonucleotides are stabilized by phosphorothioation at each linkage or by the use of locked nucleic acids and may also include 2' ribose modifications at either end of the molecule (typically the 4-5 distal nucleotides at each end). These 2' ribose modifications also improve the specificity of the antisense molecule. Oligonucleotides may further make use of morpholino nucleic acids, which are capable of hybridization but do not elict RNAse or RISC-mediated mRNA degredation. For the inhibition of apoH, an oligonucleotide with a specific sequence of nucleotides is designed so that the antisense oligonucleotide in question specifically hybridizes with apoH mRNA, usually in the open reading frame but also in the 5' and 3' untranslated regions.
Numerous different oligonucleotide sequences which will hybridize with the apoH mRNA are screened for the following properties: lack of secondary structure (i.e. hairpins) which may impede hybridization, lack of interaction with other mRNA or genomic sequences and the presence of specific motifs (e.g.
immunostimulatory CpG motifs) which may introduce unwanted secondary effects. The efficacy of these oligonucleotides is also assessed for their ability to lower the apoH mRNA in a suitable cellular system (e.g. a cell line known to express apoH such as a hepatocyte cell line).

[0064] Oligonucleotides can be introduced to cells in tissue culture with or without a delivery system designed to facilitate the entry of oligonucleotides inside the cell (e.g. a liposomal delivery agent such as oligofectamineTM) Expression of apoH in control cells and cells treated with apoH antisense compounds is assessed by any one of several accepted methods for determination of apoH mRNA concentration within treated cells (and compared to untreated cells), including with out restriction: RT-PCR, RNAse protection, northern blotting (using a probe specific for apoH mRNA), fluorescence in situ hybridization. Reductions in apoH protein can be further assessed by ELISA of treated cell lysates or of treated cellular supernatant and compared with untreated controls. The ultimate selection of the best candidate antisense sequences will be a function of a particular oligonucleotide's performance in all of these selection criterions as described above.

[0065] Continued pre clinical and clinical development of selected antisense oligonucleotides can proceed and may be accompanied by further modification consisting of 5' methylation of all cytidine bases in the sequence (to further suppress the immunostimulatory properties of nucleic acids). Any oligonucleotide which proceeds to in vivo development and beyond should be prepared as a sodium salt; however other salts may also be acceptable. In vivo development would consist of monitoring the oligonucleotide's ability to lower apoH mRNA and protein levels in vivo and to also look for the desired effects of apoH inhibition such as anti-coagulation, lowered HDL and, in surrogate models of HBV infection (e.g. DHBV infected dusks or WHV infected woodchucks) the suppression of subviral particle release which would be manifested by a reduction or clearance in serum surface antigen with minimal concomitant effect on virion production.

EXAMPLE VI
Inhibition of apoH by siRNA

[0066] The in silico and in vitro screening process for siRNA is identical for antisense oligonucleotides described in Example V with some important exceptions:

1. siRNAs are typically 20-25 nucleotides in length, are composed of ribonucleic acids, are double stranded and typically have 2 deoxyribonucleic acid overhangs at both 3' ends of the duplex;

2. siRNA stabilization is achieved by inclusion at specific locations in the duplex modifications which include without restriction: 2' ribose modification, modification of the nitrogenous base, phosphorothioation or methyl phos phonation-, 3. part of the optimization process for siRNAs is their stability to nuclease attack and hydrolysis under a number of conditions; and 4. siRNA delivery in vivo may further require more complex delivery systems such as stable nucleic acid particles (SNALPs) not only to target delivery to specific tissues but also to further reduce immunostimulatory properties.

EXAMPLE VII
Inhibition of apoH by peptide or nucleic acid aptamers [0067] Sequence-specific oligonucleotide aptamers for apoH are identified by the well known process of systemic evolution of ligands by exponential enrichment (SELEX) using apoH from any species as the target selector. Other methods for optimizing oligonucleotide sequences may also exist in the art.
Optimal nucleic acid aptamers may also be inherently unstable under physiological conditions and thus may also include the nucleic acid modifications as described in Example VI. These modifications should only be included in a manner which does not significantly alter the affinity of aptamer for apoH. Further specificity testing should be done against a variety of related proteins to minimize off-target effects. Safety and efficacy evaluation can proceed as described for pre-clinical/clinical development of antisense oligonucleotides and siRNAs.

(0068] A typical design of a peptide aptamer for apoH is a variable loop of 10-20 amino acids attached to a common peptide scaffold that has suitable solubility and compacity properties (i.e. bacterial protein Thioredoxin-A).
Peptide aptamers to apoH can be optimized using the yeast-two hybrid system with apoH as the bait. Peptide aptamers can also be derived from other structural designs. Optimization of peptide aptamers should also include the same screening for affinity, selectivity and stability as described for antisense oligonucleotides and siRNA but may utilize different modifications. Peptide aptamers may also require specific delivery systems to achieve intracellular activity in desired tissues. Pre-clinical/clinical development could proceed using the same strategies as described above for antisense oligonucleotides and siRNA.

[0069] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims (38)

1. A composition for the inhibition of apoH comprising at least one phosphorothioate oligonucleotide, wherein the oligonucleotide interacts with apoH and wherein the interaction of said oiigonucleotide is non-sequence dependent.

2. A composition for the inhibition of apoH comprising at least one phosphorothioate oligonucleotide, wherein said oligonucleotide is comprised of alternating adenosine and cytosine.

3. The composition of claim 1 or 2, wherein the at least one oligonucleotide is between 20 and 80 nucleotides in length.

4. The composition of any one of claims 1-3, wherein the at least one phosphorothioate oligonucleotide is SEQ ID NO:1.

5. A composition for the inhibition of apoH comprising at least one phosphorothioate oligonucleotide, wherein said oligonucleotide is comprised of alternating adenosine and guanosine.

6. The composition of claim 5, wherein the at least one oligonucleotide is between 20 and 80 nucleotides in length.

7. The composition of claim 5 or 6, wherein the at least one phosphorothioate oiigonucleotide is SEQ ID NO:2.

8. A composition for the inhibition of apoH comprising at least one oligonucleotide consisting of SEQ ID NO:3.

9. The composition of claim 8, wherein said oligonucleotide is phosphorothioated.

10. A composition for the inhibition of apoH comprising at least one oligonucleotide, wherein said at least one oligonucleotide comprises any of the following modified bases: 4'thiouracil, 5' thiouracil, 5; thiocytidine, 4'thiothymine or deoxy-4'thiouracil and 6'thioguanine.

11. The composition of claim 10, wherein said at least one oligonucleotide is between 20 and 80 nucleotides in length.

12. The composition of claim 10 or 11, wherein the oligonucleotide is phosphorothioated.

13. The composition of any one of claims 1-12, wherein composition comprises at least one oligonucleotide containing at least one phosphodiester linkage.

14. The composition of claim 12 or 13, wherein the oligonucleotide contains at least one unmodified thymidine, uridine, adenosine, cytosine or guanosine base.

15. The composition of any one of claims 1-14, wherein said at least one oligonucleotide has at least one 2' ribose modification.

16. The composition of any one of claims 1-14, wherein said oligonucleotide has all ribose sugars 2' modified.

17. The composition of any one of claims 1-14, wherein said oligonucleotide has at least one 2' O-methyl ribose modification.

18. The composition of any one of claims 1-14, wherein said oligonucleotide has all ribose sugars 2' O-methyl modified.

19. The composition of any one of claims 1-14, wherein said oligonucleotide has at least one 2'-O (2-methoxyethyl) ribose modification.

20. The composition of any one of claims 1-14, wherein said oligonucleotide has all ribose sugars 2'-O (2-methoxyethyl) modified.

21. The composition of any one of claims 1-14, wherein said oligonucleotide contains at least one locked nucleic acid.

22. The composition of any one of claims 1-14, wherein said oligonucleotide contains at least one methylphosphonate.

23. The composition of any one of claims 1-22, further comprising a carrier.

24. A method of inhibiting the function of apoH in a patient comprising the step of administering to said patient the composition of any one of claims 1-23.

25. The method of claim 24, wherein said patient is a human patient or a non-human animal patient.

26. The method of claim 24 or 25, further treating at least one of systemic lupus erythematosus, anti-phospholipid syndrome and hepatitis B infection.

27. Use of the composition of any one of claims 1-23 for the treatment of systemic lupus erythematosus in a human patient or a non-human animal.

28. Use of the composition of any one of claims 1-23 for the treatment of anti-phospholipid syndrome in a human patient or a non-human animal.

29. Use of the composition of any one of claims 1-23 for the treatment of hepatitis B infection in a human patient or a non-human animal.

30. Use of the composition of any one of claims 1-23 in the manufacture of a medicament for the treatment of systemic lupus erythematosus in a human patient or a non-human animal.

31. Use of the composition of any one of claims 1-23 in the manufacture of a medicament for the treatment of anti-phospholipid syndrome in a human patient or a non-human animal.

32. Use of the composition of any one of claims 1-23 in the manufacture of a medicament for the treatment of hepatitis B infection in a human patient or a non-human animal.

33. An antisense oligonucleotide complimentary to the mRNA of an isoform of apoH.

34. A siRNA complimentary to the mRNA of an isoform of apoH.

35. A morpholino antisense oligonucleotide complimentary to the mRNA of an isoform of apoH.

36. A miRNA that targets the mRNA of any isoform of apoH.

37. An oligonucleotide aptamer optimized for interaction with apoH.

38. The oligonucleotide of claim 37, wherein said oligonucleotide comprises either D- or L-nucleotides.