WO1995020043A1 - Inhibition of vacuolar h+ -atpase by phosphorothioate oligonucleotides - Google Patents

Abstract

The present invention provides a method of inhibiting vacuolar H+-ATPase activity in a cell which comprises administering to the cell an effective amount of an oligonucleotide which may be substituted or modified in its phosphate, sugar, or base, so as to thereby inhibit the vacuolar H+-ATPase activity. The invention is particularly suited for use as an assay of vacuolar H+-ATPase activity, and use with patients who are suffering from vacuolar H+-ATPase disorders associated with: osteoporosis, increased cholesterol or triglyceride, synthesis or production levels; increased acid secretion from parietal cells; or bone absorption and reabsorption.

Description

I HIBITIOK OF VACϋOIAR H÷-ATPase BY PHOSPHOROTHIOATE OLIGONUCLEOTIDES

Throughout this application, various publications are referenced by number in brackets. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

Backcrrp"τιri f>f the in-yyr-1-f-t -.τ.

The electrochemical gradient of protons (PMF) is a universal high-energy intermediate in biological systems. Many acidic secretory granules use an electrochemical proton gradient to mediate the coupled transport of cellular molecules. A novel set of intracellular proton pumps are located in the eukaryotic vacuole system, adrenal chromaffin granule (epinephrine) , the platelet dense granule (serotonin) , and the synaptic vesicles (catecholamines, and acetylcholine) [8, 15, 18, 23, 39, 47].

Most of the membrane organelles in a typical eukaryotic cell belong to the elements of the exocytic and endocytic pathways, referred to collectively as the vacuolar system

[24] • They include the endoplasmic reticulum, the golgi cσαlex, the secretory vacuoles, the endosomes, the lysosomes, and other organelles involved in biosynthesis, processing, transport, storage, release, and degradation of soluble and membrane-bound macromolecules. An important similarity among the organelles of the vacuolar system is the presence of a H⁺-ATPases responsible for generating an internal acidic environment. There are two mechanistically distinct groups of ATP dependent ion pumps. One group, the P-ATPases (E^ ATPases) operate with a phosphoenzyme intermediate and its members (i.e. Na⁺/K⁺ ATPase, gastric H⁺ATPase) are usually sensitive to low concentration of vanadate, a phosphate transition state analog. The P-ATPases are present in the cell membranes of fungi, plants, animals, sarcoplasmic reticulum of muscle cells, and the bacterial cytoplasmic membranes.

The other group, contains the families of F_JFQ ATPases (F- ATPases) and vacuolar H⁺-ATPases (V-ATPases) [17, 27, 29, 30] . F-ATPases and V-ATPases function without a phosphorylated intermediate, [28] are multisubunit protein complexes that are built of distinct catalytic and membrane sectors, are not sensitive to low vanadate concentrations, but are sensitive to bafilomycin A [5, 45, 46] .

The vacuolar proton ATPases are distinguished from the other two classes by virtue of their inhibitor specificities, lack of coupling to counter-ion transport, and intracellular distribution. V-APTase inhibitors include: N-ethylmaleimide (NEM) , 4-chloro-7-nitrobenzo-2- oxa-l,3-diazole (NBD-C1) [4, 12, 14, 33, 43], N,N'- dicyclohexylcarbodiimide, N-ethymaleimide, N0₃', bafilomycin A [5, 45], concanamycin [46], suramin [27] and fusidic acid [27] .

The family of V-ATPases are present in archaebacteria and vacuolar systems of eukaryotic cells. While the family of F-ATPases function in eubacteria and are present exclusively in the thylakoid membrane of chloroplasts, inner mitochondria membrane and bacterial cytoplasmic membranes.

V-ATPases pump protons without internal counterions and therefore are inherently electrogenic. Since they use ATP, they are also strongly oxygen-dependent in cells with low anaerobic phosphorylating capacity or stores. They energize membranes by transducing the energy from ATP hydrolysis into a proton current, which establishes an electrochemical gradient ΔμH.

The vacuolar H⁺-ATPases are ubiquitous intracellular proton pumps in cells. They serve to acidify intracellular compartments and organelles in mammalian cells such as lysozomes, golgi, and synaptic vesicles and thus lower intralumenal pH. In addition, the vacuolar H⁺-ATPases may serve to modulate cellular functions in conjunction with its external milieu.

For example, the kidneys maintains acid-base homeostasis by hydrogen ion secretion along the nephron which enables them to reabsorb filtered bicarbonate and to regenerate bicarbonate consumed by metabolic acid production. In addition, vacuolar H⁺-ATPases are critical in: osteoclast production for bone resorption, phagocytosis, aqueous humor production, receptor recycling pathways by promoting ligand-receptor dissociation, and reduction of the concentration of cholesterol carrying lipoproteins in plasma.

The vacuolar H⁺-ATPases are all 500 kD - 600 kD molecular weight (Mr) proteins with generally at least eight different component subunits. All have subunits of approximately 70 kD, 56 kD, several subunits between 30 and 50 kD, and at least one low molecular weight subunit of 17 kD. In all of the enzymes, most of the large molecular weight proteins are peripheral membrane proteins that do not have any membrane spanning portions, and the small molecular weight proteins are intrinsic membrane proteins that span the lipid bilayer. The vacuolar H⁺-ATPases have two major domains, the cytoplasmic domain and the transmembrane domain (Figure 1) . The cytoplasmic domain is the locus of the catalytic and probable regulatory sites of the enzyme, and is composed of peripheral membrane proteins. The transmembrane domain forms the channel through which protons cross the lipid bilayer and is composed of intrinsic membrane proteins which the cytoplasmic domain and anchor it on the membrane.

The cytoplasmic domain contains the ~70 kD ("A") subunit of the vacuolar H⁺-ATPases and appear to have the site where ATP is hydrolyzed during proton transport. There are 3 subunits of 70 kD in each complete proton pump. Cloning and Southern blotting of this subunit from bovine genomic DNA suggests that there is only one gene for the 73 kD subunit [32] . The sequences of the 70 kD subunit and its homolog have a highly conserved domain in the mid-coding region which comprise the nucleotide binding and catalytic site. The sequence of the bovine kidney subunit compared with the plant and the fungal enzymes show wide divergence at the amino-terminal and carboxyl- terminal domains, with no sequence conservation. It is possible that these regions have a regulatory or non- catalytic role.

There are 3 subunits of 56 kD ("B" subunit) per H⁺- ATPase. The function of this subunit is uncertain. The subunit is homologous to the α subunit of the F^ H⁺- ATPases. The α subunit does not have a catalytic ATP binding site, but is required for catalytic activity. It has a high affinity nucleotide binding site which is thought to be involved either in regulation of the enzyme, or as a non-hydrolytic part of the reaction mechanism.

The function of the 56 kD subunit of the vacuolar H⁺- ATPases is unknown although evidence from the plant enzyme suggest? that it may have an ATP binding site. Cloning of the subunit has revealed that there are at least two different isoforms of the 56 kD subunit in the kidn /, and these are encoded by different genes [31, 32] .

Vacuolar H⁺-ATPase affinity purified from a bovine kidney cortex microsomal fraction had different enzymatic properties from V-ATPase isolated from bovine kidney brush border, in addition to heterogeneity of the 56 kD and 31 kD subunits [44] . The 56 kD subunit therefore has an important role in determining the tissue specific enzymatic properties or compartmentation of the vacuolar H⁺-ATPase.

There are from 1 to 3 subunits of 31 kD per H⁺-ATPase. The cDNA has been cloned from bovine kidney [22] . monoclonal antibodies raised against heterogeneous 31 kD subunits in the renal brush border and collecting tubules are consistent with preliminary data from immunoscreening genomic DNA that suggests more than 1 gene codes for this subunit [19] . The amino acid sequence of the subunit is 98% identical between different mammalian species, far higher than for the 70 and 56 kD subunits.

The function of the other subunits of the cytosolic domain is not established, but they may constitute a "stalk" domain connecting the catalytic portion to the intrinsic membrane domain similar to the construction of the FQF_X enzymes.

The transmembrane domain forms a proton conducting channel that spans the lipid bilayer [28] . Although the entire composition of this portion of the enzyme remains in dispute, all of the vacuolar H⁺-ATPases have an approximately 17 kD (or 15 kD in the kidney) polypeptide that reacts readily with the hydrophobic α-carboxyl reagent dicyclohexy1carbodiimide.

H⁺ secretion has an important role in maintaining acid- base balance. This applies to normal conditions but increases in importance as a homeostatic compensation to metabolic acidosis. The renal response to this condition can be described as follows: (1) the complete reabsorption of filtered sodium bicarbonate; (2) the acidification of the urinary buffers; (3) the excretion of fixed anions in combination with H₄ ⁺ rather than Na⁺; and (4) the adjustment of urinary pH.

The kidneys maintains acid-base homeostasis by hydrogen ion secretion along the nephron which enables them to reabsorb filtered bicarbonate and to regenerate bicarbonate consumed by metabolic acid production. Hydrogen ion secretion in the collecting duct is carried out by the intercalated cells. Intercalating cells of the kidney collecting duct manifest V-ATPase expression in a polarized distribution on the plasma membrane enabling it to serve in transepithelial H⁺ transport. In addition, two isoforms of vacuolar H⁺-ATPase β subunit have been located in the kidney and brain.

Inhibition of H⁺ secretion by the renal tubule can cause the increased alkalinity of the urine which is accompanied by a decrease in the excretion of titratable acid and ammonia. Other inhibitory effects of H⁺ secretion are manifest in the following: gastro¬ intestinal tract, respiratory system, and the central nervous system. In the central nervous system H⁺ secretion increases cerebrospinal fluid formation and pressure as a result of increase intracranial blood flow.

V-ATPases and V-ATPase isoforms, are located in the plasma membrane at high densities forming nearly crystalline arrays. Plasma membrane forms of the V- ATPase have been identified in the osteoclast for bone resorption, which play a role in controlling hypercalcemia malignancy and in the pulmonary macrophage for phagocytosis. The enzyme may be polarized to either the apical or basolateral pole in subsets of cells, imparting a capacity either for net proton or bicarbonate secretion [6, 7] .

Active ion transport is a driving force for aqueous humor production. Ciliary epithelial transport requires primary active transp. -); proteins (ATPases) and secondary active transporters driven by electrical and ion concentration gradients. Vacuolar H⁺-ATPases located in the plasma membrane of the ciliary epithelium of the eye are responsible for the transport of protons and bicarbonate. Inhibition of the function of this enzyme leads to diminished ability of the ciliary epithelium to transfer free water and bicarbonate into the aqueous humor and lead to diminished intraocular pressure.

Acidification of a cell is accompanied by the generation of an interior positive membrane potential. Exposure to the low endosomal pH induces conformational changes which in turn can lead to the dissociation of receptor-ligand complexes, changes in ligand solubility, activation of latent activities. When endosomal acidification is inhibited by the addition of acidotropic agents or carboxylic ionophores, intracellular dissociation of the receptor-ligands complex is fully or partially blocked [10] .

Vacuolar H⁺-ATPases are important in receptor recycling pathways by lowering the pH within endocytic vesicles and thereby promoting ligand-receptor dissociation. Receptors include: the Fc receptor, the DL receptor, and the transferrin receptor. In addition, V-ATPases may have an effect on the major histocompatibility complex processing in lymphoblastoid cell surface deposition and processing of MHC Class I and II molecules.

For example, transferrin is a protein which carries iron in the blood. Cell-surface transferrin receptors deliver transferrin with its bound iron to peripheral endosomes by receptor mediated endocytosis. The low pH in the endosome induces transferrin to release its bound iron, but the iron free transferrin itself (called apotransferrin) remains bound to its receptor and is recycled back to the plasma membrane as a receptor- apotransferrin receptor complex.^' When it has returned to the neutral pH of the extracellular fluid, the apotransferrin dissociates from the receptor and is thereby freed to pick up more iron and begin the cycle again.

Further, when liver or extrahepatic tissues require cholesterol for the synthesis of new membranes, steroid hormones, or bile acids, they synthesize LDL receptors and obtain cholesterol by the receptor-mediated endocytosis of LDL. Conversely, when tissues no longer require cholesterol for cell growth or metabolic purposes, they decrease the synthesis of LDL receptors.

Reduction of the concentration of cholesterol carrying lipoproteins in plasma can diminish the risk of myocardial infarction and lead to hyperlipoproteinemias. This contributes to the deposition of cholesterol in macrophages of arterial walls (producing atheromas) and macrophages of tendons and skin (producing xanthomas) . Summary of the Invention

The present invention provides a method of inhibiting vacuolar H⁺-ATPase activity in a cell which comprises administering to the cell an effective amount of oligonucleotide which may be substituted or modified in its phosphate, sugar, or base so as to thereby inhibit the vacuolar H⁺-ATPase activity.

Further, the present invention provides a method of treating a subject suffering from vacuolar H⁺-ATPase disorders associated with: osteoporosis, increased cholesterol or triglyceride, synthesis or production levels; increased acid secretion from parietal cells; or bone absorption and reabsorption, which comprises administering to the subject an effective amount of the above oligonucleotide.

Brief Description of the Figures

Figure 1: V-ATPase subunit structure drawn according to the enzyme of chromaffin granules. The molecular weights of subunits A,B,C,D,E,a, and c are 70 kD, 57 kD, 44 kD, 30 kD, 26 kD, 20 kD, and 16 kD, respectfully.

Figure 2: Effect of phosphorothi oate oligonucleotides on the reconstituted H⁺ pumping ability. ATP-dependent proton uptake was assayed by following the absorbance changes of acridine orange at 492-540 nm 0.01 OD. Trace 1. SdT28 (5 urn) , Trace 2. SdT15 (5 urn), Trace 3. SdT5 (5 urn), Trace 4. control (no added oligonucleotides) .

Figure 3: Proton pumping capability of yeast vesicles in the presence of different oligonucleotides. H⁺ pumping activity is plotted against 5 urn SdT3, SdT5, SdT15, SdT28, 0dT5, OdT28.

Figure 4ι Concentration dependent inhibition by cytidine homopoly er SdC28 (5 urn) . The value of IC50 is 0.16 urn.

Detailed Description of the Invention

The present invention provides a method of inhibiting vacuolar H⁺-ATPase activity in a cell which comprises administering to the cell an effective amount of oligonucleotide which may be substituted or modified in its phosphate, sugar, or base so as to thereby inhibit the vacuolar H⁺-ATPase activity.

This invention is directed to a method of inhibiting V- ATPases with oligonucleotides. Oligonucleotides are compounds made up of repeating units of nucleotides. The oligonucleotide may be substituted or modified in its phosphate, sugar, or base. These compourds are also known as chimeric oligonucleotide.

Synthetic oligodeoxynucleotides have been utilized as antisense inhibitors of mRNA translation in vitro and in vivo [3, 20, 34, 38, 42] . Antisense oligonucleotides have found widespread application because of their abilities to control and/or inhibit gene expression in a selective manner in cellular systems [13, 18, 21, 35, 40, 48] . Phosphorothioate oligodeoxynucleotides are relatively nuclease resistant water soluble analogs of phosphodiester oligodeoxynucleotides. These molecules are racemic, but still hybridize well to their RNA targets [36] .

When a 20 base sequence phosphorothioate (PS) oligonucleotide was injected into the abdomens of mice, either intraperitoneally (IP) or intravenously CIV) . The highest concentrations of oligonucleotide accur ilated in the kidney and liver, with only very small amounts being found in the brain. Chain-extended oligonucleotides were alsc ^served [1] . When the PS 27-oligonucleotide αrev was g„ven IV or to rats, the initial ^tl/2α (transit out of the plasma) was 23 min, while the ^ll/2-. of total body clearance was 33.9 hours. The long β half-life of elimination maintains that dosing could be infrequent and still maintain effective, therapeutic tissue concentrations [20] .

Further, a 24-base sequence PS oligonucleotide targeted to the human c-myb mRNA was infused, through a miniosmotic pump, into scid mice bearing the human 562 chronic myeloid leukemia cell line. Mean survival times of the mice treated with the antisense oligonucleotides were six- to eightfold longer than those of mice untreated or treated with the sense controls or treated with an oligonucleotide complementary to the c-kit proto- oncogene mRNA. Furthermore, significantly less tumor burden in the brain and ovary was observed histologically compared with the controls. After injecting IP 3'-PS- modified chimeric oligonucleotides that were complementary to the initiation codon region of the NF-kB mRNA (p65) , it was observed a complete tumor involution in 13 out of 13 antisense-treated mice. Untreated or sense-treated mice died by 12 weeks, where as the treated animals had no recurrence for at least 5 months [34] .

Since many classes of oligodeoxynucleotides (e.g., phosphodiesters (PO) and phosphorothioates (PS) ) are polyanions, they cannot passively diffuse through lipophilic cell membranes [37] . However, the majority of oligonucleotide internalization is not due to receptor- mediated endocytosis, but rather results indicate that bulk internalization is predominately from pinocytosis, fluid-phase endocytosis.

Further, in HL60 cells, the net cellular accumulation of oligonucleotides may be influenced by the activity of protein kinase C (P C) , a Ca⁺- and phospholipid-dependent protein kinase. PKC phosphorylates intracellular substrates on serine and threonine residues and is the major intracellular signal transduction protein.

Throughout this application, references to specific nucleotides are to nucleotide present on the coding strand of the nucleic acid. The following standard abbreviations are used throughout the specification to indicate specific nucleotides:

C = Cytidine A = Adenosine T = Thymidine G = Guanosine

The present invention provides a method of inhibiting vacuolar H⁺-ATPase activity in a cell which comprises, administering to the cell an effective amount of oligonucleotide which may be substituted or modified in its phosphate, sugar, or base, so as to thereby inhibit the vacuolar H⁺-ATPase activity.

The oligonucleotide may be an oligodeoxynucleotide, a phosphorothioate, a phosphorodithioate, a chimeric oligonucleotide, an oligonucleotide homopolymer, or an oligonucleotide heteropolymer. In one embodiment the oligonucleotide is a chain of cytidine nucleotide sequences. In another embodiment the oligonucleotide is a chain of adenosine nucleotide sequences. In another embodiment the oligonucleotide is a chain of guanosine nucleotide sequences. In another embodiment, the oligonucleotide is a chain of thymidine nucleotide sequences. The most preferred embodiment is the cytidine nucleotide sequence. The oligonucleotide sequence may be either in a stereo regular or stereo non-regular configuration.

A ^mopolymer is a sequence of repeating cytidine, gus,..sine, adenosine, or thymidine nucleotides or other natural bases thereof. For example, SdC28 is a phosphorothioate oligonucleotide that is a homopolymer of cytidine for a 28 base length sequence. A heteropolymer is a sequence of alternating cytidine, guanosine, adenosine, or thymidine nucleotides or other natural bases thereof. For example, SdCT20 is a phosphorothioate oligonucleotide that is heteropolymer of cytidine and thymidine for a 20 base length sequence.

In one embodiment the oligonucleotide sequence is a short chain structure, 5-20 nucleotides. In another embodiment the oligonucleotide sequence is a long chain structure, 20-100 nucleotides. The most preferred structure is a long chain 28 oligonucleotide structure.

The present invention further claims a method of inhibiting vacuolar H⁺-ATPase activity in a cell which comprises, administering to the cell an effective amount of phosphorothioate so as to thereby inhibit the vacuolar H⁺-ATPase activity. Phosphorothioate may be stereo regular or stereo non-regular.

The phosphorothioate may further be linked to a 3' or 5'- cholesteryl moiety or alternatively, the phosphorothioate may be modified in the bridge wherein the one of the two oxygen atoms involved in the bridge are replaced with analogues such as NH-, CH₂- or S- .

Phoshorothioate (PS) is an oligodeoxynucleotide in which the sulfur atom replaces one of the non-bridging oxygen atoms at each interbase phosphorus atom. The phosphorothioate can occur at each PS linkage either as

Rp or Sp diastereomers. Because of phosphorothioate's nuclease resistent properties it is an effective inhibitors of replication of Human Immunodeficiency Virus

(HIV-1) in cell culture. [1, 2, 23]. PS oligonucleotide sequences are non-sequence specific and act as inhibitors of the HIV-1 cytopathic effect in de novo infected ATH8 cells. With Sd(GGC)9, maximal cytoprotection was observed at a concentration of 0.3 μM. In a series of A- T rich PS oligomers, including SdA21, SdT21, SdCT20 and SdCT19C, all, with the exception of SdA21, were of approximately equal potency. The same generalization was true in a series of non-homopolymeric PS oligomers, including the self-complementary S-d(CG)10G, S-d(GGC)7, S-d(C5T)3C3, S-d(CCT)7, S-d(CCA)7, S-d(CT)10C, S-d(CTT)7 and S-d(GTT)7. Indeed the anti-HIV effect was entirely independent of the base sequence, though for the 14 base length oligonucleotide, it was more pronounced with PS oligomers of increasing GC content, even with the phosphoroselenoate oligomer S-dC28 [1, 2] .

This invention further provides claims the oligonucleotide which may be substituted or modified in its internucleotide phosphate residue with a thioether, carbamate, carbonate, acetamidate or carboxymethyl ester.

In addition, the oligonucleotide may be substituted or modified in its sugar with a ribose, 2' allyl, glucose, sucrose, or galactose or any other sugar. Alternatively, the oligonucleotide may be substituted or modified in its 2' position; such as 2" -O-methylribonucleotide. Further, the oligonucleotide may be substituted or modified to form an α-anomeric sugar.

In addition, the oligonucleotide may be substituted or modified in its base. Apart from the bases of adenine, guanine, cytosine, and thymine, other natural bases, such as with inosine, deoxyinsosine, hypoxanthine are acceptable. In addition, isosteric purine 2'deoxy- furanoside analogues, 2' -deoxynebularine or 2'deoxyxanthosine, or other purine or pyrimidine analogues may also be used.

In addition, this invention provides the oligonucleotide which may further be linked to a 3' or 5' -cholesteryl moiety.

The present invention further provides that a 5' end of the oligonucleotide may be linked with: intercalating agents, such as acridine derivatives; cross-linkers, such as psoralen derivatives, azidophenacyl, proflavin, and azidoproflavin; artificial endonucleases which comprise those conjugates whose nuclease component is able as such to cleave DNA specifically and nonspecifically, and acquires a specificity by covalent linkage to the oligonucleotide, such as metal complexes EDTA-Fe(II), o- phenanthroline-Cu(I) , and porphyrin-Fe(II) ; and lipophilic carriers or peptide conjugates, such as long chain alcohols as phosphate esters, amino or mercapto groups, dyes or nonradioactive markers and polysine.

The present invention further provides that a 3' end of the oligonucleotide may be linked with: intercalating agents, such as 2-methoxy-6-chloroacridine, methylphosphonates, methylesters, and aminoalkyls; alkylating oligonucleotides, such as acetyl; artificial endonucleases, such as amino-1-hexanolstaphylococcal nuclease, and alkaline phosphatase; peptide conjugates, such as polylysine; and terminal transferases.

The oligonucleotides in the present invention may be conjugated to a carbohydrate, sulfated carbohydrate, or glycan. Conjugates may be regarded in such a way as to introduce a specificity into otherwise unspecific DNA binding molecules by covalently linking them to a selectively hybridizing oligonucleotide.

In addition, the present invention provides a method of inhibiting vacuolar H⁺-ATPase activity in a cell where plasma membrane hydrogen ion transport, reabsorption, or secretion is inhibited in a subject. In addition, the present invention provides a method of treating a vacuolar H⁺-ATPase disorder in a subject which comprises, administering to the subject an effective amount of an oligonucleotide which may be substituted or modified in its phosphate, sugar, or base, so as to thereby treat the vacuolar H⁺-ATPase disorder.

Further, the present invention provides a method of treating a vacuolar H⁺-ATPase disorder in a subject where the vacuolar H⁺-ATPase disorder is associated with increased cholesterol or triglyceride, synthesis or production levels. Lipoproteins, such as low density lipoprotein (LDL) receptor synthesis are decreased in a subject, thereby decreasing the cholesterol or triglyceride.

Further, the present invention provides a method of treating a vacuolar H⁺-ATPase disorder in a subject, where the vacuolar H⁺-ATPase disorder is associated with increased acid secretion from parietal cells.

Acid-base disorders include, but are not limited to; respiratory alkalosis, acute respiratory acidosis, chronic respiratory acidosis, metabolic acidosis, and metabolic alkalosis.

Further, the present invention provides a method of treating a vacuolar H⁺-ATPase disorder in a subject, where the vacuolar H⁺-ATPase disorder is associated with bone absorption and resorption. Absorption and resorption disorders may include hypercalcaemia malignancies.

Further, the present invention provides a method of treating a vacuolar H⁺-ATPase disorder in a subject, where the vacuolar H⁺-ATPase disorder is associated osteoporosis. Osteoporosis is defined as any disease process that results in reduction in the mass of bone per volume. Where the result is sufficient to interfere with the mechanical support function of the bone. Osteoporosis may effect any bone but most frequently involves the vertebrae of the lower dorsal and lumbar areas.

In addition, the oligonucleotide may be used as an assay of V-ATPase activity, comprising contacting a cell with an effective amount of the oligonucleotide which may be substituted or modified in its phosphate, sugar, or base so as to thereby inhibit V-ATPase activity.

An "effective amount" as used herein refers to that amount which inhibits vacuolar H⁺-ATPase activity in a cell of a subject. The effective amount may be in the range of 1-100 uM. The effective amount varies with the length of the oligonucleotide. For example, for the 28 base oligonucleotide the preferred effective amount is 10 uM.

Further, as is known to those of ordinary skill in the art effective amounts vary with the type of therapeutic agent. It is known to those of ordinary skill in the art how to determine an effective amount of a suitable therapeutic agent.

The subject may be a mammal or more specifically a human, dog, cat, rodent, or monkey.

Inhibition of V-ATPase activity is defined as the decrease or cessation of the proton pumping ability of the V-ATPase. One may assay the proton pumping ability in a neutral medium, pH 7-8, by employing a dye which turns a color upon protonation or by other described methods [24-27] . As used herein administration means a method of administering to a cell of a patient. Such methods are well known to those skilled in the art and include but are not limited to administration parenterally, orally, intravenously, intramuscularly or subcutaneously. Administration of the agent may be effected continuously or intermittently such that the therapeutic agent in the patient is effective to inhibit V-ATPase activity.

This invention is illustrated in the Experimental Details section which follows. These sections are set forth to aid in an understanding of the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.

EXPERIMENTAL DETAILS

Material and Methods:

The phosphorothioate oligonucleotides used for the inhibition of the vacuolar ATPase studies were synthesized by standard, published methods on an Applied Biosystems 380B DNA synthesizer using the TETD (tetraethylthiuram disulfide) sulfurizing agent. Briefly, after the coupling of each phosphoramidite synthon, the TETD reagent was used instead of the standard iodine reagent to sulfurize instead of oxidize the phosphate linkage.

Following base deblocking, when necessary, each oligonucleotide was purified from sequence by reverse phase high pressure liquid chromatography (HPLC) in a solvent system of triethyl ammonium bicarbonate (TEAB) and acetonitrile. Finally, the oligonucleotides were detritylated, precipitated as either the Na⁺ or Li⁺ salt, and quantified by ^A260 on a spectrophotometer [Applied Biosystems User Bulletin (1991) No. 58] . Further, alternative methods for synthesizing the oligonucleotide are known to one of ordinary skill in the art.

Further, to produce large quantities of the oligonucleotide, it may be synthesized chemically, such as in automated machines, or inserted into a plasmid, such as pBR322, for cloning [3] .

The gel electrophoresis protocol followed the following procedure: collect all the fractions in ~16-17 Eppendorf tubes, with a rate of about 4-5 drops/tube. Take 20 μl from each tube. Freeze the rest of the sample at -80°C. Add 10 μl of sample buffer to the 20 μl fractions in each tube. Keep at R.T. for 60 min. One skilled in the art may follow other described protocols for gel electrophoresis techniques [see, Sa brook, Molecular Cloning; A Laboratory Manual, Cold Springs Harbor Laboratory Press, 6:1-62 (1989).]

Example 1: Assay for activity of Mg/ATP vacuolar

H⁺ATPase

In 1 ml cuvette combine: 950 ul 150 mM KCL-20mM MOPS (3- [N-morpholino]propanesulfonoc acid) pH 7.0, 15 ul 1 mM Acridine Orange, 5 ul Purified chromaffin granules or yeast membranes. Monitor absorbance at 490 nm and 540 nm for 1 min. Add 10 ul 100 mM Mg-ATP. Continue to monitor absorbance until equilibrium is reached. Add 1 ul 1 mM F C C P ( c a r b o n y l c y a n i d e p - trifluoromethoxyphenylhydrazone) .

The preparation of yeast vacuolar membrane vesicles followed the method of Uchida et al. [41] . The preparation of chromaffin granule membrane and the purification of V-ATPases followed by the method of Moriyama et al. [25] .

Exam£le__2: Effects of Phosphorothioate oligonucleotides on the Reconstituted vacuolar H⁺ Pumping Ability

Chromaffin granule membranes were prepared from bovine adrenal glands by the method of Cidon and Nelson [8] . Purification of the reconstitutively active ATPase was accomplished by the method of Moriyama and Nelson [25] . The assay was performed in 1 πL of a solution containing 20 mM MOPS-Tris (pH 7.0), 0.1 M KC1, 0.1 ug valinomycin and 15 uM acridine orange. At the indicated points, 1 mM Mg/ATP and 1 uM FCCP (an uncoupler of the proton gradient) were added. ATP-dependent proton uptake was assayed by following the absorbance changes of acridine orange at 492-540 nm. Figure 2 demonstrates a length dependent inhibition by phosphorothioate homopolymers of thymidine.

Trace 1: SdT28 (5uM)

2: SdT15 (5uM)

3: SdT5 (5uM)

4: Control (no added oligo)

Example 3 Assay for vacuolar H⁺ pumping capability of yeast vesicles

Proton pumping capability of yeast vesicles in the presence of different oligodeoxynucleotides was assayed. The method was identical to that shown for bovine chromaffin granules. Plotted is vacuolar H⁺ pump activity vs. 5 uM SdT3, SdT5, SdT15, SdT28, 0dT5, OdT28. Figure 3 demonstrates a length dependent inhibition by phosphorothioate homopolymers of thymidine.

Example 4; Assay for vacuolar H⁺ pumping capability of chromaffin granules

Proton pumping capability of chromaffin granules in the presence of different oligodeoxynucleotides was assayed. The method was identical to that shown for bovine chromaffin granules. SdC28 inhibits the proton pumping capability of chromaffin granules in a concentration dependent manner. The value of IC50 is 0.16 uM. Figure 4 demonstrates a concentration dependent inhibition by homopolymer of cytidine.

Example 5: Assay of vacuolar H⁺-ATPase activity

Steady state ATPase activity from tissue and cultured cells is measured by colorometric assay of inorganic phosphate as described [10] or formation of radioactive

P; from [γ-³²P]ATP as described by Grubmeyer and Penefsky [16] . The standard mixture for inorganic phosphate assay contains 25 mM MOPS, pH 6.7, 5mM NajATP, 5 mM MgCl₂, and 0.02% asolectin in a total volume of 1.0 ml. Assays is carried out at 30°C for 5 30 min (ATP hydrolysis less than 5%) and is terminated by the addition of trichloroacetic acid to a final concentration of 1.0%. The reaction mixture for radioactive measurement of P_! contains 20 mM Tris HC1, pH 7.5, 2 mM [γ-³²P]ATP in the presence of or absence of MgCl₂ at 25°C for various times. The reaction is stopped by adding an equal volume of cold 40% trichloroacetic acid (w/v) containing 5 mM H₃P0₄ and 1 mM ATP. Then bovine serum albumin (0.7 mg/ml) is added and the mixture is kept on ice for 10 min and centrifuged for 5 min at 13000 x g. The precipitate is washed with 15% trichloroacetic acid containing 1.7 mM H₃P0₄ and 0.3 mM ATP. Finally the precipitate is dissolved in 150 ml of 1 N NaOH and its radioactivity measured in liquid scintillation counter. One unit of enzyme is defined as the amount hydrolyzing 1 μmol of ATP/min.

Examgle_£: In-vivo treatment of oligonucleotides

A 18-28 base sequence phosphorothioate (PS) oligonucleotide is injected into patients either intraperitoneally (IP) or intravenously (IV) , targeted to the V-ATPase. After injecting IP, PS oligonucleotide that are complementary to the initiation codon region of the V-ATPase, compare untreated or sense treated patients.

Oligodeoxynucleotides are prepared on an Applied Biosystems model 380B or 390Z automated synthesis instrument using published methodologies [3] . RT-PCR for detection of mRNA transcripts was carried out. After 60 cycles, 10 μl of amplified product was electrophoresed on a 4% agarose gel and then transferred to a nylon filter. Filters were prehybridized and then probed with a ³²P-end- labeled oligonucleotide probe.

REFERENCES

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Claims

What is claimed is:

1. A method of inhibiting vacuolar H⁺-ATPase activity in a cell which comprises, administering to the cell an effective amount of an oligonucleotide which may be s bstituted or modified in its phosphate, sugar, or base, so as to thereby inhibit the vacuolar H⁺- ATPase activity.

2. The method of claim 1, wherein the oligonucleotide is a oligodeoxynucleotide.

3. The method of claim 1, wherein the substituted oligonucleotide is phosphorothioate.

4. The method of claim 1, wherein the substituted oligonucleotide is a phosphorodithioate.

5. The method of claim 1, wherein the oligonucleotide is a homopolymer.

6. The method of claim 1, wherein the oligonucleotide is a heteropolymer.

7. The method of claim 3, wherein the phosphorothioate is stereo regular.

8. The method of claim 3, wherein the phosphorothioate is stereo non-regular.

9. The method of claim 1, wherein the oligonucleotide is further linked to a 3' or 5' -cholesteryl moiety.

10. The method of claim 1, wherein a 5' or a 3' end of the oligonucleotide is further linked with an intercalating agent, a cross-linker, an artificial endonuclease, a lipophilic carrier or a peptide conjugate or a combination thereof.

11. The method of claim 1, wherein the oligonucleotide is conjugated to a carbohydrate or glycan.

12. The method of claim 1, wherein the oligonucleotide is conjugated to a sulfated carbohydrate.

13. The method of claim 1, wherein the inhibition of the vacuolar H⁺-ATPase is associated with plasma membrane hydrogen ion transport, reabsorption, or secretion.

14. A method of treating a vacuolar H⁺-ATPase disorder in a subject which comprises, administering to the subject an effective amount of an oligonucleotide which may be substituted or modified in its phosphate, sugar, or base, so as to thereby treat the vacuolar H⁺-ATPase disorder.

15. The method of claim 14, wherein the vacuolar H⁺- ATPase disorder is associated with increased cholesterol or triglyceride synthesis or production levels.

16. The method of claim 15, wherein low density lipoprotein (LDL) receptor synthesis is decreased in a subject, thereby decreasing the cholesterol or triglyceride.

17. The method of claim 14, wherein the vacuolar H⁺- ATPase disorder is associated with increased acid secretion from parietal cells.

18. The method of claim 14, wherein the vacuolar H⁺- ATPase disorder is associated with bone absorption and resorption.

19. The method of claim 14, wherein the vacuolar H⁺- ATPase disorder is associated with osteoporosis.

20. The method of claim 14 wherein administering to the subject is oral, intravenous, intramuscular, intratracheal or subcutaneous.